Glyceryl butyrate attenuates enterotoxigenic Escherichia coli-induced intestinal inflammation in piglets by inhibiting the NF-κB/MAPK pathways and modulating the gut microbiota.

The aims of this study were to evaluate whether a diet supplemented with glyceryl butyrate could attenuate the immune-inflammatory response in piglets challenged with enterotoxigenic Escherichia coli (ETEC), and to explore the mechanisms of its regulation. Eighteen weaning piglets were assigned to three diets: basal diet (CON), antibiotics diet (ATB), and 0.5% glyceryl butyrate diet (GB group). Significantly lower concentrations of IL-1ß, IL-6 and TNF-α in the jejunum and IL-6 in the ileum were observed in the GB group than that in the CON group (P < 0.05). Moreover, a decreasing trend of IL-1ß (P = 0.075) and TNF-α (P = 0.070) was observed in the ileum in the GB group. Correspondingly, the GB group had significantly increased mRNA expression of porcine beta defensins (pBDs) in the jejunum (pBD1, pBD2, pBD114 and pBD129) and ileum (pBD2, pBD3, pBD114 and pBD129) (P < 0.05), and protein abundance of Claudin 1, Occludin, and ZO-1 in the jejunum and ileum (P < 0.05). Further research results showed that the improvement of beta defensins and tight junctions in the GB group was related to the decreased phosphorylation of the NFκB/MAPK pathway. In addition, the results of 16S rDNA sequencing showed that glycerol butyrate supplementation altered the ileal microbiota composition of piglets, increasing the relative abundance of Lactobacillus reuteri, Lactobacillus salivarius, and Lactobacillus agrilis. In summary, glyceryl butyrate attenuated the immune-inflammatory response in piglets challenged with ETEC by inhibiting the NF-κB/MAPK pathways and modulating the gut microbiota, and thus improved piglet intestinal health.

Early and Late Processes Driving NET Formation, and the Autocrine/Paracrine Role of Endogenous RAGE Ligands.

Neutrophil extracellular trap (NET) formation has emerged as an important response against various pathogens; it also plays a role in chronic inflammation, autoimmunity, and cancer. Despite a growing understanding of the mechanisms underlying NET formation, much remains to be elucidated. We previously showed that in human neutrophils activated with different classes of physiological stimuli, NET formation features both early and late events that are controlled by discrete signaling pathways. However, the nature of these events has remained elusive. We now report that PAD4 inhibition only affects the early phase of NET generation, as do distinct signaling intermediates (TAK1, MEK, p38 MAPK). Accordingly, the inducible citrullination of residue R2 on histone H3 is an early neutrophil response that is regulated by these kinases; other arginine residues on histones H3 and H4 do not seem to be citrullinated. Conversely, elastase blockade did not affect NET formation by several physiological stimuli, though it did so in PMA-activated cells. Among belated events in NET formation, we found that chromatin decondensation is impaired by the inhibition of signaling pathways controlling both early and late stages of the phenomenon. In addition to chromatin decondensation, other late processes were uncovered. For instance, unstimulated neutrophils can condition themselves to be poised for rapid NET induction. Similarly, activated neutrophils release endogenous proteic factors that promote and largely mediate NET generation. Several such factors are known RAGE ligands and accordingly, RAGE inbibition largely prevents both NET formation and the conditioning of neutrophils to rapidly generate NETs upon stimulation. Our data shed new light on the cellular processes underlying NET formation, and unveil unsuspected facets of the phenomenon that could serve as therapeutic targets. In view of the involvement of NETs in both homeostasis and several pathologies, our findings are of broad relevance.

gga-miR-142-3p negatively regulates Mycoplasma gallisepticum (HS strain)-induced inflammatory cytokine production via the NF-κB and MAPK signaling by targeting TAB2.

