The gut microbiota contributes to the infection of bovine viral diarrhea virus in mice.

Bovine viral diarrhea virus (BVDV) is prevalent worldwide and causes significant economic losses. Gut microbiota is a large microbial community and has a variety of biological functions. However, whether there is a correlation between gut microbiota and BVDV infection and what kind of relation between them have not been reported. Here, we found that gut microbiota composition changed in normal mice after infecting with BVDV, but mainly the low abundance microbe was affected. Interestingly, BVDV infection significantly reduced the diversity of gut microbiota and changed its composition in gut microbiota-dysbiosis mice. Furthermore, compared with normal mice of BVDV infection, there were more viral loads in the duodenum, jejunum, spleen, and liver of the gut microbiota-dysbiosis mice. However, feces microbiota transplantation (FMT) reversed these effects. The data above indicated that the dysbiosis of gut microbiota was a key factor in the high infection rate of BVDV. It is found that the IFN-I signal was involved by investigating the underlying mechanisms. The inhibition of the proliferation and increase in the apoptosis of peripheral blood lymphocytes (PBL) were also observed. However, FMT treatment reversed these changes by regulating PI3K/Akt, ERK, and Caspase-9/Caspase-3 pathways. Furthermore, the involvement of butyrate in the pathogenesis of BVDV was also further confirmed. Our results showed for the first time that gut microbiota acts as a key endogenous defense mechanism against BVDV infection; moreover, targeting regulation of gut microbiota structure and abundance may serve as a new strategy to prevent and control the disease.IMPORTANCEWhether the high infection rate of BVDV is related to gut microbiota has not been reported. In addition, most studies on BVDV focus on in vitro experiments, which limits the study of its prevention and control strategy and its pathogenic mechanism. In this study, we successfully confirmed the causal relationship between gut microbiota and BVDV infection as well as the potential molecular mechanism based on a mouse model of BVDV infection and a mouse model of gut microbiota dysbiosis. Meanwhile, a mouse model which is more susceptible to BVDV provided in this study lays an important foundation for further research on prevention and control strategy of BVDV and its pathogenesis. In addition, the antiviral effect of butyrate, the metabolites of butyrate-producing bacteria, has been further revealed. Overall, our findings provide a promising prevention and control strategy to treat this infectious disease which is distributed worldwide.

FCRL1 Regulates B Cell Receptor-Induced ERK Activation through GRB2.

Regulation of BCR signaling has important consequences for generating effective Ab responses to pathogens and preventing production of autoreactive B cells during development. Currently defined functions of Fc receptor-like (FCRL) 1 include positive regulation of BCR-induced calcium flux, proliferation, and Ab production; however, the mechanistic basis of FCRL1 signaling and its contributions to B cell development remain undefined. Molecular characterization of FCRL1 signaling shows phosphotyrosine-dependent associations with GRB2, GRAP, SHIP-1, and SOS1, all of which can profoundly influence MAPK signaling. In contrast with previous characterizations of FCRL1 as a strictly activating receptor, we discover a role for FCRL1 in suppressing ERK activation under homeostatic and BCR-stimulated conditions in a GRB2-dependent manner. Our analysis of B cells in Fcrl1 -/- mice shows that ERK suppression by FCRL1 is associated with a restriction in the number of cells surviving splenic maturation in vivo. The capacity of FCRL1 to modulate ERK activation presents a potential for FCRL1 to be a regulator of peripheral B cell tolerance, homeostasis, and activation.

Interleukin-38 Suppresses Cell Migration and Proliferation and Promotes Apoptosis of Colorectal Cancer Cell Through Negatively Regulating Extracellular Signal-Regulated Kinases Signaling.

