Innate Pattern Recognition Receptors Research Articles

Immune responses to retinal gene therapy using adeno-associated viral vectors - Implications for treatment success and safety.

Recombinant adeno-associated virus (AAV) is the leading vector for gene therapy in the retina. As non-pathogenic, non-integrating, replication deficient vector, the recombinant virus efficiently transduces all key retinal cell populations. Successful testing of AAV vectors in clinical trials of inherited retinal diseases led to the recent approval of voretigene neparvovec (Luxturna) for the treatment of RPE65 mutation-associated retinal dystrophies. However, studies applying AAV-mediated retinal gene therapy independently reported intraocular inflammation and/or loss of efficacy after initial functional improvements. Both observations might be explained by targeted removal of transduced cells via anti-viral defence mechanisms. AAV has been shown to activate innate pattern recognition receptors (PRRs) such as toll-like receptor (TLR)-2 and TLR-9 resulting in the release of inflammatory cytokines and type I interferons. The vector can also induce capsid-specific and transgene-specific T cell responses and neutralizing anti-AAV antibodies which both limit the therapeutic effect. However, the target organ of retinal gene therapy, the eye, is known as an immune-privileged site. It is characterized by suppression of inflammation and promotion of immune tolerance which might prevent AAV-induced immune responses. This review evaluates AAV-related immune responses, toxicity and inflammation in studies of retinal gene therapy, identifies influencing variables of these responses and discusses potential strategies to modulate immune reactions to AAV vectors to increase the safety and efficacy of ocular gene therapy.

Bioactive Plasma Mitochondrial DNA Is Associated With Disease Progression in Scleroderma-Associated Interstitial Lung Disease.

Systemic sclerosis-associated interstitial lung disease (SSc-ILD) is characterized by variable clinical outcomes, activation of innate immune pattern-recognition receptors (PRRs), and accumulation of α-smooth muscle actin (α-SMA)-expressing myofibroblasts. The aim of this study was to identify an association between these entities and mitochondrial DNA (mtDNA), an endogenous ligand for the intracellular DNA-sensing PRRs Toll-like receptor 9 (TLR-9) and cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING), which has yet to be determined. Human lung fibroblasts (HLFs) from normal donors and SSc-ILD explants were treated with synthetic CpG DNA and assayed for α-SMA expression and extracellular mtDNA using quantitative polymerase chain reaction for the human MT-ATP6 gene. Plasma MT-ATP6 concentrations were evaluated in 2 independent SSc-ILD cohorts and demographically matched controls. The ability of SSc-ILD and control plasma to induce TLR-9 and cGAS/STING activation was evaluated with commercially available HEK 293 reporter cells. Plasma concentrations of type I interferons (IFNs), interleukin-6 (IL-6), and oxidized DNA were measured using electrochemiluminescence and enzyme-linked immunosorbent assay-based methods. Extracellular vesicles (EVs) precipitated from plasma were evaluated for MT-ATP6 concentrations and proteomics via liquid chromatography mass spectrometry. Normal HLFs and SSc-ILD fibroblasts developed increased α-SMA expression and MT-ATP6 release following CpG stimulation. Plasma mtDNA concentrations were increased in the 2 SSc-ILD cohorts, reflective of ventilatory decline, and were positively associated with both TLR-9 and cGAS/STING activation as well as type I IFN and IL-6 expression. Plasma mtDNA was not oxidized and was conveyed by EVs displaying a proteomics profile consistent with a multicellular origin. These findings demonstrate a previously unrecognized connection between EV-encapsulated mtDNA, clinical outcomes, and intracellular DNA-sensing PRR activation in SSc-ILD. Further study of these interactions could catalyze novel mechanistic and therapeutic insights into SSc-ILD and related disorders.

Host Innate Immune Responses to Acinetobacter baumannii Infection.

Acinetobacter baumannii has emerged as a major threat to global public health and is one of the key human pathogens in healthcare (nosocomial and community-acquired)-associated infections. Moreover, A. baumannii rapidly develops resistance to multiple antibiotics and is now globally regarded as a serious multidrug resistant pathogen. There is an urgent need to develop novel vaccines and immunotherapeutics as alternatives to antibiotics for clinical management of A. baumannii infection. However, our knowledge of host immune responses to A. baumannii infection and the identification of novel therapeutic targets are significantly lacking. This review highlights the recent advances and critical gaps in our understanding how A. baumannii interacts with the host innate pattern-recognition receptors, induces a cascade of inflammatory cytokine and chemokine responses, and recruits innate immune effectors (such as neutrophils and macrophages) to the site of infection for effective control of the infection. Such knowledge will facilitate the identification of new targets for the design and development of effective therapeutics and vaccines to fight this emerging threat.

