Activation Of Toll-like Receptor Pathways Research Articles

MDSC-derived S100A8/9 contributes to lupus pathogenesis by promoting TLR7-mediated activation of macrophages and dendritic cells

Toll-like receptors (TLRs), especially TLR7, play an important role in systemic lupus erythematosus (SLE) pathogenesis. However, the regulatory mechanism underlying the abnormal activation of TLR pathways in patients with SLE has not been elucidated. Notably, accumulating evidence indicates that myeloid-derived suppressor cells (MDSCs) are important regulators of inflammation and autoimmune diseases. Compared with healthy control subjects, patients with SLE have a greater proportion of MDSCs among peripheral blood mononuclear cells (PBMCs); however, the effect of MDSCs on TLR7 pathway activation has not been determined. In the present study, lupus MDSCs significantly promoted TLR7 pathway activation in macrophages and dendritic cells (DCs), exacerbating the imiquimod-induced lupus model. RNA-sequencing analysis revealed significant overexpression of S100 calcium-binding protein A8 (S100A8) and S100A9 in MDSCs from diseased MRL/lpr mice. In vitro and in vivo studies demonstrated that S100A8/9 effectively promoted TLR7 pathway activation and that S100A8/9 deficiency reversed the promoting effect of MDSCs on TLR7 pathway activation in lupus. Mechanistically, MDSC-derived S100A8/9 upregulated interferon gamma (IFN-γ) secretion by macrophages and IFN-γ subsequently promoted TLR7 pathway activation in an autocrine manner. Taken together, these findings suggest that lupus MDSCs promote TLR7 pathway activation and lupus pathogenesis through the S100A8/9-IFN-γ axis. Our study identified an important target for SLE therapy.

The role of toll-like receptors in orchestrating osteogenic differentiation of mesenchymal stromal cells and osteoimmunology

Osteoimmunology is a concept involving molecular and cellular crosstalk between the skeletal and immune systems. Toll-like receptors (TLRs) are widely expressed both on mesenchymal stromal cells (MSCs), the hematopoietic cells, and immune cells in the osteogenic microenvironment for bone development or repair. TLRs can sense both exogenous pathogen-associated molecular patterns (PAMPs) derived from microorganisms, and damage-associated molecular patterns (DAMPs) derived from normal cells subjected to injury, inflammation, or cell apoptosis under physiological or pathological conditions. Emerging studies reported that TLR signaling plays an important role in bone remodeling by directly impacting MSC osteogenic differentiation or osteoimmunology. However, how to regulate TLR signaling is critical and remains to be elucidated to promote the osteogenic differentiation of MSCs and new bone formation for bone tissue repair. This review outlines distinct TLR variants on MSCs from various tissues, detailing the impact of TLR pathway activation or inhibition on MSC osteogenic differentiation. It also elucidates TLR pathways’ interplay with osteoclasts, immune cells, and extracellular vesicles (EVs) derived from MSCs. Furthermore, we explore biomaterial-based activation to guide MSCs’ osteogenic differentiation. Therefore, understanding TLRs’ role in this context has significant implications for advancing bone regeneration and repair strategies.

Holothuria leucospilota Polysaccharides Improve Immunity and the Gut Microbiota in Cyclophosphamide-Treated Immunosuppressed Mice.

Immunosuppression is one of the major risk factors for a series of diseases, such as tumor, rheumatoid arthritis, and microbial infection. Various natural products have attracted wide attention due to their immunomodulatory activities. Herein, the study investigates the regulation of Holothuria leucospilota polysaccharides (HLP) in immunosuppressed mice. Eight-week-old female BALB/c mice are injected intraperitoneally with cyclophosphamide (80mgkg-1 body weight day-1 ) to establish the immunosuppressive model. After 12 days of HLP treatment, the immune organ indexes, serum cytokines, and immunoglobulin levels are significantly increased in immunosuppressed mice (p < 0.05). Real-time fluorescent quantitative polymerase chain reaction and western blotting analysis find that HLP improves the immune factors, T-cell markers, and Toll-like receptors (TLR) pathway-related proteins expression. Simultaneously, HLP significantly increases the short-chain fatty acids concentration and regulates the gut microbiota composition (p < 0.05). Furthermore, the metagenomics analysis shows that HLP increases the levels of functional genes involved in amino acid metabolism, carbohydrate metabolism, and growth activity of the gut microbiota. HLP intervention improves the mice's immune function, and the beneficial effects are closely associated with intestinal homeostasis regulation and TLR pathway activation. This study suggests the potential for HLP as prebiotics in novel immunopotentiators development.

