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Intestinal macrophages arising from CCR2(+) monocytes control pathogen infection by activating innate lymphoid cells.

Seo SU, Kuffa P, Kitamoto S, Nagao-Kitamoto H, Rousseau J, Kim YG, Núñez G, Kamada N - Nat Commun (2015)

Bottom Line: Unlike resident intestinal MPs, de novo differentiated MPs are phenotypically pro-inflammatory and produce robust amounts of IL-1β (interleukin-1β) through the non-canonical caspase-11 inflammasome.Intestinal MPs from infected mice elicit the activation of RORγt(+) group 3 innate lymphoid cells (ILC3) in an IL-1β-dependent manner.Deletion of IL-1β in blood monocytes blunts the production of IL-22 by ILC3 and increases the susceptibility to infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, 1500 E Medical Center Dr Ann Arbor, Michigan 48109, USA.

ABSTRACT
Monocytes play a crucial role in antimicrobial host defence, but the mechanisms by which they protect the host during intestinal infection remains poorly understood. Here we show that depletion of CCR2(+) monocytes results in impaired clearance of the intestinal pathogen Citrobacter rodentium. After infection, the de novo recruited CCR2(+) monocytes give rise to CD11c(+)CD11b(+)F4/80(+)CD103(-) intestinal macrophages (MPs) within the lamina propria. Unlike resident intestinal MPs, de novo differentiated MPs are phenotypically pro-inflammatory and produce robust amounts of IL-1β (interleukin-1β) through the non-canonical caspase-11 inflammasome. Intestinal MPs from infected mice elicit the activation of RORγt(+) group 3 innate lymphoid cells (ILC3) in an IL-1β-dependent manner. Deletion of IL-1β in blood monocytes blunts the production of IL-22 by ILC3 and increases the susceptibility to infection. Collectively, these studies highlight a critical role of de novo differentiated monocyte-derived intestinal MPs in ILC3-mediated host defence against intestinal infection.

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Related in: MedlinePlus

Recruitment of CCR2+ monocytes activate ILCs during intestinal infection.(a) C. rodentium burden in faeces from Ccr2+/+ and Ccr2−/− mice. Data represent mean±s.d. (n=5, representative of two independent experiments). (b) Colonic lamina propria mononuclear cells (LPMCs) were isolated from at 0 and 12 days post infection (dpi), and cultured for 24 h. Produced cytokines were measured by ELISA. Data represent mean±s.d. (n=5 from two individual experiments). ND, not detected. (c,d) CCR2WT and CCR2DTR mice were infected with C. rodentium and diphtheria toxin (DT; 10 ng g−1 body weight) was injected on 5, 7, 9, 11 dpi. Bacterial burden (c) and mouse mortality (d) are shown. Dots represent individual mice. Results are representative of two independent experiments. (e) Cytokines produced by LPMC at 8 dpi. Data represent mean±s.d. (n=3, representative of three independent experiments). (f) RORγtGFP/+ mice were infected with C. rodentium. LPMCs were isolated on 8 dpi and Th17 (CD3+CD4+RORγt+) and ILC3 (CD3−RORγt+) were purified by sorting. IL-22 mRNA expression was assessed by qPCR. Data represent mean±s.d. (n=3, representative of 2 independent experiments). (g,h) CCR2WT and CCR2DTR mice were infected with C. rodentium and DT was injected on days 5 and 7. LPMCs were isolated on 8 dpi, and cultured in the presence of heat-killed C. rodentium (MOI=10) for 16 h. IL-22 production in CD4− ILCs (Lin-Thy-1+CD3-CD4−), CD4+ ILCs (Lin-Thy-1+CD3−CD4+) and CD4+ T cells (Thy-1+CD3+CD4+) was assessed by flow cytometry (g), and absolute number of IL-22-producing T cells and ILCs are shown in h. (i) Rag1−/−CCR2WT and Rag1−/−CCR2DTR mice were infected with C. rodentium and DT was injected on days 5 and 7. LPMCs were isolated on 8 dpi and cultured for 24 h. Cytokines were measured by ELISA. Data represent mean±s.d. (n=4, representative of two independent experiments). (j) Mouse mortality of Rag1−/−CCR2WT and Rag1−/−CCR2DTR mice (n=5) infected with C. rodentium. DT was injected on day 5, 7, 9 and 11 post infection. NS, not significant, *P<0.05, **P<0.01, ***P<0.001 by Mann–Whitney U-test (a–c), Student's t-test (e,h,i) and Log-rank test (d,j).
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f1: Recruitment of CCR2+ monocytes activate ILCs during intestinal infection.(a) C. rodentium burden in faeces from Ccr2+/+ and Ccr2−/− mice. Data represent mean±s.d. (n=5, representative of two independent experiments). (b) Colonic lamina propria mononuclear cells (LPMCs) were isolated from at 0 and 12 days post infection (dpi), and cultured for 24 h. Produced cytokines were measured by ELISA. Data represent mean±s.d. (n=5 from two individual experiments). ND, not detected. (c,d) CCR2WT and CCR2DTR mice were infected with C. rodentium and diphtheria toxin (DT; 10 ng g−1 body weight) was injected on 5, 7, 9, 11 dpi. Bacterial burden (c) and mouse mortality (d) are shown. Dots represent individual mice. Results are representative of two independent experiments. (e) Cytokines produced by LPMC at 8 dpi. Data represent mean±s.d. (n=3, representative of three independent experiments). (f) RORγtGFP/+ mice were infected with C. rodentium. LPMCs were isolated on 8 dpi and Th17 (CD3+CD4+RORγt+) and ILC3 (CD3−RORγt+) were purified by sorting. IL-22 mRNA expression was assessed by qPCR. Data represent mean±s.d. (n=3, representative of 2 independent experiments). (g,h) CCR2WT and CCR2DTR mice were infected with C. rodentium and DT was injected on days 5 and 7. LPMCs were isolated on 8 dpi, and cultured in the presence of heat-killed C. rodentium (MOI=10) for 16 h. IL-22 production in CD4− ILCs (Lin-Thy-1+CD3-CD4−), CD4+ ILCs (Lin-Thy-1+CD3−CD4+) and CD4+ T cells (Thy-1+CD3+CD4+) was assessed by flow cytometry (g), and absolute number of IL-22-producing T cells and ILCs are shown in h. (i) Rag1−/−CCR2WT and Rag1−/−CCR2DTR mice were infected with C. rodentium and DT was injected on days 5 and 7. LPMCs were isolated on 8 dpi and cultured for 24 h. Cytokines were measured by ELISA. Data represent mean±s.d. (n=4, representative of two independent experiments). (j) Mouse mortality of Rag1−/−CCR2WT and Rag1−/−CCR2DTR mice (n=5) infected with C. rodentium. DT was injected on day 5, 7, 9 and 11 post infection. NS, not significant, *P<0.05, **P<0.01, ***P<0.001 by Mann–Whitney U-test (a–c), Student's t-test (e,h,i) and Log-rank test (d,j).