Overview of Research Development on the Role of NF-κB Signaling in Mastitis.

Simple SummaryNF-κB signaling has been widely studied for its role in inflammatory and immunity-related diseases. Mastitis is considered one of the inflammatory and immunity associated diseases which are a serious threat to the global dairy industry. Having such a critical role in immunity and inflammation, NF-κB signaling is currently under target for therapeutic purposes in mastitis control research. The virulent factor, lipopolysaccharides (LPS), of bacteria after attachment with relevant Toll-like receptors (TLRs) on mammary epithelial cells starts its pathogenesis by using NF-κB signaling to cause mastitis. Several studies have proved that the blocking of NF-κB signaling could be a useful strategy for mastitis control.Mastitis is the inflammation of the mammary gland. Escherichia coli and Staphylococcus aureus are the most common bacteria responsible for mastitis. When mammary epithelial cells are infected by microorganisms, this activates an inflammatory response. The bacterial infection is recognized by innate pattern recognition receptors (PRRs) in the mammary epithelial cells, with the help of Toll-like receptors (TLRs). Upon activation by lipopolysaccharides, a virulent agent of bacteria, the TLRs further trigger nuclear factor-κB (NF-κB) signaling to accelerate its pathogenesis. The NF-κB has an essential role in many biological processes, such as cell survival, immune response, inflammation and development. Therefore, the NF-κB signaling triggered by the TLRs then regulates the transcriptional expression of specific inflammatory mediators to initiate inflammation of the mammary epithelial cells. Thus, any aberrant regulation of NF-κB signaling may lead to many inflammatory diseases, including mastitis. Hence, the inhibiting of NF-κB signaling has potential therapeutic applications in mastitis control strategies. In this review, we highlighted the regulation and function of NF-κB signaling in mastitis. Furthermore, the role of NF-κB signaling for therapeutic purposes in mastitis control has been explored in the current review.

Pellino1 promotes chronic inflammatory skin disease via keratinocyte hyperproliferation and induction of the T helper 17 response

Psoriasis is one of the most common immune-mediated chronic inflammatory skin diseases. However, little is known about the molecular mechanism underlying the immunological circuits that maintain innate and adaptive immune responses in established psoriasis. In this study, we found that the Pellino1 (Peli1) ubiquitin E3 ligase is activated by innate pattern-recognition receptors (PRRs), such as Toll-like receptors (TLRs), and is highly upregulated in human psoriatic skin lesions and murine psoriasis-like models. Increased Peli1 expression is strongly correlated with the immunopathogenesis of psoriasis by activating hyperproliferation of keratinocytes in the S and G2/M phases of the cell cycle and promoting chronic skin inflammation. Furthermore, Peli1-induced psoriasis-like lesions showed significant changes in the expression levels of several T helper 17 (Th17)-related cytokines, such as IL-17a, IL-21, IL-22, IL-23, and IL-24, indicating that overexpression of Peli1 resulted in the sequential engagement of the Th17 cell response. However, the overexpression of Peli1 in T cells was insufficient to trigger psoriasis, while T cells were indispensable for disease manifestation. In summary, our findings demonstrate that Peli1 is a critical cell cycle activator of innate immunity, which subsequently links Th17 cell immune responses to the psoriatic microenvironment.

Loss of Function Genetic Screen Identifies ATM Kinase as a Positive Regulator of TLR3-Mediated NF-κB Activation.

SummaryTLR3, a major innate immune pattern recognition receptor of RNA viruses, triggers inflammatory response through the transcription factor NF-κB. However, a genome-wide understanding of the genes and mechanisms regulating TLR3-mediated NF-κB activation is incomplete. We herein report the results of a human genome-wide RNAi screen that identified 591 proteins regulating TLR3-mediated NF-κB response. Bioinformatics analysis revealed several signaling modules including linear ubiquitination assembly complex and mediator protein complex network as regulators of TLR3 signaling. We further characterized the kinase ATM as a previously unknown positive regulator of TLR3 signaling. TLR3 pathway stimulation induced ATM phosphorylation and promoted interaction of ATM with TAK1, NEMO, IKKα, and IKKβ. Furthermore, ATM was determined to coordinate the assembly of NEMO with TAK1, IKKα, and IKKβ during TLR3 signaling. This study provided a comprehensive understanding of TLR3-mediated inflammatory signaling regulation and established a role for ATM in innate immune response.

