Cloning and analysis of peptidoglycan recognition protein‐LC and immune deficiency from the diamondback moth,Plutella xylostella

Peptidoglycan (PGN) exists in both Gram‐negative and Gram‐positive bacteria as a component of the cell wall. PGN is an important target to be recognized by the innate immune system of animals. PGN recognition proteins (PGRP) are responsible for recognizing PGNs. In Drosophila melanogaster, PGRP‐LC and IMD (immune deficiency) are critical for activating the Imd pathway. Here, we report the cloning and analysis of PGRP‐LC and IMD (PxPGRP‐LC and PxIMD) from diamondback moth, Plutella xylostella (L.), the insect pest of cruciferous vegetables. PxPGRP‐LC gene consists of six exons encoding a polypeptide of 308 amino acid residues with a transmembrane region and a PGRP domain. PxIMD cDNA encodes a polypeptide of 251 amino acid residues with a death domain. Sequence comparisons indicate that they are characteristic of Drosophila PGRP‐LC and IMD homologs. PxPGRP‐LC and PxIMD were expressed in various tissues and developmental stages. Their mRNA levels were affected by bacterial challenges. The PGRP domain of PxPGRP‐LC lacks key residues for the amidase activity, but it can recognize two types of PGNs. Overexpression of full‐length and deletion mutants in Drosophila S2 cells induced expression of some antimicrobial peptide genes. These results indicate that PxPGRP‐LC and PxIMD may be involved in the immune signaling of P. xylostella. This study provides a foundation for further studies of the immune system of P. xylostella.     

Functional characterization of PGRP‐LC1 of Anopheles gambiae through deletion and RNA interference

Abstract Peptidoglycan recognition proteins (PGRP) play an important role in innate immunity in insects through the activation of the Imd pathway, which has been shown to be required in the antibacterial response in insects and in the limitation of the number of Plasmodium berghei oocysts developing in mosquito midgut. The LC1 gene of the PRGP family in Anopheles gambiae produces many products through alternative splicing. In this work, we demonstrate that PGRP‐LC1a alone is sufficient to activate the Imd pathway in the A. gambiae L3–5 cell line through a combination of terminal or internal deletions, and RNA interference against endogenous PGRP‐LC products. In the absence of endogenous PGRP‐LC proteins, the integrity of the cytoplasmic domain is necessary for LC1a function, while that of the extracellular domain is not. Moreover, the shorter the extracellular domain, the higher the activity for LC1a. However, the removal of either the cytoplasmic or the extracellular PGRP‐binding domain has little impact on the activity of LC1a in the presence of endogenous PGRP‐LC proteins.     

The Protective Role of Interleukin-33 in Myocardial Ischemia and Reperfusion Is Associated with Decreased HMGB1 Expression and Up-Regulation of the P38 MAPK Signaling Pathway

Interleukin-33 (IL-33) plays a protective role in myocardial ischemia and reperfusion (I/R) injury, but the underlying mechanism was not fully elucidated. The present study was designed to investigate whether IL-33 protects against myocardial I/R injury by regulating both P38 mitogen-activated-protein kinase (P38 MAPK), which is involved in one of the downstream signaling pathways of IL-33, and high mobility group box protein 1 (HMGB1), a late pro-inflammatory cytokine. Myocardial I/R injury increased the level of IL-33 and its induced receptor (sST) in myocardial tissue. Compared with the I/R group, the IL-33 group had significantly lower cardiac injury (lower serum creatine kinase (CK), lactate dehydrogenase (LDH), and cTnI levels and myocardial infarct size), a suppressed inflammatory response in myocardial tissue (lower expression of HMGB1, IL-6, TNF-α and INF-γ) and less myocardial apoptosis (much higher Bcl-2/Bax ratio and lower cleaved caspase-3 expression). Moreover, IL-33 activated the P38 MAPK signaling pathway (up-regulating P-P38 expression) in myocardial tissue, and SB230580 partially attenuated the anti-inflammatory and anti-apoptosis effects of IL-33. These findings indicated that IL-33 protects against myocardial I/R injury by inhibiting inflammatory responses and myocardial apoptosis, which may be associated with the HMGB1 and P38 MAPK signaling pathways.     

