UNIVERSITY OF CALIFORNIA
Los Angeles
Eosinophil Infiltration in Muscular Dystrophy:
Key Characteristics and Contributions to Disease
A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy
in Molecular, Cellular & Integrative Physiology
by
Albert Chi Sek
2020
© Copyright by
Albert Chi Sek
2020
ABSTRACT OF THE DISSERTATION
Eosinophil Infiltration in Muscular Dystrophy:
Key Characteristics and Contributions to Disease
By
Albert Chi Sek
Doctor of Philosophy in Molecular, Cellular & Integrative Physiology
University of California, Los Angeles, 2020
Professor Tomas Ganz, Chair
Duchenne Muscular Dystrophy (DMD) is a genetic disorder that affects 1 in 3,500 live male births. In DMD patients, inactivating mutations in the gene encoding dystrophin prevent expression of this critical cytoskeletal protein that is essential for myofiber integrity. Mutations in the gene encoding dystrophin have also been detected in muscular dystrophy (mdx) mice, which phenocopy the disease seen in DMD patients. As a result of dystrophin deficiency, the skeletal and cardiac muscles undergo severe degeneration accompanied by infiltration with inflammatory cells. Eosinophils are prominent among the leukocytes recruited to dystrophin-deficient skeletal muscle tissues, although their contributions to disease are incompletely understood.
Eosinophils are capable of releasing cytotoxic mediators stored in cytoplasmic granules; as such, eosinophils have been viewed largely as mediators of tissue destruction. I began this work with an evaluation of the extent and impact of eosinophil infiltration on acute muscle damage in muscular dystrophy mice. Towards this end, I generated eosinophil-deficient (mdx.PHIL) and eosinophil-overabundant (mdx.IL5tg) strains of mdx mice. Despite the varied levels of eosinophil
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infiltration detected in skeletal muscle tissues, my studies revealed that there were remarkably
similar levels of muscle damage in mdx, mdx.IL5tg and mdx.PHIL mice. This was evaluated by
four key measures: histopathology scoring, distribution of myofiber sizes, proportion of centrally-
nucleated myofibers and creatine kinase levels.
To evaluate the functional characteristics of eosinophils recruited to dystrophin-deficient muscle tissue, I isolated eosinophils from the quadriceps muscles of mdx, IL5tg and mdx.IL5tg mice at 4 weeks of age, extracted RNA and performed RNA-sequencing. I identified 393 transcripts that were expressed at 5-fold higher levels in the eosinophils isolated from the muscle tissues of mdx mice when compared with those from IL5tg mice. Many of these transcripts encode tissue remodeling proteins (e.g., Ctss, Ctsk, Mmp19) as well as cell surface proteins (e.g., Cd14,
Slc15a3, Trem2). By contrast, I identified 238 transcripts at 5-fold higher levels in eosinophils from the muscle tissues of IL5tg mice when compared with those from mdx mice. Many of these latter transcripts encode eosinophil-specific proteins (e.g., Ear1, Epx, Prg2) or proteins involved with cell cycle regulation.
The findings that suggest differential expression of Trem2 are particularly intriguing, as
TREM2 has been shown to promote the proliferation, activation and survival of myeloid cells in response to tissue damage. Hence, in order to evaluate the role of Trem2 in mediating eosinophil survival within dystrophin-deficient muscle tissues, we are currently generating mdx.Trem2-/- mice
and will be in a position to evaluate eosinophil infiltration and survival in the muscle tissues of
these mice. While generation of this strain is ongoing, I have determined that Trem2 is critical for
full eosinophil development in both ex vivo and in vivo experimental studies.
Taken together, these findings add to the growing body of evidence that suggests that
eosinophils do not universally promote tissue destruction. In addition, findings presented in this
dissertation highlight the unique features of eosinophils that may be uncovered through RNA-
sequencing and analysis. Among the most intriguing findings, I established that Trem2 is critical
iii for efficient eosinophil development. Further work will elucidate the regulation and functions of
TREM2 expression in eosinophils.
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The dissertation of Albert Chi Sek is approved.
John P. Chute
Elizabeta Nemeth
Eric Pearlman
Helene F. Rosenberg
James G. Tidball
Tomas Ganz, Committee Chair
University of California, Los Angeles
2020
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For Mom, Dad
and Grace
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TABLE OF CONTENTS
Abstract of the Dissertation ii
List of tables and figures x
Acknowledgements xiii
Vita xvi
Chapter 1: Introduction 1
Duchenne Muscular Dystrophy: A Severe Genetic Disorder 2
The mdx mouse model of Duchenne Muscular Dystrophy 3
Inflammation as a critical feature of muscular dystrophy pathogenesis 5
Eosinophil infiltration in dystrophin-deficient muscle tissue 6
Study of eosinophils in vivo and ex vivo: critical reagents and mouse models 7
This dissertation 8
References 14
Chapter 2: Detection and Isolation of Mouse Eosinophils from Muscle Tissue
using Flow Cytometry 21
Preface 22
Abstract 23
Introduction 23
Methods and Results 25
Discussion 28
References 35
Chapter 3: Eosinophils Do Not Drive Acute Muscle Pathology in the mdx
Mouse Model of Duchenne Muscular Dystrophy 38
Preface 39
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Abstract 41
Introduction 41
Materials and Methods 42
Results 43
Discussion 47
References 48
Addendum 52
Chapter 4: Transcriptomic Profile of Eosinophils Recruited to Muscle Tissue
of Dystrophin-deficient (mdx) mice 59
Preface 60
Abstract 61
Introduction 62
Materials and Methods 63
Results 65
Discussion 70
References 79
Chapter 5: Role of TREM2 in Modulating the Development and Characteristics
of Eosinophils 82
Preface 83
Abstract 85
Introduction 85
Materials and Methods 87
Results 91
Discussion 96
References 106
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Chapter 6: Conclusion 109
Eosinophilic infiltration does not always promote tissue destruction 110
Eosinophil infiltration in musculoskeletal disorders 111
RNA-sequencing enables functional characterization of eosinophils 113
Future Directions – Regulation and Function of TREM2 on Eosinophils 115
Concluding Remarks 117
References 118
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LIST OF TABLES AND FIGURES
Chapter 1
Figure 1.1 The mdx mouse model of Duchenne Muscular Dystrophy 11
Figure 1.2 Eosinophil infiltration and histopathology in quadriceps muscle
tissue from mdx mice 12
Figure 1.3 Eosinophil characteristics: functional mediators and receptors 13
Chapter 2
Figure 2.1 Method for generating single cell suspensions from skeletal
muscle 31
Table 2.1 Antibodies used for detection and isolation of eosinophils
from skeletal muscle tissue 32
Figure 2.2 Flow cytometry strategy to identify eosinophils in skeletal
muscle tissue 33
Figure 2.3 Morphological evaluation of SigF+CD11c+ and SigF+Gr1+
populations 34
Chapter 3
Figure 3.1 Histopathology and quantitative determination of eosinophils in
the quadriceps muscle from wild-type, mdx, eosinophil-deficient
PHIL, and mdx.PHIL mice 43
Figure 3.2 Histopathology and quantitative determination of eosinophils in
the quadriceps muscle from IL-5 transgenic and mdx.IL5
transgenic mice 44
Figure 3.3 Quantitative indicators of muscle pathology 45
Figure 3.4 Eosinophil recruitment to muscle tissue 46
Figure 3.5 Cytokines detected in muscle tissue lysates 47
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Figure 3.S1 Additional images featuring lesions in quadriceps muscle from
mdx mice 50
Figure 3.S2 Detection of centrally-located nuclei 51
Figure A1. Quantification of myofiber integrity and neutrophil infiltration
in quadriceps muscle tissues from mice at 4 weeks of age 56
Figure A2. Histopathology and eosinophil infiltration in quadriceps muscle
tissues from mice at 8 weeks of age 57
Figure A3. Histopathology and quantification of myofiber integrity in
quadriceps muscle tissues from mice at 8 weeks of age 58
Chapter 4
Figure 4.1 Isolation of RNA from eosinophils from skeletal muscle tissues 74
Figure 4.2 Quality of RNA isolated from eosinophils from skeletal muscle
tissues 75
Figure 4.3 Transcripts and gene ontology pathways that are differentially
expressed in mdx vs IL5tg muscle eosinophils 76
Figure 4.4 Differentially expressed transcripts that are associated with
tissue remodeling 77
Figure 4.5 Differentially expressed transcripts that encode cell surface
proteins 78
Chapter 5
Figure 5.1 Differentiation of eosinophils ex vivo and detection of TREM2 100
Figure 5.2 Trem2 is required for efficient eosinophil expansion ex vivo 101
Figure 5.3 Trem2 gene-deletion alters eosinophil homeostasis in
the bone marrow 102
Figure 5.4 Trem2 is not critical for eosinophil recruitment in response
to a Th2-inflammatory stimulus 103
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Figure 5.5 Generation of mdx.Trem2-/- mice 104
Figure 5.6 Variable expression of TREM2 on human peripheral blood
eosinophils and correlation with donor BMI 105
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ACKNOWLEDGEMENTS
When I started graduate school in the fall of 2014, I had no idea of the non-linear, fast- moving and exponentially-rewarding journey that lay ahead. Through these experiences, I developed scientific and technical expertise, but also became a stronger writer, better presenter, and more critical thinker. I could not have imagined a better group of people to accompany and guide me along this remarkable period of personal and professional growth.
First and foremost, I am immensely grateful for the guidance that my adviser Dr. Helene
Rosenberg provided on a regular basis. Although I started this dissertation project well into my graduate training, Dr. Rosenberg encouraged me to pursue this project tenaciously and strategically. As a result of her spectacular mentorship, I was able to publish my key findings within a short time frame and to hone my communication skills, notably in writing and presenting.
I am also thankful for the technical guidance and support that other members of Dr.
Rosenberg’s research group provided. These include Caroline Percopo, who was immensely helpful in teaching me flow cytometry and was always prepared to discuss challenging experiments; Michelle Ma, who provided guidance on the molecular biology techniques; and fellow trainees including Wendy Geslewitz, Julia Krumholz, Jamie Redes, Gordon Wong, Ajinkya
Limkar and Eric Mai. In addition, I was fortunate during my graduate studies to continue to receive feedback and mentorship from my former supervisor, Dr. Kirk Druey, and members of his research group, including Dr. Sherry Xie and Dr. Eunice Chan.
I would like to thank members of my doctoral committee for facilitating my professional development and for their guidance and support during my graduate studies. Dr. Tomas Ganz and Dr. Elizabeta Nemeth encouraged me to continue with graduate training, despite early setbacks, and to approach projects with an inquisitive and creative mindset. Dr. James Tidball introduced me to the fascinating world of muscular dystrophy, while Dr. John Chute challenged me to pursue the most critical aspects of a research project. And, Dr. Eric Pearlman provided immensely helpful input on working with granulocytes and on succeeding in graduate school.
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I would like to thank the terrific Molecular, Cellular and Integrative Physiology (MCIP) PhD program at UCLA, which has facilitated much of my professional development. I enjoyed the coursework, the community of scholars, and the faculty that worked so tirelessly to ensure the success of its trainees. In particular, I would like to thank Dr. Mark Frye, the program chair, and
Ms. Yesenia Rayos, graduate student affairs officer of the MCIP Program; I enjoyed their company and appreciated their genuine concern and assistance throughout graduate school.
Finally, this dissertation would not be possible without the support of family, friends and classmates who provided advice, listened to my practice presentations and, of course, offered me some fun outside the laboratory. These include my parents, James and Linda Sek; my sister,
Grace Sek; my friends (in no particular order): Steven Chan, Francois de Mets, Pipat Piewgnam,
Murto Wickens, Ninad Jog, Pop Wongpalee, Chris Shen, Frank Lin, Jalal Buckley, Thomas
Calder, Duy Tran, Michael Otto, Wei-Sheng Chen, Albert Ma, Josh Norwood, Adam Moua, Emilio
Ronquillo, and Jenny Chen Link. I am proud to have completed this academic journey with my stellar cohort within the MCIP PhD program: Shuin Park, He Iris Wang, Adam Gomez, Ivan Flores,
Michael Liu, Sara Ranjbarvaziri.
Chapter 1 contains figures that have been reprinted with permission from Sicinski, P., et al., The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science,
1989. 244(4912): p. 1578-80; from Fairclough, R.J., M.J. Wood, and K.E. Davies, Therapy for
Duchenne muscular dystrophy: renewed optimism from genetic approaches. Nat Rev Genet,
2013. 14(6): p. 373-8; and from Rosenberg, H.F., K.D. Dyer, and P.S. Foster, Eosinophils: changing perspectives in health and disease. Nat Rev Immunol, 2013. 13(1): p. 9-22.
Chapter 2 contains findings which will be published in the upcoming paper “Detection of
Mouse Eosinophils from Tissue by Flow Cytometry and Isolation by Fluorescence-Activated Cell
Sorting (FACS)”, which will be published in Methods Molecular Biology, 2020 (in press). Caroline
M. Percopo, Ajinkya R. Limkar, Helene F. Rosenberg are co-authors. I designed experiments,
xiv
analyzed findings, and prepared data for publication. Caroline M. Percopo and Ajinkya R. Limkar
also designed and contributed experimental findings. Helene F. Rosenberg conceived project
design and wrote first draft. All authors reviewed the manuscript prior to publication.
Chapter 3 is a reprint of “Eosinophils Do Not Drive Acute Muscle Pathology in the mdx
Mouse Model of Duchenne Muscular Dystrophy” from Journal of Immunology 2019; 203(2):476-
484. Ian N. Moore, Margery G. Smelkinson, Katherine Pak, Mahnaz Minai, Roberta Smith,
Michelle Ma, Caroline M. Percopo, Helene F. Rosenberg are co-authors. I designed experiments,
performed most of the experimental work, coordinated collaborations, analyzed data, generated
the primary figures and methods section, and assisted with the first and subsequent drafts of the
manuscript. Ian N. Moore provided input on the design of the critical histology and
immunohistochemistry studies, performed the pathology scoring, provided critical descriptions of
the pathological findings, and reviewed the manuscript. Margery G. Smelkinson performed the analysis on muscle fiber size, central nuclei and PAX7+ cells, provided me with insight and instruction, wrote the methods section for this part of the manuscript, and reviewed the manuscript. Katherine Pak provided me with primary instruction on muscle biology, with information including, but not limited to, dissection, freezing and fixation, reagents in common use in the field, and reference to important literature; she has read and reviewed the manuscript.
Mahnaz Minai worked with Ian Moore to prepare tissue sections for analysis and evaluation and reviewed the manuscript. Roberta Smith performed and provided input on the serum creatine kinase assays and reviewed the manuscript. Michelle Ma and Caroline M. Percopo designed and assisted with experiments, prepared and reviewed the manuscript. Helene F. Rosenberg designed the project, reviewed the results on an ongoing basis, provided insight and direction, and wrote the first and subsequent drafts of the manuscript.
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VITA
Albert Chi Sek
EDUCATION
2014 – 2020 Ph.D. Candidate in Molecular, Cellular & Integrative Physiology University of California, Los Angeles National Institutes of Health Graduate Partnership Program
2008 – 2012 B.S. in Microbiology University of California, Davis
RESEARCH EXPERIENCE
2017 – present Predoctoral Fellow/PhD Candidate National Institute of Allergy and Infectious Disease (NIAID) Adviser: Helene Rosenberg, M.D., Ph.D.
2014 – 2017 Graduate Student Researcher University of California, Los Angeles Advisers: Tomas Ganz, M.D., Ph.D. & Elizabeta Nemeth, Ph.D.
2012 – 2014 Postbaccalaureate Research Fellow National Institute of Allergy and Infectious Disease Adviser: Kirk Druey, M.D.
2010 – 2012 Undergraduate Researcher University of California, Davis Adviser: Scott Dawson, Ph.D.
TEACHING & COMMUNICATION EXPERIENCE
2018 – present Staff Writer NIH Graduate Student Chronicles
2018 – 2019 Mentor to High School Students Prince George’s County, Maryland
2018 Special Volunteer Office of Strategic Planning & Evaluation, NIAID/NIH
2016 – 2017 Graduate Teaching Assistant, Department of Physiology, UCLA Foundations in Physiological Sciences, 2 sections, 17 students/year
HONORS AND AWARDS
2019 Presidential Award, Society for Leukocyte Biology
2019 NIH Graduate Student Research Award in Immunology
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2019 International Eosinophil Society Travel Award
2019 NIAID Fellows Workshop Poster Award, 1st Place
2018 NIH Fellows Award in Research Excellence: Immunology (Innate)
PROFESSIONAL ACTIVITIES
2017 – 2020 National Institutes of Health Graduate Student Council Co-Chair of Graduate Student Council, 2019-2020 Chair of Mentorship, Volunteering & Outreach Committee, 2017 – 2019
2014 – 2017 Molecular, Cellular & Integrative Physiology Graduate Program Retreat Planning Committee, 2015 – 2017 Distinguished Speaker Selection Committee, 2014 – 2016
PUBLICATIONS
1. Percopo CM, Limkar AR, Sek AC, Rosenberg HF. Detection of Mouse Eosinophils from Tissue by Flow Cytometry and Isolation by Fluorescence-Activated Cell Sorting (FACS). Methods Mol Bio, ed. G. Walsh; in press, 2020.
2. Limkar AR, Mai E, Sek AC, Percopo CM, Rosenberg HF. Frontline Science: Cytokine- mediated developmental phenotype of mouse eosinophils: IL5-associated expression of the Ly6G/Gr1 surface antigen. J Leukoc Biol; in press, 2019.
3. Redes JL, Basu T, Chan EC, Sek AC, Zhao M, Krishnan R, Rosenberg HF, Druey KM. Aspergillus fumigatus-secreted alkaline protease 1 (Alp1) mediates airways hyperresponsiveness in severe asthma. Immunohorizons, 2019. 3(8): pp.368-377.
4. Sek AC, Moore IN, Smelkinson MG, Pak K, Minai M, Smith R, Ma M, Percopo CM, Rosenberg HF. Eosinophils do not drive acute muscle pathology in the mdx mouse model of Duchenne Muscular Dystrophy. J Immunology, 2019. 203(2): pp. 476-484.
5. Ma M, Percopo CM, Sturdevant DE, Sek AC, Komarow HD, Rosenberg HF. Cytokine Diversity in Human Peripheral Blood Eosinophils: Profound Variability of Interleukin-16. J Immunology, 2019. 203(2): pp. 520-531.
6. Aschemeyer S, Qiao B, Stefanova D, Valore EV, Sek AC, Ruwe TA, Vieth KR, Jung G, Casu C, Rivella S, Jormakka M, Mackenzie B, Ganz T, Nemeth E. Structure-function analysis of ferroportin defines the binding site and an alternative mechanism of action of hepcidin. Blood, 2018. 131(8): pp.899-910.
7. Sek AC, Xie Z, Terai K, Long LM, Nelson C, Dudek AZ, Druey KM. Endothelial Expression of Endothelin Receptor A in the Systemic Capillary Leak Syndrome. Plos One, 2015. 10(7): pp. e0133266.
