Requirement of scavenger receptors for activation of the IRF-3/IFN-ß/STAT-1 pathway in TLR4-mediated production of NO by LPS-activated macrophages.

Production of nitric oxide (NO) by LPS-activated macrophages is due to a complex cellular signaling initiated by TLR4 that leads to the transcription of IFN-ß, which activates IRF-1 and STAT-1, as well as to the activation of NF-κB, required for iNOS transcription. High concentrations of LPS can also be uptaken by scavenger receptors (SRs), which, in concert with TLR4, leads to inflammatory responses. The mechanisms by which TLR4 and SRs interact, and the pathways activated by this interaction in macrophages are not elucidated. Therefore, our main goal was to evaluate the role of SRs, particularly SR-A, in LPS-stimulated macrophages for NO production. We first showed that, surprisingly, LPS can induce the expression of iNOS and the production of NO in TLR4-/- mice, provided exogenous IFN-ß is supplied. These results indicate that LPS stimulate receptors other than TLR4. The inhibition of SR-A using DSS or neutralizing antibody to SR-AI showed that SR-A is essential for the expression of iNOS and NO production in stimulation of TLR4 by LPS. The restoration of the ability to express iNOS and produce NO by addition of rIFN-ß to inhibited SR-A cells indicated that the role of SR-AI in LPS-induced NO production is to provide IFN-ß, probably by mediating the internalization of LPS/TLR4, and the differential inhibition by DSS and neutralizing antibody to SR-AI suggested that other SRs are also involved. Our results reinforce that TLR4 and SR-A act in concert in LPS activation and demonstrated that, for the production of NO, it does mainly by synthesizing IRF-3 and also by activating the TRIF/IRF-3 pathway for IFN-ß production, essential for LPS-mediated transcription of iNOS. Consequently STAT-1 is activated, and IRF-1 is expressed, which together with NF-κB from TLR4/MyD88/TIRAP, induce iNOS synthesis and NO production. SUMMARY SENTENCE: TLR4 and SRs act in concert activating IRF-3 to transcribe IFN-ß and activate STAT-1 to produce NO by LPS-activated macrophages.

STING is an intrinsic checkpoint inhibitor that restrains the TH17 cell pathogenic program.

External and intrinsic factors regulate the transcriptional profile of T helper 17 (TH17) cells, thereby affecting their pathogenic potential and revealing their context-dependent plasticity. The stimulator of interferon genes (STING), a component of the intracellular DNA-sensing pathway, triggers immune responses but remains largely unexplored in T cells. Here, we describe an intrinsic role of STING in limiting the TH17 cell pathogenic program. We demonstrate that non-pathogenic TH17 cells express higher levels of STING than those activated under pathogenic conditions. Activation of STING induces interleukin-10 (IL-10) production in TH17 cells, decreasing IL-17A and IL-23R expression in a type I interferon (IFN)-independent manner. Mechanistically, STING-induced IL-10 production partially requires aryl hydrocarbon receptor (AhR) signaling, while the decrease of IL-17A expression occurs due to a reduction of Rorγt transcriptional activity. Our findings reveal a regulatory function of STING in the TH17 cell activation program, proposing it as a valuable target to limit TH17-cell-mediated inflammation.

Natterin-Induced Neutrophilia Is Dependent on cGAS/STING Activation via Type I IFN Signaling Pathway.

Natterin is a potent pro-inflammatory fish molecule, inducing local and systemic IL-1ß/IL-1R1-dependent neutrophilia mediated by non-canonical NLRP6 and NLRC4 inflammasome activation in mice, independent of NLRP3. In this work, we investigated whether Natterin activates mitochondrial damage, resulting in self-DNA leaks into the cytosol, and whether the DNA sensor cGAS and STING pathway participate in triggering the innate immune response. Employing a peritonitis mouse model, we found that the deficiency of the tlr2/tlr4, myd88 and trif results in decreased neutrophil influx to peritoneal cavities of mice, indicative that in addition to MyD88, TRIF contributes to neutrophilia triggered by TLR4 engagement by Natterin. Next, we demonstrated that gpcr91 deficiency in mice abolished the neutrophil recruitment after Natterin injection, but mice pre-treated with 2-deoxy-d-glucose that blocks glycolysis presented similar infiltration than WT Natterin-injected mice. In addition, we observed that, compared with the WT Natterin-injected mice, DPI and cyclosporin A treated mice had a lower number of neutrophils in the peritoneal exudate. The levels of dsDNA in the supernatant of the peritoneal exudate and processed IL-33 in the supernatant of the peritoneal exudate or cytoplasmic supernatant of the peritoneal cell lysate of WT Natterin-injected mice were several folds higher than those of the control mice. The recruitment of neutrophils to peritoneal cavity 2 h post-Natterin injection was intensely impaired in ifnar KO mice and partially in il-28r KO mice, but not in ifnγr KO mice. Finally, using cgas KO, sting KO, or irf3 KO mice we found that recruitment of neutrophils to peritoneal cavities was virtually abolished in response to Natterin. These findings reveal cytosolic DNA sensors as critical regulators for Natterin-induced neutrophilia.

