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Coxiella burnetii is a bacterial pathogen that replicates within host cells by establishing a membrane-bound niche called the Coxiella -containing vacuole. Biogenesis of this compartment requires effectors of its Dot/Icm type IV secretion system. A large cohort of such effectors has been identified, but the function of most of them remain elusive. Here, by a cell-based functional screening, we identified the effector Cbu0513 (designated as CinF) as an inhibitor of NF-κB signaling. CinF is highly similar to a fructose-1,6-bisphosphate (FBP) aldolase/phosphatase present in diverse bacteria. Further study reveals that unlike its ortholog from Sulfolobus tokodaii , CinF does not exhibit FBP phosphatase activity. Instead, it functions as a protein phosphatase that specifically dephosphorylates and stabilizes IκBα. The IκBα phosphatase activity is essential for the role of CinF in C. burnetii virulence. Our results establish that C. burnetii utilizes a protein adapted from sugar metabolism to subvert host immunity., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Background: Experimental autoimmune encephalomyelitis (EAE) is an animal disease model of multiple sclerosis (MS) that involves the immune system and central nervous system (CNS). However, it is unclear how genetic predispositions promote neuroinflammation in MS and EAE. Here, we investigated how partial loss-of-function of suppressor of MEK1 (SMEK1), a regulatory subunit of protein phosphatase 4, facilitates the onset of MS and EAE., Methods: C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein 35-55 (MOG 35-55 ) to establish the EAE model. Clinical signs were recorded and pathogenesis was investigated after immunization. CNS tissues were analyzed by immunostaining, quantitative polymerase chain reaction (qPCR), western blot analysis, and enzyme-linked immunosorbent assay (ELISA). Single-cell analysis was carried out in the cortices and hippocampus. Splenic and lymph node cells were evaluated with flow cytometry, qPCR, and western blot analysis., Results: Here, we showed that partial Smek1 deficiency caused more severe symptoms in the EAE model than in controls by activating myeloid cells and that Smek1 was required for maintaining immunosuppressive function by modulating the indoleamine 2,3-dioxygenase (IDO1)-aryl hydrocarbon receptor (AhR) pathway. Single-cell sequencing and an in vitro study showed that Smek1-deficient microglia and macrophages were preactivated at steady state. After MOG 35-55 immunization, microglia and macrophages underwent hyperactivation and produced increased IL-1β in Smek1 -/+ mice at the peak stage. Moreover, dysfunction of the IDO1-AhR pathway resulted from the reduction of interferon γ (IFN-γ), enhanced antigen presentation ability, and inhibition of anti-inflammatory processes in Smek1 -/+ EAE mice., Conclusions: The present study suggests a protective role of Smek1 in autoimmune demyelination pathogenesis via immune suppression and inflammation regulation in both the immune system and the central nervous system. Our findings provide an instructive basis for the roles of Smek1 in EAE and broaden the understanding of the genetic factors involved in the pathogenesis of autoimmune demyelination.

During a 15-year period, the incidence of type 1 diabetes has doubled in Lithuania, while increasing by a third in England; however, England still has a higher incidence. Analysis of sera collected from non-diabetic schoolchildren from Lithuania and England more than 20 years ago showed a similar number of multiple autoantibody-positive schoolchildren between the populations, but a higher prevalence of islet antigen-2 autoantibodies (IA-2A) in English schoolchildren. We aimed to use recently developed, more specific islet autoantibody tests to characterize differences in humoral autoimmunity between these two general population cohorts in greater detail. Samples from 88 Lithuanian and 133 English schoolchildren previously found islet autoantibody-positive were selected for measurement of additional islet autoantibodies by radioimmunoassay. Samples were tested for autoantibodies to zinc transporter 8 (ZnT8A), GAD (96-585), the protein tyrosine phosphatase region of islet antigen-2 (PTPA) and the related IA-2βA, while autoantibodies to IA-2A were reassayed using the current harmonized method. IA-2-related autoantibodies PTPA (0·13 versus 0·45%, P = 0·027) and IA-2βA (0 versus 0·35%, P < 0·001), but not IA-2A measured using the harmonized method, were less common in Lithuanian compared to English schoolchildren. Lithuanian schoolchildren who were islet autoantibody-positive were positive for fewer biochemical autoantibodies compared with English schoolchildren (P = 0·043). Background rates of islet autoimmunity in childhood differ subtly between countries, which have different incidences of type 1 diabetes. The optimal screening strategy (age and combination of markers) for detection of islet autoimmunity may vary between countries, dependent upon the pattern of autoantibodies found in the general population., (© 2020 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.)

Stimulator of interferon genes (STING) is an essential adaptor protein of the innate DNA-sensing signaling pathway, which recognizes genomic DNA from invading pathogens to establish antiviral responses in host cells. STING activity is tightly regulated by several posttranslational modifications, including phosphorylation. However, specifically how the phosphorylation status of STING is modulated by kinases and phosphatases remains to be fully elucidated. In this study, we identified protein phosphatase 6 catalytic subunit (PPP6C) as a binding partner of Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 48 (ORF48), which is a negative regulator of the cyclic GMP-AMP synthase (cGAS)-STING pathway. PPP6C depletion enhances double-stranded DNA (dsDNA)-induced and 5'ppp double-stranded RNA (dsRNA)-induced but not poly(I:C)-induced innate immune responses. PPP6C negatively regulates dsDNA-induced IRF3 activation but not NF-κB activation. Deficiency of PPP6C greatly inhibits the replication of herpes simplex virus 1 (HSV-1) and vesicular stomatitis virus (VSV) as well as the reactivation of KSHV, due to increased type I interferon production. We further demonstrated that PPP6C interacts with STING and that loss of PPP6C enhances STING phosphorylation. These data demonstrate the important role of PPP6C in regulating STING phosphorylation and activation, which provides an additional mechanism by which the host responds to viral infection. IMPORTANCE Cytosolic DNA, which usually comes from invading microbes, is a dangerous signal to the host. The cGAS-STING pathway is the major player that detects cytosolic DNA and then evokes the innate immune response. As an adaptor protein, STING plays a central role in controlling activation of the cGAS-STING pathway. Although transient activation of STING is essential to trigger the host defense during pathogen invasion, chronic STING activation has been shown to be associated with several autoinflammatory diseases. Here, we report that PPP6C negatively regulates the cGAS-STING pathway by removing STING phosphorylation, which is required for its activation. Dephosphorylation of STING by PPP6C helps prevent the sustained production of STING-dependent cytokines, which would otherwise lead to severe autoimmune disorders. This work provides additional mechanisms on the regulation of STING activity and might facilitate the development of novel therapeutics designed to prevent a variety of autoinflammatory disorders., (Copyright © 2020 Ni et al.)

The cyclic GMP-AMP (cGAMP) synthase (cGAS) plays a critical role in host defense by sensing cytosolic DNA derived from microbial pathogens or mis-located cellular DNA. Upon DNA binding, cGAS utilizes GTP and ATP as substrates to synthesize cGAMP, leading to MITA-mediated innate immune response. In this study, we identified the phosphatase PPP6C as a negative regulator of cGAS-mediated innate immune response. PPP6C is constitutively associated with cGAS in un-stimulated cells. DNA virus infection causes rapid disassociation of PPP6C from cGAS, resulting in phosphorylation of human cGAS S435 or mouse cGAS S420 in its catalytic pocket. Mutation of this serine residue of cGAS impairs its ability to synthesize cGAMP upon DNA virus infection. In vitro experiments indicate that S420-phosphorylated mcGAS has higher affinity to GTP and enzymatic activity. PPP6C-deficiency promotes innate immune response to DNA virus in various cells. Our findings suggest that PPP6C-mediated dephosphorylation of a catalytic pocket serine residue of cGAS impairs its substrate binding activity and innate immune response, which provides a mechanism for keeping the DNA sensor cGAS inactive in the absence of infection to avoid autoimmune response.