The MAP kinase‐activated protein kinase 2 (MK2) contributes to the Shiga toxin‐induced inflammatory response

Infection with Shiga toxin (STx)‐producing bacteria can progress to a toxemic, extraintestinal injury cascade known as haemolytic uremic syndrome (HUS), the leading cause of acute renal failure in children. Mounting evidence suggests that STx activates stress response pathways in susceptible cells and has implicated the p38 mitogen‐activated protein kinase (MAPK) pathway. More importantly, some of the pathology associated with HUS is believed to be a result of a STx‐induced inflammatory response. From a siRNA screen of the human kinome adapted to a high‐throughput format, we found that knock‐down of the MAPK‐activated protein kinase 2 (MK2), a downstream target of the p38 MAPK, protected against Shiga toxicity. Further characterization of the in vitro role of MK2 revealed that STx activates the p38‐MK2 stress response pathway in both p38‐ and MK2‐dependent manners in two distinct cell lines. MK2 activation was specific to damage to the ribosome by an enzymatically active toxin and did not result from translational inhibition per se. Genetic and chemical inhibition of MK2 significantly decreased the inflammatory response to STx. These findings suggest that MK2 inhibition might play a valuable role in decreasing the immuopathological component of STx‐mediated disease.     

Reactive oxygen species‐induced activation of ERK and p38 MAPK mediates PMA‐induced NETs release from human neutrophils

Neutrophils/polymorphonuclear leukocytes (PMNs), an important component of innate immune system, release extracellular traps (NETs) to eliminate invaded pathogens; however understanding of the role of signaling molecules/proteins need to be elucidated. In the present study role of p38 MAPK and extracellular signal regulated kinase (ERK) against phorbol 12‐myristate 13‐acetate (PMA) induced reactive oxygen species (ROS) generation and NETs formation has been investigated. Human neutrophils were treated with PMA to induce free radical generation and NETs release, which were monitored by NBT reduction and elastase/DNA release, respectively. PMA treatment led to the time dependent phosphorylation of p38 MAPK and ERK in PMNs. Pretreatment of PMNs with SB202190 or U0126 did not significantly reduce PMA induce free radical generation, but prevented NETs release. Pretreatment of PMNs with NADPH oxidase inhibitor (diphenyleneiodonium chloride) significantly reduced free radical generation, p38 MAPK and ERK phosphorylation as well as NETs release, suggesting that p38 MAPK and ERK activation was downstream to free radical generation. The present study thus demonstrates ROS dependent activation of ERK and p38 MAPK, which mediated PMA induced NETs release from human neutrophils. J. Cell. Biochem. 114: 532–540, 2013. © 2012 Wiley Periodicals, Inc.     

Attenuation of pattern recognition receptor signaling is mediated by a MAP kinase kinase kinase

Pattern recognition receptors (PRRs) play a key role in plant and animal innate immunity. PRR binding of their cognate ligand triggers a signaling network and activates an immune response. Activation of PRR signaling must be controlled prior to ligand binding to prevent spurious signaling and immune activation. Flagellin perception in Arabidopsis through FLAGELLIN‐SENSITIVE 2 (FLS2) induces the activation of mitogen‐activated protein kinases (MAPKs) and immunity. However, the precise molecular mechanism that connects activated FLS2 to downstream MAPK cascades remains unknown. Here, we report the identification of a differentially phosphorylated MAP kinase kinase kinase that also interacts with FLS2. Using targeted proteomics and functional analysis, we show that MKKK7 negatively regulates flagellin‐triggered signaling and basal immunity and this requires phosphorylation of MKKK7 on specific serine residues. MKKK7 attenuates MPK6 activity and defense gene expression. Moreover, MKKK7 suppresses the reactive oxygen species burst downstream of FLS2, suggesting that MKKK7‐mediated attenuation of FLS2 signaling occurs through direct modulation of the FLS2 complex.     

