Stimulation Of Pattern Recognition Receptors Research Articles

Evidence of collective influence in innate sensing using fluidic force microscopy.

The innate immune system initiates early response to infection by sensing molecular patterns of infection through pattern-recognition receptors (PRRs). Previous work on PRR stimulation of macrophages revealed significant heterogeneity in single cell responses, suggesting the importance of individual macrophage stimulation. Current methods either isolate individual macrophages or stimulate a whole culture and measure individual readouts. We probed single cell NF-κB responses to localized stimuli within a naïve culture with Fluidic Force Microscopy (FluidFM). Individual cells stimulated in naïve culture were more sensitive compared to individual cells in uniformly stimulated cultures. In cluster stimulation, NF-κB activation decreased with increased cell density or decreased stimulation time. Our results support the growing body of evidence for cell-to-cell communication in macrophage activation, and limit potential mechanisms. Such a mechanism might be manipulated to tune macrophage sensitivity, and the density-dependent modulation of sensitivity to PRR signals could have relevance to biological situations where macrophage density increases.

The early bird catches the IL-2 in C-type lectin receptor-dependent activation of dendritic cells.

The transcriptional response of macrophages and dendritic cells to stimulation of pattern recognition receptors can be unexpectedly distinct. In this issue of Science Signaling, Watanabe etal. demonstrate that IL-2 is differentially induced by the closely related C-type lectin receptors Dectin-2 and Mincle and reveal early signaling through the adaptor protein FcRγ as a critical mechanism.

Tannic Acid Exhibits Adjuvant Activity by Enhancing Humoral and Cell-Mediated Immunity Against BSA as a Protein Antigen.

Immunization or vaccination is the process of inducing artificial immunity against an antigen taking advantage of the mechanisms of immunological memory. Current vaccines include substances known as adjuvants, which tend to improve the immunogenicity of the antigen, reduce the antigen quantity employed, and boost the immune response in weak responders. Unfortunately, only a few vaccine adjuvants are approved for human use. Thus, the objective of this study was to investigate the effect of Tannic acid on humoral and cell-mediated immunity against bovine serum albumin (BSA) as a protein antigen in Wistar rats. In order to establish the Tannic acid concentration to test it as an adjuvant, the lethal dose 50 and maximum non-toxic dose were calculated through cytotoxicity and hemolytic assays with J774 A.1 cell line and rat erythrocytes by resazurin reduction method and UV/vis spectrophotometry. Thirty Wistar rats were divided into 5 groups that included two controls without antigen and three treatment groups of adjuvants plus BSA as a protein antigen. The rats were immunized in a 30-day scheme. Blood samples were collected for humoral immunity analysis by means of immunoglobulin quantification, isotyping and antigen-antibody precipitation inhibition analysis. Rat peritoneal macrophages and splenocytes were isolated for cell-mediated immunity analysis by means of nitric oxide quantification from adjuvant stimulated peritoneal macrophages and lymphocytes proliferation assay. Tannic acid was capable of increasing the immunogenicity of the antigen; besides, it was able to stimulate cell-mediated immunity by means of increased lymphocyte proliferation. Moreover, Tannic acid improved the humoral response by means of increased specific antibodies titers. These activities may be attributed to pattern recognition receptors stimulation. Tannic acid was considered biocompatible when tested in vivo because the concentration tested did not show cytotoxicity or hemolytic effect, and there was no detrimental effect observed on the animals' health. These results show Tannic acid as a promising candidate for vaccine adjuvant.

The E3 ubiquitin ligase RNF186 and RNF186 risk variants regulate innate receptor-induced outcomes

