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NIK signaling axis regulates dendritic cell function in intestinal immunity and homeostasis

Abstract

Dendritic cells (DCs) play an integral role in regulating mucosal immunity and homeostasis, but the signaling network mediating this function of DCs is poorly defined. We identified the noncanonical NF-κB-inducing kinase (NIK) as a crucial mediator of mucosal DC function. DC-specific NIK deletion impaired intestinal immunoglobulin A (IgA) secretion and microbiota homeostasis, rendering mice sensitive to an intestinal pathogen, Citrobacter rodentium. DC-specific NIK was required for expression of the IgA transporter polymeric immunoglobulin receptor (pIgR) in intestinal epithelial cells, which in turn relied on the cytokine IL-17 produced by TH17 cells and innate lymphoid cells (ILCs). NIK-activated noncanonical NF-κB induced expression of IL-23 in DCs, contributing to the maintenance of TH17 cells and type 3 ILCs. Consistent with the dual functions of IL-23 and IL-17 in mucosal immunity and inflammation, NIK deficiency also ameliorated colitis induction. Thus, our data suggest a pivotal role for the NIK signaling axis in regulating DC functions in intestinal immunity and homeostasis.

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Fig. 1: Decreased levels of secretory IgA accompanied by increased fecal Enterococci spp. and Candidatus Savagella in Map3k14-cKO mice.
Fig. 2: DC-specific NIK deficiency impairs pIgR expression in IECs and IgA secretion.
Fig. 3: DC-specific deletion of NIK reduces the frequency of intestinal TH17 cells and ILC3s.
Fig. 4: NIK regulates intestinal homeostasis by mediating TLR-stimulated IL-23 expression in DCs.
Fig. 5: NIK-dependent noncanonical NF-κB activation contributes to TLR-stimulated IL-23 expression.
Fig. 6: TLR stimulates TRAF2 degradation in a TNFRII-dependent manner.
Fig. 7: DC-specific NIK deletion impairs host defenses against C. rodentium infection.
Fig. 8: DC-specific NIK deletion ameliorates colon inflammation in IL-10-deficient mice.

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Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank Genentech, Inc. for providing the Map3k14-flox mice, Walter and Eliza Hall Institute of Medical Research for Nfkb2Lym1 mice, and National Cancer Institute Preclinical Repository for NF-κB p100/p52 antibody. This work was supported by grants from the National Institutes of Health (GM84459, AI057555, AI104519, and AI64639). This study also used the NIH/NCI-supported resources under award number P30CA016672 at The MD Anderson Cancer Center.

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Authors

Contributions

Z.J. and J.-Y.Y. designed and performed the research, prepared the experiments, and wrote part of the manuscript; M.G., H.W., X.X., Y.L., T.L., L.Zhu, J.S., L.Zhang, X.Z., D.J., D.L., and X.C. contributed experiments; H.D.B. contributed critical reagents; Y.C. made supervising contributions to intestinal immune cells analyses; and S.-C.S. supervised the work and wrote the manuscript.

Corresponding author

Correspondence to Shao-Cong Sun.

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H.D.B. is an employee of Genentech, Inc. The other authors declare no competing interests. Genentech, Inc. provided the Map3k14-flox mice.

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Integrated supplementary information

Supplementary Figure 1 DC-specific NIK deficiency does not affect the frequency or maturation state of DCs.

a, Genotyping PCR analysis of tail DNA from wild-type (WT; NIK+/+CD11c-Cre), heterozygous (Het; NIK+/flCD11c-Cre) and Map3k14-cKO (NIKfl/flCD11c-Cre) mice, showing the Map3k14 floxed and wild-type alleles. b, qRT–PCR analysis of Map3k14 mRNA encoded by the deleted exon in Map3k14-cKO (cKO) mice using sorted DCs and control T cells (n = 3). c, Immunoblot analysis of the indicated proteins in whole-cell lysates of wild-type and Map3k14-cKO BMDCs treated for 3 h with anti-CD40 in the presence ( + ) or absence (–) of the proteasome inhibitor MG132 (MG132 was used to block NIK degradation). d,e, Flow cytometric analysis of the CD11c+MHCII+ DC population (d) and surface expression of CD80 and CD86 in DCs (e) from spleen, mesenteric lymph nodes (MLN), Peyer’s patches (PP), and the other indicated organs. f,g, Flow cytometric analysis of total lamina propria (LP) DCs (CD11c+MHCII+) (f, n = 4 mice/group) or the indicated DC subsets, including CD103+ DCs (n = 4 mice/group), myeloid DCs (CD8a+CD11bCD11c+) (n= 5 mice/group), lymphoid DCs (CD8aCD11b+CD11c+) (n= 5 mice/group), TLR5+ DCs (n= 4 mice/group), and pDCs (n= 5 mice/group) (g). h, Flow cytometric analysis of the indicated surface markers in total LP DCs. Each symbol represents an individual mouse, and small horizontal lines indicate the mean ( ± s.e.m.) (b,f,g). Data were analyzed by unpaired two-tailed Student’s t test with P values shown above the plots (b). Data were representative of two independent experiments.

