Diversity in Human Peripheral Blood : Profound Variability of IL-16

This information is current as Michelle Ma, Caroline M. Percopo, Daniel E. Sturdevant, of September 28, 2021. Albert C. Sek, Hirsh D. Komarow and Helene F. Rosenberg J Immunol 2019; 203:520-531; Prepublished online 10 June 2019; doi: 10.4049/jimmunol.1900101

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Cytokine Diversity in Human Peripheral Blood Eosinophils: Profound Variability of IL-16

Michelle Ma,* Caroline M. Percopo,* Daniel E. Sturdevant,† Albert C. Sek,* Hirsh D. Komarow,‡ and Helene F. Rosenberg*

Eosinophilic leukocytes develop in the bone marrow and migrate from peripheral blood to tissues, where they maintain homeostasis and promote dysfunction via release of preformed immunomodulatory mediators. In this study, we explore human heterogeneity with a specific focus on naturally occurring variations in cytokine content. We found that human eosinophil-associated varied on a continuum from minimally (coefficient of variation [CV] £ 50%) to moderately variable (50% < CV £ 90%). Within the moderately variable group, we detected immunoreactive IL-27 (953 6 504 pg/mg lysate), a mediator not previously associated with human eosinophils. However, our major finding was the distinct and profound variability of eosinophil-associated IL-16 (CV = 103%).

Interestingly, eosinophil IL-16 content correlated directly with body mass index (R2 = 0.60, ***p < 0.0001) in one donor subset. We found Downloaded from no direct correlation between eosinophil IL-16 content and donor age, sex, total leukocytes, , or eosinophils (cells per microliter), nor was there any relationship between IL-16 content and the characterized 2295T/C IL-16 promoter polymorphism. Likewise, although eosinophil IL-1b,IL-1a, and IL-6 levels correlated with one another, there was no direct association between any of these cytokines and eosinophil IL-16 content. Finally, a moderate increase in total dietary fat resulted in a 2.7-fold reduction in eosinophil IL-16 content among C57BL/6-IL5tg mice. Overall, these results suggest that relationships between energy metabolism, eosinophils, and IL-16 content are not direct or straightforward. Nonetheless, given our current understanding of the connections http://www.jimmunol.org/ between asthma and obesity, these findings suggest important eosinophil-focused directions for further exploration. The Journal of Immunology, 2019, 203: 520–531.

