Protein phosphatase 2A inactivation induces microsatellite instability, neoantigen production and immune response.

Microsatellite-instable (MSI), a predictive biomarker for immune checkpoint blockade (ICB) response, is caused by mismatch repair deficiency (MMRd) that occurs through genetic or epigenetic silencing of MMR genes. Here, we report a mechanism of MMRd and demonstrate that protein phosphatase 2A (PP2A) deletion or inactivation converts cold microsatellite-stable (MSS) into MSI tumours through two orthogonal pathways: (i) by increasing retinoblastoma protein phosphorylation that leads to E2F and DNMT3A/3B expression with subsequent DNA methylation, and (ii) by increasing histone deacetylase (HDAC)2 phosphorylation that subsequently decreases H3K9ac levels and histone acetylation, which induces epigenetic silencing of MLH1. In mouse models of MSS and MSI colorectal cancers, triple-negative breast cancer and pancreatic cancer, PP2A inhibition triggers neoantigen production, cytotoxic T cell infiltration and ICB sensitization. Human cancer cell lines and tissue array effectively confirm these signaling pathways. These data indicate the dual involvement of PP2A inactivation in silencing MLH1 and inducing MSI.

Microcystin-Induced Immunotoxicity in Fishes: A Scoping Review.

Cyanobacteria (blue-green algae) have been present on Earth for over 2 billion years, and can produce a variety of bioactive molecules, such as cyanotoxins. Microcystins (MCs), the most frequently detected cyanotoxins, pose a threat to the aquatic environment and to human health. The classic toxic mechanism of MCs is the inhibition of the protein phosphatases 1 and 2A (PP1 and PP2A). Immunity is known as one of the most important physiological functions in the neuroendocrine-immune network to prevent infections and maintain internal homoeostasis in fish. The present review aimed to summarize existing papers, elaborate on the MC-induced immunotoxicity in fish, and put forward some suggestions for future research. The immunomodulatory effects of MCs in fish depend on the exposure concentrations, doses, time, and routes of exposure. Previous field and laboratory studies provided strong evidence of the associations between MC-induced immunotoxicity and fish death. In our review, we summarized that the immunotoxicity of MCs is primarily characterized by the inhibition of PP1 and PP2A, oxidative stress, immune cell damage, and inflammation, as well as apoptosis. The advances in fish immunoreaction upon encountering MCs will benefit the monitoring and prediction of fish health, helping to achieve an ecotoxicological goal and to ensure the sustainability of species. Future studies concerning MC-induced immunotoxicity should focus on adaptive immunity, the hormesis phenomenon and the synergistic effects of aquatic microbial pathogens.

PP2A and Its Inhibitors in Helper T-Cell Differentiation and Autoimmunity.

Protein phosphatase 2A (PP2A) is a highly complex heterotrimeric Ser/Thr phosphatase that regulates many cellular processes. The role of PP2A as a tumor suppressor has been extensively studied and reviewed. However, emerging evidence suggests PP2A constrains inflammatory responses and is important in autoimmune and neuroinflammatory diseases. Here, we reviewed the existing literature on the role of PP2A in T-cell differentiation and autoimmunity. We have also discussed the modulation of PP2A activity by endogenous inhibitors and its small-molecule activators as potential therapeutic approaches against autoimmunity.

Recombinant Human Proteoglycan 4 Regulates Phagocytic Activation of Monocytes and Reduces IL-1ß Secretion by Urate Crystal Stimulated Gout PBMCs.