Inflammatory cytokines has been of great interest in the field of colorectal cancer (CRC) tumor immunology in recent years. As an anti-inflammatory interleukin (IL), IL-38 may contribute to the early diagnosis of CRC and improve the prognosis of CRC patients. This study was designed to investigate the role of circulating IL-38 and the regulatory mechanism of IL-38 in CRC. Expression of IL-38 were detected by ELISA and immunohistochemical staining. The influence of IL-38 on CRC were evaluated by Western blot and cell biology assays after CRC cells were treated by rhIL-38 or LM22B-10. We also verified the anti-tumor activity of IL-38 in transgenic mouse model. The expression of IL-38 was found to be correlated with progression of CRC. IL-38 inhibits CRC metastasis, proliferation and facilitates apoptosis through suppressing the activation of extracellular signal-regulated kinases (ERK) signaling pathway inducing the decrease of downstream genes, which were partially abrogated by ERK activator LM22B-10 in vitro. We also found that IL-38 overexpression inhibits tumorigenesis in vivo. Our findings indicate that IL-38 may serve as a serum prediction marker to identify the prognosis of CRC patients. IL-38 may inhibit the progression of CRC by negatively regulation on ERK signaling pathway.

Yersinia pseudotuberculosis YopJ Limits Macrophage Response by Downregulating COX-2-Mediated Biosynthesis of PGE2 in a MAPK/ERK-Dependent Manner.

Prostaglandin E2 (PGE2) is an essential immunomodulatory lipid released by cells in response to infection with many bacteria, yet its function in macrophage-mediated bacterial clearance is poorly understood. Yersinia overall inhibits the inflammatory circuit, but its effect on PGE2 production is unknown. We hypothesized that one of the Yersinia effector proteins is responsible for the inhibition of PGE2 biosynthesis. We identified that yopB-deficient Y. enterocolitica and Y. pseudotuberculosis deficient in the secretion of virulence proteins via a type 3 secretion system (T3SS) failed to inhibit PGE2 biosynthesis in macrophages. Consistently, COX-2-mediated PGE2 biosynthesis is upregulated in cells treated with heat-killed or T3SS-deficient Y. pseudotuberculosis but diminished in the presence of a MAPK/ERK inhibitor. Mutants expressing catalytically inactive YopJ induce similar levels of PGE2 as heat-killed or ΔyopB Y. pseudotuberculosis, reversed by YopJ complementation. Shotgun proteomics discovered host pathways regulated in a YopJ-mediated manner, including pathways regulating PGE2 synthesis and oxidative phosphorylation. Consequently, this study identified that YopJ-mediated inhibition of MAPK signal transduction serves as a mechanism targeting PGE2, an alternative means of inflammasome inhibition by Yersinia. Finally, we showed that EP4 signaling supports macrophage function in clearing intracellular bacteria. In summary, our unique contribution was to determine a bacterial virulence factor that targets COX-2 transcription, thereby enhancing the intracellular survival of yersiniae. Future studies should investigate whether PGE2 or its stable synthetic derivatives could serve as a potential therapeutic molecule to improve the outcomes of specific bacterial infections. Since other pathogens encode YopJ homologs, this mechanism is expected to be present in other infections. IMPORTANCE PGE2 is a critical immunomodulatory lipid, but its role in bacterial infection and pathogen clearance is poorly understood. We previously demonstrated that PGE2 leads to macrophage polarization toward the M1 phenotype and stimulates inflammasome activation in infected macrophages. Finally, we also discovered that PGE2 improved the clearance of Y. enterocolitica. The fact that Y. enterocolitica hampers PGE2 secretion in a type 3 secretion system (T3SS)-dependent manner and because PGE2 appears to assist macrophage in the clearance of this bacterium indicates that targeting of the eicosanoid pathway by Yersinia might be an adaption used to counteract host defenses. Our study identified a mechanism used by Yersinia that obstructs PGE2 biosynthesis in human macrophages. We showed that Y. pseudotuberculosis interferes with PGE2 biosynthesis by using one of its T3SS effectors, YopJ. Specifically, YopJ targets the host COX-2 enzyme responsible for PGE2 biosynthesis, which happens in a MAPK/ER-dependent manner. Moreover, in a shotgun proteomics study, we also discovered other pathways that catalytically active YopJ targets in the infected macrophages. YopJ was revealed to play a role in limiting host LPS responses, including repression of EGR1 and JUN proteins, which control transcriptional activation of proinflammatory cytokine production such as interleukin-1ß. Since YopJ has homologs in other bacterial species, there are likely other pathogens that target and inhibit PGE2 biosynthesis. In summary, our study's unique contribution was to determine a bacterial virulence factor that targets COX-2 transcription. Future studies should investigate whether PGE2 or its stable synthetic derivatives could serve as a potential therapeutic target.

