Phagocyte extracellular traps formation contributes to host defense against Clostridium perfringens infection.

Clostridium perfringens (C. perfringens) is an important Gram-positive anaerobic spore-forming pathogen that provokes life-threatening gas gangrene and acute enterotoxaemia, although it colonizes as a component of the symbiotic bacteria in humans and animals. However, the mechanisms by which C. perfringens is cleared from the host remains poorly understood, thereby impeding the development of novel strategies for control this infection. Here, we uncover a beneficial effect of extracellular traps (ETs) formation on bacterial killing and clearance by phagocytes. C. perfringens strain ATCC13124, and wild-type isolates CP1 and CP3 markedly trigger ETs formation in macrophages and neutrophils. As expected, visualization of DNA decorated with histone, myeloperoxidase (MPO) and neutrophils elastase (NE) in C. perfringens-triggered classical ETs structures. Notably, the bacteria-induced ETs formation is an ERK1/2-, P38 MAPK-, store-operated calcium entry (SOCE)-, NADPH oxidase-, histone-, NE-, and MPO-dependent process, and is independent of LDH activity. Meanwhile, the defect of bactericidal activity is mediated by impairing ETs formation in phagocytes. Moreover, In vivo studies indicated that degradation of ETs by DNase I administration leads to a defect in the protection against experimental gas gangrene, with higher mortality rates, exacerbated tissue damage, and more bacterial colonization. Together, these results suggest that phagocyte ETs formation is essential for the host defense against C. perfringens infection.

MicroRNA-146a Deficiency Protects against Listeria monocytogenes Infection by Modulating the Gut Microbiota.

The gut microbiota and microRNAs play important roles in the defense against infection. However, the role of miR-146a in L. monocytogenes infection and gut microbiota remains unclear. We tried to determine whether miR-146a controlled L. monocytogenes infection by regulating the gut microbiota. Wild-type and miR-146a-deficient mice or macrophages were used to characterize the impact of miR-146a on animal survival, cell death, bacterial clearance, and gut microbiota following L. monocytogenes challenge. We found that L. monocytogenes infection induced miR-146a expression both in vitro and in vivo. When compared to wild-type mice, miR-146a-deficient mice were more resistant to L. monocytogenes infection. MiR-146a deficiency in macrophages resulted in reduced invasion and intracellular survival of L. monocytogenes. High-throughput sequencing of 16S rRNA revealed that the gut microbiota composition differed between miR-146a-deficient and wild-type mice. Relative to wild-type mice, miR-146a-deficient mice had decreased levels of the Proteobacteria phylum, Prevotellaceae family, and Parasutterella genus, and significantly increased short-chain fatty acid producing bacteria, including the genera Alistipes, Blautia, Coprococcus_1, and Ruminococcus_1. Wild-type mice co-housed with miR-146a-deficient mice had increased resistance to L. monocytogenes, indicating that miR-146a deficiency guides the gut microbiota to alleviate infection. Together, these results suggest that miR-146a deficiency protects against L. monocytogenes infection by regulating the gut microbiota.

Liver X receptors agonists suppress NLRP3 inflammasome activation.

Inflammasomes are multiprotein complexes that control the production of IL-1ß and IL-18. NLRP3 inflammasome, the most characterized inflammasome, plays prominent roles in defense against infection, however aberrant activation is deleterious and leads to diseases. Therefore, its tight control offers therapeutic promise. Liver X receptors (LXRs) have significant anti-inflammatory properties. Whether LXRs regulate inflammasome remains unresolved. We thus tested the hypothesis that LXR's anti-inflammatory properties may result from its ability to suppress inflammasome activation. In this study, LXRs agonists inhibited the induction of IL-1ß production, caspase-1 cleavage and ASC oligomerization by NLRP3 inflammasome. The agonists also inhibited inflammasome-associated mtROS production. Importantly, the agonists inhibited the priming of inflammasome activation. In vivo data also showed that LXRs agonist prevented NLRP3-dependent peritonitis. In conclusion, LXRs agonists are identified to potently suppress NLRP3 inflammasome and the regulation of LXRs signaling is a potential therapeutic for inflammasome-driven diseases.