Objectives: To compare phagocytic activities of monocytes in peripheral blood mononuclear cells (PBMCs) from acute gout patients and normal subjects, examine monosodium urate monohydrate (MSU) crystal-induced IL-1ß secretion ± recombinant human proteoglycan 4 (rhPRG4) or interleukin-1 receptor antagonist (IL-1RA), and study the anti-inflammatory mechanism of rhPRG4 in MSU stimulated monocytes. Methods: Acute gout PBMCs were collected from patients in the Emergency Department and normal PBMCs were obtained from a commercial source. Monocytes in PBMCs were identified by flow cytometry. PBMCs were primed with Pam3CSK4 (1µg/mL) for 24h and phagocytic activation of monocytes was determined using fluorescently labeled latex beads. MSU (200µg/mL) stimulated IL-1ß secretion was determined by ELISA. Reactive oxygen species (ROS) generation in monocytes was determined fluorometrically. PBMCs were incubated with IL-1RA (250ng/mL) or rhPRG4 (200µg/mL) and bead phagocytosis by monocytes was determined. THP-1 monocytes were treated with MSU crystals ± rhPRG4 and cellular levels of NLRP3 protein, pro-IL-1ß, secreted IL-1ß, and activities of caspase-1 and protein phosphatase-2A (PP2A) were quantified. The peritoneal influx of inflammatory and anti-inflammatory monocytes and neutrophils in Prg4 deficient mice was studied and the impact of rhPRG4 on immune cell trafficking was assessed. Results: Enhanced phagocytic activation of gout monocytes under basal conditions (p<0.001) was associated with ROS generation and MSU stimulated IL-1ß secretion (p<0.05). rhPRG4 reduced bead phagocytosis by normal and gout monocytes compared to IL-1RA and both treatments were efficacious in reducing IL-1ß secretion (p<0.05). rhPRG4 reduced pro-IL-1ß content, caspase-1 activity, conversion of pro-IL-1ß to mature IL-1ß and restored PP2A activity in monocytes (p<0.05). PP2A inhibition reversed rhPRG4's effects on pro-IL-1ß and mature IL-1ß in MSU stimulated monocytes. Neutrophils accumulated in peritoneal cavities of Prg4 deficient mice (p<0.01) and rhPRG4 treatment reduced neutrophil accumulation and enhanced anti-inflammatory monocyte influx (p<0.05). Conclusions: MSU phagocytosis was higher in gout monocytes resulting in higher ROS and IL-1ß secretion. rhPRG4 reduced monocyte phagocytic activation to a greater extent than IL-1RA and reduced IL-1ß secretion. The anti-inflammatory activity of rhPRG4 in monocytes is partially mediated by PP2A, and in vivo, PRG4 plays a role in regulating the trafficking of immune cells into the site of a gout flare.

CqPP2A inhibits white spot syndrome virus infection by up-regulating antimicrobial substances expression in red claw crayfish Cherax quadricarinatus.

Protein phosphatase 2A (PP2A) is an important serine/threonine phosphatase, a highly conserved enzyme widely expressed in eukaryotic cells, which accounts for a majority of the serine/threonine phosphatase activity in cells implicated in regulation of immune signaling pathways and antiviral response. However, most of studies about PP2A have been conducted in mammals but few in crustaceans. In this study, two subunits of PP2A (named as CqPP2Ab and CqPP2Ac) were characterized to be involved in white spot syndrome virus (WSSV) infection in the haematopoietic tissue (Hpt) cells from red claw crayfish Cherax quadricarinatus. The open reading frame (ORF) of CqPP2Ab was 1341 bp encoding 446 amino acids with seven WD40 domains, and the ORF of CqPP2Ac was 930 bp encoding 309 amino acids with a PP2Ac domain. Tissue distribution analysis showed that the mRNA transcript of CqPP2Ab and CqPP2Ac were both widely expressed in all the tested tissues with the highest expression in hemocyte, followed by high expression in Hpt. The gene expressions of CqPP2Ab and CqPP2Ac were both significantly down-regulated at 6 h post WSSV infection (6 hpi) in Hpt cells. Importantly, the expression of viral immediate early gene IE1 and late viral gene envelope protein VP28 were both significantly increased post WSSV infection after gene silencing of CqPP2Ab or CqPP2Ac in Hpt cells, suggesting that CqPP2Ab and CqPP2Ac could inhibit WSSV infection in Hpt cells, probably by increasing the antimicrobial substances expression in consideration to the significantly reduced expression of anti-lipopolysaccharide factor, crustin, and lysozyme after gene silencing of CqPP2Ab or CqPP2Ac, respectively. These findings provide a new light on the mechanism of WSSV infection and the antiviral response in crustaceans.