IMPDH inhibition activates TLR-VCAM1 pathway and suppresses the development of MLL-fusion leukemia.

Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in de novo guanine nucleotide synthesis pathway. Although IMPDH inhibitors are widely used as effective immunosuppressants, their antitumor effects have not been proven in the clinical setting. Here, we found that acute myeloid leukemias (AMLs) with MLL-fusions are susceptible to IMPDH inhibitors invitro. We also showed that alternate-day administration of IMPDH inhibitors suppressed the development of MLL-AF9-driven AML invivo without having a devastating effect on immune function. Mechanistically, IMPDH inhibition induced overactivation of Toll-like receptor (TLR)-TRAF6-NF-κB signaling and upregulation of an adhesion molecule VCAM1, which contribute to the antileukemia effect of IMPDH inhibitors. Consequently, combined treatment with IMPDH inhibitors and the TLR1/2 agonist effectively inhibited the development of MLL-fusion AML. These findings provide a rational basis for clinical testing of IMPDH inhibitors against MLL-fusion AMLs and potentially other aggressive tumors with active TLR signaling.

Improving the anti-acute myeloid leukemia activity of CD123-specific engager T cells by MyD88 and CD40 costimulation.

The outcome of patients with acute myeloid leukemia remains poor, and immunotherapy has the potential to improve this. T cells expressing chimeric antigen receptors or bispecific T-cell engagers targeting CD123 are actively being explored in preclinical and/or early phase clinical studies. We have shown that T cells expressing CD123-specific bispecific T-cell engagers (CD123.ENG T cells) have anti-acute myeloid leukemia activity. However, like chimeric antigen receptor T cells, their effector function diminishes rapidly once they are repeatedly exposed to antigen-positive target cells. Here we sought to improve the effector function of CD123.ENG T cells by expressing inducible co-stimulatory molecules consisting of MyD88 and CD40 (iMC), MyD88 (iM), or CD40 (iC), which are activated by a chemical inducer of dimerization. CD123.ENG T cells expressing iMC, iM, or iC maintained their antigen specificity in the presence of a chemical inducer of dimerization, as judged by cytokine production (interferon-γ, interleukin-2) and their cytolytic activity. In repeat stimulation assays, activating iMC and iM, in contrast to iC, enabled CD123.ENG T cells to secrete cytokines, expand, and kill CD123-positive target cells repeatedly. Activating iMC in CD123.ENG T cells consistently improved antitumor activity in an acute myeloid leukemia xenograft model. This translated into a significant survival advantage in comparison to that of mice that received CD123.ENG or CD123.ENG.iC T cells. In contrast, activation of only iM in CD123.ENG T cells resulted in donor-dependent antitumor activity. Our work highlights the need for both toll-like receptor pathway activation via MyD88 and provision of co-stimulation via CD40 to consistently enhance the antitumor activity of CD123.ENG T cells.

ACK1 Contributes to the Pathogenesis of Inflammation and Autoimmunity by Promoting the Activation of TLR Signaling Pathways.

