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1.  Altered phenotype and Stat1 expression in Toll-like receptor 7/8 stimulated monocyte-derived dendritic cells from patients with primary Sjögren’s syndrome 
Arthritis Research & Therapy  2014;16(4):R166.
Dendritic cells (DC) are the most potent antigen-presenting cells of the immune system, involved in both initiating immune responses and maintaining tolerance. Dysfunctional and via toll-like receptor (TLR) ligands activated DC have been implicated in the development of autoimmune diseases, but their role in the etiology of Sjögren’s syndrome, a chronic inflammatory autoimmune disease characterized by progressive mononuclear cell infiltration in the exocrine glands, has not been revealed yet. Therefore, the aim of this study was to investigate phenotype and functional properties of immature and TLR7/8 stimulated monocyte-derived DC (moDC) of patients with primary Sjögren’s syndrome (pSS) and compare them to healthy controls.
The phenotype, apoptosis susceptibility and endocytic capacity of moDC were analyzed by flow cytometry. Secretion of cytokines was measured by enzyme-linked immunosorbent assay (ELISA) and multiplex Luminex analyses in moDC cell culture supernatants. The expression of TLR7 was analyzed by flow cytometry and real-time quantitative polymerase chain reaction (qPCR). Expression of Ro/Sjögren’s syndrome-associated autoantigen A (Ro52/SSA), interferon regulatory factor 8 (IRF-8), Bim, signal transduction and activators of transcription (Stat) 1, p-Stat1 (Tyrosin 701), p-Stat1 (Serin 727), Stat3, pStat3 (Tyrosin 705) and glyceraldehyde 3-phosphatase dehydrogenase (GAPDH) was measured by Western blotting. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) family members were quantified using the ELISA-based TransAM NF-κB family kit.
We could not detect differences in expression of co-stimulatory molecules and maturation markers such as cluster of differentiation (CD) 86, CD80, CD40 or CD83 on moDC from patients compared to healthy controls. Moreover, we could not observe variations in apoptosis susceptibility, Bim and Ro52/SSA expression and the endocytic capacity of the moDC. However, we found that moDC from pSS patients expressed increased levels of the major histocompatibility complex (MHC) class II molecule human leukocyte antigen (HLA)-DR. We also found significant differences in cytokine production by moDC, where increased interleukin (IL)-12p40 secretion in mature pSS moDC correlated with increased RelB expression. Strikingly, moDC from pSS patients matured for 48 hours with TLR7/8 ligand CL097 expressed significantly less Stat1.
Our results suggest a role for moDC in the pathogenesis of Sjögren’s syndrome.
PMCID: PMC4261979  PMID: 25113744
2.  Toll-Like Receptor 2 Impairs Host Defense in Gram-Negative Sepsis Caused by Burkholderia pseudomallei (Melioidosis) 
PLoS Medicine  2007;4(7):e248.
Toll-like receptors (TLRs) are essential in host defense against pathogens by virtue of their capacity to detect microbes and initiate the immune response. TLR2 is seen as the most important receptor for gram-positive bacteria, while TLR4 is regarded as the gram-negative TLR. Melioidosis is a severe infection caused by the gram-negative bacterium, Burkholderia pseudomallei, that is endemic in Southeast Asia. We aimed to characterize the expression and function of TLRs in septic melioidosis.
Methods and Findings
Patient studies: 34 patients with melioidosis demonstrated increased expression of CD14, TLR1, TLR2, and TLR4 on the cell surfaces of monocytes and granulocytes, and increased CD14, TLR1, TLR2, TLR4, LY96 (also known as MD-2), TLR5, and TLR10 mRNA levels in purified monocytes and granulocytes when compared with healthy controls. In vitro experiments: Whole-blood and alveolar macrophages obtained from TLR2 and TLR4 knockout (KO) mice were less responsive to B. pseudomallei in vitro, whereas in the reverse experiment, transfection of HEK293 cells with either TLR2 or TLR4 rendered these cells responsive to this bacterium. In addition, the lipopolysaccharide (LPS) of B. pseudomallei signals through TLR2 and not through TLR4. Mouse studies: Surprisingly, TLR4 KO mice were indistinguishable from wild-type mice with respect to bacterial outgrowth and survival in experimentally induced melioidosis. In contrast, TLR2 KO mice displayed a markedly improved host defenses as reflected by a strong survival advantage together with decreased bacterial loads, reduced lung inflammation, and less distant-organ injury.
Patients with melioidosis displayed an up-regulation of multiple TLRs in peripheral blood monocytes and granulocytes. Although both TLR2 and TLR4 contribute to cellular responsiveness to B. pseudomallei in vitro, TLR2 detects the LPS of B. pseudomallei, and only TLR2 impacts on the immune response of the intact host in vivo. Inhibition of TLR2 may be a novel treatment strategy in melioidosis.
Willem Wiersinga and colleagues find up-regulation of multiple Toll-like receptors (TLRs) in peripheral blood cells of patients with melioidosis. However, only TLR2 had an effect on the immune response in a mouse model.
Editors' Summary
Melioidosis is a severe tropical infection caused by the bacterium Burkholderia pseudomallei. This soil-dwelling pathogen (disease-causing organism) enters the body through cuts, by swallowed contaminated water, or by inhaled contaminated dust. Here, it can cause a severe lung infection or spread into the blood stream and around the body, where it causes widespread inflammation (sepsis) and organ failure. Untreated septic melioidosis is usually fatal. Even with antibiotic therapy, half the people who develop it in Thailand (a hot spot for melioidosis) die. B. pseudomallei is a “gram-negative” bacterium. That is, it is surrounded by a membrane that stops it taking up a stain used to detect bacteria. This membrane contains a molecule called lipopolysaccharide (LPS). Proteins on immune system cells called Toll-like receptors (TLRs), of which there are many, recognize LPS and other surface molecules common to different pathogens and tell the cells to make cytokines. These cytokines stimulate the immune system to kill the pathogen but also cause inflammation, the underlying problem in septic melioidosis and other forms of sepsis. In other words, TLRs are two-edged swords—they provide an essential first-line defense against pathogens, but cause life-threatening inflammation if overstimulated.
Why Was This Study Done?
It isn't known which TLRs are involved in melioidosis. TLR4 normally detects LPS, but the surface of B. pseudomallei also carries molecules that interact with TLR2. Understanding how B. pseudomallei interacts with TLRs might suggest new, more effective ways to treat septic melioidosis. Better remedies for this disease are badly needed because, as well as the infections it causes in the community, the US Centers for Disease Control and Prevention has identified B. pseudomallei as a potential bioterrorism agent. In this study, the researchers have characterized the expression and function of TLRs in septic melioidosis using human, in vitro (test tube), and animal approaches.
What Did the Researchers Do and Find?
The researchers isolated monocytes and granulocytes (immune system cells involved in first-line defenses against pathogens) from patients with melioidosis and from healthy people. The patients' cells made more TLR1, TLR2, TLR4, and CD14 (a protein that enhances the activation of immune system cells by LPS) than those of the healthy controls and more of the mRNAs encoding several other TLRs. Next, the researchers tested the ability of heat-killed B. pseudomallei to induce the release of TNFα (a cytokine produced in response to TLR signaling) from macrophages (immune system cells that swallow up pathogens) isolated from wild-type mice and from mice lacking TLR2 or TLR4. Macrophages isolated from wild-type mice made more TNFα than those from TLR2- or TLR4-deficient mice. In addition, a human kidney cell line engineered to express CD14/TLR2 or CD14/TLR4 but not the parent cell line released IL8 (another cytokine) when stimulated with heat-killed B. pseudomallei. Other experiments in these human cell lines showed that LPS purified from B. pseudomallei signals through TLR2 but not through TLR4. Finally, the researchers tested the ability of TLR2- and TLR4-deficient mice to survive after infection with live B. pseudomallei. Compared with TLR4-deficient or wild-type mice, the TLR2-deficient mice had a strong survival advantage, a lower bacterial load, reduced lung inflammation, and less organ damage.
What Do These Findings Mean?
These findings show that people with melioidosis have increased expression of several TLRs, any one of which might cause the sepsis associated with B. pseudomallei infection. The in vitro findings indicate that TLR2 and TLR4 both contribute to the responsiveness of immune cells to B. pseudomallei in test tubes, but that only TLR2 detects the LPS of this bacterium. This unexpected result—TLR4 normally responds to LPS—might indicate that there is something unique about the LPS of B. pseudomallei. Finally, the survival of TLR2-deficient mice after infection with B. pseudomallei suggests that TLR2-mediated dysregulation of the immune system in response to invasive B. pseudomallei might cause septic melioidosis. Although these results need confirming in people, they suggest that inhibition of TLR2 in combination with antibiotic therapy might improve outcomes for people with melioidosis.