OBJECTIVE: Mycoplasma gallisepticum (MG), a notorious avian pathogen, leads to considerable economic losses in the poultry industry. MG infection is characterized by severe, uncontrollable inflammation and host DNA damage. Micro ribonucleic acids (miRNAs) have emerged as important regulators in microbial pathogenesis. However, the role of miRNAs in MG infection is poorly characterized. In this study, we validated the functional roles of gga-miR-142-3p. METHODS: The relative expression of gga-miR-142-3p in the lungs of the MG-infected chicken embryos and the MG-infected chicken embryonic fibroblast cell line (DF-1) was determined by reverse transcription quantitative real-time PCR analysis. Bioinformatics database was used to analysis the target gene of gga-miR-142-3p. The luciferase reporter assay as well as gene expression analysis were conducted to validate the target gene. To further explore the biological functions of gga-miR-142-3p upon MG infection, the cell proliferation was quantified using Cell Counting Kit-8 (CCK-8). Meanwhile, cell cycle analysis and apoptosis were measured using a flow cytometer. RESULTS: gga-miR-142-3p was significantly upregulated in both MG-infected chicken-embryo lungs and the DF-1 cells. gga-miR-142-3p over expression significantly downregulated the expression of pro-inflammatory cytokines, including interleukin-1ß, interleukin-6 and tumor necrosis factor alpha after MG infection. Meanwhile, gga-miR-142-3p enhanced the host defense against MG infection by facilitating cell proliferation, promoting cell progression and inhibiting cell apoptosis. Interestingly, TAB2 knockdown groups show similar results, whereas, TAB2 over-expression groups and gga-miR-142-3p inhibitor groups had thoroughly opposite results. The expression of p-p65 in nuclear factor kappa B (NF-κB) and p-p38 in the mitogen-activated protein kinase (MAPK) pathway was decreased when gga-miR-142-3p was over-expressed. CONCLUSION: Upon MG infection, upregulation of gga-miR-142-3p alleviates inflammation by negatively regulating the signaling pathways of NF-κB and MAPKs by targeting TAB2 and facilitates cell proliferation by inhibiting cell apoptosis and promoting cell cycle progression to defend against MG infection.

OsVQ1 links rice immunity and flowering via interaction with a mitogen-activated protein kinase OsMPK6.

KEY MESSAGE: Knocking out OsVQ1 in rice released OsMPK6 for activation and in turn promoted H2O2 accumulation, which repressed the expression of flowering-promoting genes, thus delaying rice flowering but enhancing disease resistance. The valine-glutamine (VQ) protein family, which contains the conserved motif FxxxVQxLTG ("x" represents any amino acid), plays a crucial role in plant growth and immunity along with mitogen-activated protein kinase (MAPK) cascades. However, only a few rice VQ proteins have been functionally characterized, and the roles of the MAPK-VQ module in rice biological processes are not fully understood. Here, we investigated the role of OsVQ1 in rice disease resistance and the control of flowering time. The OsVQ1-knock out (KO) mutants exhibited increased resistance to Xanthomonas oryzae pathovars, accumulated high levels of hydrogen peroxide (H2O2), and showed a late flowering phenotype under natural long-day conditions, while the OsVQ1-overexpressing plants showed phenotypes similar to that of the wild type. Further studies revealed that OsVQ1 physically interacted with and inhibited OsMPK6 activity. In addition, OsVQ1 expression was downregulated by the pathogen-induced OsMPKK10.2-OsMPK6-OsWRKY45 cascade, suggesting a feedback loop between OsVQ1 and OsMPK6. Moreover, the OsVQ1-KO/osmpk6 double-mutant exhibited increased susceptibility to X. oryzae infection and showed an early flowering phenotype, which may partially be attributed to the reduced accumulation of H2O2 and the consequent up-expression of flowering-promoting genes. These results suggested that the OsVQ1-OsMPK6 module was involved in rice immunity and flowering.

Senkyunolide I protect against lung injury via inhibiting formation of neutrophil extracellular trap in a murine model of cecal ligation and puncture.