Viral infections trigger host innate immune responses, characterized by the production of type-I interferons (IFN) including IFNβ. IFNβ induces cellular antiviral defense mechanisms and thereby contributes to pathogen clearance. Accumulating evidence suggests that mitochondria constitute a crucial platform for the induction of antiviral immunity. Here we demonstrate that the mitochondrial protein phosphoglycerate mutase family member 5 (PGAM5) is important for the antiviral cellular response. Following challenge of HeLa cells with the dsRNA-analog poly(I:C), PGAM5 oligomers and high levels of PGAM5 were found in mitochondrial aggregates. Using immunoprecipitation, a direct interaction of PGAM5 with the mitochondrial antiviral-signaling protein (MAVS) was demonstrated. In addition, PGAM5 deficient cells showed diminished expression of IFNβ and IFNβ target genes as compared to WT cells. Moreover, PGAM5 deficient mouse embryonic fibroblasts (MEFs) exhibited decreased phosphorylation levels of IRF3 and TBK1 when challenged with poly(I:C) intracellularly. Finally, PGAM5 deficient MEFs, upon infection with vesicular stomatitis virus (VSV), revealed diminished IFNβ expression and increased VSV replication. Collectively, our study highlights PGAM5 as an important regulator for IFNβ production mediated via the TBK1/IRF3 signaling pathway in response to viral infection.

Hypoxia in adipose tissue is regarded as a trigger that induces dysregulation of the secretory profile in adipocytes. Similarly, local dysregulation of adipocytokine secretion is an initial event in the deleterious effects of obesity on metabolism. We previously reported that CXCL13 is highly produced during adipogenesis, however little is known about the roles of CXCL13 in adipocytes. Here, we found that hypoxia, as modeled by 1% O2 or exposure to the hypoxia-mimetic reagent desferrioxamine (DFO) has strong inductive effects on the expression of CXCL13 and CXCR5, a CXCL13 receptor, in both undifferentiated and differentiated adipocytes and in organ-cultured white adipose tissue (WAT). CXCL13 was also highly expressed in WAT from high fat diet-fed mice. Hypoxic profile, typified by increased expression of interleukin-6 (IL-6) and plasminogen activator inhibitor-1 (PAI-1) and decreased expression of adiponectin, was significantly induced by CXCL13 treatment during adipogenic differentiation. Conversely, the treatment of adipocytes with a neutralizing-antibody against CXCL13 as well as CXCR5 knockdown by specific siRNA effectively inhibited DFO-induced inflammation. The phosphorylation of Akt2, a protective factor of adipose inflammation, was significantly inhibited by CXCL13 treatment during adipogenic differentiation. Mechanistically, CXCL13 induces the expression of PHLPP1, an Akt2 phosphatase, through focal adhesion kinase (FAK) signaling; and correspondingly we show that CXCL13 and DFO-induced IL-6 and PAI-1 expression was blocked by Phlpp1 knockdown. Furthermore, we revealed the functional binding sites of PPARγ2 and HIF1-α within the Cxcl13 promoter. Taken together, these results indicate that CXCL13 is an adipocytokine that facilitates hypoxia-induced inflammation in adipocytes through FAK-mediated induction of PHLPP1 in autocrine and/or paracrine manner., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Ozone exposure causes irritation, airway hyperreactivity (AHR), inflammation of the airways, and destruction of alveoli (emphysema), the gas exchange area of the lung in human and mice. This review focuses on the acute disruption of the respiratory epithelial barrier in mice. A single high dose ozone exposure (1 ppm for 1 h) causes first a break of the bronchiolar epithelium within 2 h with leak of serum proteins in the broncho-alveolar space, disruption of epithelial tight junctions and cell death, which is followed at 6 h by ROS activation, AHR, myeloid cell recruitment, and remodeling. High ROS levels activate a novel PGAM5 phosphatase dependent cell-death pathway, called oxeiptosis. Bronchiolar cell wall damage and inflammation upon a single ozone exposure are reversible. However, chronic ozone exposure leads to progressive and irreversible loss of alveolar epithelial cells and alveoli with reduced gas exchange space known as emphysema. It is further associated with chronic inflammation and fibrosis of the lung, resembling other environmental pollutants and cigarette smoke in pathogenesis of asthma, and chronic obstructive pulmonary disease (COPD). Here, we review recent data on the mechanisms of ozone induced injury on the different cell types and pathways with a focus on the role of the IL-1 family cytokines and the related IL-33. The relation of chronic ozone exposure induced lung disease with asthma and COPD and the fact that ozone exacerbates asthma and COPD is emphasized., (Copyright © 2019 Sokolowska, Quesniaux, Akdis, Chung, Ryffel and Togbe.)

Neutrophils are known to adopt dynamic and distinct functional phenotypes involved in the modulation of inflammation and immune homeostasis. However, inter-cellular signaling mechanisms that govern neutrophil polarization dynamics are not well understood. Employing a novel model of PHLPP deficient mice, we examined how neutrophils deficient in PHLPP may uniquely modulate immune defense and the host response during acute colitis. We found that PHLPP -/- mice were protected from dextran sodium sulfate (DSS)-induced septic colitis characterized by minimal body weight-loss, alleviated colon tissue destruction and reduced clinical symptoms. PHLPP -/- neutrophils have enhanced immune homeostasis as compared to WT neutrophils, reflected in enhanced migratory capacity toward chemoattractants, and reduced expression of inflammatory mediators due to elevated phosphorylation of AKT, STAT1, and ERK. Further, adoptive transfer of PHLPP deficient neutrophils to WT mice is sufficient to potently alleviate the severity of DSS-induced colitis. Our data reveal that PHLPP deficient neutrophils can be uniquely reprogrammed to a state conducive to host inflammation resolution. As a consequence, PHLPP -/- neutrophils can effectively transfer immune homeostasis in mice subjected to acute colitis. Our findings hold significant and novel insights into the mechanisms by which neutrophils can be effectively reprogrammed into a homeostatic state conducive for treating acute injuries such as septic colitis.

The pathophysiological mechanisms of sepsis-induced cardiac dysfunction are largely unknown. The Toll-like receptor 4 (TLR4) is expressed in cardiac myocytes and is involved in bacterial endotoxin-mediated inflammatory disorders. TLR4 signaling leads to activation of the nuclear factor kappa B followed by increased expression of cytokines. Several protein phosphatases including PP2Cβ, PP2A or PP1 are known to act as regulators of this signaling pathway. Here, we examined the role of PP5 for the inflammatory response to the bacterial endotoxin lipopolysaccharide in the heart using a transgenic mouse model with cardiac myocyte directed overexpression of PP5. In these transgenic mice, basal cardiac contractility was reduced, in vivo as well as in vitro, but LPS-induced cardiac dysfunction was less pronounced compared to wild type mice. Quantitative RT-PCR suggested an attenuated NF-κB signaling in the heart and cardiac expression of heat shock protein 25 (HSP25) was increased in PP5 transgenic mice. From our data we assume that PP5 increases stress tolerance of cardiac myocytes by downregulation of NF-κB signaling and upregulation of HSP25 expression., Competing Interests: The authors have declared that no competing interests exist.

Th17 cells favor glycolytic metabolism, and pyruvate dehydrogenase (PDH) is the key bifurcation enzyme, which in its active dephosphorylated form advances the oxidative phosphorylation from glycolytic pathway. The transcriptional factor, inducible cAMP early repressor/cAMP response element modulator (ICER/CREM), has been shown to be induced in Th17 cells and to be overexpressed in CD4 + T cells from the patients with systemic lupus erythematosus (SLE). We found that glycolysis and lactate production in in vitro Th17-polarized T cells was reduced and that the expression of pyruvate dehydrogenase phosphatase catalytic subunit 2 (PDP2), an enzyme that converts the inactive PDH to its active form, and PDH enzyme activity were increased in Th17 cells from ICER/CREM-deficient animals. ICER was found to bind to the Pdp2 promoter and suppress its expression. Furthermore, forced expression of PDP2 in CD4 + cells reduced the in vitro Th17 differentiation, whereas shRNA-based suppression of PDP2 expression increased in vitro Th17 differentiation and augmented experimental autoimmune encephalomyelitis. At the translational level, PDP2 expression was decreased in memory Th17 cells from patients with SLE and forced expression of PDP2 in CD4 + T cells from lupus-prone MRL/ lpr mice and patients with SLE suppressed Th17 differentiation. These data demonstrate the direct control of energy production during Th17 differentiation in health and disease by the transcription factor ICER/CREM at the PDH metabolism bifurcation level., Competing Interests: The authors declare no conflict of interest.