Effects of Kumaizasa (Sasa senanensis) Leaf Extract on Innate Immune Regulation in HEK293 Cells and Macrophages.

BACKGROUND/AIM: We investigated the effect of Kumaizasa leaf extract (KLE) on innate immunity using the HEK293 and RAW 264.7 cell lines. MATERIALS AND METHODS: KLE, lipopolysaccharides (LPS), or KLE with LPS were added to RAW 264.7 cells. The TNF-α and IL-1ß mRNA expression was then quantified. The expression of MAPKs, NFĸB, TNF-α and IL-1ß proteins was also quantified. In addition, KLE was added to HEK293 cells and the IL-8 concentration was measured. RESULTS: In RAW 264.7 cells, KLE increased the levels of TNF-α and IL-1ß mRNA. By contrast, when KLE and LPS were added to RAW 264.7 cells, the increase in TNF-α and IL-1ß mRNA was ameliorated. Similarly, the expression of JNK and ERK proteins was reduced. The addition of KLE to HEK293 cells induced IL-8 production. CONCLUSION: Based on these results, a KLE-mediated mechanism may regulate immunity by suppressing the expression of JNK and ERK, which are involved in inflammatory signal transduction.

Commensal fungi and their cell-wall ß-glucans direct differential responses in human intestinal epithelial cells.

Intestinal epithelial cells (IECs) are the first to encounter luminal antigens and play an active role in intestinal immune responses. We previously reported that ß-glucans, major fungal cell-wall glycans, induced chemokine secretion by IEC lines in a Dectin-1- and Syk-dependent manner. Here, we show that in contrast to ß-glucans, stimulation of IEC lines with Candida albicans and Saccharomyces cerevisiae did not induce secretion of any of the proinflammatory cytokines IL-8, CCL2, CXCL1, and GM-CSF. Commensal fungi and ß-glucans activated Syk and ERK in IEC lines. However, only ß-glucans activated p38, JNK, and the transcription factors NF-κB p65 and c-JUN, which were necessary for cytokine secretion. Furthermore, costimulation of IEC lines with ß-glucans and C. albicans yielded decreased cytokine secretion compared to stimulation with ß-glucans alone. Finally, ex vivo stimulation of human colonic mucosal explants with zymosan and C. albicans, leads to epithelial Syk and ERK phosphorylation, implying recognition of fungi and similar initial signaling pathways as in IEC lines. Lack of cytokine secretion in response to commensal fungi may reflect IECs' response to fungal glycans, other than ß-glucans, that contribute to mucosal tolerance. Skewed epithelial response to commensal fungi may impair homeostasis and contribute to intestinal inflammation.

Inhibition of extracellular signal-regulated kinase/calpain-2 pathway reduces neuroinflammation and necroptosis after cerebral ischemia-reperfusion injury in a rat model of cardiac arrest.

BACKGROUND: Cerebral ischemia-reperfusion injury (CIRI) is the leading cause of poor neurological prognosis after cardiopulmonary resuscitation (CPR). We previously reported that the extracellular signal-regulated kinase (ERK) activation mediates CIRI. Here, we explored the potential ERK/calpain-2 pathway role in CIRI using a rat model of cardiac arrest (CA). METHODS: Adult male Sprague-Dawley rats suffered from CA/CPR-induced CIRI, received saline, DMSO, PD98059 (ERK1/2 inhibitor, 0.3 mg/kg), or MDL28170 (calpain inhibitor, 3.0 mg/kg) after spontaneous circulation recovery. The survival rate and the neurological deficit score (NDS) were utilized to assess the brain function. Hematoxylin stain, Nissl staining, and transmission electron microscopy were used to evaluate the neuron injury. The expression levels of p-ERK, ERK, calpain-2, neuroinflammation-related markers (GFAP, Iba1, IL-1ß, TNF-α), and necroptosis proteins (TNFR1, RIPK1, RIPK3, p-MLKL, and MLKL) in the brain tissues were determined by western blotting and immunohistochemistry. Fluorescent multiplex immunohistochemistry was used to analyze the p-ERK, calpain-2, and RIPK3 co-expression in neurons, and RIPK3 expression levels in microglia or astrocytes. RESULTS: At 24 h after CA/CPR, the rats in the saline-treated and DMSO groups presented with injury tissue morphology, low NDS, ERK/calpain-2 pathway activation, and inflammatory cytokine and necroptosis protein over-expression in the brain tissue. After PD98059 and MDL28170 treatment, the brain function was improved, while inflammatory response and necroptosis were suppressed by ERK/calpain-2 pathway inhibition. CONCLUSION: Inflammation activation and necroptosis involved in CA/CPR-induced CIRI were regulated by the ERK/calpain-2 signaling pathway. Inhibition of that pathway can reduce neuroinflammation and necroptosis after CIRI in the CA model rats.

