Genetic Differences in Bone Marrow-Derived Lymphoid Lineages Control Susceptibility to Experimental Autoimmune Myocarditis

This information is current as Haiyan S. Li, Davinna L. Ligons, Noel R. Rose and Mehmet of September 28, 2021. L. Guler J Immunol 2008; 180:7480-7484; ; doi: 10.4049/jimmunol.180.11.7480 http://www.jimmunol.org/content/180/11/7480 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Genetic Differences in Bone Marrow-Derived Lymphoid Lineages Control Susceptibility to Experimental Autoimmune Myocarditis1

Haiyan S. Li,2* Davinna L. Ligons,* Noel R. Rose,2*† and Mehmet L. Guler*

Bone marrow (BM) transplantation has been used to study the cellular basis of genetic control of autoimmune diseases, but conclusions remain elusive due to the contradictory findings in different animal models. In the current study, we found that BM cells from myocarditis-susceptible A.SW mice can render irradiated, myocarditis-resistant B10.S recipient mice susceptible to myosin-induced myocarditis, indicating that hematopoietic cells express the genetic differences controlling susceptibility to autoimmune myocarditis. We then sought to differentiate the role of lymphoid vs nonlymphoid components of BM in the pathogenesis of myocarditis by comparing mixed chimeras receiving BM from A.SW wild-type or RAG؊/؊ mice mixed with BM from B10.S Downloaded from wild-type mice. This experiment clearly demonstrated that T and B lymphocytes were indispensable for transferring the susceptible phenotype to disease-resistant recipients. Our findings significantly narrow the cellular expression of genetic polymorphisms controlling the EAM phenotype. The Journal of Immunology, 2008, 180: 7480–7484.

he pathogenesis of autoimmune diseases is generally at- immunopathogenic mechanisms of the disease. Like most autoim- tributed to effector functions of the adaptive immune sys- mune diseases, MHC haplotype is an important genetic factor for http://www.jimmunol.org/ T tem, but the humoral and cellular components of innate EAM susceptibility, but its effects are overshadowed by non-MHC immune responses also play a substantial role in directing the later traits (11, 12). MHC molecules are expressed prominently on he- pathogenic adaptive immune responses (1, 2). In the past, bone matopoietic cells, but varying levels of MHC class I molecules are marrow (BM)3 transplantation has been used to discriminate the also found on nonhematopoietic cells. Meanwhile, several non- contribution of hematopoietic vs nonhematopoietic cell lineages to H-2 Eam loci have been identiﬁed (13), but the cellular expression disease susceptibility. Development of some autoimmune diseases, of these genetic polymorphisms remains unclear. For example, including spontaneous diabetes or lupus nephritis (3–5), originates passive transfer of autoimmune myocardits by myosin-speciﬁc Ab from defects that reside within BM-derived stem cells of NOD or depends upon target organ polymorphism (14). In addition, muta-