osinophils have been historically perceived as end-stage, spectrum of allergic and nonallergic disorders (3–7), evolution tells cytotoxic cells with a limited range of effector responses. us that the ability to induce pathologic conditions cannot be the E Numerous observations have profoundly altered these views “raison d’eˆtre” for any existing cell lineage (1). Recent studies that and have provided a spotlight on eosinophils as complex cells with explore the role of eosinophils at homeostasis in the gastrointestinal critical immunomodulatory functions (1, 2). Whereas eosinophils tract (8, 9), which identify distinct eosinophil-mediated antimicro- by guest on September 28, 2021 are activated in and contribute to the pathophysiology of a wide bial activities (10, 11) and that reveal specific eosinophil subtypes (12, 13), suggest that there are new and profound complexities remaining to be unraveled. *Inflammation Immunobiology Section, Laboratory of Allergic Diseases, National Among the most prominent features of eosinophils are their Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; †Genomics Unit, Research Technologies Branch, National Institute of distinct cytoplasmic granules, which contain unique cationic Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840; (14) and immunomodulatory mediators (15–17). Moqbel, Lacy, and and ‡Mast Cell Biology Section, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD colleagues (18, 19) were among the first to characterize these me- 20892 diators in eosinophils, which include cytokines, , lipids, ORCIDs: 0000-0002-1497-8128 (C.M.P.); 0000-0002-8170-3749 (A.C.S.); 0000- and growth factors and their receptors (20). Physiologic roles for 0001-7012-9406 (H.D.K.). these mediators and their importance in eosinophil function was Received for publication January 24, 2019. Accepted for publication May 20, 2019. highlighted by Lee and colleagues (21) in their manuscript entitled This work was supported by the National Institute of Allergy and Infectious Diseases “Eosinophils in Health and Disease: The LIAR Hypothesis,” in which Division of Intramural Research (Z01-AI000941-14 to H.F.R.). they proposed that eosinophils were recruited to tissues to promote M.M. performed most of the experimental work, optimized the eosinophil isolation local immunity, remodeling, and repair. This hypothesis has been and cytokine profiling, coordinated the donor samples with the National Institutes of Health Clinical Center Blood Bank, and reviewed the methods for the manuscript. strongly supported by recent studies on eosinophil function in health C.M.P. performed additional experimental work and provided input on the first draft and disease in vivo [e.g., studies featured in reviews on eosinophil of the manuscript. D.E.S. performed multivariate analysis of the cytokine profiling function in helminth infection and innate immunity (22–24)]. data and reviewed the revised manuscript. A.C.S. assisted with experimental work and prepared photographic images for the manuscript. H.D.K. was instrumental in As such, a better understanding of eosinophil mediators and the obtaining normal donor samples via the Laboratory of Allergic Diseases protocol and mechanisms that promote their synthesis and release is currently a provided input on the first and subsequent drafts of the manuscript. H.F.R. concep- topic of significant interest (25–27). Toward this end, Melo and tualized the project, performed data analysis, and wrote the first and subsequent drafts of the manuscript. colleagues (28) have characterized the tubulovesicular networks Address correspondence and reprint requests to Dr. Helene F. Rosenberg, Inflamma- that serve as conduits for mediator secretion, and Lacy and col- tion Immunobiology Section, 50 South Drive, Room 6241 MSC 8014, National leagues (29, 30) have examined signaling factors that pro- Institute of Allergy and Infectious Diseases/National Institutes of Health, Bethesda, mote degranulation. The potential for distinct cytokine profiles MD 20892. E-mail address: [email protected] (i.e., differences among eosinophils from human subjects) was The online version of this article contains supplemental material. first addressed by Spencer and colleagues (31), who focused on Abbreviations used in this article: BMI, body mass index; CV, coefficient of varia- tion; LAD, Laboratory of Allergic Diseases; mpd, mean pixel density; npd, normal- seven individual cytokines identified in eosinophil lysates from 18 ized pixel density. nonallergic and allergic donors. Among their findings, the Th1 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900101 The Journal of Immunology 521 cytokine IFN-g was identified as a prominent component of hu- on-site (14BS vivarium, National Institute of Allergy and Infectious Dis- man eosinophils, although no significant differences were reported eases/National Institutes of Health). Mice were maintained on normal in comparisons between allergic versus nonallergic donors. house diets (LabDiet, Advanced Protocol 5V0T, 12.5% kcal from fat) or a diet with moderately increased fat (LabDiet, Select Mouse 5B0G, 22% In this manuscript, we build on these findings using dual-Ab kcal from fat), the latter for 5–6 wk prior to evaluation. The National proteome profiling technology. With this method we examined Institute of Allergy and Infectious Diseases Division of Intramural Re- differential expression of 36 cytokines in lysates prepared from search Animal Care and Use Committee, as part of the National Institutes peripheral blood eosinophils purified from whole blood from normal of Health Intramural Research Program, approved all the experimental procedures as per protocols LAD7 and LAD 8E. donors. As a component of this study, we identified naturally oc- curring variations in cytokine content, including the unique pattern Human eosinophil isolation displayed by the dual-function cytokine IL-16 and its unanticipated Eosinophils were isolated from whole blood using the Miltenyi Human correlation with donor body mass index (BMI) in one donor subset. Eosinophil Isolation Kit (130-104-466) according to manufacturer’s in- structions as described (34). Briefly, particle-bound Abs included in the MACS cell isolation mixture are added to whole blood, which was then Materials and Methods subjected to a magnetic field using the MACSxpress Separator (130-098- Blood donors 308). Peripheral blood erythrocytes and leukocytes other than eosinophils were bound to Abs and were held within the magnetic field, whereas un- Single samples of∼30 ml EDTA–anti-coagulated whole blood were ob- bound eosinophils were carried through in the mobile buffer phase; re- tained from de-identified adult normal donors from the National Institutes sidual erythrocytes were lysed with cold, endotoxin-free water. Isolated of Health Clinical Center (Protocol Identifier NCT00001846) and from eosinophils (typically∼2.0–8.0 3 106 from∼30 ml blood at .98% purity the Laboratory of Allergic Diseases (LAD) normal volunteer protocol and viability) were washed and resuspended at 1 3 107 cells/ml PBS with (09-I-0049). All donors agreed to donation via standard informed consent 0.1% BSA. Cytospins were prepared and stained with modified Giemsa Downloaded from protocols. Eligibility for donation via the Clinical Center protocol includes (Diff-Quik; Thermo Fisher Scientific), and a minimum of 200 cells were age $ 18 y, weight . 110 pounds, no known history of heart, lung, kidney scored per preparation. Photomicroscopy was performed with a DMI4000 disease, or bleeding disorders, and no current pregnancy; samples used in light microscope (Leica Microsystems, Wetzlar, Germany) equipped with a our study were also negative for CMV. Eligibility for donation via the LAD Retiga 2000R camera and analyzed using QCapture software (both from protocol includes age between 18 and 65, no known history of chronic QImaging, Surrey, BC, Canada). virus infection, anemia, bleeding disorder, or use of therapies that would have an impact on hematopoiesis, and no current pregnancy. Preparation of lysates from human eosinophils http://www.jimmunol.org/ Mice Isolated eosinophils were collected by centrifugation, washed, and resus- pended in lysis buffer (20 mM Tris [pH 8], 139 mM NaCL, 0.5 mM EDTA, IL-5 transgenic mice on the C57BL/6 and BALB/c backgrounds [B6-IL5tg 1% IGEPAL, and 10% glycerol with one tablet of protease inhibitor mix NJ.1638 (32) and BALB/c-IL5tg (33) mice, respectively] were maintained [04693159001; Roche] per 10 ml lysis buffer) at 1 3 107 cells/ml. After by guest on September 28, 2021

FIGURE 1. Validation of cytokine profiling and normalization to standard donor lysate. (A) Reproducibility. Lysates were prepared from eosinophils isolated from the whole blood drawn on three separate occasions from the standard donor; 100 mg from each lysate was evaluated by cytokine profiling in a single trial, and results (mpd) for a selection of seven cytokines from the full set are shown in this study. (B) Dose response. Two hundred fifty, one hundred, and seventy-five microgram samples from standard donor eosinophil lysate were evaluated in a single trial; results in mpd are as shown. (C and D) Sample data and normalization. The variability determinations presented in Tables III and IV and in Fig. 2 were generated from two experimental trials, each performed with seven unique donor lysates (100 mg each), with a single standard sample replicated in each trial. As mpds differ between trials [shown in (C) for the cytokine, IL-13], the technical replicates from each donor sample were averaged and normalized to the standard donor in each trial as follows: donor 1 (d1) is normalized via the calculation from technical replicates (error bars) (d1a + d1b)/(std a + std b) = (1423 + 1338)/(1289 + 1191) = 1.11 npd, with a and b representing technical replicates. The same calculations are performed for each donor in trial 1 and trial 2 (shown in D). Once the data are normalized (npd), the range, mean, SD, and CV percentage) are calculated. For IL-13 as shown, the CV (percentage) is 26.8%, which places it within the minimum variability group (Table III). std, standard. 522 EOSINOPHILS AND IL-16

Table I. Normal donors

Gender Number of Donorsa Age (y) Age Range (y) BMI (kg/m2) BMI Range (kg/m2) Male 22 43 6 16 22–75 28.5 6 5.4 18.9–41.3 Female 15 38 6 16 20–66 27.9 6 5.9 19.2–38.3 aEosinophil lysates were prepared from 37 normal blood donors (24 white, 6 African-American, 3 Hispanic, 1 Asian, and 3 mixed race or unrecorded). There were no statistical differences noted in any comparison of male versus female donors; data shown as 6 SD.