Balancing microbial-induced cytokines and microbial clearance is critical at mucosal sites such as the intestine. How the inflammatory bowel disease (IBD)-associated gene RNF186 regulates this balance is unclear. We found that macrophages from IBD-risk rs6426833 carriers in the RNF186 region showed reduced cytokines to stimulation through multiple pattern recognition receptors (PRRs). Upon stimulation of PRRs, the E3-ubiquitin ligase RNF186 promoted ubiquitination of signaling complex molecules shared across PRRs and those unique to select PRRs. Furthermore, RNF186 was required for PRR-initiated signaling complex assembly and downstream signaling. RNF186, along with its intact E3-ubiquitin ligase activity, was required for optimal PRR-induced antimicrobial reactive oxygen species, reactive nitrogen species, and autophagy pathways and intracellular bacterial clearance in human macrophages and for bacterial clearance in intestinal myeloid cells. Cells transfected with the rare RNF186-A64T IBD-risk variant and macrophages from common rs6426833 RNF186 IBD-risk carriers demonstrated a reduction in these RNF186-dependent outcomes. These studies identify mechanisms through which RNF186 regulates innate immunity and show that RNF186 IBD-risk variants demonstrate a loss of function in PRR-initiated outcomes.

Targeting Innate Immunity in Cancer Therapy.

The majority of current cancer immunotherapy strategies target and potentiate antitumor adaptive immune responses. Unfortunately, the efficacy of these treatments has been limited to a fraction of patients within a subset of tumor types, with an aggregate response rate of approximately 20% to date across all malignancies. The success of therapeutic inhibition of programmed death protein 1 (PD-1), protein death ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) with immune checkpoint inhibitors (ICI) has been limited to “hot” tumors characterized by preexisting T cell infiltration, whereas “cold” tumors, which lack T cell infiltration, have not achieved durable benefit. There are several mechanisms by which “cold” tumors fail to generate spontaneous immune infiltration, which converge upon the generation of an immunosuppressive tumor microenvironment (TME). The role of the innate immune system in tumor immunosurveillance and generation of antitumor immune responses has been long recognized. In recent years, novel strategies to target innate immunity in cancer therapy have emerged, including therapeutic stimulation of pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs); the DNA sensing cGAS/STING pathway; nucleotide-binding oligomerization domain-like receptors (NLRs), such as NLRP3; and the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs). In addition, therapeutic modulation of key innate immune cell types, such as macrophages and natural killer cells, has been investigated. Herein, we review therapeutic approaches to activate innate immunity within the TME to enhance antitumor immune responses, with the goal of disease eradication in “cold” tumors. In addition, we discuss rational immune-oncology combination strategies that activate both innate and adaptive immunity, with the potential to enhance the efficacy of current immunotherapeutic approaches.

Making Sense of Intracellular Nucleic Acid Sensing in Type I Interferon Activation in Sjögren's Syndrome.

Primary Sjögren’s syndrome (pSS) is a systemic autoimmune rheumatic disease characterized by dryness of the eyes and mucous membranes, which can be accompanied by various extraglandular autoimmune manifestations. The majority of patients exhibit persistent systemic activation of the type I interferon (IFN) system, a feature that is shared with other systemic autoimmune diseases. Type I IFNs are integral to anti-viral immunity and are produced in response to stimulation of pattern recognition receptors, among which nucleic acid (NA) receptors. Dysregulated detection of endogenous NAs has been widely implicated in the pathogenesis of systemic autoimmune diseases. Stimulation of endosomal Toll-like receptors by NA-containing immune complexes are considered to contribute to the systemic type I IFN activation. Accumulating evidence suggest additional roles for cytosolic NA-sensing pathways in the pathogenesis of systemic autoimmune rheumatic diseases. In this review, we will provide an overview of the functions and signaling of intracellular RNA- and DNA-sensing receptors and summarize the evidence for a potential role of these receptors in the pathogenesis of pSS and the sustained systemic type I IFN activation.

Cloning and Expression of Bovine ISG15 in Bacterial Expression System

The host response to viral infections includes stimulation of pattern recognition receptors which induces the release of type I interferons. Type I interferons further stimulate hundreds of interferons stimulated genes (ISGs). ISG15, a ubiquitin like protein, is an interferon stimulated gene implicated in antiviral response in several viral infections. Characterization of bovine ISG15 is important for studying the role of this ISG in pathogenesis of viral infections in cattle. Cloning and expression of the ISG15 in a bacterial expression system is the first step in characterization of this protein. Therefore, the present study is aimed at PCR amplification of the coding sequences of ISG15 using mRNA isolated from Foot and Mouth Disease Virus infected MDBK cells and to clone the amplified PCR product in pET32a to express in bacterial expression systems. The analysis of nucleotide and amino acid sequence of the cloned ISG15 revealed that it is highly conserved within bovine species. The cloned pET32a-ISG15 recombinant protein was successfully expressed in E. coli expression system and characterized by SDS-PAGE analysis.