Additional Information.
Please access these Web sites via the online version of this summary at
Information is available from the US Centers for Disease Control and Prevention on melioidosis (in English and Spanish)
The UK Health Protection Agency provides information for the public and health professionals on melioidosis
Wikipedia has pages on melioidosis and on Toll-like receptors (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
The MedlinePlus encyclopedia contains a page on sepsis (in English and Spanish)
PMCID: PMC1950213  PMID: 17676990
3.  Coactivation of TLR4 and TLR2/6 Coordinates an Additive Augmentation on IL-6 Gene Transcription via p38 MAPK Pathway in U937 Mononuclear Cells 
Molecular immunology  2011;49(3):423-432.
Studies have demonstrated that TLR4 and TLR2 expression by monocytes and the blood levels of TLR4 and TLR2 ligand in diabetic patients are significantly incased compared to nondiabetic patients, indicating that more monocytes in diabetic patients may have coactivation of TLR4 and TLR2. Although it has been shown that either TLR4 or TLR2 activation leads to increased expression of proinflammatory cytokines, the effect of coactivation of TLR2 and TLR4 in mononuclear cells on proinflammatory cytokine expression and the underlying molecular mechanisms remain largely unknown. In this study, we found that while TLR1, TLR2, TLR4 and TLR6 were expressed by U937 mononuclear cells, TLR4 was expressed at the highest level. Interestingly, results showed that while activation of either TLR4 or TLR2/6 (TLR2 dimerized with TLR6), but not TLR2/1 (TLR2 dimerized with TLR1), significantly increased IL-6 expression by U937 mononuclear cells, coactivation of TLR4 and TLR2/6, but not TLR4 and TLR2/1, led to a further augmentation on IL-6 expression by increasing IL-6 transcriptional activity, but not mRNA stability. To explore the signaling mechanisms involved in the augmentation, we found that p38 MAPK and NFκB pathways, but not ERK and JNK pathways, were required for the augmentation of IL-6 expression by coactivation of TLR4 and TLR2/6. Furthermore, we found that coactivation of TLR4 and TLR2/6 increased p38 phosphorylation, but not NFkB activity, as compared to activation of TLR4 or TLR2/6 alone. Taken together, this study showed that coactivation of TLR4 and TLR2/6 coordinates an additive augmentation of IL-6 gene transcription via p38 MAPK pathway in U937 mononuclear cells.
PMCID: PMC3224151  PMID: 22030478
Toll-like receptor; Interleukin 6; p38 mitogen-activated protein kinase; Inflammation
4.  The phosphoproteome of toll-like receptor-activated macrophages 
First global and quantitative analysis of phosphorylation cascades induced by toll-like receptor (TLR) stimulation in macrophages identifies nearly 7000 phosphorylation sites and shows extensive and dynamic up-regulation and down-regulation after lipopolysaccharide (LPS).In addition to the canonical TLR-associated pathways, mining of the phosphorylation data suggests an involvement of ATM/ATR kinases in signalling and shows that the cytoskeleton is a hotspot of TLR-induced phosphorylation.Intersecting transcription factor phosphorylation with bioinformatic promoter analysis of genes induced by LPS identified several candidate transcriptional regulators that were previously not implicated in TLR-induced transcriptional control.
Toll-like receptors (TLR) are a family of pattern recognition receptors that enable innate immune cells to sense infectious danger. Recognition of microbial structures, like lipopolysaccharide (LPS) of Gram-negative bacteria by TLR4, causes within hours substantial re-programming of macrophage gene expression, including up-regulation of chemokines driving inflammation, anti-microbial effector molecules and cytokines directing adaptive immune responses. TLR signalling is initiated by the adapter protein Myd88 and leads to the activation of kinase cascades that result in activation of the MAPK and NFkB pathways. Phosphorylation has an essential role in these early steps of TLR signalling, and in addition regulates critical transcription factors (TFs). Although TLR signalling has been extensively studied, a comprehensive analysis of phosphorylation events in TLR-activated macrophages is lacking. It is therefore unknown whether the canonical MAPK and NFkB pathways comprise the main phosphorylation events and which other molecular functions and processes are regulated by phosphorylation after stimulation with LPS.
Recent progress in mass spectrometry-based proteomics has opened the possibility to quantitatively investigate global changes in protein abundance and post-translational modifications. Stable isotope labelling with amino acids in cell culture (SILAC) allows highly accurate quantification, and has proved especially useful for direct comparison of phosphopeptide abundance in time-course or treatment analyses.
Here, we adapted SILAC to primary mouse macrophages, and performed a global, quantitative and kinetic analysis of the macrophage phosphoproteome after LPS stimulation. Bioinformatic analyses were used to identify kinases, pathways and biological processes enriched in the LPS-regulated phosphoproteome. To connect TF phosphorylation with transcription, we generated a parallel dataset of nascent RNA and used in silico promoter analysis to identify transcriptional regulators with binding site enrichment among the LPS-regulated gene set.
After establishing SILAC conditions for efficient labelling of primary bone marrow-derived macrophages in two independent experiments 1850 phosphoproteins with a total of 6956 phosphorylation sites were reproducibly identified. Phosphoproteins were detected from all cellular compartments, with a clear enrichment for nuclear and cytoskeleton-associated proteins. LPS caused major regulation of a large fraction of phosphopeptides, with 24% of all sites up-regulated and 9% down-regulated after stimulation (Figure 3A and B). These changes were highly dynamic, as the majority of the regulated phosphopeptides were up-regulated or down-regulated transiently or in a delayed manner (Figure 3C). Overall, the extent of changes in the phosphoproteome was comparable to the transcriptional re-programming, underscoring the importance of phosphorylation cascades in TLR signalling. Our parallel transcriptome data also showed that widespread phosphorylation precedes massive transcriptional changes.
To obtain footprints of kinase activation in response to TLR ligation, we searched phosphopeptide sequences for known linear sequence motifs of 33 kinases and identified kinase motifs enriched among LPS-regulated phosphorylation sites (compared to non-regulated phosphorylation sites) (Table I). Motif ERK/MAPK was highly enriched, in accordance with the essential role of the MAPK module in TLR signalling. Other kinases with motif enrichment have also recently been linked to TLR signalling (e.g. PKD; AKT and its targets GSK3 and mTOR). However, the DNA damage-actviated kinases ATM/ATR and the cell cycle-associated kinases AURORA and CHK1/2 have not been associated with the macrophage response to TLR activation yet. These finding shed new light on older data on the effect of TLR on macrophage proliferation in response to macrophage colony stimulating factor. Of interest, in follow-up experiments using pharmacological inhibitors of the kinases with motif enrichment, we observed that inhibition of ATM kinase activity caused increased LPS-induced expression of several cytokines and chemokines, suggesting that this pathway regulates inflammatory responses.
In further bioinformatic analyses, the Gene Ontology and signalling pathway annotations of phosphoproteins were used to identify signalling pathways and cellular processes targeted by TLR4-controlled phosphorylation (Table II). Among the expected hits, based on the known TLR pathways, were TLR signalling, MAPK and AKT as well as mTOR signalling. Of interest, the annotation terms ‘Rho GTPase cycle' and ‘cytoskeleton' were significantly enriched among LPS-regulated phosphoproteins, indicating a more prominent role for cytoskeletal proteins in the transduction of TLR signals or in the biological response to it.
We were especially interested in the phosphorylation of TFs and its regulation by LPS (Figure 6A). We hypothesised that functionally important TFs should have an increased frequency of binding sites in the promoters of LPS-regulated genes (Figure 6B). To identify transcriptionally regulated genes with high sensitivity, we isolated nascent RNA after metabolic labelling (Figure 6C–E). In silico promoter scanning using Genomatix software for binding sites for all 50 TF families with phosphorylated members was used to test for enrichment in transciptionally induced genes (Figure 6F). At the early time point, binding site enrichment for the canonical TLR-associated TF NFkB was detected, and in addition we found that several other TF families with an established role in the transcription of individual LPS-target genes showed binding site enrichment (CEBP, MEF2, NFAT and HEAT). In addition, enrichment for OCT and HOXC binding sites at the early time point and SORY matrices later after stimulation indicated an involvement of the phosphorylated members of the respective TF families in the execution of TLR-induced transcriptional responses. An initial test of the function for a few of these candidate transcriptional regulators was performed using siRNA knockdown in primary macrophages. These experiments suggested that knock down of the SORY binding phosphoprotein Capicua homolog (Cic) and to a lesser extent of the CREB family member Atf7 selectively attenuates LPS-induced expression of Il1a and Il1b.