Protein phosphatase 2A B55ß limits CD8+ T cell lifespan following cytokine withdrawal.

How T cells integrate environmental cues into signals that limit the magnitude and length of immune responses is poorly understood. Here, we provide data that demonstrate that B55ß, a regulatory subunit of protein phosphatase 2A, represents a molecular link between cytokine concentration and apoptosis in activated CD8+ T cells. Through the modulation of AKT, B55ß induced the expression of the proapoptotic molecule Hrk in response to cytokine withdrawal. Accordingly, B55ß and Hrk were both required for in vivo and in vitro contraction of activated CD8+ lymphocytes. We show that this process plays a role during clonal contraction, establishment of immune memory, and preservation of peripheral tolerance. This regulatory pathway may represent an unexplored opportunity to end unwanted immune responses or to promote immune memory.

Inhibition of protein phosphatase-2A with LB-100 enhances antitumor immunity against glioblastoma.

PURPOSE: Glioblastoma (GBM) carries a dismal prognosis despite standard multimodal treatment with surgery, chemotherapy and radiation. Immune checkpoint inhibitors, such as PD1 blockade, for treatment of GBM failed to show clinical benefit. Rational combination strategies to overcome resistance of GBM to checkpoint monotherapy are needed to extend the promise of immunotherapy to GBM management. Emerging evidence suggests that protein phosphatase 2A (PP2A) plays a critical role in the signal transduction pathways of both adaptive and innate immune cells and that inhibition of PP2A could enhance cancer immunity. We investigated the use of a PP2A inhibitor, LB-100, to enhance antitumor efficacy of PD1 blockade in a syngeneic glioma model. METHODS: C57BL/6 mice were implanted with murine glioma cell line GL261-luc or GL261-WT and randomized into 4 treatment arms: (i) control, (ii) LB-100, (iii) PD1 blockade and (iv) combination. Survival was assessed and detailed profiling of tumor infiltrating leukocytes was performed. RESULTS: Dual PP2A and PD1 blockade significantly improved survival compared with monotherapy alone. Combination therapy resulted in complete regression of tumors in about 25% of mice. This effect was dependent on CD4 and CD8 T cells and cured mice established antigen-specific secondary protective immunity. Analysis of tumor lymphocytes demonstrated enhanced CD8 infiltration and effector function. CONCLUSION: This is the first preclinical investigation of the effect of combining PP2A inhibition with PD1 blockade for GBM. This novel combination provided effective tumor immunotherapy and long-term survival in our animal GBM model.

PP2Ac Modulates AMPK-Mediated Induction of Autophagy in Mycobacterium bovis-Infected Macrophages.

Mycobacterium bovis (M. bovis) is the causative agent of bovine tuberculosis in cattle population across the world. Human beings are at equal risk of developing tuberculosis beside a wide range of M. bovis infections in animal species. Autophagic sequestration and degradation of intracellular pathogens is a major innate immune defense mechanism adopted by host cells for the control of intracellular infections. It has been reported previously that the catalytic subunit of protein phosphatase 2A (PP2Ac) is crucial for regulating AMP-activated protein kinase (AMPK)-mediated autophagic signaling pathways, yet its role in tuberculosis is still unclear. Here, we demonstrated that M. bovis infection increased PP2Ac expression in murine macrophages, while nilotinib a tyrosine kinase inhibitor (TKI) significantly suppressed PP2Ac expression. In addition, we observed that TKI-induced AMPK activation was dependent on PP2Ac regulation, indicating the contributory role of PP2Ac towards autophagy induction. Furthermore, we found that the activation of AMPK signaling is vital for the regulating autophagy during M. bovis infection. Finally, the transient inhibition of PP2Ac expression enhanced the inhibitory effect of TKI-nilotinib on intracellular survival and multiplication of M. bovis in macrophages by regulating the host's immune responses. Based on these observations, we suggest that PP2Ac should be exploited as a promising molecular target to intervene in host-pathogen interactions for the development of new therapeutic strategies towards the control of M. bovis infections in humans and animals.