Toll-like receptors (TLRs) are the first line of defense in the immune system, whose activation plays a key role in the pathogenesis of inflammation and autoimmunity. TLRs can activate a variety of immune cells such as macrophages and dendritic cells, which produce proinflammatory cytokines, chemokines, and co-stimulatory molecules that lead to the development of inflammation and autoimmune diseases. As a nonreceptor tyrosine kinase, ACK1 is involved in multiple signaling pathways and physiological processes. However, the roles of ACK1 in the activation of TLR pathways and in the pathogenesis of inflammation and autoimmune diseases have not yet been reported. We found that the expression of ACK1 could be upregulated by TLR pathways in vivo and in vitro. Intriguingly, overexpression of ACK1 significantly promoted the activation of TLR4, TLR7, and TLR9 pathways, while knockdown of ACK1 or the use of the ACK1 inhibitor AIM-100 significantly inhibited the activation of TLR4, TLR7, and TLR9 pathways. In vivo studies showed that the inhibition of ACK1 activity by AIM-100 could significantly protect mice from the TLR4 agonist lipopolysaccharide (LPS)-mediated endotoxin shock and alleviate the condition of imiquimod-mediated lupus-prone mice and MRL/lpr mice. In summary, ACK1 participates in TLR-mediated inflammation and autoimmunity and has great potential in controlling inflammation and alleviating autoimmune diseases.

The severe acute respiratory syndrome coronavirus 2 non-structural proteins 1 and 15 proteins mediate antiviral immune evasion

Infection with pathogenic viruses is often sensed by innate receptors such as Toll-Like Receptors (TLRs) which stimulate type I and III interferons (IFNs) responses, to generate an antiviral state within many cell types. To counteract these antiviral systems, many viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), encode non-structural proteins (NSPs) that mediate immune evasion. Using an overexpression system in A549 ​cells, we demonstrated a significant increase (p ​≤ ​0.0001) in Vesicular Stomatitis Virus (VSV)-EGFP reporter virus replication in cell lines overexpressing either the SARS-CoV-2 NSP1 or NSP15 when compared to control A549 ​cells. The increase in VSV-EGFP virus output was associated with a decrease in TLR2, TLR4 and TLR9 protein expression and a lack of antiviral protein production. Truncation of both NSP1 and NSP15 led to an increase in cellular TLR2, TLR4 and TLR9 as well as a decrease in TLR2 expression respectively. This observation can be attributed to the presence of a functional domain in NSP1 and NSP15 between amino acid (aa) 120–180 and aa 230–346, respectively. Both TLR3 and TLR9 ligands but not TLR2 ligand were highly effective at overcoming NSP1 and NSP15 functional interference based on significant decrease (p ​≤ ​0.0001) in VSV-EGFP virus replication. NSP1 or NSP15 intracellular interactions are likely low affinity interactions that can be easily disrupted by stimulating cells with specific TLR3 and TLR9 ligands. This report provides insights into the role of SARS-CoV-2 NSP1 and NSP15 in limiting specific TLR pathway activation, as an evasive mechanism against host innate responses.

Role of Toll-like receptors in the pathogenesis of COVID-19.

Coronavirus disease 2019 (COVID‐19), caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), has led to a pandemic since March 2020. The exact pathogenesis of SARS‐CoV‐2 and the role of each component of the innate and adaptive immune system is still unknown. However, available data from other coronavirus families, such as SARS‐CoV and the Middle East respiratory syndrome and also new findings could be useful for a better understanding of SARS‐CoV‐2. Toll‐like receptors (TLR) play an important role in recognition of viral particles and activation of the innate immune system. Activation of TLR pathways leads to secretion of pro‐inflammatory cytokines, such as interleukin‐1 (IL‐1), IL‐6, and tumor necrosis factor‐α, as well as type 1 interferon. Different TLRs, like TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, and TLR9 are potentially important in COVID‐19 infection. It is also worth mentioning that we should bear in mind both the beneficial and harmful effects of TLR in confronting COVID‐19 infection. TLRs could be a potential target in controlling the infection in the early stages of disease and production of vaccine against SARS‐CoV‐2.

Study on the relationships between molecular weights of chondroitin sulfate oligosaccharides and Aβ-induced oxidative stress and the related mechanisms.