Mentions: To assess the role of recruited monocytes in the control of intestinal infection, Citrobacter rodentium was orally administered to either wild-type (WT) mice and mice deficient in CCR2, an essential chemokine receptor for emigration of Ly6Chi monocytes from the bone marrow (BM) and recruitment to the intestine21. Compared with WT mice, the clearance of C. rodentium was markedly impaired in Ccr2−/− mice on day 14 and 18 (Fig. 1a), in agreement with previous results9. The production of IFN-γ, IL-17A and IL-22 by colonic LP cells was reduced in Ccr2−/− mice on day 12 post infection (Fig. 1b). Consistent with impaired IL-22 production, expression of RegIIIβ and RegIIIγ, two known downstream targets of IL-22 (refs 11, 16), in intestinal epithelial cells was significantly decreased in Ccr2−/− mice (Supplementary Fig. 1). Together, these results suggested that the recruitment of monocytes to the intestine regulates the activation of lymphocyte responses in response to pathogen infection. To exclude the possibility that developmental defects contributed to the phenotype in Ccr2−/− mice, we used conditional CCR2-diphtheria toxin receptor (DTR)-depleter mice in which CCR2+ monocytes and monocyte-derived CCR2+ cells are selectively deleted after diphtheria toxin (DT) administration22. To assess the role of CCR2+ monocytes in C. rodentium infection, CCR2DTR/+ and WT littermates (CCR2WT) mice were infected with the pathogen followed by administration of DT. CCR2WT mice, where monocytes were unaffected after DT treatment, cleared the pathogen from the intestine, and none of the animals succumbed to infection (Fig. 1c,d). In contrast, CCR2DTR mice where monocytes were depleted displayed impaired eradication of C. rodentium on days 14 and 16, and ∼60% succumbed by day 21 post infection (Fig. 1c,d). As observed in Ccr2−/− mice, mucosal IFN-γ, IL-17A and IL-22 production was significantly reduced in CCR2DTR mice when compared with control CCR2WT littermates (Fig. 1e). IL-22 is produced by both T cells and ILCs, and plays a critical role in protecting the host against C. rodentium1116. To identify the cellular source of IL-22 during C. rodentium infection, Th17 cells (RORγt+CD3+CD4+) and ILC3s (RORγt+CD3−) were sorted from the intestines of C. rodentium-infected mice, and IL-22 expression in these lymphoid subsets was assessed by intracellular staining. ILC3 produced greater levels of IL-22 than Th17 cells (Fig. 1f). To determine the role of monocyte recruitment in the production of IL-22 by ILC3 cells during C. rodentium infection, CCR2WT and CCR2DTR mice were injected with DT after C. rodentium infection. Consistent with the results in Fig. 1f, mucosal ILCs (Lin−Thy-1+CD3-CD4− and Lin−Thy-1+CD3−CD4+) produced IL-22 on day 8 post infection (Fig. 1g). Notably, the production of IL-22 was blunted in CCR2DTR mice compared with control CCR2WT littermates (Fig. 1g). Likewise, the absolute number of IL-22-producing ILCs was significantly decreased in CCR2DTR mice, while IL-22-producing T cells exhibited a trend, but not a significant reduction in CCR2DTR mice (Fig. 1h). To further assess the role of monocytes in the activation of ILCs, we generated CCR2DTR mice in a Rag1−/− background in which the main source of IL-22 are expected to be CD3− ILCs. Consistently, LP cells from Rag1−/−CCR2WT mice produced robust amounts of IL-22 after C. rodentium infection, suggesting that ILCs are the major source of mucosal IL-22 in response to pathogen infection (Fig. 1i). The production of IL-22, IFNγ and IL-17A were blunted in Rag1−/−CCR2DTR mice injected with DT compared with Rag1−/−CCR2WT mice, indicating that monocytes are also critical for production of these cytokines in the absence of T cells (Fig. 1i). To determine the role of monocytes for host defence in the absence of T cells, we infected Rag1−/−CCR2DTR and Rag1−/− mice, and monitored mouse survival over time. The mortality of Rag1−/−CCR2DTR pretreated with DT was accelerated compared with Rag1−/−CCR2WT mice (Fig. 1j). Collectively, these results indicate that monocytes recruited to the intestine are critical for the activation of mucosal ILC3 and host defence during C. rodentium infection.