In summary, this study provides a novel and global perspective on innate immune activation by TLR signalling (Figure 5). We quantitatively detected a large number of previously unknown site-specific phosphorylation events, which are now publicly available through the Phosida database. By combining different data mining approaches, we consistently identified canonical and newly implicated TLR-activated signalling modules. In particular, the PI3K/AKT and the related mTOR pathway were highlighted; furthermore, DNA damage–response associated ATM/ATR kinases and the cytoskeleton emerged as unexpected hotspots for phosphorylation. Finally, weaving together corresponding phophoproteome and nascent transcriptome datasets through the loom of in silico promoter analysis we identified TFs with a likely role in mediating TLR-induced gene expression programmes.
Recognition of microbial danger signals by toll-like receptors (TLR) causes re-programming of macrophages. To investigate kinase cascades triggered by the TLR4 ligand lipopolysaccharide (LPS) on systems level, we performed a global, quantitative and kinetic analysis of the phosphoproteome of primary macrophages using stable isotope labelling with amino acids in cell culture, phosphopeptide enrichment and high-resolution mass spectrometry. In parallel, nascent RNA was profiled to link transcription factor (TF) phosphorylation to TLR4-induced transcriptional activation. We reproducibly identified 1850 phosphoproteins with 6956 phosphorylation sites, two thirds of which were not reported earlier. LPS caused major dynamic changes in the phosphoproteome (24% up-regulation and 9% down-regulation). Functional bioinformatic analyses confirmed canonical players of the TLR pathway and highlighted other signalling modules (e.g. mTOR, ATM/ATR kinases) and the cytoskeleton as hotspots of LPS-regulated phosphorylation. Finally, weaving together phosphoproteome and nascent transcriptome data by in silico promoter analysis, we implicated several phosphorylated TFs in primary LPS-controlled gene expression.
PMCID: PMC2913394  PMID: 20531401
macrophage; nascent RNA; phosphoproteome; SILAC; toll-like receptors
5.  MicroRNA-3148 Modulates Allelic Expression of Toll-Like Receptor 7 Variant Associated with Systemic Lupus Erythematosus 
PLoS Genetics  2013;9(2):e1003336.
We previously reported that the G allele of rs3853839 at 3′untranslated region (UTR) of Toll-like receptor 7 (TLR7) was associated with elevated transcript expression and increased risk for systemic lupus erythematosus (SLE) in 9,274 Eastern Asians [P = 6.5×10−10, odds ratio (OR) (95%CI) = 1.27 (1.17–1.36)]. Here, we conducted trans-ancestral fine-mapping in 13,339 subjects including European Americans, African Americans, and Amerindian/Hispanics and confirmed rs3853839 as the only variant within the TLR7-TLR8 region exhibiting consistent and independent association with SLE (Pmeta = 7.5×10−11, OR = 1.24 [1.18–1.34]). The risk G allele was associated with significantly increased levels of TLR7 mRNA and protein in peripheral blood mononuclear cells (PBMCs) and elevated luciferase activity of reporter gene in transfected cells. TLR7 3′UTR sequence bearing the non-risk C allele of rs3853839 matches a predicted binding site of microRNA-3148 (miR-3148), suggesting that this microRNA may regulate TLR7 expression. Indeed, miR-3148 levels were inversely correlated with TLR7 transcript levels in PBMCs from SLE patients and controls (R2 = 0.255, P = 0.001). Overexpression of miR-3148 in HEK-293 cells led to significant dose-dependent decrease in luciferase activity for construct driven by TLR7 3′UTR segment bearing the C allele (P = 0.0003). Compared with the G-allele construct, the C-allele construct showed greater than two-fold reduction of luciferase activity in the presence of miR-3148. Reduced modulation by miR-3148 conferred slower degradation of the risk G-allele containing TLR7 transcripts, resulting in elevated levels of gene products. These data establish rs3853839 of TLR7 as a shared risk variant of SLE in 22,613 subjects of Asian, EA, AA, and Amerindian/Hispanic ancestries (Pmeta = 2.0×10−19, OR = 1.25 [1.20–1.32]), which confers allelic effect on transcript turnover via differential binding to the epigenetic factor miR-3148.
Author Summary
Systemic lupus erythematosus (SLE) is a debilitating autoimmune disease contributed to by excessive innate immune activation involving toll-like receptors (TLRs, particularly TLR7/8/9) and type I interferon (IFN) signaling pathways. TLR7 responds against RNA–containing nuclear antigens and activates IFN-α pathway, playing a pivotal role in the development of SLE. While a genomic duplication of Tlr7 promotes lupus-like disease in the Y-linked autoimmune accelerator (Yaa) murine model, the lack of common copy number variations at TLR7 in humans led us to identify a functional single nucleotide polymorphism (SNP), rs3853839 at 3′ UTR of the TLR7 gene, associated with SLE susceptibility in Eastern Asians. In this study, we fine-mapped the TLR7-TLR8 region and confirmed rs3853839 exhibiting the strongest association with SLE in European Americans, African Americans, and Amerindian/Hispanics. Individuals carrying the risk G allele of rs3853839 exhibited increased TLR7 expression at the both mRNA and protein level and decreased transcript degradation. MicroRNA-3148 (miR-3148) downregulated the expression of non-risk allele (C) containing transcripts preferentially, suggesting a likely mechanism for increased TLR7 levels in risk-allele carriers. This trans-ancestral mapping provides evidence for the global association with SLE risk at rs3853839, which resides in a microRNA–gene regulatory site affecting TLR7 expression.
PMCID: PMC3585142  PMID: 23468661
6.  Dysfunctional interferon-α production by peripheral plasmacytoid dendritic cells upon Toll-like receptor-9 stimulation in patients with systemic lupus erythematosus 
It is well known that interferon (IFN)-α is important to the pathogenesis of systemic lupus erythematosus (SLE). However, several reports have indicated that the number of IFN-α producing cells are decreased or that their function is defective in patients with SLE. We studied the function of plasmacytoid dendritic cells (pDCs) under persistent stimulation of Toll-like receptor (TLR)9 via a TLR9 ligand (CpG ODN2216) or SLE serum.
The concentrations of IFN-α were determined in serum and culture supernatant of peripheral blood mononuclear cells (PBMCs) from SLE patients and healthy controls after stimulation with CpG ODN2216 or SLE serum. The numbers of circulating pDCs were analyzed by fluoresence-activated cell sorting analysis. pDCs were treated with CpG ODN2216 and SLE serum repeatedly, and levels of produced IFN-α were measured. The expression of IFN-α signature genes and inhibitory molecules of TLR signaling were examined in PBMCs from SLE patients and healthy control individuals.
Although there was no significant difference in serum concentration of IFN-α and number of circulating pDCs between SLE patients and healthy control individuals, the IFN-α producing capacity of PBMCs was significantly reduced in SLE patients. Interestingly, the degree which TLR9 ligand-induced IFN-α production in SLE PBMCs was inversely correlated with the SLE serum-induced production of IFN-α in healthy PMBCs. Because repeated stimulation pDCs with TLR9 ligands showed decreased level of IFN-α production, continuous TLR9 stimulation may lead to decreased production of IFN-α in SLE PBMCs. In addition, PBMCs isolated from SLE patients exhibited higher expression of IFN-α signature genes and inhibitory molecules of TLR signaling, indicating that these cells had already undergone IFN-α stimulation and had become desensitized to TLR signaling.
We suggest that the persistent presence of endogenous IFN-α inducing factors induces TLR tolerance in pDCs of SLE patients, leading to impaired production of IFN-α.
PMCID: PMC2453773  PMID: 18321389
7.  Differential Expression of Toll-like Receptors in Dendritic Cells of Patients with Dengue during Early and Late Acute Phases of the Disease 
Dengue hemorrhagic fever (DHF) is observed in individuals that have pre-existing heterotypic dengue antibodies and is associated with increased viral load and high levels of pro-inflammatory cytokines early in infection. Interestingly, a recent study showed that dengue virus infection in the presence of antibodies resulted in poor stimulation of Toll-like receptors (TLRs), thereby facilitating virus particle production, and also suggesting that TLRs may contribute to disease pathogenesis.