BACKGROUND: Senkyunolide I (SEI), a component of a Chinese herb named Ligusticum Chuanxiong hort, which is included in the formulation of Xuebijing Injection, a medication used to treat sepsis in China. Our previous study showed that SEI was protective against sepsis-associated encephalopathy and the present study was performed to investigate the role of SEI in sepsis-induced lung injury in a murine model of cecal ligation and puncture (CLP). METHODS: SEI (36 mg/kg in 200 µl) or vehicle was administered immediately after CLP surgery. The lung injury was assessed 24 h later by histopathological tests, protein concentration in the bronchoalveolar lavage fluid (BALF), neutrophil recruitment in the lung tissue (myeloperoxidase fluorescence, MPO), pro-inflammatory cytokines and oxidative responses. Platelet activation was detected by CD42d/GP5 immunofluorescence and neutrophil extracellular trap (NET) were determined by immunofluorescence assays and enzyme linked immunosorbent assay (ELISA) of MPO-DNA. In vitro experiments were performed to detect the level of MPO-DNA complex released by SEI-treated neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA) or co-cultured with platelets from CLP mice. RESULTS: SEI administration relieved the injury degree in CLP mice according to the histopathological tests (P < 0.05 compared with DMSO + CLP group). Protein level in the BALF and neutrophil infiltration were remarkably reduced by SEI after CLP surgery (P < 0.05 compared with DMSO + CLP group). TNF-α, IL-1ß and IL-6 were decreased in the plasma and lung tissues from CLP mice treated with SEI (P < 0.05 compared with DMSO + CLP group). The phosphorylation of JNK, ERK, p38 and p65 were all inhibited by SEI (P < 0.05 compared with DMSO + CLP group). Immunofluorescence of MPO showed that neutrophil number was significantly lower in SEI treated CLP mice than in vehicle treated CLP mice (P < 0.05). The CD42d/GP5 staining suggested that platelet activation was significantly reduced and the NET level in the lung tissue and plasma was greatly attenuated by SEI treatment (P < 0.05 compared with DMSO + CLP group). In vitro experiments showed that the MPO-DNA level stimulated by PMA was significantly reduced by SEI treatment (P < 0.05 compared with DMSO treatment). Co-culture neutrophils with platelets from CLP mice resulted in higher level of MPO-DNA complex, while SEI partly reversed such effects of platelet on NET formation. CONCLUSIONS: SEI was protective against lung injury induced by CLP in mice. The NET formation was significantly reduced by SEI treatment, which might be involved in the mechanism of the protective effect.

Blockade of GITRL/GITR signaling pathway attenuates house dust mite-induced allergic asthma in mice through inhibition of MAPKs and NF-κB signaling.

GITRL/GITR signaling pathway plays an important role in allergy, inflammation, transplantation and autoimmunity. However, its role in asthma remains unclear. Thus, the present study aimed to investigate changes in this pathway and observe the therapeutic effect of its blocking on asthma. By using house dust mite-induced asthma model, changes of GITRL/GITR and its downstream molecules MAPKs (e.g., p38 MAPK, JNK and Erk) and NF-κB were observed. After that, GITRL in lung of mice was knocked down by recombinant adeno-associated virus to observe the impact on its downstream molecules and assess the therapeutic effect on asthma. These results showed that GITRL/GITR and its downstream molecules MAPKs/NF-κB were activated in asthmatic mice. This activation was suppressed after GITRL knockdown, and allergic airway inflammation and airway hyperresponsiveness were alleviated. These results demonstrate that GITRL/GITR-MAPKs/NF-κB signaling pathway participates in the pathogenesis of asthma. Blockade of GITRL/GITR signaling pathway exhibits protective effects in a mouse model of house dust mite-induced allergic asthma.

Deficiency of Innate Immunity against Pseudomonas aeruginosa Enhances Behavioral Avoidance via the HECW-1/NPR-1 Module in Caenorhabditis elegans.