Intestinal macrophages arising from CCR2(+) monocytes control pathogen infection by activating innate lymphoid cells.

Seo SU, Kuffa P, Kitamoto S, Nagao-Kitamoto H, Rousseau J, Kim YG, Núñez G, Kamada N - Nat Commun (2015)

Recruitment of CCR2+ monocytes activate ILCs during intestinal infection.(a) C. rodentium burden in faeces from Ccr2+/+ and Ccr2−/− mice. Data represent mean±s.d. (n=5, representative of two independent experiments). (b) Colonic lamina propria mononuclear cells (LPMCs) were isolated from at 0 and 12 days post infection (dpi), and cultured for 24 h. Produced cytokines were measured by ELISA. Data represent mean±s.d. (n=5 from two individual experiments). ND, not detected. (c,d) CCR2WT and CCR2DTR mice were infected with C. rodentium and diphtheria toxin (DT; 10 ng g−1 body weight) was injected on 5, 7, 9, 11 dpi. Bacterial burden (c) and mouse mortality (d) are shown. Dots represent individual mice. Results are representative of two independent experiments. (e) Cytokines produced by LPMC at 8 dpi. Data represent mean±s.d. (n=3, representative of three independent experiments). (f) RORγtGFP/+ mice were infected with C. rodentium. LPMCs were isolated on 8 dpi and Th17 (CD3+CD4+RORγt+) and ILC3 (CD3−RORγt+) were purified by sorting. IL-22 mRNA expression was assessed by qPCR. Data represent mean±s.d. (n=3, representative of 2 independent experiments). (g,h) CCR2WT and CCR2DTR mice were infected with C. rodentium and DT was injected on days 5 and 7. LPMCs were isolated on 8 dpi, and cultured in the presence of heat-killed C. rodentium (MOI=10) for 16 h. IL-22 production in CD4− ILCs (Lin-Thy-1+CD3-CD4−), CD4+ ILCs (Lin-Thy-1+CD3−CD4+) and CD4+ T cells (Thy-1+CD3+CD4+) was assessed by flow cytometry (g), and absolute number of IL-22-producing T cells and ILCs are shown in h. (i) Rag1−/−CCR2WT and Rag1−/−CCR2DTR mice were infected with C. rodentium and DT was injected on days 5 and 7. LPMCs were isolated on 8 dpi and cultured for 24 h. Cytokines were measured by ELISA. Data represent mean±s.d. (n=4, representative of two independent experiments). (j) Mouse mortality of Rag1−/−CCR2WT and Rag1−/−CCR2DTR mice (n=5) infected with C. rodentium. DT was injected on day 5, 7, 9 and 11 post infection. NS, not significant, *P<0.05, **P<0.01, ***P<0.001 by Mann–Whitney U-test (a–c), Student's t-test (e,h,i) and Log-rank test (d,j).
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f1: Recruitment of CCR2+ monocytes activate ILCs during intestinal infection.(a) C. rodentium burden in faeces from Ccr2+/+ and Ccr2−/− mice. Data represent mean±s.d. (n=5, representative of two independent experiments). (b) Colonic lamina propria mononuclear cells (LPMCs) were isolated from at 0 and 12 days post infection (dpi), and cultured for 24 h. Produced cytokines were measured by ELISA. Data represent mean±s.d. (n=5 from two individual experiments). ND, not detected. (c,d) CCR2WT and CCR2DTR mice were infected with C. rodentium and diphtheria toxin (DT; 10 ng g−1 body weight) was injected on 5, 7, 9, 11 dpi. Bacterial burden (c) and mouse mortality (d) are shown. Dots represent individual mice. Results are representative of two independent experiments. (e) Cytokines produced by LPMC at 8 dpi. Data represent mean±s.d. (n=3, representative of three independent experiments). (f) RORγtGFP/+ mice were infected with C. rodentium. LPMCs were isolated on 8 dpi and Th17 (CD3+CD4+RORγt+) and ILC3 (CD3−RORγt+) were purified by sorting. IL-22 mRNA expression was assessed by qPCR. Data represent mean±s.d. (n=3, representative of 2 independent experiments). (g,h) CCR2WT and CCR2DTR mice were infected with C. rodentium and DT was injected on days 5 and 7. LPMCs were isolated on 8 dpi, and cultured in the presence of heat-killed C. rodentium (MOI=10) for 16 h. IL-22 production in CD4− ILCs (Lin-Thy-1+CD3-CD4−), CD4+ ILCs (Lin-Thy-1+CD3−CD4+) and CD4+ T cells (Thy-1+CD3+CD4+) was assessed by flow cytometry (g), and absolute number of IL-22-producing T cells and ILCs are shown in h. (i) Rag1−/−CCR2WT and Rag1−/−CCR2DTR mice were infected with C. rodentium and DT was injected on days 5 and 7. LPMCs were isolated on 8 dpi and cultured for 24 h. Cytokines were measured by ELISA. Data represent mean±s.d. (n=4, representative of two independent experiments). (j) Mouse mortality of Rag1−/−CCR2WT and Rag1−/−CCR2DTR mice (n=5) infected with C. rodentium. DT was injected on day 5, 7, 9 and 11 post infection. NS, not significant, *P<0.05, **P<0.01, ***P<0.001 by Mann–Whitney U-test (a–c), Student's t-test (e,h,i) and Log-rank test (d,j).