IL-17A-Mediated Excessive Autophagy Aggravated Neuronal Ischemic Injuries via Src-PP2B-mTOR Pathway.

We previously reported that astrocyte-derived proinflammatory cytokine interleukin (IL)-17A could aggravate neuronal ischemic injuries and strength autophagy both in oxygen-glucose deprivation (OGD)/reoxygenation (R)-treated neurons and peri-infarct region of mice with middle cerebral artery occlusion (MCAO)/reperfusion (R)-simulated ischemic stroke. In this study, the role and molecular mechanism of IL-17A in autophagy were further explored under ischemic condition. We found that exogenous addition of rmIL-17A remarkably (P < 0.001) decreased cell viability, which companying with the increases of LC3 II accumulation (P < 0.05 or 0.01) and Beclin 1 levels (P < 0.05 or 0.001), and reduction of p62 levels (P < 0.01 or 0.001) in OGD/R-treated cortical neurons (n = 6). The levels of P-mTOR (Ser 2448) (P < 0.001) and P-S6 (Ser 240/244) (P < 0.01) significantly decreased without the involvement of Akt, ERK1/2 and AMPK in cortical neurons under rmIL-17A and OGD/R treatments (n = 6). Interestingly, the co-IP analysis exhibited that PP2B and mTOR could be reciprocally immunoprecipitated; and the addition of rmIL-17A increased their interactions, PP2B activities (P < 0.001), P-Src (P < 0.001), and P-PLCγ1 (P < 0.01) levels in OGD/R-treated neurons (n = 6 or 5). The PP2B inhibitor Cyclosporin A blocked the induction of excessive autophagy (P < 0.05 or <0.001) and increased cell viability (P < 0.001) after OGD/R and rmIL-17A treatments (n = 6). In addition, the ICV injection of IL-17A neutralizing mAb could attenuate autophagy levels (P < 0.01 or 0.001, n = 6) and improve neurological functions (P < 0.01 or 0.001, n = 10) of mice after 1 h MCAO/R 24 h or 7 d. These results suggested that IL-17A-mediated excessive autophagy aggravates neuronal ischemic injuries via Src-PP2B-mTOR pathway, and IL-17A neutralization may provide a potential therapeutic effect for ischemic stroke.

Phosphatase PP2A is essential for TH17 differentiation.

Phosphatase PP2A expression levels are positively correlated to the clinical severity of systemic lupus erythematosus (SLE) and IL17A cytokine overproduction, indicating a potential role of PP2A in controlling TH17 differentiation and inflammation. By generating a mouse strain with ablation of the catalytic subunit α of PP2A in peripheral mature T cells (PP2A cKO), we demonstrate that the PP2A complex is essential for TH17 differentiation. These PP2A cKO mice had reduced TH17 cell numbers and less severe disease in an experimental autoimmune encephalomyelitis (EAE) model. PP2A deficiency also ablated C-terminal phosphorylation of SMAD2 but increased C-terminal phosphorylation of SMAD3. By regulating the activity of RORγt via binding, the changes in the phosphorylation status of these R-SMADs reduced Il17a gene transcription. Finally, PP2A inhibitors showed similar effects on TH17 cells as were observed in PP2A cKO mice, i.e., decreased TH17 differentiation and relative protection of mice from EAE. Taken together, these data demonstrate that phosphatase PP2A is essential for TH17 differentiation and that inhibition of PP2A could be a possible therapeutic approach to controlling TH17-driven autoimmune diseases.