Non‐hemolytic enterotoxin of Bacillus cereus induces apoptosis in Vero cells

Bacillus cereus is an opportunistic pathogen that often causes foodborne infectious diseases and food poisoning. Non‐hemolytic enterotoxin (Nhe) is the major toxin found in almost all enteropathogenic B. cereus and B. thuringiensis isolates. However, little is known about the cellular response after Nhe triggered pore formation on cell membrane. Here, we demonstrate that Nhe induced cell cycle arrest at G₀/G₁ phase and provoked apoptosis in Vero cells, most likely associated with mitogen‐activated protein kinase (MAPK) and death receptor pathways. The influx of extracellular calcium ions and increased level of reactive oxygen species in cytoplasm were sensed by apoptosis signal‐regulating kinase 1 (ASK1) and p38 MAPK. Extrinsic death receptor Fas could also promote the activation of p38 MAPK. Subsequently, ASK1 and p38 MAPK triggered downstream caspase‐8 and 3 to initiate apoptosis. Our results clearly demonstrate that ASK1, and Fas‐p38 MAPK‐mediated caspase‐8 dependent pathways are involved in apoptotic cell death provoked by the pore‐forming enterotoxin Nhe.     

Endocytosis of Mycobacterium tuberculosis Heat Shock Protein 60 Is Required to Induce Interleukin-10 Production in Macrophages

Understanding the signaling pathways involved in the regulation of anti-inflammatory and pro-inflammatory responses in tuberculosis is extremely important in tailoring a macrophage innate response to promote anti-tuberculosis immunity in the host. Although the role of toll-like receptors (TLRs) in the regulation of anti-inflammatory and pro-inflammatory responses is known, the detailed molecular mechanisms by which the Mycobacterium tuberculosis bacteria modulate these innate responses are not clearly understood. In this study, we demonstrate that M. tuberculosis heat shock protein 60 (Mtbhsp60, Cpn60.1, and Rv3417c) interacts with both TLR2 and TLR4 receptors, but its interaction with TLR2 leads to clathrin-dependent endocytosis resulting in an increased production of interleukin (IL)-10 and activated p38 MAPK. Blockage of TLR2-mediated endocytosis inhibited IL-10 production but induced production of tumor necrosis factor (TNF)-α and activated ERK1/2. In contrast, upon interaction with TLR4, Mtbhsp60 remained predominantly localized on the cell surface due to poorer endocytosis of the protein that led to decreased IL-10 production and p38 MAPK activation. The Escherichia coli homologue of hsp60 was found to be retained mainly on the macrophage surface upon interaction with either TLR2 or TLR4 that triggered predominantly a pro-inflammatory-type immune response. Our data suggest that cellular localization of Mtbhsp60 upon interaction with TLRs dictates the type of polarization in the innate immune responses in macrophages. This information is likely to help us in tailoring the host protective immune responses against M. tuberculosis.     

Sequestosome-1/p62 Is the Key Intracellular Target of Innate Defense Regulator Peptide

Innate defense regulator-1 (IDR-1) is a synthetic peptide with no antimicrobial activity that enhances microbial infection control while suppressing inflammation. Previously, the effects of IDR-1 were postulated to impact several regulatory pathways including mitogen-activated protein kinase (MAPK) p38 and CCAAT-enhancer-binding protein, but how this was mediated was unknown. Using a combined stable isotope labeling by amino acids in cell culture-proteomics methodology, we identified the cytoplasmic scaffold protein p62 as the molecular target of IDR-1. Direct IDR-1 binding to p62 was confirmed by several biochemical binding experiments, and the p62 ZZ-type zinc finger domain was identified as the IDR-1 binding site. Co-immunoprecipitation analysis of p62 molecular complexes demonstrated that IDR-1 enhanced the tumor necrosis factor α-induced p62 receptor-interacting protein 1 (RIP1) complex formation but did not affect tumor necrosis factor α-induced p62-protein kinase ζ complex formation. In addition, IDR-1 induced p38 MAPK activity in a p62-dependent manner and increased CCAAT-enhancer-binding protein β activity, whereas NF-κB activity was unaffected. Collectively, these results demonstrate that IDR-1 binding to p62 specifically affects protein-protein interactions and subsequent downstream events. Our results implicate p62 in the molecular mechanisms governing innate immunity and identify p62 as a potential therapeutic target in both infectious and inflammatory diseases.     

