Regulation Of Innate Immune Responses Research Articles

TRIM14 is a Putative Tumor Suppressor and Regulator of Innate Immune Response in Non-Small Cell Lung Cancer

Non-small-cell lung carcinoma (NSCLC) accounts for 85% of malignant lung tumors and is the leading cause of cancer deaths. Our group previously identified Tripartite Motif 14 (TRIM14) as a component of a prognostic multigene expression signature for NSCLC. Little is known about the function of TRIM14 protein in normal or disease states. We investigated the functional and prognostic role of TRIM14 in NSCLC using in vitro and in vivo perturbation model systems. Firstly, a pooled RNAi screen identified TRIM14 to effect cell proliferation/survival in NSCLC cells. Secondly, silencing of TRIM14 expression significantly enhanced tumor growth in NSCLC xenograft mouse models, while exogenous TRIM14 expression attenuated tumorigenesis. In addition, differences in apoptotic activity between TRIM14-deficient and control tumors suggests that TRIM14 tumor suppressor activity may depend on cell death signaling pathways. TRIM14-deficient cell lines showed both resistance to hypoxia-induced cell death and attenuation of interferon response via STAT1 signaling. Consistent with these phenotypes, multivariate analyses on published mRNA expression datasets of over 600 primary NSCLCs demonstrated that low TRIM14 mRNA levels are significantly associated with poorer prognosis in early stage NSCLC patients. Our functional data therefore establish a novel tumor suppressive role for TRIM14 in NSCLC progression.

Macrophages and Mitochondria: A Critical Interplay Between Metabolism, Signaling, and the Functional Activity.

Macrophages are phagocytic cells that participate in a broad range of cellular functions and they are key regulators of innate immune responses and inflammation. Mitochondria are highly dynamic endosymbiotic organelles that play key roles in cellular metabolism and apoptosis. Mounting evidence suggests that mitochondria are involved in the interplay between metabolism and innate immune responses. The ability of these organelles to alter the metabolic profile of a cell, thereby allowing an appropriate response to each situation, is crucial for the correct establishment of immune responses. Furthermore, mitochondria act as scaffolds for many proteins involved in immune signaling pathways and as such they are able to modulate the function of these proteins. Finally, mitochondria release molecules, such as reactive oxygen species, which directly regulate the immune response. In summary, mitochondria can be considered as core components in the regulation of innate immune signaling. Here we discuss the intricate relationship between mitochondria, metabolism, intracellular signaling, and innate immune responses in macrophages.

G protein-coupled receptors mediate neural regulation of innate immune responses in caenorhabditis elegans.

G protein-coupled receptors (GPCRs) are a large family of transmembrane proteins that perceive many extracellular signals and transduce them into cellular physiological responses. GPCRs regulate immunity in both vertebrates and invertebrates. However, the mechanisms responsible for such regulation are not fully understood. Recent research using the genetically tractable model organism Caenorhabditis elegans has led to the identification of specific GPCRs, neurotransmitters, neurons and non-neural cells in the regulation of innate immunity. Several neural circuits have been demonstrated to function in GPCR-dependent immuno-regulatory pathways. Besides being essential in neural-immune interactions, GPCRs also regulate innate immune response in non-neural tissues cell-autonomously through mechanisms independent of neural circuits. Here we review GPCR-mediated neural control of innate immunity in C. elegans and briefly discuss GPCR-dependent immune regulation via non-neural mechanisms.

The Effect of Hydrolysis and Protein Source on the Efficacy of Protein Hydrolysates as Plant Resistance Inducers against Powdery Mildew

