Flagellin is recognized by both Toll-like receptor (TLR)5 and NAIP5/NLRC4 inflammasome receptors. We hypothesized that the flagellins derived from different bacteria might differentially activate TLR5 and/or NAIP5/NLRC4 signal pathways. To test this, the immune recognition of recombinant flagellins derived from pathogenic Salmonella Typhi (SF) and the nonpathogenic Escherichia coli K12 strain MG1655 (KF) were examined by the activation of TLR5 and NLRC4 pathways in various cell types. While flagellins SF and KF were not distinguishable in activating the TLR5 pathway, KF induced significantly less interleukin-1β production and pyroptotic cell death in peritoneal macrophages than SF, and showed markedly lower efficiency in activating caspase-1 through the NLRC4 pathway than SF. Macrophages may differentially recognize flagellins by intracellular sensors and thereby initiate the immune response to invading pathogenic bacteria. Our findings suggest an active role of flagellin as an important determinant in host differential immune recognition and for the control of bacteria infection.

Purpose: The objective of this study was to investigate the effect of miR-148a on the polarization and recruitment of tumor-associated macrophages (TAMs). Methods: In human monocyte THP-1 cells, M1 or M2 differentiation was induced by phorbol 12-myristate 13-acetate (PMA) with specific induction supplements and identified using flow cytometry and ELISA. To alter cellular miR-148a expression level, THP-1 cells were transfected with miR-148a mimics or inhibitors. A dual-luciferase assay was used to determine whether miR-148a could directly regulate the expression of signal regulatory protein α (SIRPα). Expression of miR-148a and SIRPα was detected with RT-PCR or Western blot. A co-culture system of THP-1 cells and colorectal cancer SW480 cells was used for TAM induction. The recruitment of macrophage to SW480 cells was measured using chemotaxis assay. In SW480 cells, apoptosis induced by macrophages was detected using flow cytometry and a xenograft assay. Macrophage infiltration was detected by immunofluorescence assay in tumor tissues. Results: miR-148a over-expression increased M1-related CD86 expression in THP-1 cells and promoted differentiation to M1-like macrophages. Inhibition of miR-148a increased M2-related CD206 expression and promoted differentiation to M2-like macrophages. In the co-culture system, THP-1 cells were induced to the M2-like state by SW480 cells. The level of miR-148a negatively correlated with the levels of M2-related cytokines. Additionally, miR-148a expression level was negatively associated with macrophage recruitment by colorectal cancer cells. Furthermore, in miR-148a over-expression, the number of macrophages recruited by SW480 cells was reduced. Meanwhile, cancer cell apoptosis induced by macrophages was enhanced. Thus, miR-148a expression was beneficial for the transformation of macrophages, from an immune-suppressive status to an immune-promoting status. These anti-cancer effects of miR-148a were related to the down-regulation of SIRPα in macrophages, directly targeted by miR-148a. A xenograft assay showed that the co-inoculation of macrophages over-expressing miR-148a reduced subcutaneous tumorigenesis and M2 macrophage infiltration. Conclusion: miR-148a promoted the differentiation of M0 macrophages into anti-tumor classical activation type M1, and reduced TAM recruitment by targeting SIRPα to inhibit colorectal cancer cell viability.

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.

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.