In the present study, we studied anti-Alzheimer's disease (AD) activities of chondroitin sulfate (CS) oligosaccharides with different molecular weights. CS from shark cartilage was degraded by a recombinant CS endolyase, chondroitinase ABC I (CHSase ABC I), and CS disaccharide (DP2), tetrasaccharide (DP4), hexasaccharide (DP6), octasaccharide (DP8), decasaccharide (DP10) and dodecasaccharide (DP12) were obtained by separation with gel filtration. Anti-AD activities of CS oligosaccharides were assessed using Aβ-injured SH-SY5Y cells and BV2 cells. It was shown that CS oligosaccharides could block Aβ-induced oxidative stress, mitochondrial dysfunction and activation of intrinsic apoptotic pathway for SH-SY5Y cells. Furthermore, these activities increased with the increase of molecular weights. For Aβ-injured BV2 cells, CS oligosaccharides inhibited oxidative stress, the production of proinflammatory cytokines and the activation of toll-like receptor pathway, and CS DP2 had the best activity among them. In conclusion, CS oligosaccharides suppressed Aβ-induced oxidative stress and relevant injury in vitro, and these effects had different relationships with the molecular weights of CS oligosaccharides for different cell lines, which might be caused by different mechanisms.

Inhibition of mTOR suppresses IFNα production and the STING pathway in monocytes from systemic lupus erythematosus patients.

Increased IFNα is important in the pathogenesis of SLE. Plasmacytoid dendritic cells are considered the main producer of IFNα upon Toll-like receptor pathway activation. However, which cells produce IFNα following stimulation with cyclic GMP-AMP synthase (cGAS) and stimulator of IFN genes (STING) in SLE remains unknown. We investigated the IFNα producing capacity of myeloid cells under cGAS-STING pathway stimulation. IFNα levels in peripheral blood mononuclear cells from SLE patients and healthy controls stimulated with 2'3'c-GAMP, a stimulator of cGAS-STING, were measured by intracellular cytokine staining and flow cytometry. STING expression and its co-localization with TBK1 were examined by flow cytometry or confocal microscopy. The effects of in vitro exposure to IFNα on IFNα production and STING expression, and in vitro rapamycin treatment on IFNα production and STING, pTBK1 and IRF3 expression were examined. IFNα was produced by monocytes, conventional dendritic cells and plasmacytoid dendritic cells upon cGAS-STING pathway activation. The frequency of IFNα-producing monocytes positively correlated with SLE disease activity. STING expression and its co-localization with TBK1 were increased in lupus monocytes. Prior exposure to IFNα enhanced the IFNα-producing capacity of monocytes. Inhibition of the mechanistic target of the rapamycin (mTOR) pathway suppressed IFNα production from monocytes and downregulated enhanced STING expression and its downstream molecules. Enhanced IFNα from lupus monocytes induced by augmented STING pathway activation is associated with SLE pathogenesis. Suppression of the mTOR pathway downregulated the enhanced STING expression and the subsequent IFNα production by monocytes.

Transcriptomic Analysis Identifies Necroptosis and Toll-Like Receptor Pathways in Pulmonary Arterial Hypertension