The substitution of synthetic chemical pesticides has become a priority in agriculture and the induction of plant resistance by protein hydrolysates may offer a sustainable alternative. The protein source, method and degree of hydrolysis, as well as the amino acid and peptide composition may affect the efficacy of protein hydrolysates in protecting plants against pathogens. The aim of this work was to clarify the effect of enzymatic and acid hydrolysis on different plant protein sources (soybean, rapeseed and guar protein meal), in terms of efficacy against the powdery mildew of courgette plants (caused by Podosphaera xanthii). The enzymatic hydrolysates were obtained by incubating each protein suspension with a measured amount of Alcalase or Flavourzyme at 50°C for 24 h, in order to obtain an enzyme/substrate ratio of 1% and 50%. The chemical hydrolysates were obtained by acid hydrolysis using 6 N H2SO4 at 121°C for 15 min (6N A), or at 100°C for 8 h (6N B) respectively. Preventive foliar treatments with guar hydrolysates produced with both enzymatic (Alcalase 50% E/S ratio) and chemical (6N B) hydrolysis significantly reduced disease symptoms compared to the non-hydrolysed protein source. A positive correlation was found between efficacy and the degree of hydrolysis of guar acid hydrolysates, suggesting that the hydrolysis method may enhance the functional properties of the original protein source. In addition, positive correlations were found between the efficacy of guar hydrolysates and concentrations of specific peptides and amino acids. In conclusion, our results showed that the biocontrol effect of plant protein hydrolysates was related to the original protein source and two specific hydrolysis processes improved the functional properties of guar, producing peptide fragments and free amino acids that may be involved in the regulation of innate immune response in plants.

Mitochondria-Endoplasmic Reticulum Contact Sites Mediate Innate Immune Responses.

Mitochondria and the endoplasmic reticulum (ER) are fundamental organelles that coordinate high-order cell functions. Mitochondria are centers of energy production, whereas the ER is responsible for folding, transport, and degradation of proteins. In addition to their specific functions, mitochondria and ER actively communicate with each other to promote a variety of cellular events, such as material transfer and signal transduction. Recent studies have shown the critical involvement of these organelles in regulation of the innate immune system, which functions in host defense. The innate immune system utilizes a wide range of germ-line-encoded pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) and induces inflammatory and antiviral responses. Contact sites between mitochondria and the ER function in assembly of the NLR family pyrin domain containing 3 (NLRP3)-inflammasome to promote the inflammatory response. The NLRP3-inflammasome is a protein complex composed of the receptor NLRP3 on the ER side and the adaptor apoptosis-associated speck-like protein containing a CARD on the mitochondrial side; it induces caspase-1-dependent maturation of proinflammatory cytokines such as interleukin (IL)-1β and IL-18. Furthermore, ER-mitochondria contact sites function in initiation and mediation of signal transduction pathways downstream of intracellular PRRs, such as retinoic acid-inducible gene I-like receptor and cyclic GMP-AMP synthase, to promote the antiviral response. Therefore, ER-mitochondria contact sites, also known as mitochondria-associated membranes, play key roles in regulation of innate immune responses.

Roles of HDACs in the Responses of Innate Immune Cells and as Targets in Inflammatory Diseases.

Histone deacetylases (HDACs) are an emerging class of molecules involved in the epigenetic regulation of innate immune responses through Toll-like receptor (TLR) and interferon (IFN) signaling pathways. HDACs are also key drivers of inflammatory diseases via epigenetic regulation through chromatin DNA and histone modification by methylation and acetylation, among other mechanisms, which control innate immune cell gene expression. Importantly, these epigenetic changes are reversible, and HDACs may also be targeted by small-molecule HDAC inhibitors, which have been used in clinical settings for cancer therapy. Here, we highlight HDACs as strong therapeutic molecules and explore HDAC-induced mechanisms regulating innate immune responses and inflammatory cytokine control, with the goal of developing personalized medicine for the treatment of human diseases, including inflammatory diseases and immune disorders. Currently, this novel class of immunomodulatory therapeutics is being evaluated in the laboratory, in preclinical models, and in the clinic.