30 min incubation at 2–8˚C, insoluble material was removed by cen- MO). The clustering was performed using Euclidean dissimilarity with trifugation. Supernatants were stored in aliquots at 270˚C until use. average linkage and gap statistics using five bootstrap iterations to produce concentration in supernatant was determined by MicroBCA the dendrograms and heat map. Missing metadata were imputed for two assay (catalog no. 23235; Thermo Fisher Scientific), typically at subjects. 600–1000 mg/ml. Isolation of DNA and amplification of IL-16 promoter sequence Cytokine profiling and analysis for identification of 2295T/C promoter polymorphism The cytokine contents in the lysates from individual human donors were Donor DNA was isolated from frozen lysates of purified eosinophils using evaluated using the Proteome Profiler Array, Panel A (ARY005B; R&D MinElute PCR Purification Kit (cat. no. 28004; QIAGEN) or from frozen Systems), which permits simultaneous detection of 36 distinct human eosinophil cell pellets after treating with DNA lysis buffer (50 mM Tris-Cl cytokines. Briefly, biotinylated detection Ab mixture was added to eosin- [pH 8], 20 mM NaCl, 1 mM EDTA, and 0.2% SDS with 200 mg/ml ophil lysates prepared as described above, which were then used to probe proteinase K.) A fragment of the promoter of the human IL-16 Downloaded from membranes embedded with anti-human cytokine capture Abs. Detection is (bp 3280–3667, GenBank accession no. AY497901) was PCR-amplified carried out with streptavidin-conjugated to IRDye 800CW (catalog no. with forward primer 59-ATT GAC AAG CAT TTT CCT GAG T-39 and 926-32230; LI-COR Biosciences), and signals were detected using a reverse primer 59-CGA GAC ACG CTT TGA TTG G-39. The resulting LI-COR Odyssey CLx and Image Studio Software version 5.2 (LI-COR). 388-bp amplicon was sequenced (GENEWIZ, South Plainfield, NJ) As information from cytokine profiling is generated as mean pixel density to identify the nucleotides at the 2295 site in the promoter region as (mpd), one donor was chosen to serve as a standard, which enabled us to described (35).

normalize data from multiple trials. http://www.jimmunol.org/ Isolation of mouse eosinophils and preparation of lysates Characterization of a standard donor lysate and normalization Eosinophils were isolated from single-cell suspensions prepared from lungs of profiling data of C57BL/6-IL5tg and BALB/c-IL5tg mice; mice were perfused, lung The cytokine profiler generates raw data in the form of mpd. As such, we tissue was excised, and single-cell suspensions were prepared for isolation of eosinophils using the FACS protocol previously described (36). Lysates were used a standard donor lysate to normalize results, which enabled us to 2 2 2 prepared from purified eosinophils (CD45+CD11c MHCII SiglecF+Gr1 /lo) compare findings from multiple trials. We demonstrated that consistent 3 7 findings (mpd detected per cytokine) were obtained in a single trial in which using the method described above for human cells at 1 10 cells/ml profiler membranes were probed with 100 mg of eosinophil lysates prepared lysate and used to determine immunoreactive IL-16 content by ELISA from three independent blood samples from the standard donor (Fig. 1A). as described below; total protein concentration was determined by MicroBCA assay (cat. no. 23235; Thermo Fisher Scientific). We also established a critical lysate concentration versus response profile by guest on September 28, 2021 (Fig. 1B). Eosinophil lysates from the standard donor were included in all ELISAs experimental trials and were used to normalize the mpd data as shown in the text. An example of the raw data obtained from the profiler for the Absolute levels of IL-16, IL-27, IL-1a,IL-1b, and IL-6 in lysates cytokine IL-13 is shown in Fig. 1C. To compare data, the technical prepared from human eosinophils and IL-16 in mouse eosinophils replicates from two independent trials are normalized as follows: for was determined using human DuoSet ELISAs from R&D Systems, example, from trial 1, technical replicates as shown, (d1a + d1b)/(s1a + including DY316 for IL-16 (15.6–1000 pg/ml range), DY2526 for s1b) = (1423 + 1338)/(1289 + 1191) = 1.11, and from trial number 2 IL-27 (156–10,000 pg/ml range), DY206 for IL-6 (9.38–600 pg/ml (d8a + d8b)/(s2a + s2b) = (780 + 740)/(703 + 740) = 0.98, and con- range), DY200 for IL-1a, (7.8–500 pg/ml range), DY201 for IL-1b tinuing, generating the data shown in Fig. 1D in units of normalized (3.91–250 pg/ml range), and mouse DuoSet ELISA DY1727 for IL-16 pixel density (npd). (46.9–3000 pg/ml range), respectively. Cluster analysis Statistics Cytokine content data normalized as above were subjected to hierarchical Statistical analyses were performed as indicated in text using algorithms in cluster analysis (Partek Genomics Suite, v 7.18.0130; Partek, St. Louis, GraphPad Prism 7 and 8.

