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Paired immunoglobulin-like receptor A is an intrinsic, self-limiting suppressor of IL-5–induced eosinophil development

Abstract

Eosinophilia is a hallmark characteristic of T helper type 2 (TH2) cell–associated diseases and is critically regulated by the central eosinophil growth factor interleukin 5 (IL-5). Here we demonstrate that IL-5 activity in eosinophils was regulated by paired immunoglobulin-like receptors PIR-A and PIR-B. Upon self-recognition of β2-microglobulin (β2M) molecules, PIR-B served as a permissive checkpoint for IL-5–induced development of eosinophils by suppressing the proapoptotic activities of PIR-A, which were mediated by the Grb2-Erk-Bim pathway. PIR-B–deficient bone marrow eosinophils underwent compartmentalized apoptosis, resulting in decreased blood eosinophilia in naive mice and in mice challenged with IL-5. Subsequently, Pirb−/− mice displayed impaired aeroallergen-induced lung eosinophilia and induction of lung TH2 cell responses. Collectively, these data uncover an intrinsic, self-limiting pathway regulating IL-5–induced expansion of eosinophils, which has broad implications for eosinophil-associated diseases.

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Figure 1: Pirb−/− LDBM cells do not differentiate into mature eosinophils in vitro.
Figure 2: PIR-B regulates eosinophil apoptosis during differentiation of LBDM eosinophils.
Figure 3: PIR-B regulates IL-5–induced but not IL-3–induced or GM-CSF–induced colony formation.
Figure 4: PIR-B regulates eosinophil apoptosis in vivo even in the presence of increased IL-5.
Figure 5: Increased eosinophil development and decreased apoptosis in the absence of MHC class I expression.
Figure 6: Neutralization of PIR-A in Pirb−/− eosinophil cultures attenuates eosinophil apoptosis.
Figure 7: Pirb−/− mice display increased BM eosinophil apoptosis as well as decreased PB and tissue eosinophilia after mucosal aeroallergen challenge.

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Acknowledgements

We thank H. Kubagawa (University of Alabama) for providing Pirb−/− mice, and S.P. Hogan, O. Mandelboim, I. Bachelet, N. Osherov and A. Ben-Baruch for critically reviewing this manuscript and helpful discussions. This work was performed in partial fulfillment of the requirements for the PhD degree of N.B.B.-M., and D.S. at Tel Aviv University. Supported by the FP7 Marie-Curie Reintegration grant (256311), the Israel Science Foundation (955/11 and 1708/11) the Israel Cancer Research Foundation Research Career Development Award and the Fritz Thyssen Foundation (to A.M.); the US-Israel Bi-national Science Foundation (2009222 to A.M. and M.E.R.); US National Institutes of Health NIAID (R01AI083450 and R37AI045898), Campaign Urging Research for Eisonophilic Disease (CURED) Foundation and Buckeye Foundation (to M.E.R.).

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N.B.B.-M., D.S., I.M., M.I., D.K.-A., C.B., P.C.F., D.R. and A.M. did in vitro and in vivo experiments and analyzed data. M.E.R and S.J. provided critical reagents and analyzed data. A.M. supervised the study, and wrote and edited the manuscript.

Corresponding author

Correspondence to Ariel Munitz.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Expression of surface activation markers and adhesion molecules in Pirb–/– LDBM cells.

Expression of surface activation markers and adhesion molecules in Pirb-/- LDBM cells. LDBM cells were obtained from wild-type (WT) and Pirb–/– mice and differentiated in-vitro into eosinophils. At day 14 of the culture, cells were stained with the indicated antibodies and isotype matched control. Assessment of surface expression was conducted by flow cytometric analysis of at least 10,000 events. Delta mean fluorescent intensity (ΔMFI) was calculated by subtracting the background isotype matched control staining. Data are representative histogram plots from 3 independent experiments.

Supplementary Figure 2 Assessment of cell proliferation in Pirb–/– LDBM cell cultures.

Assessment of cell proliferation in Pirb–/– low-density bone marrow cell cultures. (a) Low-density bone marrow (LDBM) cells were obtained from wild-type (WT) and Pirb–/– mice and differentiated in vitro-into eosinophils. At the indicated time points, cellular proliferation was assessed in eosinophil progenitors EoPs, (defined as Sca1CD34+LinC-KitintIL-5Rα+ cells) (b) or the general cell population using EdU. Data are representative of at least three independent experiments.

Supplementary Figure 3 Assessment of pro- and anti-apoptotic molecule expression in Pirb–/– LDBM cell culture.

Assessment of pro- and anti-apoptotic molecule expression in Pirb–/– low-density bone marrow cell culture. The expression of (a) BclXL, (b) Bax and (c) Bid was assessed in cDNA obtained from low-density bone marrow (LDBM) cells from wild-type (WT) and Pirb–/– mice by qPCR analysis. Gene expression was normalized to the house keeping gene hypoxanthine-guanine phosphoribosyltransferase (Hprt). Data are representative of LDBM cultures from n=3 mice.