8. Hagen KD, Hirakawa MP, House SA, Schwartz CL, Pham JK, Cipriano MJ, De La Torre MJ, Sek AC, Du G, Forsythe BM, Dawson SC. Novel Structural Components of the Ventral Disc and Lateral Crest in Giardia intestinalis. Plos Negl Trop Dis, 2011. 5(12): p. e1442
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CHAPTER 1
Introduction
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Duchenne Muscular Dystrophy: A Severe Genetic Disorder
Duchenne Muscular Dystrophy (DMD) is an X-linked recessive disorder of progressive
muscle degeneration that affects 1 in 3,500 males [1, 2]. The genetic lesion underlying this
disorder is in the gene encoding dystrophin, a large protein (427 kDa) that anchors the intracellular
cytoskeleton of muscle fibers with the surrounding extracellular matrix [1, 2]. Inactivating
mutations in the dystrophin gene result in myofiber degeneration accompanied by inflammation
[1-4]. Mutations that partially preserve the dystrophin reading frame result in reduced levels of
dystrophin protein and a milder form of the disease known as Becker Muscular Dystrophy (BMD)
[1, 5, 6].
Between the ages of 2 to 5, DMD patients will begin to exhibit symptoms such as an
unsteady gait and difficulty rising from the floor [1, 2]. Often, DMD patients will need to use both their arms and legs to push themselves up, in what is known as Gower’s sign [2]. By adolescence,
at around 12-15 years of age, DMD patients are typically wheelchair-bound and often do not
survive beyond the age of 20 typically due to failure of the respiratory or cardiac muscles [1, 2].
Treatment of this devastating disorder has relied almost exclusively on oral administration
of glucocorticoids, which function broadly to inhibit inflammation in the muscle tissue [7, 8].
However, the prognosis for DMD patients, even those on treatment remains poor; patients
typically are unable to walk unassisted by 16 years of age and typically do not survive beyond 20
years of age [6, 8]. In response to this severe medical need, in 2016 the U. S. Food and Drug
Administration (FDA) approved the use of a novel therapeutic, eteplirsen (brand name: exondys
51), for the treatment of DMD characterized by mutations in exon 51 of the dystrophin gene [9].
Eteplirsen is an antisense oligonucleotide therapy designed to restore the reading frame of the
dystrophin gene by facilitating a skip over exon 51, a region that includes deleterious mutations
in 10-15% of DMD patients [8-10]. Although eteplirsen was designed to address the underlying
mutation in the defined subset of DMD patients, the therapeutic efficacy has failed to dramatically
augment the level of dystrophin protein produced in the muscle tissues of DMD patients [9, 10].
2
In a study of 11 DMD patients with more than 3 years of open-label use of eteplirsen, Sarepta
reported extraordinarily low levels of dystrophin detected in the muscle tissues of patients treated
with eteplirsen; analysis by quantitative Western Blot revealed expression of dystrophin protein
at 0.9% ± 0.8% compared to that of healthy controls [8, 10]. Furthermore, patients using eteplirsen for 24 and 48 weeks failed to show clinical benefit in a 6-minute walk test compared with patients receiving placebo [9, 10]. Although the FDA advisory committee did not recommend approval, the therapeutic has since been released eteplirsen out of the need to exercise “the greatest flexibility possible” for this lethal genetic disease with limited treatment options [9]. Hence, improved
understanding of the pathogenesis in DMD remains an area of active research and medical need.
The mdx mouse model of Duchenne Muscular Dystrophy
Mutations in the dystrophin gene have been detected in an array of non-mammalian animal models such as Caenorhabditis elegans (nematode) [11, 12], Drosophila melanogaster
(fruit fly) [13, 14] and Danio rerio (zebrafish) [15, 16]; and in mammalian animal models such as
Mus musculus (mouse), rats [17, 18], dogs [19, 20], cats [21, 22] and pigs [23, 24]. Of these, the laboratory mouse remains a popular choice in studying muscular dystrophy because of its rapid generation time and the plethora of genetic tools available [25, 26].
In the early 1980s, an inactivating mutation in the dystrophin gene arose spontaneously in a laboratory strain of C57BL/10ScSn mice [27]. These mice, later termed mdx for muscular
dystrophy on the x chromosome, exhibited abnormally high levels of serum creatine kinase (a
biomarker for muscle damage) and presented histological evidence of muscle damage and inflammation [27]. Subsequent genetic analysis revealed a single point mutation (C T) in exon
23 of the dystrophin gene that results in a premature stop codon and prevents expression of functional, full-length dystrophin protein within the muscle tissues (Fig 1) [28-31]. Since this discovery, mdx mice (JAX Stock No. 001801) and their corresponding dystrophin-sufficient
C57BL/10ScSn parent strain (JAX Stock No. 000476) have been used widely to study the
3
pathogenesis of Duchenne Muscular Dystrophy and are a major resource used in this dissertation
[25, 26].
Skeletal muscle tissues from mdx mice exhibit distinct, well-defined periods of tissue damage, regeneration and fibrosis. Prior to three weeks of age, skeletal muscle tissues from mdx mice are histologically indistinguishable from that of dystrophin-sufficient, healthy controls [26,
32]. However, at three to four weeks of age, mdx mice exhibit the onset of acute muscle injury within the hindlimb muscles [33, 34]. This is characterized by prominent regions of myofiber
swelling and myofiber degeneration, which is accompanied by edema, leukocyte infiltration, and
mineralization [3, 4, 32, 33]. Following the onset of acute muscle injury, mdx mice maintain
successful regeneration of the skeletal muscle tissues during 6 to 12 weeks of their lifespan [33,
35]. At around 1 year of age, mdx mice will exhibit severe, irreversible fibrosis of the skeletal muscle tissues [33, 36].
Compared with DMD patients, mdx mice manifest a milder form of the disease, presenting complete ambulation, minimal wasting and fibrosis of skeletal muscle tissues, and robust
regenerative processes in the skeletal muscle tissues [26, 37]. Several hypotheses have been
raised to explain the milder disease course seen in mice, including upregulation of the compensatory, functional homologs (utrophin, α7β1 integrin) in murine muscle tissues [2, 38-40] and the background strain of mdx mice [41]. Accordingly, several research groups have generated strains of mdx mice that have mutations in modifier genes (e.g., cytidine monophosphate sialic acid hydroxylase-knockout cmah-mdx) [42], lack functional homologs (e.g., dystrophin-utrophin
double-knockout dko, α7-integrin-dystrophin double knockout α71-DkO) [43, 44], or exhibit
progressive degeneration (e.g., telomerase-deficient mdx/mTR) [45]. Despite the advent of these mdx-manipulated mouse strains, their use has not been widely adopted due to premature deaths, difficulty breeding, and the potential confounding influence of a second genetic manipulation that is not otherwise observed in DMD patients.
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Inflammation as a critical feature of muscular dystrophy pathogenesis
Although inactivating mutations in the dystrophin gene result in myofiber fragility, increasing evidence indicates that the immune response participates in key remodeling events
that influence the course of disease [3, 46, 47]. For example, administration of inflammation-
suppressing glucocorticoids has been shown to limit leukocyte infiltration in the muscle tissue [48-
50] and to improve muscle function in DMD patients [51, 52].
Among the leukocytes recruited to dystrophin-deficient skeletal muscle tissues,
macrophages are by far the most prevalent, exceeding 3 x 104 cells per cubic millimeter of tissue
[3, 34, 53-55]. In muscular dystrophy, macrophages exert distinct and multifaceted roles in both
exacerbating and ameliorating the level of muscle damage and inflammation [3]. At the onset of
acute muscle injury, dystrophin-deficient muscle tissues are dominated by pro-inflammatory, M1-
biased macrophages that release cytolytic free radicals that lyse myofiber membranes [56].
Repeated injections of mdx mice with anti-F4/80 antibody reduced infiltrating macrophage
populations and reduced the number of muscle lesions detected in the acute injury phase [34].
Following acute muscle injury, infiltrating macrophage populations are dominated by the M2-
phenotype and are distinguished by increased expression of the anti-inflammatory cytokine
Interleukin-10 [56]. Here, M2-biased macrophages limit the level of muscle damage by competing
for arginine, a substrate required for the generation of the free radical nitric oxide [57]. M2-biased
macrophages also promote muscle regeneration through increased expression of the myogenic
factors such as Klotho and Insulin-like growth factor 1 [3, 58].
Though less well-studied, leukocytes including eosinophils [53, 59, 60], neutrophils [56,
61, 62], mast cells [53, 63-67] and lymphocytes [53, 54, 68-70] are prominent within dystrophin-
deficient skeletal muscle tissues; the role of eosinophils in muscular dystrophy is described in the
following section. Neutrophils have been shown through an antibody-based depletion study to
promote muscle damage [61], although the specific cytolytic mechanism remains to be elucidated.
Meanwhile, the impact of mast cells on dystrophic muscle damage is less clear; prior studies that
5
have inhibited mast cell degranulation [71] or have injected mast cell granules directly into the
muscle tissue [72] have not achieved consistent or physiologically-meaningful results,
respectively. Lastly, cytotoxic T-lymphocytes have been shown through an antibody-based
depletion study to drive dystrophic muscle damage due in part to the release of the cytolytic
protein perforin [68, 69].
Eosinophil infiltration in the dystrophin-deficient muscle tissue
Although eosinophils are detected only rarely in healthy muscle tissues, eosinophil
infiltration is prominent in disorders associated with severe muscle damage: eosinophilia-myalgia
syndrome [73-75], eosinophilic myositits [76-79], Limb-girdle Muscular Dystrophy type 2A
(LGMD2A) [80, 81], and Becker Muscular Dystrophy [82]. Eosinophil infiltration is also detected
in the skeletal muscle tissues of Duchenne Muscular Dystrophy patients [53, 80] and muscular
dystrophy (mdx) mice (Fig 2) [53, 60]. Earlier work by Cai and colleagues [60] described eosinophils and their degranulation products in association with damaged myofibers in mdx mice at 4 weeks of age. Interestingly, and seemingly paradoxically, Wehling-Henricks and colleagues
[53] determined that deletion of the gene encoding the eosinophil granule protein Major Basic
Protein-1 (MBP), a key cytotoxic mediator, did not ameliorate the level of muscle damage in mdx
mice.
Although eosinophils can mediate tissue damage secondary to the release of granule
proteins such as MBP, eosinophils can also exert beneficial, immunomodulatory roles. For
example, eosinophil granules contain a wealth of potent, immunomodulatory mediators such as
pre-formed cytokines, chemokines and growth factors (Fig 3); for a more comprehensive list,
please see the excellent reviews by Lee and colleagues [83] and Rosenberg and colleagues [84].
Indeed, emerging evidence in the field indicates that tissue eosinophils function primarily to
regulate the “local immunity and/or remodeling/repair” processes in what is known as the LIARR
hypothesis of eosinophil function [85].
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As but one example, Heredia and colleagues reported that eosinophils were critical for the repair of muscle tissue following injury induced by experimental injection with snake cardiotoxin
[86]. Here, eosinophils were critical sources of the pro-regenerative cytokine interleukin-4, which
facilitated the proliferation of fibro-adipogenic precursors that support myogenesis. Eosinophil-
deficient mice exhibited impaired regeneration of the muscle tissue following damage.
Subsequent studies along these lines have confirmed that eosinophils mediate pro-regenerative
roles in response to experimentally induced damage to the liver [87] or the urogenital tract [88].
These studies have promoted our understanding of the pro-regenerative capabilities of
eosinophils responding to tissue damage. However, eosinophil functions in the dystrophin-
deficient muscle microenvironment may differ from those observed in the acute model of muscle
damage induced by injection of snake cardiotoxin. For instance, in muscular dystrophy, tissue
damage may or may not be mediated by eosinophils that sense cellular defects secondary to
dystrophin-deficiency; by contrast, eosinophils responding to cardiotoxin-induced muscle injury
are thought to be involved exclusively with the regenerative processes. Furthermore, muscle
tissues from muscular dystrophy (mdx) mice exhibit chronic tissue damage that may activate
eosinophils in a distinct manner compared with eosinophils activated by acute muscle damage.
Study of eosinophils in vivo and ex vivo: critical reagents and mouse models
In order to investigate the role of eosinophils in muscular dystrophy, I utilized several key
resources that permitted the identification and manipulation of eosinophils in vivo. First, to identify
eosinophils, I utilized an antibody against the eosinophil granule protein major basic protein-1 (α-
mMBP); this antibody was generously provided by the Lee Laboratories (Mayo Clinic). To
manipulate the levels of eosinophils in vivo, I utilized two key resources that were provided by the
Lee Laboratories (Mayo Clinic): the interleukin-5 transgenic (IL5tg/NJ.1638) and the PHIL
transgenic mouse. As interleukin-5 (IL-5) is critical to all aspects of eosinophil biology including
its development, IL5tg mice exhibit increased levels of eosinophils in the blood and bone marrow
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as well as eosinophil infiltration into most organ systems. By contrast, the PHIL mice harbor a
transgene encoding diphtheria toxin A expression under the control of an eosinophil lineage-
specific promoter; as a result of transgene expression, eosinophil precursors commit cyto-suicide
in the bone marrow, leading to the absence of eosinophils in vivo.
This Dissertation
In this dissertation I report new findings that relate to the identification, characterization and impact of eosinophils in muscle tissue using the mdx mouse model of muscular dystrophy. As such, I have divided my key findings into the following chapters.
Chapter 2: Detection and Isolation of Mouse Eosinophils from Muscle Tissue using Flow
Cytometry details my development of a flow cytometry protocol in order to characterize
eosinophils from the muscle tissues. Among the key findings, I determined an antigen profile for eosinophils from the muscle tissue as CD45+CD11c-Gr1-MHCIIloSigF+; I also confirmed that eosinophils in the muscle tissue do not express either CD11c or Gr1 antigen. This advance permitted us to enumerate and evaluate eosinophil populations from the muscle tissue. Much of this work was featured in a “Detection of Mouse Eosinophils from Tissue by Flow Cytometry and
Isolation by Fluorescence-Activated Cell Sorting (FACS)”, a protocol in press in Methods in
Molecular Biology; for my contribution, I was listed as a co-author.
Chapter 3: Eosinophils Do Not Drive Acute Muscle Pathology in the mdx Mouse Model of
Duchenne Muscular Dystrophy reports my study of the impact of eosinophil infiltration on the
acute muscle damage observed in muscular dystrophy (mdx) mice. Here, I generated eosinophil-
deficient (mdx.PHIL) and eosinophil-overabundant (mdx.IL5tg) mice and evaluated the level of
muscle damage. Among the key findings was that the degree of muscle damage did not
correspond with the degree of eosinophil infiltration in the muscle tissue. Rather, eosinophils can
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be present in large numbers in the muscle tissue in the absence of tissue damage, as in
Interleukin 5-transgenic (IL5tg) mice. These findings are a reprint of the research originally
published in the Journal of Immunology, with permission granted by the publisher [59].
Chapter 4: Transcriptomic Profile of Eosinophils Recruited to Muscle Tissue of
Dystrophin-deficient (mdx) mice presents my study of key characteristics in eosinophils recruited to the muscle tissues of mdx mice. Here, I utilized RNA-sequencing to develop a comprehensive, unbiased understanding of transcriptional mediators in eosinophils isolated from the muscle tissues of mdx, IL5tg and mdx.IL5tg mice. I identified distinct transcriptomic profiles among eosinophils recruited to the muscle tissues of mdx mice when compared with that of IL5tg.
Among the key differences, eosinophils from the muscle tissues of mdx mice expressed high levels of transcripts encoding tissue remodeling enzymes and cell surface proteins. Increased expression of transcripts encoding cell surface proteins suggests that eosinophils sense and
actively modulate response to the dystrophin-deficient muscle microenvironment. These findings
provide further areas of investigation: in Chapter 5, I explore the expression and function of
Trem2, a transcript which is detected at 68-fold higher levels in eosinophils from the muscle
tissues of mdx mice when compared with those from IL5tg mice.
Chapter 5: Role of TREM2 in Modulating the Development and Characteristics of
Eosinophils reports my analysis of the expression and function of TREM2 in eosinophils. Here,
I identified TREM2 expression on differentiating murine eosinophils and on human peripheral blood eosinophils. Using the Trem2-/- mouse model, I established that Trem2 promotes efficient eosinophil expansion ex vivo and in vivo, but is not required for eosinophil infiltration into the airways of allergen-challenged mice. Ongoing work will evaluate the impact of Trem2 on
eosinophil characteristics, such as recruitment to dystrophin-deficient muscle tissue and
functional responses to the fungal aeroallergen Alternaria alternata. These findings on eosinophil
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characteristics (Chapter 4) and the role of TREM2 (Chapter 5) will be prepared as a manuscript
for publication in 2020.
Chapter 6: Conclusion offers a summary and an analysis of the major findings presented in this dissertation. In addition, I discuss further promising areas of investigation revealed by this work.
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FIGURES
Figure 1. The mdx mouse model of Duchenne Muscular Dystrophy. (A) Mdx mice harbor a
null mutation (C T) in exon 23 of the dystrophin gene. Reprinted with permission from [28] (B)
Dystrophin is expressed in myofibers, where it links the intracellular cytoskeleton with the sarcoglycan complex at the membrane. Null mutations in the gene encoding dystrophin or in any
of the genes encoding sarcoglycan complex proteins have been associated with muscular
dystrophy. Reprinted with permission from [89].
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Figure 2. Eosinophil infiltration and histopathology in quadriceps muscle tissue from mdx
mice. Quadriceps muscle tissue from male mdx mice at 4-weeks of age that has been stained
with (A) anti-Major Basic Protein-1 (α-mMBP) to identify eosinophils or with (B) hematoxylin and eosin to elucidate the histological features. Eosinophil infiltration was prominent in dystrophin- deficient muscle tissue and was associated with leukocyte infiltration, edema, and myofiber damage.
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Figure 3. Eosinophil characteristics: functional mediators and receptors.
Eosinophils are distinguished by the presence of the bilobed nuclei, multiple lipid bodies, and eosin-staining granules. Eosinophil lipid bodies contain key lipid mediators, such as leukotrienes and prostaglandins. Similarly, eosinophil granules contain a wealth of pre-formed cytokines, chemokines, growth factors and enzymes. Release of key lipid mediators and granule contents is thought to mediate eosinophil functions. Eosinophils also contain many pattern recognition and chemoattractant receptors. Reprinted with permission from [84].
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CHAPTER 2
Detection and Isolation of Mouse Eosinophils
From Muscle Tissue using Flow Cytometry
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CHAPTER 2 PREFACE Flow Cytometry is a powerful technique that facilitates simultaneous, quantitative
measurements of antigen expression within a given population of cells. In addition, flow cytometry
and fluorescence-activated cell sorting (FACS) allows analysis and isolation, respectively, of
subpopulations based on antigen expression.
Earlier studies indicated that eosinophils exhibited substantial diversity in the expression of antigens, depending on the tissue and physiological context. As but two relevant examples,
Percopo and colleagues [1] identified a population of SiglecF+Gr1hi eosinophils in the lungs of allergen-challenged mice; this population exhibited a distinct cytokine profile when compared with the more numerous and morphologically-indistinguishable SiglecF+Gr1- eosinophils.