Inactivation of TMEM106A promotes lipopolysaccharide-induced inflammation via the MAPK and NF-κB signaling pathways in macrophages.

Pattern recognition receptors, such as Toll-like receptors (TLRs), play an important role in the host defense against invading microbial pathogens. Their activation must be precisely regulated, as inappropriate activation or overactivation of TLR signaling pathways may result in inflammatory disorders, such as septic shock or autoimmune diseases. TMEM106A is a type II transmembrane protein constitutively expressed in macrophages. Our current study demonstrated that TMEM106A levels were increased in macrophages upon lipopolysaccharide (LPS) stimulation, as well as in the peripheral monocytes of patients with sepsis. Tmem106a knockout mice were more sensitive to lipopolysaccharide (LPS)-induced septic shock than wild-type mice. Further experiments indicated that Tmem106a ablation enhanced the expression of CD80, CD86 and major histocompatibility complex (MHC)-II in mouse macrophages upon LPS stimulation, accompanied with up-regulation of tumor necrosis factor (TNF)-α, interleukin (IL)-6, interferon (IFN)-ß and inducible nitric oxide synthase (iNOS), indicating the activation of macrophages and polarization towards the M1 inflammatory phenotype. Moreover, elevated mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling were found to be involved in the LPS-induced inflammatory response in Tmem106a-/- macrophages. However, this effect was largely abrogated by macrophage deletion in Tmem106a-/- mice. Therefore, deficiency of Tmem106a in macrophages may enhance the M1 polarization in mice, resulting in inflammation. This suggests that TMEM106A plays an important regulatory role in maintaining macrophage homeostasis.

Interleukin 3-induced GITR promotes the activation of human basophils.

Human basophils regulate allergic reactions by secreting histamine, interleukin 4 (IL-4) and IL-13 through key surface receptors FcεRI as well as IL-3R, which are constitutively expressed on basophils. IL-3/IL-3R signaling axis plays key roles in regulating the development and activation of basophils. We and others have shown that IL-3-induced surface receptors e.g. ST2, IL-17RB and IL-2 receptors regulate the biology of basophils. However, the expression and function of IL-3-induced surface proteins on human basophils remain to be elucidated. We in this study aimed to identify new basophil activation regulators by transcriptomic analysis of IL-3-stimulated basophils. Gene expression microarray analysis of IL-3-treated basophils revealed 2050 differentially expressed genes, of which 323 genes encoded surface proteins including GITR. We identified that GITR was preferentially induced by IL-3 rather than anti-IgE, IL-33, fMLP and C5a. IL-3-induced GITR was suppressed by inhibitors targeting JAK2, PI3K and MEK1/2. Stimulation of IL-3-treated basophils by GITR enhanced the expression of IL-4 and IL-13. Moreover, IgE-mediated degranulation was enhanced by GITRL in the presence of IL-3. This transcriptomic analysis of IL-3-activated basophils helps to identify novel activation regulator. IL-3-induced GITR promoted the activation of basophils, adding new evidence supporting GITR as an important player in Th2-associated immune responses.

Luteolin ameliorates experimental colitis in mice through ERK-mediated suppression of inflammation, apoptosis and autophagy.

