NFIL3 is a regulator of IL-12 p40 in macrophages and mucosal immunity

Abstract

Regulation of innate inflammatory responses against the enteric microbiota is essential for the maintenance of intestinal homeostasis. Key participants in innate defenses are macrophages. In these studies, the basic leucine zipper protein, NFIL3, is identified as a regulatory transcription factor in macrophages, controlling IL-12 p40 production induced by bacterial products and the enteric microbiota. Exposure to commensal bacteria and bacterial products induced NFIL3 in cultured macrophages and in vivo. The Il12b promoter has a putative DNA-binding element for NFIL3. Basal and LPS-activated NFIL3 binding to this site was confirmed by chromatin immunoprecipitation. LPS-induced Il12b promoter activity was inhibited by NFIL3 expression and augmented by NFIL3-short hairpin RNA in an Il12b-bacterial artificial chromosome-GFP reporter macrophage line. Il12b inhibition by NFIL3 does not require IL-10 expression, but a C-terminal minimal repression domain is necessary. Furthermore, colonic CD11b(+) lamina propria mononuclear cells from Nfil3(-/-) mice spontaneously expressed Il12b mRNA. Importantly, lower expression of NFIL3 was observed in CD14(+) lamina propria mononuclear cells from Crohn's disease and ulcerative colitis patients compared with control subjects. Likewise, no induction of Nfil3 was observed in colons of colitis-prone Il10(-/-) mice transitioned from germ-free to a conventional microbiota. In conclusion, these experiments characterize NFIL3 as an Il12b transcriptional inhibitor. Interactions of macrophages with the enteric microbiota induce NFIL3 to limit their inflammatory capacity. Furthermore, altered intestinal NFIL3 expression may have implications for the pathogenesis of experimental and human inflammatory bowel diseases.

FIGURE 1

Nfil3 is induced by bacterial products in cultured BMDMs and the enteric microbiota in vivo. A, WT and Myd88^−/− BMDMs were stimulated with LPS (10 ng/ml) for 0.5, 1, 2, and 3 h, and Nfil3 expression was analyzed by real-time RT-PCR. Results are expressed as fold induction versus unstimulated WT BMDMs normalized to β-actin (representative from three independent experiments). B, WT BMDMs were analyzed for NFIL3 protein by Western immunoblot. WT BMDMs were stimulated with LPS (100 ng/ml) for 3, 6, and 16 h. Results are representative from four independent experiments. C, WT and Il10^−/− BMDMs were stimulated with LPS for 1, 2, and 3 h, and kinetics of Nfil3 expression was analyzed by real-time RT-PCR. Results are expressed as fold induction versus unstimulated WT BMDMs normalized to β-actin (mean ± SEM from four independent experiments). D, WT BMDMs were stimulated with LPS in the presence of anti–IL-10 Ab (10 μg/ml) or isotype control Ab (10 μg/ml) for 2, 4, and 8 h, and kinetics of Nfil3 expression was analyzed by real-time RT-PCR. Results are expressed as fold induction versus unstimulated WT BMDMs normalized to β-actin (mean ± SEM from four independent experiments). E, GF WT mice were transitioned to an SPF enteric microbiota and colonic Nfil3 expression was analyzed before and 3 and 14 d postcolonization. Results are expressed as fold induction versus GF colons normalized to β-actin (mean ± SEM from three independent experiments). *p < 0.05, **p < 0.01 versus GF.

FIGURE 2

Nfil3^−/− macrophages demonstrate increased Il12b expression. A, WT and Nfil3^−/− BMDMs were stimulated with LPS (10 ng/ml) for 4 h, and mRNA expression was quantified by PCR array (SA Biosciences). Relative mRNA expression for Il12a, Il12b, Il23a, Tnf, Il6, and Il10 in Nfil3^−/− BMDMs compared with WT BMDMs are shown. Results are representative from two independent experiments. B, WT and Nfil3^−/− BMDMs were stimulated with LPS (10 ng/ml), and Il12b mRNA (4 h post-LPS) and IL-12 p40 protein (24 h post-LPS) were analyzed by real-time RT-PCR and ELISA. Il12b results are expressed as fold induction versus unstimulated WT BMDMs. All results represent mean ± SEM of four independent experiments. *p < 0.05 versus LPS-stimulated WT BMDMs. C, WT BMDMs transduced with lentiviral expressed Nfil3 shRNA and control scrambled shRNA were stimulated with LPS for 3 h for Il12b expression analysis. Results are expressed as fold induction versus unstimulated BMDMs transduced with scrambled shRNA (mean ± SEM from four independent experiments). *p < 0.05 versus scrambled shRNA transduced BMDMs stimulated with LPS. D, WT BMDMs transduced with lentivirus to express NFIL3 or GFP (control) were stimulated with LPS (10 ng/ml). Il12b mRNA (3 h post-LPS) and IL-12 p40 protein (24 h post-LPS) were determined. Results for mRNA are expressed as fold induction versus unstimulated GFP transduced BMDMs normalized to β-actin (mean ± SEM from three independent experiments). *p < 0.05 versus LPS-stimulated GFP-transduced BMDMs. E, WT BMDMs transduced with lentivirus that expressed WT, mutated, and truncated NFIL3 were stimulated with LPS for 4 h. See text for description of protein constructs. Results are expressed as percent induction versus LPS-stimulated GFP-transduced BMDMs normalized to β-actin (mean ± SEM from three independent experiments). *p < 0.01 versus LPS-stimulated GFP-expressing BMDMs were calculated. F, WT and Nfil3^−/− BMDMs transduced with different titers of lentivirus-expressed NFIL3 constructs were stimulated with LPS for 4 h and analyzed for Il12b and Nfil3 expression. Il12b expressions are presented as percent induction versus LPS-stimulated GFP-transduced WT BMDMs normalized to β-actin. Nfil3 expression is presented as relative induction versus unstimulated GFP-transduced WT BMDMs normalized to β-actin.

