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.

An IgD-Fc-Ig fusion protein restrains the activation of T and B cells by inhibiting IgD-IgDR-Lck signaling in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by synovitis and the destruction of small joints. Emerging evidence shows that immunoglobulin D (IgD) stimulation induces T-cell activation, which may contribute to diseases pathogenesis in RA. In this study, we investigated the downstream signaling pathways by which IgD activated T cells as well as the possible role of IgD in the T-B interaction. Peripheral blood mononuclear cells were isolated from peripheral blood of healthy controls and RA patients. We demonstrated that IgD activated T cells through IgD receptor (IgDR)-lymphocyte-specific protein tyrosine kinase (Lck)-zeta-associated protein 70 (ZAP70)/phosphatidylinositol 3-kinase (PI3K)/nuclear factor kappa-B (NF-κB) signaling pathways; IgD-induced CD4+ T cells promoted the proliferation of CD19+ B cells in RA patients. A novel fusion protein IgD-Fc-Ig (composed of human IgD-Fc domain and IgG1 Fc domain, which specifically blocked the IgD-IgDR binding) inhibited the coexpression of IgDR and phosphorylated Lck (p-Lck) and the expression levels of p-Lck, p-ZAP70, p-PI3K on CD4+ T cells, and decreased NF-κB nuclear translocation in Jurkat cells. Meanwhile, IgD-Fc-Ig downregulated the expression levels of CD40L on CD4+ T cells as well as CD40, CD86 on CD19+ B cells in RA patients and healthy controls. It also decreased the expression levels of CD40L on CD4+ T cells and CD40 on CD19+ B cells from spleens of collagen-induced arthritis (CIA) mice and reduced IL-17A level in mouse serum. Moreover, administration of IgD-Fc-Ig (1.625-13 mg/kg, iv, twice a week for 4 weeks) in CIA mice dose-dependently decreased the protein expression levels of CD40, CD40L, and IgD in spleens. IgD-Fc-Ig restrains T-cell activation through inhibiting IgD-IgDR-Lck-ZAP70-PI3K-NF-κB signaling, thus inhibiting B-cell activation. Our data provide experimental evidences for application of IgD-Fc-Ig as a highly selective T cell-targeting treatment for RA.

Pra-C exerts analgesic effect through inhibiting microglial activation in anterior cingulate cortex in complete Freund's adjuvant-induced mouse model.

Chronic pain is highly prevalent worldwide and severely affects daily lives of patients and family members. Praeruptorin C (Pra-C) is a main active ingredient derived from Peucedanum praeruptorum Dunn, traditionally used as antibechic, anti-bronchitis and anti-hypertension drug. Here, we evaluated the effects of Pra-C in a chronic inflammatory pain mouse model induced by complete Freund's adjuvant (CFA) injection. Pra-C (3 mg/kg) treatment for just 3 days after CFA challenge relieved CFA-induced mechanical allodynia and hindpaw edema in mice. In the anterior cingulate cortex (ACC), Pra-C treatment inhibited microglia activation and reduced levels of proinflammatory cytokines, TNF-α and IL-1ß, and suppressed upregulation of glutamate receptors caused by CFA injection. In addition, Pra-C attenuated neuronal hyperexcitability in ACC of CFA-injected mice. In vitro studies confirmed the analgesic effect of Pra-C was due to its inhibitory ability on microglial activation. In conclusion, Pra-C administration had a certain effect on relieving chronic pain by inhibiting microglial activation, attenuating proinflammatory cytokine releasing and regulating excitatory synaptic proteins in the ACC of the CFA-injected mice.

BAFF, involved in B cell activation through the NF-κB pathway, is related to disease activity and bone destruction in rheumatoid arthritis.