Background: Inflammation and immunity play a causal role in the pathogenesis of pulmonary arterial hypertension (PAH). However, the pathways and mechanisms by which inflammation and immunity are triggered remain unknown. Methods: High-throughput RNA sequencing was used to analyze the transcriptome change in control and rats injected with monocrotaline (MCT) for 1, 2, 3 and 4 weeks. The obtained animal results were further analyzed in two independent microarray datasets derived from patients with PAH. Findings: Using the transcriptional profiling of MCT-induced PAH coupled with bioinformatics analysis, we clustered the differentially expressed genes (DEGs) and chose the increased expression patterns associated with inflammatory and immune response, we found the enrichment of toll-like receptor (TLR) pathways and identified NF-κB-mediated inflammatory and immune profiling. Pathway-based data integration and visualization showed the dysregulated TLR pathways. Further analysis revealed that the activation of TLR pathways was associated with upregulation of damage-associated molecular patterns (DAMPs) and RIPK3-mediated necroptosis was involved in the generation of DAMPs in MCT-induced PAH. Reversal of necroptosis by modern drug therapy was in accordance with attenuated expression of DAMPs, impaired activation of TLR pathways and inflammatory and immune profiling in patients with PAH. Moreover, these clinical results were validated by using currently largest clinical PAH dataset. Interpretation: We identify RIPK3-mediated necroptosis and its triggered TLR pathways in the pathogenesis of PAH, thus, providing insights into the mechanisms underlying inflammation and immunity in PAH. Funding Statement: This work was supported by grants from the National Natural Science Foundation of China (Grant No. 81873537 and 81570446). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: All procedures have been conducted in accordance with the ARRIVE guidelines and were approved by the Laboratory Animal Welfare and Ethics Committee of Fujian Medical University (Approval No. 2017–070, Fuzhou, China).

Concise Review: TLR Pathway-miRNA Interplay in Mesenchymal Stromal Cells: Regulatory Roles and Therapeutic Directions.

Mesenchymal stromal cells (MSCs) deploy Toll-like receptors (TLRs) to respond to exogenous and endogenous signals. Activation of TLR pathways in MSCs alters their inflammatory profile and immunomodulatory effects on cells from both the innate and adaptive immune systems. Micro-RNAs (miRNAs), whose expression is modulated by TLR activation, can regulate inflammatory responses by targeting components of the TLR signaling pathways either in MSCs or in the cells with which they interact. Here, we review how the miRNA-TLR pathway axis can regulate the immunomodulatory functions of MSCs, including their interactions with monocytes/macrophages and natural killer cells, and discuss the therapeutic implications for MSC-based therapies. Stem Cells 2018;36:1655-1662.

Correlation between polymorphisms in toll-like receptor genes and the activity of hepatitis B virus among treatment-na\xefve patients: a case-control study in a Han Chinese population

BackgroundBecause of the high prevalence and absence of cure for infection, chronic hepatitis B virus (HBV) infection has been acknowledged as a pressing public health issue. Toll-like receptors (TLRs) activate the human innate immune system and the polymorphisms in TLRs may alter their function. The present study aimed to investigate the association between TLR polymorphisms and disease progression of chronic HBV infection.MethodsDuring the study period, 211 treatment-naïve patients with chronic HBV infection were recruited, and blood samples were collected from each individual. Matrix-assisted laser desorption/ionization time of flight mass spectrometry was employed to genotype the selected TLR polymorphisms after human genome extraction. In addition, HbsAg, TNF-α, and IL-6 levels were quantified using enzyme linked immunosorbent assay (ELISA). Statistical analyses were conducted to investigate the association between TLR polymorphisms and hepatitis activity, liver function parameters, HbsAg level, and cytokine level.ResultsWe did not observe any mutations in rs4986790, rs4986791, and rs5743708 among all study subjects. A logistic regression revealed that mutations in rs3804099 and rs4696480 were associated with milder hepatitis activity. Consistent with the logistic regression, improved liver function parameters and reduced level of both HbsAg and cytokines were also correlated with the mutant carriers of rs3804099 and rs4696480.ConclusionsTLR mutations were significantly associated with milder hepatitis activity among patients with chronic HBV infection. Therefore, we conclude that the activation of TLR pathways may further intensify the inflammation of hepatocytes, and leads to progression of disease.

Salmeterol, a Long-Acting β2-Adrenergic Receptor Agonist, Inhibits Macrophage Activation by Lipopolysaccharide From Porphyromonas gingivalis.