Cytosolic double-stranded DNA induces nonnecroptotic programmed cell death in trophoblasts via IFI16.

The mechanisms underlying the immune defense by trophoblasts against pathogens remain ill defined. We demonstrated that placental cell death was increased upon in vivo exposure to Listeria monocytogenes. The death of infected cells is an important host innate defense mechanism. Meanwhile, double-stranded DNA (dsDNA) derived from intracellular bacteria or dsDNA viruses is emerging as a potent pathogen-associated molecular pattern recognized by host cells. We sought to characterize trophoblast death in response to cytosolic dsDNA challenge. Our results showed that dsDNA induced caspase-dependent and -independent cell death in human trophoblasts. However, necroptosis, a cell death pathway independent of caspase, could not be induced by dsDNA treatment, even in the presence of exogenously expressed RIPK3. L. monocytogenes-derived genomic DNA triggered a similar cell death pattern. Moreover, the cell death in response to dsDNA was IFI16 dependent. These data suggest that cytosolic dsDNA induces nonnecroptotic cell death in trophoblasts via IFI16, and this could contribute to placental barrier against infection.

G Protein-Coupled Receptor 120 Mediates Host Defense against Clostridium perfringens Infection through Regulating NOD-like Receptor Family Pyrin Domain-Containing 3 Inflammasome Activation.

Clostridium perfringens is a major cause of infectious foodborne disease, frequently associated with the consumption of raw and undercooked food. Despite intensive studies on clarifying C. perfringens pathogenesis, the molecular mechanisms of host-pathogen interactions remain poorly understood. In soft tissue and mucosal infection models, Gpr120-/- mice, G protein-coupled receptor 120 (GPR120), are more susceptible to C. perfringens infection. Gpr120 deficiency leads to a low survival rate (30 and 10%, p < 0.01), more bacterial loads in the muscle (2.26 × 108 ± 2.08 × 108 CFUs/g, p < 0.01), duodenum (2.80 × 107 ± 1.61 × 107 CFUs/g, p < 0.01), cecum (2.50 × 108 ± 2.05 × 108 CFUs/g, p < 0.01), and MLN (1.23 × 106 ± 8.06 × 105 CFUs/g, p < 0.01), less IL-18 production in the muscle (8.54 × 103 ± 1.20 × 103 pg/g, p < 0.01), duodenum (3.34 × 103 ± 2.46 × 102 pg/g, p < 0.01), and cecum (3.81 × 103 ± 5.29 × 102 pg/g, p < 0.01), and severe organ injury. Obviously, GPR120 facilitates IL-18 production and pathogen control via potassium efflux-dependent NOD-like receptor family pyrin domain-containing 3 (NLRP3) signaling. Mechanistically, GPR120 interaction with NLRP3 potentiates the NLRP3 inflammasome assembly. Thus, this study uncovers a novel role of GPR120 in host protection and reveals that GPR120 may be a potential therapeutic target for limiting pathogen infection.

The pseudokinase MLKL contributes to host defense against Streptococcus pluranimalium infection by mediating NLRP3 inflammasome activation and extracellular trap formation.

Host innate immunity plays a pivotal role in the early detection and neutralization of invading pathogens. Here, we show that pseudokinase mixed lineage kinase-like protein (MLKL) is required for host defence against Streptococcus pluranimalium infection by enhancing NLRP3 inflammasome activation and extracellular trap formation. Notably, Mlkl deficiency leads to increased mortality, increased bacterial colonization, severe destruction of organ architecture, and elevated inflammatory cell infiltration in murine models of S. pluranimalium pulmonary and systemic infection. In vivo and in vitro data provided evidence that potassium efflux-dependent NLRP3 inflammasome signalling downstream of active MLKL confers host protection against S. pluranimalium infection and initiates bacterial killing and clearance. Moreover, Mlkl deficiency results in defects in extracellular trap-mediated bactericidal activity. In summary, this study revealed that MLKL mediates the host defence response to S. pluranimalium, and suggests that MLKL is a potential drug target for preventing and controlling pathogen infection.