Table II. Hematology

Hematology Mean 6 SD Donor Range Normal Laboratory Range Hemoglobin (g/dl) 13.7 6 1.1 11.4–16.3 11.2–15.7 Hematocrit (%) 39.9 6 3.0 33.1–44.3 34.1–44.9 WBCs (K/ml) 5.5 6 1.1 3.1–7.50 3.98–10.04 Neutrophils (K/ml) 3.39 6 0.84 1.52–4.72 1.56–6.13 Neutrophils (%) 61 6 6.8 37.3–73.8 34–71.1 Eosinophils (K/ml)a 0.17 6 0.10 0.05–0.45 0.04–0.36 Eosinophils (%) 3.2 6 1.8 1.0–9.4 0.7–5.8 Basophils (K/ml) 0.05 6 0.03 0.01–0.12 0.01–0.08 Basophils (%) 0.94 6 0.56 0.20–3.1 0.1–1.2 (K/ml) 0.34 6 0.09 0.18–0.55 0.24–0.86 Monocytes (%) 6.2 6 1.4 3.7–9.1 4.7–12.5 Lymphocytes (K/ml) 1.55 6 0.42 0.83–2.49 1.18–3.74 Lymphocytes (%) 28.5 6 6.7 16.8–51.5 19.3–15.7 Platelets (K/ml) 239 6 57 120–374 173–369 aTwo donors with peripheral eosinophil counts above normal range. The National Institutes of Health Clinical Center Laboratory normal range is included for each parameter. The Journal of Immunology 523

Table III. Cytokines detected that display minimal variability Table IV. Cytokines detected that display moderate to profound variability Cytokine CV (%) Mean 6 SD (npd) Range (npd) Cytokine CV (%) Mean 6 SD (npd) Range (npd) MIF 19.4 1.11 6 0.22 0.80–1.52 IL-5 21.5 1.23 6 0.25 0.63–1.58 IFN-g 50.8 0.85 6 0.41 0.23–1.63 IL-13 26.8 0.94 6 0.25 0.40–1.28 IL-6 53.5 1.97 6 1.05 0.44–3.39 PAI-1 29.8 0.83 6 0.25 0.36–1.16 IL-18 54.9 1.37 6 0.75 0.39–2.56 G-CSF 30 0.86 6 0.25 0.37–1.28 IL-8 55.8 1.64 6 0.91 0.56–3.85 IL-17E 30.1 0.80 6 0.23 0.31–1.14 IL-2 56.1 1.53 6 0.86 0.47–2.82 CCL2 30.8 0.86 6 0.24 0.40–1.26 IL-10 60.3 1.23 6 0.74 0.24–2.62 CCL3/CCL4 30.8 0.87 6 0.27 0.30–1.24 IL-12p70 62.1 1.70 6 1.05 0.47–3.76 CXCL1 31.7 0.98 6 0.31 0.41–1.49 IL-1b 63.1 1.77 6 1.12 0.40–3.79 CCL1 32.5 0.91 6 0.29 0.38–1.30 IL-1a 65.7 1.31 6 0.86 0.33–3.11 IL-21 33.9 0.90 6 0.30 0.34–1.28 TREM-1 66.7 1.29 6 0.86 0.26–3.28 CXCL10 37.5 1.12 6 0.42 0.25–1.82 IL-27 72.5 1.80 6 1.30 0.30–4.11 CXCL12 38.3 1.00 6 0.37 0.41–1.64 CXCL11 73.6 1.55 6 1.14 0.31–3.94 IL-32A 39 0.98 6 0.38 0.31–1.39 IL-1Ra 84.1 1.22 6 1.03 0.33–3.10 CCL5 39.4 1.26 6 0.50 0.48–2.16 IL-16 103 6.08 6 6.3 0.49–19.1 GM-CSF 39.5 1.19 6 0.47 0.51–2.23 Thirteen of the thirty-six cytokines detected in eosinophil lysates that display ICAM-1 40.6 1.34 6 0.54 0.49–2.51 6 moderate variability (n = 14 donors). Profiling included standard donor lysate (see TNF-a 41.9 1.17 0.49 0.42–2.25 Fig. 1) as a normalization control as described in the legend to Table II. Moderate IL-17 43.1 1.57 6 0.68 0.59–2.90 variability is defined as CV .50 and #100%. See Fig. 2 for an example of how this Downloaded from C5/C5a 44.8 1.04 6 0.46 0.20–1.69 analysis was performed. One cytokine (IL-16) of the thirty-six detected displays IL-4 48.5 1.54 6 0.75 0.44–2.82 profound variability, with a CV .100%. CD40L 48.7 0.99 6 0.48 0.24–1.67 Twenty-two of the thirty-six cytokines detected in eosinophil lysates by proteome profiling display minimal variability (n = 14 donors). Profiling included the standard a component of human eosinophilic leukocytes. We confirmed donor lysate described in Fig. 1, which was included as a normalization control. See expression of immunoreactive IL-27 in the standard and 10