New Zealand White mice, respectively. Nonhematopoietic factors, tion or deletion in genes encoding cardiac Ags (15, 16) was found by guest on September 28, 2021 however, have also been shown to control disease susceptibility as in patients with dilated cardiomyopathy, suggesting genes ex- evidenced in experimental allergic encephalomyelitis (6, 7) and pressed in the target organ may contribute to disease susceptibility systemic lupus erythematosus in ␣-mannosidase II-deficient mice or severity. Our aim in the current study is to localize the cellular (1). Furthermore, in an animal model of experimental Lyme ar- mediators that harbor the major genetic differences between myo- thritis, resistance to disease induction was conserved by either he- carditis-susceptible and myocarditis-resistant mouse strains, and to matopoietic or nonhematopoietic cellular compartments (8). These further discriminate the roles of different cell lineages in genetic contradictory findings highlight the distinct pathogenic mecha- susceptibility to autoimmune myocarditis. nisms in different autoimmune diseases. Autoimmune myocarditis is an organ-specific autoimmune disease characterized by damage of the myocardium by infiltrating Materials and Methods Mice and Ags inflammatory cells. It often develops after an acute viral myocarditis in both humans (9) and rodents (10). Experimental autoim- A.SW and B10.S (both H-2s haplotype) were purchased from The Jackson mune myocarditis (EAM) induced by cardiac myosin (CM) in sus- Laboratory, and have been bred and maintained in the conventional hous- ing facilities at The Johns Hopkins University (Baltimore, MD). A.SW ceptible mouse strains provides a virus-free model to study the Ϫ Ϫ Ϫ Ϫ RAG / were generated by backcrossing A/J RAG / mice, a gift from Dr. D. Farris (University of Oklahoma, Oklahoma City, OK), to the A.SW *Department of Pathology, and †W. Harry Feinstone Department of Molecular Mi- background. Mouse CM was purified from frozen Swiss Webster white crobiology and Immunology, The Johns Hopkins University, Baltimore, MD 21205 mouse hearts (Pel-Freez Biologicals) following procedures previously de- scribed (11). Received for publication March 25, 2008. Accepted for publication March 25, 2008. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance Generation and characterization of BM chimeras with 18 U.S.C. Section 1734 solely to indicate this fact. A.SW or B10.S recipients were lethally irradiated (ϳ850 rad) and 12–24 h 1 This work is supported by Research Grants R01 HL-077611 and R01 HL-067290 later, reconstituted with 2–5 ϫ 106 donor BM cells, depleted of mature T from the National Institutes of Health. and B cells. Animals were allowed to reconstitute for 12 wk before EAM 5 2 Address correspondence and reprint requests to Dr. Noel R. Rose or Dr. Haiyan S. induction. To assess the degree of chimerism, spleen cells (5 ϫ 10 /mouse) Li, Department of Pathology, Johns Hopkins University, 720 Rutland Avenue, Bal- were stained with strain-specific markers of anti-Ly9.1 (FITC) or anti- timore, MD 21205. E-mail addresses: [email protected] or [email protected] Ly6.2 (clone 3A7), which recognize lymphocytes from A.SW or B10.S 3 Abbreviations used in this paper: BM, bone marrow; EAM, experimental autoim- origin, respectively, in combination with cell surface markers, CD4, CD8, mune myocarditis; CM, cardiac myosin; WT, wild type. and CD19. Anti-Ly6.2 is a gift from Dr. K. L. Rock (Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA) (17), and all other Abs Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 were purchased from eBioscience. www.jimmunol.org The Journal of Immunology 7481

Induction and assessment of EAM EAM was elicited by immunization with mouse CM as previously de- scribed (18). At day 21, the hearts were collected, fixed in 10% formalin, and sent to Histoseve for embedding, sectioning, and H&E staining. The degree of EAM was scored based on the percentage of the area of infil- trated myocardium in the most affected section as follows: 0 ϭ no infil- tration, 1 ϭϽ10%, 2 ϭ 10–30%, 3 ϭ 30–50%, 4 ϭ 50–80%, and 5 ϭ Ͼ80%. The presence of myosin-specific IgG in immune sera was assessed by ELISA. Statistical analysis Differences in EAM severity and OD readings of Ab ELISA between groups were assessed for statistical significance by the nonparametric Mann-Whitney U test or t test in GraphPad Prism 4 software (http://www.graphpad.com).

Results BM from susceptible mice impart EAM susceptibility to normally resistant recipients