Salmeterol is a long-acting β2-adrenergic receptor agonist used to treat chronic obstructive pulmonary disease. The authors of the current study previously showed that preincubation of primary microglial-enriched cells with salmeterol could inhibit the inflammatory response induced by Escherichia coli lipopolysaccharide (LPS), a Toll-like receptor (TLR)-4 agonist. In this study, the authors sought to determine if salmeterol had a similar inhibitory effect on the inflammatory response of the murine macrophage cell line RAW264.7 and human monocyte THP-1 to LPS from Porphyromonas gingivalis (PgLPS), an oral microbe implicated in the pathogenesis of periodontal disease. RAW264.7 and THP-1 cells were pretreated with salmeterol, followed by PgLPS, and monitored for production of inflammatory mediators by enzyme-linked immunosorbent assay. The nitric oxide concentration and nuclear factor-kappa B (NF-κB) activity were measured by Griess method and secretory alkaline phosphatase reporter activity assay, respectively. Reverse-transcriptase polymerase chain reaction and immunoblot analysis were used to measure messenger RNA and protein levels. Nuclear translocation of NF-κB was detected by immunofluorescence. Pretreatment with salmeterol significantly inhibited production of proinflammatory mediators by RAW264.7 and THP-1 cells. Salmeterol downregulated PgLPS-mediated phosphorylation of the extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase but not p38 mitogen-activated protein kinases (MAPKs). Salmeterol also attenuated activation of NF-κB via inhibition of nuclear translocation of p65-NFκB, the transcriptional activity of NF-κB and IκBα phosphorylation. Salmeterol can significantly inhibit activation of macrophage-mediated inflammation by PgLPS, suggesting that use of salmeterol may be an effective treatment in inhibiting or lessening the inflammatory response mediated through TLR pathway activation.

A novel kindred with inherited STAT2 deficiency and severe viral illness

A novel kindred with inherited STAT2 deficiency and severe viral illness

Ubiquitin Editing of a Spliceosome Auxiliary Factor By TRAF6 Links Chronic TLR Signaling with Hematopoietic Defects and Myelodysplasia

Toll-like receptors (TLR) are known for regulating myeloid homeostasis and response to infection, but chronic activation of TLR pathways can also lead to hematopoietic stem and progenitor cell (HSPC) dysfunction. Furthermore, mutations that lead to constitutive activation of TLR pathways contribute to premalignant hematologic conditions, such as myelodysplastic syndromes (MDS); however, the underlying cellular and molecular mechanisms are unknown. As a means of chronically activating TLR signaling within HSPCs, we generated a mouse model by elevating expression of TRAF6 in hematopoietic cells (Vav-TRAF6). TRAF6 is a downstream TLR-effector with ubiquitin (Ub) E3 ligase activity, and is overexpressed in MDS HSPCs. Vav-TRAF6 mice developed progressive leukopenia and anemia, and exhibited myeloid skewing and dysplasia. Eventually, over half of Vav-TRAF6 mice succumbed to a bone marrow (BM) failure associated with MDS. Despite increased frequencies of immunophenotypic HSPCs in the BM, Vav-TRAF6 HSPCs are functionally defective as evidenced by impaired colony formation and reduced in vivo competitiveness. The hematopoietic phenotype due to TRAF6 overexpression was still manifest upon transplantation, indicating that the effect is hematopoietic cell intrinsic. Consistent with the cellular effects observed with chronic TLR activation, elevated TRAF6 expression results in MDS/BMF by altering intrinsic HSPC properties.Gene expression and exon level analyses revealed that Vav-TRAF6 HSPCs exhibit discrete and durable alterations in RNA splicing patterns. The family of small G-protein GTPases emerged as a relevant pathway whose activity is affected by missplicing of Arhgap1, a GTPase activating protein. Aberrant skipping of Arhgap1 exon 2 resulted in reduced Arhgap1 protein and constitutive Cdc42 GTPase activation. Inhibition of Cdc42 activity with a pharmacological inhibitor partially reversed myeloid-biased differentiation of Vav-TRAF6 HSPCs in vivo, indicating that missplicing of Arhgap1 and increased Cdc42 activity accounts for several HSPC defects.To identify the mechanism underlying TRAF6-induced RNA splicing, we employed a global Ub-enrichment screen for novel TRAF6 substrates, and uncovered hnRNPA1, an RNA-binding protein that regulates exon usage. hnRNPA1 is ubiquitinated by TRAF6 adjacent to and within its first RNA-binding domain. hnRNPA1 binding sites were significantly enriched within misspliced exons in Vav-TRAF6 HSPCs and in primary human AML samples with elevated TRAF6 expression, indicating that TRAF6 overexpression induces exon skipping via hnRNPA1. The requirement of hnRNPA1 in TRAF6-induced exon skipping was confirmed as knockdown of hnRNPA1 significantly reduced Arhgap1 exon 2 skipping in Vav-TRAF6 HSPC. Moreover, depletion of hnRNPA1 reversed Vav-TRAF6 hematopoietic defects in vivo, unequivocally validating the importance of hnRNPA1 in TRAF6-mediated exon skipping and function of HSPCs.Our findings uncover a novel mechanism by which sustained TLR signaling, via TRAF6-mediated ubiquitination of hnRNPA1, alters RNA splicing and contributes to MDS-associated HSPC defects in part by activating Cdc42. These results indicate a novel function for Ub signaling in coordinating transcriptional initiation and alternative splicing by TLR signaling pathway within the immune system and in premalignant hematologic diseases, such as MDS. DisclosuresStarczynowski:Celgene Corporation: Research Funding.