CARD9 mediates T cell inflammatory response in Coxsackievirus B3-induced acute myocarditis

Cardiac inflammation in Coxsackievirus B3 (CVB3)-induced myocarditis is a consequence of viral-related cardiac injury and immune response. Caspase-associated recruitment domain 9 (CARD9) is a critical adaptor protein involved in transduction of signals from various innate pattern recognition receptors. In this study, the role of CARD9 in acute viral myocarditis was evaluated. CARD9−/− and C57BL/6 mice were infected with CVB3. On day 7 postinfection, myocardial tissue and blood samples were collected and examined. After CARD9 knockout, mRNA and protein levels of transforming growth factor-β(TGF-β), interleukin-17A(IL-17A), and CARD domain of B-cell CLL/lymphoma 10(BCL-10) in the myocardium were markedly lower in CARD9−/− mice than in C57BL/6 mice with CVB3-induced viral myocarditis. This trend was similar for the pathological scores for inflammation and serum levels of cytokines interleukin-6(IL-6), interleukin-10(IL-10), interferon -γ(IFN-γ), TGF-β, and IL-17A. These results suggest that the CARD9-mediated secretion of pro-inflammatory cytokines plays an important role in the immune response to acute viral myocarditis.

The Mechanism of Contrast-Induced Acute Kidney Injury and Its Association with Diabetes Mellitus.

Contrast-induced acute kidney injury (CI-AKI) is the third most common hospital-acquired AKI after AKI induced by renal perfusion insufficiency and nephrotoxic drugs, taking great adverse effects on the prognosis and increasing hospital stay and medical cost. Diabetes nephropathy (DN) is a common chronic complication of DM (diabetes mellitus), and DN is an independent risk factor for chronic kidney disease (CKD) and CI-AKI. The incidence of CI-AKI significantly increases in patients with renal injury, especially in DM-related nephropathy. The etiology of CI-AKI is not fully clear, and research studies on how DM becomes a facilitated factor of CI-AKI are limited. This review describes the mechanism from three aspects. ① Pathophysiological changes of CI-AKI in kidney under high-glucose status (HGS). HGS can enhance the oxidative stress and increase ROS which next causes stronger vessel constriction and insufficient oxygen supply in kidney via vasoactive substances. HGS also aggravates some ion pump load and the latter increases oxygen consumption. CI-AKI and HGS are mutually causal, making the kidney function continue to decline. ② Immunological changes of DM promoting CI-AKI. Some innate immune cells and pattern recognition receptors (PRRs) in DM and/or DN may respond to some damage-associated molecular patterns (DAMPs) formed by CI-AKI. These effects overlap with some pathophysiological changes in hyperglycemia. ③ Signaling pathways related to both CI-AKI and DM. These pathways involved in CI-AKI are closely associated with apoptosis, inflammation, and ROS production, and some studies suggest that these pathways may be potential targets for alleviating CI-AKI. In conclusion, the pathogenesis of CI-AKI and the mechanism of DM as a predisposing factor for CI-AKI, especially signaling pathways, need further investigation to provide new clinical approaches to prevent and treat CI-AKI.

Innate Immune Response against Hepatitis C Virus: Targets for Vaccine Adjuvants.

Despite successful treatments, hepatitis C virus (HCV) infections continue to be a significant world health problem. High treatment costs, the high number of undiagnosed individuals, and the difficulty to access to treatment, particularly in marginalized susceptible populations, make it improbable to achieve the global control of the virus in the absence of an effective preventive vaccine. Current vaccine development is mostly focused on weakly immunogenic subunits, such as surface glycoproteins or non-structural proteins, in the case of HCV. Adjuvants are critical components of vaccine formulations that increase immunogenic performance. As we learn more information about how adjuvants work, it is becoming clear that proper stimulation of innate immunity is crucial to achieving a successful immunization. Several hepatic cell types participate in the early innate immune response and the subsequent inflammation and activation of the adaptive response, principally hepatocytes, and antigen-presenting cells (Kupffer cells, and dendritic cells). Innate pattern recognition receptors on these cells, mainly toll-like receptors, are targets for new promising adjuvants. Moreover, complex adjuvants that stimulate different components of the innate immunity are showing encouraging results and are being incorporated in current vaccines. Recent studies on HCV-vaccine adjuvants have shown that the induction of a strong T- and B-cell immune response might be enhanced by choosing the right adjuvant.

