CSF-1R+ Macrophages Control the Gut Microbiome-Enhanced Liver Invariant NKT Function through IL-18

Abstract

The gut microbiome is an important modulator of the host immune system. In this study, we found that altering the gut microbiome by oral vancomycin increases liver invariant NKT (iNKT) cell function. Enhanced iNKT cytokine production and activation marker expression were observed in vancomycin-treated mice following both Ag-specific and Ag-independent in vivo iNKT stimulations, with a more prominent effect in the liver than in the spleen. Fecal transplantation studies demonstrated that the iNKT functional regulation is mediated by altering the gut microbiome but uncoupled from the modulation of iNKT cell population size. Interestingly, when stimulated in vitro, iNKT cells from vancomycin-treated mice did not show increased activation, suggesting an indirect regulation. iNKT cells expressed high levels of IL-18 receptor, and vancomycin increased the expression of IL-18 in the liver. Blocking IL-18 by neutralizing Ab or using genetically deficient mice attenuated the enhanced iNKT activation. Liver macrophages were identified as a major source of IL-18. General macrophage depletion by clodronate abolished this iNKT activation. Using anti-CSF-1R depletion or LyzCrexCSF-1RLsL-DTR mice identified CSF-1R+ macrophages as a critical modulator of iNKT function. Vancomycin treatment had no effect on iNKT cell function in vivo in IL-18 knockout macrophage reconstituted mice. Together, our results demonstrate that the gut microbiome controls liver iNKT function via regulating CSF-1R+ macrophages to produce IL-18.

Figure.1. Oral vancomycin treatment increases liver iNKT activity.

(A-F) C57BL/6 mice (age 7 to 10 weeks) were kept on vancomycin or control H2O for 3 weeks. 10⁶ αGalCer-loaded EL4 cells plus brefeldin A (250ug/mouse) were injected i.v. in mice. Two hours after injection, mice were euthanized, and liver mononuclear cells were prepared for flow cytometry. (A) Experimental setup. (B) Representative IFNγ and IL-4 staining of liver iNKT cells gated on TCRβ⁺CD1d-tetramer⁺. Liver iNKT cells IFNγ⁺% (C), IL-4⁺% (D), composition of iNKT cells expressing IFNγ or IL-4 (E), and CD69 MFI (F) are shown. Results are presented as mean+/− SEM of two independent experiments. n=8, p<0.05, student t test. (G-I). Concanavalin A (ConA) 50 ug/mouse plus brefeldin A (250ug/mouse) was injected into mice kept on vancomycin or H2O. Two hours later, liver mononuclear cells were prepared. (G) Experimental setup. Liver iNKT cells IFNγ⁺% (H) and CD69 MFI (I) are shown. Results are presented as mean+/− SEM of two independent experiments. n=9, p<0.05, student t test.

Figure.2. Vancomycin altered gut microbiota causes the enhanced iNKT function.

(A-C) Male germ-free BALB/c mice (age ~6 weeks) were kept under germ-free condition or transferred to a SPF facility to colonize commensal microbiome (GF->SPF). Four weeks later, the GF->SPF mice received vancomycin or control H2O for 3 weeks. iNKT cell stimulation was performed by i.v. injection of 10⁶ αGalCer-loaded A20 cells plus brefeldin A (250ug/mouse). Two hours later, liver mononuclear cells were prepared for flow cytometry. (A) Experimental setup. Liver iNKT cells IFNγ⁺ % (B) and IL-4⁺ % (C) are shown. Results are presented as mean+/− SEM of two independent experiments. n=8 for GF, 8 for GF->SPF H2O, 4 for GF->SPF Vanco, p<0.05, one-way ANOVA. (D-F) Germ-free BALB/c mice (age 7~10 weeks) received vancomycin in their drinking water or FMT of feces from vancomycin treated SPF mice by oral gavage. Three weeks later mice were challenged by i.v. injection of 10⁶ αGalCer-loaded A20 cells plus brefeldin A (250ug/mouse). (D) Experimental setup. Liver iNKT cells IFNγ⁺% (E) and IL-4⁺% (F) are shown. Results are presented as mean+/− SEM of one experiment. n=5, p<0.05, one-way ANOVA.

Figure.3. IL-18 controls gut microbiota dependent increase in liver iNKT cell function.