B cell activating factor of TNF family (BAFF) is a member of TNF ligand superfamily and plays a key role in B cell homeostasis, proliferation, maturation, and survival. In this study, we detected BAFF level, the expressions of BAFF receptors and important molecules in NF-κB pathway in rheumatoid arthritis (RA) patients and analyzed the correlation between BAFF level and clinical variables, laboratory parameters or X-ray scores in order to elucidate the roles of BAFF in RA. A total of 50 RA patients and 50 healthy controls (HCs) were enrolled. We showed that the serum BAFF level in RA patients was significantly higher than that of HCs, and the percentages of B cell subsets (CD19+ B cells, CD19+CD27+ B cells, CD19+CD20+CD27+ B cells, and CD19+CD20-CD27+ B cells) in the serum of RA patients were significantly increased compared with those of HCs. The percentages of CD19+BAFFR+ B cells, CD19+ BCMA+ B cells, and CD19+ TACI+ B cells in RA patients were significantly increased compared with those in HCs. The expression of important molecules in the NF-κB pathway (MKK3, MKK6, p-P38, p-P65, TRAF2, and p52) was significantly higher in RA patients than in HCs, but p100 level in RA patients was lower than that in HCs. The serum BAFF level was positively correlated with C-reactive protein, rheumatoid factor, disease activity score (in 28 joints), swollen joint counts, tender joint counts, and X-ray scores. When normal B cells were treated with BAFF in vitro, the percentages of the B cell subset and the expression of BAFF receptors were significantly upregulated. BAFF also promoted the expression of MKK3, MKK6, p-P38, p-P65, TRAF2, and p52. In conclusion, this study demonstrates that BAFF level is correlated with the disease activity and bone destruction of RA. BAFF is involved in the differentiation, proliferation, and activation of B cells in RA through NF-κB signaling pathway, suggesting that BAFF might be an ideal therapeutic target for RA.

IgD-Fc-Ig fusion protein, a new biological agent, inhibits T cell function in CIA rats by inhibiting IgD-IgDR-Lck-NF-κB signaling pathways.

IgD-Fc-Ig fusion protein, a new biological agent, is constructed by linking a segment of human IgD-Fc with a segment of human IgG1-Fc, which specifically blocks the IgD-IgDR pathway and selectively inhibits the abnormal proliferation, activation, and differentiation of T cells. In this study we investigated whether IgD-Fc-Ig exerted therapeutic effects in collagen-induced arthritis (CIA) rats. CIA rats were treated with IgD-Fc-Ig (1, 3, and 9 mg/kg) or injected with biological agents etanercept (3 mg/kg) once every 3 days for 40 days. In the PBMCs and spleen lymphocytes of CIA rats, both T and B cells exhibited abnormal proliferation; the percentages of CD3+ total T cells, CD3+CD4+ Th cells, CD3+CD4+CD25+-activated Th cells, Th1(CD4+IFN-γ+), and Th17(CD4+IL-17+) were significantly increased, whereas the Treg (CD4+CD25+Foxp3+) cell percentage was decreased. IgD-Fc-Ig administration dose-dependently decreased the indicators of arthritis; alleviated the histopathology of spleen and joint; reduced serum inflammatory cytokines levels; decreased the percentages of CD3+ total T cells, CD3+CD4+ Th cells, CD3+CD4+CD25+-activated Th cells, Th1 (CD4+IFN-γ+), and Th17(CD4+IL-17+); increased Treg (CD4+CD25+Foxp3+) cell percentage; and down-regulated the expression of key molecules in IgD-IgDR-Lck-NF-κB signaling (p-Lck, p-ZAP70, p-P38, p-NF-κB65). Treatment of normal T cells with IgD (9 µg/mL) in vitro promoted their proliferation. Co-treatment with IgD-Fc-Ig (0.1-10 µg/mL) dose-dependently decreased IgD-stimulated T cell subsets percentages and down-regulated the IgD-IgDR-Lck-NF-κB signaling. In summary, this study demonstrates that IgD-Fc-Ig alleviates CIA and regulates the functions of T cells through inhibiting IgD-IgDR-Lck-NF-κB signaling.

Benzophenone and Benzoylphloroglucinol Derivatives from Hypericum sampsonii with Anti-Inflammatory Mechanism of Otogirinin A.

Three new compounds, 4-geranyloxy-2-hydroxy-6-isoprenyloxybenzophenone (1), hypericumone A (2) and hypericumone B (3), were obtained from the aerial parts of Hypericum sampsonii, along with six known compounds (4-9). The structures of these compounds were determined through spectroscopic and MS analyses. Hypericumone A (2), sampsonione J (8) and otogirinin A (9) exhibited potent inhibition (IC50 values ≤ 40.32 µM) against lipopolysaccharide (LPS)-induced nitric oxide (NO) generation. Otogirinin A (9) possessed the highest inhibitory effect on NO production with IC50 value of 32.87 ± 1.60 µM. The well-known proinflammatory cytokine, tumor necrosis factor-alpha (TNF-α) was also inhibited by otogirinin A (9). Western blot results demonstrated that otogirinin A (9) downregulated the high expression of inducible nitric oxide synthase (iNOS). Further investigations on the mechanism showed that otogirinin A (9) blocked the phosphorylation of MAPK/JNK and IκBα, whereas it showed no effect on the phosphorylation of MAPKs/ERK and p38. In addition, otogirinin A (9) stimulated anti-inflammatory M2 phenotype by elevating the expression of arginase 1 and Krüppel-like factor 4 (KLF4). The above results suggested that otogirinin A (9) could be considered as potential compound for further development of NO production-targeted anti-inflammatory agent.