The innate immune pattern-recognition receptor CD14 influences osteoclastogenesis and the persistence of synovial leukocyte infiltration after joint injury in mice

Purpose: CD14 is a macrophage co-receptor that facilitates Toll-like receptor signaling. Deficiency of CD14 protects mice from subchondral bone pathology and progression of cartilage degeneration in a murine model of post-traumatic OA. CD14 is highly expressed by myeloid cells including monocytes, macrophages, and osteoclast precursors and can influence activity of these cell types. Binding of TLR ligands to the CD14/TLR complex activates inflammatory signaling cascades and promotes chronic inflammation, and many TLR ligands have been implicated in OA pathogenesis.

Inter-relation analysis of signaling adaptors of porcine innate immune pathways

To study the interrelationship between the signaling adaptors of innate pattern recognition receptor (PRR) pathways including toll-like receptor (TLR), retinoic acid-inducible gene-1-like receptor (RLR), nucleotide-binding oligomerization domain-like receptor (NLR), and cytoplasmic DNA recognition receptors (CDR) pathways. The coding genes of porcine TRIF, MAVS, STING, MyD88, RIPK2, and ASC were isolated from PK15 cells. Phylogenetic analysis of the six adaptor proteins in pig, cattle, goat, horse, human, mouse, chicken, and duck performed by MEGA 5.05 showed that these adaptors have slightly different similarity across species. The expression of these proteins in transfected cells were detected by both Western blotting and confocal microscopy. All six adaptors were visualized in cytoplasm but with different distribution patterns. The activities of the six adaptors triggering NF-κB and ISRE signaling and downstream gene productions were examined by dual-luciferase reporter assay and real-time RT-PCR, respectively. The results showed that STING has an ability to activate ISRE signaling, MyD88, RIPK2 and ASC possess NF-κB signal activity, while TRIF and MAVS can activate both. Furthermore, the mutual signaling effects were assessed by NF-κB and ISRE dual-luciferase reporter assay in the co-expression experiments. STING was shown to enhance MAVS activated NF-κB signaling and MyD88 could heighten STING activated ISRE signaling. However, all other adaptors inhibited each other to varying degrees. The work provides a global insight of porcine innate immune signaling pathways and their interaction network.

RHOA-mediated mechanical force generation through Dectin-1.

Dendritic cell-associated C-type lectin 1 (Dectin-1, also known as CLEC7A) is an innate immune pattern recognition receptor that recognizes β-glucan on the Candida albicans cell wall. Recognition of β-glucan by immune cells leads to phagocytosis, oxidative burst, cytokine and chemokine production. We looked for specific mechanisms that coordinate phagocytosis downstream of Dectin-1 leading to actin reorganization and internalization of fungus. We found that stimulation of Dectin-1 by soluble β-glucan leads to mechanical force generation and areal contraction in Dectin-1-transfected HEK-293 cells and M1 macrophages. With inhibitor studies, we found this force generation is a spleen tyrosine kinase (SYK)-independent, but SRC family kinase (SFK)-dependent process mediated through the RHOA-ROCK-myosin light chain (MLC) pathway. We confirmed activation of RHOA downstream of Dectin-1 using activity assays and stress fiber formation. Through phagocytosis assays, we found direct evidence for the importance of RHOA-ROCK-MLC signaling in the process of phagocytosis of C. albicans.

Genetic variants of innate immunity receptors are associated with mortality in cirrhotic patients with bacterial infection.

