IL-4Ralpha-independent expression of mannose receptor and Ym1 by macrophages depends on their IL-10 responsiveness

Abstract

IL-4Ralpha-dependent responses are essential for granuloma formation and host survival during acute schistosomiasis. Previously, we demonstrated that mice deficient for macrophage-specific IL-4Ralpha (LysM(cre)Il4ra(-/lox)) developed increased hepatotoxicity and gut inflammation; whereas inflammation was restricted to the liver of mice lacking T cell-specific IL-4Ralpha expression (iLck(cre)Il4ra(-/lox)). In the study presented here we further investigated their role in liver granulomatous inflammation. Frequencies and numbers of macrophage, lymphocyte or granulocyte populations, as well as Th1/Th2 cytokine responses were similar in Schistosoma mansoni-infected LysM(cre)Il4ra(-/lox) liver granulomas, when compared to Il4ra(-/lox) control mice. In contrast, a shift to Th1 responses with high IFN-gamma and low IL-4, IL-10 and IL-13 was observed in the severely disrupted granulomas of iLck(cre)Il4ra(-/lox) and Il4ra(-/-) mice. As expected, alternative macrophage activation was reduced in both LysM(cre)Il4ra(-/lox) and iLck(cre)Il4ra(-/lox) granulomas with low arginase 1 and heightened nitric oxide synthase RNA expression in granuloma macrophages of both mouse strains. Interestingly, a discrete subpopulation of SSC(high)CD11b+I-A/I-E(high)CD204+ macrophages retained expression of mannose receptor (MMR) and Ym1 in LysM(cre)Il4ra(-/lox) but not in iLck(cre)Il4ra(-/lox) granulomas. While aaMphi were in close proximity to the parasite eggs in Il4ra(-/lox) control mice, MMR+Ym1+ macrophages in LysM(cre)Il4ra(-/lox) mice were restricted to the periphery of the granuloma, indicating that they might have different functions. In vivo IL-10 neutralisation resulted in the disappearance of MMR+Ym1+ macrophages in LysM(cre)Il4ra(-/lox) mice. Together, these results show that IL-4Ralpha-responsive T cells are essential to drive alternative macrophage activation and to control granulomatous inflammation in the liver. The data further suggest that in the absence of macrophage-specific IL-4Ralpha signalling, IL-10 is able to drive mannose receptor- and Ym1-positive macrophages, associated with control of hepatic granulomatous inflammation.

Figure 1. Flow cytometry analysis of liver granuloma during acute schistosomiasis.

BALB/c mice were infected with 100 S. mansoni cercariae, killed 8 weeks later and liver granuloma-associated leukocytes isolated before 4-colour flow cytometry analysis. (A) Gating strategy for analysis of surface expression of CD11b and I-A/I-E on SSC^high and live (7-AAD⁻) liver granuloma-associated leukocytes. Outlined regions define CD11b⁺I-A/I-E⁻ and CD11b⁺I-A/I-E^high, respectively. (B) CD11b⁺I-A/I-E⁻, and CD11b⁺I-A/I-E^high gated populations in A were analyzed for F4/80, Gr-1, Siglec-F, CD204, MMR, Dectin-1, CD68, CD80, CD86 and IL-4Rα expression, respectively. Greyscale histograms show relevant isotype control. (C) 4-colour flow cytometry analysis of liver granuloma-associated leukocytes gated on CD11b⁺I-A/I-E⁻ cells as described in A. F4/80 and Gr-1 double staining contour plot is shown. Outlined regions were sorted for cytospin and morphological analysis. (D) 4-colour flow cytometry analysis of liver granuloma-associated leukocytes gated on CD11b⁺F4/80⁺ cells as described in A. MHC-II (I-A/I-E) and scavenger receptor-A (CD204) double staining contour plot is shown. Outlined region was sorted for cytospin and morphological analysis. Data are representative of three independent experiments with similar results.

Figure 2. IL-4Rα-expressing macrophages do not affect cytokine responses in granulomas.

Il4ra^−/lox, LysM^creIl4ra^−/lox, iLck^creIl4ra^−/lox and Il4ra^−/− mice were infected with 100 S. mansoni cercariae and killed 8 weeks later. Mesenteric lymph node (mLN) cells or liver granuloma-associated leukocytes (Liver) were then isolated and restimulated overnight with 20 µg/ml SEA before analyzed for ex vivo cytokine production capability. Staining of surface CD4 was followed by detection of IL-4, IL-10 and IFN-γ secretion in a cytokine catch assay or detection of intracellular levels of IL-13 after 4h of monensin treatment, as described in Methods. Live gate was placed on lymphocytes according to their forward- and side-scatter by flow cytometry. (A) Representative contour plots of IFN-γ, IL-4, IL-10 and IL-13-producing lymphocytes. Quadrant bars were set up on unstimulated cells and numbers represent percent cells in each quadrant. Data represent one out of three independent experiments with similar results. (B) Percentages of IFN-γ, IL-4, IL-10 or IL-13-producing cells were determined in gated CD4⁺ or non-CD4⁺ cell populations from mLN or liver granuloma-associated lymphocytes (Liver) (mean±SEM, n = 4). Data are representative of three independent experiments. *p<0.05; **p<0.001; ***p<0.001 compared to control Il4ra^−/lox mice.

Figure 3. Mannose receptor and Ym1 expression in granuloma macrophages of LysM^creIl4ra^−/lox mice.