A hot water extract of Aralia cordata activates bone marrow‐derived macrophages via a myeloid differentiation protein 88‐dependent pathway and protects mice from bacterial infection

In traditional Asian medicine, Aralia cordata (AC) is a known as a pain reliever and anti‐inflammatory drug. Although several of its biological activities have been reported, the immunomodulatory effects of a hot water extract of AC (HAC) have not yet been described. The aim of this study was to investigate whether HAC modulates the activation of macrophages, which play important roles in innate immune responses against microbial pathogens, and if so, to determine the molecular mechanisms by which HAC mediates this process. It was found that HAC activates bone marrow‐derived macrophages (BMDM) and increases amounts of nitric oxide and proinflammatory cytokines in a dose‐dependent manner. In addition, HAC was found to induce phosphorylation of NF‐κB and mitogen‐activated protein kinases (MAPKs), including c‐Jun N‐terminal kinases, extracellular signal‐regulated kinases and p38. Interestingly, these effects were absent in BMDM prepared from myeloid differentiation protein 88‐knockout mice. Polysaccharides from HAC exerted stronger immunostimulatory effects than HAC itself. Furthermore, orally administered HAC clearly enhanced clearance of the intracellular pathogen Listeria monocytogenes by boosting innate immune responses. These results demonstrate that HAC exerts immunostimulatory effects through the TLR/MyD88 and NF‐κB/MAPK signal transduction pathways.     

Viral induction of inflammatory cytokines in human epithelial cells follows a p38 mitogen-activated protein kinase-dependent but NF-kappa B-independent pathway

The initial step in an immune response toward a viral infection is the induction of inflammatory cytokines. This innate immune response is mediated by expression of a variety of cytokines exemplified by TNF-alpha and IL-1beta. A key signal for the recognition of intracellular viral infections is the presence of dsRNA. Viral infections and dsRNA treatment can activate several signaling pathways including the protein kinase R pathway, mitogen-activated protein kinase (MAPK) pathways, and NF-kappaB, which are important in the expression of inflammatory cytokines. We previously reported that activation of protein kinase R was required for dsRNA induction of TNF-alpha, but not for IL-1beta. In this study, we report that activation of the p38 MAPK pathway by respiratory viral infections is necessary for induction of inflammatory cytokines in human bronchial epithelial cells. Inhibition of p38 MAPK by two different pharmacological inhibitors showed that expression of both TNF-alpha and IL-1beta required activation of this signaling pathway. Interestingly, inhibition of NF-kappaB did not significantly reduce viral induction of either cytokine. Our data show that, during the initial infections of epithelial cells with respiratory viruses, activation of the p38 MAPK pathway is associated with induction of inflammation, and NF-kappaB activation may be less important than previously suggested.     

Crystal structure of peptidoglycan recognition protein SA in Apis mellifera (Hymenoptera: Apidae)

Peptidoglycan recognition protein SA (PGRP‐SA) is a key pattern recognition receptor in the insect innate immune system. PGRP‐SA can bind to bacterial PGN and activate the Toll pathway, which triggers the expression and release of antimicrobial peptides to prevent bacterial infection. Here, we report the first structure of Apis mellifera PGRP‐SA from Hymenoptera at 1.86 Å resolution. The overall architecture of Am‐PGRP‐SA was similar to the Drosophila PGRP‐SA; however, the residues involved in PGN binding groove were not conserved, and the binding pocket was narrower. This structure gives insight into PGN binding characteristics in honeybees.     

Paralytic peptide activates insect humoral immune response via epidermal growth factor receptor

Paralytic peptide (PP) activates innate immunity of silkworm Bombyx mori, inducing production of anti-microbial peptides (AMPs) and phagocytosis-related proteins; however the signal pathways of PP-dependent immune responses are not clear. In present study, we characterized BmE cells as a PP-responsive cell line by examining the expression of AMP genes and activation of p38 mitogen-activated protein kinase (p38 MAPK) under PP stimulation, and we also found PP directly binds to BmE cell membrane. Then we found that PP-dependent expression of AMP genes is suppressed by tyrosine kinase inhibitor (genistein) both in BmE cells and in fat body of silkworm larvae. Moreover, the specific tyrosine kinase epidermal growth factor receptor (EGFR) inhibitor (AG1478) attenuates PP-induced expression of AMP genes in BmE cells and fat body of silkworm and RNA interference (RNAi) to BmEGFR also suppresses PP-induced expression of AMP genes. Furthermore, the PP-induced p38 MAPK phosphorylation is inhibited by AG1478. Our results suggest that BmE cells can be used as a cell model to investigate the signal pathway of PP-dependent humoral immune response and receptor tyrosine kinase EGFR/p38 MAPK pathway is involved in the production of AMPs induced by PP.     

