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CARD9S12N facilitates the production of IL-5 by alveolar macrophages for the induction of type 2 immune responses

Nature Immunology volume 19, pages 547–560 (2018)Cite this article

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Abstract

The adaptor CARD9 functions downstream of C-type lectin receptors (CLRs) for the sensing of microbial infection, which leads to responses by the TH1 and TH17 subsets of helper T cells. The single-nucleotide polymorphism rs4077515 at CARD9 in the human genome, which results in the substitution S12N (CARD9S12N), is associated with several autoimmune diseases. However, the function of CARD9S12N has remained unknown. Here we generated CARD9S12N knock-in mice and found that CARD9S12N facilitated the induction of type 2 immune responses after engagement of CLRs. Mechanistically, CARD9S12N mediated CLR-induced activation of the non-canonical transcription factor NF-κB subunit RelB, which initiated production of the cytokine IL-5 in alveolar macrophages for the recruitment of eosinophils to drive TH2 cell–mediated allergic responses. We identified the homozygous CARD9 mutation encoding S12N in patients with allergic bronchopulmonary aspergillosis and revealed activation of RelB and production of IL-5 in peripheral blood mononuclear cells from these patients. Our study provides genetic and functional evidence demonstrating that CARD9S12N can turn alveolar macrophages into IL-5-producing cells and facilitates TH2 cell–mediated pathologic responses.

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Fig. 1: Characterization of type 2 responses in wild-type and CARD9S12N KI mice.
Fig. 2: Characterization of TH2 differentiation in CARD9S12N KI mice.
Fig. 3: Alveolar macrophages expressing CARD9S12N are critical for Af-induced type 2 responses.
Fig. 4: Transfer of alveolar macrophages expressing CARD9S12N facilitates Af-induced type 2 responses.
Fig. 5: CARD9S12N reprograms alveolar macrophages to secret IL-5 for the promotion of type 2 responses in mice.
Fig. 6: CARD9S12N mediates the activation of RelB in macrophages.
Fig. 7: CARD9S12N mediates the activation of RelB to regulate Il5 expression in macrophages.
Fig. 8: The CARD9 S12N polymorphism is associated with the development of ABPA.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (31622023 and 81571611 to X.M.J, 81630058 and 91542107 to X.L.), Outstanding academic leader program of Shanghai health and Family Planning Commission (2017BR024 to X.M.J.),Shanghai laboratory animal research fund (16140902600 to X.M.J), Shuguang Program of Shanghai Education Development Foundation and Shanghai Municipal Education Commission (17SG24 to X.M.J.),a start-up fund from the Tsinghua University-Peking University Joint Center for Life Sciences (with grants from Tsinghua University to X.L.) and Shanghai Leading Talent Program (2016036 to J.F.X).

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  1. These authors contributed equally: Xia Xu, Jin-Fu Xu.

Authors and Affiliations

  1. Clinical Translational Research Center, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China

    Xia Xu, Jie-Lin Duan, Jian-Hong Guan, Quan-Zhen Lv, Chun-Xia Chen & Xin-Ming Jia

  2. Institute for Immunology, Tsinghua University School of Medicine, Tsinghua University-Peking University Joint Center for Life Sciences, Beijing, China

    Xia Xu, Guoxing Zheng, Wei Rui, Li-Qing Cheng, Dan-Dan Yang, Ming-Chao Wang, Xueqiang Zhao & Xin Lin

  3. Department of Respiratory Medicine, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China

    Jin-Fu Xu, Hai-Wen Lu & Jian-Xiong Li

  4. Medical Statistics Department, Children’s Hospital of Fudan University, Shanghai, China

    Peng Shi

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Contributions

X.X., G.Z., J.-L.D., W.R., J.-H.G., Q.-Z.L., J.-X.L. and C.-X.C. performed experiments; J.-F.X. and H.-W.L. collected samples from patients; L.-Q.C., D.-D.Y., M.-C.W. and X.Z. contributed critical reagents; P.S. performed statistical analysis; X.X., J.-F.X.., X-M.J. and X.L. designed the study; and X.X, X-M. J., and X.L. wrote the paper.

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Correspondence to Xin-Ming Jia or Xin Lin.

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Integrated Supplementary Information

Supplementary Figure 1 Characterization of CARD9S12N knock-in mice.

(a) Raw data of sequencing CARD9 gene amplified from the genome of F0 and F1 offsprings. (b) Raw data of sequencing CARD9 gene amplified from the genome of WT, heterozygous (Het) and homozygous CARD9S12N KI mice. (c) CARD9 protein levels in BMDMs from WT and KI mice. (d) ELISA results for TNF, IL-6 and IL-12p40 in the supernatant of BMDMs from WT, Het and KI mice, which were stimulated with Af SC (MOI = 5) for 6 hours. (e) CFU assay in lungs of WT and KI mice at day 2 and 6 after single exposure to Af conidia (1×107). *P < 0.05, **P < 0.01 and ***P < 0.001, by one-way ANOVA and post hoc Tukey test (d) and two-tailed unpaired t-test (e). Data shown are representative of three independent experiments.

Supplementary Figure 2 Characterization of type-2 responses in wide-type and CARD9S12N KI mice in chronic asthma models.