CP-25 reverses prostaglandin E4 receptor desensitization-induced fibroblast-like synoviocyte dysfunction via the G protein-coupled receptor kinase 2 in autoimmune arthritis.

Paeoniflorin-6'-O-benzene sulfonate (CP-25) is a novel compound derived from paeoniflorin that has been demonstrated to have therapeutic effects in a rat model of rheumatoid arthritis (RA). However, the underlying mechanism has not been elucidated to date. We explored this mechanism in the present study by treating rats with adjuvant arthritis (AA) with CP-25. We found that the membrane EP4 protein level was downregulated; whereas, GRK2 was upregulated, in fibroblast-like synoviocyte (FLS)s of AA rats. Prostaglandin (PGE)2 stimulated FLS proliferation and enhanced the membrane EP4 receptor protein level; the latter was reversed by the administration of an EP4 receptor agonist, whereas the membrane GRK2 protein level gradually increased. The changes in the EP4 receptor and GRK2 expression were enhanced by TNF-α, and the former was accompanied by an alteration in the cyclic (c)AMP level. The EP4 receptor agonist stimulation increased the association between GRK2 and the EP4 receptor. GRK2 knockdown abrogated the abnormalities in FLS proliferation. The CP-25 treatment (100 mg/kg) suppressed joint inflammation with an efficacy that was similar to that of methotrexate. This finding was associated with EP4 upregulation and GRK2 downregulation in FLSs. Thus, GRK2 plays an important role in the abnormal FLS proliferation observed in AA possibly by promoting EP4 receptor desensitization and decreasing the cAMP level. Our results demonstrate that CP-25 has therapeutic potential for the treatment of human RA via GRK2 regulation.

Paeoniflorin-6'-O-benzene sulfonate alleviates collagen-induced arthritis in mice by downregulating BAFF-TRAF2-NF-κB signaling: comparison with biological agents.

Paeoniflorin-6'-O-benzene sulfonate (CP-25) is a new ester derivative of paeoniflorin with improved lipid solubility and oral bioavailability, as well as better anti-inflammatory activity than its parent compound. In this study we explored whether CP-25 exerted therapeutic effects in collagen-induced arthritis (CIA) mice through regulating B-cell activating factor (BAFF)-BAFF receptors-mediated signaling pathways. CIA mice were given CP-25 or injected with biological agents rituximab or etanercept for 40 days. In CIA mice, we found that T cells and B cells exhibited abnormal proliferation; the percentages of CD19+ total B cells, CD19+CD27+-activated B cells, CD19+BAFFR+ and CD19+TACI+ cells were significantly increased in PBMCs and spleen lymphocytes. CP-25 suppressed the indicators of arthritis, alleviated histopathology, accompanied by reduced BAFF and BAFF receptors expressions, inhibited serum immunoglobulin levels, decreased the B-cell subsets percentages, and prevented the expressions of key molecules in NF-κB signaling. Furthermore, we showed that treatment with CP-25 reduced CD19+TRAF2+ cell expressions stimulated by BAFF and decreased TRAF2 overexpression in HEK293 cells in vitro. Thus, CP-25 restored the abnormal T cells proliferation and B-cell percentages to the normal levels, and normalized the elevated levels of IgA, IgG2a and key proteins in NF-κB signaling. In comparison, rituximab and etanercept displayed stronger anti-inflammatory activities than CP-25; they suppressed the elevated inflammatory indexes to below the normal levels in CIA mice. In summary, our results provide evidence that CP-25 alleviates CIA and regulates the functions of B cells through BAFF-TRAF2-NF-κB signaling. CP-25 would be a soft immunomodulatory drug with anti-inflammatory effect.

Regulatory effects of paeoniflorin-6'-O-benzene sulfonate (CP-25) on dendritic cells maturation and activation via PGE2-EP4 signaling in adjuvant-induced arthritic rats.

Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease. Dendritic cells (DCs) are one of the most powerful antigen-presenting cells, and they play an important role in RA pathogenesis. Prostaglandin E2 (PGE2) is a potent lipid mediator that can regulate the maturation and activation of DCs, but the molecular mechanisms have not been elucidated. In this study, both in vitro and in an RA rat model, we investigated the mechanisms involved by focusing on PGE2-mediated signaling and using a novel anti-inflammatory compound, paeoniflorin-6'-O-benzene sulfonate (CP-25). PGE2 combined with tumor necrosis factor-α promoted DC maturation and activation through EP4-cAMP signaling. Treatment with CP-25 increased the endocytic capacity of DCs induced by PGE2. CP-25 inhibited the potency of DCs induced by the EP4 receptor agonist, CAY10598, to stimulate allogeneic T cells. Consistent with these findings, the CAY10598-induced upregulation of DC surface activation markers and production of IL-23 was significantly inhibited by CP-25 in a concentration-dependent manner. In vivo administration of CP-25 alleviated adjuvant arthritis (AA) in rats through inhibition of DC maturation and activation. Our results indicate that PGE2-EP4-cAMP signal hyperfunction can lead to abnormal activation of DC functions, which correlates with the course of disease in AA rats and provides a possible treatment target. The inhibition of DC maturation and activation by CP-25 interference of the PGE2-EP4 pathway may significantly contribute to the immunoregulatory profile of CP-25 when used to treat RA and other immune cell-mediated disorders.

The immunoglobulin D Fc receptor expressed on fibroblast-like synoviocytes from patients with rheumatoid arthritis contributes to the cell activation.

Immunoglobulin IgD might play an important role in autoimmune diseases, but the function of IgD has remained elusive, despite multiple attempts to define its biological function. Fibroblast-like synoviocytes (FLSs) are specialized cells of the synovium that play a key role in the pathogenesis of rheumatoid arthritis (RA). In this study we explored the possible roles of excessive IgD expression on the function of FLSs from RA patients (RA-FLSs). We showed that IgD Fc receptor (IgDR) was constitutively expressed on FLSs, and was significantly elevated in RA-FLSs compared with FLSs prepared from synovial tissues of healthy controls (HC-FLSs). Furthermore, IgDR was mainly detected on the cell surface and in the cytoplasm. We further detected the intrinsic binding affinity of IgD to IgDR on HC-FLSs with an equilibrium dissociation constant (KD) of 0.067 nmol/L. Incubation of RA-FLSs with IgD (1-10 µg/mL) for 48 h dose-dependently promoted the expression of IgDR, and stimulated the production of inflammatory cytokines and chemokines, such as IL-1ß, IL-6, monocyte chemotactic protein (MCP)-1, TNF-α and receptor activator of nuclear factor-κB ligand (RANKL), thus potentially contributing to IgD-IgDR crosslinking. Moreover, incubation with IgD (0.1-10 µg/mL) for 48 h dose-dependently enhanced viability for both HC-FLSs and RA-FLSs. Our results demonstrate that IgDR is expressed on RA-FLSs and contributes to the activation of FLSs, and suggest that IgD-IgDR is a potential novel immunotherapeutic target for the management of RA.

BAFF upregulates CD28/B7 and CD40/CD154 expression and promotes mouse T and B cell interaction in vitro via BAFF receptor.

AIM: B cell-activating factor belonging to the TNF family (BAFF) is a member of TNF family and required for peripheral B cell survival and homeostasis. BAFF has been shown to promote the proliferation of T and B cells. In this study we examined whether and how BAFF mediated the interaction between mouse T and B cells in vitro. METHODS: BAFF-stimulated B or T cells were co-cultured with T or B cells. The interactions between T and B cells were analyzed by measuring the expression of co-stimulatory molecules (CD28/CD80 or CD40/CD154), the proliferation and secretion of T and B cells and other factors. Two siRNAs against the transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and BAFF receptor (BAFF-R) were used to identify the receptors responsible for the actions of BAFF. RESULTS: BAFF-stimulated B cells significantly promoted the proliferation and activity of co-cultured T cells, and increased the percentages of CD4(+)CD28(+) and CD4(+)CD154(+) T cells. Similarly, BAFF-stimulated T cells significantly promoted the proliferation and activity of co-cultured B cells, and increased CD19(+)CD80(+) and CD19(+)CD40(+)B cell subpopulations. BAFF-R siRNA-silenced B cells showed significantly lower expression of CD40 and CD80 than the control B cells. When the BAFF-R siRNA-silenced B cells were stimulated with BAFF, then co-cultured with T cells, the expression of CD28 and CD154 on T cells was not increased. TACI siRNA-silenced B cells exhibited higher expression of CD40 and CD80 than the control B cells. When the TACI siRNA-silenced B cells were stimulated with BAFF, then co-cultured with T cells, the expression of CD28 and CD154 on T cells was significantly increased. CONCLUSION: BAFF upregulates CD28/B7 and CD40/CD154 expression, and promotes the interactions between T and B cells in a BAFF-R-dependent manner.