FIGURE 3

NFIL3 inhibition of Il12b is independent of IL-10. A, WT and Nfil3^−/− BMDMs were stimulated with LPS (10 ng/ml), and Il10 mRNA (4 h post-LPS) and IL-10 protein (24 h post-LPS) were analyzed by real-time RT-PCR and ELISA. Il10 results are expressed as fold induction versus unstimulated WT BMDMs. Results represent mean ± SEM of four independent experiments. B, Il10^−/− BMDMs transduced with lentivirus to express NFIL3 or GFP were stimulated with LPS (10 ng/ml). Il12b mRNA (3 h post-LPS, left panel) and IL-12 p40 protein (24 h post-LPS, right panel) were determined. Results for mRNA are expressed as fold induction versus unstimulated GFP-transduced BMDMs normalized to β-actin (mean ± SEM from three independent experiments). **p < 0.01 versus LPS-stimulated GFP-transduced BMDMs. C, Il10^−/− BMDMs transduced with lentivirus that expressed WT, mutated (Mut), and truncated (Trunc) NFIL3 were stimulated with LPS for 4 h. Results are expressed as percent induction versus LPS-stimulated GFP-transduced BMDMs normalized to β-actin (mean ± SEM from three independent experiments). *p < 0.01 versus LPS-stimulated GFP-expressing BMDMs. D, Il10^−/− BMDMs transduced with lentiviral that expressed Nfil3 shRNA and control scrambled shRNA were stimulated with LPS for 3 h for Il12b expression analysis. Results are expressed as fold induction versus unstimulated BMDMs transduced with scrambled shRNA (mean ± SEM from four independent experiments). *p < 0.05 versus scrambled shRNA-transduced BMDMs stimulated with LPS by paired t test.

FIGURE 4

NFIL3 inhibits Il12b through interactions with the promoter. A, Recruitment of NFIL3 on the Il12b promoter was analyzed by ChIP. Induction of NFIL3-binding activity to the Il12b promoter was confirmed by ChIP with PCR products visualized on a 2% agarose gel. WT BMDMs were stimulated with or without LPS (100 ng/ml) for 3 h. Polyclonal goat IgG (first lane) is a negative control. This result is representative of three independent chromatin preparations. B, In separate experiments, WT and Nfil3^−/− BMDMs were stimulated with or without LPS (100 ng/ml) for 3 h. Quantitative results are presented as enrichment (percentage input DNA) of NFIL3 binding to the Il12b promoter and are representative of three independent chromatin preparations. C, Il12b-GFP transformed macrophages transduced with empty (control), WT, 2-aa mutated (Mut) or truncated (Trunc) NFIL3 were stimulated with 100 ng/ml LPS for 24 h and analyzed by FACS. Representative histograms with mean fluorescent intensity are given. Percentage of GFP⁺ cells relative to control is also shown (mean ± SEM from three independent experiments). *p < 0.05 versus control. D, Il12b-GFP macrophages were transduced with scrambled or NFIL3-shRNA. Cells were stimulated with 10 ng/ml LPS for 24 h and analyzed by FACS. Representative histograms with mean fluorescent intensity are given. Data are presented as percentage of GFP⁺ cells relative to scrambled shRNA-treated cells (mean ± SEM from three independent experiments). *p < 0.05 versus scrambled shRNA.

FIGURE 5

Induction of NFIL3 by commensal bacteria is impaired in murine and human IBD. A, Colonic CD11b⁺ and CD11b⁻ LPMCs from WT and Nfil3^−/− mice were analyzed for Il12b expression. Results are presented as expression relative to WT 11b⁺ LPMCs normalized to β-actin (mean ± SEM from three independent experiments). B, GF WT and colitis-prone Il10^−/− mice were transitioned to an SPF enteric microbiota, and colonic Nfil3 expression was analyzed 3 d postcolonization. Results are expressed as fold induction versus WT GF colons normalized to β-actin (mean ± SEM from three independent experiments). *p < 0.05 versus GF. C, NFIL3 expression in human CD14⁺ LPMCs was examined (control [cont], n = 8; Crohn’s disease [CD], n = 8; ulcerative colitis [UC], n = 8). Results are presented as relative expression compared with controls normalized to β-actin (mean ± SEM). *p < 0.05, **p < 0.01 versus control (Dunnett’s multiple-comparison test).