Mentions: To assess the role of recruited monocytes in the control of intestinal infection, Citrobacter rodentium was orally administered to either wild-type (WT) mice and mice deficient in CCR2, an essential chemokine receptor for emigration of Ly6Chi monocytes from the bone marrow (BM) and recruitment to the intestine21. Compared with WT mice, the clearance of C. rodentium was markedly impaired in Ccr2−/− mice on day 14 and 18 (Fig. 1a), in agreement with previous results9. The production of IFN-γ, IL-17A and IL-22 by colonic LP cells was reduced in Ccr2−/− mice on day 12 post infection (Fig. 1b). Consistent with impaired IL-22 production, expression of RegIIIβ and RegIIIγ, two known downstream targets of IL-22 (refs 11, 16), in intestinal epithelial cells was significantly decreased in Ccr2−/− mice (Supplementary Fig. 1). Together, these results suggested that the recruitment of monocytes to the intestine regulates the activation of lymphocyte responses in response to pathogen infection. To exclude the possibility that developmental defects contributed to the phenotype in Ccr2−/− mice, we used conditional CCR2-diphtheria toxin receptor (DTR)-depleter mice in which CCR2+ monocytes and monocyte-derived CCR2+ cells are selectively deleted after diphtheria toxin (DT) administration22. To assess the role of CCR2+ monocytes in C. rodentium infection, CCR2DTR/+ and WT littermates (CCR2WT) mice were infected with the pathogen followed by administration of DT. CCR2WT mice, where monocytes were unaffected after DT treatment, cleared the pathogen from the intestine, and none of the animals succumbed to infection (Fig. 1c,d). In contrast, CCR2DTR mice where monocytes were depleted displayed impaired eradication of C. rodentium on days 14 and 16, and ∼60% succumbed by day 21 post infection (Fig. 1c,d). As observed in Ccr2−/− mice, mucosal IFN-γ, IL-17A and IL-22 production was significantly reduced in CCR2DTR mice when compared with control CCR2WT littermates (Fig. 1e). IL-22 is produced by both T cells and ILCs, and plays a critical role in protecting the host against C. rodentium1116. To identify the cellular source of IL-22 during C. rodentium infection, Th17 cells (RORγt+CD3+CD4+) and ILC3s (RORγt+CD3−) were sorted from the intestines of C. rodentium-infected mice, and IL-22 expression in these lymphoid subsets was assessed by intracellular staining. ILC3 produced greater levels of IL-22 than Th17 cells (Fig. 1f). To determine the role of monocyte recruitment in the production of IL-22 by ILC3 cells during C. rodentium infection, CCR2WT and CCR2DTR mice were injected with DT after C. rodentium infection. Consistent with the results in Fig. 1f, mucosal ILCs (Lin−Thy-1+CD3-CD4− and Lin−Thy-1+CD3−CD4+) produced IL-22 on day 8 post infection (Fig. 1g). Notably, the production of IL-22 was blunted in CCR2DTR mice compared with control CCR2WT littermates (Fig. 1g). Likewise, the absolute number of IL-22-producing ILCs was significantly decreased in CCR2DTR mice, while IL-22-producing T cells exhibited a trend, but not a significant reduction in CCR2DTR mice (Fig. 1h). To further assess the role of monocytes in the activation of ILCs, we generated CCR2DTR mice in a Rag1−/− background in which the main source of IL-22 are expected to be CD3− ILCs. Consistently, LP cells from Rag1−/−CCR2WT mice produced robust amounts of IL-22 after C. rodentium infection, suggesting that ILCs are the major source of mucosal IL-22 in response to pathogen infection (Fig. 1i). The production of IL-22, IFNγ and IL-17A were blunted in Rag1−/−CCR2DTR mice injected with DT compared with Rag1−/−CCR2WT mice, indicating that monocytes are also critical for production of these cytokines in the absence of T cells (Fig. 1i). To determine the role of monocytes for host defence in the absence of T cells, we infected Rag1−/−CCR2DTR and Rag1−/− mice, and monitored mouse survival over time. The mortality of Rag1−/−CCR2DTR pretreated with DT was accelerated compared with Rag1−/−CCR2WT mice (Fig. 1j). Collectively, these results indicate that monocytes recruited to the intestine are critical for the activation of mucosal ILC3 and host defence during C. rodentium infection.