The Intrinsically Disordered W Protein Is Multifunctional during Henipavirus Infection, Disrupting Host Signalling Pathways and Nuclear Import.

Nipah and Hendra viruses are highly pathogenic, zoonotic henipaviruses that encode proteins that inhibit the host’s innate immune response. The W protein is one of four products encoded from the P gene and binds a number of host proteins to regulate signalling pathways. The W protein is intrinsically disordered, a structural attribute that contributes to its diverse host protein interactions. Here, we review the role of W in innate immune suppression through inhibition of both pattern recognition receptor (PRR) pathways and interferon (IFN)-responsive signalling. PRR stimulation leading to activation of IRF-3 and IFN release is blocked by henipavirus W, and unphosphorylated STAT proteins are sequestered within the nucleus of host cells by W, thereby inhibiting the induction of IFN stimulated genes. We examine the critical role of nuclear transport in multiple functions of W and how specific binding of importin-alpha (Impα) isoforms, and the 14-3-3 group of regulatory proteins suggests further modulation of these processes. Overall, the disordered nature and multiple functions of W warrant further investigation to understand henipavirus pathogenesis and may reveal insights aiding the development of novel therapeutics.

Inflammaging as the basis of age-associated diseases

Aging is one of the most complex biological phenomena that affects all human physiological systems, including the immune system. Immunosenescence is understood as structural and functional changes in both adaptive and innate immunity systems. The so-called inflammaging is among manifestations of immune aging. It is an age-related increase in inflammatory mediators and development of an inflammatory phenotype. An important role in development of inflammaging is assigned to chronic stimulation of immune system by exogenous and endogenous danger signals (pathogen-associated molecular pattern, PAMP and damage-associated molecular pattern, DAMP), which include viruses, microbiota of the gastrointestinal tract, free radicals, etc. PAMP and DAMP are recognized by the innate immunity system cells through the pattern recognition receptors (PRR), e.g., Toll-like receptors (TLR), RIG-I-like receptors (RLR), NODlike receptors (NLR), lectin receptors. Stimulation of PRR leads to activation of intracellular signaling and increased expression of pro-inflammatory factors. PAMPs are the most powerful activators of PRR and inflammation triggers; DAMPs can activate the same receptors and signaling pathways, causing the development of a sterile inflammatory response. The NF-kB signaling pathway is considered as a key signaling pathway for inflammaging. NLR stimulation also leads to formation of inflammasome. Its function is to transform the pro-inflammatory cytokines to a biologically active form, which is an important for the formation of a pro-inflammatory phenotype and development of inflammaging. This process is considered an important risk factor for morbidity and mortality among older people. Chronic inflammation underlies pathogenesis of many age-related diseases, such as osteoporosis, atherosclerosis, Alzheimer’s disease, Parkinson’s disease, type 2 diabetes. Various chronic diseases associated with age are directly related to PAMP and DAMP-induced TLR or NLRP3-mediated inflammatory response. Hence, these ligands and their receptors can be suggested as biomarkers and interventional targets for age-related disorders. Despite numerous studies in age-associated pathology, there are only few works on the contribution of innate immunity in healthy aging. It remains unclear whether the inflammatory phenotype is a manifestation of healthy aging, or it is associated with development of age-related pathology. Further study of the mechanisms of inflammatory aging will reveal biomarkers of healthy aging and potential targets for the treatment of age-associated diseases.

