TRIM14 Is a Key Regulator of the Type I IFN Response during Mycobacterium tuberculosis Infection

Abstract

Tripartite motif-containing proteins (TRIMs) play a variety of recently described roles in innate immunity. Although many TRIMs regulate type I IFN expression following cytosolic nucleic acid sensing of viruses, their contribution to innate immune signaling and gene expression during bacterial infection remains largely unknown. Because Mycobacterium tuberculosis is an activator of cGAS-dependent cytosolic DNA sensing, we set out to investigate a role for TRIM proteins in regulating macrophage responses to M. tuberculosis In this study, we demonstrate that TRIM14, a noncanonical TRIM that lacks an E3 ubiquitin ligase RING domain, is a critical negative regulator of the type I IFN response in Mus musculus macrophages. We show that TRIM14 interacts with both cGAS and TBK1 and that macrophages lacking TRIM14 dramatically hyperinduce IFN stimulated gene (ISG) expression following M. tuberculosis infection, cytosolic nucleic acid transfection, and IFN-β treatment. Consistent with a defect in resolution of the type I IFN response, Trim14 knockout macrophages have more phospho-Ser754 STAT3 relative to phospho-Ser727 and fail to upregulate the STAT3 target Socs3, which is required to turn off IFNAR signaling. These data support a model whereby TRIM14 acts as a scaffold between TBK1 and STAT3 to promote phosphorylation of STAT3 at Ser727 and resolve ISG expression. Remarkably, Trim14 knockout macrophages hyperinduce expression of antimicrobial genes like Nos2 and are significantly better than control cells at limiting M. tuberculosis replication. Collectively, these data reveal an unappreciated role for TRIM14 in resolving type I IFN responses and controlling M. tuberculosis infection.

Graphical abstract

FIGURE 1.

TRIMs are players in the innate immune response to M. tuberculosis. (A) Heatmap of significant (p < 0.05) gene expression differences (log₂ fold-change) in TRIM family genes in uninfected BMDMs versus BMDMs infected with WT M. tuberculosis. MOI = 10. (B) RT-qPCR of fold-change in Trim14 transcripts in BMDMs infected with WT M. tuberculosis or stimulated with ISD (n = 3 biological replicates). (C) RAW 264.7 cells infected with mCherry M. tuberculosis (Mtb, red) for 6 h and immunostained for TRIM14 (green); nuclei stained with DAPI (blue). (D) As in (C) with the addition of costaining for endogenous selective autophagy markers (purple). Quantification shows the proportion of TRIM14+ bacilli that are also positive for indicated selective autophagy marker. Statistical significance was determined using two-tailed Student t test. ***p < 0.001.

FIGURE 2.

TRIM14 interacts with both cGAS and TBK1 in the DNA sensing pathway. (A) Model of DNA sensing pathway during M. tuberculosis infection. Model created using BioRender software (B) Immunofluorescence microscopy of MEFs expressing 3xFLAG-TRIM14 with HA-cGAS, HA-STING, or HA-TBK1 costained with α-HA and α-FLAG Abs. Nuclei stained with DAPI (blue). (C) Immunoblot analysis of coimmunoprecipitation of 3xFLAG-TRIM14 coexpressed with HA-cGAS, HA-STING, HA-TBK1, or HA-IRF3 in HEK293T cells. Blot is representative of >3 independent biological replicates. (D) Diagram of mTRIM14, mTBK1, hTBK1, mIRF3 gene domains and truncations used in SPR studies. (E) Equilibrium binding study of mTRIM14 and mTBK1 by SPR. mIRF3 was used as a negative control. Dissociation constant (K_d = 24.3 μM) was derived by fitting of the equilibrium binding data to a one-site binding model. (F) As in (E) but with mTRIM14 and hTBK1. Dissociation constant (K_d = 42.6 μM) was derived by fitting of the equilibrium binding data to a one-site binding model.

FIGURE 3.

Loss of TRIM14 leads to hyperinduction of Ifnb in response to M. tuberculosis and cytosolic DNA. (A) Sequencing chromatogram depicting mutations in Trim14 gRNA CRISPR-Cas9 RAW 264.7 macrophages compared with GFP gRNA control (WT). (B) Immunoblot analysis and immunofluorescence microscopy of Trim14 in WT versus Trim14 KO RAW 264.7 macrophages using an endogenous α-TRIM14 Ab. (C) RT-qPCR of Ifnb transcripts in WT and Trim14 KO RAW 264.7 macrophages infected with M. tuberculosis at specified times postinfection. (D) RT-qPCR of Ifnb transcripts in WT and Trim14 KO RAW 264.7 macrophages treated with ISD at specified times after treatment. (E) RT-qPCR of Ifnb transcripts in WT and Trim14 KO RAW 264.7 macrophages treated with poly(I:C) at specified times after treatment. (F) IFN stimulated response element reporter cells expressing luciferase with relative light units measured as a readout for IFN-β protein. (G) IFN-β protein ELISA of WT and Trim14 KO RAW 264.7 macrophages treated with ISD. All RT-qPCRs represent n = 3 biological replicates. Statistical significance was determined using two-tailed Student t test. ***p < 0.001.

