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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.

No MeSH data available.


Related in: MedlinePlus

Citrobacter rodentium elicits IL-1β production by intestinal macrophages via caspase-11 inflammasome.(a) BM-derived macrophages (BMDMs) were obtained from WT, Nlrp3−/−, Casp11−/− mice and stimulated with C. rodentium (C. rod) or Salmonella (Sal; MOI=25) for 1 h without antibiotics and then cultured additional 17 h in the presence of 100 μg ml−1 gentamicin. Cytokines in the culture supernatant were analysed by ELISA. Data are given as mean±s.d. (n=3, representative of three independent experiments). (b) WT, Nlrp3−/− and Casp11−/− mice were infected with C. rodentium. On day 8 post infection, LPMCs were isolated from the infected mice, and 2 × 106 cells ml−1 LPMCs were cultured in the presence of heat-killed C. rodentium (MOI=10) for 24 h. Cytokines in the culture supernatant were analysed by ELISA. Data are given as mean±s.d. of 3 independent experiments. *P<0.05; **P<0.01; NS, not significant by Bonferroni test. (c) Isolated LPMCs in b were 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-) and CD4+ ILCs (Lin-Thy-1+CD3-CD4+) was assessed by flow cytometry. Data are representative of four individual mice. (d) LPMCs were isolated from uninfected and C. rodentium-infected (day 8 post infection) WT and Casp11−/− mice and cultured in the presence of heat-killed C. rodentium (MOI=10) for 24 h. IL-22 in the culture supernatant was analysed by ELISA. Data are given as mean±s.e.m (n=4–6). **P<0.01; ***P<0.001; NS, not significant by Bonferroni test. (e) CD45+MHC-II+CD11b+CD11c+CD103−Gr-1− MP1 subset was sorted from naive WT mice and C. rodentium-infected (day 8) WT and Casp11−/− mice. 2 × 105 of MP1 cells were loaded with SDS–polyacrylamide gel electrophoresis, and blotted with anti-mouse-caspase-11 antibody. As positive and negative controls, BMDMs from WT and Casp11−/− mice with or without LPS priming (6 h) were used. The original gel images are shown in Supplementary Fig. 12.
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f5: Citrobacter rodentium elicits IL-1β production by intestinal macrophages via caspase-11 inflammasome.(a) BM-derived macrophages (BMDMs) were obtained from WT, Nlrp3−/−, Casp11−/− mice and stimulated with C. rodentium (C. rod) or Salmonella (Sal; MOI=25) for 1 h without antibiotics and then cultured additional 17 h in the presence of 100 μg ml−1 gentamicin. Cytokines in the culture supernatant were analysed by ELISA. Data are given as mean±s.d. (n=3, representative of three independent experiments). (b) WT, Nlrp3−/− and Casp11−/− mice were infected with C. rodentium. On day 8 post infection, LPMCs were isolated from the infected mice, and 2 × 106 cells ml−1 LPMCs were cultured in the presence of heat-killed C. rodentium (MOI=10) for 24 h. Cytokines in the culture supernatant were analysed by ELISA. Data are given as mean±s.d. of 3 independent experiments. *P<0.05; **P<0.01; NS, not significant by Bonferroni test. (c) Isolated LPMCs in b were 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-) and CD4+ ILCs (Lin-Thy-1+CD3-CD4+) was assessed by flow cytometry. Data are representative of four individual mice. (d) LPMCs were isolated from uninfected and C. rodentium-infected (day 8 post infection) WT and Casp11−/− mice and cultured in the presence of heat-killed C. rodentium (MOI=10) for 24 h. IL-22 in the culture supernatant was analysed by ELISA. Data are given as mean±s.e.m (n=4–6). **P<0.01; ***P<0.001; NS, not significant by Bonferroni test. (e) CD45+MHC-II+CD11b+CD11c+CD103−Gr-1− MP1 subset was sorted from naive WT mice and C. rodentium-infected (day 8) WT and Casp11−/− mice. 2 × 105 of MP1 cells were loaded with SDS–polyacrylamide gel electrophoresis, and blotted with anti-mouse-caspase-11 antibody. As positive and negative controls, BMDMs from WT and Casp11−/− mice with or without LPS priming (6 h) were used. The original gel images are shown in Supplementary Fig. 12.

