PtdIns3P phosphatases MTMR3 and MTMR4 negatively regulate innate immune responses to DNA through modulating STING trafficking.

The innate immune system plays an essential role in initial recognition of pathogen infection by producing inflammatory cytokines and type I interferons. cGAS is a cytoplasmic sensor for DNA derived from DNA viruses. cGAS binding with DNA induces the production of cGAMP, a second messenger that associates with STING in endoplasmic reticulum (ER). STING changes its cellular distribution from ER to perinuclear Golgi, where it activates the protein kinase TBK1 that catalyzes the phosphorylation of IRF3. Here we found that STING trafficking is regulated by myotubularin-related protein (MTMR) 3 and MTMR4, members of protein tyrosine phosphatases that dephosphorylate 3' position in phosphatidylinositol (PtdIns) and generate PtdIns5P from PtdIns3,5P2 and PtdIns from PtdIns3P. We established MTMR3 and MTMR4 double knockout (DKO) RAW264.7 macrophage cells and found that they exhibited increased type I interferon production after interferon-stimulatory DNA (ISD) stimulation and herpes simplex virus 1 infection concomitant with enhanced IRF3 phosphorylation. In DKO cells, STING rapidly trafficked from ER to Golgi after ISD stimulation. Notably, DKO cells exhibited enlarged cytosolic puncta positive for PtdIns3P and STING was aberrantly accumulated in this puncta. Taken together, these results suggest that MTMR3 and MTMR4 regulate the production of PtdIns3P, which plays a critical role in suppressing DNA-mediated innate immune responses via modulating STING trafficking.

Phosphoinositol 3-phosphate acts as a timer for reactive oxygen species production in the phagosome.

Production of reactive oxygen species (ROS) in the phagosome by the NADPH oxidase is critical for mammalian immune defense against microbial infections and phosphoinositides are important regulators in this process. Phosphoinositol 3-phosphate (PI(3)P) regulates ROS production at the phagosome via p40phox by an unknown mechanism. This study tested the hypothesis that PI(3)P controls ROS production by regulating the presence of p40phox and p67phox at the phagosomal membrane. Pharmacologic inhibition of PI(3)P synthesis at the phagosome decreased the ROS production both in differentiated PLB-985 cells and human neutrophils. It also releases p67phox, the key cytosolic subunit of the oxidase, and p40phox from the phagosome. The knockdown of the PI(3)P phosphatase MTM1 or Rubicon or both increases the level of PI(3)P at the phagosome. That increase enhances ROS production inside the phagosome and triggers an extended accumulation of p67phox at the phagosome. Furthermore, the overexpression of MTM1 at the phagosomal membrane induces the disappearance of PI(3)P from the phagosome and prevents sustained ROS production. In conclusion, PI(3)P, indeed, regulates ROS production by maintaining p40phox and p67phox at the phagosomal membrane.

Missense Variant in MAPK Inactivator PTPN5 Is Associated with Decreased Severity of Post-Burn Hypertrophic Scarring.

BACKGROUND: Hypertrophic scarring (HTS) is hypothesized to have a genetic mechanism, yet its genetic determinants are largely unknown. The mitogen-activated protein kinase (MAPK) pathways are important mediators of inflammatory signaling, and experimental evidence implicates MAPKs in HTS formation. We hypothesized that single-nucleotide polymorphisms (SNPs) in MAPK-pathway genes would be associated with severity of post-burn HTS. METHODS: We analyzed data from a prospective-cohort genome-wide association study of post-burn HTS. We included subjects with deep-partial-thickness burns admitted to our center who provided blood for genotyping and had at least one Vancouver Scar Scale (VSS) assessment. After adjusting for HTS risk factors and population stratification, we tested MAPK-pathway gene SNPs for association with the four VSS variables in a joint regression model. In addition to individual-SNP analysis, we performed gene-based association testing. RESULTS: Our study population consisted of 538 adults (median age 40 years) who were predominantly White (76%) males (71%) admitted to our center from 2007-2014 with small-to-moderate-sized burns (median burn size 6% total body surface area). Of 2,146 SNPs tested, a rare missense variant in the PTPN5 gene (rs56234898; minor allele frequency 1.5%) was significantly associated with decreased severity of post-burn HTS (P = 1.3×10-6). In gene-based analysis, PTPN5 (P = 1.2×10-5) showed a significant association and BDNF (P = 9.5×10-4) a borderline-significant association with HTS severity. CONCLUSIONS: We report PTPN5 as a novel genetic locus associated with HTS severity. PTPN5 is a MAPK inhibitor expressed in neurons, suggesting a potential role for neurotrophic factors and neuroinflammatory signaling in HTS pathophysiology.