To antagonize infection of pathogenic bacteria in soil and confer increased survival, Caenorhabditis elegans employs innate immunity and behavioral avoidance synchronously as the two main defensive strategies. Although both biological processes and their individual signaling pathways have been partially elucidated, knowledge of their interrelationship remains limited. The current study reveals that deficiency of innate immunity triggered by mutation of the classic immune gene pmk-1 promotes avoidance behavior in C. elegans and vice versa. Restoration of pmk-1 expression using the tissue-specific promoters suggested that the functional loss of both intestinal and neuronal pmk-1 is necessary for the enhanced avoidance. Additionally, PMK-1 colocalized with the E3 ubiquitin ligase HECW-1 in OLL neurons and regulated the expressional level of the latter, which consequently affected the production of NPR-1, a G-protein-coupled receptor (GPCR) homologous to the mammalian neuropeptide Y receptor, in RMG neurons in a non-cell-autonomous manner. Collectively, our study illustrates that once the innate immunity is impaired when C. elegans antagonizes bacterial infection, the other defensive strategy of behavioral avoidance can be enhanced accordingly via the HECW-1/NPR-1 module, suggesting that GPCRs in neural circuits may receive the inputs from the immune system and integrate those two systems for better adapting to the real-time status.

T-Cell Activation-Inhibitory Assay to Screen Caloric Restriction Mimetics Drugs for Drug Repositioning.

We previously reported a screening method for caloric restriction mimetics (CRM), a group of plant-derived compounds capable of inducing good health and longevity. In the present study, we explored the possibility of using this method to screen CRM drugs for drug repositioning. The method, T-cell activation-inhibitory assay, is based on inductive logic. Most of CRM such as resveratrol have been reported to suppress T-cell activation and have anti-inflammatory functions. Here, we assessed the activity of 12 antiallergic drugs through T-cell activation-inhibitory assay and selected four that showed the lowest IC50 values-ibudilast (IC50 0.97 µM), azelastine (IC50 7.2 µM), epinastine (IC50 16 µM), and amlexanox (IC50 33 µM)-for further investigation. Because azelastine showed high cytotoxicity, we selected only the remaining three drugs to study their biological functions. We found that all the three drugs suppressed the expression of interleukin (IL)-6, an inflammatory cytokine, in lipopolysaccharide-treated macrophage cells, with ibudilast being the strongest suppressor. Ibudilast also suppressed the secretion of another inflammatory cytokine, tumor necrosis factor (TNF)-α, and the expression of an inflammatory enzyme, cyclooxygenase-2, in the cells. These results suggest that T-cell activation-inhibitory assay can be used to screen potential CRM drugs having anti-inflammatory functions for the purpose of drug repositioning.

NLK suppresses MAVS-mediated signaling in black carp antiviral innate immunity.

Mammalian Nemo-like kinase (NLK) plays important roles in multiple biological processes including immune response; however, the roles of teleost NLK remain largely unknown. In the present study, the NLK homolog (bcNLK) of black carp (Mylopharyngodon piceus) has been cloned and characterized. The coding region of bcNLK consists of 1427 nucleotides and encodes 476 amino acid, including two low complexity region (LCR) domains at the N-terminus and a serine/threonine protein kinase catalytic (S-TKc) domain in the middle region. The transcription of bcNLK are promoted after spring viremia of carp virus (SVCV) infection and poly (I:C) stimulation in host cells, but not post LPS treatment. bcNLK exhibits weak impact on the transcription of interferon (IFN) promoter in the reporter assay, however, black carp MAVS (bcMAVS)-mediated IFN promoter transcription is remarkably dampened by bcNLK. The interaction between bcNLK and bcMAVS is detected through the co-immunoprecipitation assay. Accordingly, the plaque assay results show that bcMAVS-mediated antiviral ability is impaired by bcNLK. Moreover, knockdown of bcNLK in host cells leads to the enhanced antiviral ability against SVCV. All these data support the conclusion that black carp NLK associates with MAVS and inhibited MAVS-mediated antiviral signaling.

SUMO enables substrate selectivity by mitogen-activated protein kinases to regulate immunity in plants.