The serine/threonine protein phosphatase 2A controls autoimmunity.

Protein phosphatase 2A (PP2A) is the first serine/threonine phosphatase recognized to contribute to human and murine lupus immunopathology. PP2A expression in SLE is controlled both epigenetically and genetically, and it is increased in patients with SLE, which contributes to decreased IL-2 production, decreased CD3ζ and increased FcRγ expression on the surface of T cells, increased CREMα expression, hypomethylation of genes associated with SLE pathogenesis, and increased IL-17 production. ß regulatory subunit of PP2A regulates IL-2 deprivation-induced T cell death and is decreased in SLE patients. A mouse overexpressing PP2Ac in T cells displays peripheral granulocytosis, elevated IL-17 production, and develops glomerulonephritis when challenged. A mouse which lacks PP2Ac only in regulatory T cells develops severe autoimmunity and multiorgan inflammation because of loss of restraint on mTORC1 and inability of Foxp3+ cells to regulate conventional T cells. Targeting PP2A in T cell subsets may be therapeutic for SLE and other autoimmune diseases.

Green Tea Polyphenol EGCG Upregulates Tollip Expression by Suppressing Elf-1 Expression.

TLR signaling is critical to innate immune system regulation; however, aberrant TLR signaling is involved in several diseases, including insulin resistance, Alzheimer's disease, and tumor metastasis. Moreover, a recent study found that TLR-4 signaling pathway inhibition might be a target for the suppression of chronic inflammatory disorders. In this article, we show that the green tea polyphenol epigallocatechin-3-O-gallate (EGCG) increases the expression of Toll interacting protein, a strong inhibitor of TLR4 signaling, by suppressing the expression of E74-like ETS transcription factor 1 (Elf-1). A mechanistic study revealed that EGCG suppressed Elf-1 expression via protein phosphatase 2A/cyclic GMP (cGMP)-dependent mechanisms. We also confirmed that orally administered EGCG and a cGMP inducer upregulated Toll interacting protein expression, increased intracellular levels of cGMP in macrophages, and suppressed Elf-1 expression. These data support EGCG and a cGMP inducer as potential candidate suppressors of TLR4 signaling.

Self-adjuvanting C18 lipid vinil sulfone-PP2A vaccine: study of the induced immunomodulation against Trichuris muris infection.

Despite the importance of the adjuvant in the immunization process, very few adjuvants merge with the antigens in vaccines. A synthetic self-adjuvant oleic-vinyl sulfone (OVS) linked to the catalytic region of recombinant serine/threonine phosphatase 2A from the nematode Angiostrongylus costaricensis (rPP2A) was used for intranasal immunization in mice previously infected with Trichuris muris The animal intranasal immunization with rPP2A-OVS showed a reduction of 99.01% in the number of the nematode eggs and 97.90% in adult. The immunohistochemical analysis of the intestinal sections showed that in immunized animals with lipopeptide the mucus was significantly higher than in the other experimental groups. Also, these animals presented significantly different chemokine, CCL20 and CCL11, levels. However, although the number and size of Tuft cells did not vary between groups, the intensity of fluorescence per cell was significant in the group immunized with the rPP2A-OVS. The results of the present study suggest that mice immunized with the lipopeptide are capable of activating a combined Th17/Th9 response. This strategy of immunization may be of great applicability not only in immunotherapy and immunoprophylaxis to control diseases caused by nematodes but also in pathologies necessitating action at the level of the Th9 response in the intestinal mucosa.

A Novel Function of F-Box Protein FBXO17 in Negative Regulation of Type I IFN Signaling by Recruiting PP2A for IFN Regulatory Factor 3 Deactivation.

