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A role for the ITAM signaling module in specifying cytokine-receptor functions

Nature Immunology volume 15, pages 333–342 (2014)Cite this article

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Abstract

Diverse cellular responses to external cues are controlled by a small number of signal-transduction pathways, but how the specificity of functional outcomes is achieved remains unclear. Here we describe a mechanism for signal integration based on the functional coupling of two distinct signaling pathways widely used in leukocytes: the ITAM pathway and the Jak-STAT pathway. Through the use of the receptor for interferon-γ (IFN-γR) and the ITAM adaptor Fcγ as an example, we found that IFN-γ modified responses of the phagocytic antibody receptor FcγRI (CD64) to specify cell-autonomous antimicrobial functions. Unexpectedly, we also found that in peritoneal macrophages, IFN-γR itself required tonic signaling from Fcγ through the kinase PI(3)K for the induction of a subset of IFN-γ-specific antimicrobial functions. Our findings may be generalizable to other ITAM and Jak-STAT signaling pathways and may help explain signal integration by those pathways.

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Figure 1: IFN-γR is coupled with the FcγRI signalosome in phagocytes.
Figure 2: FcγR and IFN-γR are dispensable for each other's expression and canonical signaling.
Figure 3: Induction of subset of IFN-γ-regulated genes in peritoneal macrophages requires expression of Fcγ.
Figure 4: Induction of a subset of IFN-γ-regulated genes in peritoneal macrophages requires tonic PI(3)K signaling through FcγR and Fcγ.
Figure 5: Early IFN-γ-mediated control of infection with L. monocytogenes in vivo requires Fcγ.
Figure 6: IFN-γR and FcγRI function by the 'coincidence detection principle' to induce a specific transcriptional program in phagocytes.

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Acknowledgements

We thank D. Stetson (University of Washington, Seattle) for modified retroviral vector pMSCV.hCD2 and vectors MIGR2 and pMSCV.IRES-GFP; members of the Medzhitov laboratory and S. Joyce for discussions and reading of the manuscript; and C. Annicelli and S. Cronin for assistance with animal work. Supported by the Howard Hughes Medical Institute (R.M. and J.S.B.), the US National Institutes of Health (1R56AI087725-01 AI046688, AI055502, AI089771 and DK071754 to R.M.) and the Damon Runyon Cancer Research Foundation (DRG-1968-08 to J.S.B.).

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  1. Igor Brodsky

    Present address: Present address: Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.,

Authors and Affiliations

  1. Howard Hughes Medical Institute and Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut, USA

    Jelena S Bezbradica, Rachel K Rosenstein, Igor Brodsky & Ruslan Medzhitov

  2. Amnis at EMD Millipore, Seattle, Washington, USA

    Richard A DeMarco

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Contributions

J.S.B. and R.M. designed research and wrote manuscript; J.S.B. did and analyzed experiments; R.K.R. and J.S.B. cloned and initially characterized Fcγ and its mutants; I.B. and J.S.B. did in vitro L. monocytogenes infection experiments; and R.A.D. analyzed and interpreted ImageStream data.

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Correspondence to Ruslan Medzhitov.

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Integrated supplementary information

Supplementary Figure 1 Immunofluorescence analysis of actin, IFN-γRI and FcγRI on macrophages.

(a, b) Immunofluorescence analysis of IFN-γRI localization in BM macrophages from wild-type and indicated KO mice, stimulated with IgG2a-opsonised SRBC for 15 min in the absence in a or presence in b of IFN-γ. Cells were fixed, permeabilized and probed with IFN-γRI antibody (clone C-20). Uptake of SRBC (N) by macrophages is extremely low, and so is uptake of SRBC (O) by Fcγ-deficient macrophages. Thus, arrows were added to those images to indicate location of few phagocytosed SRBC. (c) ImageStream analysis of actin recruitment to the FcγR phagocytic cup of BM DC stimulated with rabbit IgG-opsonized, Alexa fluor 488-labeled beads for 5 or 10 min. Cells were fixed, permeabilized and stained with Phalloidin Alexa fluor 647. (d) ImageStream analysis showing surface distribution of IFN-γRI and FcγRI in BM DC stained with antibodies against IFN-γRI (biotinilated clone 2E2) and FcγRI. Cells were not fixed nor permeabilized because, unlike clone C-20 (used in a and b), clone 2E2 against IFN-γRI and antibody against FcγRI do not work well under those conditions. BDS score is shown as histogram on top right and representative images with BDS>2 are shown at the bottom. (e and f) Flow cytometry analysis of IFN-γRI and FcγRI in wild-type and indicated KO macrophages, indicating the specificity of antibodies used in (d). For un-stained control, mix of wild-type and KO cells were used. (g) Schematic representation of IFN-γR and FcγRI with their key signaling components.

Supplementary Figure 2 Serine-phosphorylation of STAT1 by IFN-γR and FcγRI

(a-d) Immunoblot analysis of signaling in wild-type BM macrophages stimulated as indicated on the top with SRBC (O) (in synchronized phagocytosis assay) or with IFN-γ.Cells were lysed and analyzed by IB.

Supplementary Figure 3 Gene expression analysis of Fcγ-deficient peritoneal macrophages after IFN-γ and IL-4 stimulation.