STRUCTURAL FEATURES OF SELECTIVE AND NONSELECTIVE NOD RECEPTOR AGONISTS

Muramyl peptides are fragments of bacterial peptidoglycan playing an important role in the activation of innate immune system. Core structure of muramyl peptides is composed of N-acetylmuramic acid residue (MurNAc) covalently bound to a peptide consisting of 2-5 amino acid residues. The first residue in the peptide chain is usually L-alanine, the 2nd is D-isoglutamine or D-isoglutamic acid, the 4th and 5th are D-alanines. The 3rd residue is most variable being represented by L-lysine or L-ornithine in Gram-positive bacteria, or by meso-diaminopimelic acid (meso-DAP) in Gram-negative bacteria. Muramyl peptides are potential immunostimulants and adjuvants. Two receptors, NOD1 and NOD2, are known to play a central role in muramyl peptide recognition. However, the data on NOD receptor specificity for several common muramyl peptides are not available or incomplete. The goal of this study was to extend knowledge on possible relationships between structure of natural, or chemically modified muramyl peptides, and their selectivity towards NOD1 and NOD2 receptors. We investigated ability of natural muramyl peptides and their derivatives to activate NOD1 and NOD2. To this end, we used a novel biological test system, which represents modified HEK293T cells with NF-κB-driven reporter gene (luciferase) expression and knock-outs of endogenous NOD1 and/or NOD2 genes. As a result, we have shown that NOD2 has a broader spectrum of natural agonists than previously thought. In particular, NOD2 recognizes muramyl peptides from Gram-negative bacteria containing a meso-DAP residue, which have been considered inactive towards NOD2. NOD1 recognizes only muramyl peptides containing a mesoDAP residue; however, this molecule should not be exposed as a terminal residue, as thought earlier, but it may also be present within the peptide chain. This data imply that natural muramyl peptides containing mesoDAP have a dual receptor specificity (NOD1 and NOD2), i.e. they are non-selective NOD receptor agonists. Destruction of the MurNAc residue eliminates ability of these muramyl peptides to activate NOD2, but has no effect on their NOD1 agonism. Hence, modified muramyl peptides containing meso-DAP and a destroyed MurNAc residue are selective NOD1 agonists. Natural muramyl peptides featuring an intact MurNAc and no meso-DAP are selective NOD2 agonists. Conclusions: 1) we have characterized key structural features of selective NOD1 and NOD2 agonists as well as non-selective NOD receptors agonists; 2) we show the importance of these new data for understanding innate immune response regulation, and for development of novel muramyl peptide immunostimulants.

Divergent Epigenomes in Pediatric and Adult Acute Myeloid Leukemia Implicate Cell of Origin and Transcriptional Silencing of Immune Responses As Sources of Clinically Relevant Heterogeneity: A Report from the Children's Oncology Group and NCI/COG Therapeutically Applicable Research to Generate Effective Treatments (TARGET) Initiative