The versatility of mitogen-activated protein kinases (MAPKs) in translating exogenous and endogenous stimuli into appropriate cellular responses depends on its substrate specificity. In animals, several mechanisms have been proposed about how MAPKs maintain specificity to regulate distinct functional pathways. However, little is known of mechanisms that enable substrate selectivity in plant MAPKs. Small ubiquitin-like modifier (SUMO), a posttranslational modification system, plays an important role in plant development and defense by rapid reprogramming of cellular events. In this study we identified a functional SUMO interaction motif (SIM) in Arabidopsis MPK3 and MPK6 that reveals a mechanism for selective interaction of MPK3/6 with SUMO-conjugated WRKY33, during defense. We show that WRKY33 is rapidly SUMOylated in response to Botrytis cinerea infection and flg22 elicitor treatment. SUMOylation mediates WRKY33 phosphorylation by MPKs and consequent transcription factor activity. Disruption of either WRKY33 SUMO or MPK3/6 SIM sites attenuates their interaction and inactivates WRKY33-mediated defense. However, MPK3/6 SIM mutants show normal interaction with a non-SUMOylated form of another transcription factor, SPEECHLESS, unraveling a role for SUMOylation in differential substrate selectivity by MPKs. We reveal that the SUMO proteases, SUMO PROTEASE RELATED TO FERTILITY1 (SPF1) and SPF2 control WRKY33 SUMOylation and demonstrate a role for these SUMO proteases in defense. Our data reveal a mechanism by which MPK3/6 prioritize molecular pathways by differentially selecting substrates using the SUMO-SIM module during defense responses.

The elevated expression of IL-38 serves as an anti-inflammatory factor in osteoarthritis and its protective effect in osteoarthritic chondrocytes.

The objective of this study is to investigate the role of IL-38 in osteoarthritis (OA). IL-38 levels in serum and synovial fluid (SF) of patients with OA were examined to identify the correlation between IL-38 expression and OA activity and to determine its anti-inflammatory effects in IL-1ß-induced chondrocytes. A total of 75 patients with OA who underwent joint replacement surgery and 25 age- and sex-matched healthy volunteers were recruited. The levels of IL-38 in serum and SF are shown to be significant elevated in OA patients compared with that of healthy controls. Serum and SF IL-38 levels of OA patients are positively correlated with Kellgren-Lawrence (K-L) grades 2 to 3, as well as with pro-inflammatory cytokines IL-6, IL-23, and TNF-α, but are negatively correlated with the anti-inflammatory cytokine IL-10 in K-L grades 3 to 4. Furthermore, overexpression of IL-38 in vitro is shown to attenuate the expression of pro-inflammatory cytokines such as COX-2, IL-6, IL-8, IL-36Ra, IL-36α/ß/γ, iNOS, and TNF-α, as well as matrix degrading enzymes such as MMP3, MMP13, and ADAMTS5, and apoptosis-related indicators Bax/Bcl-2, cleaved caspase 3/pro-caspase 3, and cleaved caspase 9/pro-caspase 9. IL-38 overexpression also reduces expression of the signaling proteins p-p38, p-p65, p-JNK, and RhoA significantly. Taken together, our results show that expression of IL-38 is increased in OA tissues and OA rat chondrocytes, and is positively correlated with early disease activity. This increased IL-38 expression lead to the inactivation of MAPK, NF-κB, JNK, and RhoA signaling pathways, which might have impletion on OA chondrocytes apoptosis, degradation and inflammatory effect. Thus, IL-38 probably serves as a novel therapeutic target for the treatment of OA.

Anti-Inflammatory Effects of Ellagic Acid on Keratinocytes via MAPK and STAT Pathways.

Atopic dermatitis (AD) is a chronic inflammatory skin disease that is characterized by an impaired skin barrier and intense itchiness, which decreases the individual's quality of life. No fully effective therapeutic agents have prevailed for AD due to an insufficient grasp of the complex etiology. Ellagic acid (EA), a natural compound, has anti-inflammatory properties in chronic diseases. The effects of EA on AD have not yet been explored. The present study investigated the effects of EA on TNF-α/IFN-γ-stimulated HaCaT keratinocytes and house dust mite-induced AD-like skin lesions in NC/Nga mice. Treatment with EA suppressed inflammatory responses in keratinocytes by regulating critical inflammatory signaling pathways, such as mitogen-activated protein kinases and signal transducers and activators of transcription. In vivo studies using a DfE-induced AD mouse model showed the effects of EA administration through ameliorated skin lesions via decremented histological inflammatory reactions. These results suggest that EA could be a potential therapeutic alternative for the treatment of AD by inhibiting inflammatory signaling pathways.