FIGURE 4.

Loss of TRIM14 leads to hyperinduction of ISGs in response to multiple innate immune agonists. (A) RT-qPCR of Ifit1, Isg15, and Irf7 transcripts in WT and Trim14 KO RAW 264.7 macrophages infected with M. tuberculosis at specified times postinfection. (B) RT-qPCR of Ifit1, Isg15, and Irf7 transcripts in WT and Trim14 KO RAW 264.7 macrophages transfected with 1 μg ISD. (C) As in (B) but with transfection of 1 μg poly(I:C). (D) As in (B) but with IFN-β treatment (200 IU). All RT-qPCRs represent n = 3 biological replicates. Statistical significance was determined using two-tailed Student t test. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5.

Loss of TRIM14 promotes inhibitory phosphorylation of STAT3 at Ser754. (A) Diagram of STAT3 phosphorylation sites. (B) Immunoblot of phospho-Ser754 STAT3, phospho-Ser727 STAT3, and phospho-Y701 STAT1 in WT and Trim14 KO RAW 264.7 macrophages at 1, 2, 4, 6, and 8 h after ISD transfection. ACTIN is shown as a loading control. (C) Cellular fractionation showing nuclear STAT3 in WT and Trim14 KO RAW 264.7 macrophages treated with ISD at specified times after treatment. Histone 3 shown as loading/nuclear control. (D) WT and Trim14 KO RAW 264.7 macrophages immunostained for STAT3 6 h after ISD transfection. Nuclear translocation calculated by automated image analysis of nuclear STAT3 relative to total cellular STAT3 (n > 800 cells per genotype/condition). (E) Coimmunoprecipitation and immunoblot analysis of HEK293T cells cotransfected with FLAG-STAT3 and HA-TRIM14. (F) Coimmunoprecipitation and immunoblot analysis of TBK1 and STAT3 in RAW 264.7 macrophages stably expressing 3xFLAG-TRIM14 over a time course of ISD treatment. A total of 1% total cell lysate loaded, 16% immunoprecipitation loaded. (G) RT-qPCR of Ifit1, Isg15, and Irf7 transcripts in control (Tbk1^+/−Tnfr^−/−) and Tbk1^−/−Tnfr^−/− BMDMs treated with IFN-β (200 IU). All immunoblots are representative of >3 independent biological replicates. Statistical significance was determined using two-tailed Student t test. **p < 0.01, ***p < 0.001.

FIGURE 6.

Trim14 KO macrophages fail to induce expression of the negative regulator of the type I IFN response, Socs3. (A) RT-qPCR of Socs3, Socs1, and Usp18 transcripts in WT and Trim14 KO RAW 264.7 macrophages infected with M. tuberculosis at specified times postinfection. (B) RT-qPCR of Socs3, Socs1, and Usp18 transcripts in WT and Trim14 KO RAW 264.7 macrophages treated with ISD at specified times after treatment. (C) Quantitative PCR (qPCR) primers designed to tile Socs3 gene for ChIP experiments. (D) ChIP qPCR of STAT3-associated genomic DNA from the Socs3 locus in WT and Trim14 KO RAW 264.7 macrophages transfected with 1 μg ISD. (E) RT-qPCR of Socs3 transcript in RAW 264.7 macrophages stably expressing siRNA to either control or Socs3. (F) RT-qPCR of Ifnb transcript in RAW 264.7 macrophages stably expressing siRNA to either control or Socs3 treated with ISD at specified times after treatment. (G) RT-qPCR of Ifnb transcript in RAW 264.7 macrophages stably expressing siRNA to either control or Socs3 infected with M. tuberculosis at specified times postinfection. Statistical significance was determined using two-tailed Student t test. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 7.

Trim14 KO macrophages restrict M. tuberculosis replication via hyperinduction of iNOS. (A) Fold replication of M. tuberculosis luxBCADE in WT and Trim14 KO RAW 264.7 macrophages. (B) CFUs in WT and Trim14 KO RAW 264.7 macrophages at specified times postinfection. (C) RT-qPCR of Nos2, Gbp1, and Gbp5 transcript levels in M. tuberculosis–infected WT and Trim14 KO RAW 264.7 macrophages. (D) Fold replication of M. tuberculosis luxBCADE in WT and Trim14 KO RAW 264.7 macrophages ± 1400W dihydrochloride (iNOS inhibitor) at indicated concentration. (E) CFUs in WT and Trim14 KO RAW 264.7 macrophages ± 1400W dihydrochloride. (F) Proposed model of TRIM14’s dual role in regulating cytosolic DNA sensing. TRIM14/cGAS interaction is required to inhibit proteasomal degradation of cGAS. TRIM14/TBK1 interaction is required to promote TBK1-dependent phosphorylation of STAT3 at Ser727 and activate transcription of negative regulators of the type I IFN response like Socs3. Model created using BioRender software. **p < 0.01; ***p < 0.001.