Methodology/Principal Findings
We evaluated the expression levels of TLR2, 3, 4 and 9 and the co-stimulatory molecules CD80 and CD86 by flow cytometry. This was evaluated in monocytes, in myeloid and plasmacytoid dendritic cells (mDCs and pDCs) from 30 dengue patients with different clinical outcomes and in 20 healthy controls. Increased expression of TLR3 and TLR9 in DCs of patients with dengue fever (DF) early in infection was detected. In DCs from patients with severe manifestations, poor stimulation of TLR3 and TLR9 was observed. In addition, we found a lower expression of TLR2 in patients with DF compared to DHF. Expression levels of TLR4 were not affected. Furthermore, the expression of CD80 and CD86 was altered in mDCs and CD86 in pDCs of severe dengue cases. We show that interferon alpha production decreased in the presence of dengue virus after stimulation of PBMCs with the TLR9 agonist (CpG A). This suggests that the virus can affect the interferon response through this signaling pathway.
These results show that during dengue disease progression, the expression profile of TLRs changes depending on the severity of the disease. Changes in TLRs expression could play a central role in DC activation, thereby influencing the innate immune response.
Author Summary
Dengue virus (DENV) infections cause a broad spectrum of clinical manifestations, ranging from self-limited fever to severe disease, such as dengue hemorrhagic fever (DHF) that can be fatal. The pathogenesis of severe dengue is associated with an inadequate immune response characterized by the over-production of cytokines and other inflammatory components. However, little is known about the role of the innate immune response in the progression to hemorrhagic manifestations. TLRs are among the most important components of innate immunity and are responsible for initiating a response against a variety of pathogens, including viruses. Recent studies suggest that TLRs may contribute to disease pathogenesis. Here we aimed to explore the role of these receptors in dengue disease progression. To this end, we examined the expression of several TLRs and of co-stimulatory molecules in monocytes and DCs from dengue patients. A link between TLRs expression and the severity of dengue was observed: patients with dengue fever express higher levels of TLR3 and TLR9 than patients with DHF. This could be crucial for the host defense against dengue virus or disease progression. In addition, expression of CD80 and CD86 was altered in DCs of severe dengue cases. We show that interferon type I production is also altered in vitro through TLR9. This suggests that dengue virus affects the interferon response through this signaling pathway.
PMCID: PMC3585035  PMID: 23469297
8.  Activation of the Alternative NFκB Pathway Improves Disease Symptoms in a Model of Sjogren's Syndrome 
PLoS ONE  2011;6(12):e28727.
The purpose of our study was to understand if Toll-like receptor 9 (TLR9) activation could contribute to the control of inflammation in Sjogren's syndrome. To this end, we manipulated TLR9 signaling in non-obese diabetic (NOD) and TLR9−/− mice using agonistic CpG oligonucleotide aptamers, TLR9 inhibitors, and the in-house oligonucleotide BL-7040. We then measured salivation, inflammatory response markers, and expression of proteins downstream to NF-κB activation pathways. Finally, we labeled proteins of interest in salivary gland biopsies from Sjogren's syndrome patients, compared to Sicca syndrome controls. We show that in NOD mice BL-7040 activates TLR9 to induce an alternative NF-κB activation mode resulting in increased salivation, elevated anti-inflammatory response in salivary glands, and reduced peripheral AChE activity. These effects were more prominent and also suppressible by TLR9 inhibitors in NOD mice, but TLR9−/− mice were resistant to the salivation-promoting effects of CpG oligonucleotides and BL-7040. Last, salivary glands from Sjogren's disease patients showed increased inflammatory and decreased anti-inflammatory biomarkers, in addition to decreased levels of alternative NF-κB pathway proteins. In summary, we have demonstrated that activation of TLR9 by BL-7040 leads to non-canonical activation of NF-κB, promoting salivary functioning and down-regulating inflammation. We propose that BL-7040 could be beneficial in treating Sjogren's syndrome and may be applicable to additional autoimmune syndromes.
PMCID: PMC3235165  PMID: 22174879
9.  Distinct Dictation of Japanese Encephalitis Virus-Induced Neuroinflammation and Lethality via Triggering TLR3 and TLR4 Signal Pathways 
PLoS Pathogens  2014;10(9):e1004319.
Japanese encephalitis (JE) is major emerging neurologic disease caused by JE virus. To date, the impact of TLR molecules on JE progression has not been addressed. Here, we determined whether each TLR modulates JE, using several TLR-deficient mouse strains (TLR2, TLR3, TLR4, TLR7, TLR9). Surprisingly, among the tested TLR-deficient mice there were contrasting results in TLR3−/− and TLR4−/− mice, i.e. TLR3−/− mice were highly susceptible to JE, whereas TLR4−/− mice showed enhanced resistance to JE. TLR3 ablation induced severe CNS inflammation characterized by early infiltration of inflammatory CD11b+Ly-6Chigh monocytes along with profoundly increased viral burden, proinflammatory cytokine/chemokine expression as well as BBB permeability. In contrast, TLR4−/− mice showed mild CNS inflammation manifested by reduced viral burden, leukocyte infiltration and proinflammatory cytokine expression. Interestingly, TLR4 ablation provided potent in vivo systemic type I IFN innate response, as well as ex vivo type I IFN production associated with strong induction of antiviral PRRs (RIG-I, MDA5), transcription factors (IRF-3, IRF-7), and IFN-dependent (PKR, Oas1, Mx) and independent ISGs (ISG49, ISG54, ISG56) by alternative activation of IRF3 and NF-κB in myeloid-derived DCs and macrophages, as compared to TLR3−/− myeloid-derived cells which were more permissive to viral replication through impaired type I IFN innate response. TLR4 ablation also appeared to mount an enhanced type I IFN innate and humoral, CD4+ and CD8+ T cell responses, which were mediated by altered immune cell populations (increased number of plasmacytoid DCs and NK cells, reduced CD11b+Ly-6Chigh monocytes) and CD4+Foxp3+ Treg number in lymphoid tissue. Thus, potent type I IFN innate and adaptive immune responses in the absence of TLR4 were closely coupled with reduced JE lethality. Collectively, these results suggest that a balanced triggering of TLR signal array by viral components during JE progression could be responsible for determining disease outcome through regulating negative and positive factors.
Author Summary
Japanese encephalitis (JE) is major emerging encephalitis, and more than 60% of global population inhabits JE endemic areas. The etiological virus is currently spreading to previously unaffected regions due to rapid changes in climate and demography. However, the impact of TLR molecules on JE progression has not been addressed to date. We found that the distinct outcomes of JE progression occurred in TLR3 and TLR4-dependent manner, i.e. TLR3−/− mice were highly susceptible, whereas TLR4−/− mice showed enhanced resistance to JE. TLR3 ablation induced severe CNS inflammation manifested by early CD11b+Ly-6Chigh monocyte infiltration, high expression of proinflammatory cytokines, as well as increased BBB permeability. In contrast, TLR4 ablation provided potent type I IFN innate response in infected mice, as well as in myeloid-derived cells closely associated with strong induction of antiviral ISG genes, and also resulted in enhanced humoral, CD4+, and CD8+ T cell responses along with altered plasmacytoid DC and CD4+Foxp3+ Treg number. Thus, potent type I IFN innate and adaptive immune responses in the absence of TLR4 were coupled with reduced JE lethality. Our studies provide an insight into the role of each TLR molecule on the modulation of JE, as well as its mechanism of neuroinflammation control during JE progression.
PMCID: PMC4154777  PMID: 25188232
10.  TLR9, TACI and CD40 synergize in causing B cell activation 
B cells receive activating signals from T cells via CD40, from microbial DNA via TLR9, and from dendritic cells (DCs) via transmembrane activator calcium modulator and cyclophilin ligand interactor (TACI). TLR9 and CD40 ligation augment TACI driven B cell activation, but only the mechanism of synergy between CD40 and TACI has been explored. Synergy between CD40 and TLR9 in B cell activation is controversial.
To examine the mechanisms by which TLR9 modulates CD40- and TACI-mediated activation of B cells, and to determine whether all three receptors synergize to activate B cells.
Naïve mouse B cells and human peripheral blood mononuclear cells (PBMCs) were stimulated with combinations of anti-CD40, CpG and APRIL in the presence of IL-4. Proliferation was measured by 3H-thymidine incorporation. Immuniglobulin production was measured by ELISA. Class switch recombination (CSR) was examined by measuring mRNA for germ line transcripts, activation-induced cytidine deaminase (AICDA) and mature Ig transcripts. Plasma cell differentiation was examined by syndecan-1/CD138 staining and mRNA expression of B lymphocyte-induced maturation protein 1 (Blimp-1).