Antifibrotics Modify B-cell Induced Fibroblast Migration and Activation in IPF Patients

Abstract Background Acute respiratory exacerbations in idiopathic pulmonary fibrosis (IPF), often triggered by infection, are life threatening events characterized by rapid deterioration of lung function and associated with poor prognosis. Since microbial antigens modulate immunity by activating different pattern recognition receptors (PRR) expressed by innate immune cells (including B-cells), we sought to investigate how the activation of human B-cells through PRR stimulation by different microbial (CpG and β-glucan) and non-microbial (Mitochondrial DAMPs; MTDs) antigens resulted in the release of an inflammatory/fibrotic milieu that contributed to the pathogenesis of fibrosis in patients with IPF. Results Our results show that an inflammatory milieu is induced by stimulating circulating B-cells with both noninfectious (MTDs) and infectious antigens (CpG and β-glucan) commonly found in bacteria and fungi. Moreover, we show that the inflammatory milieu produced was specific to the inducing antigen, and that the microbial antigens (CpG and β-glucan) triggered distinct signaling pathways in B-cells. Specifically, CpG induced mTOR-activation while β-glucan was mTOR-independent and activated Src and p38. Furthermore, our results also show that B-cell aggregates are present within fibrotic areas of the lung in IPF patients and activated B-cells induce fibroblast migration, which was modified by antifibrotics (nintedanib and pirfenidone). Conclusion Our results highlight the potential contribution of activated B-cells through PRRs to the proinflammatory and profibrotic milieu seen in patients with idiopathic pulmonary fibrosis.

Symmetric Arginine Dimethylation Is Selectively Required for mRNA Splicing and the Initiation of Type I and Type III Interferon Signaling

Alternative splicing is well understood to enhance proteome diversity as cells respond to stimuli. However, mechanistic understanding for how the spliceosome processes precursor messenger RNA (mRNA) transcripts to achieve template diversification is incomplete. We use recently developed enzymatic inhibitors of protein arginine methyltransferase 5 (PRMT5) and human naive T lymphocyte activation as a model system to uncover a precise set of mRNA transcripts that require symmetric arginine dimethylation. This methylation-dependent splicing selectivity is associated with a limited set of signaling pathways that are affected when PRMT5 is inhibited. Specifically, we identify a conserved role for symmetric arginine dimethylation in the induction of antiviral type I and type III interferon signaling following Tcell receptor and pattern recognition receptor stimulation in human T lymphocytes and undifferentiated human THP-1 monocytes. Altogether, these findings reveal a mechanism by which cells may be enabled toprecisely modulate transcript heterogeneity to orchestrate specific functional outcomes.

Twist1 and Twist2 Induce Human Macrophage Memory upon Chronic Innate Receptor Treatment by HDAC-Mediated Deacetylation of Cytokine Promoters.

Intestinal tissues are continuously exposed to microbial products that stimulate pattern-recognition receptors (PRRs). Ongoing PRR stimulation can confer epigenetic changes in macrophages, which can then regulate subsequent immune outcomes and adaptation to the local environment. Mechanisms leading to these changes are incompletely understood. We found that short-term stimulation of the PRR NOD2 in primary human monocyte-derived macrophages resulted in increased H3 and H4 acetylation of cytokine promoters, consistent with the increased cytokine secretion observed. However, with prolonged NOD2 stimulation, both the acetylation and cytokine secretion were dramatically decreased. Chronic NOD2 stimulation upregulated the transcription factors Twist1 and Twist2, which bound to the promoters of the histone deacetylases HDAC1 and HDAC3 and induced HDAC1 and HDAC3 expression. HDAC1 and HDAC3 then mediated histone deacetylation at cytokine promoters and, in turn, cytokine downregulation under these conditions. Similar regulation was observed upon chronic stimulation of multiple PRRs. Consistent with the chronic microbial exposure in the intestinal environment, TWIST1, TWIST2, HDAC1, and HDAC3 were upregulated in human intestinal relative to peripheral macrophages. Importantly, complementing HDAC1 and HDAC3 in Twist1/Twist2-deficient monocyte-derived macrophages restored the reduced histone acetylation on cytokine promoters and the decreased cytokine secretion with chronic NOD2 stimulation. Taken together, we identify mechanisms wherein Twist1 and Twist2 promote chromatin modifications, resulting in macrophage instruction and adaptation to conditions in the intestinal microenvironment.

IRF1 Maintains Optimal Constitutive Expression of Antiviral Genes and Regulates the Early Antiviral Response.