Multiple sclerosis (MS) is a long-lasting autoimmune disease of the central nervous system. Currently, the etiology of MS is not known. Experimental autoimmune encephalomyelitis (EAE), has been recognized as the most widely used animal models to study the molecular mechanisms underlying MS and the efficacy of potential drugs for treatment of MS. In the present study, we found that Dl-3-n-butylphthalide (NBP), a neuroprotective drug in ischemic brain injury, prevented development of disease in experimental autoimmune encephalomyelitis (EAE) and significantly reduced inflammatory factors and necroptosis-associated genes, including PGAM5 in the spinal cord tissues. Similarly, silence of PGAM5 in spinal cord also ameliorated the disease severity in the mice with EAE. Moreover, re-expression of PGAM5 counteracted the protective effect of NBP on the pathogenesis of EAE. Importantly, we found that both NBP and silence of PGAM5 inhibited cellular necroptosis and inflammation in microglia induced by TNFα plus zVAD-fmk. Meanwhile, overexpression of PGAM5 reactivated cellular necroptosis and inflammation suppressed by NBP in vitro. Taken together, our findings provide evidence that NBP can attenuate the progression of EAE by suppressing PGAM5-induced necroptosis and inflammation in microglia and represents a new therapeutic strategy for treating autoimmune diseases., (Copyright © 2018. Published by Elsevier Inc.)

Salmonella enterica serovar Typhimurium exploits the host's type I interferon (IFN-I) response to induce receptor-interacting protein (RIP) kinase-mediated necroptosis in macrophages. However, the events that drive necroptosis execution downstream of IFN-I and RIP signaling remain elusive. In this study, we demonstrate that S Typhimurium infection causes IFN-I-mediated up-regulation of the mitochondrial phosphatase Pgam5 through RIP3. Pgam5 subsequently interacts with Nrf2, which sequesters Nrf2 in the cytosol, thereby repressing the transcription of Nrf2-dependent antioxidative genes. The impaired ability to respond to S Typhimurium-induced oxidative stress results in reactive oxygen species-mediated mitochondrial damage, energy depletion, transient induction of autophagy, and autophagic degradation of p62. Reduced p62 levels impair interaction of p62 with Keap1, which further decreases Nrf2 function and antioxidative responses to S Typhimurium infection, eventually leading to cell death. Collectively, we identify impaired Nrf2-dependent redox homeostasis as an important mechanism that promotes cell death downstream of IFN-I and RIP3 signaling in S Typhimurium-infected macrophages., (© 2017 Hos et al.)

Mitochondrial antiviral signaling platform protein (MAVS) acts as a central hub for RIG-I receptor proximal signal propagation. However, key components in the assembly of the MAVS mitochondrial platform that promote RIG-I mitochondrial localization and optimal activation are still largely undefined. Employing pooled RNAi and yeast two-hybrid screenings, we report that the mitochondrial adaptor protein tripartite motif (TRIM)14 provides a docking platform for the assembly of the mitochondrial signaling complex required for maximal activation of RIG-I-mediated signaling, consisting of WHIP and protein phosphatase PPP6C. Following viral infection, the ubiquitin-binding domain in WHIP bridges RIG-I with MAVS by binding to polyUb chains of RIG-I at lysine 164. The ATPase domain in WHIP contributes to stabilization of the RIG-I-dsRNA interaction. Moreover, phosphatase PPP6C is responsible for RIG-I dephosphorylation. Together, our findings define the WHIP-TRIM14-PPP6C mitochondrial signalosome required for RIG-I-mediated innate antiviral immunity., (Copyright © 2017 Elsevier Inc. All rights reserved.)

We have previously reported that bacterial endotoxin LPS attenuates expression of PHLPP, a ser/thr phosphatase, at both transcript and protein levels in different immune cells, however the underlying molecular mechanism is unknown and is of significant interest. Here, in line with the decreased transcript levels upon LPS treatment, we observed that LPS caused significant reduction in PHLPP promoter activity. We observed that SP1, a transcription factor frequently associated with inflammation, was recruited to the PHLPP promoter region. Ectopic expression of SP1 enhanced both transcript and protein levels of PHLPP while knockdown of SP1 or pharmacological inhibition of SP1 DNA binding by mithramycin reduced PHLPP expression. Moreover, over-expression of SP1 co-activators CBP/p300 augmented SP1 driven PHLPP promoter activity. Of note, LPS treatment depleted SP1 and CBP protein levels due to which recruitment of SP1 to PHLPP promoter was reduced. Further, we found that re-introduction of SP1 restored promoter activity and transcript levels of PHLPP in LPS stimulated cells. Collectively, our data revealed the molecular mechanism underlying the regulation of PHLPP expression during LPS induced macrophage inflammatory response., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Plants recognize pathogen-associated molecular patterns (PAMPs) via cell surface-localized pattern recognition receptors (PRRs), leading to PRR-triggered immunity (PTI). The Arabidopsis cytoplasmic kinase BIK1 is a downstream substrate of several PRR complexes. How plant PTI is negatively regulated is not fully understood. Here, we identify the protein phosphatase PP2C38 as a negative regulator of BIK1 activity and BIK1-mediated immunity. PP2C38 dynamically associates with BIK1, as well as with the PRRs FLS2 and EFR, but not with the co-receptor BAK1. PP2C38 regulates PAMP-induced BIK1 phosphorylation and impairs the phosphorylation of the NADPH oxidase RBOHD by BIK1, leading to reduced oxidative burst and stomatal immunity. Upon PAMP perception, PP2C38 is phosphorylated on serine 77 and dissociates from the FLS2/EFR-BIK1 complexes, enabling full BIK1 activation. Together with our recent work on the control of BIK1 turnover, this study reveals another important regulatory mechanism of this central immune component.

Leishmaniasis is an important parasitic disease found in the tropics and subtropics. Cutaneous and visceral leishmaniasis affect an estimated 1.5 million people worldwide. Despite its human health relevance, relatively little is known about the cell death pathways that control Leishmania replication in the host. Necroptosis is a recently identified form of cell death with potent antiviral effects. Receptor interacting protein kinase 1 (RIPK1) is a critical kinase that mediates necroptosis downstream of death receptors and TLRs. Heme, a product of hemoglobin catabolism during certain intracellular pathogen infections, is also a potent inducer of macrophage necroptosis. We found that human visceral leishmaniasis patients exhibit elevated serum levels of heme. Therefore, we examined the impact of heme and necroptosis on Leishmania replication. Indeed, heme potently inhibited Leishmania replication in bone marrow-derived macrophages. Moreover, we found that inhibition of RIPK1 kinase activity also enhanced parasite replication in the absence of heme. We further found that the mitochondrial phosphatase phosphoglycerate mutase family member 5 (PGAM5), a putative downstream effector of RIPK1, was also required for inhibition of Leishmania replication. In mouse infection, both PGAM5 and RIPK1 kinase activity are required for IL-1β expression in response to Leishmania However, PGAM5, but not RIPK1 kinase activity, was directly responsible for Leishmania-induced IL-1β secretion and NO production in bone marrow-derived macrophages. Collectively, these results revealed that RIPK1 and PGAM5 function independently to exert optimal control of Leishmania replication in the host., (Copyright © 2016 by The American Association of Immunologists, Inc.)