Ulcerative colitis (UC) is a chronic inflammatory disease with increasing incidence and prevalence worldwide. Currently used treatments of UC are unsatisfactory, while natural bioactive compounds are considered to be emerging therapeutic agents. Luteolin (Lut) is a natural compound with beneficial effects in a variety of diseases, however, its effect in UC has been poorly studied. In this study we investigated the effect of Lut in posttreatment and cotreatment of dextran sulfate sodium (DSS)-induced experimental colitis in mice. In addition, the role of extracellular signal-regulated kinases 1/2 (ERK1/2) in the mechanism of action of Lut in experimental colitis was investigated using the ERK inhibitor PD0325901. Lut attenuated symptoms of DSS-induced colitis in mice, ameliorated colon tissue damage and reduced inflammation, apoptosis and autophagy. The effect was more pronounced if Lut was administered simultaneously with DSS. The administration of ERK inhibitor exacerbated DSS-induced colitis symptoms and prevented the protective effects of Lut. The results provide new mechanistic details underlying the anti-inflammatory, anti-apoptotic and anti-autophagic effects of Lut through the activation of the ERK signaling pathway. This suggested that Lut can be used as a novel therapeutic candidate in the treatment of UC or could be used as a supplement to existing therapy.

Immunostimulatory efficacy and protective potential of putative TgERK7 protein in mice experimentally infected by Toxoplasma gondii.

The extracellular signal-regulated kinases (ERKs) serve as important determinants of cellular signal transduction pathways, and hence may play important roles during infections. Previous work suggested that putative ERK7 of Toxoplasma gondii is required for efficient intracellular replication of the parasite. However, the antigenic and immunostimulatory properties of TgERK7 protein remain unknown. The objective of this study was to produce a recombinant TgERK7 protein in vitro and to evaluate its effect on the induction of humoral and T cell-mediated immune responses against T. gondii infection in BALB/c mice. Immunization using TgERK7 mixed with Freund's adjuvants significantly increased the ratio of CD3e+CD4+ T/CD3e+CD8a+ T lymphocytes in spleen and elevated serum cytokines (IFN-γ, IL-2, IL-4, IL-10, IL-12p70, IL-23, MCP-1, and TNF-α) in immunized mice compared to control mice. On the contrary, immunization did not induce high levels of serum IgG antibodies. Five predicted peptides of TgERK7 were synthesized and conjugated with KLH and used to analyze the antibody specificity in the sera of immunized mice. We detected a progressive increase in the antibody level only against TgERK7 peptide A (DEVDKHVLRKYD). Antibody raised against this peptide significantly decreased intracellular proliferation of T. gondii in vitro, suggesting that peptide A can potentially induce a protective antibody response. We also showed that immunization improved the survival rate of mice challenged with a virulent strain and significantly reduced the parasite cyst burden within the brains of chronically infected mice. Our data show that TgERK7-based immunization induced TgERK7 peptide A-specific immune responses that can impart protective immunity against T. gondii infection. The therapeutic potential of targeting ERK7 signaling pathway for future toxoplasmosis treatment is warranted.

Anti-neuroinflammatory Effect of Short-Chain Fatty Acid Acetate against Alzheimer's Disease via Upregulating GPR41 and Inhibiting ERK/JNK/NF-κB.

Alzheimer's disease (AD) is a high-incidence neurodegenerative disease in the elderly. Acetate (Ace) is a short-chain fatty acid (SCFA) with neuroprotective activity. The purpose of this study was to investigate the effects and its possible mechanisms of SCFA Ace on AD. A male APP/PS1 transgenic mouse was given intragastric administration Ace for 4 weeks. Cognitive function and microglia activation in mice were assessed. Furthermore, Ace pretreated amyloid-ß (Aß)-induced BV2 microglia, and the levels of CD11b, COX-2, and G-protein-coupled receptor 41 (GPR41) and phosphorylation of ERK, JNK, and NF-κB p65 were determined. Our results revealed that Ace significantly attenuated the cognitive impairment and decreased the CD11b level in the APP/PS1 mice. Moreover, Ace inhibited the phosphorylation of NF-κB p65, ERK, and JNK and decreased the levels of COX-2 and interleukin 1ß in the Aß-stimulated BV2 microglia. Finally, Ace increased the GPR41 level in the Aß-stimulated BV2 cells. The finding indicated that Ace exerted antineuroinflammatory effects via the upregulation of GPR41 and suppression of the ERK/JNK/NF-κB pathway, which might provide an alternative therapy strategy of AD.