The F-box proteins were originally identified as the key component of SKP1-Cullin1-F-box E3 ligase complexes that control the stability of their specific downstream substrates essential for cell growth and survival. However, the involvement of these proteins in type I IFN (IFN-I) signaling during innate immunity has not been investigated. In this study we report that the F-box protein FBXO17 negatively regulates IFN-I signaling triggered by double-strand DNA, RNA, or viral infection. We found that FBXO17 specifically interacts with IFN regulatory factor 3 (IRF3) and decreases its dimerization and nuclear translocation. The decrease of IRF3 dimerization and nuclear translocation is due to the recruitment of protein phosphatase 2 (PP2A) mediated by FBXO17, resulting in IRF3 dephosphorylation. Interestingly, PP2A recruitment does not require the F-box domain but instead the F-box associated region of the protein; thus, the recruitment is independent of the canonical function of the SKP1-Cullin1-F-box family of E3 ligase. Together, our studies identify a previously unreported role of FBXO17 in regulating IFN-I signaling and further demonstrate a novel mechanism for IRF3 deactivation by F-box protein-mediated recruitment of PP2A.

Peripheral Lipopolysaccharide Challenge Induces Long-Term Changes in Tyrosine Hydroxylase Regulation in the Adrenal Medulla.

Immune activation can alter the activity of adrenal chromaffin cells. The effect of immune activation by lipopolysaccharide (LPS) on the regulation of tyrosine hydroxylase (TH) in the adrenal medulla in vivo was determined between 1 day and 6 months after LPS injection. The plasma levels of eleven cytokines were reduced 1 day after LPS injection, whereas the level for interleukin-10 was increased. The levels of all cytokines remained at control levels until 6 months when the levels of interleukin-6 and -4 were increased. One day after LPS injection, there was a decrease in TH-specific activity that may be due to decreased phosphorylation of serine 31 and 40. This decreased phosphorylation of serine 31 and 40 may be due to an increased activation of the protein phosphatase PP2A. One week after LPS injection, there was increased TH protein and increased phosphorylation of serine 40 that this was not accompanied by an increase in TH-specific activity. All TH parameters measured returned to basal levels between 1 month and 3 months. Six months after injection there was an increase in TH protein. This was associated with increased levels of the extracellular regulated kinase isoforms 1 and 2. This work shows that a single inflammatory event has the capacity to generate both short-term and long-term changes in TH regulation in the adrenal medulla of the adult animal. J. Cell. Biochem. 118: 2096-2107, 2017. © 2016 Wiley Periodicals, Inc.

[Interleukin-2 signaling pathway regulating molecules in systemic lupus erythematosus].

Systemic lupus erythematosus (SLE) is a prototypic systemic autoimmune disease, which characterized by complex immunological abnormalities and multiple tissue and organ damages. The etiology and pathogenesis of SLE have not been entirely recognized. Genetic, environmental and viral infections and other factors might be related to the pathogenetic mechanisms of SLE. Interleukin-2 (IL-2) is a critical cytokine produced by T cells upon activation and is important for the generation of T regulatory cells and activation-induced cell death. In SLE patients, T cells display decreased capacity to produce IL-2. Impaired IL-2 expression resulted in decreased generation of regulatory T lymphocytes, and defect of activation-induced cell death. Former researches indicated that IL-2 deficiency in SLE is important for the pathogenesis and treatment of SLE. Several regulating molecules can affect the transcription of IL-2 gene and had an important role in the pathogenesis of SLE. These molecules include cyclic AMP-responsive element modulator (CREM), protein phosphatase 2A (PP2A), E-74 like factor 1 (Elf-1), B lymphocyte induced maturation protein-1 (Blimp-1) and interferon regulator factor 5 (IRF-5). CREM is a transcriptional inhibitor that can repress the transcription of the IL-2 gene by binding to the promoter of the IL-2 gene. PP2A is a Ser/Thr phosphatase that expressed in eukaryotic cells ubiquitously, it represents a negative regulator of the IL-2 gene promoter activity. Elf-1 belongs to the Ets family of transcription factors and can promote the expression of IL-2. Blimp-1 is a crucial transcription factors for regulating B lymphocyte terminal differentiation, an important function of Blimp-1 in T cells is to repress IL-2 gene transcription directly. Interferon regulatory factors (IRFs) are distinctive transcriptional regulators of type I interferons (IFNs) and IFN inducible genes, IRF-5 is a member of the IRFs family. IRF-5 is found to be increased in SLE and can regulate the production of IL-2 negatively. PP2A can inhibit the synthesis of IL-2 in two ways: on the one hand, activating the IL-2 transcription inhibitory factor CREMα, on the other hand, inhibiting IL-2 stimulating transcription factor Elf-1. While IRF-5 can activate the IL-2 transcription negative regulator Blimp-1 as to inhibit IL-2 expression. These molecules participate in the regulation of IL-2 through different pathways. This paper reviews the current knowledge of IL-2 signaling pathway regulating molecules in SLE.