A bioassay for Lafora disease and laforin glucan phosphatase activity.

OBJECTIVES: Lafora disease is a rare yet invariably fatal form of progressive neurodegenerative epilepsy resulting from mutations in the phosphatase laforin. Several therapeutic options for Lafora disease patients are currently being explored, and these therapies would benefit from a biochemical means of assessing functional laforin activity following treatment. To date, only clinical outcomes such as decreases in seizure frequency and severity have been used to indicate success of epilepsy treatment. However, these qualitative measures exhibit variability and must be assessed over long periods of time. In this work, we detail a simple and sensitive bioassay that can be used for the detection of functional endogenous laforin from human and mouse tissue. DESIGN AND METHODS: We generated antibodies capable of detecting and immunoprecipitating endogenous laforin. Following laforin immunoprecipitation, laforin activity was assessed via phosphatase assays using para-nitrophenylphosphate (pNPP) and a malachite green-based assay specific for glucan phosphatase activity. RESULTS: We found that antibody binding to laforin does not impede laforin activity. Furthermore, the malachite green-based glucan phosphatase assay used in conjunction with a rabbit polyclonal laforin antibody was capable of detecting endogenous laforin activity from human and mouse tissues. Importantly, this assay discriminated between laforin activity and other phosphatases. CONCLUSIONS: The bioassay that we have developed utilizing laforin antibodies and an assay specific for glucan phosphatase activity could prove valuable in the rapid detection of functional laforin in patients to which novel Lafora disease therapies have been administered.

[Molecular genetics of type 1 diabetes mellitus: achievements and future trends].

Type 1 diabetes mellitus (T1DM) is a widespread, severe disease which results from the immunologically mediated destruction of the beta-cells of pancreatic Langerhans islets. To date the several loci involved to the T1DM development have been reliably identified by means of a number of approaches: MHC locus, VNTR within 5'-nontranscibed region of insulin (INS) gene, CTLA4 gene, encoding surface receptor of T cells, PTPN22 and PTPN2 genes, encoding tyrosine phosphatases of T lymphocytes, interleukin 2 (IL2) gene and alpha-chain of its receptor gene (IL2RA), as well as KIAA0350 gene (unknown function) and IFIH1 gene, encoding receptor of double-stranded DNA generated during viral infections. The functional analysis of proteins encoded by the genes, which are involved to the T1DM development, performed to confirm the hypothesis that on the one hand the origin of T1DM development is founded on the some deregulation of mechanisms of the immune tolerance formation and on the other hand the cause is founded on the formation of destructive immune response against own proteins of organism after virus infection or some other immune stress. Thus the protein products of MHC, INS, PTPN22 and PTPN2 genes involve in the formation in thymus of T-lymphocyte repertoire, which provides the immune defense of organism. On the other hand the nonspecific activation of T cells, from that starts the autoimmune destruction of beta-cells of Langerhans islets of pancreas, in all probability, connects with the protein products of CTLA4, IL2, IL2RA genes, and, perhaps, PTPN22 and PTPN2 genes. The only exception, if not considering the genes with unknown function,--is the IFIH1 gene, but its association with T1DM confirms that fact, that the certain types of virus infection can lead to the activation of autoreactive T cells and T1DM development.