Anti-inflammatory effects of a p38 mitogen-activated protein kinase inhibitor during human endotoxemia

The p38 mitogen-activated protein kinase (MAPK) participates in intracellular signaling cascades resulting in inflammatory responses. Therefore, inhibition of the p38 MAPK pathway may form the basis of a new strategy for treatment of inflammatory diseases. However, p38 MAPK activation during systemic inflammation in humans has not yet been shown, and its functional significance in vivo remains unclear. Hence, we exposed 24 healthy male subjects to an i.v. dose of LPS (4 ng/kg), preceded 3 h earlier by orally administered 600 or 50 mg BIRB 796 BS (an in vitro p38 MAPK inhibitor) or placebo. Both doses of BIRB 796 BS significantly inhibited LPS-induced p38 MAPK activation in the leukocyte fraction of the volunteers. Cytokine production (TNF-alpha, IL-6, IL-10, and IL-1R antagonist) was strongly inhibited by both low and high dose p38 MAPK inhibitor. In addition, p38 MAPK inhibition diminished leukocyte responses, including neutrophilia, release of elastase-alpha(1)-antitrypsin complexes, and up-regulation of CD11b with down-regulation of L-selectin. Finally, blocking p38 MAPK decreased C-reactive protein release. These data identify p38 MAPK as a principal mediator of the inflammatory response to LPS in humans. Furthermore, the anti-inflammatory potential of an oral p38 MAPK inhibitor in humans in vivo suggests that p38 MAPK inhibitors may provide a new therapeutic option in the treatment of inflammatory diseases.     

Osteopontin stimulates autophagy via integrin/CD44 and p38 MAPK signaling pathways in vascular smooth muscle cells

Osteopontin (OPN) exerts pro‐inflammatory effect and is associated with the development of abdominal aortic aneurysm (AAA). However, the molecular mechanism underlying this association remains obscure. In the present study, we compared gene expression profiles of AAA tissues using microarray assay, and found that OPN was the highest expressed gene (>125‐fold). Furthermore, the expression of LC3 protein and autophagy‐related genes including Atg4b, Beclin1/Atg6, Bnip3, and Vps34 was markedly upregulated in AAA tissues. To investigate the ability of OPN to stimulate autophagy as a potential mechanism involved in the pathogenesis of this disease, we treated vascular smooth muscle cells (SMCs) with OPN, and found that OPN significantly increased the formation of autophagosomes, expression of autophagy‐related genes and cell death, whereas blocking the signal by anti‐OPN antibody markedly inhibited OPN‐induced autophagy and SMC death. Furthermore, inhibition of integrin/CD44 and p38 MAPK signaling pathways markedly abrogated the biological effects of OPN on SMCs. These data for the first time demonstrate that OPN sitmulates autophagy directly through integrin/CD44 and p38 MAPK‐mediated pathways in SMCs. Thus, inhibition of OPN‐induced autophagy might be a potential therapeutic target in the treatment of AAA disease. J. Cell. Physiol. 227: 127–135, 2012. © 2011 Wiley Periodicals, Inc.     

Vesicular stomatitis virus matrix protein impairs CD1d-mediated antigen presentation through activation of the p38 MAPK pathway

Natural killer T (NKT) cells are unique T lymphocytes that recognize CD1d-bound lipid antigens and play an important role in both innate and acquired immune responses against infectious diseases and tumors. We have already shown that a vesicular stomatitis virus (VSV) infection results in the rapid inhibition of murine CD1d-mediated antigen presentation to NKT cells. In the present study, it was found that the VSV matrix (VSV-M) protein is an important element in this decrease in antigen presentation postinfection. The VSV-M protein altered the intracellular distribution of murine CD1d molecules, resulting in qualitative (but not quantitative) changes in cell surface CD1d expression. The M protein was distributed throughout the infected cell, and it was found to activate the mitogen-activated protein kinase (MAPK) p38 very early postinfection. Infection of CD1d⁺ cells with a temperature-sensitive VSV-M mutant at the nonpermissive temperature both substantially reversed the inhibition of antigen presentation by CD1d and delayed the activation of p38. Thus, the VSV-M protein plays an important role in permitting the virus to evade important components of the innate immune response by regulating specific MAPK pathways.