(a) Strategy of developing chronic murine asthma model, in which WT and KI mice were sensitized with 5×106 Af conidia, challenged with 1×106 Af conidia for eight times, and sacrificed for sequent assay at day 18. (b–e) Eosinophil (SiglecF+, b and c) and Neutrophil (Ly6G+, d and e) counts in lungs of the above disposed mice. (f) ELISA results for lung IL-4 and IL-5 and serum IgE in the above disposed mice. *P < 0.05, **P < 0.01 and ***P < 0.001, by two-tailed unpaired t-test (c, e-f). Data shown are representative of three independent experiments.

Supplementary Figure 3 Determining T cell responses in wide-type and CARD9S12N KI mice in response to Af challenging.

(a) Gating strategy for flow assay of Th2 (CD3+CD4+ICOS+ST2+) cell recruitment in the lungs of WT and CARD9S12N KI mice at day 6 after single intratracheal exposure to Af conidia (1×107). (b) Gating strategy for flow assay of T-bet and GATA3 proteins in CD3+CD4+T cells from the lungs of the above disposed mice. (c) Gating strategy for flow assay of IL-4-producing CD3+CD4+T cells in the mediastinal lymph node (MLN) from the above disposed mice. (d) Gating strategy for flow assay of IL-17A-producing and IFN-γ-producing CD4+T cells in the MLN of the above disposed mice.

Supplementary Figure 4 Depletion of CD4+ T cell in CARD9S12N KI mice.

(a and b) T cell counts in lungs of KI mice, which were intravenously injected with 200 μg anti-CD4 or control IgG per mouse at 24 hours before challenge and assayed at day 6 after single intratracheal exposure to Af conidia (1×107). (c and d) Neutrophil (Ly6G+) and eosinophil (SiglecF+) counts in lungs of the above disposed mice. *P < 0.05, **P < 0.01 and ***P < 0.001. By one-way ANOVA and post hoc Tukey test. Data shown are representative of three independent experiments.

Supplementary Figure 5 ILC2s decreased in WT and CARD9S12N KI mice after infection.

(a) Gating strategy for flow assay of ILC2 (CD90.2+Lin-RORγt-GATA-3+) counts in lungs from WT and KI mice at day 2 after single intratracheal exposure to Af conidia (1×107). (b and c) ILC2 counts in the lung of the above disposed mice. *P < 0.05; **P < 0.01 and ***P < 0.001. Data shown are representative of three independent experiments. By two-tailed unpaired t-test (c), in a-c, n = 6 mice per group.

Supplementary Figure 6 Determining cellular responses in wide-type and CARD9S12N KI mice in response to Af challenging.

(a) Gating strategy for flow assay of alveolar macrophage (AM, CD11c+SiglecF+), eosinophil (SiglecF+) and neutrophil (Ly6G+) counts in lungs of WT, Het and KI mice at the indicated day after single intratracheal exposure to Af conidia (1 × 107). (b, c) AM, eosinophil and neutrophil counts in lungs of the above disposed mice.

Supplementary Figure 7 Intracellular staining for IL-4 expression in wide-type and CARD9S12N KI mice.

(a) Gating strategy of flow assay for sorting alveolar macrophage (CD11c+ SiglecF+) in lungs of WT and CARD9S12N KI mice at day 2 after single exposure to Af conidia (1 × 107). (b) Gating strategy of flow assay for sorting DCs (CD11c+, Q1), alveolar macrophage (CD11c+SiglecF+, Q2), CD11c-SiglecF-cells (Q3) and eosinophil (SiglecF+, Q4) in lungs of the above disposed mice. (c) Quantitative real-time PCR results for IL-4 expression normalized to GAPDH in DCs and AMs sorted from the above disposed mice at day 2. Data are mean ± s.e.m. *P < 0.05, **P < 0.01 and ***P < 0.001, by two-tailed t-test. Data shown are representative of three independent experiments.

Supplementary Figure 8 IL-5-producing cells in ABPA patients.

(a) Gating strategy and (b) raw data of flow assay for IL-5-producing PBMCs from ABPA patients carrying wild-type or homozygous S12N mutation of CARD9, which were determined by flow cytometry.

Supplementary Figure 9 Nuclear translocation of RelB in PBMCs from ABPA patients.

(a) Gating strategy and (b) raw data of RelB nuclear translocation in PBMCs (CD14+CD11b+ cells) from ABPA patients carrying wild-type or homozygous S12N mutation of CARD9, which were determined by Millipore-Amnis FlowSight.

Supplementary Figure 10 Nuclear translocation of p65 in PBMCs from ABPA patients.

(a) Gating strategy and (b) raw data of p65 nuclear translocation in PBMCs (CD14+CD11b+ cells) from ABPA patients carrying wild-type or homozygous S12N mutation of CARD9, which were determined by Millipore-Amnis FlowSight.

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Xu, X., Xu, JF., Zheng, G. et al. CARD9S12N facilitates the production of IL-5 by alveolar macrophages for the induction of type 2 immune responses. Nat Immunol 19, 547–560 (2018). https://doi.org/10.1038/s41590-018-0112-4

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