KP-10/Gpr54 attenuates rheumatic arthritis through inactivating NF-κB and MAPK signaling in macrophages.

Rheumatoid arthritis (RA) is an autoimmune disease mainly characterized as chronic inflammation of joint. Both genetic and environmental factors play important roles in RA progression. G protein-coupled receptor 54 (GPR54) and Kisspeptins (KPs), the natural GRP54 ligands encoded by Kiss-1 gene are known to play important roles in immune regulation but the precise role of KP-10/GPR54 in RA remains elusive. Kiss1/Gpr54 expression was determined by immunohistochemistry on protein and real-time PCR on RNA from isolated RA-patient synovial tissue and PBMC. Collagen-induced arthritis (CIA) mouse models were used to investigate the effect of KP-10/Gpr54 on the rheumatic arthritis severity in the mice. The signaling pathway involved in KP-10/GPR54 was assessed by western blot and immunofluorescence.In the present study, we demonstrated that GPR54 upregulation in bone marrow-derived macrophages (BMDM) was associated with the severity of RA. In addition, Gpr54-/- increased the inflammatory cytokines induced by lipopolysaccharide (LPS) in BMDM and diseased severity of CIA (n = 10), while KP-10 reduced the LPS-induced inflammatory cytokines in vitro and ameliorated the CIA symptoms in vivo. Furthermore, we demonstrated that KP-10/GPR54 binds to PP2A-C to suppressed LPS induced NF-κB and MAPK signaling in BMDM. All these findings suggest that KP-10/GPR54 may be a novel therapeutic target for the diagnosis and treatment of RA.

Differential Role of Threonine and Tyrosine Phosphorylation in the Activation and Activity of the Yeast MAPK Slt2.

The Mitogen-Activated Protein Kinase (MAPK) Slt2 is central to signaling through the yeast Cell Wall Integrity (CWI) pathway. MAPKs are regulated by phosphorylation at both the threonine and tyrosine of the conserved TXY motif within the activation loop (T190/Y192 in Slt2). Since phosphorylation at both sites results in the full activation of MAPKs, signaling through MAPK pathways is monitored with antibodies that detect dually phosphorylated forms. However, most of these antibodies also recognize monophosphorylated species, whose relative abundance and functionality are diverse. By using different phosphospecific antibodies and phosphate-affinity (Phos-tag) analysis on distinct Slt2 mutants, we determined that Y192- and T190-monophosphorylated species coexist with biphosphorylated Slt2, although most of the Slt2 pool remains unphosphorylated following stress. Among the monophosphorylated forms, only T190 exhibited biological activity. Upon stimulation, Slt2 is first phosphorylated at Y192, mainly by the MAPKK Mkk1, and this phosphorylation is important for the subsequent T190 phosphorylation. Similarly, dephosphorylation of Slt2 by the Dual Specificity Phosphatase (DSP) Msg5 is ordered, with dephosphorylation of T190 depending on previous Y192 dephosphorylation. Whereas Y192 phosphorylation enhances the Slt2 catalytic activity, T190 is essential for this activity. The conserved T195 residue is also critical for Slt2 functionality. Mutations that abolish the activity of Slt2 result in a high increase in inactive Y192-monophosphorylated Slt2. The coexistence of different Slt2 phosphoforms with diverse biological significance highlights the importance of the precise detection of the Slt2 phosphorylation status.

Purification, characterization and immunostimulatory effects of polysaccharides from Anemarrhena asphodeloides rhizomes.