Fig. 2 for an example of how this analysis was performed. Minimal variability is additional donor lysates at 953 6 504 pg/mg lysate, with a http://www.jimmunol.org/ defined as CV #50%. coefficient of variation (CV) = 53%. Numerous and complex immunomodulatory activities have been described for IL-27, pri- Results marily those directed at regulating the activities of T subsets (38, 39). Normal donors As shown in Table III, 22 of the 36 mediators detected by Eosinophils were isolated from EDTA-anticoagulated whole blood profiling display minimal variability, defined in this study as from normal adult volunteer donors. The eligibility and exclusion those with a calculated CV # 50%. In addition to IL-13 (see criteria for donation are described in the Materials and Methods. Fig. 1D), this group also includes IL-5, CCL2, CCL1, IL-4, As shown in Table I, the full donor group, including metadata TNF-a, and IL-17. by guest on September 28, 2021 (n = 37), included 22 males and 15 females between the ages of 22 The 13 cytokines detected in eosinophil lysates at levels that and 75 and 20 and 66 y of age, respectively. The mean BMI was vary more moderately (CV . 50% but , 90%) are listed in 2 2 determined as 28.5 6 5.4 kg/m and 27.9 6 5.9 kg/m for male TableIV.AmongtheseareIFN-g, IL-8, IL-10, and near the and female donors, respectively. These average values are within higher end of this group, CXCL11 and IL-1Ra, the latter two the range defined as overweight (25 , BMI # 29.9) by the U.S. with CVs at 74 and 84%, respectively. Centers for Disease Control, which are findings consistent with the By contrast, IL-16 was detected in eosinophil lysates at con- current prevalence of overweight body habitus as well as obesity centrations that varied profoundly, over a range of 0.49–19.1 npd, (BMI $ 30) in the United States (37). Hematologic parameters at a mean 6 SD of 6.08 6 6.3, with a CV = 103%. for all donors were within or near our laboratory normal range Eosinophil IL-16 content was examined by cluster analysis with (Table II). Of note, two donors had peripheral eosinophil counts each cytokine content (npd) displayed for each donor (n =14) that were slightly higher than the laboratory normal range, al- (Fig. 2). This analysis highlights the remarkable variability of though both values were ,500 eosinophils/ml and thus not within eosinophil IL-16; the content pattern is unique and showed no the definition of peripheral eosinophilia. The donor selected to correlations to those of any other cytokine or cytokines. Principal be the source of eosinophil lysate standard as described in the component analysis revealed no associations with donor age, Materials and Methods and Fig. 1 was female, white, age 60, BMI gender, or BMI group (data not shown). Equally intriguing, the 3 19.2, and hematocrit 42%, with total leukocytes at 6.8 3 10 /ml cluster analysis suggested coordinate regulation of eosinophil- and 4.8% eosinophils. associated cytokines (see Discussion) and revealed direct cor- relations among several in the moderately variable group (see Relative expression of eosinophil cytokines Table IV), including IL-1b,IL-1a, and IL-6 as discussed further Lysates were generated from eosinophils isolated from an initial 14 below. normal donors, and relative expression of 36 cytokines was de- termined by profiling in two separate trials, each including the Absolute quantitation of eosinophil-derived IL-16 standard lysate; the method for evaluating profiling data with this To continue our exploration of eosinophil IL-16 content, ELISA standard and presentation of data in units of npd was described analysis was performed on the lysates from the original 15 do- in the Materials and Methods and the legend to Fig. 1. These nors (14 donors and the standard) together with lysates prepared findings were used to determine range (minimum and maxi- from eosinophils from an additional 22 donors (total n = 37) mum) and mean 6 SD for all cytokines evaluated. All 36 (see Tables I and II). First, as shown in Fig. 3A, we demonstrated cytokines and soluble mediators included on the profiler were that the absolute concentration of IL-16 (picograms per milligram detected in the donor eosinophil lysates, including IL-27, which, eosinophil lysate) correlated directly with the mpd readings from to the best of our knowledge, has not been identified previously as lysates from the original donor set. These findings provide direct 524 EOSINOPHILS AND IL-16

FIGURE 2. Cytokine variability in eosinophil lysates: heat map. Variability (npd) determined as indicated in the legend in Fig. 1 via clustering analysis. Donor (1–14) characteristics listed in columns include race (see legend to Table I), BMI class (normal [n], overweight [o], obese [b], by standard criteria; Downloaded from age group (young [y] 18–35 y; middle aged [m] 36–55 y; old [o] $56 y), and sex (male [M], female [F]). Asterisk (*) indicates donor group in which one can detect coordinate upregulation of a cohort of moderately variable proinflammatory cytokines. confirmation and validation of the findings from the cytokine at 4970 pg/mg lysate; as shown, these eosinophils are pure profiling methodology. Immunoreactive IL-16 (picograms per preparations with normal morphology.

milligram eosinophil lysate) for the complete set of eosinophil http://www.jimmunol.org/ lysates is shown in Fig. 3B. As anticipated, immunoreactive Eosinophil IL-16 content correlates with BMI for a subset IL-16 content varies dramatically among donors, from a low of of donors 274 pg/mg lysate to a high of 13,300 pg/mg lysate; the mean 6 As shown, this analysis permits us to divide the donors into three SD calculates to 2683 6 3026 pg/mg lysate and a CVof 113%. groups. The first group (group A) includes donors with BMI #22 The eosinophils shown Fig. 3C were isolated from the donor (n = 8). Interestingly, all donors with this low BMI maintain low indicated at the arrow in Fig. 2C, one of the donors with a levels of eosinophil-derived IL-16 (,2500 pg/mg lysate). The sec- relatively high concentration of eosinophil-associated IL-16, ond, group B (n = 18), are donors with a higher BMI (BMI $ 23) by guest on September 28, 2021

FIGURE 3. Cytokine variability in eosinophil lysates: IL-16. (A) Concentration of IL-16 determined by ELISA (picograms per milligram lysate) correlates with data from cytokine profiling (mpd). R2 = 0.80, ***p , 0.0001. (B) Immunoreactive IL-16 (picograms per milligram lysate) evaluated standard donor (pink bar) and extended donor set (n = 38); CV = 113%. (C) Modified Giemsa staining of eosinophils isolated from donor indicated at arrow in (B), IL-16 = 4971 pg/mg lysate. Original magnification 340. The Journal of Immunology 525 Downloaded from http://www.jimmunol.org/