PGE2 deficiency predisposes to anaphylaxis by causing mast cell hyperresponsiveness.

BACKGROUND: Reduced levels of prostaglandin E2 (PGE2) contribute to aspirin-induced hypersensitivity. COX inhibitors are also frequent cofactors in anaphylaxis. Whether alterations in the PGE2 system contribute to anaphylaxis independently of COX inhibitor intake is unclear. OBJECTIVE: Our aim was to test the hypothesis that relative PGE2 deficiency predisposes to anaphylaxis. METHODS: Sera from 48 patients with anaphylaxis and 27 healthy subjects were analyzed for PGE2 levels and correlated against severity; 9α,11ß-PGF2 and PGI2 metabolites were measured for control purposes. PGE2 stabilization by 15-hydroxyprostaglandin dehydrogenase inhibitor or EP2 or EP4 receptor agonists were used in a murine model of passive systemic anaphylaxis. FcεRI-triggered mediator release was determined in bone marrow-derived cultured mast cells (MCs) and human skin-derived MCs. Signaling was studied by Western blot analysis. RESULTS: Patients with anaphylaxis were characterized by markedly reduced PGE2 levels vis-à-vis healthy subjects, whereas prostacyclin metabolite levels were diminished only weakly, and 9α,11ß-PGF2 levels conversely increased. PGE2 was negatively correlated with severity. Lower PGE2 levels and higher susceptibility to anaphylaxis were also found in C57BL/6 mice vis-à-vis in Balb/c mice. Stabilization of PGE2 level by 15-hydroxyprostaglandin dehydrogenase inhibitor protected mice against anaphylaxis. Exogenous PGE2 attenuated bone marrow-derived cultured MC activation through EP2 and EP4 receptors. EP2 and EP4 agonism also curbed FcεRI-mediated degranulation of human MCs. Mechanistically, PGE2 interfered with the phosphorylation of phospholipase C gamma-1 and extracellular signal-regulated kinase. CONCLUSIONS: Homeostatic levels of PGE2 attenuate MC activation via EP2/EP4 and protect against anaphylaxis. Relative deficiency of PGE2 predisposes to anaphylaxis in humans and mice, whereas PGE2 stabilization protects against anaphylactic reactions.

IFN-κ suppresses the replication of influenza A viruses through the IFNAR-MAPK-Fos-CHD6 axis.

Type I interferons (IFNs) are the first line of defense against viral infection. Using a mouse model of influenza A virus infection, we found that IFN-κ was one of the earliest responding type I IFNs after infection with H9N2, a low-pathogenic avian influenza A virus, whereas this early induction did not occur upon infection with the epidemic-causing H7N9 virus. IFN-κ efficiently suppressed the replication of various influenza viruses in cultured human lung cells, and chromodomain helicase DNA binding protein 6 (CHD6) was the major effector for the antiviral activity of IFN-κ, but not for that of IFN-α or IFN-ß. The induction of CHD6 required both of the type I IFN receptor subunits IFNAR1 and IFNAR2, the mitogen-activated protein kinase (MAPK) p38, and the transcription factor c-Fos but was independent of signal transducer and activator of transcription 1 (STAT1) activity. In addition, we showed that pretreatment with IFN-κ protected mice from lethal influenza viral challenge. Together, our findings identify an IFN-κ-specific pathway that constrains influenza A virus and provide evidence that IFN-κ may have potential as a preventative and therapeutic agent against influenza A virus.

Alleviating effect of paeoniflorin-6'-O-benzene sulfonate in antigen-induced experimental Sjögren's syndrome by modulating B lymphocyte migration via CXCR5-GRK2-ERK/p38 signaling pathway.