Supplementary Figure 4 Siglec-F+CCR3int and Siglec-F+CCR3hi BM cells display eosinophil morphology and granule proteins.

Siglec-F+CCR3int and Siglec-F+CCR3hi bone marrow cells display eosinophil morphology and granule proteins. (a) Bone marrow cells from naïve wild-type mice were obtained and stained with anti-Siglec-F and anti-CCR3. Thereafter, Siglec-F+CCR3int and Siglec-F+CCR3hi cells were sorted, cytospins prepared and stained with modified Wright Giemsa stain. (b) The expression of eosinophil major basic protein (Mbp) was determined using qPCR. Gene expression was normalized to the house keeping gene hypoxanthine-guanine phosphoribosyltransferase (Hprt). Data are representative of LDBM cultures from n=3.

Supplementary Figure 5 Correlation analysis between PIR-B and Bim expression.

Correlation analysis between PIR-B and Bim expression. The expression of PIR-B was assessed throughout the low-density bone marrow (LDBM)-derived eosinophil culture using flow cytometric analysis at the indicated time points. Pearson correlation between PIR-B and Bim expression is shown (r = 0.47, P = 0.31).

Supplementary Figure 6 Miniphosphorpoteomics and assessment of phospho JNK and p38 in BM eosinophils.

Miniphosphorpoteomics and assessment of phospho JNK and p38 in BM eosinophils. (a) A custom-made membrane coated with various antibodies recognizing several kinases and/or adaptor molecules was used to determine the interactions between PIR-A and (b) selected phosphorylated downstream signaling intermediates. (c-d) Wild-type (WT) and Pirb–/– BM cells were stained with anti-Siglec-F, Annexin-V and anti-phospho(p)-JNK or anti-phospho(p)-p38. Thereafter, the mean fluorescence intensity of (c) phospho-Jnk 1/2 (P-Jnk 1/2) and (d) phospho-p38 (P-p38) was assessed by flow cytometry.

Supplementary Figure 7 PIR-B is required for development allergic airway inflammation.

PIR-B is required for development allergic airway inflammation. (a) Wild-type (WT) and Pirb–/– mice were sensitized with Alum+OVA on days 0 and 14. On day 24, serum was obtained; serially diluted and total IgE was assessed. (b-e) WT and Pirb–/– mice were challenged with allergen extracts of Aspergillus fumigatus (Asp) (f-g) or house dust mite (HDM). Thereafter, (b) the frequencies of CD3+CD4+ T cells and expression of (c) IL-4, (d) IL-13 and (e) CCL17 were assessed. Bronchoalveolar lavage fluid was obtained from HDM-challenged mice and (g) eosinophil percentages and (h) total numbers assessed. (i-l) Mixed bone marrow (BM) chimeric mice that harbor a specific deletion of PIR-B in their CD11c+ cell component were generated. Following adoptive transfer, engraftment of mixed BM cells from WT, Pirb–/– and CD11c-diphteria toxin receptor (CD11c-DTR) mice were assessed using anti-CD45.1 and anti-CD45.2 staining. Ablation of lung CD11c+ cells following diphtheria toxin treatment was examined. Single cell suspensions from the lungs of chimeric BM mice were obtained and (i) PIR-A/B expression as well as (j) frequencies of CD45.2+ cells assessed. Chimeric BM mice were intranasally challenged with saline or Aspergillus fumigatus (Asp) and (k) CCL17 was measured using ELISA and (l) lung IL-4 expression was assessed using qPCR analysis. Data are representative of at least two independent experiments using seven mice per group; NS-non significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary Figure 8 Schematic presentation of the proposed function of PIRs in eosinophil expansion.

Schematic presentation of the proposed function of PIRs in eosinophil expansion. Exposure of eosinophils to IL-5 in the bone marrow induces eosinophil growth and expansion by delivering survival signals. In contrast to IL-5, self-recognition via PIR-A induces pro-apoptotic signaling in eosinophils and counter regulates IL-5-driven responses. PIR-B, which is expressed in eosinophils in higher levels than PIR-A, suppresses the pro-apoptotic signaling driven by PIR-A, via a pathway that likely involves activation of Bim and ERK. Thus, PIR-B serves as a permissive checkpoint for IL-5 induced eosinophil expansion.

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Ben Baruch-Morgenstern, N., Shik, D., Moshkovits, I. et al. Paired immunoglobulin-like receptor A is an intrinsic, self-limiting suppressor of IL-5–induced eosinophil development. Nat Immunol 15, 36–44 (2014). https://doi.org/10.1038/ni.2757

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