TLR9 synergized with CD40 and TACI in driving CSR and inducing IgG1 and IgE secretion by naïve murine B cells, and synergized with TACI in driving B cell proliferation and plasma cell differentiation. All three receptors synergized together in driving mouse B cell proliferation, CSR, plasma cell differentiation and IgG1 and IgE secretion. TLR9 synergized with CD40 and TACI in driving IgG secretion in IL-4 stimulated human B cells.
Signals from TLR9, TACI and CD40 are integrated to promote B cell activation and differentiation.
Clinical Implications
Stimulation of B cells via CD40, TLR9 and TACI results in their optimal activation. Defects in these pathways may co-operate to impair B cell immunoglobulin production in patients with common variable immunodeficiency (CVID).
PMCID: PMC3164940  PMID: 21741080
TLR9; CD40; TACI; CSR; plasma cell; immunoglobulin; B cells
11.  Effects of Separate and Concomitant TLR-2 and TLR-4 Activation in Peripheral Blood Mononuclear Cells of Newborn and Adult Horses 
PLoS ONE  2013;8(6):e66897.
Deficient innate and adaptive immune responses cause newborn mammals to be more susceptible to bacterial infections than adult individuals. Toll-like receptors (TLRs) are known to play a pivotal role in bacterial recognition and subsequent immune responses. Several studies have indicated that activation of certain TLRs, in particular TLR-2, can result in suppression of inflammatory pathology. In this study, we isolated peripheral blood mononuclear cells (PBMCs) from adult and newborn horses to investigate the influence of TLR-2 activation on the inflammatory response mediated by TLR-4. Data were analysed in a Bayesian hierarchical linear regression model, accounting for variation between horses. In general, cytokine responses were lower in PBMCs derived from foals compared with PBMCs from adult horses. Whereas in foal PBMCs expression of TLR-2, TLR-4, and TLR-9 was not influenced by separate and concomitant TLR-2 and TLR-4 activation, in adult horse PBMCs, both TLR ligands caused significant up-regulation of TLR-2 and down-regulation of TLR-9. Moreover, in adult horse PBMCs, interleukin-10 protein production and mRNA expression increased significantly following concomitant TLR-2 and TLR-4 activation (compared with sole TLR-4 activation). In foal PBMCs, this effect was not observed. In both adult and foal PBMCs, the lipopolysaccharide-induced pro-inflammatory response was not influenced by pre-incubation and co-stimulation with the specific TLR-2 ligand Pam3-Cys-Ser-Lys4. This indicates that the published data on other species cannot be translated directly to the horse, and stresses the necessity to confirm results obtained in other species in target animals. Future research should aim to identify other methods or substances that enhance TLR functionality and bacterial defence in foals, thereby lowering susceptibility to life-threatening infections during the first period of life.
PMCID: PMC3686748  PMID: 23840549
12.  HIV Type 1 Infection Up-Regulates TLR2 and TLR4 Expression and Function in Vivo and in Vitro 
AIDS Research and Human Retroviruses  2012;28(10):1313-1328.
Toll-like receptors (TLRs) play a critical role in innate immunity against pathogens. Their stimulation induces the activation of NF-κB, an important inducer of HIV-1 replication. In recent years, an increasing number of studies using several cells types from HIV-infected patients indicate that TLRs play a key role in regulating the expression of proinflammatory cytokines and viral pathogenesis. In the present study, the effect of HIV-1 stimulation of monocyte-derived macrophage (MDM) and peripheral blood mononuclear cell (PBMC) subpopulations from healthy donors on the expression and functions of TLR2 and TLR4 was examined. In addition, and to complete the in vitro study, the expression pattern of TLR2 and TLR4 in 49 HIV-1-infected patients, classified according to viral load and the use of HAART, was determined and compared with 25 healthy subjects. An increase of TLR expression and production of proinflammatory cytokines were observed in MDMs and PBMCs infected with HIV-1 in vitro and in response to TLR stimulation, compared to the mock. In addition, an association between TLR expression and up-regulation of CD80 in plasmacytoid dendritic cells (pDCs) was observed. The ex vivo analysis indicated increased expression of TLR2 and TLR4 in myeloid dendritic cells (mDCs), but only of TLR2 in monocytes obtained from HIV-1-infected patients, compared to healthy subjects. Remarkably, the expression was higher in cells from patients who do not use HAART. In monocytes, there was a positive correlation between both TLRs and viral load, but not CD4+ T cell numbers. Together, our in vitro and ex vivo results suggest that TLR expression and function can be up-regulated in response to HIV-1 infection and could affect the inflammatory response. We propose that modulation of TLRs represents a mechanism to promote HIV-1 replication or AIDS progression in HIV-1-infected patients.
PMCID: PMC3482876  PMID: 22280204
13.  Macrophages exposed continuously to lipopolysaccharide and other agonists that act via toll-like receptors exhibit a sustained and additive activation state 
BMC Immunology  2001;2:11.
Macrophages sense microorganisms through activation of members of the Toll-like receptor family, which initiate signals linked to transcription of many inflammation associated genes. In this paper we examine whether the signal from Toll-like receptors [TLRs] is sustained for as long as the ligand is present, and whether responses to different TLR agonists are additive.
RAW264 macrophage cells were doubly-transfected with reporter genes in which the IL-12p40, ELAM or IL-6 promoter controls firefly luciferase, and the human IL-1β promoter drives renilla luciferase. The resultant stable lines provide robust assays of macrophage activation by TLR stimuli including LPS [TLR4], lipopeptide [TLR2], and bacterial DNA [TLR9], with each promoter demonstrating its own intrinsic characteristics. With each of the promoters, luciferase activity was induced over an 8 hr period, and thereafter reached a new steady state. Elevated expression required the continued presence of agonist. Sustained responses to different classes of agonist were perfectly additive. This pattern was confirmed by measuring inducible cytokine production in the same cells. While homodimerization of TLR4 mediates responses to LPS, TLR2 appears to require heterodimerization with another receptor such as TLR6. Transient expression of constitutively active forms of TLR4 or TLR2 plus TLR6 stimulated IL-12 promoter activity. The effect of LPS, a TLR4 agonist, was additive with that of TLR2/6 but not TLR4, whilst that of lipopeptide, a TLR2 agonist, was additive with TLR4 but not TLR2/6. Actions of bacterial DNA were additive with either TLR4 or TLR2/6.
These findings indicate that maximal activation by any one TLR pathway does not preclude further activation by another, suggesting that common downstream regulatory components are not limiting. Upon exposure to a TLR agonist, macrophages enter a state of sustained activation in which they continuously sense the presence of a microbial challenge.
PMCID: PMC58839  PMID: 11686851
14.  Mycobacterium tuberculosis Induces Interleukin-32 Production through a Caspase- 1/IL-18/Interferon-γ-Dependent Mechanism 
PLoS Medicine  2006;3(8):e277.
Interleukin (IL)–32 is a newly described proinflammatory cytokine that seems likely to play a role in inflammation and host defense. Little is known about the regulation of IL-32 production by primary cells of the immune system.
Methods and Findings
In the present study, freshly obtained human peripheral blood mononuclear cells were stimulated with different Toll-like receptor (TLR) agonists, and gene expression and synthesis of IL-32 was determined. We demonstrate that the TLR4 agonist lipopolysaccharide induces moderate (4-fold) production of IL-32, whereas agonists of TLR2, TLR3, TLR5, or TLR9, each of which strongly induced tumor necrosis factor α and IL-6, did not stimulate IL-32 production. However, the greatest amount of IL-32 was induced by the mycobacteria Mycobacterium tuberculosis and M. bovis BCG (20-fold over unstimulated cells). IL-32-induced synthesis by either lipopolysaccharide or mycobacteria remains entirely cell-associated in monocytes; moreover, steady-state mRNA levels are present in unstimulated monocytes without translation into IL-32 protein, similar to other cytokines lacking a signal peptide. IL-32 production induced by M. tuberculosis is dependent on endogenous interferon-γ (IFNγ); endogenous IFNγ is, in turn, dependent on M. tuberculosis–induced IL-18 via caspase-1.
In conclusion, IL-32 is a cell-associated proinflammatory cytokine, which is specifically stimulated by mycobacteria through a caspase-1- and IL-18-dependent production of IFNγ.
Synthesis of IL-32, a cell-associated proinflammatory cytokine, was promoted by Mycobacterium tuberculosis and M. bovis, suggesting a role in a role in inflammation and host defense against tuberculosis.
