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.

Figure 1. Human myeloid cells from rs7554511 C risk allele carriers demonstrate less cytokine secretion upon PRR stimulation compared with AA carriers.

Human MDMs (n = 15/genotype) were treated for 24 hours with the indicated doses of MDP (recognized by NOD2), Pam₃Cys (recognized by TLR2), polyI:C (recognized by TLR3), lipid A (recognized by TLR4), flagellin (recognized by TLR5), CL097 (recognized by TLR7), or CpG DNA (recognized by TLR9). Shown is TNF secretion. *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.

Figure 2. INAVA is required for optimal secretion of cytokines in MDMs upon stimulation through a broad range of PRRs.

Human MDMs (n = 4) were transfected with scrambled or INAVA siRNA. Cells were treated with (A) 100 μg/ml MDP or (B) 10 μg/ml Pam₃Cys (recognized by TLR2), 100 μg/ml polyI:C (recognized by TLR3), 0.1 μg/ml lipid A (recognized by TLR4), 5 ng/ml flagellin (recognized by TLR5), 1 μg/ml CL097 (recognized by TLR7), or 10 μg/ml CpG DNA (recognized by TLR9) for 24 hours. Mean cytokine secretion + SEM is shown. Similar results were observed in an additional n = 16 for A and n = 8 for B. Tx, treatment; Scr, scrambled. ^#P < 0.01; ^§P < 0.001; ^†P < 1 × 10^–4; ^‡P < 1 × 10^–5; determined by 2-tailed Student’s t test.

Figure 3. INAVA is expressed in peripheral and intestinal myeloid-derived cells, and INAVA expression increases with PRR stimulation.

(A) Human MDMs (n = 10) were stimulated for the indicated times with 100 μg/ml MDP. INAVA mRNA expression was normalized to untreated MDMs and GAPDH. Mean + SEM is shown. GAPDH expression was stable at each of these time points and similar INAVA mRNA regulation was observed with normalization to a different housekeeping gene, ACTB (data not shown). (B) MDMs (n = 8) were treated with 100 μg/ml MDP for the indicated times and INAVA protein levels were assessed by flow cytometry. Left: Representative flow cytometry plots with INAVA mean fluorescence intensity (MFI) values. Right: Summary of INAVA protein expression + SEM. Similar results were observed in an additional n = 8. (C) MDMs were transfected with scrambled or INAVA siRNA and assessed for INAVA protein expression. Representative flow cytometry plot with summary graph (n = 8) for MFI + SEM are shown. (D) MDMs were treated with 100 μg/ml MDP for 24 hours and assessed for INAVA protein expression by Western blot (representative for 1 of 7 individuals). GAPDH was used as a loading control; GAPDH levels were stable over the time course examined (data not shown). (E) INAVA mRNA expression was assessed in intestinal (n = 7) and peripheral (n = 7) myeloid-derived cells and normalized to CD11c. Mean + SEM. Tx, treatment; scr, scrambled. *P < 0.05; ^#P < 0.01; ^§P < 0.001; ^†P < 1 × 10^–4; determined by 2-tailed Student’s t test.

Figure 4. MDMs from rs7554511 C risk carriers express less INAVA.

(A and B) MDMs from rs7554511 AA, CA, and CC carriers (n = 15/genotype, similar results were seen in an additional n = 8/genotype) were left untreated or treated with 100 μg/ml MDP for 8 hours (A) or 24 hours (B). (A) INAVA mRNA expression (expressed as change in Ct values normalized to GAPDH and represented as a linear scale) + SEM. (B) INAVA protein expression with (left) representative flow cytometry and mean fluorescence intensity (MFI) values shown, and (right) summarized data for mean + SEM. 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.

Figure 5. The INAVA rs7554511 C risk variant results in lower intron 6–mediated transcription and lower TBP-mediated transcription within intron 6.

(A–C) INAVA intron 6 (842 bp) expressing the indicated rs7554511 variants or the control pGL4.17 vector were transfected into HEK293 cells along with Renilla and NOD2 vector. Cells were additionally transfected with (A) scrambled or TBP siRNA, or (C) empty vector or TBP vector. Cells were then treated with 100 μg/ml MDP for 24 hours and luciferase activity was assessed in 6 replicates. The INAVA intron 6 TCCCCT mutant (mut) (see Supplemental Figure 11A) served as a specificity control for the transcription factor TBP. Representative of 3 independent experiments. Significance is shown for (A) scrambled compared with TBP siRNA for the same conditions in the intron 6–transfected cells or (B and C) rs7554511 C compared with A variants for the same conditions or as indicated. Tx, treatment; NS, not significant. ^#P < 0.01; ^§P < 0.001; ^†P < 1 × 10^–4; ^‡P < 1 × 10^–5; determined by 2-tailed Student’s t test.

Figure 6. INAVA is required for optimal NOD2-initiated MAPK and NF-κB signaling in MDMs and is decreased in MDMs from INAVA rs7554511 C risk carriers.

