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Inflammation-induced formation of fat-associated lymphoid clusters

Nature Immunology volume 16, pages 819–828 (2015)Cite this article

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Abstract

Fat-associated lymphoid clusters (FALCs) are a type of lymphoid tissue associated with visceral fat. Here we found that the distribution of FALCs was heterogeneous, with the pericardium containing large numbers of these clusters. FALCs contributed to the retention of B-1 cells in the peritoneal cavity through high expression of the chemokine CXCL13, and they supported B cell proliferation and germinal center differentiation during peritoneal immunological challenges. FALC formation was induced by inflammation, which triggered the recruitment of myeloid cells that expressed tumor-necrosis factor (TNF) necessary for signaling via the TNF receptors in stromal cells. Natural killer T cells (NKT cells) restricted by the antigen-presenting molecule CD1d were likewise required for the inducible formation of FALCs. Thus, FALCs supported and coordinated the activation of innate B cells and T cells during serosal immune responses.

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Figure 1: Distribution of FALCs in visceral AT.
Figure 2: CXCL13 expression by FALC stromal cells is necessary for B cell recruitment.
Figure 3: Activation and differentiation of B cells in FALCs after peritoneal immunological challenge.
Figure 4: Factors that control the development of FALCs.
Figure 5: TNF is required for FALC formation after inflammation is elicited.
Figure 6: CD1d-restricted NKT cells are required for FALC formation.
Figure 7: Activation of iNKT cells induces FALC formation.
Figure 8: Formation of FALCs is dependent on signaling via IL-4Rα.

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Acknowledgements

We thank the personnel of the Biomedical Services Unit of the University of Birmingham for animal colony care; E. Jenkinson for support; G. Anderson, P. Lane, and A. Rot for comments on the manuscript; C. Buckley for facilitating animal procedures; R. Bird for cell sorting; K. Pfeffer (Heinrich Heine University) for Ltbr−/− mice; G. Eberl (Institut Pasteur) for tissues from germ-free mice; D. Finke (University of Basel) for tissues from Cxcr5−/− mice; A. Diefenback (University of Mainz) for tissues from Id2CreErt2/+Gt(ROSA)26SorYFP/+Gata3f/f mice; and the US National Institutes of Health Tetramer Facility for CD1d-PBS-57 loaded tetramers. Supported by the European Union Framework Programme 7 integrated project INFLACARE (J.H.C.); the Biotechnology and Biological Sciences Research Council (BB/K004900/1 to J.H.C.) and the College of Medical and Dental Sciences of the University of Birmingham (J.H.C.); the American Asthma Foundation, the UK Medical Research Council and the Wellcome Trust (100963/Z/13/Z) for work in the A. McKenzie laboratory; the Biotechnology and Biological Sciences Research Council institute synergy programme, the European Research Council (280307) and the Agency for Science, Technology and Research (to Y.L.) for work in the M. Veldhoen laboratory; Deutsche Forschungsgemeinschaf (NE1466/2-1), the Russian Science Foundation (14-50-00060) and the Russian Foundation for Basic Research (13-04-40268 to A.A.K.) for work in the S. Nedospasov laboratory; the European Research Council (340217-MCs_inTEST) for work in the G. Kollias laboratory; the Medical Research Council (S.N.); the UK Medical Research Council (MRC/K01207X/1 to L.H.J.); and the Lifelong Health and Wellbeing cross council initiative (Topjabs G1001390 for K.M.T., Y.Z. and J.M.).