PH domain leucine-rich repeats protein phosphatase 1(PHLPP1) belongs to a novel family of Ser/Thr protein phosphatases: PHLPP serves as tumor suppressor in several cancers. However, little knowledge about the expression of PHLPP1 in human glioma tumor tissue and its role in inflammation response in glioma cells was known. Glioma samples were obtained from a total of 37 patients including 16 males and 21 females with surgical removal of the brain tumor. PHLPP1 protein and inflammatory cytokines were measured by Western blot analysis and immunohistochemistry while mRNA was determined by RT-PCR. The levels of inflammatory cytokines including TNF-α, IL-17, IL-1β in U251 glioma cells were evaluated by siRNA PHLPP1 and PHLPP1 addition. The loss of PHLPP1 expression occurs at high frequency in human gliomas. The highest mean values of PHLPP1 mRNA and protein were found in non-glioma brain tissues whereas the lowest mean values were found in those in glioblastoma with an increase of TNF-α, IL-17, IL-1β (p<0.05). PHLPP1 expression in human glioma was associated negatively with the severity of the tumor and inflammatory cytokines. siRNA PHLPP1 could increase the levels of inflammatory cytokines in U251 glioma cells while PHLPP1 addition could inhibit significantly inflammatory cytokines. We concluded that PHLPP1 played a suppression role in inflammatory response of glioma. The present study indicated that PHLPP1 could be used as a predictor for the prediction of the patients or as a therapeutic target for the treatment of human glioma., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Endotoxin tolerance protects the host by limiting excessive 'cytokine storm' during sepsis, but compromises the ability to counteract infections in septic shock survivors. It reprograms Toll-like receptor (TLR) 4 responses by attenuating the expression of proinflammatory cytokines without suppressing anti-inflammatory and antimicrobial mediators, but the mechanisms of reprogramming remain unclear. In this study, we demonstrate that the induction of endotoxin tolerance in human monocytes, THP-1 and MonoMac-6 cells inhibited lipopolysaccharide (LPS)-mediated phosphorylation of Lyn, c-Src and their recruitment to TLR4, but increased total protein phosphatase (PP) activity and the expression of protein tyrosine phosphatase (PTP) 1B, PP2A, PTP nonreceptor type (PTPN) 22 and mitogen-activated protein kinase phosphatase (MKP)-1. Chemical PP inhibitors, okadaic acid, dephostatin and cantharidic acid markedly decreased or completely abolished LPS tolerance, indicating the importance of phosphatases in endotoxin tolerization. Overexpression of PTPN22 decreased LPS-mediated nuclear factor (NF)-x03BA;B activation, p38 phosphorylation and CXCL8 gene expression, while PTPN22 ablation upregulated LPS-induced p65 NF-x03BA;B and p38 phosphorylation and the expression of TNF-α and pro-IL-1β mRNA, indicating PTPN22 as an inhibitor of TLR4 signaling. Thus, LPS tolerance interferes with TLR4 signaling by inhibiting Lyn and c-Src phosphorylation and their recruitment to TLR4, while increasing the phosphatase activity and expression of PP2A, PTPN22, PTP1B and MKP1., (© 2015 S. Karger AG, Basel.)

The effective recognition of viral infection and subsequent type I IFN production is essential for the host antiviral innate immune responses. The phosphorylation and activation of kinase TANK-binding kinase 1 (TBK1) plays crucial roles in the production of type I IFN mediated by TLR and retinoic acid-inducible gene I-like receptors. Type I IFN expression must be tightly regulated to prevent the development of immunopathological disorders. However, how the activated TBK1 is negatively regulated by phosphatases remains poorly understood. In this study, we identified a previously unknown role of protein phosphatase (PP)4 by acting as a TBK1 phosphatase. PP4 expression was upregulated in macrophages infected with RNA virus, vesicular stomatitis virus, and Sendai virus in vitro and in vivo. Knockdown of PP4C, the catalytic subunit of PP4, significantly increased type I IFN production in macrophages and dentritic cells triggered by TLR3/4 ligands, vesicular stomatitis virus, and Sendai virus, and thus inhibited virus replication. Similar results were also found in peritoneal macrophages with PP4C silencing in vivo and i.p. infection of RNA virus. Accordingly, ectopic expression of PP4C inhibited virus-induced type I IFN production and promoted virus replication. However, overexpression of a phosphatase-dead PP4C mutant abolished the inhibitory effects of wild-type PP4C on type I IFN production. Mechanistically, PP4 directly bound TBK1 upon virus infection, then dephosphorylated TBK1 at Ser(172) and inhibited TBK1 activation, and subsequently restrained IFN regulatory factor 3 activation, resulting in suppressed production of type I IFN and IFN-stimulated genes. Thus, serine/threonine phosphatase PP4 functions as a novel feedback negative regulator of RNA virus-triggered innate immunity., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Background: Noninvasive sputum sampling has enabled the identification of biomarkers in asthmatic patients. Studies of discrete cell populations in sputum can enhance measurements compared with whole sputum in which changes in rare cells and cell-cell interactions can be masked., Objective: We sought to enrich for sputum-derived human bronchial epithelial cells (sHBECs) and sputum-derived myeloid type 1 dendritic cells (sDCs) to describe transcriptional coexpression of targets associated with a type 2 immune response., Methods: A case-control study was conducted with patients with mild asthma (asthmatic cases) and healthy control subjects. Induced sputum was obtained for simultaneous enrichment of sHBECs and sDCs by using flow cytometry. Quantitative PCR was used to measure mRNA for sHBEC thymic stromal lymphopoietin (TSLP), IL33, POSTN, and IL25 and downstream targets in sDCs (OX40 ligand [OX40L], CCL17, PPP1R14A, CD1E, CD1b, CD80, and CD86)., Results: Final analyses for the study sample were based on 11 control subjects and 13 asthmatic cases. Expression of TSLP, IL33, and POSTN mRNA was increased in sHBECs in asthmatic cases (P = .001, P = .05, and P = .04, respectively). Expression of sDC OX40L and CCL17 mRNA was increased in asthmatic cases (P = .003 and P = .0001, respectively). sHBEC TSLP mRNA expression was strongly associated with sDC OX40L mRNA expression (R = 0.65, P = .001) and less strongly with sDC CCL17 mRNA expression. sHBEC IL33 mRNA expression was associated with sDC OX40L mRNA expression (R = 0.42, P = .04) but not sDC CCL17 mRNA expression., Conclusions: Noninvasive sampling and enrichment of select cell populations from sputum can further our understanding of cell-cell interactions in asthmatic patients with the potential to enhance endotyping of asthmatic patients., (Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Stimulator of interferon genes (STING, also known as MITA and ERIS) is critical in protecting the host against DNA pathogen invasion. However, the molecular mechanism underlying the regulation of STING remains unclear. Here, we show that PPM1A negatively regulates antiviral signaling by targeting STING in its phosphatase activity-dependent manner, and in a line with this, PPM1A catalytically dephosphorylates STING and TBK1 in vitro. Importantly, we provide evidence that whereas TBK1 promotes STING aggregation in a phosphorylation-dependent manner, PPM1A antagonizes STING aggregation by dephosphorylating both STING and TBK1, emphasizing that phosphorylation is crucial for the efficient activation of STING. Our findings demonstrate a novel regulatory circuit in which STING and TBK1 reciprocally regulate each other to enable efficient antiviral signaling activation, and PPM1A dephosphorylates STING and TBK1, thereby balancing this antiviral signal transduction.