Viral defense at mucosal sites depends on interferons (IFN) and IFN stimulated genes (ISGs), either of which may be constitutively expressed to maintain an “antiviral state” (AVS). However, the mechanisms that govern the AVS are poorly defined. Using a BEAS-2B respiratory epithelial cell line deficient in IRF1, we demonstrate higher susceptibility to infection with vesicular stomatitis virus (VSV) and influenza virus. IRF1-mediated restriction of VSV is IFN-independent, as blockade of types I and III IFNs and JAK-STAT signaling before infection did not affect VSV infection of either parent or IRF1 KO cells. Transcriptome analysis revealed that IRF1 regulates constitutive expression of ~300 genes, including antiviral ISGs: OAS2, BST2, and RNASEL and knockdown of any of these IRF1-dependent genes increased VSV infection. Additionally, IRF1 enhances rapid expression of IFNβ and IFNλ after stimulation with poly I:C and also regulates ISG expression. Mechanistically, IRF1 enhances recruitment of BRD4 to promotor-enhancer regions of ISGs for rapid expression and maintains levels of histone H3K4me1 for optimal constitutive expression. Finally, IRF1 also regulates constitutive expression of TLR2 and TLR3 and promotes signaling through these pattern recognition receptors (PRR). These data reveal multiple roles for IRF1 toward effective anti-viral responses by maintaining IFN-independent constitutive expression of anti-viral ISGs and supporting early IFN-dependent responses to PRR stimulation.

Quantifying Human Innate Cytokine and Chemokine Responses Ex Vivo via Pattern Recognition Receptor Stimulation.

Linkages between human innate immune capacity, the environment in which we live, and the development of clinical tolerance versus a spectrum of disease phenotypes are a major focus of inflammatory disease research. While extensive epidemiologic evidence indicates key roles for the microbiome and other environmental factors, the underlying mechanisms that explain how these stimuli lead to a given clinical phenotype remain speculative. Here we review strategies for characterizing human cytokine production ex vivo in response to innate immune receptor stimulation with defined ligands. Human cytokine and chemokine biomarker data provides a tool to test hypotheses on the relationship between innate immune capacity in vivo and expression of current or future clinical phenotypes. The most important limitations of experimental strategies that have been used to date are reviewed. Detailed experimental protocols are provided for characterization of pattern recognition receptor (PRR)-driven stimulation with a panel of bacterial (TLR4, TLR5) and viral (TLR3, TLR7/8, RIG-I/MDA5) ligands to assess the role played by human pro-inflammatory, anti-inflammatory, Th1-like, and Th2-like responses. The importance of characterizing human innate immune phenotypes extends beyond discovery-based research to development of improved strategies for prevention or inhibition of chronic inflammatory diseases, improved design of immunization programs, and more effective cancer immunotherapy.

Cocaine evokes a profile of oxidative stress and impacts innate antiviral response pathways in astrocytes

HIV-1 and Zika virus (ZIKV) represent RNA viruses with neurotropic characteristics. Infected individuals suffer neurocognitive disorders aggravated by environmental toxins, including drugs of abuse such as cocaine, exacerbating HIV-associated neurocognitive disorders through a combination of astrogliosis, oxidative stress and innate immune signaling; however, little is known about how cocaine impacts the progression of ZIKV neural perturbations. Impaired innate immune signaling is characterized by weakened antiviral activation of interferon signaling and alterations in inflammatory signaling, factors contributing to cognitive sequela associated with cocaine in HIV-1/ZIKV infection. We employed cellular/molecular biology techniques to test if cocaine suppresses the efficacy of astrocytes to initiate a Type 1 interferon response to HIV-1/ZIKV, in vitro. We found cocaine activated antiviral signaling pathways and type I interferon in the absence of inflammation. Cocaine pre-exposure suppressed antiviral responses to HIV-1/ZIKV, triggering antiviral signaling and phosphorylation of interferon regulatory transcription factor 3 to stimulate type I interferon gene transcription. Our data indicate that oxidative stress is a major driver of cocaine-mediated astrocyte antiviral immune responses. Although astrocyte antiviral signaling is activated following detection of foreign pathogenic material, oxidative stress and increased cytosolic double-stranded DNA (dsDNA) can drive antiviral signaling via stimulation of pattern recognition receptors. Pretreatment with the glial modulators propentofylline (PPF) or pioglitazone (PIO) reversed cocaine-mediated attenuation of astrocyte responses to HIV-1/ZIKV. Both PPF/PIO protected against cocaine-mediated generation of reactive oxygen species (ROS), increased dsDNA, antiviral signaling pathways and increased type I interferon, indicating that cocaine induces astrocyte type I interferon signaling in the absence of virus and oxidative stress is a major driver of cocaine-mediated astrocyte antiviral immunity. Lastly, PPF and PIO have therapeutic potential to ameliorate cocaine-mediated dysregulation of astrocyte antiviral immunity possibly via a myriad of protective actions including decreases in reactive phenotype and damaging immune factors.