Author information

Author notes

  1. Cécile Bénézech & Lucy H Jones

    Present address: Present address: BHF/UoE Centre for Cardiovascular Sciences, University of Edinburgh, Edinburgh, UK.,

  2. Nguyet-Thin Luu and Jennifer A Walker: These authors contributed equally to this work.

Authors and Affiliations

  1. School of Immunity and Infection, IBR-MRC Centre for Immune Regulation, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK

    Cécile Bénézech, Nguyet-Thin Luu, Kyoko Nakamura, Yang Zhang, Saba Nayar, Adriana Flores-Langarica, Alistair McIntosh, Jennifer Marshall, Francesca Barone, Adam F Cunningham, David R Withers, Kai Michael Toellner, Nick D Jones & Jorge H Caamaño

  2. MRC Laboratory of Molecular Biology, Cambridge, UK

    Jennifer A Walker & Andrew N J McKenzie

  3. Inflammation Biology, German Rheumatism Research Center, Leibniz Institute, Berlin, Germany

    Andrei A Kruglov & Sergei A Nedospasov

  4. Engelhardt Institute of Molecular Biology, Moscow, Russia

    Andrei A Kruglov & Sergei A Nedospasov

  5. Lomonosov Moscow State University, Moscow, Russia

    Andrei A Kruglov & Sergei A Nedospasov

  6. Lymphocyte Signalling and Development Programme, The Babraham Institute, Cambridge, UK

    Yunhua Loo & Marc Veldhoen

  7. Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh, UK

    Lucy H Jones & Judith E Allen

  8. College of Life and Environmental Sciences, University of Birmingham, Birmingham, UK

    Gurdyal Besra

  9. Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK

    Katherine Miles & Mohini Gray

  10. Division of Immunology, Biomedical Sciences Research Center 'Alexander Fleming', Vari, Greece

    George Kollias

  11. Departament of Experimental Physiology, National and Kapodistrian University of Athens, Athens, Greece

    George Kollias

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  1. Cécile Bénézech
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Contributions

C.B., N.-T.L., J.A.W., A.A.K., Y.L., K.N., Y.Z., S.N., L.H.J. and J.H.C. designed and performed the research and collected and analyzed the data; A.F.-L., A.M., J.M., F.B., G.B., K.M., J.E.A., M.G., G.K., A.F.C., D.R.W., K.M.T., N.D.J., M.V., S.A.N. and A.N.J.M. facilitated the research; and C.B. and J.H.C. wrote the manuscript.

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Correspondence to Cécile Bénézech or Jorge H Caamaño.

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Integrated supplementary information

Supplementary Figure 1 FALC morphology.

Whole mount immunofluorescence staining showing a representative array of FALCs harvested from the mesenteries of a single C57BL/6J (WT) mouse with CD11b+ myeloid cells (blue), CD45+ hematopoietic cells (green), IgM+ B cells (red), and CD4+ T cells (white) on the top row and without CD45 staining on the bottom row. Pictures are representative of 6 mice. Scale bar 50 μm.

Supplementary Figure 2 B cell activation in FALCs upon peritoneal immunological challenge.

(a) Flow cytometry of the mesenteries of QM/QM (IgHNP/NP Igκ−/−) mice 24 hours post PBS (left panel) and NP-Ficoll (right panel) IP injection. In the CD45+ hematopoietic fraction, the different B cell subsets were identified according to the expression of CD19 and CD11b (first row), CD19 and CD5 (second row). The percentages of NP+Ki67+ cells in the B2 (CD19+CD11b−CD5-), B1c (CD19+CD11b−CD5+), B1a (CD19+CD11b+CD5+) and B1b (CD19+CD11b+CD5-) cell subsets are shown (third and forth row). Image representative of 2 independent experiments (n=6, 5). (b) Quantification of the percentage of NP+Ki67+ activated B cells in the mesenteries, peritoneal cavity lavage (PCL) and spleen of QM/QM mice 24 hours after PBS or NP-Ficoll injections. Data are representative of 2 independent experiments (n=6, 5). Each symbol represents an individual mouse. Mann-Whitney nonparametric two-tailed test, *p < 0.05, **p < 0.01, ***p< 0.001, ****p < 0.0001, and NS not significant. (c) Whole mount immunofluorescence staining showing FALCs from QM/QM mice after PBS or NP-Ficoll injection with CD11b+ myeloid cells (blue), CD45+ hematopoietic cells (green) and IgM+ B cells (red). Pictures representative of 8 clusters from 4 mice for each condition. Scale bar 50 μm.

Supplementary Figure 3 ILC2 cells in visceral AT and FALCs.