T cell development and homeostasis are both regulated by TCR signals. Protein phosphorylation and dephosphorylation, which are catalyzed by protein kinases and phosphatases, respectively, serve as important switches controlling multiple downstream pathways triggered by TCR recognition of Ags. It has been well documented that protein tyrosine phosphatases are involved in negative regulation of proximal TCR signaling. However, how TCR signals are terminated or attenuated in the distal TCR signaling pathways is largely unknown. We investigated the function of Ser/Thr protein phosphatase (PP) 6 in TCR signaling. T cell lineage-specific ablation of PP6 in mice resulted in enhanced thymic positive and negative selection, and preferential expansion of fetal-derived, IL-17-producing Vγ6Vδ1(+) T cells. Both PP6-deficient peripheral CD4(+) helper and CD8(+) cytolytic cells could not maintain a naive state and became fast-proliferating and short-lived effector cells. PP6 deficiency led to profound hyperactivation of multiple distal TCR signaling molecules, including MAPKs, AKT, and NF-κB. Our studies demonstrate that PP6 acts as a critical negative regulator, not only controlling both αβ and γδ lineage development, but also maintaining naive T cell homeostasis by preventing their premature activation before Ag stimulation., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Interleukin-33 (IL-33) is a novel member of the IL-1 family of cytokines that plays diverse roles in the regulation of immune responses. IL-33 exerts its effects through a heterodimeric receptor complex resulting in the production and release of proinflammatory cytokines. A detailed understanding of the signaling pathways activated by IL-33 is still unclear. To gain insights into the IL-33-mediated signaling mechanisms, we carried out a SILAC-based global quantitative phosphoproteomic analysis that resulted in the identification of 7191 phosphorylation sites derived from 2746 proteins. We observed alterations in the level of phosphorylation in 1050 sites corresponding to 672 proteins upon IL-33 stimulation. We report, for the first time, phosphorylation of multiple protein kinases, including mitogen-activated protein kinase activated protein kinase 2 (Mapkapk2), receptor (TNFRSF) interacting serine-threonine kinase 1 (Ripk1), and NAD kinase (Nadk) that are induced by IL-33. In addition, we observed IL-33-induced phosphorylation of several protein phosphatases including protein tyrosine phosphatase, nonreceptor-type 12 (Ptpn12), and inositol polyphosphate-5-phosphatase D (Inpp5d), which have not been reported previously. Network analysis revealed an enrichment of actin binding and cytoskeleton reorganization that could be important in macrophage activation induced by IL-33. Our study is the first quantitative analysis of IL-33-regulated phosphoproteome. Our findings significantly expand the understanding of IL-33-mediated signaling events and have the potential to provide novel therapeutic targets pertaining to immune-related diseases such as asthma where dysregulation of IL-33 is observed. All MS data have been deposited in the ProteomeXchange with identifier PXD000984 (http://proteomecentral.proteomexchange.org/dataset/PXD000984)., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Intestinal ischemia/reperfusion (I/R) injury is a pathophysiology involving local tissue injury and organ dysfunction. Accumulating evidence has confirmed that the infiltration of neutrophils is of central importance in mediating intestinal I/R injury. On the other hand, adequate neutrophils in the intestine could also benefit the antibacterial translocation and tissue repair. Consequently, regulation of neutrophil immunity after intestinal I/R might be a promising therapy for controlling intestinal injury. Wip1 is a serine/threonine protein phosphatase that acts as the master regulator of tumorigenesis. However, emerging evidence highlights the importance of Wip1 in regulating neutrophil development, maturation, migration and neutrophil pro-inflammatory cytokine productions. Our recent studies showed that Wip1 negatively regulates neutrophil inflammatory responses and plays a protective role in intestinal I/R injury. In light of this discovery, we believe that Wip1 might be a new therapeutic target for treating intestinal I/R injury.

There have been many attempts for regeneration of peripheral nerve injury. In this study, we examined the in vivo effects of non-differentiated and neuronal differentiated adipose-derived stem cells (ADSCs) in inducing the neuronal regeneration in the Sprague-Dawley (SD) rats undergoing nerve defect bridged with the PCL nanotubes. Then, we performed immunohistochemical and histopathologic examinations, as well as the electromyography, in three groups: the control group (14 sciatic nerves transplanted with the PCL nanotube scaffold), the experimental group I (14 sciatic nerves with the non-differentiated ADSCs at a density of 7×10(5) cells/0.1 mL) and the experimental group II (14 sciatic nerves with the neuronal differentiated ADSCs at 7×10(5) cells/0.1 mL). Six weeks postoperatively, the degree of the neuronal induction and that of immunoreactivity to nestin, MAP-2 and GFAP was significantly higher in the experimental group I and II as compared with the control group. In addition, the nerve conduction velocity (NCV) was significantly higher in the experimental group I and II as compared with the control group (P=0.021 and P=0.020, respectively). On the other hand, there was no significant difference in the NCV between the two experimental groups (P>0.05). Thus, our results will contribute to treating patients with peripheral nerve defects using PCL nanotubes with ADSCs.

Objective: Although ankylosing spondylitis (AS) is driven by immune-mediated processes, little is known about the presence and role of autoantibodies in this disease. This study was undertaken to investigate whether autoantibodies occur in and are involved in AS., Methods: We performed human protein microarray analysis of sera derived from patients with AS or other autoimmune disorders to identify autoantibodies associated specifically with AS, and identified autoantibody targeting of protein phosphatase magnesium-dependent 1A (PPM1A) in AS. We performed enzyme-linked immunosorbent assay (ELISA) analysis of sera from 2 independent AS cohorts to confirm autoantibody targeting of PPM1A, and to assess associations between levels of anti-PPM1A antibodies and AS disease severity or response to anti-tumor necrosis factor (anti-TNF) therapy (as measured by Bath AS Disease Activity Index [BASDAI] score). Levels of anti-PPM1A antibodies were also evaluated in sera from rats transgenic for HLA-B27 and human β2 -microglobulin. The expression of PPM1A was assessed by immunohistochemistry in synovial tissue samples from patients with AS, rheumatoid arthritis, or osteoarthritis. The role of PPM1A in osteoblast differentiation was investigated by gene knockdown and overexpression., Results: AS was associated with autoantibody targeting of PPM1A, and levels of anti-PPM1A autoantibodies were significantly higher in patients with more advanced sacroiliitis and correlated positively with BASDAI score after treatment with anti-TNF agents. The levels of anti-PPM1A autoantibodies were also higher in the sera of transgenic rats that are prone to develop spondyloarthritis than in those that are not. PPM1A was expressed in AS synovial tissue, and PPM1A overexpression promoted osteoblast differentiation, whereas PPM1A knockdown suppressed it., Conclusion: Anti-PPM1A autoantibodies are present in AS, and our findings suggest that PPM1A may contribute to the pathogenic bone ankylosis characteristic of AS., (Copyright © 2014 by the American College of Rheumatology.)

PP4 is a serine/threonine phosphatase required for immunoglobulin (Ig) VDJ recombination and pro-B/pre-B cell development in mice. To elucidate the role of PP4 in mature B cells, we ablated the catalytic subunit of murine PP4 in vivo utilizing the CD23 promoter and cre-loxP recombination and generated CD23(cre)PP4(F/F) mice. The development of follicular and marginal zone B cells was unaffected in these mutants, but the proliferation of mature PP4-deficient B cells stimulated by in vitro treatment with either anti-IgM antibody (Ab) or LPS was partially impaired. Interestingly, the induction of CD80 and CD86 expression on these stimulated B cells was normal. Basal levels of serum Igs of all isotypes were strongly reduced in CD23(cre)PP4(F/F) mice, and their B cells showed a reduced efficiency of class switch recombination (CSR) in vitro upon stimulation by LPS or LPS plus IL-4. When CD23(cre)PP4(F/F) mice were challenged with either the T cell-dependent antigen TNP-KLH or the T cell-independent antigen TNP-Ficoll, or by H1N1 virus infection, the mutant animals failed to form germinal centers (GCs) in the spleen and the draining mediastinal lymph nodes, and did not efficiently mount antigen-specific humoral responses. In the resting state, PP4-deficient B cells exhibited pre-existing DNA fragmentation. Upon stimulation by DNA-damaging drug etoposide in vitro, mutant B cells showed increased cleavage of caspase 3. In addition, the mutant B cells displayed impaired CD40-mediated MAPK activation, abnormal IgM-mediated NF-κB activation, and reduced S phase entry upon IgM/CD40-stimulation. Taken together, our results establish a novel role for PP4 in CSR, and reveal crucial functions for PP4 in the maintenance of genomic stability, GC formation, and B cell-mediated immune responses.

Crosslinking BCR in the immature B cell line WEHI-231 causes apoptosis. We found that Bcl-xL was degraded by polyubiquitination upon BCR crosslinking and in this study explored the mechanism that controls the degradation of Bcl-xL. Ser(62) of Bcl-xL was phosphorylated by JNK to trigger polyubiquitination, and this was opposed by serine/threonine protein phosphatase 6 (PP6) that physically associated with Bcl-xL. We show BCR crosslinking decreased PP6 activity to allow Ser(62) phosphorylation of Bcl-xL. CD40 crosslinking rescues BCR-induced apoptosis, and we found PP6 associated with CD40 and PP6 activation in response to CD40. Our data suggest that PP6 activity is regulated to control apoptosis by modulating Ser(62) phosphorylation of Bcl-xL, which results in its polyubiquitination and degradation., (Copyright © 2014 by The American Association of Immunologists, Inc.)