Acute myeloid leukemia (AML) carries a poor prognosis across age groups. In children, AML has become the leading cause of leukemia mortality, with only 60% of cases securing long-term remission. In adults, outcomes are far worse, with 5 year survival approaching 24%. The mutational and transcriptional characterization of AML1has not yet translated into improved outcomes for most patients.The TARGET AML project is an effort of Children's Oncology Group (COG) and the National Cancer Institute to characterize molecular abnormalities in pediatric AML. 197 cases were selected for whole genome sequencing (WGS) of diagnostic specimens, 284 cases for mRNA sequencing, 289 cases for DNA methylation arrays, and 721 cases for targeted sequencing (182 assayed by WGS). Most patients (93%) were uniformly treated on COG study AAML0531 or AAML03P1. The Cancer Genome Atlas (TCGA) AML project1characterized 177 comparable adult AMLs with identical assays.DNA methylation changes radically during differentiation of blood cells2, and recurrent pre-leukemic mutations in adult AML3affect DNA methylation and chromatin modifiers. We thus investigated whether differences in cell-of-origin, immune signalling, and regulatory aberrations were captured by focal or regional differences in DNA methylation, within or between adult and pediatric AML patients.In cytogenetically similar TARGET and TCGA AML cases, striking differences in DNA methylation emerge (fig. 1). Pediatric FLT3-mutant AMLs dominate a cluster with normal-progenitor-like DNA methylation. Mutant DNMT3A, RUNX1, and TP53, which selectively favor preleukemic hematopoietic stem cells3,4,5 (HSCs), are common in adult AML, rare in pediatric AML, and tend towards HSC-like hypermethylation. Transcriptional & epigenetic signatures of the cell of origin persist even after leukemic transformation6. Thus we sought to identify the most likely cell of origin for each case. Previous studies of mRNA7 and DNA methylation8 differences in HSCs and progenitor cells (HSPCs), leukemic stem cells (LSCs), and AML blasts allowed us to model these differences in TCGA and TARGET AMLs. RNAseq results revealed many LSC-like cases with aberrant β-catenin signaling and TP53 regulation, distinct from blasts and normal HSPCs (fig. 2a). DNA methylation segregated cases resembling granulocyte/monocyte progenitors (GMPs) from those resembling other HSPC subsets (fig. 2b). DNMT3A mutants strongly associated with HSC/LSC-like mRNA expression, as did most MLL-rearranged AMLs. Nearly all TP53 and RUNX1 mutants presented LSC-like mRNA expression and retained HSC-like methylomes. These results suggest that decades of selective HSC attrition enable cooperating adult-specific mutations to initiate leukemia, while the timescales in pediatric AML favor fusion genes capable of transforming progenitors as well as HSCs.With matched mRNA expression & DNA methylation data from 256 TARGET cases and 156 TCGA cases, we found over 100 genes where DNA methylation accompanied loss of transcription (silencing) in AML but not in normal HSPCs (fig. 3a). Many such genes lie in regions affected by recurrent copy number aberrations, most notably chromosome arms 5q and 19q. Recurrently mutated or deleted genes such as DNMT3A, TET2, SPRY4, and CDKN2A/B are silenced, some mutually exclusively with mutations or CNV. Functional enrichment analyses of silenced genes with DAVID9revealed 4 clusters: NK-cell signaling, innate immune response regulation, transcriptional regulation, and (on chromosome 19q) zinc finger genes involved in Toll-like receptor signaling. Some silencing co-occurs with specific molecular features, but no event was perfectly predicted by any molecular or cytogenetic feature (fig. 3b).Drug-gene interaction mining with DGIDb10 suggests silencing may inform treatment. Silencing of the mitotic checkpoint gene CHFR may confer sensitivity to microtubule inhibitors11, silencing of MGMT suggests greater benefit from alkylating agents12, and demethylating agents may benefit cases with silenced immune response13. Biomarker driven clinical trials will be needed to evaluate these and other markers in pediatric and adult AML, but evidence of independent genetic and epigenetic evolution in AML14supports their continued investigation.This work is dedicated to the late Robert J. Arceci, without whom none of this would have been possible. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Adaptively introgressed Neandertal haplotype at the OAS locus functionally impacts innate immune responses in humans.

BackgroundThe 2’-5’ oligoadenylate synthetase (OAS) locus encodes for three OAS enzymes (OAS1-3) involved in innate immune response. This region harbors high amounts of Neandertal ancestry in non-African populations; yet, strong evidence of positive selection in the OAS region is still lacking.ResultsHere we used a broad array of selection tests in concert with neutral coalescent simulations to demonstrate a signal of adaptive introgression at the OAS locus. Furthermore, we characterized the functional consequences of the Neandertal haplotype in the transcriptional regulation of OAS genes at baseline and infected conditions. We found that cells from people with the Neandertal-like haplotype express lower levels of OAS3 upon infection, as well as distinct isoforms of OAS1 and OAS2.ConclusionsWe present evidence that a Neandertal haplotype at the OAS locus was subjected to positive selection in the human population. This haplotype is significantly associated with functional consequences at the level of transcriptional regulation of innate immune responses. Notably, we suggest that the Neandertal-introgressed haplotype likely reintroduced an ancestral splice variant of OAS1 encoding a more active protein, suggesting that adaptive introgression occurred as a means to resurrect adaptive variation that had been lost outside Africa.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-1098-6) contains supplementary material, which is available to authorized users.

A role for intestinal TLR4-driven inflammatory response during activity-based anorexia.

