An inflammatory bowel disease–risk variant in INAVA decreases pattern recognition receptor–induced outcomes
Jie Yan, Matija Hedl, Clara Abraham
Jie Yan, Matija Hedl, Clara Abraham
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Research Article Immunology

An inflammatory bowel disease–risk variant in INAVA decreases pattern recognition receptor–induced outcomes

Abstract

Inflammatory bowel disease (IBD) is characterized by dysregulation in both cytokines and responses to intestinal microbes, and proper regulation of pattern recognition receptor (PRR) signaling is critical for intestinal immune homeostasis. Altered functions for the IBD risk locus containing rs7554511, which encompasses the C1orf106 gene (recently named INAVA), and roles for the protein encoded by the INAVA gene are unknown. Here, we investigated the role of INAVA and INAVA genotype in regulating PRR-initiated outcomes in primary human cells. Both peripheral and intestinal myeloid cells expressed INAVA. Upon PRR stimulation, INAVA was required for optimal MAPK and NF-κB activation, cytokine secretion, and intracellular bacterial clearance. INAVA recruited 14-3-3τ, thereby contributing to recruitment of a signaling complex that amplified downstream signals and cytokines. Further, INAVA enhanced bacterial clearance by regulating reactive oxygen, reactive nitrogen, and autophagy pathways. Macrophages from rs7554511 C risk carriers expressed lower levels of INAVA RNA and protein. Lower expression was attributed in part to decreased transcription mediated directly by the intronic region containing the rs7554511 C variant. In rs7554511 C risk carrier macrophages, lower INAVA expression led to decreased PRR-induced activation of MAPK and NF-κB pathways, cytokines, and bacterial clearance pathways. Thus, IBD-associated polymorphisms in INAVA modulate PRR-initiated signaling, cytokines, and intracellular bacterial clearance, likely contributing to intestinal immune homeostasis.

Authors

Jie Yan, Matija Hedl, Clara Abraham

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Figure 7

14-3-3τ recruitment to INAVA contributes to optimal assembly of a signaling complex and to INAVA modulation of PRR-induced signaling and cytokine secretion.

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14-3-3τ recruitment to INAVA contributes to optimal assembly of a signal...
(A) MDMs were treated with 100 μg/ml MDP for 15 minutes. INAVA was immunoprecipitated and recruitment of NOD2, RIP2, and IRAK1 was assessed by Western blot. Equivalent expression for the respective proteins is shown in whole-cell lysates (WCLs). (B) Sequence alignments for putative 14-3-3 binding regions within INAVA from select species. (C) MDMs were treated with 100 μg/ml MDP for 15 minutes. INAVA was immunoprecipitated and recruitment of 14-3-3τ, p-ERK, ERK, p-p38, p38, and p-IκBα was assessed by Western blot. Equivalent expression for the respective proteins is shown in WCLs. Data are representative of n = 9 for 14-3-3τ, n = 9 for p-ERK, n = 4 for ERK, n = 3 for p-p38, n = 4 for p38, and n = 3 for p-IκBα. (D–F) MDMs were transfected with scrambled or 14-3-3τ siRNA. (D) Transfected MDMs were treated with 100 μg/ml MDP for 15 minutes. INAVA was immunoprecipitated and recruitment of 14-3-3τ and p-ERK was assessed by Western blot. Representative Western blot for 1 of 6, and 1 of 4 individuals, respectively. (E) Transfected cells were treated with 100 μg/ml MDP for 15 minutes and assessed for ERK activation by phospho-flow. Fold p-ERK induction was normalized to untreated, scrambled siRNA–transfected cells + SEM (n = 8). Similar results were observed in an additional n = 8. (F) Transfected cells were treated with 100 μg/ml MDP for 24 hours. Mean cytokines + SEM (n = 4). Similar results were observed in an additional n = 8. (G) HEK293 cells were transfected with NOD2 + the indicated HA-INAVA variants, and then treated with 100 μg/ml MDP for 15 minutes. HA-INAVA was immunoprecipitated with an anti-HA antibody and recruitment of 14-3-3τ and p-ERK was assessed by Western blot. Representative of n = 6 (14-3-3τ) and n = 3 (p-ERK). (H and I) HEK293 cells were transfected with NOD2, a Renilla reporter, AP-1 or NF-κB luciferase reporters, and empty vector or the indicated INAVA variants. (H) Transfected cells were treated with 100 μg/ml MDP for 6 hours and activation of AP-1 and NF-κB luciferase reporters was assessed and normalized to Renilla. Mean + SEM for triplicates. Representative of 3 independent experiments. (I) Transfected cells were treated with 100 μg/ml MDP for 24 hours and secreted IL-6 was assessed + SEM for triplicates. Representative of 3 independent experiments. Tx, treatment. *P < 0.05; #P < 0.01; §P < 0.001; †P < 1 × 10–4; ‡P < 1 × 10–5; determined by 2-tailed Student’s t test. IP, immunoprecipitated; IB, immunoblotted.