PHLPP1 is a novel tumor suppressor, but its role in the regulation of innate immune responses, which are frequently dysregulated in cancer, is unexplored. Here, we report that LPS attenuated PHLPP1 expression at mRNA and protein levels in immune cells, suggesting its involvement in immune responses. To test this, we overexpressed PHLPP1 in RAW 264.7 macrophages and observed a dramatic reduction in LPS/IFN-γ-induced iNOS expression. Conversely, silencing of PHLPP1 by siRNA or by shRNA robustly augmented LPS/IFN-γ-induced iNOS expression. qPCR and iNOS promoter reporter experiments showed that PHLPP1 inhibited iNOS transcription. Mechanistic analysis revealed that PHLPP1 suppressed LPS/IFN-γ-induced phosphorylation of ser 727 STAT1; however, the underlying mechanisms differed. PHLPP1 reduced IFN-γ-stimulated but not LPS-induced ERK1/2 phosphorylation, and inhibition of ERK1/2 abolished IFN-γ-induced ser 727 STAT1 phosphorylation and iNOS expression. In contrast, PHLPP1 knockdown augmented LPS-induced but not IFN-γ-elicited p38 phosphorylation. Blockade of p38 abolished LPS-stimulated phosphorylation of ser 727 STAT1 and iNOS expression. Furthermore, PHLPP1 suppressed LPS-induced phosphorylation of tyr 701 STAT1 by dampening p38-dependent IFN-β feedback. Collectively, our data demonstrate for the first time that PHLPP1 plays a vital role in restricting innate immune responses of macrophages, and further studies may show it to be a potential therapeutic target within the context of dysregulated macrophage activity., (© 2014 Society for Leukocyte Biology.)

Thymic epithelial cells (TECs) are a key cell type in the thymic microenvironment essential for T cell development. However, intrinsic molecular mechanisms controlling TEC differentiation and activities are poorly defined. In this study, we found that deficiency of p53-induced phosphatase 1 (Wip1) in mice selectively caused severe medullary TEC (mTEC) maturation defects in an intrinsic manner. Wip1 knockout (KO) mice had decreased mature epithelial cell adhesion molecule⁺Ulex europaeus agglutinin-1 (UEA-1)⁺mTECs, including UEA-1⁺MHC class II(high), UEA-1⁺CD80⁺, UEA-1⁺CD40⁺, and UEA-1⁺Aire⁺ cells, but not decreased numbers of cortical epithelial cell adhesion molecule⁺BP-1⁺ TECs, in the postnatal stage but not in the fetal stage. Wip1-deficient mTECs express fewer tissue-restricted Ags and UEA-1⁺involucrin⁺ terminal-differentiated cells. Animal models, including grafting fetal Wip1-deficient thymic tissue into T cell-deficient nude mice and reconstitution of lethally irradiated Wip1KO mouse recipients with wild-type bone marrow cells, also showed the impaired mTEC components in Wip1KO thymi, indicating the intrinsic regulatory role of Wip1 in mTEC maturation. Furthermore, thymus regeneration was significantly less efficient in adult Wip1KO mice than in wild-type mice after cyclophosphamide treatment. Wip1 deficiency resulted in elevated p38 MAPK activity in mTECs. Activated p38 MAPK has the ability to suppress CD40 expression on mTECs. Wip1-deficient thymi displayed poor response to CD40L in the fetal thymus organ culture system. Thus, Wip1 positively controls mTEC maturation, homeostasis, and regeneration through limiting the p38 MAPK pathway.

Follicular helper T cells (T(FH) cells) provide critical help to B cells during humoral immune responses. Here we report that mice with T cell-specific deletion of the miR-17∼92 family of microRNAs (miRNAs) had substantially compromised T(FH) differentiation, germinal-center formation and antibody responses and failed to control chronic viral infection. Conversely, mice with T cell-specific expression of a transgene encoding miR-17∼92 spontaneously accumulated T(FH) cells and developed a fatal immunopathology. Mechanistically, the miR-17∼92 family controlled the migration of CD4(+) T cells into B cell follicles by regulating signaling intensity from the inducible costimulator ICOS and kinase PI(3)K by suppressing expression of the phosphatase PHLPP2. Our findings demonstrate an essential role for the miR-17∼92 family in T(FH) differentiation and establish PHLPP2 as an important mediator of their function in this process.

Protein phosphatase 2Cβ (PP2Cβ) was found to act as a negative regulator of NF-κB-mediated inflammatory signaling; however, its regulatory mechanism has not been examined. Here, we show that protein kinase A (PKA) phosphorylates the PP2Cβ, which was inhibited by PKA-specific inhibitor, H89. Mutation analysis of serine residues in PP2Cβ revealed that Ser-195 in PP2Cβ is phosphorylated by PKA. Importantly, PKA inhibition by H89 abrogated the Forskolin-induced destabilization of PP2Cβ against ubiquitin-dependent proteosomal degradation pathway. Furthermore, H89 treatment efficiently reversed the negative effect of Forskolin on the anti-inflammatory function of PP2Cβ. Collectively, these data suggest that PKA destabilizes PP2Cβ upon inflammatory stimuli via phosphorylation of Ser-195 in PP2Cβ., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Adoptive cell therapy with tumor-infiltrating lymphocytes (TILs) represents an effective treatment for patients with metastatic melanoma. However, most of the Ag targets recognized by effective melanoma-reactive TILs remain elusive. In this study, patient 2369 experienced a complete response, including regressions of bulky liver tumor masses, ongoing beyond 7 y following adoptive TIL transfer. The screening of a cDNA library generated from the autologous melanoma cell line resulted in the isolation of a mutated protein phosphatase 1, regulatory (inhibitor) subunit 3B (PPP1R3B) gene product. The mutated PPP1R3B peptide represents the immunodominant epitope recognized by tumor-reactive T cells in TIL 2369. Five years following adoptive transfer, peripheral blood T lymphocytes obtained from patient 2369 recognized the mutated PPP1R3B epitope. These results demonstrate that adoptive T cell therapy targeting a tumor-specific Ag can mediate long-term survival for a patient with metastatic melanoma. This study also provides an impetus to develop personalized immunotherapy targeting tumor-specific, mutated Ags.

Neutrophils are critically involved in host defense and tissue damage. Intrinsic molecular mechanisms controlling neutrophil differentiation and activities are poorly defined. Herein we found that p53-induced phosphatase 1(Wip1) is preferentially expressed in neutrophils among immune cells. The Wip1 expression is gradually up-regulated during the differentiation of myeloid precursors into mature neutrophils. Wip1-deficient mice and chimera mice with Wip1(-/-) hematopoietic cells had an expanded pool of neutrophils with hypermature phenotypes in the periphery. The in vivo and in vitro studies showed that Wip1 deficiency mainly impaired the developing process of myeloid progenitors to neutrophils in an intrinsic manner. Mechanism studies showed that the enhanced development and maturation of neutrophils caused by Wip1 deficiency were mediated by p38 MAPK-STAT1 but not p53-dependent pathways. Thus, our findings identify a previously unrecognized p53-independent function of Wip1 as a cell type-specific negative regulator of neutrophil generation and homeostasis through limiting the p38 MAPK-STAT1 pathway.

Mining of the literature and high-throughput mass spectrometry data from both healthy and diseased tissues and from body fluids reveals evidence that various extracellular proteins can exist in phosphorylated states. Extracellular kinases and phosphatases (ectokinases and ectophosphatases) are active in extracellular spaces during times of sufficiently high concentrations of adenosine triphosphate. There is evidence for a role of extracellular phosphorylation in various physiological functions, including blood coagulation, immune cell activation, and the formation of neuronal networks. Ectokinase activity is increased in some diseases, including cancer, Alzheimer's disease, and some microbial infections. We summarize the literature supporting the physiological and pathological roles of extracellularly localized protein kinases, protein phosphatases, and phosphorylated proteins and provide an analysis of the available mass spectrometry data to annotate potential extracellular phosphorylated proteins.