Etanercept attenuates collagen-induced arthritis by modulating the association between BAFFR expression and the production of splenic memory B cells.

Anti-tumour necrosis factor-α (TNF-α) drugs are approved for the treatment of rheumatoid arthritis (RA). Many studies have investigated the effect of these drugs on the T cell response; however, some clues have indicated that it may also target B cells. This study was carried out to explore the potential effects and mechanisms of etanercept, a soluble TNF-α receptor, on the function of B cells and their development into memory B cells in type II collagen (CII)-induced arthritis (CIA). Beginning on day 24 after CII immunisation, the mice were evaluated every 2-3 days to determine two clinical parameters: their arthritis global assessment and swollen joint count (SJC). The serum concentrations of IgG1, IgG2a and anti-CII antibodies and the splenic pathology and proliferation of B cells were measured. The percentage of total memory B cells in the spleen was analysed with flow cytometry. BAFFR was detected by immunohistochemistry. In CIA mice, etanercept markedly suppressed the arthritis global assessment and the SJC, reduced the production of anti-CII, IgG1 and IgG2a antibodies, and prevented spleen histopathology to varying degrees; however, it had no obvious effect on splenic B cell proliferation. Etanercept also decreased the percentage of total CD27(+) memory B cells in the spleen. Treatment with etanercept was associated with a further increase in BAFFR expression, a significant reduction in CD27 expression, and a negative correlation between the levels of BAFFR and the percentage of memory B cells. Our findings showed that increased BAFFR expression has a regulatory effect on the activation of B cells and the generation of memory B cells, which may be one of the mechanisms of the therapeutic effects of etanercept.

Paeoniflorin inhibits inflammatory responses in mice with allergic contact dermatitis by regulating the balance between inflammatory and anti-inflammatory cytokines.

OBJECTIVE: Paeoniflorin (Pae) was previously reported to inhibit inflammation in the skin of mice with allergic contact dermatitis (ACD); however, the mechanism remains unclear. The primary purpose of this study was to investigate the effect of Pae on the regulation of cytokine production in a murine model of ACD. METHODS: ACD was induced in the mice by repeated application of dinitrochlorobenzene (DNCB) to their skin. Cutaneous inflammation was evaluated by measuring ear swelling and by histological examination. The cytokine levels were measured by enzyme-linked immunosorbent assays. RESULTS: The results showed that topical application of DNCB caused obvious swelling and inflammatory cell infiltration. Treatment with Pae (70 or 140 mg/kg/d) significantly inhibited the cutaneous inflammation and decreased thymocyte proliferation in the mice with ACD. Additional data indicated that Pae increased interleukin-4 (IL-4) and IL-10 production but reduced IL-2 and IL-17 levels in the serum as well as in thymocyte and splenocyte culture supernatants. As expected, IL-2 and IL-17 levels in the serum displayed a significant positive correlation with the severity of skin inflammation. In contrast, IL-4 and IL-10 levels were negatively correlated with the inflammation. CONCLUSIONS: The anti-inflammatory action of Pae in the murine model of ACD may be related to its regulation of an imbalanced cytokine production.

BF02, a recombinant TNFR2 fusion protein, alleviates adjuvant arthritis by regulating T lymphocytes in rats.