FIGURE 4. Eosinophil IL-16 content correlates with BMI in a subset of normal donors. (A) IL-16 versus BMI, Group A (pink symbols), BMI #22; Group B (black symbols), BMI $23, IL-16 ,2500 pg/mg lysate; Group C (blue symbols), BMI $23, IL-16 .2500 pg/mg lysate. Correlation (dashed line). R2 = 0.60, ***p , 0.0001. (B) Distribution of normal donors as in (A). (IL-16 versus BMI), documenting male (filled symbol) versus female (open symbol). No sex prevalence in either group (AC or AB) as per x2 test (total group 22 male, 15 female, see text). (C) Eosinophil IL-16 content (picograms per milligram lysate) versus donor age. Groups color-coded as indicated in (A). ns, no significance. by guest on September 28, 2021 but who also maintain IL-16 ,2500 pg/mg eosinophil lysate. The CV of 49.7% (Fig. 5B). If group A is included with group C (as in final group, group C (n = 9), is most interesting, as these donors Fig. 4A), the normal distribution remains, and the CV increases to also have higher BMIs, in the same general range as group B 85.6% (data not shown). Evaluated as two distinct distributions, (BMI $ 23) but have markedly elevated eosinophil IL-16 content IL-16 is among the high minimal to low moderately variable cy- (IL-16 $ 2500 pg/mg lysate). As shown in Fig. 4A, there is a sig- tokines as described in Tables III and IV. nificant correlation between eosinophil IL-16 content and donor However, even when divided into these two groups (IL-16 . BMI for donors in combined groups A/C (dotted line; R2 = 0.60, 2500, group C and IL-16 , 2500, group A/B), no distinct corre- ***p , 0.0001); there is also a correlation between eosinophil lations with any hematologic parameters were revealed. No cor- IL-16 content and BMI for donors in group C evaluated alone relations were observed between eosinophil IL-16 content and (R2 = 0.39, *p , 0.05, data not shown). By contrast, no corre- total leukocytes (cells per microliter; (Fig. 6A, 6B)). Likewise, lation between eosinophil IL-16 content and BMI was observed despite characterization of IL-16 as a lymphocyte chemoattractant when evaluating combined group A/B or group B alone. (40), no correlations were observed between eosinophil IL-16 content Interestingly, donor sex does not factor into these observations. and total lymphocytes (cells per microliter; (Fig. 6C, 6D)), nor was As shown in Fig. 4B, there are 16 males and 10 females in group there any correlation between eosinophil number (cells per mi- A/B (62 and 38%, respectively) and 9 males and 10 females in croliter) and IL-16 content (Fig. 6E, 6F). Donor sex does not skew group A/C (47 and 53%, respectively); although there are fewer or contribute to any of these results (see Supplemental Fig. 1). males in the latter group, in neither group do findings deviate Eosinophil IL-16 content does not correlate with IL-1b, IL-1a, significantly from the null hypothesis (p . 0.05, x2 test), which or IL-6 was calculated given expected values of 59% males (22) and 41% females (15). Likewise, there was no significant correlation between Cytokines IL-1b, IL-1a, and IL-6 play critical roles in modulating eosinophil IL-16 levels and donor age (Fig. 4C). energy metabolism (41–45). As suggested by the findings pre- sented in the cluster analysis in Fig. 2 and shown graphically in Eosinophil IL-16 content: two distinct normal distributions and Fig. 7A–C, eosinophil levels of IL-1b, IL-1a, and IL-6 correlate no correlations with hematologic parameters directly with one another (R2 = 0.67, 0.72 and 0.85, respectively); As shown in Fig. 5A, group A and group B as defined above, which these correlations were confirmed on another set of samples by comprise all donors with IL-16 #2500 pg/mg lysate generate a ELISA (Fig. 7D, 7E; picograms per milligram lysate, R2 = 0.73 normal distribution, with a mean and SD of 1138 6 656 pg/mg and 0.86, respectively). As suggested by the findings in Fig. 2, and IL-16, and a CV of 57.6%. Likewise, group C (all donors with confirmed in this study graphically (Fig. 7F) and on another set of IL-16 . 2500 pg/mg lysate) also generates a distinct normal samples by ELISA (Fig. 7G, 7H), eosinophil IL-16 content does distribution, with a higher mean (6554 6 3259 pg/mg lysate), and not correlate directly with any of these mediators; a minor inverse 526 EOSINOPHILS AND IL-16

FIGURE 5. Normal distributions and statistical evaluations. (A) Groups A and B (pink and black symbols), IL-16 ,2500; normal distribution with pa- rameters as indicated, CV = 57.6%. (B) Group C (blue symbols), IL-16 .2500; normal distribution with pa- rameters as indicated, CV = 49.7%. Downloaded from http://www.jimmunol.org/