Signal transduction to nuclear factor-kappa B (NF-κB) involves multiple kinases and phosphorylated target proteins, but little is known about signal termination by dephosphorylation. By RNAi screening, we have identified protein phosphatase 4 regulatory subunit 1 (PP4R1) as a negative regulator of NF-κB activity in T lymphocytes. PP4R1 formed part of a distinct PP4 holoenzyme and bridged the inhibitor of NF-κB kinase (IKK) complex and the phosphatase PP4c, thereby directing PP4c activity to dephosphorylate and inactivate the IKK complex. PP4R1 expression was triggered upon activation and proliferation of primary human T lymphocytes and deficiency for PP4R1 caused sustained and increased IKK activity, T cell hyperactivation, and aberrant NF-κB signaling in NF-κB-addicted T cell lymphomas. Collectively, our results unravel PP4R1 as a previously unknown activation-associated negative regulator of IKK activity in lymphocytes whose downregulation promotes oncogenic NF-κB signaling in a subgroup of T cell lymphomas., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Many fungal and oomycete pathogens differentiate a feeding structure named the haustorium to extract nutrition from the plant epidermal cell. The atypical resistance (R) protein RPW8.2 activates salicylic acid (SA)-dependent, haustorium-targeted defenses against Golovinomyces spp., the causal agents of powdery mildew diseases on multiple plant species. How RPW8.2 activates defense remains uncharacterized. Here, we report that RPW8.2 interacts with the phytochrome-associated protein phosphatase type 2C (PAPP2C) in yeast and in planta as evidenced by co-immunoprecipitation and bimolecular fluorescence complementation assays. Down-regulation of PAPP2C by RNA interference (RNAi) in Col-0 plants lacking RPW8.2 leads to leaf spontaneous cell death and enhanced disease resistance to powdery mildew via the SA-dependent signaling pathway. Moreover, down-regulation of PAPP2C by RNAi in the RPW8.2 background results in strong HR-like cell death, which correlates with elevated RPW8.2 expression. We further demonstrate that hemagglutinin (HA)-tagged PAPP2C prepared from tobacco leaf cells transiently transformed with HA-PAPP2C possesses phosphatase activity. In addition, silencing a rice gene (Os04g0452000) homologous to PAPP2C also results in spontaneous cell death in rice. Combined, our results suggest that RPW8.2 is functionally connected with PAPP2C and that PAPP2C negatively regulates SA-dependent basal defense against powdery mildew in Arabidopsis.

Brucellosis is an important zoonotic disease of almost worldwide distribution. One significant immune phenomenon of this disease is the ability of the pathogen to hide and survive in the host, establishing long lasting chronic infections. Brucella was found to have the ability to actively modulate the host immune response in order to establish chronic infections, but the mechanism by which the pathogen achieves this remains largely unknown. In our screening for protective antigens of Brucella abortus, 3 proteins (BAB1&#95;0597, BAB1&#95;0917, and BAB2&#95;0431) were found to induce significantly higher levels of gamma interferon (IFNγ) in splenocytes of PBS immunized mice than those immunized with S19. This finding strongly implied that these three proteins inhibit the production of IFNγ. Previous studies have shown that LPS, PrpA, and Btp1/TcpB are three important immunomodulatory molecules with the capacity to interfere with host immune response. They have been shown to have the ability to inhibit the secretion of IFNγ, or to increase the production of IL-10. Due to the role of these proteins in virulence and immunomodulation, they likely offer significant potential as live, attenuated Brucella vaccine candidates. Understanding the mechanisms by which these proteins modulate the host immune responses will deepen our knowledge of Brucella virulence and provide important information on the development of new vaccines against Brucellosis.

Interleukin-1β (IL-1β) is a pro-inflammatory cytokine that regulates inflammatory responses to injury and infection. IL-1β secretion requires the protease caspase-1, which is activated following recruitment to inflammasomes. Endogenous danger-associated molecular patterns (DAMPs) released from necrotic cells activate caspase-1 through an NLRP3-inflammasome. Here, we show that the endogenous lipid metabolite sphingosine (Sph) acts as a DAMP by inducing the NLRP3-inflammasome-dependent secretion of IL-1β from macrophages. This process was dependent upon serine/threonine protein phosphatases since the PP1/PP2A inhibitors okadaic acid and calyculin A inhibited Sph-induced IL-1β release. IL-1β release induced by other well-characterized NLRP3-inflammasome activators, such as ATP and uric acid crystals, in addition to NLRC4 and AIM2 inflammasome activators was also blocked by these inhibitors. Thus, we propose Sph as a new DAMP, and that a serine/threonine phosphatase (PP1/PP2A)-dependent signal is central to the endogenous host mechanism through which diverse stimuli regulate inflammasome activation., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Protein phosphorylation plays central roles in a wide variety of signal transduction pathways and most phosphorylated proteins contain multi-phosphorylated sites. PPM1 type Ser/Thr protein phosphatase family is known to show rigid substrate specificity unlike other Ser/Thr phosphatase PPP family including PP1, PP2A and PP2B. PPM1 type phosphatases are reported to play important roles in growth regulation and in cellular stress signalling. In this study, we developed a phosphatase assay of PPM1D using phosphatase motif-specific antibody. PPM1D is a member of PPM1 type Ser/Thr phosphatase and known to dephosphorylate Ser(P)-Gln sequence. The gene amplification and overexpression of PPM1D were reported in many human cancers. We generated the monoclonal antibody specific for the Ser(P)-Gln sequence, named 3G9-H11. The specificity of this method using ELISA enables the convenient measurement of the dephosphorylation level of only PPM1D target residues of substrate peptides with multiple phosphorylated sites in the presence of multiple phosphatases. In addition, the antibody was applicable to immunoblotting assay for PPM1D function analysis. These results suggested that this method should be very useful for the PPM1D phosphatase assay, including high-throughput analysis and screening of specific inhibitors as anti-cancer drugs. The method using phosphatase motif-specific antibody can be applied to other PPM1 phosphatase family.

T cell activity is controlled in large part by the T cell receptor (TCR). The TCR detects the presence of foreign pathogens and activates the T cell-mediated immune reaction. Numerous intracellular signaling pathways downstream of the TCR are involved in the process of T cell activation. Negative regulation of these pathways helps prevent excessive and deleterious T cell responses. Two homologous proteins, Sts-1 and Sts-2, have been shown to function as critical negative regulators of TCR signaling. The phosphoglycerate mutase-like domain of Sts-1 (Sts-1(PGM)) has a potent phosphatase activity that contributes to the suppression of TCR signaling. The function of Sts-2(PGM) as a phosphatase has been less clear, principally because its intrinsic enzyme activity has been difficult to detect. Here, we demonstrate that Sts-2 regulates the level of tyrosine phosphorylation on targets within T cells, among them the critical T cell tyrosine kinase Zap-70. Utilizing new phosphorylated substrates, we demonstrate that Sts-2(PGM) has clear, albeit weak, phosphatase activity. We further pinpoint Sts-2 residues Glu-481, Ser-552, and Ser-582 as specificity determinants, in that an Sts-2(PGM) triple mutant in which these three amino acids are altered to their counterparts in Sts-1(PGM) has substantially increased activity. Our results suggest that the phosphatase activities of both suppressor of TCR signaling homologues cooperate in a similar but independent fashion to help set the threshold for TCR-induced T cell activation.

The immune system is vital for the immediate survival of multicellular organisms by protecting them from the damaging effects of bacterial infections, viruses, and toxic molecules. It has been hypothesized that the immune system plays a pivotal role in determining longevity. We investigated the efficiency of the innate immune system in Drosophila carrying the longevity extending mutations puc (JNK signaling pathway, stress response) and chico (insulin signaling pathway), as well as animals subjected to dietary restriction (DR), which also extends lifespan. We found that puc heterozygous animals, as well as chico homozygous and heterozygous flies, have enhanced pathogen resistance. Surprisingly, diet manipulation did not reproducibly alter pathogen resistance, despite its significant effect on the expression of many immunity-related genes. Considering that chronic or frequent activation of the immune system results in reduced longevity, we postulate that the longevity extending potential of the above mutations may be partially obscured by parallel activation of the immune system. Such upregulation is not observed during DR, suggesting the presence of a mechanism that suppresses immune activity in diet-restricted animals.