Primary Sjögren's syndrome (pSS) is an autoimmune disease of unresolved aetiology that affects the exocrine glands. Clinical symptoms frequently also involve skin, liver, kidney and neurovascular components. The pathogenesis of pSS is still unclear but B cell hyperactivity has been identified as a hallmark of pSS. Currently, a curative therapeutic agent is lacking. In this study, we explored whether paeoniflorin-6'-O-benzene (CP-25) exerted therapeutic effects through regulating B lymphocyte migration via CXCR5-GRK2-MAPK mediated signaling pathways in a mouse model of antigen-induced, experimental Sjögren's syndrome (ESS). We found that CP-25 increased the salivary flow and alleviated the histopathology of ESS. Furthermore, CP-25 reduced the viability of B lymphocyte and limited the target organs index. In the peripheral blood and salivary gland of ESS mice, CP-25 down-regulated the proportion of total B cells, CXCR5+ B cells and PDCA1 + CD19- and limited the presence of phosphorylated (p-) p38 and ERK (p-ERK). Besides, CP-25 increased the percentage of memory B cells in the peripheral blood and reduced it in salivary gland. Furthermore, in vitro, CP-25 down-regulated p-p38, p-ERK, CXCR5 and membrane GRK2, and increased cytoplasm GRK2 in Maver-1 cells, a mantle cell lymphoma cell line, causing a lower migration ability of Maver-1 cells. Thus, we define CP-25 as a novel compound that is a potent therapeutic agent for pSS which modulates B lymphocyte subsets and impacts the migration of B lymphocytes through regulating the CXCR5-GRK2-ERK/p38 signaling pathway.

Ets2 suppresses inflammatory cytokines through MAPK/NF-κB signaling and directly binds to the IL-6 promoter in macrophages.

Proper activation of Toll-like receptor (TLR)-mediated signaling and production of proinflammatory cytokines are critical for the initiation of innate immunity, while the specific mechanism maintaining inflammatory homeostasis remains mostly unknown. Here, we show that Ets2 is upregulated following LPS and VSV stimulation. Ets2 knockdown or knockout leads to increased IL-6, TNF-α, and IFN-ß production in macrophages. Consistently, Ets2-deficient mice show exacerbated inflammatory cytokine production and are more susceptible to CLP-induced sepsis. Mechanistically, Ets2 inhibits the LPS- and VSV-induced activation of ERK1/2, JNK, p38, and p65. Ets2 also binds to the promoter of IL-6 to inhibit transcription. Collectively, the results of the present study show the negative regulatory role of Ets2 in LPS- and VSV-induced inflammation through the suppression of MAPK/NF-κB signaling, direct binding to the IL-6 promoter and inhibition of transcription.

MiRNA Regulation of MIF in SLE and Attenuation of Murine Lupus Nephritis With miR-654.

Objective: Macrophage Migration Inhibitory Factor (MIF) is involved in the pathogenesis of systemic lupus erythematosus (SLE) and lupus nephritis (LN). MicroRNAs (miRNAs) play important roles in LN but whether specific miRNAs regulate the expression of MIF in LN is unknown. We explore specific miRNAs that can regulate MIF expression, and investigate miR-654 for the treatment of experimentally-induced murine lupus nephritis. Methods: Sera samples from 24 SLE patients and 24 controls were collected to measure the MIF concentration and its correlation with disease activity. A luciferase reporter assay was used to explore the target of miR-654. ELISA was used to detect the downstream cytokines regulated by miR-654 and MIF. Western blot was applied to measure the impact of miR-654 inhibition on downstream MIF signaling. The therapeutic efficacy of miR-654 was tested in the pristine-induced lupus mouse model. We further measured miR-654 expression and analyzed its relationship with MIF expression in SLE patients. Results: The serum MIF level was increased in SLE patients (p < 0.001) and positively correlated with the SLEDAI score (r = 0.5473; p = 0.0056). MiR-654 inhibited MIF and downstream inflammatory cytokine production by selectively inhibiting the phosphorylation of ERK and AKT. Activation of miR-654 reduced IL-1ß, IL-6, IL-8, and TNF-α production, reduced gomerulonephritis, and decreased MIF, IgG, and C3 expression in murine lupus glomeruli. Furthermore, MIF was negatively correlated with miR-654 expression (r = -0.4644; p = 0.0222) in SLE patients. Conclusion: MiR-654 negatively correlated with MIF and disease activity in patients with SLE. MiR-654 inhibits MIF expression via binding to MIF 3'UTR, selectively suppresses the phosphorylation of ERK and AKT, and reduces downstream inflammatory cytokine production. In vivo miR-654 treatment decreases MIF and downstream cytokine production and ameliorates murine lupus nephritis.