Mitochondrial DNA as an inflammatory mediator in cardiovascular diseases.

Mitochondria play a central role in multiple cellular functions, including energy production, calcium homeostasis, and cell death. Currently, growing evidence indicates the vital roles of mitochondria in triggering and maintaining inflammation. Chronic inflammation without microbial infection — termed sterile inflammation — is strongly involved in the development of heart failure. Sterile inflammation is triggered by the activation of pattern recognition receptors (PRRs) that sense endogenous ligands called damage-associated molecular patterns (DAMPs). Mitochondria release multiple DAMPs including mitochondrial DNA, peptides, and lipids, which induce inflammation via the stimulation of multiple PRRs. Among the mitochondrial DAMPs, mitochondrial DNA (mtDNA) is currently highlighted as the DAMP that mediates the activation of multiple PRRs, including Toll-like receptor 9, Nod-like receptors, and cyclic GMP–AMP synthetase/stimulator of interferon gene pathways. These PRR signalling pathways, in turn, lead to the activation of nuclear factor-κB and interferon regulatory factor, which enhances the transcriptional activity of inflammatory cytokines and interferons, and induces the recruitment of inflammatory cells. As the heart is an organ comprising abundant mitochondria for its ATP consumption (needed to maintain constant cyclic contraction and relaxation), the generation of massive amounts of mitochondrial radical oxygen species and mitochondrial DAMPs are predicted to occur and promote cardiac inflammation. Here, we will focus on the role of mtDNA in cardiac inflammation and review the mechanism and pathological significance of mtDNA-induced inflammatory responses in cardiac diseases.

Regulation of Cytokine Production by the Unfolded Protein Response; Implications for Infection and Autoimmunity.

Protein folding in the endoplasmic reticulum (ER) is an essential cell function. To safeguard this process in the face of environmental threats and internal stressors, cells mount an evolutionarily conserved response known as the unfolded protein response (UPR). Invading pathogens induce cellular stress that impacts protein folding, thus the UPR is well situated to sense danger and contribute to immune responses. Cytokines (inflammatory cytokines and interferons) critically mediate host defense against pathogens, but when aberrantly produced, may also drive pathologic inflammation. The UPR influences cytokine production on multiple levels, from stimulation of pattern recognition receptors, to modulation of inflammatory signaling pathways, and the regulation of cytokine transcription factors. This review will focus on the mechanisms underlying cytokine regulation by the UPR, and the repercussions of this relationship for infection and autoimmune/autoinflammatory diseases. Interrogation of viral and bacterial infections has revealed increasing numbers of examples where pathogens induce or modulate the UPR and implicated UPR-modulated cytokines in host response. The flip side of this coin, the UPR/ER stress responses have been increasingly recognized in a variety of autoimmune and inflammatory diseases. Examples include monogenic disorders of ER function, diseases linked to misfolding protein (HLA-B27 and spondyloarthritis), diseases directly implicating UPR and autophagy genes (inflammatory bowel disease), and autoimmune diseases targeting highly secretory cells (e.g., diabetes). Given the burgeoning interest in pharmacologically targeting the UPR, greater discernment is needed regarding how the UPR regulates cytokine production during specific infections and autoimmune processes, and the relative place of this interaction in pathogenesis.

Improvement of In Vivo Expression of Genes Delivered by Self-Amplifying RNA Using Vaccinia Virus Immune Evasion Proteins.

