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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

Activation of RORγt+ ILCs requires IL-1β produced by monocyte-derived intestinal macrophages during C. rodentium (C. rod) infection.(a) CD3-RORγt+ ILCs from uninfected RORγtGFP/+ reporter mice and MP1 cells from C. rodentium-infected CD115GFP were isolated. ILCs and MP1 cells (1 × 106 cells per ml) were cultured alone or co-cultured with or without stimulation with heat-killed C. rodentium (MOI=10) for 24 h. Neutralizing antibodies for IL-23 (10 μg ml−1), IL-1β (10 μg ml−1), or isotype controls were used to block cytokines. Data are given as mean±s.d. (n=4). *P<0.05; NS, not significant by Dunnett's test (compared with isotype control). (b) Schematic illustrating experimental protocol of mixed BM chimera for IL-1β monocyte/macrophage-conditional depletion. Lethally irradiated C57BL/6 recipient mice were reconstituted with mixed bone-marrows from Ccr2WT or Ccr2DTR and Il1b−/− mice (1:1 ratio) for 8 weeks. After 8 weeks, mice were infected with C. rodentium, and CCR2+ monocytes and monocyte-derived MP1 cells were depleted by DT injection (10 ng g−1 body weight) on days 5, 7, 9 and 11 post infection. (c) Survival of chimeric mice infected with C. rodentium (n=7). **P<0.01 by Log-rank test. Results are pooled data of two independent experiments with 3–4 mice each. (d) CCR2WT/Il1b−/− control chimera (IL-1βWT) and CCR2DTR/Il1b−/− chimera (IL-1βΔMo/MP) were infected with C. rodentium and DT was injected (10 ng g−1 body weight) on days 5 and 7. LPMCs were isolated on day 8 post infection. 2 × 106 cells per ml LPMCs were cultured with or without stimulation with 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. (n=4, representative of two independent experiments). *P<0.05; **P<0.01; ***P<0.001 by Student's t-test. (e) Isolated LPMCs in d 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.
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f4: Activation of RORγt+ ILCs requires IL-1β produced by monocyte-derived intestinal macrophages during C. rodentium (C. rod) infection.(a) CD3-RORγt+ ILCs from uninfected RORγtGFP/+ reporter mice and MP1 cells from C. rodentium-infected CD115GFP were isolated. ILCs and MP1 cells (1 × 106 cells per ml) were cultured alone or co-cultured with or without stimulation with heat-killed C. rodentium (MOI=10) for 24 h. Neutralizing antibodies for IL-23 (10 μg ml−1), IL-1β (10 μg ml−1), or isotype controls were used to block cytokines. Data are given as mean±s.d. (n=4). *P<0.05; NS, not significant by Dunnett's test (compared with isotype control). (b) Schematic illustrating experimental protocol of mixed BM chimera for IL-1β monocyte/macrophage-conditional depletion. Lethally irradiated C57BL/6 recipient mice were reconstituted with mixed bone-marrows from Ccr2WT or Ccr2DTR and Il1b−/− mice (1:1 ratio) for 8 weeks. After 8 weeks, mice were infected with C. rodentium, and CCR2+ monocytes and monocyte-derived MP1 cells were depleted by DT injection (10 ng g−1 body weight) on days 5, 7, 9 and 11 post infection. (c) Survival of chimeric mice infected with C. rodentium (n=7). **P<0.01 by Log-rank test. Results are pooled data of two independent experiments with 3–4 mice each. (d) CCR2WT/Il1b−/− control chimera (IL-1βWT) and CCR2DTR/Il1b−/− chimera (IL-1βΔMo/MP) were infected with C. rodentium and DT was injected (10 ng g−1 body weight) on days 5 and 7. LPMCs were isolated on day 8 post infection. 2 × 106 cells per ml LPMCs were cultured with or without stimulation with 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. (n=4, representative of two independent experiments). *P<0.05; **P<0.01; ***P<0.001 by Student's t-test. (e) Isolated LPMCs in d 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.