Ochratoxin A-Triggered Chicken Heterophil Extracellular Traps Release through Reactive Oxygen Species Production Dependent on Activation of NADPH Oxidase, ERK, and p38 MAPK Signaling Pathways.

Ochratoxin A (OTA) is a mycotoxin which could cause strong immunosuppressive toxicological effects in animals and humans. Heterophil extracellular traps (HETs) as a novel defense of chicken heterophils play an important role against pathogen infection. It has been reported that OTA can weaken the phagocytosis function of neutrophils. However, whether or not OTA shows immunosuppressive effects on HET release remains unclear. In the present study, we aim to first investigate the effects of OTA on HET release and then try to clarify the mechanisms in this process. OTA-induced HET structures were observed and analyzed by fluorescence confocal microscopy. The quantitative determination of OTA-induced HETs was measured by PicoGreen and a fluorescence microplate. The results clearly showed that OTA obviously induced the release of HET-like structures in heterophils, and these extracellular networks were composed by chromatin decorated with histones and neutrophil elastase. Reactive oxygen species (ROS) production was also increased in the process of OTA-induced HET formation. Furthermore, the inhibitors of NADPH oxidase, ERK [Formula: see text], and p38 MAPK signaling pathways significantly decreased OTA-induced HET formation. The abovementioned results suggest that OTA-induced HET formation is related to ROS production dependent on the activation of NADPH oxidase, ERK [Formula: see text], and p38 MAPK signaling pathways. Taken together, this study first shows that OTA possesses the ability to trigger HET formation, which provides our understanding of the host that continuously suffered OTA exposure leading to the hyporeactivity of the immune system against infection.

Sorafenib attenuated the function of natural killer cells infiltrated in HCC through inhibiting ERK1/2.

Sorafenib has been systemically utilized to therapy the advanced hepatocellular carcinoma (HCC). Natural killer cells (NKs) are important cytotoxic innate lymphocytes, which can exert effector functions especially in liver and tumor. However, how Sorafenib affects the function of NKs remains to be elucidated. Here, we utilized the subcutaneous and in situ tumor bearing mice with mouse hepatoma cell line hepa 1-6. At the endpoint, the number and function of NKs in blood, liver, TDLN, and tumor were explored using FACS, ELISA, WB, etc. To confirm the direct effects of Sorafenib on NKs, the NKs were sorted using FACS, which were then stimulated with Sorafenib to detect the functions and the relevant mechanisms using qPCR, western blot, and FACS in vitro. Finally, we found that Sorafenib led to a significant block of tumor progression, but reduced the number of NK cells through suppressing the proliferation of NK cells. This phenotype made us study the terminal function of NK cells, revealing that Sorafenib could decrease the production of effector molecules and cytokines, such as perforin, granzyme B, TNF-α, IFN-γ, etc. Besides, p-ERK1/2 in NK cells was inhibited after treatment with Sorafenib, and a similar tendency of NK cells could be achieved using ERK1/2 inhibitor. Collectively, our data suggested that Sorafenib functioned as a critical inhibitor that controlled the number and function of NK cells through inhibiting ERK1/2.

Extracellular signal-regulated kinase activation of self-healing Langerhans cell histiocytosis: A case report.

A 3-month-old boy developed small papules on his trunk. After the papules increased in number, the patient was diagnosed with Langerhans cell histiocytosis based on the pathological findings. He was referred to our department for further examination. Upon initial examination, the papules and nodules were scattered on his back, abdomen and lumbar region. Because he did not present with any organ involvement except the skin, he was diagnosed with single-system and skin-limited Langerhans cell histiocytosis. Skin rashes were treated with a topical steroid and started regressing 3 months after onset. All papules disappeared 6 months after onset. In this boy, the Langerhans cell histiocytosis tumor cells expressed phosphorylated extracellular signal-regulated kinases. In Langerhans cell histiocytosis, BRAF V600E and other genes are known to mutate to act as driver mutations in stem cells of the myeloid dendritic cell lineage. Consequently, extracellular signal-regulated kinases are continuously activated, which contributes to Langerhans cell histiocytosis carcinogenesis.