Furthermore, in more recent work (Limkar, Mai, Sek and colleagues [2]), we demonstrated that
the eosinophil-activating cytokine, interleukin-5, drives the expression of the Gr1 antigen on
eosinophils.
This chapter reports the development of a protocol to identify muscle eosinophils by flow
cytometry. Towards this end, we adapted a protocol to prepare muscle single cell suspensions
from Liu et al. [3] and we ultimately identified muscle eosinophils as live CD45+CD11c-Gr1-
MHCIIloSigF+ cells. Subsequently, Kastenschmidt and colleagues [4] published a protocol in which eosinophils in dystrophic muscle were identified as live F4/80+CD11b+SigF+ cells.
The protocol described in this chapter was among those included in a recent publication that I co-authored, Percopo CM, et al. Detection of Mouse Eosinophils from Tissue by Flow
Cytometry and Isolation by Fluorescence-Activated Cell Sorting (FACS). Methods Mol Bio, ed. G.
Walsh; in press, 2020.
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ABSTRACT
While eosinophil infiltration in the muscle tissue is a prominent feature of Duchenne muscular
dystrophy, the contributions of eosinophils to disease pathology are not fully understood. Earlier
studies using the dystrophin-deficient mdx mouse model suggested that eosinophils could
promote characteristic muscle damage, but this hypothesis had not been addressed directly by
an evaluation of dystrophin-deficiency in mice lacking eosinophils. As a first and essential step
towards the study of eosinophils and their role in this disorder, we devised a means to detect,
quantify, and isolate eosinophils from infiltrates identified in quadriceps muscles of mdx mice. To
do this, we generated single cell suspensions from muscle tissue and adapted the flow cytometric
protocol currently in use in our lab for identification of eosinophils in lung and bone marrow. Using
this method, we determined that eosinophils from muscle tissue can be defined as CD45+CD11c-
Gr1-SigF+MHC-IIlo cells. When stained with modified Wright-Giemsa dyes, > 99% of the cells with this antigen profile exhibited ring-shaped nuclei and eosin-staining cytoplasmic granules characteristic of eosinophils. At four weeks of age, leukocyte infiltrates in quadriceps muscles of mdx mice contain a significant fraction of eosinophils (7.7% ± 1.9% of total CD45+ cells). There is also a very small population of SigF+Gr1+ cells (0.11 ± 0.05% of total CD45+ cells), which is similar to the population previously identified in the lungs of allergen-challenged mice; by contrast, no eosinophils were detected in the SigF+CD11c+ fraction. Taken together, these findings represent a crucial technical advance in our ability to enumerate eosinophils and to perform
downstream analyses, including characterization of eosinophil contents and gene expression in
this unique tissue microenvironment.
INTRODUCTION Although rarely detected in healthy muscle tissue, eosinophil infiltration is prominent in
disorders with significant muscle damage (e.g., muscular dystrophy [5-8], eosinophil-myalgia syndrome [9-11]) and in experimental animal models (e.g., injection with cobra cardiotoxin [12]).
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Earlier work examining eosinophil function in the muscle tissue has relied in large part on
immunohistochemical detection using an antibody against the eosinophil granule protein, major
basic protein-1 (α-mMBP1). While this technique is important for determining eosinophil localization and their association with muscle lesions in situ, it is not suited for evaluating the cellular and biochemical properties of these leukocytes.
Multiple studies have demonstrated the heterogeneous nature of tissue eosinophils, including distinct granule contents [1, 13, 14], transcriptomic profiles [15], and even unique expression of cell surface antigens [1, 15-18]. It is thought that cues provided by the tissue
microenvironment, such as cytokines, chemokines and damage-associated molecular patterns
(DAMPs), prime eosinophils to assume unique, functional characteristics. These cues are
abundantly detected in the muscle tissue of dystrophin-deficient mdx mice [19-22] and may influence the characteristics of infiltrating eosinophils.
Flow cytometry is a powerful technique to quantify populations expressing specific antigen profiles and to analyze characteristics such as the level of immunoreactive protein expression.
Similarly, fluorescence-activated cell sorting (FACS) can facilitate isolation of cells with unique antigen profiles for downstream analyses. When applied to the study of eosinophils in muscular dystrophy, flow cytometry and fluorescence-activated cell sorting can be used to i) quantify the level of eosinophils in the muscle tissue across disease models; ii) measure the expression of specific proteins in eosinophils; and iii) isolate eosinophils for downstream applications. Although eosinophils in the bone marrow and in the lung have been identified as CD45+CD11c-Gr1-
SigF+MHC-IIlo cells [23-25], eosinophils have been shown to express distinct cell surface antigens depending on their activation state and tissue source. For example, eosinophils in the thymus and gastrointestinal tract express the CD11c antigen [26, 27] and a subset of eosinophils isolated from the lungs of allergen-challenged mice express the Gr1 antigen [1]. At the start of this study, the
antigen profile of eosinophils infiltrating the muscle tissue had not been fully characterized.
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Here, we describe a protocol that facilitates preparation of single cell suspensions from
muscle tissue followed by procedures to identify and to isolate eosinophils by flow cytometry and
FACS, respectively. We provide a preliminary characterization of eosinophils from the muscle tissue, including their prevalence, antigen profile and cytological features. Finally, we note the impact that this technical advance has on our ability to perform subsequent characterization of eosinophils in the muscle tissue.
METHODS AND RESULTS
Mice mdx mice (C57BL/10ScSn-Dmdmdx/J, 001801) were obtained from Jackson laboratory and bred
under pathogen-free conditions in the National Institute of Allergy and Infectious Diseases (NIAID)
14B South Animal Facility. All experimental procedures were conducted in accordance with
approved animal study protocol LAD 7.
Preparation of Single Cell Suspension of Muscle Tissue
Mice were anesthetized, exsanguinated, and exposed to a lethal dose of isoflurane via inhalation
prior to cervical dislocation. Hindlimb skeletal muscle tissues were carefully dissected to remove
the surrounding fascia and excess fat. Muscle tissues were stored in a 6-well plate containing
Roswell Park Memorial Institute (RPMI) 1640 media, on ice, until completion of tissue collection.
As outlined in Figure 1, muscle tissues were then minced using dissection scissors and transferred to a 50 mL conical tube containing 10 mL of collagenase-containing muscle
dissociation media [RPMI, 10% fetal bovine serum, 1000 U/mL type II Collagenase, 1x Penicillin-
Streptomycin]; tubes were incubated in a rotating incubator at 37°C for 90 minutes. Cells were
pelleted and re-suspended in 10 mL of collagenase and dispase-containing muscle dissociation
media [RPMI, 10% fetal bovine serum, 100 U/mL type II Collagenase, 1 U/mL Dispase, 1x
Penicillin-Streptomycin]; tubes were returned to the rotating incubator and incubated for 37oC for
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30 minutes. Muscle fragments were subjected to 10-successive passages through a 20-gauge
needle before filtration using a cell strainer with 40 µM pores. Cells were examined and counted
using a Luna-Fl Dual Fluorescent Counter (Logos Biosystems). Most cells appeared well-
separated and exhibited > 90% viability when assessed by propidium iodide-uptake. For
fluorescence-activated cell sorting (FACS), single cell suspensions were stained as described in
the following section; for flow cytometry, single-cell suspensions were frozen at 1-10 x 106
cells/mL in freezing media (90% FBS, 10% DMSO) prior to staining and analysis.
Flow Cytometric Analysis of Leukocyte Populations in Muscle Tissue To identify eosinophils in skeletal muscle tissue, we adapted our existing flow cytometry
strategy which had been used previously to identify eosinophils in the lung and bone marrow. Our
strategy utilizes a viability dye (Live/Dead Aqua), the pan-leukocyte marker (CD45), and a specific marker for mature mouse eosinophils (Siglec-F); the evaluation also includes markers commonly found on macrophages, dendritic cell and neutrophils (MHC-II, CD11c, and Gr1). Detailed information regarding antibody concentrations used and vendor information is shown in Table 1.
Fluorescent-labeled cells in suspension were evaluated using an LSR-II flow cytometer
(BD Biosciences) and further analyzed using the FlowJo software, version 10.6 (TreeStar). A
minimum of 200,000 events were recorded per sample. As shown in Figure 2, we set gates for
(i) single cells using a combination of forward scatter-height and forward scatter-area; (ii) non-
debris cells that were >50,000 side scatter-area and >50,000 forward scatter-area; (iii) live cells;
and (iv) CD45+ leukocytes. Within the CD45+ leukocyte population, we identified eosinophils from the (v) myeloid cells, which have relatively higher granularity, using forward scatter-area and side scatter-area; (vi) CD11c-Gr1- cells; (vii) and MHC-IIloSiglec-F+ cells. Although we identified muscle
eosinophils as CD11c-Gr1-SigF+ cells, we also explored the possibility that the smaller populations
of SigF+ CD11c+ or SigF+Gr1+ cells might be eosinophil variants; these findings are presented in the following section.
26
Wright-Giemsa Staining Provides Cytochemical confirmation of CD45+CD11c-Gr1-
SigF+MHC-IIlo cells as Eosinophils
Recent studies have demonstrated remarkable heterogeneity in eosinophil features,
including variations in antigen expression [1, 27, 28] and cytokine content [1, 15, 25] that relate
in part to the activation state and tissue source. Indeed, level of Siglec-F expression has been
considered to be a marker of eosinophil activation [29-31] and has been associated with altered cytokine profiles [15, 32], changes in eosinophil morphology [25], and antigen expression [15].
Likewise, eosinophils from the gastrointestinal tract and thymus express the antigen CD11c [26,
27], and, a subset of lung eosinophils from allergen-challenged mice express the antigen Gr1 [1].
We therefore considered the possibility that some or all the SigF+CD11c+ and/or SigF+Gr1+ cells infiltrating dystrophin-deficient skeletal muscle might also be eosinophils.
Fluorescence-activated cell sorting (FACS) is a specialized application of flow cytometry that permits isolation of cells expressing unique antigen profiles. We utilized FACS to isolate and evaluate the SigF+CD11c-Gr1-, SigF+CD11c+, SigF+Gr1+ populations from the muscle tissues of mdx mice at 4 weeks of age. As shown in Figure 2, panel viii and in Figure 3A, virtually all
(>99%) of the SigF+CD11c-Gr1- cells exhibited prominent eosinophil features when stained with modified Wright-Giemsa dyes (Diff-Quik, Thermo Fisher Scientific), including eosin staining of the granules and ring-shaped nuclei [31, 33, 34]. Flow cytometric analysis revealed robust levels of
Siglec-F in CD11c-Gr1-SigF+ cells, with a median fluorescence intensity (MFI) of 2250. This predominant population of Siglec-F+ cells was detected among the leukocytes infiltrating the muscle tissues of mdx mice at 4 weeks of age (7.7 ± 1.9% of total CD45+ cells). By contrast,
CD11c+SigF+ cells were detected at significantly lower levels within the dystrophic muscle tissue
(Figure 3B, 0.21 ± 0.12% of total CD45+ cells) and did not exhibit eosin staining of the granules
and lacked ring-shaped nuclei.
Interestingly, SigF+Gr1+ cells (0.11 ± 0.05% of total CD45+ cells, Figure 3C) exhibited
segmented nuclei but lacked robust granule staining. While nuclear segmentation is a feature that
27
eosinophils share with neutrophils, Percopo et al. [1] demonstrated that a subset of lung
eosinophils from allergen-challenged mice express the Gr1 antigen; unlike those identified in
muscle, these lung eosinophils were morphologically indistinguishable from the dominant
SigF+Gr1- population. Likewise, Limkar and colleagues [2] found that the Th2 cytokine, IL-5, drives
expression of Gr1 on eosinophils isolated from mouse bone marrow. Thus, by visual inspection
alone, we are unable to define the lineage of the SigF+Gr1+ cells. It may be possible that SigF+Gr1+
cells are a minor population of eosinophils that have undergone degranulation, and thus stain poorly with anionic eosin dyes.
DISCUSSION
In this study, we developed a protocol to identify eosinophils from skeletal muscle infiltrates detected in the mdx mouse model of Duchenne muscular dystrophy. We adapted a protocol from Liu et al. [3] for preparing single cell suspensions of muscle tissue. Using flow cytometry, we identified eosinophils isolated from the skeletal muscle tissue as liveCD45+CD11c-
Gr1-SigF+MHC-IIlo and identified eosinophils as a prominent component of the leukocyte infiltration in muscle tissue from mdx mice at 4 weeks of age. We confirmed that this population exhibited cytological features characteristic of eosinophils and we explored the possibility of eosinophils within the smaller SigF+Gr1+ and SigF+CD11c+ cell populations in the muscle tissue.
More recently, Kastenschmidt et al. [4] identified eosinophil populations from mdx muscle
infiltrates as F4/80+CD11b+SigF+ cells; further study of the cellular features of this population, such
as antigen expression, eosin staining, morphology, is needed to clarify potential similarities and
differences with the antigen profile we have described in this chapter.
Our strategy to identify eosinophils utilizes an antibody targeting the Sialic acid-binding
immunoglobulin-type lectin F (Siglec-F). Expressed on all mouse eosinophils, Siglec-F is a
convenient target for identification of eosinophils by flow cytometry [23]. However, administration
of antibodies targeting Siglec-F have been linked to reduced eosinophil levels in vivo [35] and
28
eosinophil apoptosis ex vivo [36]; a monoclonal antibody targeting the functional paralog in humans, Siglec 8, is currently under clinical exploration as a therapeutic agent in eosinophil- associated disorders [37-39]. Hence, use of an antibody targeting Siglec-F may affect eosinophil viability. As such, we ultimately identified alternative means for identifying eosinophils in lungs from IL5tg and allergen-challenged mice by gating on a combination of granularity, as determined by side scatter, and the antigens CD11c, Gr1, and MHC-II [40]. Unfortunately, this strategy was unsuccessful in identifying eosinophils in the muscle tissues (data not shown). Alternative markers for the identification of eosinophils, such as the IL-5 receptor alpha-subunit (CD125) and the chemokine receptor CCR3, were not ubiquitously or highly co-expressed on SigF+ eosinophils detected in the muscle tissue (data not shown). Hence, use of anti-Siglec-F appears to be necessary to discriminate between eosinophils and other, more numerous leukocyte populations detected in mdx muscle infiltrates. Despite the use of Siglec-F in our flow cytometry and fluorescence-activated cell sorting strategy, eosinophils (CD45+CD11c-Gr1-SigF+MHC-IIlo cells)
were largely viable and appeared intact morphologically (Figure 2).
Eosinophil expression of the CD11c and Gr1 antigens has been demonstrated in specific
tissue contexts. For example, eosinophils residing the thymus and gastrointestinal tract express
the CD11c antigen [26, 27] while a subset of eosinophils recruited to the airways of allergen-
challenged mice express the Gr1 antigen [1]. In this study, we identified a prominent population
of SigF+ eosinophils recruited to the muscle tissue (7.7 ± 1.9% of CD45+ cells); this population did not express the CD11c or Gr1 antigens. By contrast, we detected two minor populations of SigF+
cells that express the CD11c (0.21 ± 0.12% of CD45+ cells) or Gr1 antigens (0.11 ± 0.05% of
CD45+ cells). While the SigF+CD11c+ cells lacked clear cytological evidence of eosinophil features, the SigF+Gr1+ presented some features that have been observed in eosinophils and,
more broadly, in granulocytes. Cytological examination suggests that this population of SigF+Gr1+
cells may belong the neutrophil lineage, a hypothesis that is supported by the association of the
Gr1 antigen with the neutrophil lineage [41]. Definitive exclusion of this minor population of
29
SigF+Gr1+ from the eosinophil lineage may be obtained through genetic analyses, for example,
by measuring the expression of genes specific for neutrophils (e.g., myeloperoxidase, mpo; neutrophil elastase, elane) and eosinophils (e.g., eosinophil peroxidase, epx; major basic protein, prg2). Taken together, our findings indicate that the predominant population of eosinophils in the
muscle tissue lack expression of the CD11c and Gr1 antigens and can be identified as
CD45+CD11c-Gr1-SigF+MHC-IIlo cells.
Critically, flow cytometry and fluorescence-activated cell sorting (FACS) have enabled us
to quantify the level of eosinophil infiltration, to characterize the expression of immunoreactive
antigens on eosinophils, and to isolate eosinophils for downstream analyses. As described in
Chapter 3, we utilized this method to evaluate the level of eosinophil infiltration in the muscle
tissue of eosinophil-deficient and eosinophil-overabundant mdx mouse models; and we utilized
this method to evaluate the expression of the chemokine receptor, CCR3, on eosinophils [7].
Subsequently, in Chapter 4, we described use of this method to isolate eosinophil RNA for
sequencing; this approach has been instrumental in identifying unique, functional characteristics
in this infiltrating leukocyte population. Finally, in Chapter 5, we utilized this approach to
characterize expression of a novel cell surface protein that has not been previously reported on
eosinophils, the triggering receptor expressed on myeloid cells -2 (TREM-2).
30
FIGURES
Figure 1. Method for generating single cell suspensions from mouse skeletal muscle. As described in [3], single cell suspensions were generated from skeletal muscle tissue by enzymatic digestion using 1000 U/mL of type II collagenase followed by a digestion with 1000 U/mL type II collagenase and 11 U/mL dispase. Samples were further separated by 10 successive passages through a 20-gauge needle before filtration with a 40-µM cell strainer.
31
Antibody Vendor & Antibody Fluorophore Target Details Target Catalog Number Dilution
Membrane-permeable dye Thermo Fisher, Live/Dead Aqua 1:100 that enters nonviable cells #L34957
Fc Block BD Biosciences, none non-specific Fc binding 1:100 (CD16/32) #553142 Thermo Fisher, CD45 eF450 detected on leukocytes 1:100 #48-0451-82 detected usually on Thermo Fisher, CD11c Alexa 700 macrophage, dendritic 1:100 #56-0114-82 cells detected primarily on BD Biosciences, Gr1 APC neutrophils but also on 1:100 #553129 some eosinophils
BD Biosciences, SigF PE detected on eosinophils 1:100 #552126
detected primarily on dendritic cells, Thermo Fisher, MHC-II Pe-Cy7 1:100 macrophages, B-cells, T- #25-5321-82 cells
Table 1. Antibodies used for detection and isolation of eosinophils from skeletal muscle tissue.
32
Figure 2. Flow cytometry strategy to identify eosinophils in skeletal muscle tissue.
Eosinophils were identified in the skeletal muscle tissue as LiveCD45+CD11c-Gr1-SigF+MHC-IIlo
cells. Briefly, single cell suspensions of skeletal muscle tissues were gated (1) on forward scatter-
height and forward scatter-area to identify single cells and (2) side scatter-area and forward scatter-area to eliminate debris. Cells were then gated for (3) no staining with the membrane-
permeable Live/Dead dye, (4) presence of the leukocyte marker CD45 and (5) granularity based
on side scatter-area. Of the myeloid population, we identified eosinophils as (6) lacking CD11c,
Gr1 and (7) expressing Siglec-F and low levels of MHC-II. (8) Staining this population with modified Wright-Giemsa dyes (Diff-Quik) confirmed hallmark eosinophil characteristics such as eosin staining of the granules and the ring-form nuclear morphology. Figure reprinted with permission from [7].