Mentions: Given that IL-1β and IL-23 are critical activators of ILCs2027, we hypothesized that the monocyte-derived MP1 subset activates ILCs. We first tested the role of IL-1β and IL-23 in the activation of ILC3s isolated from the intestine. RORγt+CD3+CD4+ Th17 cells and RORγt+CD3− ILC3s were isolated from the intestinal LP. About half of the ILC3s expressed NKp46, a marker found specifically on ILC3 but not on Th17 cells (Supplementary Fig. 5a). Purified Th17 cells and ILC3s were then stimulated with recombinant IL-1β, IL-23 and IL-1β plus IL-23. Consistent with previous reports2027, both IL-23 and IL-1β induced IL-22 secretion by ILC3s (Supplementary Fig. 5b). Moreover, these cytokines synergistically induced IL-22 production in ILC3s (Supplementary Fig. 5b). Additionally, co-stimulation with IL-23 and IL-1β induced ILC3s to produce robust amounts of IFN-γ, while stimulation with IL-23 or IL-1β alone did not (Supplementary Fig. 5b). In contrast, Th17 cells produced higher amounts of IL-17A than ILC3s but minimal amounts of IFN-γ and IL-22, even after dual stimulation with IL-23 and IL-1β (Supplementary Fig. 5b). To address whether the monocyte-derived intestinal MP1 cells can activate ILCs through IL-23 and IL-1β, RORγt+CD3− ILC3 and the MP1 subset were purified from the intestine, and then co-cultured ex vivo. Individually cultured ILC3 and MP1 cells did not produce IL-22, even after stimulation with heat-killed C. rodentium (Fig. 4a). However, co-culture of ILC and MP1 induced IL-22 production, which was further enhanced in the presence of C. rodentium (Fig. 4a). While ILC3-MP1 co-culture did not promote IFN-γ or IL-17A production, stimulation with C. rodentium robustly induced IFN-γ production by ILC3s in the presence of MP1 cells (Fig. 4a). Notably, the production of both IL-22 and IFN-γ by ILC3s co-cultured with MP1 was diminished by neutralizing antibody against IL-1β, but not IL-23p19 (Fig. 4a). These results demonstrate that IL-1β produced by monocyte-derived MPs is a key inducer of IL-22 and IFN-γ production by ILC3.


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)