Our experiments investigated associations of specific isoforms of protein kinase C (PKC) with individual proteins in the cardiac troponin complex. Troponin I (cTnI) associated with PKCepsilon and zeta and troponin T (cTnT) associated with PKC alpha, delta, and epsilon. Based on its association with cTnI, we hypothesized that PKCzeta is a major regulator of myofilament protein phosphorylation. To test this, we infected adult cardiac myocytes with adenoviral constructs containing DsRed monomer-tagged wild type (WT) and the following constitutively active forms of PKCzeta: the pseudo-substrate region (A119E), 3'-phospho-inositide-dependent kinase-1 (T410E), and auto-phosphorylation (T560E). The A119E and T410E mutants displayed increased localization to the Z-discs compared with WT and T560E. Immunoprecipitations were performed in myocytes expressing PKCzeta using PKC phospho-motif antibodies to determine the phosphorylation of cTnI, cTnT, tropomyosin, myosin-binding protein C, and desmin. We did not find serine (Ser) phosphorylation of cTnI or cTnT. However, we observed a significant decrease in threonine (Thr) phosphorylation of cTnI and cTnT notably by PKCzeta T560E. Ser phosphorylation of tropomyosin was increased by all three active mutants of PKCzeta. Ser/Thr phosphorylation of myosin-binding protein C increased primarily by PKCzeta A119E. Both PKCzeta A119E and T410E mutants increased desmin Ser/Thr phosphorylation. To explain the apparent Thr dephosphorylation of cTnI and cTnT, we hypothesized that PKCzeta exists as a complex with p21-activated kinase-1 (Pak1) and protein phosphatase 2A (PP2A), and this was confirmed by immunoprecipitation Western blot. Our data demonstrate that PKCzeta is a novel regulator of myofilament protein phosphorylation.

Chronic inflammatory diseases often have residual CD8(+) T-cell infiltration despite treatment with systemic corticosteroids, which suggests divergent steroid responses between CD4(+) and CD8(+) cells. To examine steroid sensitivity, dexamethasone (DEX)-induced histone H4 lysine 5 (K5) acetylation and glucocorticoid receptor alpha (GCR alpha) translocation were evaluated. DEX treatment for 6 hours significantly induced histone H4 K5 acetylation in normal CD4(+) cells (P = .001) but not in CD8(+) cells. DEX responses were functionally impaired in CD8(+) compared with CD4(+) cells when using mitogen-activated protein kinase phosphatase (1 hour; P = .02) and interleukin 10 mRNA (24 hours; P = .004) induction as a readout of steroid-induced transactivation. Normal DEX-induced GCR alpha nuclear translocation and no significant difference in GCR alpha and GCR beta mRNA expression were observed in both T-cell types. In addition, no significant difference in SRC-1, p300, or TIP60 expression was found. However, activating transcription factor-2 (ATF2) expression was significantly lower in CD8(+) compared with CD4(+) cells (P = .009). Importantly, inhibition of ATF2 expression by small interfering RNA in CD4(+) cells resulted in inhibition of DEX-induced transactivation in CD4(+) cells. The data indicate refractory steroid-induced transactivation but similar steroid-induced transrepression of CD8(+) cells compared with CD4(+) cells caused by decreased levels of the histone acetyltransferase ATF2.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) belongs to the TNF superfamily of proteins. It is highly expressed on natural killer cells, cytotoxic T lymphocytes, and monocytes after stimulation, and plays a critical role in immune surveillance. Two splice variants of TRAIL were identified recently that show no proapoptotic activity. Phosphorylation level in splicing factors, serine-arginine-rich (SR) and heterogeneous ribonucleoproteins (hnRNPs) govern the mRNA splicing of several apoptosis-related genes. We characterized the apoptotic stimuli-mediated alternative splicing pattern of TRAIL and investigated the possible underlying mechanism of alternative splicing. Etoposide and cycloheximide induced alternative splicing, whereas staurosporine (a broad kinase inhibitor) blocked both constitutive and alternative splicing. De novo ceramide synthesis and subsequent protein phosphatase-1 (PP-1) activation enhanced the alternative splicing, as did TNF-alpha but not interferon alpha (IFN-alpha) stimulation. We demonstrated that TRAIL alters gene expression through mRNA splicing and may change proapoptotic potential in response to cytokine stimulation.

Background: The release of adenosine triphosphate (ATP) from the airway epithelial cells during the inflammatory process is considered to play an important role in the pathophysiology of asthma and chronic obstructive pulmonary disease., Objective: This study was designed to determine whether extracellular ATP is involved in the bronchial hyperresponsiveness as an interaction between epithelium and smooth muscle in the airways., Methods: We examined the contractile response to methacholine (MCh) before and after exposure to low concentrations (< or = 10 microm) of ATP in isolated, epithelium-denuded guinea-pig tracheal smooth muscle by measuring isometric tension. Intracellular Ca2+ concentrations ([Ca2+]i) were assessed by fluorescent intensities of fura-2., Results: MCh-induced contractile force was increased with no change in [Ca2+]i after exposure to 10 microm ATP for 15 min. The ability of ATP to enhance the MCh-induced contraction was markedly attenuated by suramin, a non-selective P2 receptor inhibitor. Pre-incubation with ATPgammaS, a non-hydrolysable analogue of ATP and alpha,beta-meATP, a P2X agonist, also enhanced the MCh-induced contraction. In contrast, uracil triphosphate, a P2Y agonist, did not affect the MCh-induced contraction. Y-27632, a Rho-kinase inhibitor, suppressed the ability of ATP to enhance the MCh-induced contraction. Moreover, PP1 and PP2, Src tyrosin kinase inhibitors, suppressed the enhancement of MCh-induced contraction by ATP., Conclusion: Pre-treatment with ATP induces hyperresponsiveness to MCh mediated by Ca2+ sensitization via the P2X receptor in airway smooth muscle. The present findings suggest the possible involvement of both the Rho-kinase and Src pathways in the intracellular mechanism of this phenomenon.

The mechanism underlying targeting of the nuclear membrane to chromatin at the end of mitosis was studied using an in vitro cell-free system comprising Xenopus egg membrane and cytosol fractions, and sperm chromatin. The mitotic phase membrane, which was separated from a mitotic phase extract of Xenopus eggs and could not bind to chromatin, became able to bind to chromatin on pretreatment with a synthetic phase cytosol fraction of Xenopus eggs. When the cytosol fraction was depleted of protein phosphatase 1 (PP1) with anti-Xenopus PP1gamma1 antibodies, this ability was lost. The addition of recombinant xPP1gamma1 to the PP1-depleted cytosol fraction restored the ability. These and other results suggested that dephosphorylation of mitotic phosphorylation sites on membranes by PP1 in the synthetic phase cytosol fraction promoted targeting of the membranes to chromatin. On the other hand, a fragment containing the chromatin-binding domain of lamin B receptor (LBR) but not emerin inhibited targeting of membrane vesicles. It was also shown that PP1 dephosphorylates a phosphate group(s) responsible for regulation of the binding of LBR to chromatin. A possible mechanism involving PP1 and LBR for the regulation of nuclear membrane targeting to chromatin was discussed.

Throughout evolution, mammals have developed an elaborate network of positive and negative regulatory mechanisms, which provide balance between defensive measures against bacterial and viral pathogens and protective measures against unwarranted destruction of the host by the activated immune system. Kinases and phosphatases encompassing the MAPK pathway are key players in the orderly action of pro- and anti-inflammatory processes, forming numerous promiscuous interactions. Several lines of evidence demonstrate that the phosphorylation and activation status of kinases in the MAPK system has crucial impact on the outcome of downstream events that regulate cytokine production. At least 13 members of the family of dual-specificity phosphatases (DUSP) display unique substrate specificities for MAPKs. Despite the considerable amount of information obtained about the contribution of the different DUSP to MAPK-mediated signaling and innate immunity, the interpretation of available data remains problematic. The in vitro and ex vivo findings are often complicated by functional redundancy of signaling molecules and do not always accurately predict the situation in vivo. Until recently, DUSP research has been hampered by the lack of relevant mammalian knockout (KO) models, which is a powerful tool for delineating in vivo function and redundancy in gene families. This situation changed dramatically over the last year, and this review integrates recent insights into the precise biological role of the DUSP family in innate immunity gained from a comprehensive analysis of mammalian KO models.