Erratum: Mixed lineage kinase-like protein protects against Clostridium perfringens infection by enhancing NLRP3 inflammasome-extracellular traps axis.

[This corrects the article DOI: 10.1016/j.isci.2022.105121.].

Mixed lineage kinase-like protein protects against Clostridium perfringens infection by enhancing NLRP3 inflammasome-extracellular traps axis.

Despite intense research in understanding Clostridium perfringens (C. perfringens) pathogenesis, the mechanisms by which it is cleared from the host are largely unclarified. In C. perfringens gas gangrene and enterocolitis model, Mlkl -/- mice, lacking mixed lineage kinase-like protein (MLKL), are more susceptible to C. perfringens infection. Mlkl deficiency results in a defect in inflammasome activation, and IL-18 and IL-1ß releases. Exogenous administration of recombinant IL-18 is able to rescue the susceptibility of Mlkl -/- mice. Notably, K+ efflux-dependent NLRP3 inflammasome signaling downstream of active MLKL promotes bacterial killing and clearance. Interestingly, the defect of bactericidal activity is also mediated by decreased classical extracellular trap formation in the absence of Mlkl. Our results demonstrate that MLKL mediates extracellular trap formation in a NLRP3 inflammasome-dependent manner. These findings highlight the requirement of MLKL for host defense against C. perfringens infection through enhancing NLRP3 inflammasome-extracellular traps axis.

DNA Sensor IFI204 Contributes to Host Defense Against Staphylococcus aureus Infection in Mice.

Interferon-inducible protein (IFI204) (p204, the murine homolog of human IFI16) is known as a cytosolic DNA sensor to recognize DNA viruses and intracellular bacteria. However, little is known about its role during extracellular bacterial infection. Here we show that IFI204 is required for host defense against the infection of Staphylococcus aureus, an extracellular bacterial pathogen. IFI204 deficiency results in decreased survival, increased bacterial loads, severe organs damage, and decreased recruitment of neutrophils and macrophages. Production of several inflammatory cytokines/chemokines including IFN-ß and KC is markedly decreased, as well as the related STING-IRF3 and NF-κB pathways are impaired. However, exogenous administration of recombinant KC or IFN-ß is unable to rescue the susceptibility of IFI204-deficient mice, suggesting that other mechanisms rather than KC and IFN-ß account for IFI204-mediated host defense. IFI204 deficiency leads to a defect in extracellular bacterial killing in macrophages and neutrophils, although bacterial engulf, and intracellular killing activity are normal. Moreover, the defect of bactericidal activity is mediated by decreased extracellular trap formation in the absence of IFI204. Adoptively transferred WT bone marrow cells significantly protect WT and IFI204-deficient recipients against Staphylococcus infection compared with transferred IFI204-deficient bone marrow cells. Hence, this study suggests that IFI204 is essential for the host defense against Staphylococcus infection.

Salmonella Outer Protein B Suppresses Colitis Development via Protecting Cell From Necroptosis.

Salmonella effectors translocated into epithelial cells contribute to the pathogenesis of infection. They mediate epithelial cell invasion and subsequent intracellular replication. However, their functions in vivo have not been well-identified. In this study, we uncovered a role for Salmonella outer protein B (SopB) in modulating necroptosis to facilitate bacteria escape epithelial cell and spread to systemic sites through a Salmonella-induced colitis model. Mice infected with SopB deleted strain ΔsopB displayed increased severity to colitis, reduced mucin expression and increased bacterial translocation. In vitro study, we found there was an increased goblet cell necroptosis following ΔsopB infection. Consistently, mice infected with ΔsopB had a strong upregulation of mixed lineage kinase domain-like (MLKL) phosphorylation. Deletion of MLKL rescued severity of tissue inflammatory, improved mucin2 expression and abolished the increased bacterial translocation in mice infected with ΔsopB. Intriguingly, the expression of sopB in LS174T cells was downregulated. The temporally regulated SopB expression potentially switched the role from epithelial cell invasion to bacterial transmission. Collectively, these results indicated a role for SopB in modulating the onset of necroptosis to increased bacteria pathogenesis and translocated to systemic sites.