Among nucleic acid–based delivery platforms, self-amplifying RNA (saRNA) vectors are of increasing interest for applications such as transient expression of recombinant proteins and vaccination. saRNA is safe and, due to its capability to amplify intracellularly, high protein levels can be produced from even minute amounts of transfected templates. However, it is an obstacle to full exploitation of this platform that saRNA induces a strong innate host immune response. In transfected cells, pattern recognition receptors sense double-stranded RNA intermediates and via activation of protein kinase R (PKR) and interferon signaling initiate host defense measures including a translational shutdown. To reduce pattern recognition receptor stimulation and unleash suppressed saRNA translation, this study co-delivered non-replicating mRNA encoding vaccinia virus immune evasion proteins E3, K3, and B18. It was shown that E3 is far superior to K3 or B18 as a highly potent blocker of PKR activation and of interferon (IFN)-β upregulation. B18, in contrast, is superior in controlling OAS1, a key IFN-inducible gene involved in viral RNA degradation. By combining all three vaccinia proteins, the study achieved significant suppression of PKR and IFN pathway activation in vitro and enhanced expression of saRNA-encoded genes of interest both in vitro and in vivo. This approach promises to overcome key hurdles of saRNA gene delivery. Its application may improve the bioavailability of the encoded protein, and reduce the effective dose and correspondingly the cost of goods of manufacture in the various fields where saRNA utilization is envisioned.

Transflammation: Innate immune signaling in nuclear reprogramming.

Induction of pluripotency in somatic cells by retroviral overexpression of four transcription factors has revolutionized the field of stem cell biology and regenerative medicine. The efficient induction of pluripotency requires the activation of innate immune signaling in a process termed "transflammation" (Lee et al., 2012). Specifically, the stimulation of pattern recognition receptors (PRRs) causes global alterations in the expression and activity of epigenetic modifiers to favor an open chromatin configuration. Activation of toll-like receptors (TLR) or RIG-1-like receptors (RLR) (Sayed et al. 2017) trigger signaling cascades that result in NFκB or IRF-3 mediated changes in epigenetic plasticity that facilitate reprogramming. Another form of nuclear reprogramming is so-called direct reprogramming or transdifferentiation of one somatic cell to another lineage. We have shown that transdifferentiation of human fibroblasts to endothelial cells also involves transflammation (Sayed et al., 2015). Recently, we also identified reactive oxygen species (ROS) (Zhou et al. 2016) and reactive nitrogen species (RNS) (Meng et al., 2016) as mediators of innate immune signaling in nuclear reprogramming. Innate immune signaling plays a key role in nuclear reprogramming by regulating DNA accessibility (Fig. 1). Here, we review recent progress of innate immunity signaling in nuclear reprogramming and epigenetic plasticity.

Lung epithelial cells: therapeutically inducible effectors of antimicrobial defense.

Lung epithelial cells are increasingly recognized to be active effectors of microbial defense, contributing to both innate and adaptive immune function in the lower respiratory tract. As immune sentinels, lung epithelial cells detect diverse pathogens through an ample repertoire of membrane-bound, endosomal, and cytosolic pattern-recognition receptors (PRRs). The highly plastic epithelial barrier responds to detected threats via modulation of paracellular flux, intercellular communications, mucin production, and periciliary fluid composition. Epithelial PRR stimulation also induces production of cytokines that recruit and sculpt leukocyte-mediated responses, and promotes epithelial generation of antimicrobial effector molecules that are directly microbicidal. The epithelium can alternately enhance tolerance to pathogens, preventing tissue damage through PRR-induced inhibitory signals, opsonization of pathogen-associated molecular patterns, and attenuation of injurious leukocyte responses. The inducibility of these protective responses has prompted attempts to therapeutically harness epithelial defense mechanisms to protect against pneumonias. Recent reports describe successful strategies for manipulation of epithelial defenses to protect against a wide range of respiratory pathogens. The lung epithelium is capable of both significant antimicrobial responses that reduce pathogen burdens and tolerance mechanisms that attenuate immunopathology. This manuscript reviews inducible lung epithelial defense mechanisms that offer opportunities for therapeutic manipulation to protect vulnerable populations against pneumonia.