Mentions: Given that the intestinal MP1 subset from infected mice produces significant amounts of IL-1β, C. rodentium may be capable of activating an inflammasome during infection (Fig. 3). To delineate the mechanism by which monocyte-derived MP1 cells acquire the ability to produce IL-1β during C. rodentium infection, we first investigated the expression of inflammasome proteins in intestinal mononuclear phagocytes. The expression of Nlrp3, Nlrc4, Casp1 and Casp11 mRNAs were higher in MP1 than in DC1 cells, even in the steady state (Supplementary Fig. 8), suggesting that expression of inflammasome proteins may contribute to the enhanced production of IL-1β by the MP1 subset. To identify which inflammasome mediates C. rodentium-induced IL-1β production in MPs, BM-derived MPs (BMDMs) were stimulated with C. rodentium, and IL-1β production was measured in the culture supernatants. C. rodentium-induced IL-1β and TNF-α in BMDMs (Fig. 5a). IL-1β levels induced by C. rodentium were dramatically reduced in BMDMs derived from Nlrp3−/− or Casp11−/− mice, while TNF-α production was unaffected (Fig. 5a). In contrast, Salmonella-induced IL-1β, which has been shown to be dependent on the NLRC4 inflammasome28, was intact in Nlrp3−/− or Casp11−/− BMDMs (Fig. 5a). Given that caspase-11 acts upstream of the NLRP3 inflammasome29, these results indicated that C. rodentium may induce IL-1β production via caspase-11-mediated non-canonical inflammasome activation. To address the role of caspase-11 in IL-1β production in vivo, Casp11−/− and Nlrp3−/− mice were infected with C. rodentium, and the production of cytokines by LP cells was evaluated ex vivo in the absence and presence of pathogen stimulation. Production of IL-1β, TNF-α and IL-6 by LP cells was enhanced by stimulation of LP cells with heat-killed C. rodentium (Fig. 5b). Importantly, the production IL-1β, but not TNF-α or IL-6, by LP cells was impaired in LP cells from infected Casp11−/− and Nlrp3−/− mice when compared with WT mice (Fig. 5b). To address the link between caspase-11 and mucosal ILC activation, we next analysed IL-22 production by colonic ILCs during C. rodentium infection in Casp11−/− mice. Consistent with our results, IL-22 production by ILCs was significantly impaired in Casp11−/− mice (Fig. 5c). Since IL-22 induction was compromised in C. rodentium-infected Casp11−/− mice, but not in naive Casp11−/− mice (Fig. 5d), we examined the expression of caspase-11 in colonic MPs isolated from naive and C. rodentium-infected WT mice. Notably, the expression of caspase-11 was low or undetected in naive mice, but was clearly detected in colonic MPs of C. rodentium-infected mice (Fig. 5e). These results indicate that C. rodentium infection induces caspase-11 expression in intestinal macrophages, and caspase-11 contributes to the production of IL-1β by MPs, which promotes mucosal defence by activating ILCs.


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)