(A-C) Liver mononuclear cells were prepared from C57BL/6 mice kept on vancomycin or H2O, and incubated with αGalCer-loaded EL4 cells in the present of Brefeldin A for two hours. Liver iNKT cell IFNγ⁺% (B) and IL-4⁺% (C) are shown. Results are presented as mean+/− SEM of two independent experiments. n=12, p<0.05, student t test. (D) Representative IL-18 receptor staining of liver iNKT cells. (E) IL-18R⁺% of liver or spleen iNKT cells. Results are presented as mean+/− SEM of one experiment, n=5. (F) Liver IL-18 mRNA from mice kept on vancomycin or H2O was measured by RT-PCR. Results are presented as mean+/− SEM of two independent experiments. n=10, p<0.05, student t test. (G, H) BALB/c mice (age 7~10 weeks) were kept on vancomycin or H2O for 3 weeks. Anti-IL18 or IgG were given by i.p. injections twice before i.v. injection of αGalCer-loaded A20 cells with brefeldin A. (G) Experimental setup. (H) Liver iNKT cell IFNγ⁺% is shown. Results are presented as mean+/− SEM from two independent experiments. n=8 for IgG H2O, 9 for IgG vanco, 4 for αIL-18 H2O, 9 for αIL-18 vanco, p<0.05, two-way ANOVA. (I, J) IL-18KO or Wt C57BL/6 mice (age 7~10 weeks) were kept on vancomycin or H2O for 3 weeks. Then mice were challenged i.v. with αGalCer-loaded EL4 cells plus brefeldin A. (I) Experimental setup. (J) Liver iNKT cell IFNγ⁺% is shown. Results are presented as mean+/− SEM of one experiment. n=5, p<0.05, two-way ANOVA.

Figure.4. CSF-1R⁺ macrophages control liver iNKT cell function.

(A) Hepatocytes, macrophages or stellate cells were isolated from C57BL/6 mice kept on vancomycin or H2O. IL-18 mRNA in each cell type was determined by RT-PCR. Results are presented as mean+/− SEM of one experiment. n=5, p<0.05, two-way ANOVA. (B) Vancomycin or H2O treated C57BL/6 mice were treated with clodronate liposomes to deplete macrophages. One day later, IL-18 mRNA expression in liver tissue was measured by RT-PCR. Results are presented as mean+/− SEM from two independent experiments. n=7, p<0.05, two-way ANOVA. (C,D) Vancomycin or H2O treated C57BL/6 mice were treated with clodronate liposome to deplete macrophages followed by i.v. injection of αGalCer-loaded EL4 cells plus brefeldin A. (C) Experimental setup. (D) Liver iNKT cell IFNγ⁺% was measured by flow cytometry. Results are presented as mean+/− SEM from two independent experiments. n=8, p<0.05, two-way ANOVA. (E,F) BALB/c mice (age 7~10 weeks) were kept on vancomycin or H2O for 3 weeks. Mice received two doses of αCSF-1R or IgG i.p. injections before i.v. injection of αGalCer-loaded A20 cells plus brefeldin A. (E) Experimental setup. (F) Liver iNKT cell IFNγ⁺% is shown. Results are presented as mean+/− SEM of one experiment, n=4 for IgG H2O and IgG Vanco, n=5 for αCSF-1R H2O and αCSF-1R Vanco, p<0.05, two-way ANOVA. (G,H) MM^DTR mice or littermates (age 7~10 weeks) were kept on vancomycin or H2O for 3 weeks. Then mice were s.c. injected 200 ng diphtheria toxin (DT) to deplete CSF-1R⁺ macrophage one day before given i.v. injection of αGalCer-loaded EL4 cells with brefeldin A. (G) Experimental setup. (H) Liver iNKT cell IFNγ⁺% is shown. Results are presented as mean+/− SEM from two independent experiment, n=5 for littermate H2O, 4 for littermate Vanco, 7 for MM^DTR H2O, 8 for MM^DTR Vanco, p<0.05, two-way ANOVA. (I, J) CD45.1 recipient mice (age ~6 weeks) were irradiated with 900 rads followed by adoptive transfer of donor bone marrow cells from either CD45.2 WT or IL-18 KO mice, followed by clodronate treatment. Mice were kept on vancomycin or H2O for 3 weeks, and αGalCer-loaded EL4 cells plus brefeldin A was injected i.v.. (G) Experimental setup. (H) Liver iNKT cell IFNγ⁺% is shown. Results are presented as mean+/− SEM from two independent experiment, n=10, p<0.05, two-way ANOVA.