correlation was detected in a comparison between IL-16 content with BMI but only among one donor subset. There is no evidence and IL-1a (picograms per milligram lysate, R2 = 0.29, *p , 0.05) to date suggesting a cause and effect relationship even in this donor although not when comparing npd data (data not shown). As such, subset, and, despite the growing mouse literature (51–54), there is at by guest on September 28, 2021 these findings reveal no clear relationships between IL-16, body present no clear evidence regarding a role of human eosinophils in weight, inflammation, and metabolism. the immunomodulation of adipose tissue. Nevertheless, as an initial consideration, hypereosinophilic mice 2 The 295T/C promoter polymorphism has no impact on from two distinct background strains (BALB/c and C57BL/6) were eosinophil IL-16 content maintained for 5–6 wk on enriched (22% fat) diets (see Materials Variants at the 2295 position in the leukocyte promoter of the and Methods). Eosinophils were isolated from these mice and human gene encoding IL-16 have been associated with varied IL-16 content was compared with those from matched mice responses to several human disorders, including asthma, atopy, maintained on baseline diets. As shown, this acute dietary change and gastrointestinal diseases (35, 46, 47). Burkart and colleagues had no impact on detection of eosinophils in the lung in either of (35) identified the C codon at this position as promoting 6-fold in these mouse strains (Fig. 9A). However, among mice main- more promoter activity when evaluated in human bronchial epi- tained on the normal diet, IL-16 content in eosinophils isolated thelial cells. To determine whether our findings relate in any way from the C57BL/6-IL5tg strain was significantly higher (5.2-fold) to donor genotype, we isolated DNA from a series of donors, and than that measured in eosinophils from the BALB/c-IL5tg strain amplified a 388-bp promoter fragment for direct sequencing. As (Fig. 9B). More interesting, the IL-16 content in eosinophils isolated shown in Fig. 8A, the ratios we obtained, 0.53 TT:0.44 CT:0.03 from C57BL/6-IL5tg mice maintained on the 22% fat diet underwent a CC were consistent with those reported in multiple studies for 2.7-fold reduction, from 2380 6 650 pg/mg lysate to 870 6 360 pg/mg similar human donor populations [see data available at https:// lysate. These results are consistent with the findings shown in Fig. 7, as www.snpedia.com/index.php/Rs4778889]. However, as shown in they suggest that, whereas eosinophil IL-16 content in human subjects Fig. 8B, there are no statistically significant differences in eosin- may correlate with BMI in one donor subset, this finding may not be ophil IL-16 content when comparing donors of the 2295TT ge- directly related to energy metabolism. notype to those of the 2295CT/CC genotype. Findings from these data suggest that a fully unbiased approach to the question of mice, diet, and eosinophil cytokines may be IL-16 content of mouse eosinophils: strain and diet required. Given the findings above, it was not possible to predict how mice might respond to a diet that is higher in fat content. High-fat diets Discussion are typically used to promote weight gain in susceptible mice, In this work, we used a proteome profiling method to examine including those on the C57BL/6 background strain (48, 49); in natural cytokine variability in eosinophils isolated from normal contrast, human subjects are substantially more complex and do human subjects. As eosinophils play important roles in maintaining not necessarily gain weight in response to similar regimens (50). homeostasis and promoting tissue repair (1, 2), differences in Furthermore, our data suggest that eosinophil IL-16 content correlates cytokine content may have a direct impact on an individual’s The Journal of Immunology 527 Downloaded from http://www.jimmunol.org/

FIGURE 6. Eosinophil IL-16 content and hematologic parameters. Groups A and B and Group C, color coded as in Figs. 4 and 5. No correla- tions determined for eosinophil lysate IL-16 (picograms per milligram lysate) versus (A and B) total WBCs (3103/ml); (C and D) total lymphocytes (3103/ml). (E and F) Total eosinophils (3103/ml). ns, no significance.

capacity to defend against infection, respond to medication, and In this study, we also examined the 2295T/C polymorphism in by guest on September 28, 2021 recover from disease. Among our prominent discoveries was the the leukocyte promoter of IL-16, a region that has been implicated profound variability in IL-16 content and the correlation between in differential transcription in cell culture studies [C . T (35)]. IL-16 content and donor BMI in a subset of phenotypically normal The 2295T/C polymorphism has been associated with several donors. eosinophil-related disorders, notably, asthma, atopic dermati- IL-16 was first identified by Center and Cruikshank (55) as a tis, and Crohn’s disease (35, 46, 47), although in most cases no T lymphocyte chemoattractant factor, the first of such molecules direct associations between polymorphisms and cytokine lev- isolated from mitogen-stimulated human PBMCs. IL-16 is a tet- els have been reported. The genotypes of the donors in our rameric glycoprotein with high interspecies study were distributed in a manner consistent with others in our that is expressed widely in both neuronal (56) and nonneuronal region and included a ratio of 0.53 TT:0.44 TC:0.03 CC (n =34). cells, the latter including CD8+ and CD4+ T cells, mast cells, Despite the aforementioned tissue culture findings, this specific eosinophils, fibroblasts, and bronchial epithelial cells (57, 58). promoter polymorphism had no direct association with the IL-16 Initially of interest as a biological means to suppress replication of content of the human eosinophil lysates. We cannot rule out HIV (59), IL-16 has also been associated with the pathogenesis of the possibility that eosinophil IL-16 content is regulated by allergic disorders (60) and promoting cell growth in a wide array an unrelated transcriptional mechanism (69, 70) or by epigenetic/ of neoplastic diseases (61). Biochemically, IL-16 has features in posttranscriptional means. Interestingly, we note that the human common with dual-function cytokines IL-1a, IL-33, and HMGB1, eosinophils evaluated by Lim and colleagues (63) showed marked including N-terminal DNA binding domains with nuclear locali- variation in levels of the amplified transcript encoding this cytokine zation signals separated by protease activation sites from regions (see Fig. 4). capable of interacting with extracellular receptors (62); as a group, As part of this study, we found a significant correlation between these proteins, also known as alarmins, function to promote sterile eosinophil IL-16 content and donor BMI (Fig. 3A) in a subset of inflammation in response to internal stress and cell necrosis. normal donors. BMI, which is a ratio of weight to height in metric Lim, Weller and colleagues (63) reported immunoreactive IL-16 units, was a measure devised by the Belgian sociologist and protein in and release from the cytoplasmic granules of human statistician Adolphe Que´teletaspartofanefforttogenerate eosinophils. Interestingly, IL-16isalsoaneosinophilche- standard data on human populations (71). Although not a fully moattractant, eliciting responses via CD4 on the eosinophil perfect means to evaluate weight or health (72), the BMI re- cell membrane (64, 65). Interestingly, IL-16 does not promote mains in widespread use. Likewise, although the roles of cy- eosinophil degranulation or production of reactive oxidants. Given tokines, inflammation, and obesity is a field of significant interest its role as a chemoattractant for both eosinophils and CD4+ (reviewed in Ref. 73–75), the specific role of IL-16 and its cor- T cells, IL-16 blockade in respiratory allergies and asthma has relation with weight in human subjects has been the subject of only been explored (66–68). limited evaluation. In one such study, Lichtenauer and colleagues 528 EOSINOPHILS AND IL-16 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 7. Eosinophil cytokine content: no correlations with IL-1a,IL-1b, or IL-6. Correlations between (A)IL-1a and IL-1b (npd). R2 =0.67,***p = 0.0004. (B) IL-1a versus IL-6 (npd). R2 = 0.72, ***p = 0.0001. (C) IL-6 versus IL-1b (npd). R2 = 0.85, ***p , 0.0001. (D) IL-1a versus IL-1b (picograms per milligram lysate). R2 = 0.73, ***p , 0.005. (E)IL-6versusIL-1b (picograms per milligram lysate). R2 = 0.86, ***p , 0.005. (F) IL-16 versus IL-6 (npd). ns, no significance. (G) IL-16 versus IL-1b (picograms per milligram lysate). ns, no significance. (H) IL-16 versus IL-1a (picograms per milligram lysate). R2 = 0.29, *p , 0.05.