33
Figure 3. Morphological evaluation of SigF+CD11c+ and SigF+Gr1+ populations. Specific populations of mdx muscle cells were isolated using fluorescence-activated cell sorting (FACS) and stained with modified Wright-Giemsa (Diff-Quik, Thermo Fisher Scientific). (A) SiglecF+ cells
that lack both the CD11c and Gr1 antigens (CD45+CD11c-Gr1-SigF+MHC-IIlo) comprised the dominant population (7.7% ± 1.9% of CD45+ cells) and exhibited hallmark eosinophil features, such as eosin staining and a ring-shaped nucleus. (B) By contrast, very few cells were detected as SigF+CD11c+Gr1- (0.21 ± 0.12% of total CD45+) and those that were detected were not morphologically eosinophils . (C) Likewise, the small population of SiglecF+Gr1+ cells (0.11 ±
0.05% of total CD45+ cells) exhibited ring-shaped nuclei but only minimal eosin staining of the
granules.
34
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CHAPTER 3
Eosinophils Do Not Drive Acute Muscle Pathology in the mdx Mouse Model of Duchenne Muscular Dystrophy
38
Chapter Preface In this chapter, I present my findings on the contribution of eosinophils to acute muscle
damage that is a characteristic of muscular dystrophy. This includes a reprint of our study that
was originally published in the Journal of Immunology.
Although eosinophil infiltration has been observed as a prominent feature of muscular
dystrophy, their contribution to disease pathophysiology is not well-understood. Traditionally, eosinophils have been viewed as tissue-destructive; this perception arises from their infiltration in pathophysiological states and from the presence of cationic granule contents that have characterized cytotoxicity in in vitro experimental systems. However, recent studies have illustrated beneficial immunomodulatory roles for these granulocytes in an array of disease contexts [35, 36, 49, 50].
Given this reconsideration of eosinophil function, we investigated their contribution to acute muscle damage in mdx mice, which model Duchenne Muscular Dystrophy by harboring null mutations in the dystrophin gene. Towards this end, we generated novel strains of dystrophin- deficient mdx mice, including those in which eosinophils are genetically-ablated (mdx.PHIL) or are present in large numbers (mdx.IL5tg). We also examined factors that might promote eosinophil infiltration in dystrophin-deficient muscle tissue.
Our major finding was that eosinophil infiltration did not drive the acute muscle damage in quadriceps muscle tissue in the setting of dystrophin-deficiency. We determined that eosinophil infiltration in this model was not associated with systemic eosinophilia or with any detectable type
2 immune response. However, we detected increased expression of canonical eosinophil chemo- attractants within the dystrophin-deficient skeletal muscle. As eosinophils are potent immunomodulatory cells, their contribution to muscle repair and inflammation in muscular dystrophy remains to be further elucidated.
These findings provided the impetus for subsequent investigations into the functional characteristics of eosinophils recruited to dystrophin-deficient skeletal muscle tissue (Chapter 4)
39 and into the mechanisms by which eosinophils are recruited to dystrophin-deficient skeletal muscle tissue (Chapter 6).
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42
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44
45
46
47
48
49
50
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ADDENDUM
During the oral defense of the dissertation, I received excellent feedback regarding the contributions of eosinophil infiltration and its role in promoting muscle damage in the mdx mouse model of muscular dystrophy. Many of these points can be clarified by additional studies that have already been performed but were not included in the Sek AC et al. J Immunol, 2019 manuscript contained within this chapter. Hence, findings from these studies are included here as an addendum to this chapter.
Further evaluation of myofiber integrity and inflammation during the acute injury phase of muscular dystrophy
Measuring the distribution of myofiber sizes provides a quantitative assessment of the proportion of myofibers that have been damaged. In our initial assessment (Figure 3B, 3C of this chapter), we observed a normal bell-shaped distribution of myofiber sizes in quadriceps muscle tissues from wild-type and eosinophil-deficient PHIL mice at 4 weeks of age. By contrast, muscle tissues from mdx and mdx.PHIL mice did not exhibit the normal bell-shaped distribution of myofiber sizes. When compared with their respective controls, muscle tissues from mdx and mdx.PHIL mice included sizable proportions of enlarged (> 5000 µm2) or shrunken (< 1000 µm2) myofibers, indicating significant myofiber damage in both groups. Accordingly, we presented our data by comparing the distribution of myofiber sizes in mdx mice and wild-type controls and, separately, and presented the distribution of myofiber sizes in mdx.PHIL and PHIL controls.
These comparisons facilitated a binary understanding of myofiber damage that occurs both in the presence (mdx) and absence (mdx.PHIL) of eosinophil infiltration in dystrophin- deficient muscle tissues. However, these comparisons did not facilitate direct analysis of the reduction in myofiber damage that might accompany eosinophil depletion in muscular dystrophy.
To address this issue, we compared directly the distribution of myofiber sizes in quadriceps muscle tissues from male mdx and mdx.PHIL mice at 4 weeks of age (Figure A1a). The
52
distributions of myofiber sizes were remarkably similar between mdx and mdx.PHIL genotypes
and there were no statistically significant differences in any of the myofiber size bins. In order to
quantify the impact of eosinophil depletion on myofiber damage, we evaluated the proportion of
myofibers within the normal size range observed in quadriceps muscle tissues from wild-type
mice (1001 – 5000 µm2, 87 ± 3.5% of myofibers). Myofibers occurring within this size range can be distinguished from damaged myofibers that are swollen (>5000 µm2) or shrunken (<1000 µm2).
Using this metric, we detected similar proportions of myofibers of normal size in mdx and mdx.PHIL mice (62 ± 5.5 % vs 56 ± 4.6%, Figure A1b). The proportion of myofibers of normal sizes in mdx and mdx.PHIL mice differed significantly from that of their respective wild-type (87 ±
3.5% of myofibers, p = 0.0054) and PHIL (79 ± 6.6, p = 0.0041) controls. Intriguingly, the proportions of myofibers within this normal size range were similar to the proportions of healthy
myofibers detected by nuclear positioning (Figure 3D of this chapter), which were 72 ± 2.3% in mdx mice, 70 ± 1.8% in mdx.PHIL mice. Taken together, these data indicate that eosinophil infiltration has no apparent impact on quadriceps muscle damage at 4 weeks of age in dystrophin- deficient mdx mice.
Next, we asked if manipulation of the eosinophil lineage might have an impact on the other leukocyte populations recruited to dystrophin-deficient muscle tissue. We focused our attention on neutrophils, a subset of myeloid cells that shares many features in common with eosinophils.
As shown in Figure A1c, neutrophils (CD45+CD11c-Gr1+SigF-) were detected at similar levels in preparations of skeletal muscle tissues from mdx (0.90 ± 0.45% of live cells), mdx.PHIL (0.90 ±
1.0% of live cells) and mdx.IL5tg (0.58 ± 0.41% of live cells) mice at 4 weeks of age. Although these data suggest that eosinophil infiltration does not affect the degree of neutrophil infiltration in dystrophin-deficient muscle tissues, eosinophil infiltration may have an impact on the infiltration
of other leukocyte populations, including mast cells and T-lymphocytes.
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Eosinophil Infiltration during the regenerative phase of muscular dystrophy
Muscular dystrophy (mdx) mice exhibit well-defined stages of skeletal muscle
pathophysiology; these include acute muscle injury at 3-4 weeks of age and ongoing regeneration
at 6-12 weeks of age. In our earlier work, we focused exclusively on the 4-week timepoint in order to evaluate the impact of eosinophil infiltration on acute muscle damage in mdx mice. Since eosinophils have been described as critical to successful tissue regeneration in several experimental models, we evaluated the impact of eosinophil infiltration during the regenerative phase of muscular dystrophy. Quadriceps muscle tissues from mdx mice at 8 weeks of age exhibited histological features consistent with ongoing regeneration, including regions with intact myofibers of intermediate size and regions with limited myofiber necrosis (Figure A2a). Major basic protein-1 (MBP)-positive eosinophils were detected in quadriceps muscle tissues from mdx mice at 8 weeks of age (Figure A2b), often near sites of myofiber necrosis. Flow cytometric analysis identified populations of eosinophils (CD45+CD11c-Gr1-SigF+MHC-IIlo) among
preparations of skeletal muscle tissues from mdx and eosinophil-enriched mdx.IL5tg mice at 8
weeks of age; unsurprisingly, eosinophils were rarely detected in preparations of skeletal muscle
tissues from eosinophil-deficient mdx.PHIL mice (Figure A2c). Interestingly, the fraction of eosinophils (% of live cells) was significantly lower in preparations of skeletal muscle tissues from mdx mice at 8 weeks of age compared with 4 weeks of age (0.58 ± 0.31% of live cells versus 1.2
± 0.34% of live cells, respectively; p = 0.007).
Evaluation of muscle pathology during the regenerative phase of muscular dystrophy
As eosinophil infiltration is detected in skeletal muscle tissues from mdx mice during the regenerative phase (8 weeks of age), I asked if eosinophil infiltration might contribute to the successful regeneration of damaged dystrophin-deficient muscle tissue. Towards this end, I evaluated the histopathology of quadriceps muscle tissues from mdx, eosinophil-deficient mdx.PHIL, and eosinophil-enriched mdx.IL5tg mice at 8 weeks of age. Histological evaluation of
54
quadriceps muscle tissue from eosinophil-deficient mdx.PHIL mice at 8 weeks of age revealed
prominent myofiber regeneration, indicated by central positioning of nuclei within myofibers, along
with limited edema and myofiber necrosis (Figure A3a). Similarly, quadriceps muscle tissue from
eosinophil-enriched mdx.IL5tg mice at 8 weeks of age revealed prominent myofiber regeneration,
as indicated by central positioning of nuclei within myofibers, along with limited myofiber necrosis
and robust leukocyte infiltration (Figure A3b).
In order to evaluate quantitatively the impact of eosinophil infiltration on the regeneration
of damaged, dystrophin-deficient muscle tissues, I assessed features such as myofiber integrity
and myofiber regeneration. Quadriceps muscle tissues from eosinophil-deficient mdx.PHIL mice
at 8 weeks of age exhibited similar distribution of myofiber sizes when compared with the mdx
parent strain at 8 weeks of age (Figure A3c), including similar proportions of small myofibers less
than 1000 µm2 (15 ± 3.2 % vs 15 ± 1.5%, respectively) and large myofibers greater than 5000
µm2 (19 ± 5.7% vs 19 ± 3.1%, respectively). Compared with the normal myofiber size range at 8 weeks of age (1001 – 6000 µm2, 80 ± 2.7% of wild-type myofibers), substantial proportions of
myofibers of normal size were detected in mdx (70 ± 1.6%) and mdx.PHIL (70 ± 3.2%) mice;
these data indicate ongoing myofiber regeneration within the muscle tissues of both mdx and
mdx.PHIL mice at 8 weeks of age. Furthermore, quadriceps muscle tissues from eosinophil-
deficient mdx.PHIL and eosinophil-enriched mdx.IL5tg mice at 8 weeks of age exhibited similar
proportions of centrally-nucleated myofibers when compared with the mdx parent strain at 8
weeks of age (Figure A3d; 37 ± 3.9%, 41 ± 7.34% vs 37 ± 3.5%, respectively). Of note, the proportions of centrally-nucleated myofibers detected across dystrophin-deficient mouse strains at 8 weeks were slightly higher than those at 4 weeks of age (Figure 3D; 27 ± 2.4%, mdx; 30 ±
1.9%, mdx.PHIL). Taken together, these data indicate that the presence of eosinophil had no significant impact on the integrity or regeneration of myofibers in 8-week mice during the regenerative phase of muscular dystrophy.
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FIGURES
Figure A1. Quantification of myofiber integrity and neutrophil infiltration in quadriceps
muscle tissues from mice at 4 weeks of age. (A) Distribution of myofiber sizes in quadriceps muscle tissue; data reproduced from Figures 3B and 3C, with modifications. n = 3.0 X 104
myofibers (mdx), 3.2 X 104 myofibers (mdx.PHIL); 2-3 mice per genotype. None of the differences
observed were statistically significant between mdx and mdx.PHIL groups, two-way ANOVA with
multiple comparisons. (B) Quantification of myofibers of intermediate size (1001 – 5000 µm2) in
quadriceps muscle tissues; data reproduced from Figures 3B and 3C with modifications. n = 2-3 mice per genotype, ** p < 0.01, one-way ANOVA with multiple comparisons. (C) Neutrophils
(CD45+CD11c-Gr1+SigF-) as a percent of the total live cells in preparations of quadriceps muscle tissues from male mice at 4 weeks of age. n = 2 – 15 mice per genotype, n.s. = no significant difference, one-way ANOVA with multiple comparisons.
56
Figure A2. Histopathology and eosinophil infiltration in quadriceps muscle tissues from
mice at 8 weeks of age. (A) H&E-stained cross-section of quadriceps muscle tissue from a male
mdx mouse at 8 weeks of age. (B) Immunohistochemical staining with anti-MBP documenting
eosinophil infiltration in quadriceps muscle tissue from a male mdx mouse at 8 weeks of age. (C)
Eosinophils (CD45+CD11c-Gr1-SigF+MHC-IIlo) as a percent of the total live cells in preparations
of quadriceps muscle tissues from male mice at 8 weeks of age. n = 4 – 5 mice per genotype, **** p < 0.0001, n.s. = no significant difference, one-way ANOVA with multiple comparisons.
57
Figure A3. Histopathology and quantification of myofiber integrity in quadriceps muscle
tissues from mice at 8 weeks of age. (A) H&E-stained cross-section of quadriceps muscle
tissue from a male mdx.PHIL mouse at 8 weeks of age. (B) H&E-stained longitudinal section of quadriceps muscle tissue from a male mdx.IL5tg mouse at 8 weeks of age. (C) Distribution of
myofiber sizes in quadriceps muscle tissues from mdx and mdx.PHIL mice at 8 weeks of age; n
= 3 per genotype. None of the myofiber sizes were statistically significant between mdx and
mdx.PHIL groups, two-way ANOVA with multiple comparisons. (D) Proportion of centrally
nucleated myofibers in quadriceps muscle tissues from male mice at 8 weeks of age. ** p < 0.01,
*** p < 0.001, one-way ANOVA with multiple comparisons.
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CHAPTER 4
Transcriptomic Profile of Eosinophils Recruited to Muscle Tissue of Dystrophin-deficient (mdx) mice
59
CHAPTER 4 PREFACE
RNA-sequencing is a powerful technique that allows for simultaneous detection and
quantification of all transcripts contained within a population of cells. As such, RNA-sequencing
provides a comprehensive, unbiased view of the genetic processes underlying key pathways and
functional mediators within cells.
To date, there have been fewer than 7 publications that have utilized RNA-sequencing to
interrogate eosinophil characteristics [1-6]. There are several reasons for the paucity of studies utilizing this approach; chief among them is the perception that eosinophils do not respond transcriptionally to the tissue microenvironment. However, increasing evidence indicates that eosinophils exhibit unique properties, including distinct transcriptomic profiles, that depend in part on the tissue from which they are isolated [1, 4, 5, 7-9]. It is thought that cues provided by the
tissue microenvironment prime eosinophils to assume distinct characteristics that potentiate their function [10].
In muscular dystrophy (mdx) mice, eosinophils are detected in inflammatory infiltrates that are associated with severe muscle damage [11-13]. To develop a comprehensive understanding of the characteristics of eosinophils recruited to skeletal muscle and to explore the role of these cells in the pathogenesis of muscular dystrophy, we examined gene expression with RNA-sequencing.
This chapter represents our successful efforts towards obtaining high-quality RNA from eosinophils, followed by sequencing and analysis of genes that are differentially expressed in eosinophils from distinct microenvironments. Among the differentially expressed genes, we detected high levels of the transcript encoding Triggering Receptor Expressed on Myeloid Cells-
2 (TREM2) in eosinophils from mdx muscle tissue; investigation into the regulation and function of TREM2 on eosinophils forms the basis of subsequent investigation (Chapter 5).
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ABSTRACT
Eosinophils are among the leukocytes recruited to the muscle lesions in muscular dystrophy
(mdx) mice, although their contribution to disease remains incompletely understood. In our earlier
work, we demonstrated that eosinophils do not drive the acute muscle damage in mdx mice but
do contribute immunomodulatory mediators including IL-4 and IL-1RA. To uncover additional
mediators of eosinophil function in the muscle tissue, we isolated and sequenced eosinophil RNA
from the muscle tissues of mdx, IL5tg and mdx.IL5tg mice at 4 weeks of age. Despite abundant
ribonucleases contained within eosinophil granules, we were able to isolate high-quality RNA
(RIN: 8.2 – 9.7) for sequencing. Analysis of our sequencing results revealed distinct transcriptomic profiles among eosinophils isolated from the muscle tissues of mdx and IL5tg mice. We identified
393 unique transcripts that were detected at significantly higher levels in eosinophils from muscle tissues from mdx mice when compared with those from IL5tg; by contrast, we detected 238 unique transcripts that were detected at significantly higher levels in eosinophils from muscle tissue from
IL5tg mice when compared with those from the mdx strain. Among the set of 393 transcripts increased in eosinophils from mdx muscle tissue, we detected many transcripts encoding enzymes associated with tissue remodeling (Ctsk, Ctss, Mmp2, Mmp12) as well as transcripts encoding cell surface receptors (Cd14, Slc15a3, Trem2). Gene ontology pathway confirmed that the set of 393 transcripts elevated in eosinophils from mdx muscle tissue was significantly associated with tissue remodeling pathways such as serine-type peptidase activity (p = 2.2e-07) and collagen binding (p = 9.4e-10). Taken together, these findings challenge the perception of eosinophils as static and end-stage and highlight unique features for further investigation. As such, ongoing work will elucidate the impact of receptors, such as TREM2, on the functions and characteristics of eosinophils in health and disease.
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INTRODUCTION
Eosinophils are granulocytes that are recruited to dystrophin-deficient muscle tissue, where their contributions to disease pathophysiology remain incompletely understood. Recently, we demonstrated that eosinophil infiltration does not drive the acute muscle damage in muscular dystrophy (mdx) mice [13]. These findings are consistent with earlier work by Wehling-Henricks and colleagues, who demonstrated that deletion of the gene encoding the cytotoxic eosinophil granule protein Major Basic Protein-1 (MBP-1), did not have any impact on the level of muscle damage in mdx mice [12].
Rather, we uncovered elevated levels of immunomodulatory mediators associated with
eosinophil infiltration in dystrophin-deficient muscle tissue; these include the pro-regenerative
cytokine interleukin-4 (IL-4) as well as the anti-inflammatory mediator interleukin-1 receptor antagonist (IL-1RA) [13]. Increased expression and release of these eosinophil-derived cytokines
may affect the pathophysiology in muscular dystrophy. While important, IL-4 and IL-1RA
represent but a small fraction of the known repertoire of mediators that are contained within
eosinophil granules and lipid bodies [14]. As such, IL-4 and IL-1RA may work in conjunction with
other eosinophil-derived cytokines to mediate key eosinophil functions in muscular dystrophy.