The crude polysaccharide was extracted from A. asphodeloides rhizomes and further purified to produce two fractions F1 (50.0%) and F2 (19.6%). The chemical constitutions of the polysaccharides were neutral sugars (51.4%-89.7%), uronic acids (1.0%-30.2%) and sulfate esters (3.4%-8.1%), with various ratios of monosaccharides including rhamnose (1.4%-6.1%), arabinose (7.1%-21.2%), xylose (0.2%-4.8%), mannose (39.9%-79.0%), glucose (6.0%-11.1%) and galactose (2.6%-22.0%). The molecular properties of the polysaccharides were investigated by the HPSEC-UV-MALLS-RI system, revealing the Mw 130.0 × 103-576.5 × 103 g/moL, Rg 87.6-382.6 nm and SVg 0.3-54.3 cm3/g. The polysaccharides stimulated RAW264.7 cells to produce considerable amounts of NO and up-regulate the expression of TNF-α, IL-1 and COX-2 genes. Polysaccharides exhibited the growth inhibitory effects on cancer cells lines of AGS, MKN-28 and MKN-45, in which F2 fraction exhibited prominent bioactivities. The AGS cells treated with F2 experienced condensed cytoplasm, shrinkage of nucleus and chromatin marginalization with the highest number of cells at early-stage apoptosis reaching 54.6%. The inhibitory effect of F2 polysaccharide on AGS cells was through MAPKs and STAT3 signaling pathways. The backbone of the F2 was mainly linked by (1 â†’ 4)-linked mannopyranosyl and (1 â†’ 3)-linked galactopyranosyl. Taken together, the polysaccharide from A. asphodeloides rhizomes could be utilized as medicinal, pharmacological and functional food ingredients.

Deciphering the Role of Leptospira Surface Protein LigA in Modulating the Host Innate Immune Response.

Leptospira, a zoonotic pathogen, is known to infect various hosts and can establish persistent infection. This remarkable ability of bacteria is attributed to its potential to modulate (activate or evade) the host immune response by exploiting its surface proteins. We have identified and characterized the domain of the variable region of Leptospira immunoglobulin-like protein A (LAV) involved in immune modulation. The 11th domain (A11) of the variable region of LigA (LAV) induces a strong TLR4 dependent innate response leading to subsequent induction of humoral and cellular immune responses in mice. A11 is also involved in acquiring complement regulator FH and binds to host protease Plasminogen (PLG), there by mediating functional activity to escape from complement-mediated killing. The deletion of A11 domain significantly impaired TLR4 signaling and subsequent reduction in the innate and adaptive immune response. It also inhibited the binding of FH and PLG thereby mediating killing of bacteria. Our study discovered an unprecedented role of LAV as a nuclease capable of degrading Neutrophil Extracellular Traps (NETs). This nuclease activity was primarily mediated by A11. These results highlighted the moonlighting function of LigA and demonstrated that a single domain of a surface protein is involved in modulating the host innate immune defenses, which might allow the persistence of Leptospira in different hosts for a long term without clearance.

Inappropriate activation of invariant natural killer T cells and antigen-presenting cells with the elevation of HMGB1 in preterm births without acute chorioamnionitis.

PROBLEM: Acute chorioamnionitis (aCAM) associated with microbial infection is a primary cause of preterm birth (PB). However, recent studies have demonstrated that innate immunity and sterile inflammation are causes of PB in the absence of aCAM. Therefore, we analyzed immune cells in the decidua of early to moderate PB without aCAM. METHOD OF STUDY: Deciduas were obtained from patients with PB at a gestational age of 24+0 to 33+6  weeks without aCAM in pathological diagnosis. The patients were divided into two groups as follows: patients with labor and/or rupture of membrane (ROM) (no aCAM with labor and/or ROM: nCAM-w-LR), and patients without labor and/or ROM (no aCAM without labor and/or ROM: nCAM-w/o-LR). The immune cells and high mobility group box 1 (HMGB1) levels in the decidua were analyzed using flow cytometry. Co-culture of CD56+ cells with dendritic cells (DCs) and macrophages obtained from the decidua was also performed in the presence of HMGB1. RESULTS: The nCAM-w-LR group demonstrated an accumulation of iNKT cells, and increased expression of HMGB1, TLR4, receptors for advanced glycation end products, and CD1d on DCs and macrophages. HMGB1 facilitated the proliferation of iNKT cells co-cultured with DCs and macrophages, which was found to be inhibited by heparin. CONCLUSIONS: Inappropriate activation of innate immune cells and increased HMGB1 expression may represent parturition signs in human pregnancy. Therefore, control of these cells and HMGB1 antigenicity may be represent a potential therapeutic target for the prevention of PB.