Citrobacter rodentium elicits IL-1β production by intestinal macrophages via caspase-11 inflammasome.(a) BM-derived macrophages (BMDMs) were obtained from WT, Nlrp3−/−, Casp11−/− mice and stimulated with C. rodentium (C. rod) or Salmonella (Sal; MOI=25) for 1 h without antibiotics and then cultured additional 17 h in the presence of 100 μg ml−1 gentamicin. Cytokines in the culture supernatant were analysed by ELISA. Data are given as mean±s.d. (n=3, representative of three independent experiments). (b) WT, Nlrp3−/− and Casp11−/− mice were infected with C. rodentium. On day 8 post infection, LPMCs were isolated from the infected mice, and 2 × 106 cells ml−1 LPMCs were cultured in the presence of heat-killed C. rodentium (MOI=10) for 24 h. Cytokines in the culture supernatant were analysed by ELISA. Data are given as mean±s.d. of 3 independent experiments. *P<0.05; **P<0.01; NS, not significant by Bonferroni test. (c) Isolated LPMCs in b were 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-) and CD4+ ILCs (Lin-Thy-1+CD3-CD4+) was assessed by flow cytometry. Data are representative of four individual mice. (d) LPMCs were isolated from uninfected and C. rodentium-infected (day 8 post infection) WT and Casp11−/− mice and cultured in the presence of heat-killed C. rodentium (MOI=10) for 24 h. IL-22 in the culture supernatant was analysed by ELISA. Data are given as mean±s.e.m (n=4–6). **P<0.01; ***P<0.001; NS, not significant by Bonferroni test. (e) CD45+MHC-II+CD11b+CD11c+CD103−Gr-1− MP1 subset was sorted from naive WT mice and C. rodentium-infected (day 8) WT and Casp11−/− mice. 2 × 105 of MP1 cells were loaded with SDS–polyacrylamide gel electrophoresis, and blotted with anti-mouse-caspase-11 antibody. As positive and negative controls, BMDMs from WT and Casp11−/− mice with or without LPS priming (6 h) were used. The original gel images are shown in Supplementary Fig. 12.
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f5: Citrobacter rodentium elicits IL-1β production by intestinal macrophages via caspase-11 inflammasome.(a) BM-derived macrophages (BMDMs) were obtained from WT, Nlrp3−/−, Casp11−/− mice and stimulated with C. rodentium (C. rod) or Salmonella (Sal; MOI=25) for 1 h without antibiotics and then cultured additional 17 h in the presence of 100 μg ml−1 gentamicin. Cytokines in the culture supernatant were analysed by ELISA. Data are given as mean±s.d. (n=3, representative of three independent experiments). (b) WT, Nlrp3−/− and Casp11−/− mice were infected with C. rodentium. On day 8 post infection, LPMCs were isolated from the infected mice, and 2 × 106 cells ml−1 LPMCs were cultured in the presence of heat-killed C. rodentium (MOI=10) for 24 h. Cytokines in the culture supernatant were analysed by ELISA. Data are given as mean±s.d. of 3 independent experiments. *P<0.05; **P<0.01; NS, not significant by Bonferroni test. (c) Isolated LPMCs in b were 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-) and CD4+ ILCs (Lin-Thy-1+CD3-CD4+) was assessed by flow cytometry. Data are representative of four individual mice. (d) LPMCs were isolated from uninfected and C. rodentium-infected (day 8 post infection) WT and Casp11−/− mice and cultured in the presence of heat-killed C. rodentium (MOI=10) for 24 h. IL-22 in the culture supernatant was analysed by ELISA. Data are given as mean±s.e.m (n=4–6). **P<0.01; ***P<0.001; NS, not significant by Bonferroni test. (e) CD45+MHC-II+CD11b+CD11c+CD103−Gr-1− MP1 subset was sorted from naive WT mice and C. rodentium-infected (day 8) WT and Casp11−/− mice. 2 × 105 of MP1 cells were loaded with SDS–polyacrylamide gel electrophoresis, and blotted with anti-mouse-caspase-11 antibody. As positive and negative controls, BMDMs from WT and Casp11−/− mice with or without LPS priming (6 h) were used. The original gel images are shown in Supplementary Fig. 12.
Mentions: Given that the intestinal MP1 subset from infected mice produces significant amounts of IL-1β, C. rodentium may be capable of activating an inflammasome during infection (Fig. 3). To delineate the mechanism by which monocyte-derived MP1 cells acquire the ability to produce IL-1β during C. rodentium infection, we first investigated the expression of inflammasome proteins in intestinal mononuclear phagocytes. The expression of Nlrp3, Nlrc4, Casp1 and Casp11 mRNAs were higher in MP1 than in DC1 cells, even in the steady state (Supplementary Fig. 8), suggesting that expression of inflammasome proteins may contribute to the enhanced production of IL-1β by the MP1 subset. To identify which inflammasome mediates C. rodentium-induced IL-1β production in MPs, BM-derived MPs (BMDMs) were stimulated with C. rodentium, and IL-1β production was measured in the culture supernatants. C. rodentium-induced IL-1β and TNF-α in BMDMs (Fig. 5a). IL-1β levels induced by C. rodentium were dramatically reduced in BMDMs derived from Nlrp3−/− or Casp11−/− mice, while TNF-α production was unaffected (Fig. 5a). In contrast, Salmonella-induced IL-1β, which has been shown to be dependent on the NLRC4 inflammasome28, was intact in Nlrp3−/− or Casp11−/− BMDMs (Fig. 5a). Given that caspase-11 acts upstream of the NLRP3 inflammasome29, these results indicated that C. rodentium may induce IL-1β production via caspase-11-mediated non-canonical inflammasome activation. To address the role of caspase-11 in IL-1β production in vivo, Casp11−/− and Nlrp3−/− mice were infected with C. rodentium, and the production of cytokines by LP cells was evaluated ex vivo in the absence and presence of pathogen stimulation. Production of IL-1β, TNF-α and IL-6 by LP cells was enhanced by stimulation of LP cells with heat-killed C. rodentium (Fig. 5b). Importantly, the production IL-1β, but not TNF-α or IL-6, by LP cells was impaired in LP cells from infected Casp11−/− and Nlrp3−/− mice when compared with WT mice (Fig. 5b). To address the link between caspase-11 and mucosal ILC activation, we next analysed IL-22 production by colonic ILCs during C. rodentium infection in Casp11−/− mice. Consistent with our results, IL-22 production by ILCs was significantly impaired in Casp11−/− mice (Fig. 5c). Since IL-22 induction was compromised in C. rodentium-infected Casp11−/− mice, but not in naive Casp11−/− mice (Fig. 5d), we examined the expression of caspase-11 in colonic MPs isolated from naive and C. rodentium-infected WT mice. Notably, the expression of caspase-11 was low or undetected in naive mice, but was clearly detected in colonic MPs of C. rodentium-infected mice (Fig. 5e). These results indicate that C. rodentium infection induces caspase-11 expression in intestinal macrophages, and caspase-11 contributes to the production of IL-1β by MPs, which promotes mucosal defence by activating ILCs.

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