Editors' Summary
Tuberculosis (TB) is a serious infectious disease that is becoming more common. Worldwide it causes around 2 million deaths every year, mostly in developing countries. Some 2 billion people—or one-third of the world's population—are chronically infected without active symptoms. In humans the disease is usually caused by a bacterium called Mycobacterium tuberculosis. Another related bacterium, Mycobacterium bovis, causes TB in cattle and sometimes in people.
The immune system, which defends the body against infections, involves a number of cells (for example, the white blood cells) and also chemicals. People with defects in their immune system are more likely to suffer from infectious diseases. Cytokines are one class of chemicals in the immune system. A particular cytokine called interleukin-32 (IL-32) has been shown to play a role in the development of inflammation, which is a part of the body's response to infection. Previous research has suggested that IL-32 might be of particular importance in the defenses against TB. In recent years scientists have discovered a lot about the processes involved in the “switching on” of the individual parts of the immune system in response to infection. However, very little is known about the factors influencing the switching on of production of IL-32.
Why Was This Study Done?
It would be useful to know more about the production of IL-32 because it would advance understanding of the immune system in general and, more specifically, how the body protects itself against bacteria, such as those that cause TB.
What Did the Researchers Do and Find?
Working with eight healthy volunteers, the researchers took white blood cells of a particular type (peripheral blood mononuclear cells) and exposed them to substances known as TLR agonists. Toll-like receptors (TLRs) are receptors on the surface of leukocytes that recognize specific components of microorganisms. Upon recognition of these microbial components, which function as TLR stimuli (or TLR agonists), signals are transmitted that activate the immune system and thus the host defense. Using a complex series of laboratory procedures, they found that one type of TLR agonist (known as LPS) produced a big increase in IL-32 production, whereas all the other types of TLR agonists that they used produced only small increases. The researchers tested M. tuberculosis and M. bovis bacteria to see whether they increased IL-32 production and they found that they did so, to a greater degree even than LPS. The researchers also learned other details about IL-32 and the pathway of chemical changes that eventually leads to its production.
What Do These Findings Mean?
The researchers say their study provides several important insights into the biology of IL-32. The findings confirm that IL-32 is an important factor in the body's defenses against TB. This information will help in understanding how the disease spreads and who is most vulnerable to it. Ultimately, it may assist in the search for new ways of treating and preventing the disease.
Additional Information.
Please access these Web sites via the online version of this summary at
The online encyclopedia Wikpedia has useful information on tuberculosis
Wikipedia also has useful information on the immune system
More detailed information about international efforts to control TB may be found at the Web sites of the International Union Against Tuberculosis and Lung Disease and the World Health Organization's Stop TB Department
PMCID: PMC1539091  PMID: 16903774
15.  Increased Responsiveness to Toll-Like Receptor 4 Stimulation in Peripheral Blood Mononuclear Cells from Patients with Recent Onset Rheumatoid Arthritis 
Mediators of Inflammation  2008;2008:132732.
Background. Cell signaling via Toll-like receptors (TLRs) leads to synovial inflammation in rheumatoid arthritis (RA). We aimed to assess effects of TLR2 and TLR4 stimulation on proinflammatory cytokine production by peripheral blood mononuclear cells (PBMCs) from patients with recent-onset RA, osteoarthrosis (OA), and healthy control (HC). Methods. PBMCs were stimulated with LPS, biglycan and cytokine mix. Cytokines were analyzed in supernatants with ELISA. Expression of toll-like receptors mRNA in leukocytes was analyzed using real-time qPCR. Results. PBMCs from RA patients spontaneously produced less IL-6 and TNFα than cells from OA and HC subjects. LPS increased cytokines' production in all groups. In RA patients increase was dramatic (30 to 48-fold and 17 to 31-fold, for respective cytokines) compared to moderate (2 to 8-fold) in other groups. LPS induced 15-HETE generation in PBMCs from RA (mean 251%) and OA patients (mean 43%), although only in OA group, the increase was significant. TLR2 and TLR4 gene expressions decreased in response to cytokine mix, while LPS enhanced TLR2 expression in HC and depressed TLR4 expression in OA patients. Conclusion. PBMCs from recent-onset RA patients are overresponsive to stimulation with bacterial lipopolysaccharide. TLR expression is differentially regulated in healthy and arthritic subjects.
PMCID: PMC2435281  PMID: 18584044
16.  Toll-like receptor 4–, 7–, and 8–activated myeloid cells from patients with X-linked agammaglobulinemia produce enhanced inflammatory cytokines 
Bruton tyrosine kinase (BTK) is a component of signaling pathways downstream from Toll-like receptors (TLRs) 2, 4, 7, 8, and 9. Previous work in BTK-deficient mice, cell lines, and cultured cells from patients with X-linked agammaglobulinemia (XLA) suggested defective TLR-driven cytokine production.
We sought to compare TLR-4–, TLR-7–, and TLR-8–induced cytokine production of primary cells from patients with XLA with that seen in control cells.
PBMCs from patients with XLA, freshly isolated plasmacytoid dendritic cells, monocytes, and monocytoid dendritic cells were activated with TLR-4, TLR-7, and TLR-8 agonists. Signaling intermediates and intracellular and secreted cytokine levels were compared with those seen in control cells.
Although TLR-4, TLR-7, and TLR-8 activation of nuclear factor κB and mitogen-activated protein kinase pathways in cells from patients with XLA and control cells were comparable, TLR-activated freshly isolated monocytes and monocytoid dendritic cells from patients with XLA produced significantly more TNF-α, IL-6, and IL-10 than control cells. TLR-7/8–activated plasmacytoid dendritic cells produced normal amounts of IFN-α. In murine models BTK regulates the degradation of Toll–IL-1 receptor domain–containing adaptor protein, terminating TLR-4–induced cytokine production. Although this might explain the heightened TLR-4–driven cytokine production we observed, Toll–IL-1 receptor domain–containing adaptor protein degradation is intact in cells from patients with XLA, excluding this explanation.
In contrast to previous studies with BTK-deficient mice, cell lines, and cultured cells from patients with XLA suggesting impaired TLR-driven cytokine production, these data suggest that BTK inhibits TLR-induced cytokine production in primary human cells.
PMCID: PMC3428022  PMID: 22088613
Bruton tyrosine kinase; X-linked agammaglobulinemia; Toll-like receptors; MyD88 adapter-like protein; Toll–IL-1 receptor domain–containing adapter protein
17.  B cell-activating factor of the tumor necrosis factor family (BAFF) is expressed under stimulation by interferon in salivary gland epithelial cells in primary Sjögren's syndrome 
B cell-activating factor (BAFF) has a key role in promoting B-lymphocyte activation and survival in primary Sjögren's syndrome (pSS). The cellular origin of BAFF overexpression in salivary glands of patients with pSS is not fully known. We investigated whether salivary gland epithelial cells (SGECs), the main targets of autoimmunity in pSS, could produce and express BAFF. We used quantitative RT-PCR, ELISA and immunocytochemistry in cultured SGECs from eight patients with pSS and eight controls on treatment with IL-10, tumor necrosis factor α (TNF-α), IFN-α and IFN-γ. At baseline, BAFF expression in SGECs was low in pSS patients and in controls. Treatment with IFN-α, IFN-γ and TNF-α + IFN-γ increased the level of BAFF mRNA in pSS patients (the mean increases were 27-fold, 25-fold and 62-fold, respectively) and in controls (mean increases 19.1-fold, 26.7-fold and 17.7-fold, respectively), with no significant difference between patients and controls. However, in comparison with that at baseline, stimulation with IFN-α significantly increased the level of BAFF mRNA in SGECs of pSS patients (p = 0.03) but not in controls (p = 0.2), which suggests that SGECs of patients with pSS are particularly susceptible to expressing BAFF under IFN-α stimulation. Secretion of BAFF protein, undetectable at baseline, was significantly increased after IFN-α and IFN-γ stimulation both in pSS patients (40.8 ± 12.5 (± SEM) and 47.4 ± 18.7 pg/ml, respectively) and controls (24.9 ± 8.0 and 9.0 ± 3.9 pg/ml, respectively), with no significant difference between pSS and controls. Immunocytochemistry confirmed the induction of cytoplasmic BAFF expression after stimulation with IFN-α and IFN-γ. This study confirms the importance of resident cells of target organs in inducing or perpetuating autoimmunity. Demonstrating the capacity of SGECs to express and secrete BAFF after IFN stimulation adds further information to the pivotal role of these epithelial cells in the pathogenesis of pSS, possibly after stimulation by innate immunity. Our results suggest that an anti-BAFF therapeutic approach could be particularly interesting in pSS.