EP4 Receptor-Associated Protein in Macrophages Protects against Bleomycin-Induced Pulmonary Inflammation in Mice.

Excessive activation of inflammatory macrophages drives the pathogenesis of many chronic diseases. EP4 receptor-associated protein (EPRAP) has been identified as a novel, anti-inflammatory molecule in macrophages. In this study, we investigated the role of EPRAP using a murine model of bleomycin (BLM)-induced pulmonary inflammation. When compared with wild-type mice, EPRAP-deficient mice exhibited significantly higher mortality, and increased accumulation of macrophages and proinflammatory molecules in the lung 7 d post-BLM administration. Accordingly, the levels of phosphorylated p105, MEK1/2, and ERK1/2 were elevated in EPRAP-deficient alveolar macrophages following BLM administration. In contrast, macrophage-specific EPRAP overexpression decreased the production of proinflammatory cytokines and chemokines, suggesting that EPRAP in macrophages plays a key role in attenuating BLM-induced pulmonary inflammation. As EPRAP is phosphorylated after translation, we examined the role of posttranslational modifications in cellular inflammatory activation using mouse embryo fibroblasts (MEFs) expressing mutant EPRAP proteins. Expression of mutant EPRAP, in which serine-108 and serine-608 were replaced with alanine (EPRAP S108A/S608A), markedly suppressed TNF-α production in LPS-treated MEFs. Conversely, the serine phosphatase 2A (PP2A) inhibitor, cantharidic acid, increased LPS-induced TNF-α production in MEFs expressing wild-type EPRAP, but not in MEFs expressing EPRAP S108A/S608A. Immunoprecipitation analyses demonstrated that EPRAP associated with PP2A in both MEFs and alveolar macrophages from BLM-treated mice. Our data suggest that PP2A dephosphorylates EPRAP, which may be a crucial step in exertion of its anti-inflammatory properties. For these reasons, we believe the EPRAP-PP2A axis in macrophages holds the key to treating chronic inflammatory disorders.

Phosphatase PP2A is requisite for the function of regulatory T cells.

Homeostasis of the immune system depends on the proper function of regulatory T cells (T(reg) cells). Compromised suppressive activity of T(reg) cells leads to autoimmune disease and graft rejection and promotes anti-tumor immunity. Here we report a previously unrecognized requirement for the serine-threonine phosphatase PP2A in the function of T(reg) cells. T(reg) cells exhibited high PP2A activity, and T(reg) cell-specific ablation of the PP2A complex resulted in a severe, multi-organ, lymphoproliferative autoimmune disorder. Mass spectrometry revealed that PP2A associated with components of the mTOR metabolic-checkpoint kinase pathway and suppressed the activity of the mTORC1 complex. In the absence of PP2A, T(reg) cells altered their metabolic and cytokine profile and were unable to suppress effector immune responses. Therefore, PP2A is required for the function of T(reg) cells and the prevention of autoimmunity.

Glutamic acid decarboxylase autoantibodies are dominant but insufficient to identify most Chinese with adult-onset non-insulin requiring autoimmune diabetes: LADA China study 5.