(a) Flow cytometry of the mesenteries showing CD45+Lin-c-Kit+Sca-1+IL-7Rα+ ILC2 in WT (first row) and Rag2−/− (second row) mice (NK1.1 and CD4 present in Lin cocktail). Data representative of 4 mice (b) Flow cytometric analysis of the mesenteries showing CD45+Lin-Sca-1+NK1.1- ILC2 and CD45+Lin-Sca-1-+NK1.1+ NK cells in Rag2−/− mice. Data representative of 4 mice (c) Whole mount immunofluorescence staining showing FALCs in Rag2−/− with CD11b+ myeloid cells (blue) and CD90+ ILC (green). The picture is representative of 8 clusters from 4 mice. Scale bar 50 μm.

Supplementary Figure 4 Myeloid cells are recruited to FALCs when inflammation is elicited.

Whole mount immunofluorescence staining showing FALCs containing CD11b+ myeloid cells (blue) and Ly6C+ monocytes (first row, green), Gr1+ granulocytes (second row, green) or F4/80+ macrophages (third row, green) after zymosan injection. Pictures are representative of 8 clusters from 4 mice. Scale bar 50 μm.

Supplementary Figure 5 IFN-γ is not necessary for the formation of FALCs when inflammation is elicited.

Number of clusters found in the mesenteries of Ifng−/− mice and whole mount immunofluorescence staining showing FALCs with CD11b+ myeloid cells (blue), CD45+ hematopoietic cells (green), IgM+ B cells (red), and CD4+ T cells (white) 72 hours after IP injection of PBS or zymosan. Data corrrespond to control (n=6) and treated (n=4) mice pooled from 2 independent experiments. Each symbol represents an individual mouse Mann-Whitney nonparametric two-tailed test, **p < 0.01. Pictures are representative of 8 clusters from 4 mice. Scale bar 50 μm. Scale bar 50 μm.

Supplementary Figure 6 Partial ‘rescue’ of inflamation-induced FALC formation with IL-4c in the absence of TNF signaling.

(a) Number of clusters found in the mesenteries of C57BL/6J (WT) mice and whole mount immunofluorescence staining showing FALCs with CD11b+ myeloid cells (blue), CD45+ hematopoietic cells (green), IgM+ B cells (red), and CD4+ T cells (white) four days after PBS, TNF, IL-4c and both TNF and IL4c injections. Data correspond to control (n=6) and TNF (n=6), IL-4c (n=7) and TNF+IL-4c (n=7) treated mice, pooled from 2 independent experiments. Mann-Whitney nonparametric two-tailed test, PBS and TNF p=0.4248, PBS and IL-4c p=0.4156, PBS and TNF+IL-4c p= 0.7964. Pictures are representative of 8 clusters from 4 mice for each condition. (b) Number of clusters found in the mesenteries of C57BL/6J (WT), Il4r a−/− and Tnfrsf1a−/− Tnfrsf1b−/− mice and whole mount immunofluorescence staining as in a, 4 days after PBS, zymosan, zymosan+TNF, and zymosan+IL-4c injections. Data correspond to PBS (n=13), zymosan (n=15) and zymosan+TNF (n=9) treated WT mice pooled from 3 independent experiments, and to PBS (n=7), zymosan (n=7) and zymosan+TNF (n=8) treated Il4ra−/− mice pooled from 2 independent experiments, and to zymosan (n=7) and zymosan+IL-4c (n=7) treated Tnfrsf1a−/− Tnfrsf1b−/− mice pooled from 2 independent experiments. Each symbol represents an individual mouse. Mann-Whitney nonparametric two-tailed test. *p < 0.05, **p < 0.01, ***p< 0.001 and ****p <0.0001, NS not significant. Pictures are representative of 8 clusters from 4 mice for each condition. Scale bar 50 μm.

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Bénézech, C., Luu, NT., Walker, J. et al. Inflammation-induced formation of fat-associated lymphoid clusters. Nat Immunol 16, 819–828 (2015). https://doi.org/10.1038/ni.3215

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