PMCID: PMC1526588  PMID: 16507175
18.  Involvement of TLR7 MyD88-dependent signaling pathway in the pathogenesis of adult-onset Still's disease 
The objective of this study was to investigate the potential role of the Toll-like receptor 7 (TLR7) signaling pathway in the pathogenesis of adult-onset Still's disease (AOSD).
Frequencies of TLR7-expressing precursor of myeloid dendritic cells (pre-mDCs) and mDCs in 28 AOSD patients, 28 patients with systemic lupus erythematosus (SLE) and 12 healthy controls (HC) were determined by flow cytometry analysis. Transcript and protein levels of TLR7 signaling molecules in peripheral blood mononuclear cells (PBMCs) were evaluated by quantitative PCR and western blotting respectively. Serum cytokines levels were measured by ELISA.
Significantly higher median frequencies of TLR7-expressing pre-mDCs and mDCs were observed in AOSD patients (65.5% and 14.9%, respectively) and in SLE patients (60.3% and 14.4%, respectively) than in HC (42.8% and 8.8%, respectively; both P <0.001). Transcript and protein levels of TLR7-signaling molecules, including MyD88, TRAF6, IRAK4 and IFN-α, were upregulated in AOSD patients and SLE patients compared with those in HC. Disease activity scores were positively correlated with the frequencies of TLR7-expressing mDCs and expression levels of TLR7 signaling molecules in both AOSD and SLE patients. TLR7 ligand (imiquimod) stimulation of PBMCs resulted in significantly enhanced levels of interleukin (IL)-1β, IL-6, IL-18 and IFN-α in AOSD and SLE patients. Frequencies of TLR7-expressing mDCs and expression levels of TLR7 signaling molecules significantly decreased after effective therapy.
Elevated levels of TLR7 signaling molecules and their positive correlation with disease activity in AOSD patients suggest involvement of the TLR7 signaling pathway in the pathogenesis of this disease. The overexpression of TLR7 MyD88-dependent signaling molecules may be a common pathogenic mechanism for both AOSD and SLE.
PMCID: PMC3672755  PMID: 23497717
19.  Differential effects of cytokines and corticosteroids on Toll-like receptor 2 expression and activity in human airway epithelia 
Respiratory Research  2009;10(1):96.
The recognition of microbial molecular patterns via Toll-like receptors (TLRs) is critical for mucosal defenses.
Using well-differentiated primary cultures of human airway epithelia, we investigated the effects of exposure of the cells to cytokines (TNF-α and IFN-γ) and dexamethasone (dex) on responsiveness to the TLR2/TLR1 ligand Pam3CSK4. Production of IL-8, CCL20, and airway surface liquid antimicrobial activity were used as endpoints.
Microarray expression profiling in human airway epithelia revealed that first response cytokines markedly induced TLR2 expression. Real-time PCR confirmed that cytokines (TNF-α and IFN-γ), dexamethasone (dex), or cytokines + dex increased TLR2 mRNA abundance. A synergistic increase was seen with cytokines + dex. To assess TLR2 function, epithelia pre-treated with cytokines ± dex were exposed to the TLR2/TLR1 ligand Pam3CSK4 for 24 hours. While cells pre-treated with cytokines alone exhibited significantly enhanced IL-8 and CCL20 secretion following Pam3CSK4, mean IL-8 and CCL20 release decreased in Pam3CSK4 stimulated cells following cytokines + dex pre-treatment. This marked increase in inflammatory gene expression seen after treatment with cytokines followed by the TLR2 ligand did not correlate well with NF-κB, Stat1, or p38 MAP kinase pathway activation. Cytokines also enhanced TLR2 agonist-induced beta-defensin 2 mRNA expression and increased the antimicrobial activity of airway surface liquid. Dex blocked these effects.
While dex treatment enhanced TLR2 expression, co-administration of dex with cytokines inhibited airway epithelial cell responsiveness to TLR2/TLR1 ligand over cytokines alone. Enhanced functional TLR2 expression following exposure to TNF-α and IFN-γ may serve as a dynamic means to amplify epithelial innate immune responses during infectious or inflammatory pulmonary diseases.
PMCID: PMC2772856  PMID: 19835594
20.  Increase in peripheral blood mononuclear cell Toll-like receptor 2/3 expression and reactivity to their ligands in a cohort of patients with wet age-related macular degeneration 
Molecular Vision  2013;19:1826-1833.
To investigate Toll-like receptor (TLR) expression and reactivity in patients with the wet form age-related macular degeneration (AMD).
Blood samples were collected from 25 patients with wet AMD and 25 age-matched healthy controls. Peripheral blood mononuclear cells (PBMCs) were isolated with Ficoll-Hypaque density gradient centrifugation. Expression of TLR1 to TLR10 mRNAs in PBMCs from 15 patients with wet AMD and 15 controls was assessed with real-time PCR. TLR2 and TLR3 protein levels in PBMCs from six patients with wet AMD and six controls were measured with flow cytometry. After PBMCs were stimulated with peptidoglycan (PGN) and poly(I:C), the specific ligands of TLR2 and TLR3, cytokines interleukin-6 (IL-6), IL-8, VEGF, and monocyte chemoattractant protein-1 (MCP-1) production in 11 patients with wet AMD and 11 controls were assessed.
TLR2 and TLR3 mRNA and protein expression in the PBMCs of the patients with wet AMD was significantly higher than that in the controls. However, the difference in TLR1 and TLR4–10 mRNA expression between the two groups was not significant. The PBMCs of the patients with wet AMD produced more IL-6 and IL-8 proteins than the controls in response to PGN, a ligand for TLR2, and more IL-6 protein than the controls in response to poly(I:C), the ligand for TLR3. However, there was no significant difference in vascular endothelial growth factor and monocyte chemoattractant protein-1 production between the wet AMD group and the control group when the PBMCs were stimulated with PGN or poly(I:C).
Our data suggested that upregulation of TLR2 and TLR3 may be associated with the pathogenesis of wet AMD.
PMCID: PMC3742118  PMID: 23946637
21.  Toll-like receptor 7 and 9 defects in common variable immunodeficiency 
Common variable immunodeficiency (CVID) is characterized by hypogammaglobulinemia, reduced numbers of peripheral blood isotype-switched memory B cells, and loss of plasma cells.
Because Toll-like receptor (TLR) activation of B cells can initiate and potentially sustain normal B cell functions, we examined functional outcomes of TLR7 and TLR9 signaling in CVID B cells.
TLR7-mediated, TLR7/8-mediated, and TLR9-mediated cell proliferation, isotype switch, and immunoglobulin production by control and CVID B cells or isolated naive and memory B cell subsets were examined. We quantitated TNF-α, IL-6, and IL-12 production in response to TLR1-9 ligands and measured IFN-α production by TLR7-stimulated PBMCs and isolated plasmacytoid dendritic cells (pDCs). IFN-β mRNA expression by TLR3-stimulated fibroblasts was assessed. Results: Unlike CD27+ B cells of controls, TLR7-activated, TLR7/8-activated, or TLR9-activated CVID B cells or isolated CD27+ B cells did not proliferate, upregulate CD27, or shed surface IgD. TLR-stimulated CVID B cells failed to upregulate activation-induced cytosine deaminase mRNA or produce IgG and IgA. TLR7-stimulated PBMCs and pDCs produced little or no IFN-α. Reconstituting IFN-α in TLR7-stimulated CVID B-cell cultures facilitated proliferation, CD27 upregulation, and isotype switch. These TLR defects are restricted because CVID PBMCs stimulated with TLR ligands produced normal amounts of TNF-α, IL-6, and IL-12; TLR3-mediated expression of IFN-β by CVID fibroblasts was normal.
Defective TLR7 and TLR9 signaling in CVID B cells and pDCs, coupled with deficient IFN-α, impairs CVID B cell functions and prevents TLR-mediated augmentation of humoral immunity in vivo.
PMCID: PMC2908501  PMID: 19592080
Common variable immunodeficiency; Toll-like receptor; memory B cell; plasmacytoid dendritic cell; isotype switch; IFN-α
22.  Systems Biology Analysis of Sjögren’s Syndrome and MALT Lymphoma Development in Parotid Glands 
Arthritis and rheumatism  2009;60(1):81-92.
To identify key target genes and activated signal pathways associated with the disease pathogenesis by conducting a systems analysis of parotid gland manifesting primary Sjögren’s syndrome (pSS) and pSS/mucosa-associated lymphoid tissue (pSS/MALT) lymphoma phenotypes.
A systems biologic approach was used to analyze parotid gland tissues obtained from non-pSS, pSS and pSS/MALT lymphoma patients. Concurrent expression microarray profiling and proteomic analysis were performed followed by weighted gene co-expression network analysis (WGCNA).