Along these lines, recent studies have reported unique functional characteristics in
eosinophils responding to pathophysiological contexts. For example, Arnold and colleagues [1]
described distinct transcriptomic profiles in eosinophils isolated from the gastrointestinal tract
following bacterial infection when compared with eosinophils from the gastrointestinal tracts of
uninfected mice. Furthermore, eosinophils from the gastrointestinal tract of infected mice
exhibited higher levels of programmed death ligand-1 (PDL-1) which directly suppressed the
bacterially induced Th1/Th17 response [1].
By extension, eosinophils recruited to the dystrophin-deficient muscle microenvironment may
exhibit unique, as yet undetermined, characteristics that help promote its function within muscular
dystrophy. To uncover these characteristics, we utilized an unbiased, comprehensive approach
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by sequencing eosinophil RNA isolated from the muscle tissues of mdx, IL5tg, and mdx.IL5tg. We compared the transcriptomic profiles of eosinophils isolated from the muscle tissues of mdx mice with those from IL5tg in order to identify key functional mediators for further investigation.
MATERIALS AND METHODS
Mice
Mdx mice (C57BL/10ScSn-Dmdmdx/J, 001801) were obtained from Jackson Laboratory. IL5tg mice were obtained from the Lee Laboratories (Mayo Clinic). Mdx-IL5tg mice were generated by crossing female mdx mice with male IL5tg mice; male offspring were serotyped to determine IL-
5 expression. Only male mice were used in all experiments. All mice were bred and maintained under pathogen-free conditions in the National Institute of Allergy and Infectious Diseases (NIAID)
14B South Animal Facility; all experimental procedures were conducted in accordance with approved animal study protocol LAD7.
Isolation of Eosinophils by Fluorescent-Activated Cell Sorting
As described in Chapter 2, single cell suspensions were generated from hind limb muscle tissue.
These cells were stained with a viability dye (Live/Dead Aqua, ThermoFisher), an Fc receptor blocking antibody (anti-CD16/32, BD Biosciences) and fluorochrome-conjugated antibodies including anti-CD45 and anti-CD11c (eBioscience); anti-Gr1 and anti-Siglec-F (BD Biosciences).
Eosinophils (liveCD45+CD11c-Gr1-SigF+MHC-IIlo cells) were isolated and sorted directly into
TRIZOL-LS (Life Technologies) by fluorescence-activated cell sorting (FACS).
Isolation and Purification of Eosinophil RNA
RNA was isolated from eosinophils that had been FACS sorted into TRIZOL-LS (Life
Technologies). Following the manufacturer’s protocol, the 3:1 ratio of TRIZOL-LS to sample was maintained prior to addition of chloroform. RNA was purified from the aqueous phase, combined
63
with equal volumes of 70% ethanol, and subjected to the RNeasy cleanup kit (Qiagen) to remove
potential contaminants. RNA samples were resuspended in 20-25 µL of RNase-free water and
stored at -80oC prior to quality control analysis.
RNA Quality
RNA samples were loaded onto an RNA Pico Chip (Agilent Technologies) and the electrophoretic trace was analyzed using an the 2100 Bioanalyzer (Agilent Technologies). Based on the ratio of the 28S:18S RNA detected, the 2100 Bioanalyzer assigned RNA integrity number (RIN) scores, which range from 0 (most degraded) to 10 (most intact).
RNA-Sequencing and Analysis
RNA libraries were prepared using the NEBnext Ultra Low Input Total RNA Library Prep Kit (New
England BioLabs) following the manufacturer’s instructions. Paired end-reads of the RNA libraries were sequenced using an Illumina HiSeq 4000 Next Generation sequencer, with more than 100
Million reads per sample. Using the Cutadapt software, reads were trimmed to remove adapters and low-quality sequences before aligning to the mouse reference genome mm10. Transcript abundance was quantified using the RSEM software package and reported as fragments per kilobase of transcript per million reads (FPKM).
Identification of Differentially Expressed Genes
Voom-normalized gene counts from eosinophils from mdx muscle tissue were compared against those from eosinophils from muscle tissue from IL5tg mice. Transcripts that were significantly upregulated more than 5-fold in eosinophils from the muscle tissues of mdx vs. IL5tg mice were identified; statistical significance was determined using limma empirical bayes t-test with multiple
test correction.
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RESULTS
Successful isolation of high-quality RNA from muscle eosinophils
Within their cytoplasmic granules, eosinophils contain abundant ribonucleases that can
complicate the isolation of high-quality RNA for sequencing. As eosinophils represent a
subpopulation of the leukocytes that are recruited to skeletal muscle tissue, efforts to isolate RNA
from this population must be specific for the eosinophil population and rapid to avoid RNA
degradation secondary to endogenous ribonuclease activity. Here, eosinophil RNA was obtained
by isolating eosinophils (CD45+CD11c-Gr1-SigF+MHC-IIlo cells; described in Chapter 2) from the muscle tissues and subsequently extracting RNA from this population using the phenol-based
TRIZOL-LS reagent (Figure 1A). To remove contaminants, eosinophil RNA was further purified using the RNeasy Mini Kit (Qiagen); RNA purification has been utilized in prior studies examining
eosinophil transcriptomes [6, 15, 16]. Furthermore, all experimental procedures were conducted on the same day to limit potential changes to the transcriptome, as well as nucleic acid degradation, that can be associated with prolonged experimental procedures.
A total of 9 RNA samples were collected; these samples represent biological replicates from each of the three relevant genotypes (mdx, IL5tg, mdx.IL5tg). Although 3 - 5 mice were used for each biological sample, the number of eosinophils varied depending on the genotype. For example, approximately 400,000 eosinophils were collected from each mdx sample while >
1,000,000 eosinophils were collected for each mdx.IL5tg sample (Figure 1B). Therefore, the total
RNA yield per sample varied from 12 ng to 547 ng (Figure 1B); these yields were similar to those reported by Wacht and colleagues who successfully isolated ~100 ng of RNA from the peripheral blood eosinophils of healthy volunteers [15].
Eosinophil RNA isolated from the muscle tissues using the method outlined above exhibited minimal degradation. All RNA samples submitted for sequencing had an RNA Integrity Number
(RIN) greater than 8.20, with most samples having a RIN greater than 9.0 (Figure 2). These RIN
65
values are similar to those of Wacht et al. [15] and Barnig et al. [16] who reported RIN scores
between 9.2 - 9.4 in RNA isolated from peripheral blood eosinophils from healthy volunteers.
Distinct transcriptomic profiles in muscle eosinophils
Because eosinophils contain pre-formed cytokines within their cytoplasmic granules,
eosinophils were viewed traditionally as end-stage effector cells incapable of responding
transcriptionally to distinct stimuli. However, analysis of the transcriptome in isolated eosinophils
indicated abundant expression of transcripts: in eosinophils from mdx muscle tissue, a total of
9,525 distinct transcripts were detected at ≥ 2 fragments per kilobase of transcript per million
reads (FPKM); similarly, 9,258 transcripts were detected in eosinophils from muscle tissue from
IL5tg mice and 8,870 transcripts were detected in eosinophils from mdx.IL5tg muscle tissue.
To identify transcripts that may encode key eosinophil characteristics, we compared the transcriptome of eosinophils from the skeletal muscle tissues of mdx mice against the transcriptome of eosinophils from the skeletal muscle tissues of IL5tg mice. We reasoned that eosinophils detected in the presence of severe muscle damage associated with dystrophin- deficiency would differ significantly from eosinophils detected in the absence of muscle damage, as in IL5tg mice [13]. Consistent with our hypothesis, eosinophils from the mdx muscle tissue expressed 393 unique transcripts that were detected at >5-fold higher levels when compared with
eosinophils from the muscle tissues of IL5tg mice (mdx > IL5tg; Figure 3A). Many of the mdx >
IL5tg transcripts encode proteins involved with tissue remodeling such as the cysteine protease
family of cathepsins (e.g., Ctsb, Ctsk, Ctss) and the zinc endopeptidase family of matrix
metalloproteinases (e.g., Mmp19, Mmp14, Mmp2). In addition, many of the mdx > IL5tg
transcripts highly detected in eosinophils from the mdx muscle tissue encode cell surface proteins
such as inflammatory and chemotactic receptors (e.g., Cd14, Ccr3, Nlrp3, Trem2) as well as
transporters (e.g., Slc15a3, Slc22a4). Analysis of the mdx > IL5tg set of transcripts indicated
significant association with tissue remodeling pathways (Figure 3B), such as collagen binding
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(GO ID: 0005518, p = 9.41e-10), extracellular matrix structural constituent (GO ID: 0005201, p =
3.17e-07) and metalloendopeptidase activity (GO ID: 0004222, p = 3.60e-04).
By contrast, eosinophils from the muscle tissues of IL5tg mice expressed 238 unique
transcripts that were detected at >5-fold higher levels when compared with eosinophils from the
muscle tissues of mdx mice (IL5tg > mdx; Figure 3A). Many of the IL5tg > mdx transcripts encode
eosinophil-specific proteins such as eosinophil-associated ribonucleases (e.g., Ear1, Ear2, Ear6) and eosinophil granule proteins such as eosinophil peroxidase (Epx) and major basic protein-1
(Prg2). In addition, many of the IL5tg > mdx transcripts encode proteins involved with cell cycle processes such as histones (e.g., Hist1h2bn, Hist1h3c). Analysis of the IL5tg > mdx set of transcripts indicated significant association with eosinophil-specific pathways such as defense response to nematode (GO ID: 0002215, p = 7.54e-05); as well as cellular proliferation pathways such as chromatin assembly (GO ID: 0031497, p = 1.71e-04).
Eosinophils recruited to mdx muscle tissue exhibit increased expression of transcripts associated with tissue remodeling
Given the association between the mdx transcriptome and tissue remodeling, we examined the level of key transcripts in eosinophils from the muscle tissues of mdx, IL5tg and mdx.IL5tg mice (Figure 4). We identified a set of 32 key transcripts that encode proteins involved with tissue remodeling. In eosinophils from the muscle tissues of mdx mice, these transcripts were detected at relatively abundant levels; this may have profound physiologic impact. For example, 23 of these transcripts were expressed at FPKM values >10 in eosinophils from the muscle tissues of mdx mice; by contrast, only 4 of these transcripts were expressed at FPKM values >10 in eosinophils from the muscle tissues of IL5tg mice.
Of the 32 key transcripts, many encoded enzymes associated with tissue remodeling, including the cysteine protease family of cathepsins as well as the zinc endopeptidase family of matrix metalloproteinases (MMPs). For example, transcripts encoding cathepsin S (Ctss), were
67
detected at 198-fold higher levels in eosinophils from the mdx muscle tissue when compared with
that of IL5tg (p = 1.57e-03). Similarly, transcripts encoding the enzyme, matrix metalloproteinase
12 (Mmp12), were detected at 172-fold higher levels in mdx eosinophils when compared with that
of IL5tg eosinophils (p = 5.17e-04). Both cathepsin S and matrix metalloproteinase 12 have been
associated with the degradation of components of the extracellular matrix [17-21].
In addition, many of the key transcripts encode structural components of the extracellular
matrix in muscle tissue. For example, transcripts of Col22a1, which encodes the alpha 1 chain of type XXII collagen, were detected at 142-fold higher levels in eosinophils from mdx mice when compared with that of IL5tg (p = 2.41e-05). Similarly, transcripts of Col3a1, which encode the alpha 1 chain of type III collagen, were detected at 11-fold higher levels in eosinophils from mdx mice when compared with that that of IL5tg (p = 6.76e-04). Collagens, together with laminins, fibronectins and proteoglycans, form the extracellular matrix that surrounds myofibers [22].
Eosinophils recruited to mdx muscle tissue exhibit increased expression of transcripts encoding cell surface proteins
Given the association between the mdx transcriptome and cell surface proteins, we examined the expression of key transcripts in eosinophils from the muscle tissues of mdx, IL5tg and mdx.IL5tg mice (Figure 5). We identified 17 key transcripts that encode cell surface proteins.
Expression of these transcripts were relatively high: 9 of the transcripts were expressed at FPKM values >10 in eosinophils from the muscle tissues of mdx mice; by contrast, only 1 of the transcripts was expressed at FPKM values >10 in eosinophils from the muscle tissues of IL5tg.
The relative abundance of these key transcripts in eosinophils from the muscle tissues of mdx mice suggests they may encode proteins at level sufficient to produce a physiologically relevant impact.
Of the 17 key transcripts, many encoded inflammatory receptors that may potentiate eosinophil characteristics within dystrophin-deficient muscle tissue. For example, transcripts of
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Cd14, which encode the CD14 receptor, were detected at 6.8-fold higher levels in eosinophils
from the muscle tissues of mdx mice when compared with eosinophils from the muscle tissues of
IL5tg mice (p = 3.81e-04). Expression of the CD14 receptor has not been previously detected on
eosinophils [23]; CD14 has been reported as a co-receptor for toll-like receptor 4 (TLR4) on
macrophages [24, 25]. Similarly, transcripts of Nlrp3, which encodes the NALP3 inflammasome
receptor, were detected at 18.51-fold higher levels in eosinophils from the muscle tissues of mdx
mice when compared with that of IL5tg (p = 2.22e-03). In macrophages, NALP3 receptor
recognizes damage-associated molecular patterns to initiate the release of the pro-inflammatory
cytokine interleukin-1 beta (IL1β) [26]; however, expression and function of NALP3 on eosinophils
has not been reported previously.
In addition to inflammatory receptors, many of the key transcripts encoded transporters that
may have an impact on the bioenergetic state of eosinophils in the muscle tissues of mdx mice.
For example, transcripts encoding the peptide/histidine transporter SLC15A3 (Slc15a3) were
detected at 15.5-fold higher levels in eosinophils from the muscle tissues of mdx mice when
compared with that of IL5tg (p = 4.74e-04). Expression of SLC15A3 has not been previously
reported on eosinophils; however, increased expression of SLC15A3 has been reported in
macrophages following stimulation of the TLR2, TLR4, TLR7 or TLR9 signaling pathways [27].
Similarly, transcripts encoding the ergothioneine transporter SLC22A4 (Slc22a4), were detected
at 11.3-fold higher levels in eosinophils from the muscle tissues of mdx mice when compared with that of IL5tg (p = 3.35e-04). Expression of SLC22A4 transporter has not been previously reported on eosinophils; furthermore, the physiological relevance to transport of ergothioneine, a key antioxidant, remains unclear [28].
Intriguingly, levels of the transcript encoding TREM2 (Trem2) were detected at 68-fold higher levels in eosinophils from the muscle tissues of mdx mice when compared with that detected in the IL5tg (p = 5.23 e-03). Trem2 encodes the triggering receptor expressed on myeloid cells-2
(Trem2) which has been linked to the activation, proliferation, and survival of microglial cells
69
encountering tissue damage [29]. Expression of Trem2 has not been reported on eosinophils;
detailed examination of the function and regulation of this receptor is presented in Chapter 5.
Discussion
In this study, we examined the transcriptional profile of eosinophils recruited to the muscle
tissues of mdx, IL5tg and mdx.IL5tg mice at 4 weeks of age. Although eosinophils contain abundant ribonucleases within their cytoplasmic granules, we were able to isolate high-quality
RNA for sequencing (RIN Scores: 8.2-9.7). Analysis of our RNA-sequencing experiment identified distinct transcriptomic features among eosinophils recruited to the muscle tissues of mdx and
IL5tg mice, including 393 unique transcripts that were detected at significantly higher levels in eosinophils from the muscle tissues of mdx mice and 238 unique transcripts that were detected at significantly higher levels in eosinophils from the muscle tissues of IL5tg mice. Many of the 393 unique transcripts identified in eosinophils from the muscle tissues of mdx mice encode proteins involved with tissue remodeling, suggesting a potential role for eosinophils in the remodeling of muscle damage associated with dystrophin-deficiency. Intriguingly, many of the 393 unique transcripts encode cell surface receptors, suggesting that eosinophils may be actively sensing and responding to cues provided by the dystrophin-deficient muscle microenvironment.
Our findings challenge the perception of eosinophils as static and end-stage by demonstrating that eosinophils recruited to dystrophin-deficient (mdx) muscle tissue exhibit distinct transcriptomic features compared with eosinophils recruited to hyper-eosinophilic (IL5tg) muscle tissue. These findings add to the growing body of evidence that demonstrate heterogeneity in the characteristics of eosinophils. For example, Mesnil and colleagues used mRNA microarrays to identify differential expression of transcripts among populations of inflammatory and resident eosinophils in allergen-challenged airways [8]. In another instance, Barnig and colleagues used mRNA microarrays to describe distinct transcriptomic features among peripheral blood eosinophils from asthmatic patients compared with that of healthy donors [16].
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The distinct transcriptomic features observed in eosinophils isolated from the muscle tissues
of mdx and IL5tg mice may derive in part from unique signals provided by the tissue microenvironment. In mdx mice, the dystrophin-deficient muscle microenvironment contains
abundant damage-associated molecular patterns (DAMPs) such as the high mobility group box 1
(HMGB1, [30]); and also contains abundant cytokines and chemokines. By contrast, eosinophils
isolated from the skeletal muscle tissues of IL5-transgenic mice may have been primed by
constitutively high levels of the eosinophilopoietic cytokine, IL-5. Extensive work among
eosinophil researchers has identified a wealth of receptors that are expressed on the surface of
eosinophils; activation of these receptors may contribute to initiation of key pathways involved
with gene transcription. Among the receptors described on eosinophils are adhesion receptors
(e.g., CD44, CVLA4) and pattern recognition receptors (e.g., TLR2, TLR4) as well as receptors
that modulate responses to chemoattractants (e.g., CCR3, CCR5), lipid mediators (e.g.,
Leukotriene B4 receptor, EP4 prostaglandin receptor), and cytokines (e.g., IL-4R, IL-5R); a more
comprehensive list of receptors expressed on eosinophils was compiled by Lee and colleagues
[31].
Current understanding of eosinophil transcriptional pathways that occur downstream of
receptor activation is limited by the relatively few studies examining this topic. Stokes and
colleagues demonstrated that the transcription factor stat6, which is activated by IL-4/IL-13
signaling, is required for eosinophil migration to allergen-challenged airways; however, loss of
stat6 did not affect the immunoreactive levels of key cytokines in eosinophils [32]. Changes in the
eosinophil transcriptome that occur as a result of receptor activation may be mediated through
other transcription factors. For example, Fairfax and colleagues identified 9 transcription factors
(e.g., Ikzf3, Nod2, Bcl3, Foxo1) present in mature eosinophils [33]; activation of any combination
of these transcription factors may in turn propagate and impart the unique transcriptomic features
observed in eosinophils isolated from the muscle tissues of mdx and IL5tg mice. Despite limited
understanding of transcriptional regulation in eosinophils, this remains an area of active research
71 with important clinical relevance. Glucocorticoids, which are a mainstay in the treatment of eosinophilic and inflammatory disorders, affect the transcriptional profile of eosinophils [2].