Anorexia nervosa (AN) is associated with low-grade systemic inflammation and altered gut microbiota. However, the molecular origin of the inflammation remains unknown. Toll-like receptors are key regulators of innate immune response and their activation seems also to be involved in the control of food intake. We used activity-based anorexia (ABA) model to investigate the role of TLR4 and its contribution in anorexia-associated low-grade inflammation. Here, we found that ABA affected early the intestinal inflammatory status and the hypothalamic response. Indeed, TLR4 was upregulated both on colonic epithelial cells and intestinal macrophages, leading to elevated downstream mucosal cytokine production. These mucosal changes occurred earlier than hypothalamic changes driving to increased levels of IL-1β and IL-1R1 as well as increased levels of plasma corticosterone. Paradoxically, TLR4-deficient mice exhibited greater vulnerability to ABA with increased mortality rate, suggesting a major contribution of TLR4-mediated responses during ABA-induced weight loss.

Emerging roles of orphan nuclear receptors in regulation of innate immunity

Innate immunity constitutes the first line of defense against pathogenic and dangerous insults. However, it is a double-edged sword, as it functions in both clearance of infection and inflammatory damage. It is therefore important that innate immune responses are tightly controlled to prevent harmful excessive inflammation. Nuclear receptors (NRs) are a family of transcription factors that play critical roles in various physiological responses. Orphan NRs are a subset of NRs for which the ligands and functions are unclear. Accumulating evidence has revealed that orphan NRs play essential roles in innate immune responses to prevent pathogenic inflammatory responses and to enhance antimicrobial host defenses. In this review, we describe current knowledge on the roles and mechanisms of orphan NRs in the regulation of innate immune responses. Discovery of new functions of orphan NRs would facilitate development of novel preventive and therapeutic strategies against human inflammatory diseases.

Widespread Shortening of 3' Untranslated Regions and Increased Exon Inclusion Are Evolutionarily Conserved Features of Innate Immune Responses to Infection.

The contribution of pre-mRNA processing mechanisms to the regulation of immune responses remains poorly studied despite emerging examples of their role as regulators of immune defenses. We sought to investigate the role of mRNA processing in the cellular responses of human macrophages to live bacterial infections. Here, we used mRNA sequencing to quantify gene expression and isoform abundances in primary macrophages from 60 individuals, before and after infection with Listeria monocytogenes and Salmonella typhimurium. In response to both bacteria we identified thousands of genes that significantly change isoform usage in response to infection, characterized by an overall increase in isoform diversity after infection. In response to both bacteria, we found global shifts towards (i) the inclusion of cassette exons and (ii) shorter 3’ UTRs, with near-universal shifts towards usage of more upstream polyadenylation sites. Using complementary data collected in non-human primates, we show that these features are evolutionarily conserved among primates. Following infection, we identify candidate RNA processing factors whose expression is associated with individual-specific variation in isoform abundance. Finally, by profiling microRNA levels, we show that 3’ UTRs with reduced abundance after infection are significantly enriched for target sites for particular miRNAs. These results suggest that the pervasive usage of shorter 3’ UTRs is a mechanism for particular genes to evade repression by immune-activated miRNAs. Collectively, our results suggest that dynamic changes in RNA processing may play key roles in the regulation of innate immune responses.

A plasma microRNA signature as a biomarker for acquired aplastic anemia.

Aplastic anemia is an acquired bone marrow failure characterized by marrow hypoplasia, a paucity of hematopoietic stem and progenitor cells, and pancytopenia of the peripheral blood, due to immune attack on the bone marrow. In aplastic anemia, a major challenge is to develop immune biomarkers to monitor the disease. We measured circulating microRNAs in plasma samples of aplastic anemia patients in order to identify disease-specific microRNAs. A total of 179 microRNAs were analyzed in 35 plasma samples from 13 aplastic anemia patients, 11 myelodysplastic syndrome patients, and 11 healthy controls using the Serum/Plasma Focus microRNA Polymerase Chain Reaction Panel. Subsequently, 19 microRNAs from the discovery set were investigated in the 108 plasma samples from 41 aplastic anemia patients, 24 myelodysplastic syndrome patients, and 43 healthy controls for validation, confirming that 3 microRNAs could be validated as dysregulated (>1.5-fold change) in aplastic anemia, compared to healthy controls. MiR-150-5p (induction of T-cell differentiation) and miR-146b-5p (involvement in the feedback regulation of innate immune response) were elevated in aplastic anemia plasma, whereas miR-1 was decreased in aplastic anemia. By receiver operating characteristic curve analysis, we developed a logistic model with these 3 microRNAs that enabled us to predict the probability of a diagnosis of aplastic anemia with an area under the curve of 0.86. Dysregulated expression levels of the microRNAs became normal after immunosuppressive therapy at 6 months. Specifically, miR-150-5p expression was significantly reduced after successful immunosuppressive therapy, but did not change in non-responders. We propose 3 novel plasma biomarkers in aplastic anemia, in which miR-150-5p, miR-146b-5p, and miR-1 can serve for diagnosis and miR-150-5p for disease monitoring. Clinicaltrials.gov identifiers:00260689, 00217594, 00961064.