Activation of RORγt+ ILCs requires IL-1β produced by monocyte-derived intestinal macrophages during C. rodentium (C. rod) infection.(a) CD3-RORγt+ ILCs from uninfected RORγtGFP/+ reporter mice and MP1 cells from C. rodentium-infected CD115GFP were isolated. ILCs and MP1 cells (1 × 106 cells per ml) were cultured alone or co-cultured with or without stimulation with heat-killed C. rodentium (MOI=10) for 24 h. Neutralizing antibodies for IL-23 (10 μg ml−1), IL-1β (10 μg ml−1), or isotype controls were used to block cytokines. Data are given as mean±s.d. (n=4). *P<0.05; NS, not significant by Dunnett's test (compared with isotype control). (b) Schematic illustrating experimental protocol of mixed BM chimera for IL-1β monocyte/macrophage-conditional depletion. Lethally irradiated C57BL/6 recipient mice were reconstituted with mixed bone-marrows from Ccr2WT or Ccr2DTR and Il1b−/− mice (1:1 ratio) for 8 weeks. After 8 weeks, mice were infected with C. rodentium, and CCR2+ monocytes and monocyte-derived MP1 cells were depleted by DT injection (10 ng g−1 body weight) on days 5, 7, 9 and 11 post infection. (c) Survival of chimeric mice infected with C. rodentium (n=7). **P<0.01 by Log-rank test. Results are pooled data of two independent experiments with 3–4 mice each. (d) CCR2WT/Il1b−/− control chimera (IL-1βWT) and CCR2DTR/Il1b−/− chimera (IL-1βΔMo/MP) were infected with C. rodentium and DT was injected (10 ng g−1 body weight) on days 5 and 7. LPMCs were isolated on day 8 post infection. 2 × 106 cells per ml LPMCs were cultured with or without stimulation with 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. (n=4, representative of two independent experiments). *P<0.05; **P<0.01; ***P<0.001 by Student's t-test. (e) Isolated LPMCs in d 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.
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f4: Activation of RORγt+ ILCs requires IL-1β produced by monocyte-derived intestinal macrophages during C. rodentium (C. rod) infection.(a) CD3-RORγt+ ILCs from uninfected RORγtGFP/+ reporter mice and MP1 cells from C. rodentium-infected CD115GFP were isolated. ILCs and MP1 cells (1 × 106 cells per ml) were cultured alone or co-cultured with or without stimulation with heat-killed C. rodentium (MOI=10) for 24 h. Neutralizing antibodies for IL-23 (10 μg ml−1), IL-1β (10 μg ml−1), or isotype controls were used to block cytokines. Data are given as mean±s.d. (n=4). *P<0.05; NS, not significant by Dunnett's test (compared with isotype control). (b) Schematic illustrating experimental protocol of mixed BM chimera for IL-1β monocyte/macrophage-conditional depletion. Lethally irradiated C57BL/6 recipient mice were reconstituted with mixed bone-marrows from Ccr2WT or Ccr2DTR and Il1b−/− mice (1:1 ratio) for 8 weeks. After 8 weeks, mice were infected with C. rodentium, and CCR2+ monocytes and monocyte-derived MP1 cells were depleted by DT injection (10 ng g−1 body weight) on days 5, 7, 9 and 11 post infection. (c) Survival of chimeric mice infected with C. rodentium (n=7). **P<0.01 by Log-rank test. Results are pooled data of two independent experiments with 3–4 mice each. (d) CCR2WT/Il1b−/− control chimera (IL-1βWT) and CCR2DTR/Il1b−/− chimera (IL-1βΔMo/MP) were infected with C. rodentium and DT was injected (10 ng g−1 body weight) on days 5 and 7. LPMCs were isolated on day 8 post infection. 2 × 106 cells per ml LPMCs were cultured with or without stimulation with 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. (n=4, representative of two independent experiments). *P<0.05; **P<0.01; ***P<0.001 by Student's t-test. (e) Isolated LPMCs in d 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.
Mentions: Given that IL-1β and IL-23 are critical activators of ILCs2027, we hypothesized that the monocyte-derived MP1 subset activates ILCs. We first tested the role of IL-1β and IL-23 in the activation of ILC3s isolated from the intestine. RORγt+CD3+CD4+ Th17 cells and RORγt+CD3− ILC3s were isolated from the intestinal LP. About half of the ILC3s expressed NKp46, a marker found specifically on ILC3 but not on Th17 cells (Supplementary Fig. 5a). Purified Th17 cells and ILC3s were then stimulated with recombinant IL-1β, IL-23 and IL-1β plus IL-23. Consistent with previous reports2027, both IL-23 and IL-1β induced IL-22 secretion by ILC3s (Supplementary Fig. 5b). Moreover, these cytokines synergistically induced IL-22 production in ILC3s (Supplementary Fig. 5b). Additionally, co-stimulation with IL-23 and IL-1β induced ILC3s to produce robust amounts of IFN-γ, while stimulation with IL-23 or IL-1β alone did not (Supplementary Fig. 5b). In contrast, Th17 cells produced higher amounts of IL-17A than ILC3s but minimal amounts of IFN-γ and IL-22, even after dual stimulation with IL-23 and IL-1β (Supplementary Fig. 5b). To address whether the monocyte-derived intestinal MP1 cells can activate ILCs through IL-23 and IL-1β, RORγt+CD3− ILC3 and the MP1 subset were purified from the intestine, and then co-cultured ex vivo. Individually cultured ILC3 and MP1 cells did not produce IL-22, even after stimulation with heat-killed C. rodentium (Fig. 4a). However, co-culture of ILC and MP1 induced IL-22 production, which was further enhanced in the presence of C. rodentium (Fig. 4a). While ILC3-MP1 co-culture did not promote IFN-γ or IL-17A production, stimulation with C. rodentium robustly induced IFN-γ production by ILC3s in the presence of MP1 cells (Fig. 4a). Notably, the production of both IL-22 and IFN-γ by ILC3s co-cultured with MP1 was diminished by neutralizing antibody against IL-1β, but not IL-23p19 (Fig. 4a). These results demonstrate that IL-1β produced by monocyte-derived MPs is a key inducer of IL-22 and IFN-γ production by ILC3.

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