Our findings indicate that eosinophils recruited to dystrophin-deficient muscle tissue express higher levels of transcripts that encode proteins associated with tissue remodeling. These include enzymes that remodel components of the extracellular matrix such as matrix metalloproteinases
2, 12, 14, and 19; as well as cathepsin b, k, s. This is consistent with earlier reports indicating that eosinophils express matrix metalloproteinase and cathepsin family members within eosinophil granules [14, 34, 35]. However, our findings indicate significantly higher levels of these remodeling transcripts in eosinophils recruited to, and possibly activated by, the dystrophin- deficient muscle microenvironment. Furthermore, our findings identify expression of two key remodeling enzymes that have not been previously reported in eosinophil granules or associated with eosinophil disorders: matrix metalloproteinase 19 and cathepsin K. Highlighting their complexity in vivo, expression of cathepsin and matrix metalloproteinase family members have been shown both to limit and to promote muscle damage in muscular dystrophy mice [18, 21].
In addition, we detected increased expression of transcripts encoding collagen subunits in mdx eosinophils; these factors are critical components of the extracellular matrix. Increased collagen deposition also results in tissue fibrosis, which have been linked with specific instances of eosinophil infiltration. However, eosinophil-mediated tissue fibrosis has been described through the release of the pro-fibrotic factor Transforming Growth Factor-Beta (TGF-β) [36]; we are unaware of any earlier reports documenting the storage and secretion of collagen from eosinophil granules. Hence, we investigated the possibility that mdx eosinophils may be engulfing exogeneous nucleic acids, such as those provided by the myofibers. Through electron microscopic studies of eosinophils from mdx mice, there was no ultrastructural evidence for the bulk uptake of exogeneous nucleic acids (Elizabeth R. Fischer, RML/NIAID, personal communication).
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Intriguingly, we detected increased expression of transcripts encoding cell surface proteins in
eosinophils from mdx mice. These include the inflammatory receptors CD14 and NLRP3, the
peptide/histidine transporter SLC15A3 and the ergothioneine transporter SLC22A4. Increased
expression of these cell surface proteins may potentiate the functional characteristics of
eosinophils in dystrophin-deficient muscle tissue. For example, increased expression of
transporters may promote the uptake of metabolites critical for prolonged cell survival, especially
in the presence of severe extracellular damage seen in dystrophin-deficient muscle tissue.
Similarly, expression of inflammatory receptors may potentiate eosinophil characteristics, for
example, by promoting transcription and/or release of key mediators from the granules. Given the
role of inflammatory receptors in mediating key eosinophil characteristics, we focus in chapter 5
on the function and regulation of a key receptor, TREM2, that is expressed at significantly higher
levels in mdx muscle eosinophils.
In summary, we extracted and sequenced eosinophil RNA from the muscle tissues of mdx,
IL5tg, and mdx.IL5tg mice. Eosinophils from the mdx muscle tissue exhibited distinct
transcriptomic features when compared with eosinophils from the IL5tg muscle tissue. In mdx muscle eosinophils, increased expression of transcripts associated with tissue remodeling as well as transcripts encoding cell surface proteins may potentiate specific eosinophil functional characteristics. As such, further studies will evaluate the impact of eosinophil infiltration and eosinophil-derived mediators on the regeneration of damaged, dystrophin-deficient muscle tissue.
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FIGURES
Figure 1. Isolation of RNA from eosinophils from skeletal muscle tissues. (A) As described
in Chapter 2, hind limb skeletal muscles were enzymatically digested to generate single cell suspensions that were subjected to fluorescence-activated cell sorting (FACS). Isolated eosinophils (CD45+CD11c-Gr1-SigF+MHC-IIlo cells) were sorted directly into TRIZOL-LS and RNA
was isolated following the manufacturer’s protocol. To remove potential contaminants, isolated
RNA was subjected to cleanup using the Qiagen RNeasy Kit. Following library preparation, RNA
was sequenced using an Illumina HiSeq 4000 Next Generation sequencer with more than 100
Million reads per sample. Trimmed reads were aligned to the mouse reference genome and
transcript abundance quantified using the RSEM program. (B) A total of 9 eosinophil RNA
samples were submitted for sequencing: 3 from mdx, 3 from IL5tg, and 3 from mdx.IL5tg mice.
RNA samples ranged from 12 – 547 ng and were collected from 3-5 male mice at 4 weeks of age;
between 329,000 and 4,940,0000 eosinophils were used for RNA isolation.
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Figure 2. Quality of RNA isolated from eosinophils from skeletal muscle tissues. The electrophoretic trace of each RNA was analyzed using an RNA PicoChip and Agilent BioAnalyzer
2100, which calculates the RNA Integrity Number (RIN) score based on the ratio of the 28s:18s ribosomal RNA subunits. RIN scores range from 0 (most degraded) to 10 (most intact). All RNA samples submitted for sequencing exhibited two distinct electrophoretic peaks and had RIN scores ≥ 8.20.
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Figure 3. Transcripts and gene ontology pathways that are differentially expressed in mdx
vs IL5tg muscle eosinophils. (A) In black circle, transcripts that were detected at significantly
higher levels (> 5-fold) in eosinophils from mdx muscle tissue when compared with that of IL5tg.
Many of the mdx > IL5tg transcripts encode proteins involved with tissue remodeling (e.g., collagens, cathepsins, and matrix metalloproteinases) or cell surface proteins (e.g., CD74,
SLC15A3, TREM2). In blue circle, transcripts that were detected at significantly higher levels ( >
5-fold) in eosinophils from IL5tg muscle tissue when compared with that of mdx. Many of the IL5tg
> mdx transcripts encode eosinophil-specific proteins (e.g., eosinophil-associated ribonucleases, eosinophil peroxidase) or cell cycle processes. (B) Select gene ontology terms that were significantly associated with mdx > IL5tg transcripts. Gene ontology terms shown here were those associated with tissue remodeling processes.
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Figure 4. Differentially expressed transcripts that are associated with tissue remodeling.
Expression of select transcripts that were detected at significantly higher levels in eosinophils from the mdx muscle tissue when compared with that of IL5tg (mdx > IL5tg, > 5-fold). Transcripts shown here encode structural components of the extracellular matrix or encode proteins involved with remodeling of the muscle tissue. Transcript abundance is expressed as fragments per kilobase of transcript per million reads (FPKM); values are represented as mean ± standard deviation. n = 3 per genotype.
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Figure 5. Differentially expressed transcripts that encode cell surface proteins. Expression of select transcripts that were detected at significantly higher levels in eosinophils from the mdx muscle tissue when compared with that of IL5tg (mdx > IL5tg, > 5-fold). Transcripts shown here encode cell surface proteins. Transcript abundance is expressed as fragments per kilobase of transcript per million reads (FPKM); values are represented as mean ± standard deviation. n = 3 per genotype.
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CHAPTER 5
Role of TREM2 in Modulating The
Development and Characteristics of Eosinophils
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CHAPTER 5 PREFACE
In our earlier work (Chapter 4), we described the use of RNA-sequencing to identify transcripts that were differentially expressed in eosinophils isolated from the muscle tissues of mdx, IL5tg and mdx.IL5tg mice. Among our key findings, we detected high levels of transcripts encoding tissue remodeling enzymes and cell surface receptors in eosinophils isolated from the muscle tissues of mdx mice; by contrast, eosinophils from the muscle tissues of IL5tg mice exhibited relatively low levels of these transcripts.
We were intrigued by increased expression of transcripts encoding cell surface proteins, as it suggests that eosinophils recruited to the dystrophin-deficient muscle tissue may be promoting mechanisms to enable them to sense and to adapt to this unique microenvironment. We focused our attention on the differential expression of these transcripts in eosinophils isolated from the muscle tissues of mdx, IL5tg and mdx.IL5tg mice. In general, we observed that eosinophils from mdx mice exhibit high levels of these transcripts while eosinophils from mdx.IL5tg and IL5tg mice exhibited intermediate and low expression of these transcripts, respectively. These trends in transcript expression indicate that eosinophils respond transcriptionally and can increase expression of specific pathways or classes of genes. Furthermore, it is likely that cues provided by the dystrophin-deficient and/or IL5-overabundant microenvironment mediate the distinct transcriptional profiles observed in these eosinophils.
One transcript in particular caught our attention – Trem2, which was detected at 68-fold higher levels in eosinophils from the muscle tissues of mdx mice when compared with eosinophils from the IL5tg mice. Trem2 encodes the Triggering Receptor Expressed on Myeloid cell-2 (TREM2) receptor that is expressed on myeloid cells such as microglia and osteoclasts [1-3]. Importantly,
TREM2 has been shown to mediate the activation, proliferation and survival of myeloid cells, particularly in response to tissue damage [3-5]. To the best of our knowledge, there are no published reports documenting expression of Trem2 in either mouse or human eosinophils at the transcript or at the protein level. Given the function of TREM2 in other myeloid populations, we
83 hypothesized that increased expression of Trem2 may be critical for the activation, proliferation and survival of eosinophils recruited to dystrophin-deficient (mdx) muscle tissue. In this chapter, we explore the expression and regulation of TREM2 on eosinophils, as well as the impact on key eosinophil characteristics, such as development and proliferation.
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ABSTRACT
Eosinophils exhibit a wealth of cell surface proteins that modulate key responses to the tissue
microenvironment. Through RNA-sequencing, we identified relatively high expression of Trem2
in eosinophils from dystrophin-deficient (mdx) muscle tissue. Trem2 encodes the Triggering
Receptor Expressed on Myeloid Cell-2 (TREM2), which modulates the activation, proliferation
and survival of myeloid cells including osteoclasts and microglia. Expression and function of
Trem2 has not been reported previously in eosinophils. In this study, we investigated the
expression and function of TREM2 in eosinophils in health and in disease. Using an ex vivo
differentiation system, we detected expression of TREM2 on 4 – 21% of bone marrow-derived
cultured eosinophils. Expression reached a maximum at day 8, a timepoint that corresponded
with a 5-fold increase in the level of eosinophils in culture. Interestingly, Trem2-deficient cultures
failed to expand and instead exhibited increased cell death. Loss of Trem2 reduced the
homeostatic levels of eosinophils in the bone marrow but did not abrogate eosinophil recruitment
to the airways of allergen-challenged mice. In addition, we are generating a mouse model
(mdx.Trem2-/-) to evaluate the impact of Trem2-deficiency on eosinophil recruitment in a Th2- independent setting, such as that seen in muscular dystrophy. Expression of TREM2 was highly variable on peripheral blood eosinophils from healthy donors and was correlated with the body mass index (BMI) of the donor. Taken together, these findings demonstrate that TREM2 is expressed on human and murine eosinophils and that expression of this receptor mediates key characteristics such as proliferation and survival. Ongoing work will elucidate the regulation of
TREM2 expression on eosinophils as well as the impact of TREM2 receptor activation on eosinophil characteristics.
INTRODUCTION
Despite the perception of eosinophils as merely ‘end-stage effector cells’ [6], emerging evidence has highlighted remarkable heterogeneity in the characteristics and functions of
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eosinophils [7-10]. In line with these studies, we identified distinct transcriptomic profiles among eosinophils recruited to the muscle tissues of mdx, IL5tg and mdx.IL5tg mice (Chapter 4). These include differential expression of transcripts encoding tissue remodeling enzymes and, intriguingly, differential expression of transcripts encoding cell surface proteins. We noted that eosinophils isolated from the muscle tissues of mdx mice exhibited 68-fold higher levels of the
Trem2 transcript when compared with eosinophils from IL5tg mice.
Trem2 encodes Triggering Receptor Expressed on Myeloid Cells-2 (TREM-2), a single
transmembrane receptor of the immunoglobulin family that signals through the DAP10 and
DAP12 adapters [5]. Variants in the Trem2 gene are associated with increased risk of developing
Alzheimer’s Disease; these variants result in decreased expression of TREM2 on the cell surface
as well as decreased signaling response [5]. Inactivating mutations in the Trem2 gene result in
Nasu-Hakola Disease, an autosomal recessive disorder distinguished by the presence of early-
onset dementia and recurrent bone fractures [5, 11]. As a result of these disease associations,
Trem2 has been studied extensively in myeloid cells involved with bone resorption (i.e.,
osteoclasts) and with the clearance of Aβ plaques (i.e., microglial cells).
Expression of Trem2 has been shown to promote the activation, proliferation and survival of
myeloid cells. For example, in the 5XFAD mouse model of Alzheimer’s Disease, Trem2-deficiency
was associated with reduced viability, recruitment and activation of microglia in response to Aβ
plaques [4, 12]. Furthermore, Trem2-/-5XFAD mice exhibited increased Aβ burden in the hippocampus, suggesting that Trem2 mediates beneficial processes that limit the pathology in
Alzheimer’s Disease [12]. Accordingly, monoclonal antibody-based therapeutics that seek to augment the activity of TREM2 in microglial cells are currently being explored for the treatment of
Alzheimer’s Disease; examples include the compound AL002 developed jointly by AbbVie, Inc. and Alector, Inc. (www.abbvie.com/our-science/pipeline/al002.html) as well as the compound
ATV:TREM2 developed jointly by Denali Therapeutics, Inc. and Takeda Pharmaceutical
Company, Ltd. (https://denalitherapeutics.com/pipeline).
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TREM2 can be activated in vitro by a range of phospholipids such as phosphatidic acid (PA),
phosphatidylinositol (PI) and phosphatidylserine (PS) [12]. However, the relevance of these
ligands in vivo remains to be determined; detection of phosphatidylserine displayed on apoptotic
cells may activate the TREM2 receptor in vivo [5, 12]. Nonetheless, activation of TREM2 results in enhanced cellular energetics and activation of key pathways such as mechanistic target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK) [4, 5, 13]. Interestingly, loss of
Trem2 in macrophages results in enhanced cytokine release following treatment with diverse stimuli such as bacterial lipopolysaccharides, zymosan and CpG oligodeoxynucleotides [14].
Together, these findings indicate critical roles for TREM2 in mediating intracellular characteristics and in modulating the release of inflammatory cytokines.
The pleiotropic roles of Trem2 suggest that its expression in eosinophils may mediate critical functions. To our knowledge, there are no prior reports of Trem2 expression in eosinophils or, more broadly, in granulocytes. Similar to the role described in other myeloid populations, Trem2 may mediate the activation, proliferation and survival of eosinophils, particularly in response to tissue damage such as that seen in muscular dystrophy. In this study, we evaluated the expression patterns of TREM2 in eosinophils as well as the impact of this receptor on eosinophil- mediated processes in health and disease. Central to our exploration of Trem2 function in eosinophils is the use of the Trem2-/- mouse model, which contains a 175 base pair deletion in
the Trem2 gene that results in a premature stop codon (www.jax.org/strain/027197).
MATERIALS AND METHODS
Mice
All mice were bred and maintained under pathogen-free conditions in the National Institute of
Allergy and Infectious Diseases (NIAID) Animal Facility 14b South. mdx (C57BL/10ScSn-
Dmdmdx/J, 001801) and Trem2-/- mice (C57BL/6J-TREM2em2Adiuj, 027197) were obtained from
Jackson Laboratory and wild-type controls (C57BL/6NCr, 556) were obtained from Charles River 87
Laboratory. To generate muscular dystrophy mice that lack Trem2 (mdx.Trem2-/-), parental mdx and Trem2-/- strains were intercrossed to generate F1 hybrids that were backcrossed onto the
Trem2-/- parental strain. The presence of the mdx and Trem2 mutations were confirmed by
polymerase chain reaction (PCR) as recommended by Jackson Laboratory.
Ex vivo culturing of Bone Marrow-Derived Eosinophils
As described in [15], bone marrow cells were collected by flushing the femurs and tibiae of adult
mice with RPMI-1640 medium (Thermo Fisher). Bone marrow cells were subjected to hypotonic
lysis to remove erythrocytes before culturing in media at 1 x 106 cells/mL. Cells were cultured in
RPMI-1640 medium containing 20% Fetal Bovine Serum (Atlanta Biologicals), 25 mM HEPES
(Life Technologies), 100 IU/mL penicillin and 10 µg/mL streptomycin (Cellgro), 2 mM glutamine
(Life Technologies), 1 X nonessential amino acids, 1 mM sodium pyruvate (Life Technologies),
50 µM β-mercaptoethanol (Sigma-Aldrich).
From day 0 to 4, growth media was supplemented with 100 ng/mL recombinant mouse Stem Cell
Factor (rm-CSF) and recombinant mouse FMS-like tyrosine kinase 3 ligand (rm-FLT3L). Starting on day 4, growth media was supplemented with 10 ng/mL of recombinant mouse Interleukin-5
(rm-IL5) only. Every 2 days, half of the growth medium was replaced with fresh growth medium containing supplemented factors (rm-CSF and rm-FLT3L; or rm-IL5) and cells were maintained at 1e6/mL. Cell Number and viability were assessed using the Luna-FL dual fluorescence cell counter (Logos Bio).
Evaluation of Bone Marrow-Derived Eosinophils
The morphological features of cultured cells were evaluated using Wright-Giemsa staining. As described in [15], approximately 50,000 cells were centrifuged onto slides using the Shandon
Cytospin 4 (Thermo Fisher). Cells were fixed with methanol before staining with modified Giemsa
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Dyes (Diff-Quik). Images were acquired using the Leica DMI 4000B microscope equipped with
Image Pro Plus software (Media Cybernetics).
TREM2 expression and eosinophil maturity were evaluated by flow cytometric analysis. Aliquots
of cells (1 x 106) were collected every two days of culture and frozen at -80°C in 90% fetal bovine
serum, 10% dimethylsulfoxide. Samples were stained with an Fc receptor blocking antibody (anti-
CD16/32, BD Biosciences), a viability dye (Live/Dead Aqua, ThermoFisher) and a fluorochrome-
conjugated antibody against TREM2 (R&D Systems, Cat No. FAB17291A). Samples were
evaluated using the LSR-II Flow Cytometer (BD Biosciences), with > 150,000 events were
recorded per sample; flow cytometry data was analyzed using FlowJo (Tree Star). Mature
eosinophils were identified as live SSC-Ahi SigF+ cells and were expressed as a percent of the
total live cells in each sample. TREM2+ populations were identified using fluorescent minus one
(FMO) and isotype controls. TREM2+ populations were expressed as a percent of the total number of eosinophils (live SSC-Ahi SigF+ cells) in each sample.
Evaluation of Eosinophils in Bone Marrow
Bone marrow cells were collected by flushing the tibia and femur of mice with RPMI-1640 media.