Background & AimsAcute‐on‐chronic liver failure (ACLF) is characterized by acute decompensation of cirrhosis (AD), organ failure(s) and high risk of short‐term mortality with bacterial infection frequently as precipitating event. Innate immune pattern recognition receptors and members of the lectin pathway of complement activation are crucial to the innate immune response to pathogens. The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) of innate immune components are associated with the occurrence of bacterial infections or mortality in patients with cirrhosis hospitalized for AD or ACLF.MethodsTwenty‐one innate immunity SNPs with known functional implications were genotyped in 826 AD/ACLF patients included in the CANONIC study. Associations between baseline characteristics of the patients, the occurrence of bacterial infections and survival rate at 90 days of follow‐up in relation to the innate immunity genetic variants were analysed.ResultsThe NOD2‐G908R genetic variant was associated with mortality (HR 2.25, P = .004) independently of age and MELD Score. This association was also found in a predefined subgroup analysis in patients with bacterial infections (HR 2.78, P < .001) along with MBL_Yx (HR 1.72, P = .008) and MASP2_371 (HR 1.67, P = .012) genetic variants. None of the analysed SNPs were significantly associated with the occurrence of acute bacterial infections or spontaneous bacterial peritonitis in particular.ConclusionsInnate immune system‐specific NOD2‐G908R, MBL_Yx and MASP2_371 genetic variants were independently associated with increased risk of short‐term mortality in AD/ACLF patients with bacterial infection.

Effect of TLR agonist on infections bronchitis virus replication and cytokine expression in embryonated chicken eggs

Avian infectious bronchitis (IB) is an acute, highly infectious and contagious viral disease of chickens caused by avian infectious bronchitis virus (IBV) belonging to the genus Coronavirus and family Coronaviridae. It can affect all age groups of birds. The toll-like receptors (TLRs) are a major class of innate immune pattern recognition receptors that have a key role in immune response and defense against various infections.The TLRs are essential for initiation of innate immune responses and in the development of adaptive immune responses. An in ovo model was employed to study the antiviral activity of TLR ligands (Pam3CSK4, LPS and CpG ODN) on replication of IBV. It was hypothesized that optimum dose and specific timing of TLR ligands may reduce viral load of IBV in specific pathogen free (SPF) embryonated chicken eggs (ECEs). Further, the mechanism involved in the TLR-mediated antiviral response in chorioallantoic membrane (CAM) of ECEs was investigated. The ECEs of 9–11 days old were treated with different doses (high, intermediate and low) of TLR-2 (Pam3CSK4), TLR-4 (LPS) and TLR-21 (CpG ODN) ligands. In addition, to know the timing of TLR ligand treatment, six time intervals were analyzed viz. 36, 24 and 12 h prior to infection, time of infection (co-administration of TLR ligands and avian IBV) and 12 and 24 h post-IBV infection. For studying the relative expression of immuno-stimulatory genes (IFN-α, IFN-β, IFN-γ, IL-1β, iNOS and OAS) in CAM, TLR ligands were administered through intra-allantoicroute and CAM were collected at 4, 8 and 16 h post treatment. The results demonstrated that intermediate dose of all the three TLR ligands significantly reduced virus titers and used in the present study. However, the LPS reduced virus titer pre- and post-IBV infection but Pam3CSK4 and CpG ODN reduced only pre-IBV infection. Further analysis showed that TLR ligands induced IFN-γ, IL-1β and IFN stimulated genes viz. iNOS and OAS genes in CAM. The present study pointed towards the novel opportunities for rational design of LPS as immuno-stimulatory agent in chickens with reference to IBV. It may be speculated that in ovo administration of these TLR ligands may enhance resistance against viral infection in neonatal chicken and may contribute towards the development of more effective and safer vaccines including in ovo vaccines.

Effect of Toll-like receptor 4 on depressive-like behaviors induced by chronic social defeat stress.

IntroductionA growing body of evidence suggests that stress is an important factor in depression, and pro‐inflammatory cytokines contribute to the occurrence and development of depression in both animal models and human patients. Toll‐like receptor 4 (TLR4) has been shown to be a key innate immune pattern recognition receptor involved in the regulation of stress responses and inflammation. However, the exact effects of TLR4 on depressive‐like behaviors induced by chronic social defeat stress (CSDS) are not known.MethodsIn this study, the effects of TLR4 on depressive‐like behaviors were investigated in an animal model of depression induced by CSDS. The depressive‐like behaviors were assessed by forced swimming test (FST), social interaction test (SIT), and light–dark box test (LDT). The protein expressions of TLR4 and tumor necrosis factor‐α (TNF‐α) in the hippocampus were measured using Western blotting.ResultsWe found that CSDS increased TLR4 protein levels in the hippocampus and induced behavioral despair in FST, social avoidance in SIT, and anxiety‐like behavior in LDT. Fluoxetine normalized the increased expression of TLR4 and reversed behavioral despair, social avoidance, as well as anxiety‐like behavior induced by CSDS. However, directly blocking TLR4, by using either TLR4 inhibitor TAK‐242 or knockout of TLR4, only inhibited behavioral despair, but not social avoidance or anxiety‐like behavior induced by CSDS.ConclusionsThese results demonstrate a specific modulating role of TLR4 in behavioral despair induced by CSDS and suggest that TAK‐242 may be a beneficial treatment for patients with behavioral despair.