Bottom Line: Unlike resident intestinal MPs, de novo differentiated MPs are phenotypically pro-inflammatory and produce robust amounts of IL-1β (interleukin-1β) through the non-canonical caspase-11 inflammasome.Intestinal MPs from infected mice elicit the activation of RORγt(+) group 3 innate lymphoid cells (ILC3) in an IL-1β-dependent manner.Deletion of IL-1β in blood monocytes blunts the production of IL-22 by ILC3 and increases the susceptibility to infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, 1500 E Medical Center Dr Ann Arbor, Michigan 48109, USA.

ABSTRACT
Monocytes play a crucial role in antimicrobial host defence, but the mechanisms by which they protect the host during intestinal infection remains poorly understood. Here we show that depletion of CCR2(+) monocytes results in impaired clearance of the intestinal pathogen Citrobacter rodentium. After infection, the de novo recruited CCR2(+) monocytes give rise to CD11c(+)CD11b(+)F4/80(+)CD103(-) intestinal macrophages (MPs) within the lamina propria. Unlike resident intestinal MPs, de novo differentiated MPs are phenotypically pro-inflammatory and produce robust amounts of IL-1β (interleukin-1β) through the non-canonical caspase-11 inflammasome. Intestinal MPs from infected mice elicit the activation of RORγt(+) group 3 innate lymphoid cells (ILC3) in an IL-1β-dependent manner. Deletion of IL-1β in blood monocytes blunts the production of IL-22 by ILC3 and increases the susceptibility to infection. Collectively, these studies highlight a critical role of de novo differentiated monocyte-derived intestinal MPs in ILC3-mediated host defence against intestinal infection.

No MeSH data available.


Related in: MedlinePlus