Non-Hematopoietic MLKL Protects Against Salmonella Mucosal Infection by Enhancing Inflammasome Activation.

The intestinal mucosal barrier is critical for host defense against pathogens infection. Here, we demonstrate that the mixed lineage kinase-like protein (MLKL), a necroptosis effector, promotes intestinal epithelial barrier function by enhancing inflammasome activation. MLKL-/- mice were more susceptible to Salmonella infection compared with wild-type counterparts, with higher mortality rates, increased body weight loss, exacerbated intestinal inflammation, more bacterial colonization, and severe epithelial barrier disruption. MLKL deficiency promoted early epithelial colonization of Salmonella prior to developing apparent intestinal pathology. Active MLKL was predominantly expressed in crypt epithelial cells, and experiments using bone marrow chimeras found that the protective effects of MLKL were dependent on its expression in non-hematopoietic cells. Intestinal mucosa of MLKL-/- mice had impaired caspase-1 and gasdermin D cleavages and decreased interleukin (IL)-18 release. Moreover, administration of exogenous recombinant IL-18 rescued the phenotype of increased bacterial colonization in MLKL-/- mice. Thus, our results uncover the role of MLKL in enhancing inflammasome activation in intestinal epithelial cells to inhibit early bacterial colonization.

Cirtical role for Salmonella effector SopB in regulating inflammasome activation.

OBJECTIVE: Salmonella is known to evolve many mechanisms to avoid or delay inflammasome activation which remain largely unknown. In this study, we investigated whether the SopB protein critical to bacteria virulence capacity was an effector that involved in the regulation of inflammasome activation. METHODS: BMDMs from NLRC4-, NLRP3-, caspase-1/-11-, IFI16- and AIM2-deficient mice were pretreated with LPS, and subsequently stimulated with a series of SopB-related strains of Salmonella, inflammasome induced cell death, IL-1ß secretion, cleaved caspase-1 production and ASC speckle formation were detected. RESULTS: We found that SopB could inhibit host IL-1ß secretion, caspase-1 activation and inflammasome induced cell death using a series of SopB-related strains of Salmonella; however the reduction of IL-1ß secretion was not dependent on sensor that contain PYD domain, such as NLRP3, AIM2 or IFI16, but dependent on NLRC4. Notably, SopB specifically prevented ASC oligomerization and the enzymatic activity of SopB was responsible for the inflammasome inhibition. Furthermore, inhibition of Akt signaling induced enhanced inflammasome activation. CONCLUSIONS: These results revealed a novel role in inhibition of NLRC4 inflammasome for Salmonella effector SopB.

Decidual Stromal Cell Necroptosis Contributes to Polyinosinic-Polycytidylic Acid-Triggered Abnormal Murine Pregnancy.

Infectious agents can reach the placenta either via the maternal blood or by ascending the genito-urinary tract, and then initially colonizing the maternal decidua. Decidual stromal cells (DSCs) are the major cellular component of the decidua. Although DSCs at the maternal-fetal interface contribute to the regulation of immunity in pregnancy in the face of immunological and physiological challenges, the roles of these DSCs during viral infection remain ill defined. Here, we characterized the response of DSCs to a synthetic double-stranded RNA molecule, polyinosinic-polycytidylic acid [poly(I:C)], which is a mimic of viral infection. We demonstrated that both transfection of cells with poly(I:C) and addition of extracellular (non-transfected) poly(I:C) trigger the necroptosis of DSCs and that this response is dependent on RIG-I-like receptor/IPS-1 signaling and the toll-like receptor 3/TIR-domain-containing adapter-inducing interferon-ß pathway, respectively. Furthermore, following poly(I:C) challenge, pregnant mixed lineage kinase domain-like protein-deficient mice had fewer necrotic cells in the mesometrial decidual layer, as well as milder pathological changes in the uterine unit, than did wild-type mice. Collectively, our results establish that necroptosis is a contributing factor in poly(I:C)-triggered abnormal pregnancy and thereby indicate a novel therapeutic strategy for reducing the severity of the adverse effects of viral infections in pregnancy.

RCAN1 deficiency protects against Salmonella intestinal infection by modulating JNK activation.