Il4ra^−/lox, LysM^creIl4ra^−/lox, iLck^creIl4ra^−/lox and Il4ra^−/− mice were infected with 100 S. mansoni cercariae, killed 8 weeks later and liver granuloma-associated leukocytes purified. (A) CD11b⁺I-A/I-E^highCD204⁺ granuloma macrophages were sorted to high purity (>98%) and RNA extracted before analysis of expression levels of genes associated with macrophage activation by real time RT-PCR: IL-4Rα (Il4ra), nitric oxide synthase (Nos2), arginase 1 (Arg1), FIZZ1 (Retnla), macrophage mannose receptor (Mrc1), and Ym1 (Chi3l3). Relative expression levels normalized to r12S house-keeping gene are shown as fold-change over Il4ra^−/lox mice values. Data is representative of two independent experiments with analysis of pooled samples (n = 4). Bars show mean±SD of analyses performed in triplicates. (B) 4-colour flow cytometry analysis of granuloma leukocytes gated on CD11b⁺I-A/I-E^highCD204⁺ cells. Representative monoparametric histograms show stainings of surface macrophage mannose receptor (MMR) or intracellular Ym1 expression. Data is representative of four independent experiments of pooled samples (n = 4). Greyscale histograms show relevant isotype control. (C) Mean fluorescent intensities of MMR and Ym1 stainings based on analysis by flow cytometry as presented in B. Data show results of individual mice for 2 independent experiments. (D) 4-colour flow cytometry analysis of granuloma leukocytes gated on CD11b⁺I-A/I-E^high cells. Representative contour plots show MMR/Ym1 double staining and outlined regions show MMR-positive staining. Data is representative of two independent experiments of pooled samples (n = 4). (E) 4-colour flow cytometry analysis of granuloma leukocytes. Analysis gate (R1) was placed on SSC^highCD11b⁺MMR⁺7-AAD⁻ and histograms show isotype IgG2a (upper panel) and anti-IL-4Rα (lower panel) stainings. Data is representative of two independent experiments of pooled samples (n = 4). Black, Il4ra^−/lox; grey, LysM^creIl4ra^−/lox. * p<0.05, **p<0.01; ***p<0.001.

Figure 4. Localisation of mannose receptor and Ym1-expressing granuloma macrophages in close contact with S. mansoni eggs depends on their IL-4Rα signalling.

Livers were collected at 8 weeks p.i. from Il4ra^−/lox, LysM^creIl4ra^−/lox, iLck^creIl4ra^−/lox and Il4ra^−/− mice and immunofluorescent stainings performed on cryosections. (A) Representative micrographs of MMR (panels v–viii), Ym1 (panels xiii–xvi), CD204 (panels xxi–xxiv) and iNOS (panels xxix–xxxii) stainings of liver cryosections as described in Methods. Stainings with secondary antibody (MMR, iNOS), streptavidin alone (Ym1) or isotype-control (CD204) is shown for each corresponding staining (MMR = i–iv, Ym1 = ix–xii, CD204 = xvii–xx, iNOS = xxv–xxviii). Note that MMR⁺ and Ym1⁺ cells are restricted to the periphery of LysM^creIl4ra^−/lox granulomas. Outlined regions represent the areas magnified in B. (B) Representative micrographs of liver cryosections stained with scavenger receptor (CD204) for macrophages detection (panels i–viii, green) or iNOS for classically activated macrophages detection (panels ix–xvi, green); and co-stained for MMR (panels i–iv and ix–xii, red) or Ym1 (panels v–viii and xiii–xvi, red). Note the low frequency of CD204⁺ macrophages co-expressing MMR⁺ or Ym1⁺ cells around the parasite eggs (panels ii and vi) but the high levels of iNOS⁺ cells (panels x and xiv) in LysM^creIl4ra^−/lox mice, suggesting these macrophages to be classically activated. White arrows indicate the parasite eggs. Original magnification: 400×. Data represent one of three independent experiments.

Figure 5. IL-10 signalling drives mannose receptor and Ym1 expression in macrophages independently of their IL-4Rα expression.

Peritoneal cells were harvested 7 days after injection of 3000 S. mansoni purified eggs in the peritoneum of Il4ra^−/lox, LysM^creIl4ra^−/loxor Il4ra^−/− mice. (A) Representative histograms of macrophage mannose receptor (MMR) or Ym1 expression by peritoneal macrophages gated on FSC^highSSC^lowCD11b⁺F4/80⁺ cells after exclusion of peritoneal eosinophils (FSC^lowSSC^highCD11b⁺F4/80⁺) . Black, isotype control; red, Il4ra^−/lox; blue, LysM^creIl4ra^−/lox; green, Il4ra^−/−. Broken lines show threshold for positive signal. Data are representative of two independent experiments of pooled samples (n = 3). (B) Anti-IL-10 receptor treatment. Four mice out of eight in each group received 4µg of anti-IL-10R i.p. at day 0, 4 and 6 post-injection. Overlays of representative monoparametric histograms of macrophage mannose receptor (MMR) or Ym1 expression by peritoneal macrophages are shown as described in A. Bracketed line indicates positive signal. Dotted line, isotype control; greyscale, untreated mice; bold line, mice treated with anti-IL-10R. (C) Percent and mean fluorescent intensities of peritoneal macrophages expressing MMR or Ym1 based on flow cytometry analysis in B. Data is representative of two independent experiments with mean±SEM (n = 3). *p<0.05, ** p<0.01, *** p<0.001.