Nod-like receptors are critical for gut-brain axis signalling in mice.

•Nucleotide binding oligomerization domain (Nod)-like receptors regulate cognition, anxiety and hypothalamic-pituitary-adrenal axis activation. •Nod-like receptors regulate central and peripheral serotonergic biology. •Nod-like receptors are important for maintenance of gastrointestinal physiology. •Intestinal epithelial cell expression of Nod1 receptors regulate behaviour. Gut-brain axis signalling is critical for maintaining health and homeostasis. Stressful life events can impact gut-brain signalling, leading to altered mood, cognition and intestinal dysfunction. In the present study, we identified nucleotide binding oligomerization domain (Nod)-like receptors (NLR), Nod1 and Nod2, as novel regulators for gut-brain signalling. NLR are innate immune pattern recognition receptors expressed in the gut and brain, and are important in the regulation of gastrointestinal physiology. We found that mice deficient in both Nod1 and Nod2 (NodDKO) demonstrate signs of stress-induced anxiety, cognitive impairment and depression in the context of a hyperactive hypothalamic-pituitary-adrenal axis. These deficits were coupled with impairments in the serotonergic pathway in the brain, decreased hippocampal cell proliferation and immature neurons, as well as reduced neural activation. In addition, NodDKO mice had increased gastrointestinal permeability and altered serotonin signalling in the gut following exposure to acute stress. Administration of the selective serotonin reuptake inhibitor, fluoxetine, abrogated behavioural impairments and restored serotonin signalling. We also identified that intestinal epithelial cell-specific deletion of Nod1 (VilCre+ Nod1f/f ), but not Nod2, increased susceptibility to stress-induced anxiety-like behaviour and cognitive impairment following exposure to stress. Together, these data suggest that intestinal epithelial NLR are novel modulators of gut-brain communication and may serve as potential novel therapeutic targets for the treatment of gut-brain disorders.

Toll-like receptor 2–dependent endosomal signaling by Staphylococcus aureus in monocytes induces type I interferon and promotes intracellular survival

Pathogen activation of innate immune pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) stimulates cellular signaling pathways. This often leads to outcomes that contribute to pathogen clearance. Alternatively, activation of specific PRR pathways can aid pathogen survival. The human pathogen Staphylococcus aureus is a case in point, employing strategies to escape innate immune recognition and killing by the host. As for other bacteria, PRR-stimulated type I interferon (IFN-I) induction has been proposed as one such immune escape pathway that may favor S. aureus. Cell wall components of S. aureus elicit TLR2-dependent cellular responses, but the exact signaling pathways activated by S. aureus–TLR2 engagement and the consequences of their activation for the host and bacterium are not fully known. We previously showed that TLR2 activates both a cytoplasmic and an endosome-dependent signaling pathway, the latter leading to IFN-I production. Here, we demonstrate that S. aureus infection of human monocytes activates a TLR2-dependent endosomal signaling pathway, leading to IFN-I induction. We mapped the signaling components of this pathway and identified roles in IFN-I stimulation for the Toll-interleukin-1 receptor (TIR) adaptor Myd88 adaptor-like (Mal), TNF receptor-associated factor 6 (TRAF6), and IκB kinase (IKK)-related kinases, but not for TRIF-related adaptor molecule (TRAM) and TRAF3. Importantly, monocyte TLR2-dependent endosomal signaling enabled immune escape for S. aureus, because this pathway, but not IFN-I per se, contributed to intracellular bacterial survival. These results reveal a TLR2-dependent mechanism in human monocytes whereby S. aureus manipulates innate immune signaling for its survival in cells.

Impairment of Th cell response in Card9 knockout mice with cutaneous mucormycosis caused by Rhizopus arrhizus.