PTP1B preferentially regulates IRF7-ISRE-IFN pathway and negatively impacts host defense against Pseudomonas aeruginosa infection. (INM7P.348)

Abstract Pseudomonas aeruginosa is a major opportunistic pathogen in immune-compromised individuals. Toll-like receptors (TLRs) contribute to innate immunity against P. aeruginosa through activation of transcription factors IRF7/IRF3 and NFκB. However, mechanisms involved in the regulation of P. aeruginosa-induced TLR pathway activation remain incompletely defined. Here, we demonstrate that protein tyrosine phosphatase-1B (PTP1B) is a critical negative regulator in P. aeruginosa infection. PTP1B-deficient mice display greatly enhanced bacterial clearance which is accompanied with increased neutrophil infiltration and cytokine production. Interestingly, PTP1B-deficiency mainly up-regulates the production of IFN-stimulated response elements (ISRE)-regulated cytokines and chemokines including CCL5 (RANTES), CXCL10 (IP-10) and IFN-β production. Further studies reveal that PTP1B-deficiency leads to increased IRF7 activation. Importantly, PTP1B is physically associated with IRF7 in dendritic cells. These findings demonstrate a novel regulatory mechanism of the immune response to P. aeruginosa infection through PTP1B-IRF7 interaction. This novel PTP1B-IRF7-ISRE pathway may have broader implications in TLR-mediated innate immunity.

Regulation of B lymphocyte responses to Toll-like receptor ligand binding during diabetes prevention in non-obese diabetic (NOD) mice.

Interactions between genetic risk factors and the environment drive type 1 diabetes (T1D). The system of Toll-like receptors (TLR) detects these environmental triggers; however, the target cell that intermediates these interactions to drive T1D remains unknown. We investigated the effect of TLR pathway activation (myeloid differentiation primary response 88 [MyD88] vs TIR-domain-containing adapter-inducing interferon-β [TRIF]) on B cell subsets via flow cytometry, including their activation, survival, proliferation, and cytoskeletal mobilization. The effect of polyinosinic-polycytidylic acid (poly(I:C)) on diabetes development was addressed, including the B cell-dependent activation of diabetes-protective DX5+ cells, using genetic models and adoptive transfer. B lymphocytes from non-obese diabetic (NOD) mice expressed enhanced levels of TLR-responsive proteins. Ex vivo analysis of B lymphocyte subsets demonstrated that TLR3 stimulation via TRIF deletes cells exhibiting a marginal zone phenotype, whereas MyD88-dependent ligands enhance their survival. In vivo, marginal zone B cells were activated by poly(I:C) and were unexpectedly retained in the spleen of NOD mice, in contrast with the mobilization of these cells in non-autoimmune mice, a phenotype we traced to defective actin cytoskeletal dynamics. These activated B cells mediated TLR3-induced diabetes protection. Immunotherapies must account for both B cell location and activation, and these properties may differ in autoimmune and healthy settings.