Supplementary Figure 2 16 S rRNA sequencing analysis of IgA+ and IgA microbiota.

a,b, 16 S rRNA sequencing analysis of IgA+ and IgA fecal bacteria, isolated from multiple mice (n= 6 per genotype) by magnetic-activated cell sorting (MACS), showing trends in beta diversity based on principal-coordinate analysis (PCoA; a) and taxa abundances (b). Each symbol represents an individual mouse (a,b), and b is presented as box plots (where the middle line indicates the mean value and all individual plots are shown). Data were analyzed by multiple comparisons using the Holm–Sidak method (b), with P values shown above the plots. Data are representative of three independent experiments.

Supplementary Figure 3 DC-specific NIK deficiency has no effect on serum IgA concentration, B cell frequency, or Peyer’s patch size and numbers.

a, ELISA of IgA concentration in the sera of wild-type (WT; n= 8) and Map3k14-cKO (cKO; n= 8) mice with the dilution factors indicated. b,c, Flow cytometric analysis of B cell frequency in the spleen, mesenteric lymph nodes (MLN) and Peyer’s patches (PP) of age-matched wild-type and Map3k14-cKO mice. Data are presented as a representative plot (b) and summary graph (c) based on multiple mice (each circle represents a mouse, n= 6–7 mice/group). d, Representative images of Peyer’s patches from age-matched wild-type and Map3k14-cKO mice. eg, Summary graphs of the size (e, wild type, n= 68; cKO, n= 58), numbers (f, wild type, n= 27; cKO, n= 24) and cell numbers (g, n= 5 mice/group) of Peyer’s patches of age-matched wild-type and Map3k14-cKO mice. Each symbol represents an individual mouse (a,c,f,g) or an individual Peyer’s patch (e); small horizontal lines indicate the mean ( ± s.e.m.). Data were analyzed by unpaired two-tailed Student’s t test and are representative of three independent experiments.

Supplementary Figure 4 Microbiota composition and fecal concentration of IgA and pIgR in co-housed wild-type and NIK-DKO mice.

a, 16 S rRNA sequencing analysis of the genus levels of microbiota composition from age-matched and co-housed wild-type (WT, n= 8) and Map3k14-cKO (n= 8) mice. b, Analysis of Enterococcus spp. in the fecal extracts of age-matched and co-housed wild-type (n= 5) and Map3k14-cKO (n= 5) mice using selective agar plates. c,d, ELISA of IgA (c) and pIgR (d) concentrations in fecal extracts of age-matched and co-housed wild-type (n= 7) and Map3k14-cKO (n= 7) mice. Each symbol (ad) represents an individual mouse. Small horizontal lines (bd) indicate the mean ( ± s.e.m.), and a is presented as box plots (where the middle line indicates the mean value and all individual plots are shown). Data were analyzed by unpaired two-tailed Student’s t test (bd) or analyzed for multiple comparisons using the Holm–Sidak method (a), with P values shown above the plots. Data are representative of three independent experiments.