To determine whether the genetic control of EAM was mainly Downloaded from mediated by cells of hematopoietic or nonhematopoietic origin, we generated BM chimeric mice from myocarditis-susceptible A.SW mice and myocarditis-resistant B10.S mice (both having the same H-2s haplotype). Regardless of whether BM transfer was per- formed with syngeneic or allogeneic donors, there was little evidence of graft-vs-host disease. Animals displaying any sign of FIGURE 1. Phenotype of susceptibility to EAM is transferable through http://www.jimmunol.org/ graft-vs-host disease, such as weight loss or skin lesions, were BM. After 12 wk of reconstitution, BM chimeric mice were subjected to excluded from the study. Twelve weeks following BM transplan- EAM induction (A) and tested for repopulation from donor BM (B). A, The tation, chimeric mice were tested for EAM susceptibility by im- EAM score was assigned based on a grading scale describing the severity munization with CM. Twenty-one days later, hearts were collected of myocarditis as shown in Fig. 2. Data are presented as individual values and histologically examined for the presence of myocardial le- with mean (horizontal line). The differences between groups were analyzed sions. At the same time, spleen cells were harvested and stained by nonparametric Mann-Whitney U test in GraphPad Prism. B, Percentage with Ly9.1 or Ly6A.2, which recognize lymphocytes of A.SW or of chimerism of spleen cells in each experimental group is expressed as B10.S origin, respectively, to test the repopulation of chimeric individual values with mean (horizontal line). mice by donor BM. As shown in Fig. 1B, in B10.S mice receiving by guest on September 28, 2021 BM from A.SW donor (A3B) or conversely transplanted A.SW mice (B3A), ϳ80% of spleen cells were derived from donor BM, 3 whereas 20% were repopulated from residual host BM that sur- planted with BM from the following: 1) WT B10.S (B B), 2) WT 3 vived after irradiation. In mice receiving autologous BM (A3Aor A.SW (A B), or 3) a 50/50 mixture of WT A.SW and B10.S B3B), 100% of spleen cells were either A- or B-derived, respec- (A/B3 B). The purpose of cotransfer of B10.S WT BM with Ϫ/Ϫ tively. When myocarditis was examined, we found that BM-de- RAG was to render irradiated recipients immunocompetent, rived hematopoietic cells from A.SW were fully capable of trans- i.e., having normal T and B cell pools. In this experiment, re- ferring disease susceptibility to normally resistant B10.S mice population of the chimeric mice by different cell types was ex- (Figs. 1A and 2). Approximately 68% of B10.S mice receiving BM amined in the spleen. Like in Fig. 1B, chimeric status in A3 B ϩ from A.SW donor (A3B mice) developed EAM with mean score or B3 A mice was ϳ80:20 of donor to host in CD4 T cells ϩ of 1.22 Ϯ 1.14, and both incidence and severity were comparable (Fig. 3). In A/B3 B mice, approximately half of CD4 spleen to control A3A mice. Interestingly, in the converse experiment, cells came from A.SW origin and the other half from B10.S ϩ Ϫ Ϫ A.SW mice receiving BM from B10.S mice (B3A mice) devel- origin. CD4 population in A / /B3 B mice represent similar ϩ oped slightly, but significantly, more EAM vs B3B controls profile as B3 B mice (Fig. 3). In all groups, CD8 T cell and ϩ (EAM with mean score of 0.70 Ϯ 0.88 vs 0.21 Ϯ 0.46, p ϭ 0.013). CD19 B cell in spleens express similar profiles as observed in ϩ This slight increase in EAM induction in B3A mice could be due CD4 T cells (Fig. 3B). to the hematopoietic cells originated from remaining A.SW BM These chimeras were also tested for their susceptibility to EAM. after lethal irradiation (Fig. 1B) or minor contributions from non- As shown in Fig. 4, B3 B mice remained low responders, hematopoietic cells. whereas A3 B and A/B3 B were almost equally susceptible to myocarditis. Approximately 80% of A3 B and A/B3 B mice The susceptibility to EAM is mainly determined by BM-derived developed myocarditis with mean EAM score of 1.65 Ϯ 1.14 vs lymphoid compartments 1.45 Ϯ 1.15, respectively. AϪ/Ϫ/B3 B developed EAM with The finding that A.SW BM could transfer disease susceptibility to intermediate severity, but not significantly differ from B3 B the normally resistant B10.S mice provides conclusive evidence control and A/B3 B, suggesting that disease susceptibility is that the predominant EAM susceptibility genes are expressed in determined by lymphocytes and possibly other hematopoietic BM-derived cells. To determine whether the lymphoid or nonlym- cells such as NK cells and mast cells contributed by the RAG- phoid compartment within BM-derived cells express these suscep- deficient mice. In agreement with previous findings in the SCID tibility genes, irradiated B10.S were transplanted with 50/50 BM mouse (19), RAGϪ/Ϫ mouse itself and B10.S transplanted with mixture of A.SW RAGϪ/Ϫ and B10.S wild-type (WT) mice (AϪ/Ϫ/ RAGϪ/Ϫ BM did not develop myocarditis following CM im- B3B). As controls, irradiated B10.S recipients were also trans- munization (data not shown). 7482 T AND B LYMPHOCYTES CONTROL GENETIC SUSCEPTIBILITY TO EAM Downloaded from

FIGURE 3. BM reconstitution of recipient mice transplanted with syngeneic or allogeneic donor BM. Recipient mice were lethally irradiated and injected i.v. with 2–5 ϫ 106 BM cells from A.SW, B10.S, 50/50 mixture A.SW/B10.S, or 50/50 mixture A.SW RAGϪ/Ϫ/B10.S mice. After 12 wk, reconstitution of lymphocyte population in the spleen was analyzed by http://www.jimmunol.org/ FACS. A, Surface expression of strain markers Ly9.1 (A.SW) and Ly6A.2 (B10.S) on CD4ϩ spleen cells from representative mouse of each experimental group is shown. The experimental population (gray shaded histogram) and isotype control (open histogram) are indicated. B, Histogram represents the mean percentage of chimerism of CD4ϩ, CD8ϩ, CD19ϩ lymphocyte population of individual mice in each experimental group.