OBJECTIVE: RCAN1 (regulator of calcineurin 1) has been shown to be involved in various physiological and pathological processes. However, the biological implications of RCAN1 during gastrointestinal tract infection remain unclear. In this study, we tried to determine the role of RCAN1 in acute Salmonella infectious colitis. METHODS: Wild type and RCAN1-deficient mice or macrophages were used to characterize the impacts of RCAN1 on intestinal inflammation, inflammatory cytokines production, animal survival, and pathogen clearance following Salmonella challenge. RESULTS: Histologic and quantitative assessments showed increased inflammation and elevated proinflammatory cytokines production in RCAN1-deficient mice. The aberrant inflammatory response was recapitulated in primary bone marrow-derived macrophages. In addition, we reveal a novel regulatory role for RCAN1 in the proinflammatory JNK signaling both in vitro and in vivo. Further analysis showed that the increased inflammation in RCAN1-deficient mice contributed to pathogen clearance and host survival. CONCLUSIONS: The present study demonstrates that RCAN1 deficiency protects against Salmonella intestinal infection by enhancing proinflammatory JNK signaling.

IFI16 mediates soluble Flt-1 and endoglin production by trophoblast cells.

OBJECTIVES: Preeclampsia is a serious pregnancy-specific hypertensive syndrome that is characterized by widespread maternal endothelial dysfunction. Previous studies have shown that increased levels of circulating cell-free fetal DNA in women with preeclampsia correspond to the degree of disease severity; however, it is unknown whether this DNA is a key signal that contributes to the development of preeclampsia. The detection of DNA is critical to appropriate innate immune responses. The interferon-inducible protein 16 (IFI16) - a member of the HIN-200 family - is an innate immune receptor for intracellular DNA, which is implicated in the control of cell growth, apoptosis, angiogenesis, and immunomodulation; however, its role in preeclampsia remains unresolved. Here, we tested the hypothesis that this DNA can activate IFI16 in the placentas of women with preeclampsia and is sufficient to induce soluble fms-like tyrosine kinase 1 (sFlt-1) and soluble endoglin (sEng) production. METHODS: We characterized IFI16 in severe preeclamptic placentas and assessed whether DNA increased the release of sFlt-1 and sEng from trophoblast cells and placental explants. Furthermore, we determined whether IFI16 was involved in DNA-induced sFlt-1 and sEng production. RESULTS: Placental immunoreactivity and protein levels of IFI16 were significantly increased in women with preeclampsia compared to matched control women. Treatment of human trophoblasts with the IFI16 agonist poly(dA:dT) significantly increased IFI16 levels. Furthermore, poly(dA:dT) induced sFlt-1 and sEng production by human trophoblasts in an IFI16-dependent manner. CONCLUSIONS: We conclude that trophoblast cells respond to cell-free fetal DNA through the IFI16 receptor, resulting in the production of the preeclampsia-related antiangiogenic factors sFlt-1 and sEng.

Genipin inhibits NLRP3 and NLRC4 inflammasome activation via autophagy suppression.

Inflammasomes are cytoplasmic, multiprotein complexes that trigger caspase-1 activation and IL-1ß maturation in response to diverse stimuli. Although inflammasomes play important roles in host defense against microbial infection, overactive inflammasomes are deleterious and lead to various autoinflammatory diseases. In the current study, we demonstrated that genipin inhibits the induction of IL-1ß production and caspase-1 activation by NLRP3 and NLRC4 inflammasomes. Furthermore, genipin specifically prevented NLRP3-mediated, but not NLRC4-mediated, ASC oligomerization. Notably, genipin inhibited autophagy, leading to NLRP3 and NLRC4 inflammasome inhibition. UCP2-ROS signaling may be involved in inflammasome suppression by genipin. In vivo, we showed that genipin inhibited NLRP3-dependent IL-1ß production and neutrophil flux in LPS- and alum-induced murine peritonitis. Additionally, genipin provided protection against flagellin-induced lung inflammation by reducing IL-1ß production and neutrophil recruitment. Collectively, our results revealed a novel role in inhibition of inflammatory diseases for genipin that has been used as therapeutics for centuries in herb medicine.