Supplementary Figure 5 Fecal IgA and pIgR concentrations and microbiota composition in wild-type and Rorc–/– mice.

a,b, ELISA analysis of IgA (a) and pIgR (b) concentrations in fecal extracts of age-matched wild-type (WT, n= 10) and Rorc–/– (n= 7) mice. c, Analysis of the commensal anaerobic bacteria Enterococcus spp. in the fecal extracts of age-matched wild-type (n= 10) and Rorc–/– (n= 7) mice by using selective agar plates. d, 16 s rRNA sequencing analysis of the genus levels of microbiota composition from age-matched wild-type (n= 5) and Rorc–/– (n= 3) mice. Each symbol (ad) represents an individual mouse. Small horizontal lines (ac) indicate the mean ( ± s.e.m.), and d is presented as box plots (where the middle line indicates the mean value and all individual plots are shown). Data were analyzed by unpaired two-tailed Student’s t test (ac) or analyzed for multiple comparisons using the Holm–Sidak method (d), with P values shown above the plots. Data are representative of three independent experiments.

Supplementary Figure 6 NIK mediates TLR-stimulated IL-23 expression in lamina propria DCs.

a,b, qRT–PCR analysis of the indicated mRNAs in sorted wild-type (WT, n= 3) and Map3k14-cKO (n= 3) lamina propria DCs (CD45+CD11c+MHCII+) that were either incubated in medium (NT) or stimulated with the indicated TLR ligands for 6 h (a) or 12 h (b). Each symbol represents an individual mouse, and small horizontal lines indicate the mean ± s.e.m. (a,b). Data were analyzed by unpaired two-tailed Student’s t test, with P values shown above the plots. Data are representative of two independent experiments.

Supplementary Figure 7 Defective mucosal immunity and altered microbiota composition in Nfkb2Lym1/+ mice.

a, qRT–PCR analysis of Il23a mRNA in freshly sorted and untreated lamina propria DCs of wild-type (WT, n= 6) and Nfkb2Lym1/+ (n= 6) mice. b,c, ELISA of IgA (b) and pIgR (c) concentration in the feces of wild-type and Nfkb2Lym1/+ mice. d, Flow cytometric analysis of IL-17-producing CD4+ T (TH17) cells, presented as a representative FACS plot (left) and a summary graph (right) (n= 5 mice/group). e, Flow cytometric analysis of IL-17- and IL-22-producing ILC population, presented as a representative FACS plot (left) and summary graphs (right) (n = 3 mice/group). f, Analysis of commensal anaerobic bacteria Enterococcus spp. in the fecal extracts of age-matched wild-type (n= 7) and Nfkb2Lym1/+ (n= 7) mice. g, 16 S rRNA sequencing analysis of the genus levels of microbiota composition from age-matched wild-type and Nfkb2Lym1/+ mice (n= 3 mice/group). Each symbol (ag) represents an individual mouse. Small horizontal lines (af) indicate the mean ( ± s.e.m.), and g is presented as box plots (where the middle line indicates the mean value and all individual plots are shown). Data were analyzed by unpaired two-tailed Student’s t test (af) or analyzed for multiple comparisons using the Holm–Sidak method (g), with P values shown above the plots. Data are representative of three independent experiments.

Supplementary Figure 8 Nfkb2Lym1/+ mice have impaired host defense against C. rodentium infection.

ad, Body weight change (a), colon length (b), C. rodentium bacterial load in spleen, liver and feces (c), and histological analysis of H&E-stained colon tissue (d) of wild-type (WT) and Nfkb2Lym1/+ mice infected orally with 4 × 109 CFUs of C. rodentium (n= 4 per genotype). Scale bar, 200 μm. Each symbol represents an individual mouse, and small horizontal lines indicate the mean ( ± s.e.m.) (b,c). Data were analyzed by two-way ANOVA followed by Sidak’s multiple-comparisons test (a) or unpaired two-tailed Student’s t test (b,c). Data are representative of two independent experiments. P values are shown above plots. For a, **P= 0.006, ***P< 0.001 and ###P< 0.001.

Supplementary Figure 9 Gating strategy used in flow cytometry analysis experiments.

Live immune cell populations were first gated based on the FSC-A and SSC-A, and single cells were gated based on FSC-A and FSC-H. Immune cells were further gated based on CD45 expression. The subpopulations of the indicated immune cells were gated based on specific surface markers as indicated in the individual panels.

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Jie, Z., Yang, JY., Gu, M. et al. NIK signaling axis regulates dendritic cell function in intestinal immunity and homeostasis. Nat Immunol 19, 1224–1235 (2018). https://doi.org/10.1038/s41590-018-0206-z

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