(data not shown). Interestingly, IgG1 levels were signiﬁcantly by guest on September 28, 2021 3 3 ϭ higher in EAM-susceptible A B and A/B B (OD450 nm 2.96 Ϯ 0.77 and 2.58 Ϯ 0.90, respectively, p Ͻ 0.01), but not in Ϫ/Ϫ 3 ϭ Ϯ resistant A /B B mice (OD450 nm 2.30 0.72), in compar- 3 ϭ Ϯ ison to B B mice (OD 450 nm 1.56 1.14). IgG2a and IgG2c isotypes, encoded by different Ig H chain alleles, are expressed in A.SW and B10.S mice exclusively (20). Therefore, it is not sur- prising that a large amount of IgG2a was detected in B10.S recip- ϭ ients reconstituted with B cells of A.SW origin (OD450 nm Ϯ 3 ϭ Ϯ 2.12 0.96 in A B mice and OD450 nm 1.02 0.89 in A/B3B mice). IgG2c, although at a much lower amount with OD FIGURE 2. Representative histological appearance of EAM severity readings ranging from 0.17 to 0.21, was only detected in B10.S score. BM chimeric mice were immunized with CM in CFA at day 0 and day 7, with additional dose of pertussis toxin at day 0, to promote EAM induction. Twenty-one days later, hearts were collected for histological examination. A and B, Normal heart, EAM score ϭ 0. C and D, Minimal myocarditis with Ͻ10% of myocardium involved, EAM ϭ 1. E and F, Moderate myocarditis with 10–30% of myocardium involved, EAM ϭ 2. G and H, Severe myocarditis with 30–50% of myocardium involved, EAM ϭ 3. I and J, Extensive myocarditis with Ͼ50% of myocardium involved, EAM ϭ 4. Magniﬁcation is ϫ5(left column) and ϫ100 (right column).

Ab responses in BM chimeric mice The production of CM-specific total IgG and its isotypes in these FIGURE 4. Phenotype of susceptibility to EAM is transferable through chimeric mice was measured by ELISA to determine their corre- BM-derived lymphoid compartment. Similar to experiment in Fig. 3, chimeric lation with myocarditis phenotype. At total IgG level, a small dif- mice were created by transferring BM from WT or RAGϪ/Ϫ A.SW alone or 3 3 ference was observed between A B and control B B mice mixed with WT B10.S mice to irradiated WT B10.S recipients. EAM induc- (OD ϭ 2.97 Ϯ 0.57 vs 2.35 Ϯ 0.63, p Ͻ 0.05), but not in tion in these mice was tested after 12 wk of reconstitution, and represented as 450 nm Ϫ Ϫ A/B3B and A / /B3B mice. There was also no difference ob- individual values with mean (horizontal line). EAM severity between groups served in WT A.SW and B10.S mice following EAM induction was compared by nonparametric Mann-Whitney U test in GraphPad Prism. The Journal of Immunology 7483 recipients reconstituted with B cells of B10.S origin (B3B, predominance in the myocardial lesions, their capability to transfer A3B, and A/B3B). Among the IgG isotypes, IgG1 and IgG2b disease following stimulation with CM, and the amelioration of were predominant Abs detected at large amounts, suggesting that EAM after their depletion with mAb in A/J mice (19, 29). Mono- there may be a Th2 component of EAM in the A strain mice (21). clonal Ab-mediated depletion of CD8ϩ population reduced EAM severity but not incidence in A/J mice (19), whereas both disease Discussion incidence and severity were significantly enhanced in mice ho- Clinical studies have reported that BM cells can transfer disease- mozygous for CD8 mutation (CD8Ϫ/Ϫ) following immunization causing autoreactive clones from donors to patients (22–24). This with CM (30). These paradoxical findings indicate that the CD8ϩ approach was used by us and other investigators to examine the T cell may mediate both effector and suppressor functions in EAM cellular basis of autoimmune diseases in animal models. In accor- via distinct pathways. dance with previous findings in diabetes and lupus (3–5, 25), ge- Other cells or cellular products including NK cells (2), mast netic susceptibility of myocarditis was shown to be controlled by cells (31), and complement (2) also influence disease susceptibil- donor-derived hematopoietic cells, not by the host background. ity. Interestingly, a subset of TCR␣␤ϩ CD4ϪCD8Ϫ double neg- Different findings were observed in other autoimmune diseases in ative cells were found in lesions of autoimmune myocarditis (30), which disease susceptibility or resistance was either mediated by arthritis (32), and encephalomyelitis (33) developed in CD4-defi- nonhematopoietic factors (6, 7) or by both hematopoietic and non- cient mice, suggesting these cells may be capable of obviating the hematopoietic factors (8). To further localize the EAM-controlling requirement for CD4ϩ T cells and subserve the effector function. factors to lymphoid vs nonlymphoid compartment, mixed BM chi- B cells are generally considered not essential for EAM induction Ϫ Ϫ meras were created using BM mixture of RAG / A.SW and (34). The pathogenic mechanisms of the particular cell type may Downloaded from B10.S mice in the current study. Our results demonstrate that both be altered in BM chimeras and their function will be investigated lymphoid and nonlymphoid components of hematopoietic cells in future studies by creating chimeras using BM from mice defi- were the restriction elements for EAM susceptibility. These cell cient in CD4, CD8, or Btk (B cell) or depleting Ab. lineages may harbor polymorphic genes that are required for EAM Autoimmune myocarditis has been linked to both MHC haplo- pathogenesis but differ between strains. type and non-MHC genes, with non-MHC genes having strong