(76) evaluated plasma cytokine levels among 79 adolescents and and any one of these mediators, levels of IL-1a,IL-1b, and IL-6 found elevated levels of IL-16 among those who were overweight in eosinophils from normal donors all correlate directly with one (as determined by BMI) compared with those who were at normal another. Furthermore, levels of these eosinophil-derived cytokines weight. At the cellular level, Alomar and colleagues (77) iden- are all relatively higher (together with IL-2, IL-12p70, IL-18, and tified IL-16 as synthesized by human preadipocytes, although the IL-27, among others) in one specific cluster of primary donors proinflammatory mediator IL-1b hadnoimpactonitsrelease (see asterisk in Fig. 2). Given our ongoing interests in natural from these cells. variation in eosinophils, it will be intriguing to explore this obser- By contrast, there is a large and growing literature on the role vation further and to identify unique features among this potentially of mouse eosinophils in promoting metabolic homeostasis and the important donor cohort. immunomodulation of adipose tissue (51–54, 78, 79). Among At this writing, there is only limited information on obesity these studies, Wu and colleagues (52) characterized eosinophil in human subjects and its impact on eosinophils and, likewise, migration to adipose tissue in wild-type mice, and found that the impact of eosinophils on human metabolic homeostasis and eosinophil-deficient mice gain weight, develop impaired glucose regulation of adipose tissue in disease states. Kim and colleagues tolerance, and develop insulin resistance when maintained on a (80) and Takeda and colleagues (81) reviewed the evidence link- high-fat diet. Rao and colleagues (53) and Qiu and colleagues ing obesity, asthma, eosinophilic inflammation, and leptin, the (54) found that eosinophils are critical in promoting anti-inflammatory proinflammatory mediator synthesized by and released from adi- programs in adipose tissue, specifically, generating beige fat via pose cells and detected in serum at levels corresponding with BMI release of IL-4. in human subjects (82). Human eosinophils express the There is considerable literature focused on the roles of the for leptin (83) and respond to exogenous leptin by undergoing largely proinflammatory cytokines IL-1b,IL-1a, and IL-6 in , adhesion, release of cytokines, and prolonged via- immunoregulation of metabolism and control of adipose function bility (83, 84). Although it would be premature to speculate too far at (41–44). Although there was no direct correlation (suggesting this juncture, it is interesting to consider the possibility that chronic potential cross-regulation) between eosinophil-content of IL-16 elevations in serum leptin may have an impact not only on acute The Journal of Immunology 529

In summary, we have performed proteome profiling on eo- sinophil lysates prepared from normal donors and characterized eosinophil-associated cytokines that varied on a continuum from minimally (CV # 50%) to moderately (50% , CV , 90%) vari- able. Among the latter group, we identified IL-27, a member of the IL-12 family, as a (to our knowledge) new and previously un- recognized component of eosinophils from normal donors. By contrast, eosinophil IL-16 content varied profoundly and via a unique pattern with no correlations to levels of other eosinophil- derived cytokines. The dataset generated two distinct normal dis- tributions, with one donor subset demonstrating a direct correlation between eosinophil IL-16 content and donor BMI. Given our current understanding of the links between eosinophils, asthma, and obesity, an improved understanding of eosinophils and IL-16 content may provide important connections worthy of further exploration.

Acknowledgments We gratefully acknowledge the efforts of the clinical staff of the LAD, Downloaded from including Michael Young, Linda Scott, Robin Eisch, Daly Cantave, and FIGURE 8. Genotyping and impact of 2295T/C IL-16 promoter poly- Hyejeong Bolan, for assistance with normal blood donors via the LAD normal morphisms. (A). Fractions of the donor population with the given genotype donor protocol. We also thank Thomas Lewis at the National Institutes of and example of sequence shown. (B) Eosinophil IL-16 content versus Health Clinical Center Blood Bank for assistance in obtaining normal donor genotype (2295T/T or 2295C/T or C/C); ns, no significant difference. samples and de-identified donor data. We also thank Kristi Chu (Miltenyi Biotech) and Dr. Paige Lacy (University of Alberta) for technical advice http://www.jimmunol.org/ responses but also on eosinophil development, resulting in differences and Michael M. Rosenberg (Wayfair, Inc.) for additional assistance with statistical analysis and data presentation. This manuscript is dedicated in cytokine content in a subset of otherwise normal donors. Of to the memories of Dr. Redwan Moqbel and Dr. Jamie Lee, eosinophil interest, Suzukawa and colleagues (85) found that leptin promoted researchers extraordinaire. cytokine synthesis in isolated human basophils. Disclosures The authors have no financial conflicts of interest.

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