Natterin-induced neutrophilia is dependent on cGAS/STING activation via type i IFN signaling pathway

Natterin is a potent pro-inflammatory fish molecule, inducing local and systemic IL-1β/IL-1R1-dependent neutrophilia mediated by non-canonical NLRP6 and NLRC4 inflammasome activation in mice, independent of NLRP3. In this work, we investigated whether Natterin activates mitochondrial damage, resulting in self-DNA leaks into the cytosol, and whether the DNA sensor cGAS and STING pathway participate in triggering the innate immune response. Employing a peritonitis mouse model, we found that the deficiency of the tlr2/tlr4, myd88 and trif results in decreased neutrophil influx to peritoneal cavities of mice, indicative that in addition to MyD88, TRIF contributes to neutrophilia triggered by TLR4 engagement by Natterin. Next, we demonstrated that gpcr91 deficiency in mice abolished the neutrophil recruitment after Natterin injection, but mice pre-treated with 2-deoxy-d-glucose that blocks glycolysis presented similar infiltration than WT Natterin-injected mice. In addition, we observed that, compared with the WT Natterin-injected mice, DPI and cyclosporin A treated mice had a lower number of neutrophils in the peritoneal exudate. The levels of dsDNA in the supernatant of the peritoneal exudate and processed IL-33 in the supernatant of the peritoneal exudate or cytoplasmic supernatant of the peritoneal cell lysate of WT Natterin-injected mice were several folds higher than those of the control mice. The recruitment of neutrophils to peritoneal cavity 2 h post-Natterin injection was intensely impaired in ifnar KO mice and partially in il-28r KO mice, but not in ifnγr KO mice. Finally, using cgas KO, sting KO, or irf3 KO mice we found that recruitment of neutrophils to peritoneal cavities was virtually abolished in response to Natterin. These findings reveal cytosolic DNA sensors as critical regulators for Natterin-induced neutrophilia.

PARP inhibitor niraparib as a radiosensitizer promotes antitumor immunity of radiotherapy in EGFR-mutated non-small cell lung cancer.

BACKGROUND: Poly-(ADP-Ribose)-Polymerase inhibitors (PARPi) were reported as radiosensitizers in non-small cell lung cancer (NSCLC) with wide-type epidermal growth factor receptor (EGFR), but the effects of radiation combined with PARPi were not investigated in EGFR-mutated NSCLC. Moreover, the underlying mechanisms were not well examined. This study aimed to study the efficacy of radiation combined with niraparib in EGFR-mutated NSCLC and explore their influence on the immune system. METHODS: Clone formation and apoptosis assay were conducted to explore the effects of niraparib and radiation. Immunofluorescence was conducted to detect the double-strand DNA breaks. Real-time PCR and immunoblotting were employed to evaluate the activation of STING/TBK1/TRF3 pathway and the expression levels of interferon ß, CCL5 and CXCL10. Immunocompetent mice model bearing with subcutaneous Lewis lung cancer was established to confirm the results in vivo. RESULTS: Niraparib and radiation were synergistic to inhibit tumor both in vitro and in vivo. Radiation plus niraparib could activate anti-tumor immunity, which appeared as increased CD8+ T lymphocytes and activated STING/TBK1/IRF3 pathway. CONCLUSION: PARPi not only as a radiosensitizer inhibited EGFR-mutated NSCLC tumor growth, but also cooperated with radiation to promote anti-tumor immune responses.

Association of an IRF3 putative functional uORF variant with resistance to Brucella infection: A candidate gene based analysis of InDel polymorphisms in goats.