The PB2 Subunit of the Influenza A Virus RNA Polymerase Is Imported into the Mitochondrial Matrix.

ABSTRACTThe polymerase basic 2 (PB2) subunit of the RNA polymerase complex of seasonal human influenza A viruses has been shown to localize to the mitochondria. Various roles, including the regulation of apoptosis and innate immune responses to viral infection, have been proposed for mitochondrial PB2. In particular, PB2 has been shown to inhibit interferon expression by associating with the mitochondrial antiviral signaling (MAVS) protein, which acts downstream of RIG-I and MDA-5 in the interferon induction pathway. However, in spite of a growing body of literature on the potential roles of mitochondrial PB2, the exact location of PB2 in mitochondria has not been determined. Here, we used enhanced ascorbate peroxidase (APEX)-tagged PB2 proteins and electron microscopy to study the localization of PB2 in mitochondria. We found that PB2 is imported into mitochondria, where it localizes to the mitochondrial matrix. We also demonstrated that MAVS is not required for the import of PB2 into mitochondria by showing that PB2 associates with mitochondria in MAVS knockout mouse embryo fibroblasts. Instead, we found that amino acid residue 9 in the N-terminal mitochondrial targeting sequence is a determinant of the mitochondrial import of PB2, differentiating the localization of PB2 of human from that of avian influenza A virus strains. We also showed that a virus encoding nonmitochondrial PB2 is attenuated in mouse embryonic fibroblasts (MEFs) compared with an isogenic virus encoding mitochondrial PB2, in a MAVS-independent manner, suggesting a role for PB2 within the mitochondrial matrix. This work extends our understanding of the interplay between influenza virus and mitochondria.IMPORTANCE The PB2 subunit of the influenza virus RNA polymerase is a major determinant of viral pathogenicity. However, the molecular mechanisms of how PB2 determines pathogenicity remain poorly understood. PB2 associates with mitochondria and inhibits the function of the mitochondrial antiviral signaling protein MAVS, implicating PB2 in the regulation of innate immune responses. We found that PB2 is imported into the mitochondrial matrix and showed that amino acid residue 9 is a determinant of mitochondrial import. The presence of asparagine or threonine in over 99% of all human seasonal influenza virus pre-2009 H1N1, H2N2, and H3N2 strains is compatible with mitochondrial import, whereas the presence of an aspartic acid in over 95% of all avian influenza viruses is not, resulting in a clear distinction between human-adapted and avian influenza viruses. These findings provide insights into the interplay between influenza virus and mitochondria and suggest mechanisms by which PB2 could affect pathogenicity.

MiRNA in Macrophage Development and Function.