(a) Flow cytometry of peritoneal cells from wild-type (B6 or BALB/c) and Fcγ-deficient mice stained with macrophage and B cells specific markers. (b and c) Quantitative PCR analysis of gene transcription in wild-type and Fcγ-deficient peritoneal macrophages stimulated with IFN-γ in b or IL-4 in c for 0-6 h. (d and e) Immunoblot and flow cytometry analysis of wild-type Fcγ and its mutant protein expression upon retroviral transduction into Fcγ-deficient BM. Expression was monitored in differentiated macrophages using Flag immunoblot in d, of Flag surface staining in e. (f) Outline of the microarray data analysis of global gene expression changes in peritoneal macrophages after IFN-γ or IL-4 stimulation for 6 h. (g) Cluster analysis of genes differently expressed between wild type and Fcγ-deficient peritoneal macrophages relative to wild-type unstimulated sample after stimulation with IL-4 for 6 h. Data were analyzed and presented as in Fig. 3f. (h) Summary of the results in g. (i) Quantitative PCR analysis confirmation of selected genes of interest from g, results are presented relative to KO unstimulated sample, set as 1, because expression in wild-type was low to undetectable for Ifi202b. (j) Comparison of two arrays: Genes differently expressed between wild-type and Fcγ-deficient peritoneal macrophages relative to wild-type unstimulated sample in response to IFN-γ or IL-4, from the two microarrays shown in Fig. 3f and Supplementary fig. 3g, respectively, were compared. Gene groups A, B, C, E, F, and G from each of those arrays (i.e., where cluster profiles were different between WT STIM and KO STIM) were aligned and compared. Shared genes in two arrays were identified and clustered in two main groups: Group A, where expression patterns are similar between two arrays and Group B, where expression patterns are distinct.

Supplementary Figure 4 Peritoneal macrophage signaling in the presence of pathway inhibitors.

(a) Two models describing how Fcγ adapter could be linked to IFN-γR functions. (b) Griess assay measuring NO production in the sup of STAT1-deficient and wild type BM macrophages stimulated with SRBC (O) with or without IFN-γ for ~16-20 h. (c) Immunoblot analysis of signaling in BM macrophages pretreated with DMSO, NFAT inhibitor (Cyclosporine, 1 and 10 uM), PI3K inhibitor (Ly294002, 10 and 20 uM) or ERK inhibitor (PD98059, 20 and 40 uM) for 60 min and then stimulated with SRBC (O) for 15 min. (d) Immunoblot analysis of signaling in peritoneal macrophages pretreated with DMSO, PI3K inhibitor (Wortmannin or AktVIII), pan Jak inhibitor, ERK inhibitor (PD98059) or NFkB inhibitor (Bay11) for 30 min and then stimulated with SRBC (O) and IFN-γ or LPS Concentrations tested for Wortmannin and panJak: 1 uM, 500 nM, 100 nM; for AKTVIII, PD98059 and Bay11: 10 uM, 1 uM, 100 nM. LPS was used for Bay11 testing because it is more robust stimulator of NFkB pathway than SRBC (O). (e and f) Quantitative PCR analysis of gene transcription in peritoneal macrophages pretreated with DMSO, Bay11, PD98059 (ERK inh) or CyclosporineA (NFAT inh) in e or 100 nM panJak inhibitor in f for 30 min prior to IFN-γ stimulation for 6 h.

Supplementary Figure 5 L. monocytogenes uptake by wild-type, Fcγ-deficient and IFN-γRI-deficient macrophages.

B6, Fcγ-deficient and IFN-γRI-deficient BM macrophages were replated in 24 well TC dishes in antibiotic free media, stimulated in triplicates with IFN-γ, where indicated, for 16 h to induce iNOS expression before being infected with L. monocytogenes at MOI of 10. To synchronize infection bacteria were spun over macrophages for 5 min, infection was carried for 30 min at 37oC, and extracellular bacteria were killed by adding gentamycin to cultures at c=50 ug/ml for 1 h. Cells were then lysed in H2O, lysates were plated on streptomycin containing LB plates and bacterial loads calculated after 24 h.

Supplementary Figure 6 IFN-γ functional 'collaboration' with FcγRs during phagocytosis.

(a) BM macrophages microarray data analysis outline. (b) Primers used for quantitative PCR in this study. (c) To validate hybridoma sups used for opsonization, SRBC were opsonized for 1 h at RT. Cells were lysed in 1X SDS buffer and analyzed by IB. Normal mouse IgG was run on the left as control. (d) To validate that SRBC (O) signaling is Fcγ-dependent BM macrophages were stimulated with SRBC (N) (SRBC incubated with either in 10% DMEM, left, or with control hybridoma conditional media, middle) or with SRBC (O) (right) for 15 min. Cells were analyzed by immunoblot. (e, f) To validate that SRBC (O) responses are Fcγ-dependent BM macrophages were stimulated as indicated for 16 h. NO was measured in the culture supernatant using Griess assay.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 (PDF 6790 kb)

Supplementary Table 1

Peritoneal macrophage IFN-γ array (XLSX 149 kb)

Supplementary Table 2

Peritoneal macrophage IL-4 array (XLSX 128 kb)

Supplementary Table 3

Shared genes between IFN-γ and IL-4 array (XLSX 88 kb)

Supplementary Table 4

BM macrophages SRBC (O) and IFN-γ array (XLSX 77 kb)

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Bezbradica, J., Rosenstein, R., DeMarco, R. et al. A role for the ITAM signaling module in specifying cytokine-receptor functions. Nat Immunol 15, 333–342 (2014). https://doi.org/10.1038/ni.2845

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