Brucellosis is an important zoonotic disease caused by infection with Brucella spp. It generates major economic losses in livestock production worldwide. Goats are the principal hosts of B. melitensis, the main infection agent of caprine and human brucellosis. The selection of resistance-related genes is considered one of the best long-term means to improve control to bacterial infection in domestic ruminants. We performed a candidate gene association study to test if six short insertion/deletion polymorphisms (InDels) at bacterial-infection related genes influence the resistance to Brucella infection in female creole goats. InDels (IRF3-540: rs660531540, FKBP5-294: rs448529294, TIRAP-561: rs657494561, PTPRT-588: rs667380588, KALRN-989: rs667660989 and RAB5a-016: rs661537016) were resolved by PCR-capillary electrophoresis in samples from 64 cases and 64 controls for brucellosis. Allelic frequencies were significantly different between cases and controls at IRF3-540 and KALRN-989 (p = 0.001 and 0.005). Indeed, the minor alleles (a and k) at InDels IRF3-540 and KALRN-989 were more frequent among controls than cases, providing evidence that these alleles confer protection against Brucella infection. Moreover, IRF3-540 a-containing genotypes (Aa and aa) were associated with absence of Brucella-specific antibodies in goats (p = 0.003; OR = 3.52; 95% CI = 1.55-7.96), and more specifically, a-allele was associated with resistance to Brucella infection in a dose-dependent manner. Also, we observed that the IRF3-540 deletion (a-allele) extends a conserved upstream ORF by 75 nucleotides to the main ORF, and thus it may decrease gene expression by reducing translation efficiency from the main ORF. These results suggest a potential functional role of IRF3-540 deletion in genetic resistance to Brucella infection in goats.

Respiratory Syncytial Virus Persistence in Murine Macrophages Impairs IFN-ß Response but Not Synthesis.

Type-I interferon (IFN-I) production is an early response to viral infection and pathogenic viruses have evolved multiple strategies to evade this cellular defense. Some viruses can establish and maintain persistent infections by altering the IFN-I signaling pathway. Here, we studied IFN-I synthesis and response in an in vitro model of persistent infection by respiratory syncytial virus (RSV) in a murine macrophage-like cell line. In this model, interferon regulatory factor 3 was constitutively active and located at nuclei of persistently infected cells, inducing expression of IFN-beta mRNA and protein. However, persistently infected macrophages did not respond in an autocrine manner to the secreted-IFN-beta or to recombinant-IFN-beta, since phosphorylated-STAT1 was not detected by western blot and transcription of the interferon-stimulated genes (ISGs) Mx1 and ISG56 was not induced. Treatment of non-infected macrophages with supernatants from persistently infected cells induced STAT1 phosphorylation and ISGs expression, mediated by the IFN-I present in the supernatants, because blocking the IFN-I receptor inhibited STAT1 phosphorylation. Results suggest that the lack of autocrine response to IFN-I by the host cell may be one mechanism for maintenance of RSV persistence. Furthermore, STAT1 phosphorylation and ISGs expression induced in non-infected cells by supernatants from persistently infected macrophages suggest that RSV persistence may trigger a proinflammatory phenotype in non-infected cells as part of the pathogenesis of RSV infection.

Interferon regulatory factor 3 as key element of the interferon signature in plasmacytoid dendritic cells from systemic lupus erythematosus patients: novel genetic associations in the Mexican mestizo population.

Many genetic studies have found an association between interferon regulatory factors (IRF) single nucleotide polymorphisms (SNPs) and systemic lupus erythematosus (SLE); however, specific dendritic cell (DC) alterations have not been assessed. The aim of the present study was to address the expression of IRF3 and IRF5 on different DC subsets from SLE patients, as well as their association with interferon (IFN)-α production and novel SNPs. For the genetic association analyses, 156 SLE patients and 272 healthy controls from the Mexican mestizo population were included. From these, 36 patients and 36 controls were included for functional analysis. Two IRF3 SNPs - rs2304206 and rs2304204 - were determined. We found an increased percentage of circulating pDC in SLE patients in comparison to controls (8.04 ± 1.48 versus 3.35 ± 0.8, P = 0.032). We also observed enhanced expression of IRF3 (64 ± 6.36 versus 36.1 ± 5.57, P = 0.004) and IRF5 (40 ± 5.25 versus 22.5 ± 2.6%, P = 0.010) restricted to this circulating pDC subset from SLE patients versus healthy controls. This finding was associated with higher IFN-α serum levels in SLE (160.2 ± 21 versus 106.1 ± 14 pg/ml, P = 0.036). Moreover, the IRF3 rs2304206 polymorphism was associated with increased susceptibility to SLE [odds ratio (OR), 95% confidence interval (CI) = 2.401 (1.187-4.858), P = 0.021] as well as enhanced levels of serum type I IFN in SLE patients who were positive for dsDNA autoantibodies. The IRF3 rs2304204 GG and AG genotypes conferred decreased risk for SLE. Our findings suggest that the predominant IRF3 expression on circulating pDC is a key element for the increased IFN-α activation based on the interplay between the rs2304206 gene variant and the presence of dsDNA autoantibodies in Mexican mestizo SLE patients.