BM transplantation created chimeric mouse consisting of donor effects (13). MHC molecules modulate adaptive immune re- http://www.jimmunol.org/ BM-derived components on the host genetic background, which sponses, including autoimmune responses, via their unique role in provides an ideal system to differentiate the role of hematopoietic Ag presentation. Yet, at least seven non-MHC loci have been re- vs nonhematopoietic cells. Good reconstitution status, i.e., 80:20 ported to influence susceptibility to viral or immunization-induced chimerism of donor to host, was achieved in these mice after 12 wk autoimmune myocarditis in mouse (18, 35, 36). Linkage of these (Fig. 1). In all experiments, sham control B3B mice developed loci to apoptosis or tolerance induction was suggested; however, minimal myocarditis compared with their WT counterparts, indi- the mechanisms by which they modulate disease susceptibility re- cating the damage to the heart was caused by autoimmune remain to be investigated. In addition, polymorphisms in genes en- sponses following CM immunization, but not transplantation- coding cardiac Ags (15, 16) and CD45 (37) were found in patients induced immunologic dysregulation. The degree of EAM with dilated cardiomyopathy, indicating potential association of by guest on September 28, 2021 susceptibility was positively correlated with the percentage of chi- these polymorphisms with myocarditis. The current findings sig- merism achieved, as A3A mice (100% of A strain) developed nificantly narrow our focus on identification of the predominant EAM with highest EAM incidence and severity, B3B mice genetic polymorphisms responsible for EAM phenotype. Future (100% of B strain) have lowest disease and A/B3B mice (ϳ50/50 studies will further subclassify the cells of interest by using ge- for A to B strain) display intermediate phenotype. Coexistence of netically engineered animals that are deficient in particular subsets donor and host BM in mixed chimeras (A/B3B) has been sug- of T and B cells. This approach can also be used in other autoim- gested to facilitate the reciprocal induction of tolerance between mune disease models to dissect the cellular expression of a par- donor and host, which minimize graft-vs-host and host-vs-graft ticular genetic trait. disease (26, 27). We also showed that the autoimmune phenotype of A.SW recipients can be rescued by BM derived from resistant Acknowledgments B10.S donor, as was previously shown in other models (5, 24, 28). We thank Monica V. Talor and Dongfeng Zheng for technical assistance. These observations highlight the primary role of immune system (donor), but not the target organ (host), in the pathogenesis of Disclosures autoimmune diseases. However, the contribution of the residual The authors have no financial conflict of interest. host BM to this process cannot be excluded because ϳ20% of lymphocytes were host-derived. References Hematopoietic cells of both innate and adaptive immune re- 1. Green, R. S., E. L. Stone, M. Tenno, E. Lehtonen, M. G. Farquhar, and sponses have been suggested to play role in the pathogenic process J. D. Marth. 2007. Mammalian N-glycan branching protects against innate immune self-recognition and inflammation in autoimmune disease pathogenesis. of EAM (2, 29). 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