RESUMO
BACKGROUND & AIMS: Gut bacterial translocation contributes to immune dysfunction and spontaneous bacterial peritonitis (SBP) in cirrhosis. We hypothesized that exposure of peritoneal macrophages (PMs) to bacterial DNA results in type-I interferon (IFN) production, shaping subsequent immune responses, inflammasome activation, and the release of damage-associated molecular patterns (DAMPs). METHODS: PMs from patients with cirrhosis were stimulated with E. coli single-stranded DNA (ssDNA), lipopolysaccharide and IFN, or infected with E. coli, S. aureus, and Group B streptococcus in vitro. Cytokine release, inflammasome activation, and DAMP release were quantified by quantitative-PCR, ELISA, western blots, and reporter cells employing primary PMs, monocytes, and caspase-deficient THP-1 macrophages. Serum progranulin concentration was correlated with transplant-free survival in 77 patients with SBP. RESULTS: E. coli ssDNA induced strong type-I IFN activity in PMs and monocytes, priming them for enhanced lipopolysaccharide-mediated tumor necrosis factor production without inducing toll-like receptor 4 tolerance. During in vitro macrophage bacterial infection, type-I IFN release aligned with upregulated expression of IFN-regulatory factors (IRF)1/2 and guanylate binding proteins (GBP)2/5. PMs upregulated inflammasome-associated proteins and type-I IFN upon E. coli ssDNA exposure and released interleukin-1ß upon bacterial infection. Proteomic screening in mouse macrophages revealed progranulin release as being caspase-11-dependent during E. coli infection. PMs and THP-1 macrophages released significant amounts of progranulin when infected with S. aureus or E. coli via gasdermin D in a type-I IFN- and caspase-5-dependent manner. During SBP, PMs upregulated IRF1, GBP2/5 and caspase-5 and higher serum progranulin concentrations were indicative of lower 90-day transplant-free survival after SBP. CONCLUSIONS: Type-I IFN shapes peritoneal immune responses and regulates caspase-5-mediated progranulin release during SBP. IMPACT AND IMPLICATIONS: Patients with cirrhosis exhibit impaired immune responses and increased susceptibility to bacterial infections. This study reveals that type-I interferon responses, triggered by pathogen-associated molecular patterns, are crucial in regulating macrophage activation and priming them for inflammatory responses. Additionally, we elucidate the mechanisms by which type-I interferons promote the release of progranulin from macrophages during spontaneous bacterial peritonitis. Our findings enhance understanding of how bacterial translocation affects immune responses, identify novel biomarkers for inflammasome activation during infections, and point to potential therapeutic targets.
RESUMO
TLRs mediate the recognition of microbial and endogenous insults to orchestrate the inflammatory response. TLRs localize to the plasma membrane or endomembranes, depending on the member, and rely critically on ER-resident chaperones to mature and reach their subcellular destinations. The chaperone canopy FGF signaling regulator 3 (CNPY3) is necessary for the proper trafficking of multiple TLRs including TLR1/2/4/5/9 but not TLR3. However, the exact role of CNPY3 in inflammatory signalling downstream of TLRs has not been studied in detail. Consistent with the reported client specificity, we report here that functional loss of CNPY3 in engineered macrophages impairs downstream signalling by TLR2 but not TLR3. Unexpectedly, CNPY3-deficient macrophages show reduced IL-1ß and IL-18 processing and production independent of the challenged upstream TLR species, demonstrating a separate, specific role for CNPY3 in inflammasome activation. Mechanistically, we document that CNPY3 regulates caspase-1 localization to the apoptosis speck and autoactivation of caspase-1. Importantly, we were able to recapitulate these findings in macrophages from an early infantile epileptic encephalopathy (EIEE) patient with a novel CNPY3 loss-of-function variant. Summarizing, our findings reveal a hitherto unknown, TLR-independent role of CNPY3 in inflammasome activation, highlighting a more complex and dedicated role of CNPY3 to the inflammatory response than anticipated.