Gene co-expression modules related to pSS and pSS/MALT lymphoma are significantly enriched with genes known to be involved in immune/defense response, apoptosis, cell signaling, gene regulation, and oxidative stress. A detailed functional pathway analysis indicates that the pSS-associated modules are enriched with genes involved in proteasome degradation, apoptosis, signal peptides (MHC) class I, complement activation, cell growth and death, and integrin-mediated cell adhesion. The pSS/MALT-associated modules are enriched with genes involved in translation, ribosome, protease degradation, signal peptides (MHC) class I, G13 signaling pathway, complement activation, and Integrin-mediated cell adhesion. The combined analysis of gene expression and proteomics data implicates six highly connected hub genes for distinguishing pSS from non-pSS controls, and eight hub genes for distinguishing pSS/MALT lymphoma from pSS.
Systems biologic analysis of pSS and pSS/MALT parotid glands reveals pathways and molecular targets associated with the disease pathogenesis. The identified gene modules/pathways provide further insights into the molecular mechanisms of pSS and pSS/MALT lymphoma. The identified disease hub genes represent promising targets for therapeutic intervention, diagnosis, and prognosis.
PMCID: PMC2718690  PMID: 19116902
23.  Elevated expression of the toll like receptors 2 and 4 in obese individuals: its significance for obesity-induced inflammation 
Expression profile of the toll like receptors (TLRs) on PBMCs is central to the regulation of proinflammatory markers. An imbalance in the TLRs expression may lead to several types of inflammatory disorders. Furthermore, the dynamic regulation of inflammatory activity and associated impaired production of cytokines by peripheral blood mononuclear cells (PBMCs) in obese individulas remain poorly understood. Therefore, we determined the perturbation in TLRs (TLR2 and TLR4), their adaptor proteins (MyD88, IRAK1 and TRAF6) expression in PBMCs/subcutaneous adipose tissue (AT) as well as inflammatory cytokines changes in obese individuals.
mRNA expression levels of TLR2, TLR4, IL-6, TNF-α and adaptor proteins were determined by RT-PCR. TLR2, TLR4 and adaptor proteins expression in AT was determined by immunohistochemistry.
Obese and overweight individuals showed significantly increased expression of TLR2, TLR4 and MyD88 in both PBMCs and AT as compared with lean individuals (P < 0.05). Interestingly, we found a remarkably higher expression of TLRs in obese and overweight individuals with type 2 diabetes (P < 0.05). Increased expression of TLR2, TLR4, MyD88 and IRAK1 correlated with body mass index (BMI) (TLR2: r = 0.91; TLR4: r = 0.88, P <0.0001; MyD88: r = 0.95, P < 0.0001; IRAK1 r = 0.78, P < 0.002). TLRs’ expression was also correlated with fasting blood glucose (FBG) (TLR2: r = 0.61, P < 0.002; TLR4: r = 0.52, P < 0.01) and glycated haemoglobin (HbA1c) ( TLR2: r = 0.44, P <0.03; TLR4: r = 0.48, P < 0.03). Transcript levels of IL-6 and TNF-α were highly elevated in obese subjects compared to lean subjects. There was a strong association of TLRs’ expression in PBMCs with TNF-α (TLR2: r = 0.92; TLR4: r = 0.92; P < 0.0001) and IL-6 (TLR2: r = 0.91, P < 0.0001; TLR4: r = 0.81; P < 0.001). Similarly adaptor proteins were significantly correlated with TNF-α (MyD88: r = 0.9, P < 0.0001; IRAK1: r = 0.86; P < 0.0002) and IL-6 (MyD88: r = 0.91, P < 0.0001; IRAK1: 0.77; P < 0.002).
TLRs and adapter proteins were overexpressed in PBMCs from obese subjects, which correlated with increased expression of TNF-α and IL-6. This association may explain a potential pathophysiological link between obesity and inflammation leading to insulin resistance.
PMCID: PMC3542010  PMID: 23191980
24.  Differential expression of Toll-like receptors on human alveolar macrophages and autologous peripheral monocytes 
Respiratory Research  2010;11(1):2.
Toll-like receptors (TLRs) are critical components in the regulation of pulmonary immune responses and the recognition of respiratory pathogens such as Mycobacterium Tuberculosis (M.tb). Through examination of human alveolar macrophages this study attempts to better define the expression profiles of TLR2, TLR4 and TLR9 in the human lung compartment which are as yet still poorly defined.
Sixteen healthy subjects underwent venipuncture, and eleven subjects underwent additional bronchoalveolar lavage to obtain peripheral blood mononuclear and bronchoalveolar cells, respectively. Surface and intracellular expression of TLRs was assessed by fluorescence-activated cell sorting and qRT-PCR. Cells were stimulated with TLR-specific ligands and cytokine production assessed by ELISA and cytokine bead array.
Surface expression of TLR2 was significantly lower on alveolar macrophages than on blood monocytes (1.2 ± 0.4% vs. 57 ± 11.1%, relative mean fluorescence intensity [rMFI]: 0.9 ± 0.1 vs. 3.2 ± 0.1, p < 0.05). The proportion of TLR4 and TLR9-expressing cells and the rMFIs of TLR4 were comparable between alveolar macrophages and monocytes. The surface expression of TLR9 however, was higher on alveolar macrophages than on monocytes (rMFI, 218.4 ± 187.3 vs. 4.4 ± 1.4, p < 0.05) while the intracellular expression of the receptor and the proportion of TLR9 positive cells were similar in both cell types. TLR2, TLR4 and TLR9 mRNA expression was lower in bronchoalveolar cells than in monocytes.
Pam3Cys, LPS, and M.tb DNA upregulated TLR2, TLR4 and TLR9 mRNA in both, bronchoalveolar cells and monocytes. Corresponding with the reduced surface and mRNA expression of TLR2, Pam3Cys induced lower production of TNF-α, IL-1β and IL-6 in bronchoalveolar cells than in monocytes. Despite comparable expression of TLR4 on both cell types, LPS induced higher levels of IL-10 in monocytes than in alveolar macrophages. M.tb DNA, the ligand for TLR9, induced similar levels of cytokines in both cell types.
The TLR expression profile of autologous human alveolar macrophages and monocytes is not identical, therefore perhaps contributing to compartmentalized immune responses in the lungs and systemically. These dissimilarities may have important implications for the design and efficacy evaluation of vaccines with TLR-stimulating adjuvants that target the respiratory tract.
PMCID: PMC2817655  PMID: 20051129
25.  Pneumococcal Lipoteichoic Acid (LTA) Is Not as Potent as Staphylococcal LTA in Stimulating Toll-Like Receptor 2  
Infection and Immunity  2003;71(10):5541-5548.
Streptococcus pneumoniae is a leading cause of gram-positive sepsis, and lipoteichoic acid (LTA) may be important in causing gram-positive bacterial septic shock. Even though pneumococcal LTA is structurally distinct from the LTA of other gram-positive bacteria, the immunological properties of pneumococcal LTA have not been well characterized. We have investigated the ability of LTAs to stimulate human monocytes by using highly pure and structurally intact preparations of pneumococcal LTA and its two structural variants. The variants were pneumococcal LTA with only one acyl chain (LTA-1) and completely deacylated LTA (LTA-0). The target cells used in the study were peripheral blood mononuclear cells (PBMCs) and two model cell lines (CHO/CD14/TLR2 and CHO/CD14/TLR4) that express human CD25 protein in response to Toll-like receptor 2 (TLR2) and TLR4 stimulation, respectively. Intact pneumococcal LTA and LTA-1 stimulated PBMC and CHO/CD14/TLR2 cells in a dose-dependent manner but did not stimulate CHO/CD14/TLR4 cells. Pneumococcal LTA was about 100-fold less potent than Staphylococcus aureus LTA in stimulating the CHO/CD14/TLR2 cells and PBMCs. LTA-0 (or pneumococcal teichoic acid) stimulated neither CHO/CD14/TLR2 nor CHO/CD14/TLR4 cells even at high concentrations. Excess teichoic acid, LTA-0, antibodies to phosphocholine, or antibodies to TLR4 did not inhibit the LTA-induced TLR2 stimulation. However, antibodies to CD14, TLR1, or TLR2 suppressed tumor necrosis factor alpha (TNF-α) production by PBMCs in response to LTA or LTA-1. These results suggest that pneumococcal LTA with one or both acyl chains stimulates PBMCs primarily via TLR2 with the help of CD14 and TLR1.
PMCID: PMC201083  PMID: 14500472

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