AIM: To investigate the therapeutic effects of BF02 on adjuvant arthritis (AA) in rats and the regulatory effects of BF02 on T lymphocyte function. METHODS: SD rats received a single intradermal injection of Freund's complete adjuvant emulsion into the right hind metatarsal footpad. After the onset of AA, the rats were injected BF02 (1, 3, or 9 mg/kg, sc) every 3 d for a total of 15 d. Intragastric administration of methotrexate (MTX, 0.5 mg/kg, every 3 d for a total of 15 d) was taken as the positive control drug. Arthritis index, swollen joint count, ankle joint histopathology, spleen histopathology and the paw radiography were used for evaluating the drug effects on AA rats. T lymphocyte function was assessed by measuring T lymphocyte cytokine levels, IL17 and TNF-α mRNA expression levels, and percentage of T lymphocyte subsets. RESULTS: In the AA rats, remarkable secondary inflammatory responses exhibited, accompanied by significantly higher levels of IL-1, IL-6, TNF-α, IL-17, LTα, RANKL, and MMP-13. The expression of IL17 and TNF-α mRNAs was also substantially higher than in normal rats. The percentages of CD3(+)CD4(+) and CD4(+)CD25(+) T lymphocytes were increased, whereas the percentages of CD4(+)CD62L(+) and CD4(+)CD25(+)FoxP3(+) T lymphocytes were decreased. Treatment of the AA rats with BF02 (9 mg/kg) or MTX significantly decreased the arthritis index, swollen joint count and arthritis global assessment. Moreover, both BF02 (9 mg/kg) and MTX significantly inhibited T lymphocyte proliferation, and blocked the above mentioned aberrance in T lymphocyte cytokine levels, IL17 and TNF-α mRNA expression, and percentages of T lymphocyte subsets. CONCLUSION: BF02 exerts therapeutic effects on AA rats via the regulation of T lymphocytes.

New insights into the Lck-NF-κB signaling pathway.

Lck is essential for the development, activity, and proliferation of T cells, which may contribute to pathological progression and development of human diseases, such as autoimmune disorders and cancers when functioning aberrantly. Nuclear factor-κB (NF-κB) was initially discovered as a factor bound to the κ light-chain immunoglobulin enhancer in the nuclei of activated B lymphocytes. Activation of the nuclear factor-κB pathway controls expression of several genes that are related to cell survival, apoptosis, and inflammation. Abnormal expression of Lck and nuclear factor-κB has been found in autoimmune diseases and malignancies, including rheumatoid arthritis, systemic lupus erythematosus, acute T cell lymphocytic leukemia, and human chronic lymphocytic leukemia, etc. Nuclear factor-κB inhibition is effective against autoimmune diseases and malignancies through blocking inflammatory responses, although it may lead to serious adverse reactions that are unexpected and unwanted. Further investigation of the biochemical and functional interactions between nuclear factor-κB and other signaling pathways may be helpful to prevent side-effects. This review aims to clarify the Lck-nuclear factor-κB signaling pathway, and provide a basis for identification of new targets and therapeutic approaches against autoimmune diseases and malignancies.

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.].

Prostanoid EP1 receptor as the target of (-)-epigallocatechin-3-gallate in suppressing hepatocellular carcinoma cells in vitro.

AIM: To investigate the effects of (-)-epigallocatechin-3-gallate (EGCG), an active compound in green tea, on prostaglandin E(2) (PGE(2))-induced proliferation and migration, and the expression of prostanoid EP(1) receptors in hepatocellular carcinoma (HCC) cells. METHODS: HCC cell line HepG2, human hepatoma cell lines MHCC-97L, MHCC-97H and human hepatocyte cell line L02 were used. Cell viability was analyzed using MTT assay. PGE(2) production was determined with immunoassay. Wound healing assay and transwell filter assay were employed to assess the extent of HCC cell migration. The expression of EP(1) receptor and Gq protein were examined using Western blot assay. RESULTS: PGE(2) (4-40000 nmol/L) or the EP(1) receptor agonist ONO-DI-004 (400-4000 nmol/L) increased the viability and migration of HepG2 cells in concentration-dependent manners. EGCG (100 µg/mL) significantly inhibited the viability and migration of HepG2 cells induced by PGE(2) or ONO-DI-004. HepG2 cells secreted an abundant amount of PGE(2) into the medium, and EGCG (100 µg/mL) significantly inhibited the PGE(2)production and EP(1) receptor expression in HepG2 cells. EGCG (100 µg/mL) also inhibited the viability of MHCC-97L cells, but not that of MHCC-97H cells. Both EGCG (100 µg/mL) and EP(1) receptor antagonist ONO-8711 inhibited PGE(2) 4 µmol/L and ONO-DI-004 400 nmol/L-induced growth and migration of HepG2 cells. Both EGCG (100 µg/mL) and ONO-8711 210 nmol/L inhibited PGE(2)- and ONO-DI-004-induced EP(1) expression. EGCG and ONO-8711 had synergistic effects in inhibiting EP(1) receptor expression. PGE(2), ONO-DI-004, ONO-8711, and EGCG had no effects on Gq expression in HepG2 cells, respectively. CONCLUSION: These findings suggest that the anti-HCC effects of EGCG might be mediated, at least partially, through the suppressing EP(1) receptor expression and PGE(2) production.

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.