Card9 is a signalling adaptor protein in the downstream of many innate pattern recognition receptors (PRRs) and exerts a significant role in antifungal immunity. To date, Card9 deficiency has been reported to be related to increased susceptibility to many fungal infections. In this study, we established mucormycosis murine model of Rhizopus arrhizus (R.arrhizus) using wild-type (WT) mice and Card9 knockout (Card9-/- ) mice to investigate the antifungal effect of Card9 against R.arrhizus infection. Card9-/- mice were more susceptible to R.arrhizus infection than WT mice, which could be related to the impaired NF-κB pathway activation, local cytokine production and Th cell responses in Card9-/- mice.

Toll-like receptor 2 stimulation augments esophageal barrier integrity.

Germline-encoded innate immune pattern recognition receptors (PRR) are expressed at epithelial surfaces and modulate epithelial defenses. Evidence suggests that stimulation of the Toll-like receptor (TLR) family of PRR may regulate epithelial barrier integrity by upregulating tight junction (TJ) complex protein expression, but it is not known whether this mechanism is utilized in esophageal epithelial cells. TJ complex proteins maintain intact barrier function and are dysregulated in atopic disorders including eosinophilic esophagitis. Pattern recognition receptors expression was assessed in EoE and control primary esophageal epithelial cells, demonstrating robust expression of TLR2 and TLR3. The three-dimensional air-liquid interface culture (ALI) model was used to test whether TLR2 or TLR3 stimulation alters epithelial barrier function using an in vitro model of human epithelium. Transepithelial electrical resistance (TEER) and FITC-Dextran permeability were evaluated to assess membrane permeability. ALI cultures were evaluated by histology, immunohistochemistry, Western blotting, and chromatin immunoprecipitation (ChIP). TLR3 stimulation did not change TEER in the ALI model. TLR2 stimulation increased TEER (1.28- to 1.31-fold) and decreased paracellular permeability to FITC-Dextran, and this effect was abolished by treatment with anti-TLR2 blocking antibody. TJ complex proteins claudin-1 and zonula occludens-1 were upregulated following TLR2 stimulation, and ChIP assay demonstrated altered histone 4 acetyl binding at the TJP1 enhancer and CLDN1 enhancer and promoter following zymosan treatment, implying the occurrence of durable chromatin changes. Our findings implicate the TLR2 pathway as a potential regulator of esophageal epithelial barrier function and suggest that downstream chromatin modifications are associated with this effect.

NOD2 signaling pathway is involved in fibronectin fragment-induced pro-catabolic factor expressions in human articular chondrocytes

The nucleotide-binding and oligomerization domain (NOD) is an innate pattern recognition receptor that recognizes pathogen- and damage-associated molecular patterns. The 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f) is a matrix degradation product found in the synovial fluids of patients with osteoarthritis (OA). We investigated whether NOD2 was involved in 29-kDa FN-f-induced pro-catabolic gene expression in human chondrocytes. The expression of mRNA and protein was measured using quantitative real-time polymerase chain reaction (qrt-PCR) and Western blot analysis. Small interfering RNAs were used for knockdown of NOD2 and toll-like receptor 2 (TLR-2). An immunoprecipitation assay was performed to examine protein interactions. The NOD2 levels in human OA cartilage were much higher than in normal cartilage. NOD1 and NOD2 expression, as well as pro-inflammatory cytokines, including interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α), were upregulated by 29-kDa FN-f in human chondrocytes. NOD2 silencing showed that NOD2 was involved in the 29-kDa FN-f-induced expression of TLR-2. Expressions of IL-6, IL-8, matrix metalloproteinase (MMP)-1, -3, and -13 were also suppressed by TLR-2 knockdown. Furthermore, NOD2 and TLR-2 knockdown data demonstrated that both NOD2 and TLR-2 modulated the expressions of their adaptors, receptor-interacting protein 2 (RIP2) and myeloid differentiation 88, in 29-kDa FN-f-treated chondrocytes. 29-kDa FN-f enhanced the interaction of NOD2, RIP2 and transforming growth factor beta-activated kinase 1 (TAK1), an indispensable signaling intermediate in the TLR-2 signaling pathway, and activated nuclear factor-κB (NF-κB), subsequently leading to increased expressions of pro-inflammatory cytokines and cartilage-degrading enzymes. These results demonstrate that 29-kDa FN-f modulated pro-catabolic responses via cross-regulation of NOD2 and TLR-2 signaling pathways.