MicroRNAs (miRNAs) control cellular gene expression via primarily binding to 3' or 5' untranslated region of the target transcript leading to translational repression or mRNA degradation. In most cases, miRNAs have been observed to fine-tune the cellular responses and, therefore, act as a rheostat rather than an on/off switch. Transcription factor PU.1 is a master switch that controls monocyte/macrophage development from hematopoietic stem cells. Recent Advances: PU.1 induces a specific set of miRNAs while suppressing the miR17-92 cluster to regulate monocyte/macrophage development. In addition to development, miRNAs tightly control the macrophage polarization continuum from proinflammatory M1 or proreparative M2 by regulating expression of key transcription factors involved in the process of polarization. miRNAs are intricately involved with fine-tuning fundamental macrophage functions such as phagocytosis, efferocytosis, inflammation, tissue repair, and tumor promotion. Macrophages are secretory cells that participate in intercellular communication by releasing regulatory molecules and microvesicles (MVs). MVs are bilayered lipid membranes packaging a hydrophilic cargo, including proteins and nucleic acids. Macrophage-derived MVs carry functionally active miRNAs that suppress gene expression in target cells via post-transcriptional gene silencing, thus regulating cell function. In summary, miRNAs fine-tune several major facets of macrophage development and function. Such fine-tuning is critical in preventing exaggerated macrophage response to endogenous or exogenous stimuli. A critical role of miRNAs in the regulation of innate immune response and macrophage biology, including development, differentiation, and activation, has emerged. A clear understanding of such regulation on macrophage function remains to be elucidated. Antioxid. Redox Signal. 25, 795-804.

Proteasome function shapes innate and adaptive immune responses.

The proteasome system degrades more than 80% of intracellular proteins into small peptides. Accordingly, the proteasome is involved in many essential cellular functions, such as protein quality control, transcription, immune responses, cell signaling, and apoptosis. Moreover, degradation products are loaded onto major histocompatibility class I molecules to communicate the intracellular protein composition to the immune system. The standard 20S proteasome core complex contains three distinct catalytic active sites that are exchanged upon stimulation with inflammatory cytokines to form the so-called immunoproteasome. Immunoproteasomes are constitutively expressed in immune cells and have different proteolytic activities compared with standard proteasomes. They are rapidly induced in parenchymal cells upon intracellular pathogen infection and are crucial for priming effective CD8(+) T-cell-mediated immune responses against infected cells. Beyond shaping these adaptive immune reactions, immunoproteasomes also regulate the function of immune cells by degradation of inflammatory and immune mediators. Accordingly, they emerge as novel regulators of innate immune responses. The recently unraveled impairment of immunoproteasome function by environmental challenges and by genetic variations of immunoproteasome genes might represent a currently underestimated risk factor for the development and progression of lung diseases. In particular, immunoproteasome dysfunction will dampen resolution of infections, thereby promoting exacerbations, may foster autoimmunity in chronic lung diseases, and possibly contributes to immune evasion of tumor cells. Novel pharmacological tools, such as site-specific inhibitors of the immunoproteasome, as well as activity-based probes, however, hold promises as innovative therapeutic drugs for respiratory diseases and biomarker profiling, respectively.

TLR10 is a Negative Regulator of Human Monocyte Innate Immune Responses

Abstract Toll-like receptors (TLRs) are important pattern recognition receptors involved in the activation of innate immune responses against foreign pathogens. Humans possess ten TLRs, of which TLR10 is the only remaining TLR without a known ligand, signaling pathway or cellular function. Recent data by our lab has shown for the first time a suppression of cellular innate immune responses in primary human monocytes that is mediated by a TLR10 monoclonal antibody. This study sought to expand on our initial observations by investigating the signaling pathways, gene expression and long-term consequences of TLR10 engagement on primary human monocytes. Our data shows that TLR10 engagement is sufficient to suppress both IL-6 and TNF-α induction in response to TLR4, TLR7/8 or CD40 stimulation. Cellular lysates show that the primary target of TLR10 signaling may be to inhibit phosphorylation of Akt and its downstream targets. To further this hypothesis, we have performed RNA sequencing on cellular extracts to determine which pathways are targeted by TLR10 signaling. Our data also suggests that these events also affect long-term differentiation of monocytes into macrophages and dendritic cells. Macrophages and dendritic cells differentiated in the presence of a TLR10 antibody exhibit suppressed differentiation markers such as CD80/CD86 and HLA-DR/CD16 respectively after four days of differentiation. These data show that TLR10 is a novel regulator of innate immune responses on primary human monocytes and may have eventual therapeutics benefits in chronic inflammatory and auto-immune diseases.

The role of MAVS signaling in humoral immune responses to West Nile virus.