Following hypotonic lysis, bone marrow cells were enumerated and frozen at -80°C at a concentration of 1-10 x 106 cells/mL of freezing media (90% fetal bovine serum, 10%
dimethylsulfoxide). As described in Chapter 2, samples were stained with a viability dye
(Live/Dead Aqua, ThermoFisher), an Fc receptor blocking antibody (anti-CD16/32, BD
Biosciences) and fluorochrome-conjugated antibodies including anti-CD45 and anti-CD11c
(eBioscience); anti-Gr1 and anti-Siglec-F (BD Biosciences). Samples were evaluated using the
LSR-II Flow Cytometer (BD Biosciences), with > 400,000 events were recorded per sample; flow
cytometry data was analyzed using FlowJo (Tree Star). Eosinophils were identified as
liveCD45+CD11c-Gr1-SigF+MHC-IIlo cells and expressed as a percent of the total live cells in each sample.
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Alternaria alternata challenge and evaluation of disease parameters
Wild-type and Trem2-/- mice were inoculated intranasally with a filtrate of Alternaria alternata
(Greer Allergy Immunotherapy; 50 µg/mL, 50 µL per mouse) at day 0, 3, 6, 10. At day 14, mice
were euthanized and the bronchoalveolar lavage fluid (BALf) was collected using 0.8-1.2 mL of
PBS containing 0.1% bovine serum albumin. Cells recovered from the BALf of each mouse were enumerated using the Luna-FL dual fluorescence cell counter (Logos Bio). Approximately 50,000 cells from the BALf fluid were used to evaluate the fraction of eosinophils. Briefly, cells were centrifuged onto slides (Shandon Cytospin 4, ThermoFisher), fixed in methanol and stained with modified Wright-Giemsa dyes (Diff-Quik). Eosinophils were identified based on cytological features including eosin staining of the granules and ring-shaped nuclei. Eosinophils were enumerated relative to other cell types and expressed as a percent of the total cells detected in the BALf fluid for each sample. Total number of eosinophils in the BALf was calculated using the total number of cells recovered in the BALf and the proportion of eosinophils detected among
BALf cells.
Blood Donors
Samples of whole, EDTA-anti-coagulated blood were obtained from the National Institutes of
Health Clinical Center (Protocol Identifier NCT00001846). All donors consented to donation of blood sample and met the eligibility criteria: ≥ 18 years of age, ≥ 110 pounds, no known history of heart, lung, kidney, hematological disorders, and were not pregnant at time of donation. Donor samples were de-identified by the National Institutes of Health Clinical Center and all samples used in this study were negative for cytomegalovirus.
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Isolation of Human Blood Eosinophils and Detection of TREM2
Eosinophils were isolated from human blood samples via negative selection using the Miltenyi
Human Eosinophil Isolation Kit (Catalog Number 130-104-466) as described previously [7].
Following the manufacturer’s protocol, each blood sample was combined with a cocktail of
particle-bound antibodies that target non-eosinophil populations. Subsequently, each sample was subjected to a magnetic field using the MACSxpress Separator (Catalog Number 130-098-308), which impeded the passage of antibody-bound leukocytes. Isolated eosinophils were stained with modified Wright-Giemsa Dyes (Diff-Quik, Thermo Fisher Scientific) and analyzed for eosin staining and nuclear morphology; staining confirmed that eosinophils isolated using this protocol were ≥ 98% pure.
Isolated eosinophils were stained with an Fc receptor blocking antibody (anti-CD16/32, BD
Biosciences), a viability dye (Live/Dead Aqua, ThermoFisher) and a fluorochrome-conjugated
antibody against TREM2 (R&D Systems, Cat No. FAB17291A) prior to analysis via flow
cytometry. Samples were evaluated using the LSR-II Flow Cytometer (BD Biosciences), with >
60,000 events were recorded per sample; flow cytometry data was analyzed using FlowJo (Tree
Star). TREM2+ populations were identified by comparing TREM2-staining with isotype and
fluorescent minus one (FMO) controls; TREM2+ eosinophils were expressed as a percent of the
total number of eosinophils (live SSC-Ahi SigF+ cells) in each sample.
RESULTS
TREM2 Expression Patterns on Bone Marrow-Derived Eosinophils
TREM2 has been detected on myeloid cells including microglia and osteoclasts, where it has
been shown to mediate cell survival through the activation of the Wnt/β-Catenin pathway [1, 3,
16]. As the expression and function of TREM2 on eosinophils has not been reported, we began
with an ex vivo system to investigate the expression of TREM2 during the differentiation and
proliferation of eosinophils. In this protocol [15], unselected bone marrow cells are cultured for 4
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days in media containing Stem Cell Factor (SCF) and FMS-like Tyrosine Kinase-3 Ligand (Flt3l)
before differentiation in media containing Interleukin-5 (IL-5). As a result, cultures exhibit an
expansion in the number of mature eosinophils, from approximately 8% of the cultured cells at
day 0 to more than 94% of the cultured cells at day 16 (Fig 1A). Eosinophils differentiated ex vivo
exhibit characteristic morphologic features, such as eosin staining of the granules and ring-form
nuclei (Fig 1B).
Because TREM2 is an inducible receptor, we examined its expression patterns in cultures of
bone marrow-derived eosinophils sampled every 2 days; this allowed us to evaluate TREM2
during eosinophil development and maturation. Immunoreactive TREM2 was detected on the
surface of a small population (~5%) of eosinophils at the earliest time points during differentiation
in response to IL-5 (days 4-6 of culture). However, a prominent population of eosinophils (20 ±
1.4%, red arrow) expressed immunoreactive TREM2 at day 8; expression then decreased, returning to baseline levels (~5% of the total eosinophils) by day 12 (Fig 1C). Interestingly, the peak expression of TREM2 at day 8 coincided with the 5-fold expansion in the number of eosinophils: at day 6, eosinophils represent ~5% of the cultured cells while at day 8, eosinophils represent ~25% of the cultured cells (Fig 1A, red arrow). Increased expression of TREM2 during eosinophil expansion suggests that this receptor may be associated with eosinophil expansion and proliferation, similar to the role described in osteoclasts and microglial cells [1, 3, 4, 11, 12,
16].
Trem2-Deficiency Limits Expansion of Eosinophils ex vivo and in vivo
Given that TREM2 can be detected on eosinophils cultured ex vivo, we evaluated the impact of Trem2 on the differentiation, proliferation, and survival of eosinophils. We utilized the ex vivo system described in Fig 1 to examine eosinophil expansion and proliferation in Trem2-/- mice.
While similar numbers of cells were observed between day 0 and day 10, cultures from Trem2-/-
bone marrow did not exhibit similar robust expansion observed in cultures from wild-type bone
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marrow (Fig 2A). Furthermore, between day 2 and day 17, cultures of Trem2-/- bone marrow- derived eosinophils exhibited slightly lower viability when compared with those derived from the wild-type (Fig 2B). Nonetheless, when evaluated at day 14, cultures of Trem2-/- cells exhibited
features characteristic of eosinophils, including eosin staining of the granules and ring-form nuclei,
but also exhibited increased evidence of cell-death (Fig 2C, arrows).
Next, we asked if Trem2 might modulate the levels of eosinophils at homeostasis. Towards this end, we evaluated the level of eosinophils in the bone marrow of wild-type and Trem2-/- mice.
While eosinophils constitute approximately 6% of the bone marrow cells in wild-type mice, we detected significantly lower levels of eosinophils in the bone marrow of Trem2-/- mice (Fig 3A; 3.7
± 0.25%, ** p < 0.01). Furthermore, eosinophils in the bone marrow of Trem2-/- expressed
significantly lower median fluorescent intensity (MFI) for Siglec-F when compared with those of
wild-type (Fig 3B; 727 ± 14 vs 857 ± 16, respectively; ****p < 0.0001). Taken together, these findings indicate that Trem2 is not required for the differentiation of eosinophils, but suggest that it may contribute to the potential for proliferation and their ongoing survival.
Trem2 Is Not Required for Eosinophil Recruitment to the Airways of Allergen-challenged mice
As Trem2 is critical for expansion of eosinophils ex vivo and in vivo, we asked if Trem2 might
contribute to the efficient expansion and mobilization of eosinophils associated with
pathophysiological states. Accordingly, we utilized a mouse model of airway inflammation, in
which eosinophils are recruited to the airways following challenge with the fungal allergens such
as Alternaria alternata. We challenged both wild-type and Trem2-/- mice with four doses of
Alternaria alternata (day 0, 3, 6, 10) and then evaluated eosinophil infiltration at day 14 (Fig 4A).
Surprisingly, we detected similar levels of total cells (Fig 4B) and eosinophils (Fig 4C) recruited
to the airways of wild-type and Trem2-/- mice. In addition, eosinophils recruited to the airways of
Trem2-/- mice appeared morphologically indistinguishable from those recruited to the airways of
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wild-type mice (Fig 4D); we did not observe any evidence for increased cell death associated with
eosinophils recruited to the airways of Trem2-/- mice. Taken together, these findings indicate that
Trem2 gene-deletion does not result in impaired recruitment of eosinophils to the airways of
allergen-challenged mice. Rather, signals associated with Alternaria alternata challenge may be
sufficient to overcome the decreased eosinophil proliferation associated with Trem2-deficiency.
Generation of Trem2-deficient muscular dystrophy mice (mdx.Trem2-/-)
We also initiated a study to evaluate the impact of Trem2 on the persistence and characteristics of eosinophils detected in muscle infiltrates as a feature of muscular dystrophy. As described in Chapter 4, we detected a striking, 68-fold increase in the expression of Trem2 transcripts in eosinophils isolated from the muscle tissues of mdx mice when compared with eosinophils from the muscle tissues of IL5tg mice. Increased expression of Trem2 in these eosinophils may promote an activated state in which eosinophils exhibit increased persistence and also exhibit unique functional characteristics. Hence, in order to evaluate the impact of Trem2
on eosinophil persistence and characteristics in muscular dystrophy, we generated muscular
dystrophy mice that lack Trem2 (mdx.Trem2-/-) by intercrossing the mdx (C57BL/10ScSn-
Dmdmdx/J, 001801) and Trem2-/- strains (C57BL/6J-TREM2em2Adiuj, 027197).
As shown in Figure 5, F1 offspring generated during the first cross include female mice that are heterozygous for both the mdx and the Trem2 mutations. Subsequently, F1 hybrid females
(mdx+/-Trem2+/-) were bred with Trem2-/- males to generate male offspring that are hemizygous for the mdx mutation and homozygous for the Trem2 mutation (mdxx/-Trem2-/-); these mice
represent our desired genotype and are expected to occur at the Mendelian frequency of 1:4 male
mice. Subsequent mating between F2 mice will most likely augment the expected frequency of
the desired genotype to 1:2 in the F3 male offspring, while mating of the F3 mice can produce
offspring that exclusively express both alleles of the mdx and Trem2 mutations (mdx-/-Trem2-/-).
94
While generation of these mice is ongoing, we have successfully obtained F2 offspring in
January 2020; here, the desired genotype (mdxx/-Trem2-/-) is to be expected in 1:4 males.
Previously, we have utilized polymerase chain reaction (PCR) to confirm the genotypes of the mdx and Trem2-/- parental strains; we expect that confirmation of the mdx and Trem2 mutations should proceed seamlessly in F2 mice. Using PCR-confirmed mdx.Trem2-/- mice, we will evaluate
the degree of eosinophil infiltration, the functional characteristics of eosinophils in the muscle
tissue and the impact on muscle pathology. We note here that while loss of Trem2 did not
abrogate eosinophil recruitment to the airways of allergen-challenged mice, dystrophin-deficient
muscle tissue represents a distinct context in which eosinophils are recruited and activated.
Eosinophil infiltration in muscular dystrophy occurs in the absence of a systemic, Th2-mediated
response [17] in contrast to the Th2-driven, allergen-challenged model we utilized (Fig 4; [18,
19]); hence, investigation of the potential impact of Trem2 on eosinophils recruited in muscular
dystrophy may yield intriguing insights into the effects of Trem2 activity in non Th2-mediated
settings.
Variability of surface TREM2 expression on Human Peripheral Blood Eosinophils
Given the association between variants in the Trem2 gene and disorders such as Alzheimer’s
Disease [20-22] and Nasu-Hakola disease (NHD) [23, 24], we asked if TREM2 might also be
detected on human eosinophils. Towards this end, we isolated eosinophils from the peripheral
blood of healthy donors using negative selection, in which antibodies bound and selectively
removed non-eosinophil populations. Eosinophils were isolated at high purity (>98%) and
analyzed for the expression of surface TREM2 by flow cytometry. We detected TREM2
expression on the surface of 0.7 – 63% of peripheral blood eosinophils from normal human
subjects; the MFI varied from 343 – 538 (Fig 6A). In Figure 6B, representative flow cytometry
plots are shown from donors with low (donor 15) or high (donor 5) proportions of TREM2+
eosinophils.
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Next, we asked if the variability in TREM2 expression on peripheral blood eosinophils might
correlate with any of the clinical parameters provided on de-identified reports. Intriguingly, we
observed a significant correlation between the proportion of TREM2 expressing eosinophils and
the body mass index (BMI) of the donor (R2 = 0.56, p = 0.01; Fig 6C). Taken together, these findings provide evidence of TREM2 expression on human eosinophils and indicate that
expression may be affected by unique pathophysiological conditions such as adiposity or
variations in diet.
DISCUSSION
In this study, we evaluated the expression of TREM2 on human and murine eosinophils, and
identified a key role for TREM2 in the expansion of the eosinophil lineage ex vivo. We extended
these findings in vivo and determined that Trem2-deficient mice exhibited a reduced number of
eosinophils in bone marrow at baseline. Despite lower levels of eosinophils in the bone marrow
at baseline, Trem2-/- mice mobilized eosinophils to the airways following challenge with a Th2 stimulus; these results suggest that Trem2-mediated signaling may have an impact on eosinophils via a mechanism that is independent of Th2 signaling. We intend to investigate Trem2-mediated
effects on eosinophil recruitment in a non Th2-mediated signaling such as muscular dystrophy
[17]; as such, we are actively generating muscular dystrophy mice that lack Trem2 (mdx.Trem2-/-
). Finally, we described variable expression of TREM2 on peripheral blood eosinophils that correlated with increased body mass index of the donor.
These findings, while intriguing, represent an initial exploration towards elucidating the function and regulation of Trem2 in eosinophils. As such, these data invite many new areas of
investigation that are discussed below.
Expression and function of TREM2 has been identified on multiple cell types, nearly all of
which belong to the monocyte/macrophage lineage: osteoclasts [1, 11], microglia [3, 4, 12, 25],
Kupffer cells [26], alveolar macrophages [27, 28], adipose macrophages [29] and dendritic cells
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[30]. Hence, to the best of our knowledge, this is the first report of TREM2 expression on
eosinophils and, more broadly, on cells of the granulocyte lineage. Earlier work demonstrated that
neutrophilic infiltrates express high levels of TREM1 – a receptor thought to oppose the activity
of TREM2 – in response to pathogens such as bacteria and fungi [25, 31]. In addition, several groups reported an inverse relationship between Trem1 and Trem2 expression in macrophages and microglia, notably in response to bacterial lipopolysaccharides or interleukin-4 (IL-4) [25, 32].
These seminal studies, while important, have led to the perception that cells of the monocyte/macrophage lineage largely express TREM2 but can also express TREM1 under specific inflammatory circumstances; and that granulocytes, including eosinophils, largely express TREM1 but not TREM2 [2].
Our findings identify TREM2 expression on eosinophils differentiated ex vivo and demonstrate that Trem2 is required for the efficient development of eosinophils under ex vivo and in vivo conditions. These findings are consistent with earlier reports, in which Trem2 has been shown to promote the survival of microglia and osteoclasts through activation of the β-catenin/Wnt pathway
[1, 3, 16] and increased intracellular metabolism [4, 33]. Ongoing work will evaluate these features in wild-type and Trem2-/- eosinophils as potential mechanisms by which Trem2 exerts pro-survival and proliferative effects.
We detected peak TREM2 expression on eosinophils at day 8 of ex vivo culturing, a timepoint that corresponded with the 5-fold expansion in the number of eosinophils in culture. Increased expression of TREM2 at this timepoint highlights the potential role of TREM2 in mediating eosinophil proliferation. However, we note that eosinophils continue to proliferate following day 8 and that Trem2-deficiency abrogates the dramatic eosinophil expansion observed at later timepoints, notably at day 13. One possibility is that Trem2 might be acting very early to promote the survival and expansion of eosinophils, the effects of which are magnified after several cycles of cell proliferation. Another possibility is that while peak TREM2 is detected at day 8, its effects may be mediated at subsequent timepoints through the actions of cleaved or soluble Trem2
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(sTREM2) [34]. Indeed, multiple reports have demonstrated that TREM2 is cleaved by proteases such as ADAM10/17 [35-38] and that sTREM2 promotes the survival of cells such as microglia and alveolar macrophages [27, 39, 40]. Ongoing work will evaluate the extent to which sTREM2 can be detected in the supernatants of eosinophil cultures and the effect that sTREM2 depletion
– for example, through specific protease inhibitors – might have on eosinophil proliferation.
Finally, our findings raise intriguing questions about the regulation and function of TREM2 in disease. As described in Chapter 4, we noted a 68-fold increase in the level of Trem2 transcripts detected in eosinophils from the muscle tissues of mdx mice when compared with eosinophils from the muscle tissues of IL5tg mice. Increased expression of Trem2 in eosinophils may be attributed to activation by factors found in dystrophin-deficient muscle microenvironment such as
IL-4, IL-1RA and Eotaxin-1/CCL11. In support of this, Turnball and colleagues [14] demonstrated that administration of IL-4 or thioglycollate augments the level of TREM2 expressed on peritoneal macrophages. As such, we have initiated aspects of this study to identify factors that promote
TREM2 expression on eosinophils; similar to the findings by Turnball and colleagues [14], we observed dramatic upregulation of TREM2 expression on bone marrow-derived eosinophils stimulated with IL-4 (data not shown).
Along these lines, we observed increased expression of TREM2 on peripheral blood eosinophils that was correlated with the body mass index (BMI) of the donor. Since adiposity is associated with a chronic inflammatory state [41, 42], circulating factors in donors with higher
BMIs may promote the expression of TREM2 on eosinophils. Interestingly, Jaitin and colleagues
[29] reported increased expression of TREM2 on macrophages recruited to the adipose tissue of mice fed a high-fat diet. Although Trem2 was not required for macrophage infiltration into the adipose tissue, Trem2 mediated a key transcriptional program in recruited macrophages. Hence, further work is needed to investigate the functional differences between TREM2+ and TREM2- eosinophils and their impact in metabolic syndromes such as adiposity.
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In summary, we evaluated TREM2 expression patterns on human and mouse eosinophils and identified contexts in which TREM2 expression is increased. Among these are eosinophils undergoing expansion, eosinophils recruited to dystrophin-deficient muscle tissue and eosinophils from donors with increased body mass index. Ongoing work will evaluate the role of specific factors in promoting TREM2 expression as well as the intracellular pathways by which
TREM2 regulate eosinophil expansion.