AIMS: Adult-onset autoimmune diabetes is prevalent in China, in contrast to childhood-onset type 1 diabetes mellitus. Islet autoantibodies are the most important immune biomarkers to diagnose autoimmune diabetes. We assayed four different islet autoantibodies in recently diagnosed adult non-insulin-requiring diabetes Chinese subjects to investigate the best antibody assay strategy for the correct diagnosis of these subjects. METHODS: LADA China study is a nation-wide multicenter study conducted in diabetes patients from 46 university-affiliated hospitals in China. Non-insulin-treated newly diagnosed adult diabetes patients (n = 2388) were centrally assayed for glutamic acid decarboxylase autoantibody (GADA), protein tyrosine phosphatase-2 autoantibody (IA-2A), and zinc transporter 8 autoantibody (ZnT8A) by radioligand assay and insulin autoantibody (IAA) by microtiter plate radioimmunoassay. Clinical data were determined locally. RESULTS: Two hundred and six (8.63 %) subjects were autoantibody positive, of which GADA identified 5.78 % (138/2388) of the total, but only 67 % (138/206) of the autoimmune cases. IA-2A, ZnT8A, and IAA were found in 1.51, 1.84, and 1.26 % of the total study subjects, respectively. When assaying three islet autoantibodies, the most effective strategy was the combination of GADA, ZnT8A, and IAA, which could identify 92.2 % (190/206) autoimmune diabetes patients. The clinical data showed that those subjects with positive GADA had lower random C-peptide than autoantibody negative subjects (P < 0.05). CONCLUSIONS: As with Europeans, GADA is the dominant autoantibody in this form of autoimmune diabetes in China, but in contrast to Europeans, screening should include other diabetes-associated autoantibodies.

MicroRNA-9 regulates steroid-resistant airway hyperresponsiveness by reducing protein phosphatase 2A activity.

BACKGROUND: Steroid-resistant asthma is a major clinical problem that is linked to activation of innate immune cells. Levels of IFN-γ and LPS are often increased in these patients. Cooperative signaling between IFN-γ/LPS induces macrophage-dependent steroid-resistant airway hyperresponsiveness (AHR) in mouse models. MicroRNAs (miRs) are small noncoding RNAs that regulate the function of innate immune cells by controlling mRNA stability and translation. Their role in regulating glucocorticoid responsiveness and AHR remains unexplored. OBJECTIVE: IFN-γ and LPS synergistically increase the expression of miR-9 in macrophages and lung tissue, suggesting a role in the mechanisms of steroid resistance. Here we demonstrate the role of miR-9 in IFN-γ/LPS-induced inhibition of dexamethasone (DEX) signaling in macrophages and in induction of steroid-resistant AHR. METHODS: MiRNA-9 expression was assessed by means of quantitative RT-PCR. Putative miR-9 targets were determined in silico and confirmed in luciferase reporter assays. miR-9 function was inhibited with sequence-specific antagomirs. The efficacy of DEX was assessed by quantifying glucocorticoid receptor (GR) cellular localization, protein phosphatase 2A (PP2A) activity, and AHR. RESULTS: Exposure of pulmonary macrophages to IFN-γ/LPS synergistically induced miR-9 expression; reduced levels of its target transcript, protein phosphatase 2 regulatory subunit B (B56) δ isoform; attenuated PP2A activity; and inhibited DEX-induced GR nuclear translocation. Inhibition of miR-9 increased both PP2A activity and GR nuclear translocation in macrophages and restored steroid sensitivity in multiple models of steroid-resistant AHR. Pharmacologic activation of PP2A restored DEX efficacy and inhibited AHR. MiR-9 expression was increased in sputum of patients with neutrophilic but not those with eosinophilic asthma. CONCLUSION: MiR-9 regulates GR signaling and steroid-resistant AHR. Targeting miR-9 function might be a novel approach for the treatment of steroid-resistant asthma.