(A and B) MDMs (n = 8) were transfected with scrambled or INAVA siRNA. Cells were then treated with 100 μg/ml MDP for 15 minutes and assessed for (A) p-ERK, p-p38, p-JNK, or (B) p-IκBα. Left: Representative flow cytometry plots with mean fluorescence intensity (MFI) values. Right: Fold phospho-protein induction normalized to untreated, scrambled siRNA–transfected cells + SEM. (C and D) MDMs from rs7554511 AA and CC (n = 8/genotype) carriers were treated with 100 μg/ml MDP for 15 minutes and assessed for (C) p-ERK, p-p38, p-JNK, or (D) p-IκBα. Left: Representative flow cytometry plots. Right: Summarized data + SEM. Tx, treatment. *P < 0.05; ^§P < 0.001; ^†P < 1 × 10^–4; ^‡P < 1 × 10^–5; determined by 2-tailed Student’s t test.

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.

(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.

Figure 8. INAVA is required for optimal intracellular bacterial clearance and this clearance is decreased in MDMs from INAVA rs7554511 C risk carriers.

(A) MDMs (n = 4) were transfected with scrambled or INAVA siRNA, then left untreated or treated with 100 μg/ml MDP for 48 hours, and then cocultured with AIEC, S. aureus, or E. faecalis. Shown are bacterial colony forming units (CFU) + SEM. Significance is compared with scrambled siRNA–transfected cells for the corresponding treatment or as indicated. Similar results were observed in an additional n = 4. (B) MDMs from rs7554511 AA, CA, CC carriers (n = 15/genotype) were left untreated or treated with 100 μg/ml MDP for 48 hours and then cocultured with AIEC, S. aureus, or E. faecalis. Shown are the CFU + SEM. (C and D) MDMs from rs7554511 CC or AA carriers (n = 15/genotype) were transfected with (C) scrambled or INAVA siRNA, or (D) empty vector (EV) or INAVA vector, treated with 100 μg/ml MDP for 48 hours, and then cocultured with the indicated bacteria. Shown are CFU + SEM. NS, not significant; scr, scrambled; 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.

Figure 9. PRR-induced ROS, RNS, and autophagy are required for INAVA-mediated bacterial clearance.

(A–E) MDMs were transfected with scrambled or INAVA siRNA and then left untreated or treated with 100 μg/ml MDP for 48 hours. (A and D) Cells were analyzed by flow cytometry utilizing (A) the ROS-detecting dye H₂DCDFDA (n = 4) or (D) LC3II antibody (n = 4). Shown are representative flow cytometry plots with mean fluorescence intensity (MFI) values as indicated and a summary graph with MFI + SEM. (B, C, and E) mRNA expression was assessed by quantitative reverse transcription PCR. Data are represented as the fold mRNA induction compared with scrambled siRNA-transfected, untreated cells (n = 8, similar results seen in an additional n = 4) + SEM. (F) MDMs (n = 6) were transfected with scrambled or INAVA siRNA ± p47phox-, p67phox-, NOS2-, or ATG5-expressing vectors alone or in combination or with empty vector, then left untreated or treated with 100 μg/ml MDP for 48 hours. Cells were cocultured with AIEC at 10:1 MOI. Shown are bacterial CFU + SEM. Scr, scrambled; vec, vector. ^#P < 0.01; ^§P < 0.001; ^†P < 1 × 10^–4; ^‡P < 1 × 10^–5; determined by 2-tailed Student’s t test.

Figure 10. PRR-induced ROS, RNS, and autophagy are modulated by INAVA rs7554511 genotype.

(A–C) MDMs from rs7554511 genotype carriers (n = 15/genotype) were left untreated or treated with 100 μg/ml MDP for 48 hours. Shown is summarized mean fluorescence intensity (MFI) of ROS or the indicated proteins assessed by flow cytometry + SEM. (D–F) MDMs from rs7554511 CC or AA carriers (n = 15/genotype) were transfected with scrambled or INAVA siRNA, and then treated with 100 μg/ml MDP for 48 hours. Shown is summarized MFI of ROS or the indicated proteins + SEM. (G–I) MDMs from rs7554511 CC or AA carriers (n = 15/genotype) were transfected with empty vector (EV) or INAVA vector, and then treated with 100 μg/ml MDP for 48 hours. Shown is summarized MFI of ROS or the indicated proteins + SEM. NS, not significant; scr, scrambled; 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.

Figure 11. Model of INAVA mechanisms for regulating PRR-induced signaling, cytokines, and bacterial clearance.

NOD2 stimulation in MDMs results in the assembly of a complex that includes NOD2, RIP2, IRAK1, INAVA, and 14-3-3τ. INAVA contains 3 distinct 14-3-3 binding domains, and 14-3-3τ recruitment, in turn, modulates the recruitment of additional signaling molecules to INAVA, including p-ERK, p-p38, and p-IκBα, which then contribute to activation of downstream signaling pathways and cytokine secretion. In addition to the induction of cytokines, INAVA is required for induction of a broad range of NOD2-dependent transcripts, as well as for optimal bacterial clearance pathways and intracellular bacterial clearance in MDMs. Importantly, INAVA is required for optimal signaling and cytokine secretion downstream of multiple PRRs. MDMs from IBD rs7554511 C risk carriers in INAVA demonstrate lower INAVA expression, and consistently, decreased MAPK and NF-κB signaling, cytokine secretion, and bacterial clearance.