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FIGURES
Figure 1. Differentiation of eosinophils ex vivo and detection of TREM2. (A) Eosinophils can undergo ex vivo expansion and differentiation from unselected mouse bone marrow cells using our published protocol [15]. Following expansion of eosinophil progenitors by Stem Cell Factor
(SCF) and FMS-like tyrosine kinase-3 ligand (Flt3l), IL-5 results in the generation of mature eosinophils (SSChiSigF+) which have expanded to ~25% of the cells in culture by day 8 as indicated by the red arrow. Mature eosinophils constitute > 80% of the cultured cells by day 12, and > 94% by day 16. (B) At day 14, cultured cells exhibited features characteristic of eosinophils, including eosin staining of the granules and ring-form nuclei. (C) Immunoreactive TREM2 was
detected on 4 - 7% of bone marrow-derived eosinophils (SSChiSigF+) on days 4 - 6 and days 12
- 16 of culture. Immunoreactive TREM2 was detected on 18 - 21% of bone marrow-derived
eosinophils at day 8 of culture as indicated by the red arrow. (A, C) Mean ± Standard Deviation
of three separate cultures.
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Figure 2. Trem2 is required for efficient eosinophil expansion ex vivo. (A) Total live cells and
(B) viability in ex vivo bone marrow cultures from wild-type (black bars) and Trem2-/- mice (blue bars). Cultured cells were differentiated in stem cell factor (SCF), Fms-like tyrosine kinase-3
Ligand (Flt3l) and Interleukin-5 (IL-5) as indicated. Mean ± Standard Deviation of three separate cultures; ** p < 0.01, * p < 0.05, 2-way ANOVA with multiple comparisons. (C) At day 14, cultured cells from Trem2-/- mice were morphologically eosinophils, and displayed eosin staining within cytoplasmic granules and ring-form nuclei. Cells undergoing apoptosis are indicated by the black
arrows.
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Figure 3. Trem2 gene-deletion alters eosinophil homeostasis in the bone marrow. (A)
Levels of mature eosinophils (CD45+CD11c-Gr1-SigF+MHC-IIlo cells) in the bone marrow of wild- type and Trem2-/- mice. (B) Median Fluorescent Intensity (MFI) of Siglec-F expression on eosinophils in the bone marrow of wild-type and Trem2-/- mice; data shown are mean ± standard
deviation, n= 4 per genotype; **** p < 0.0001, ** p < 0.01, unpaired t-test.
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Figure 4. Trem2 is not critical for eosinophil recruitment in response to a Th2-inflammatory stimulus. (A) Wild-type and Trem2-/- mice were challenged with four inoculations of a filtrate of
Alternaria alternata on days 0, 3, 6, 10 (50 µg / mL, 50 µL per mouse) and evaluated on day 14 as previously described [19]. Similar number of (B) cells and (C) eosinophils were recovered from the bronchoalveolar lavage fluid of wild-type and Trem2-/- mice at day 14 post challenge. (D) At day 14 post challenge, eosinophils recovered from the bronchoalveolar lavage fluid of wild-type
and Trem2-/- mice were morphologically indistinguishable; n = 6 per genotype; n.s., no significant
differences.
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Figure 5. Generation of mdx.Trem2-/- mice. Male Trem2-/- mice (C57BL/6J-TREM2em2Adiuj,
027197) were crossed with female mdx mice (C57BL/10ScSn-Dmdmdx/J, 001801) to generate
female mice heterozygous for mutations in both the mdx and Trem2 genes. Mdx+/-Trem2+/- female
mice were crossed with male Trem2-/- mice to produce offspring in which 25% of the males are
hemizygous for the mdx mutation and homozygous for the Trem2 mutation. Skeletal muscle
tissues from male mdx.Trem2-/- mice will be evaluated for the degree of eosinophil infiltration and the level of muscle damage.
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Figure 6. Variable expression of TREM2 on human peripheral blood eosinophils and correlation with donor BMI. (A) Peripheral blood eosinophils from healthy donors exhibit variable expression of TREM2 (0.7 – 63.7%) and variable median fluorescence intensity (MFI;
343 – 538). Significant correlation (p = 0.05, R2 = 0.39) between the MFI of TREM2 and percent of eosinophils expressing TREM2. (B) Flow cytometric analysis of TREM2 expression on peripheral blood eosinophils from a donor with low TREM2 expression (0.7%, Donor #5) compared to a donor with high TREM2 expression (63%, Donor #15). SSC-A, side scatter area.
(C) Significant correlation (p = 0.012, R2 = 0.57) between percent of TREM2+ eosinophils and the body mass index of donor.
105
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CHAPTER 6
Conclusion
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Eosinophil infiltration does not always promote tissue destruction
The view of eosinophils as primary mediators of tissue destruction arose initially from their
association with innate immunity and host defense [1]. Eosinophils can release granule proteins
that have prominent anti-parasitic [2, 3], anti-viral [4-7] and bactericidal [8, 9] properties. While
the contributions of eosinophils to host defense in vivo remains an area of active investigation,
the most recent findings are highly controversial. For example, while eosinophil granule proteins
are toxic to the schistosomulae and larvae of the helminth S. mansoni in experiments carried out
in vitro, eosinophil ablation did not significantly alter disease progression in vivo [10]. As but
another example, eosinophils were shown to promote the growth of Trichinella spiralis larvae by
suppressing inflammation and promoting glucose uptake in the muscle tissue [11, 12].
Eosinophil infiltration into the tissues in the absence of known pathogens was viewed as an
extension of their role in host defense [1]. In this paradigm, recruited eosinophils release cytotoxic
mediators such as the cationic protein major basic protein-1 (MBP-1) and reactive oxygen species
(ROS) by eosinophil peroxidase (EPX) and the NADPH oxidase system. Supporting this view are
a number of associations between eosinophils and tissue damage, for instance, in asthma [13,
14] and in the autoimmune skin disease bullous pemphigoid [15].
Despite their association with tissue damage, eosinophils have also been detected in the
absence of tissue damage, notably in the gastrointestinal tract and in the mammary gland. Here,
eosinophils have been shown to mediate critical homeostatic functions; eosinophils maintain the mucosal barrier by secreting Immunoglobulin-A (IgA) [16, 17] and facilitate ductal branching of the mammary gland [18]. These associations indicate that eosinophils are not universally associated with tissue destruction and that their infiltration into the tissues should not necessarily be regarded as pathological features of the disease process.
In this dissertation, we determined that eosinophil infiltration is associated with, but does not drive, acute muscle damage in muscular dystrophy (mdx) mice. We utilized eosinophil overabundant (mdx.IL5tg) and eosinophil-deficient (mdx.PHIL) mice to demonstrate that the
110 degree of eosinophil infiltration did not correspond with the degree of acute muscle damage. For instance, muscle tissues from eosinophil-sufficient (mdx) and eosinophil-deficient (mdx.PHIL) mice exhibited similar proportions of centrally-nucleated myofibers (27 ± 2.3% vs 29.5 ± 1.8%, respectively) at 4 weeks of age; central positioning of myofiber nuclei identifies recently-damaged myofibers undergoing regeneration (Chapter 3). In fact, we observed that eosinophils were present in high numbers in the absence of any muscle damage in hyper-eosinophilic IL5tg parent mouse strain.
These findings add to the growing body of evidence that challenges the view of eosinophils as purely tissue-destructive. Rather, eosinophils may be recruited to sites of existing tissue damage by elevated levels of damage-associated molecular patterns (DAMPs) such as sphingosine 1-phosphate (S1P) and high mobility group box 1 (HMGB1); indeed, eosinophils exhibit chemotactic properties towards HMGB1 [19] and S1P [20]. Once recruited, eosinophils may modulate the local immunity and/or tissue repair processes, an idea first advanced by Lee and colleagues [21]. Consistent with this idea, recent studies have demonstrated that eosinophil infiltration is critical for the remodeling of acutely damaged tissues in the muscle [22], liver [23], or urogenital tract [24]. Similarly, eosinophils recruited to dystrophin-deficient muscle tissue may mediate one or more aspects of tissue repair; of note, we have detected prominent expression of transcripts encoding tissue remodeling proteins in eosinophils recruited to the muscle tissues of mdx mice (Chapter 4).
Eosinophil infiltration in musculoskeletal disorders
Although we established that eosinophils do not promote muscle damage in mdx mice
(Chapter 3, [25]), the roles of eosinophils in DMD as well as in other musculoskeletal disorders remain to be determined. Earlier work by Wehling-Henricks and colleagues [26] demonstrated that CCR3 antibody-based depletion of eosinophils mitigated the level of muscle damage in mdx mice; however, CCR3 expression has also been detected on lymphocytes [27-30], which are
111 recruited to dystrophin-deficient muscle tissues [26, 31]. More specific targeting of the eosinophil population, for example through the deletion of the gene encoding the eosinophil granule protein
Major Basic Protein-1, did not ameliorate the level of muscle damage in muscular dystrophy mice
[26].
Eosinophil infiltration is prominent in the skeletal muscle tissues of individuals with eosinophilia-myalgia syndrome, a multisystem disorder distinguished by peripheral eosinophilia, myalgia, and the absence of infectious or neoplastic etiology. Epidemiological evidence has linked cases of eosinophilia-myalgia syndrome with the consumption of contaminated L-tryptophan, for example, 1,1’-ethylidenebis L-tryptophan (EBT) [32-34]. However, the mechanisms by which contaminated L-tryptophan lead to myalgia, as well as the role of eosinophils in this disorder, remain unclear. Accordingly, several groups have tried unsuccessfully to replicate features of eosinophilia-myalgia syndrome in animal models [35]. Notably, Silver and colleagues [36, 37] administered contaminated tryptophan (EBT) to wild-type mice, which exhibited prominent inflammation and fibrosis in response to EBT; however, features such as peripheral eosinophilia were conspicuously absent, in contrast with the disease seen in humans. The absence of a suitable animal model suggests that other factors, in addition to the consumption of contaminated
L-tryptophan, may contribute to eosinophil infiltration that is a characteristic of this disorder.
Eosinophil infiltration in skeletal muscle tissues has been reported in patients with limb girdle muscular dystrophy type 2A (LGMD2A) [38-42], an inherited disorder of muscle wasting caused by mutations in the gene encoding calpain-3 (CAPN3) [43]. Expression of calpain-3 is limited to skeletal muscle tissues, where it binds structural proteins including titin [43]. Recent studies indicate that calpain-3 is not required for maintaining the integrity of the sarcolemma but may be critical for the expansion in muscle mass following periods of atrophy [44]. Furthermore, histological analysis revealed rare and limited necrosis in the skeletal muscle tissues of LGMD2A patients and Capn3-/- mice [45-47]. The contributions of eosinophils to the pathophysiology of
LGMD2A remains unclear. Given current concepts that feature a role for eosinophils in
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remodeling damaged muscle tissue [22], it is reasonable to speculate that recruited eosinophils might promote the expansion of pro-myogenic populations in calpain-3-deficient muscle tissue.
Together, these disorders illustrate the distinct etiology and pathophysiology accompanying disorders associated with eosinophil infiltration in the muscle tissue. As such, infiltrating eosinophils may exhibit distinct features and modulate unique functions within the muscle tissue that would merit further investigation. In the following section, we review our use of RNA- sequencing methods in evaluating the characteristics of eosinophils recruited in muscular dystrophy; use of this approach may reveal unique insights into the role of eosinophils recruited in other musculoskeletal disorders.
RNA-sequencing enables functional characterization of eosinophils
In Chapter 4 of this dissertation, we provided evidence indicating that eosinophils isolated from the muscle tissues of mdx, IL5tg, and mdx.IL5tg mice exhibit distinct transcriptomic features.
We note that eosinophils from the muscle tissues of mdx mice exhibit robust expression of transcripts encoding remodeling enzymes and cell surface proteins; by contrast, eosinophils from the muscle tissues of IL5tg mice preferentially express transcripts encoding eosinophil-specific and cell cycle-related proteins. Taken together, these findings are consistent with current
concepts regarding eosinophils and their capacity to respond transcriptionally to the tissue
microenvironment in order to modify their cellular features and functionality.
These findings directly challenge the perception of eosinophils as purely “end-stage effector
cells” with limited transcriptional activity. This view derived in large part to the dominant presence
of pre-formed granule contents, which would abrogate the need for any de novo synthesis of
functional mediators. Furthermore, isolating intact, high-quality RNA from eosinophils is
complicated by limited abundance and the high concentration of ribonucleases stored in the
cytoplasmic granules. As a result of these factors, only a limited number of studies (<7
publications) have pursued RNA-sequencing to analyze eosinophil characteristics.
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While recognizing these challenges, we believe that our findings indicate that sequencing and
analysis of the eosinophil transcriptome provides an invaluable approach for the characterization
of these leukocytes. First, although eosinophils contain pre-formed mediators stored within their
granules, there may be a need to replenish granule contents through de novo synthesis.
Eosinophil granules may be rapidly depleted in response to inflammatory insults and/or increased
persistence within tissues. For example, eosinophils recruited to the gastrointestinal tract exhibit
prolonged survival (t1/2 = 6 days) when compared with eosinophils in circulation (t1/2 = 3-24 hours)
[48]. Hence, analysis of the transcriptomic profiles in eosinophils will help to identify unique cellular characteristics and potential mediators of eosinophil function within the tissues.
By utilizing RNA-sequencing, we were able to develop a comprehensive, unbiased view of the characteristics of eosinophils from three distinct microenvironments. Analysis of the eosinophil transcriptome revealed differential expression of several transcripts encoding proinflammatory cytokines and chemokines (e.g., Il15, Ccl7, Ccl8), but the most prominent class of transcripts that were differentially expressed in eosinophils were those encoding tissue remodeling and cell surface proteins. As such, these findings are consistent with the view that eosinophils recruited to the muscles of mdx mice function primarily to regulate remodeling and local immune responses.
Similarly, use of RNA-sequencing in other settings may further challenge preconceived notions regarding eosinophil function and orient investigators towards critical themes for subsequent investigation.
Analysis of our RNA-sequencing results revealed differential expression of the transcript encoding Trem2, which was detected at relatively higher levels in eosinophils recruited to the muscle tissues of mdx mice. In Chapter 5 of this dissertation, we engaged in critical studies aimed at elucidating the expression and function of Trem2 in eosinophils. Trem2 transcripts encode
Triggering Receptor Expressed on Myeloid Cells-2 (TREM2), a receptor that has been shown to promote the proliferation, activation and survival of myeloid cells in the setting of tissue damage
[49-53]. Importantly, we identified a key role for Trem2 in modulating the expansion of the
114
eosinophil lineage; as such, expression of TREM2 may be critical for the expansion and
persistence of eosinophils in dystrophin-deficient muscle tissue. As discussed in the following
section, these findings raise intriguing questions about the regulation of Trem2 expression as well
as its potential role in mediating eosinophil activation.
Future Directions – Regulation and Function of TREM2 on Eosinophils
In this dissertation, we identified prominent expression of Trem2 transcripts in eosinophils
isolated from the muscle tissues of mdx mice (Chapter 4) as well as prominent expression of
immunoreactive TREM2 on mouse eosinophils cultured ex vivo and on their human eosinophil
counterparts isolated from peripheral blood (Chapter 5). These findings are both novel and
intriguing, as expression of TREM2 was widely considered to be limited to cells of the
monocyte/macrophage lineage [54, 55]. Moving forward, it will be critical to elucidate the signals
that promote TREM2 expression in eosinophils. Earlier work has demonstrated that pro-
inflammatory mediators (e.g., bacterial lipopolysaccharides) limit, while anti-inflammatory
mediators (e.g., interleukin-4) promote, expression of immunoreactive TREM2 in macrophages
and microglial cells [56, 57]. Given the increased expression of Trem2 in eosinophils isolated from the muscle tissues of dystrophin-deficient (mdx) mice, we will evaluate the impact of specific inflammatory mediators on the expression of TREM2 on eosinophils; notably, we will examine the impact of IL-4, IL-1RA, Eotaxin-1/CCL11 and RANTES/CCL5, all of which are elevated in dystrophin-deficient muscle tissue (Chapter 3, [25]) and have known impact on mouse
eosinophils. As part of our investigation into the regulation of TREM2 expression in eosinophils,
we will also examine the expression of other TREM and TREM-like family members (e.g., Trem1,
Trem3, Trem4, Trem5, Treml1, Treml2) in cultured eosinophils at baseline and in response to
stimuli.
Our findings indicate that Trem2 is required for efficient development of eosinophils at
baseline, but is not required for their mobilization into the airways in response to allergen- 115
challenge. Intriguingly, Trem2-/- mice challenged intranasally with components of the fungal allergen, Alternaria alternata exhibited similar levels of eosinophils in both the airways and in the
bone marrow when compared with findings generated in wild-type mice (data not shown). These
results suggest that conditions secondary to allergen challenge, such as Th2-mediated
inflammation, may overcome defects in eosinophil development associated with Trem2-
deficiency. As such, it will be critical to evaluate the inter-connecting pathways and cellular
features critical to Trem2-mediated development of eosinophils. To start, we will evaluate
activation of key signaling pathways (e.g., mTOR, Akt) as well as the activity of key cellular
processes (e.g., autophagic flux, metabolic activity) in cultures of wild-type and Trem2-/-
eosinophils; these features are impaired in Trem2-/- microglial cells [51, 53]. Furthermore, we will
evaluate the impact of Th2 cytokines (e.g., IL-4, IL-13) on these cellular features; findings from
this study may help explain the eosinophil recruitment and expansion observed in both wild-type
and Trem2-/- mice in response to challenge with Alternaria alternata.
Finally, we intend to investigate the role of Trem2 in disorders associated with eosinophil
infiltration. Because Trem2 is detected at high levels in eosinophils isolated from the muscle
tissues of mdx mice, we have generated mdx.Trem2-/- mice and will utilize these mice to analyze
the degree of eosinophil infiltration in the muscle tissues. Given the decreased viability that is a
characteristic of Trem2-/- eosinophils cultured ex vivo, we expect mdx.Trem2-/- mice to exhibit
decreased eosinophil infiltration in the muscle tissues when compared with that observed in mdx
mice. In addition, we intend to continue our investigation of Trem2-mediated functions in
association with Alternaria alternata-driven airways hyperreactivity. In our earlier work (Chapter
5), we established that Trem2-deficiency did not abrogate eosinophil recruitment to the airways.
However, it may have an impact on eosinophil function in this microenvironment. Further work will elucidate the impact of Trem2-/- eosinophils on airway histopathology following allergen
challenge.
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Concluding Remarks
This dissertation examined the function and characteristics of eosinophils recruited to the skeletal muscle tissues of muscular dystrophy (mdx) mice. Towards this end, I developed novel protocols to identify and to isolate eosinophils from muscle tissue and to isolate eosinophil RNA for sequencing and analysis. These tools may be applicable to the characterization of eosinophils in other disorders; for examples, eosinophils are prominent in the muscle tissues associated with
Limb-Girdle Muscular Dystrophy Type 2A (LGMD2A) and eosinophilia-myalgia syndrome. Using these tools, I established that eosinophils do not drive the acute muscle damage in muscular dystrophy (mdx) mice, and that eosinophils exhibit unique features that may potentiate their function. In particular, I focused on Trem2, which encodes a receptor critical for the efficient development of eosinophils. Further work is needed in order to elucidate the regulation and functions of Trem2 in eosinophils and eosinophil-mediated disorders.
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