Role of Toll-like receptors 2 and 4 in mediating endothelial dysfunction and arterial remodeling in primary arterial antiphospholipid syndrome.

To assess the role of Toll-like receptors (TLRs) in antiphospholipid antibody (aPL)-mediated vascular abnormalities in patients with primary arterial antiphospholipid syndrome (APS). Forty-eight subjects participated in the study. Arterial function and structure and TLR pathway activation were determined in patients with primary arterial APS and matched controls. The pathogenic effects of aPL isolated from patients were assessed in wild-type (WT) and TLR-knockout mice. APS patients had endothelial dysfunction, arterial stiffening, and hypertrophy, as evidenced by decreased brachial artery endothelium-dependent flow-mediated dilation (FMD) and increased aortic pulse wave velocity and carotid intima-media thickness (IMT), as compared with controls. Plasma samples from APS patients revealed decreased nitric oxide (NO) availability and a pro-oxidative, proinflammatory, and prothrombotic state illustrated by a decrease in nitrite and an increase in lipid peroxidation, tumor necrosis factor α levels, and tissue factor (TF) levels. Furthermore, TLR pathway activation was found in APS patients with increased TLR-2 and TLR-4 messenger RNA expression and increased protein levels of the activated TLR transduction protein interleukin-1 receptor-associated kinase 1 in peripheral blood mononuclear cells. Moreover, agonist-stimulated cell-surface expression of TLR-2 and TLR-4 in circulating monocytes was higher in APS patients than in controls. These changes were positively associated with IMT and negatively associated with FMD. Finally, aPL injection decreased mesenteric endothelium-dependent relaxation and increased TF expression in WT mice but not in TLR-2- or TLR-4-knockout mice. This translational study supports the notion that TLR-2 and TLR-4 play a role in mediating vascular abnormalities in patients with primary arterial APS. TLRs thus constitute a promising pharmacologic target for preventing cardiovascular complications in APS.

Epstein–Barr Virus Infection Induces Aberrant TLR Activation Pathway and Fibroblast–Myofibroblast Conversion in Scleroderma

Scleroderma (SSc) is a complex and heterogeneous connective tissue disease mainly characterized by autoimmunity, vascular damage, and fibrosis that mostly involve the skin and lungs. Epstein–Barr virus (EBV) is a lymphotropic γ-herpesvirus that has co-evolved with human species, infecting >95% of the adult population worldwide, and has been a leading candidate in triggering several autoimmune diseases. Here we show that EBV establishes infection in the majority of fibroblasts and endothelial cells in the skin of SSc patients, characterized by the expression of the EBV noncoding small RNAs (EBERs) and the increased expression of immediate-early lytic and latency mRNAs and proteins. We report that EBV is able to persistently infect human SSc fibroblasts in vitro, inducing an aberrant innate immune response in infected cells. EBV–Toll-like receptor (TLR) aberrant activation induces the expression of selected IFN-regulatory factors (IRFs), IFN-stimulated genes (ISGs), transforming growth factor-β1 (TGFβ1), and several markers of fibroblast activation, such as smooth muscle actin and Endothelin-1, and all of these genes play a key role in determining the profibrotic phenotype in SSc fibroblasts. These findings imply that EBV infection occurring in mesenchymal, endothelial, and immune cells of SSc patients may underlie the main pathological features of SSc including autoimmunity, vasculopathy, and fibrosis, and provide a unified disease mechanism represented by EBV reactivation.