Mitogen-Activated Protein Kinase Cascade and Reactive Oxygen Species Metabolism are Involved in Acibenzolar-S-Methyl-Induced Disease Resistance in Apples.

Apple fruits were subjected to dipping treatment to explore the effects of acibenzolar-S-methyl (ASM) and the mitogen-activated protein kinase (MAPK) inhibitor PD98059 on lesion growth in fruits inoculated with Penicillium expansum. We investigated the roles of the MAPK cascade and reactive oxygen species metabolism in disease resistance in apples. ASM treatment inhibited lesion growth; suppressed catalase (CAT) activity; increased H2O2 content; reduced glutathione and ascorbic acid contents; and increased glutathione reductase, ascorbate peroxidase, peroxidase, superoxide dismutase, and NADPH oxidase activities. Moreover, ASM upregulated MdSOD, MdPOD, MdGR, MdAPX, MdMAPK4, MdMAPK2, and MdMAPKK1 expressions and downregulated MdCAT and MdMAPK3 expressions. PD98059 + ASM treatment increased CAT activity and MdCAT and MdMAPK3 expressions; inhibited MdSOD, MdPOD, MdGR, MdAPX, MdMAPK4, MdMAPK2, and MdMAPKK1 expressions; reduced superoxide dismutase, peroxidase, ascorbate peroxidase, and glutathione reductase activities; and reduced glutathione content in apples. These findings indicate that ASM induces disease resistance in apples by regulating the expressions of key genes involved in reactive oxygen species metabolism and the MAPK cascade.

Chicken avian ß-defensin 8 modulates immune response via the mitogen-activated protein kinase signaling pathways in a chicken macrophage cell line.

Defensins are antimicrobial peptides composed of 3 conserved disulfide bridges, a ß-sheet, and both hydrophobic and cationic amino acids. In this study, we aimed to demonstrate the immunomodulation role of avian ß-defensin 8 (AvBD8) in a chicken macrophage cell line. Chicken AvBD8 stimulated the expression of proinflammatory cytokines (IL-1ß, interferon gamma, and IL-12p40) and chemokines (CCL4, CXCL13, and CCL20) in macrophages. Furthermore, by Western blotting and immunocytochemistry, we confirmed that AvBD8 activated the mitogen-activated protein kinase signaling pathway via extracellular regulated kinases 1/2 and p38 signaling molecules. Overall, AvBD8 plays a crucial role in host defense as not only an antimicrobial peptide but also an immunomodulator by activating the mitogen-activated protein kinase signaling pathway and inducing the expression of proinflammatory cytokines and chemokines.

Hispidulin alleviates imiquimod-induced psoriasis-like skin inflammation by inhibiting splenic Th1/Th17 cell population and keratinocyte activation.

Psoriasis is a chronic inflammatory skin disease characterized by hyperproliferation and abnormal differentiation of epidermal keratinocytes accompanied by increased infiltration of immune cells. Previous studies have demonstrated that hispidulin (4',5,7-trihydroxy-6-methoxyflavone, HPD) has various pharmacological benefits such as anti-fungal, anti-inflammation, and anti-allergic effects. This study investigated the effectiveness of HPD to treat psoriasis using an imiquimod (IMQ)-induced mouse model and activated keratinocytes. IMQ was topically applied to the back skin of mice for six consecutive days, and the mice were orally administered HPD. Based on the histological observation and immunological analysis, oral administration of HPD suppressed psoriatic characteristics including skin thickness, psoriasis area severity index, transepidermal water loss, and neutrophil infiltration. HPD alleviated pathologically increased levels of immunoglobulin G2a, myeloperoxidase, and tumor necrosis factor-α. Splenic Th1 and Th17 cell populations were also reduced by HPD in the murine model. In addition, in activated keratinocytes, HPD inhibited gene expression of Th1- and Th17-associated cytokines and chemokines, and phosphorylation of mitogen-activated protein kinases and nuclear factor-κB. In summary, HPD alleviates psoriasis skin inflammation in vivo and in vitro. Therefore, we suggest that HPD would be a potent therapeutic candidate for the treatment of psoriasis.