Abstract MAVS is a key regulator of innate immune responses to RNA viruses, such as West Nile virus (WNV). Previous work identified that MAVS is important for the development of adaptive immunity to WNV. MAVS KO mice are highly susceptible to infection with lethal WNV-Tx. MAVS KO mice produced significantly increased levels of WNV-specific IgM and IgG compared to WT mice, however, a corresponding increase in neutralizing antibodies (NAbs) was not observed. As MAVS KO mice succumb rapidly to WNV-Tx, it was not possible in our initial study to define the role of MAVS in the development of the germinal centers (GCs) as well as long-term plasma cell (PC) and memory B cell responses. Using the less pathogenic WNV-MAD, we examined the development of humoral responses in the absence of MAVS. MAVS KO mice survived infection with WNV-MAD, and as with WNV-Tx infection, produced increased levels of IgM and IgG but comparatively low levels of NAbs. This increase in serum Ab levels was maintained up to 9 weeks post-infection (pi). Moreover, the number of WNV-specific Ab secreting cells in the spleen and bone marrow were increased in MAVS KO mice compared to WT mice. Using flow cytometry we found that MAVS KO mice have a slight delay in expansion of GL7+ GC B cells, but by day 10 pi the number of GC B cells was about 10-fold higher than in WT mice. MAVS KO mice also showed a rapid expansion of IgM−CD138hi PCs that was significantly increased compared to WT mice. We are currently testing the hypothesis that these results are due to an intrinsic dysregulation of MAVS KO B cells rather than simply due to an increase in viral load. We are also examining both extrafollicular and GC responses to identify the basis for the rapid IgG antibody responses in MAVS KO mice. (Supported by NIH grant AI83019).

Platelets constitutively express IL-33 protein and modulate eosinophilic airway inflammation

Although platelets play a key role in allergic inflammation in addition to their well-established role in hemostasis, the precise mechanisms of how platelets modulate allergic inflammation are not fully understood. IL-33 is an essential regulator of innate immune responses and allergic inflammation. We sought to determine the expression of IL-33 protein by platelets and its functional significance in airway inflammation. IL-33 protein in human platelets, the human megakaryocyte cell line MEG-01, and bone marrow-derived mouse megakaryocytes was detected by using Western blot analysis and fluorescent immunostaining. We examined the functional relevance of IL-33 protein in platelets by comparing platelet-intact and platelet-depleted groups in a murine model of IL-33-dependent airway eosinophilia elicited by intranasal administration of papain. We further compared the additive effect of administration of platelets derived from wild-type versus IL-33-deficient mice on the papain-induced eosinophilia. Platelets and their progenitor cells, megakaryocytes, constitutively expressed IL-33 protein (31kDa). Papain-induced IL-33-dependent airway eosinophilia in mice was significantly attenuated by platelet depletion. Conversely, concomitant administration of platelets derived from wild-type mice but not IL-33-deficient mice enhanced the papain-induced airway eosinophilia. Our novel findings suggest that platelets might be important cellular sources of IL-33 protein invivo and that platelet-derived IL-33 might play a role in airway inflammation. Therefore platelets might become an attractive novel therapeutic target for asthma and probably allergic inflammation.

H3K4me3 Demethylase Kdm5a Is Required for NK Cell Activation by Associating with p50 to Suppress SOCS1.

The H3K4me3 demethylase Kdm5a regulates gene transcription and is implicated in carcinogenesis. However, the role of Kdm5a in innate immune response remains poorly understood. Here, we demonstrate that Kdm5a deficiency impairs activation of natural killer (NK) cells, with decreased IFN-γ production. Accordingly, Kdm5a(-/-) mice are highly susceptible to Listeria monocytogenes (Lm) infection. During NK cell activation, loss of Kdm5a profoundly impairs phosphorylation and nuclear localization of STAT4, along with increased expression of suppressor of cytokine signaling 1 (SOCS1). Mechanistic studies reveal that Kdm5a associates with p50 and binds to the Socs1 promoter region in resting NK cells, leading to a substantial decrease in H3K4me3 modification and repressive chromatin configuration at the Socs1 promoter. Thus, Kdm5a is required for priming activation of NK cells by suppressing the suppressor, SOCS1. Our study provides insights into the epigenetic regulation of innate immune response of NK cells.