Zhenyu Li

The Salmonella enterica serovar Typhimurium (ST) mutant lacking the msbB gene (ΔmsbB) has been widely studied as a candidate for attenuated bacterial vectors in therapeutic applications. Deletion of msbB results in LPS with under-acylated lipid A, which lowers endotoxicity while maintaining structural integrity. This attenuation has traditionally been attributed to reduced TLR4 activation due to weaker interaction between the modified lipid A and TLR4. In our study, we confirmed that ΔmsbB ST was less lethal than wild-type (WT) ST in a mouse sepsis model. However, this difference persisted even in TLR4- and caspase-11-deficient mice, suggesting that LPS signaling is not the primary determinant of virulence. In vitro, bone marrow–derived macrophages (BMDMs) from TLR4- or caspase-11-deficient mice showed only modest reductions in ST-induced cell death and cytokine production. Importantly, ΔmsbB ST behaved similarly to WT ST in these assays, further indicating that LPS-mediated signaling is not central to the observed attenuation. Our previous studies showed that ST-induced mortality in mice is primarily mediated through NLRC4 activation. Using qPCR and immunoblotting, we found that expression of NLRC4 activators was diminished in the ΔmsbB strain. Additionally, the mutant exhibited increased outer membrane permeability—likely contributing to its heightened antibiotic sensitivity—and reduced motility due to lower flagellin protein levels. In summary, the attenuation of virulence observed in the ΔmsbB strain is not directly due to altered LPS–TLR4 interactions, but rather an indirect effect of diminished expression of virulence factors that activate the NLRC4 inflammasome.

Toll-like receptor 3 (TLR3) initiates antiviral and inflammatory responses exclusively through the adaptor protein TRIF (TIR-domain-containing adapter-inducing interferon-β). In contrast, MyD88 (myeloid differentiation primary response 88), a central adaptor for most other TLRs, is traditionally considered dispensable for TLR3 signaling. Here, we demonstrate that MyD88 directly contributes to TLR3-mediated NF-κB activation and cytokine production in macrophages. Bone marrow-derived macrophages (BMDMs) from MyD88 deficient mice exhibited significantly attenuated NF-κB activation in response to the TLR3 agonist polyinosinic–polycytidylic acid (poly(I:C)) compared to wild-type cells, as evidenced by the reduced phosphorylation of NF-κB p65 and IκBα, as well as IκBα degradation. Consistently, pro-inflammatory cytokine production, including IL-6, TNF-α, and IFN-β, was attenuated in MyD88-deficient BMDMs in vitro following stimulation by poly(I:C) or poly(A:U), another TLR3 agonist. Blood concentrations of IL-6, TNF-α, and IFN-β were significantly reduced in both TRIF-deficient mice and MyD88-deficient mice challenged by the i.p. injection of poly(I:C). Mechanistic analyses revealed that MyD88 physically associates with activated TLR3 upon poly(I:C) stimulation, and that TLR3 engagement triggered MyD88 oligomerization, which was absent in TLR3 or TRIF deficient macrophages. Our findings highlight a previously unrecognized dual-adaptor mechanism for TLR3, wherein MyD88 recruitment amplifies NF-κB signaling dynamics by bridging TLR3 to the canonical NF-κB activation cascade and robust cytokine induction. This study expands the paradigm of TLR3 signaling by establishing MyD88 as a direct contributor to TLR3-driven innate immune responses, offering new insight into cross-talk between MyD88-dependent and -independent pathways.

Introduction: Klebsiella pneumoniae is a Gram-negative bacterium and the third most commonly isolated microorganism in blood cultures from septic patients. Despite extensive research, the mechanisms underlying K. pneumoniae-induced sepsis and its pathogenesis remain unclear. Acute respiratory failure is a leading cause of mortality in systemic K. pneumoniae infections, highlighting the need to better understand the host immune response and bacterial clearance mechanisms.Method: To investigate the impact of K. pneumoniae infection on organ function and immune response, we utilized a systemic infection model through intraperitoneal injection in mice. Bacterial loads in key organs were quantified, and lung injury was assessed. Survival analysis was performed in wild-type (WT) and gene deficient mice. Mitochondrial damage and reactive oxygen species (ROS) production, as well as cytokine levels were measured in macrophages isolated from these mice to evaluate their contribution to bacterial clearance capacity.Results: Our findings demonstrate that K. pneumoniae systemic infection results in severe lung injury and significant bacterial accumulation in multiple organs, with the highest burden in the lungs. Deficiency of caspase-11 or NLRP3 led to prolonged survival, a reduction in pulmonary bacterial load, increased blood oxygen levels, and decreased IL-6 levels in the lungs compared to WT controls. Furthermore, caspase-11- and NLRP3-deficient macrophages exhibited elevated mitochondrial ROS production in response to K. pneumoniae, which correlated with more effective bacterial clearance.Discussion: These results suggest that caspase-11 and NLRP3 contribute to K. pneumoniae-induced sepsis by impairing mitochondrial function and reducing ROS production in macrophages, thereby compromising bacterial clearance. The observed reduction in lung injury and increased survival in caspase-11- and NLRP3-deficient mice indicate that targeting these pathways may offer potential therapeutic strategies to improve host defense against systemic K. pneumoniae infection.

Pyroptosis is a form of proinflammatory cell death characterized by inflammasome activation, pore formation, and the release of pro-inflammatory cytokines such as interleukin-1β (IL-1β) and IL-18 upon cell rupture. Nuclear factor-κB (NF-κB), a prototypical pro-inflammatory transcription factor, plays a critical role in immune system regulation. Recent research highlights the multifaceted roles of NF-κB signaling in pyroptosis. Various immunologically relevant ligands and their receptors can activate the NF-κB pathway to promote pyroptosis, with Toll-like receptors (TLRs), IL-1 receptors (IL-1Rs), and TNF receptors (TNFRs) being the most prominent. NF-κB regulates the transcription of key components of inflammasomes involved in pyroptosis, particularly the NLRP3 inflammasome. Recent studies also indicate that NF-κB modulates the activation of NLRC4 and AIM2 inflammasomes through distinct pathways in diverse inflammatory conditions, such as acute lung injury and neuroinflammation. Additionally, the NF-κB pathway mediates the production of inflammatory cytokines, including IL-1β, IL-33, and TNF-α, which further regulate pyroptosis. This review examines recent advances in understanding the role of the NF-κB signaling pathway in regulating pyroptosis during infection and inflammation.

Histones are cationic nuclear proteins that are essential for the structure and functions of eukaryotic chromatin. However, extracellular histones trigger inflammatory responses and contribute to death in sepsis by unknown mechanisms. We recently reported that inflammasome activation and pyroptosis trigger coagulation activation through a tissue-factor (TF)-dependent mechanism. We used a combination of various deficient mice to elucidate the molecular mechanism of histone-induced coagulation. We showed that histones trigger coagulation activation in vivo, as evidenced by coagulation parameters and fibrin deposition in tissues. However, histone-induced coagulopathy was neither dependent on intracellular inflammasome pathways involving caspase 1/11 and gasdermin D (GSDMD), nor on cell surface receptor TLR2- and TLR4-mediated host immune response, as the deficiency of these genes in mice did not protect against histone-induced coagulopathy. The incubation of histones with macrophages induced lytic cell death and phosphatidylserine (PS) exposure, which is required for TF activity, a key initiator of coagulation. The neutralization of TF diminished the histone-induced coagulation. Our findings revealed lytic cell death as a novel mechanism of histone-induced coagulation activation and thrombosis.