Lower peripheral blood Toll-like receptor 3 expression is associated with an unfavorable outcome in severe COVID-19 patients.

The role of innate immunity in COVID-19 is not completely understood. Therefore, this study explored the impact of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection on the expression of Pattern Recognition Receptors (PRRs) in peripheral blood cells and their correlated cytokines. Seventy-nine patients with severe COVID-19 on admission, according to World Health Organization (WHO) classification, were divided into two groups: patients who needed mechanical ventilation and/or deceased (SEVERE, n = 50) and patients who used supplementary oxygen but not mechanical ventilation and survived (MILD, n = 29); a control group (CONTROL, n = 17) was also enrolled. In the peripheral blood, gene expression (mRNA) of Toll-like receptors (TLRs) 3, 4, 7, 8, and 9, retinoic-acid inducible gene I (RIGI), NOD-like receptor family pyrin domain containing 3 (NLRP3), interferon alpha (IFN-α), interferon beta (IFN-ß), interferon gamma (IFN-γ), interferon lambda (IFN-λ), pro-interleukin(IL)-1ß (pro-IL-1ß), and IL-18 was determined on admission, between 5-9 days, and between 10-15 days. Circulating cytokines in plasma were also measured. When compared to the COVID-19 MILD group, the COVID-19 SEVERE group had lower expression of TLR3 and overexpression of TLR4.

Variety of endosomal TLRs and Interferons (IFN-α, IFN-ß, IFN-γ) expression profiles in patients with SLE, SSc and MCTD.

We investigated Toll-like receptor (TLR)-3/-7/-8/-9 and interferon (IFN)-α/ß/γ mRNA expression in whole blood and serum IFN-α/ß/γ levels in patients with mixed connective tissue disease (MCTD), systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) and in healthy subjects to assess the association between the TLR-IFN expression and severity of and susceptibility to diseases, and identify potential biomarkers. Expression of the IFN-γ, TLR-3 and TLR-8 was detected only in SLE patients. TLR-7, IFN-α and IFN-ß expression was highest in SLE, while TLR-9 expression was highest in SSc patients. In SLE and MCTD patients a strong correlation was observed between TLR-7 and IFN-α expression and IFN-ß and IFN-α expression. In MCTD patients, negative correlation between IFN-α and TLR-9 and TLR-7 and TLR-9 was revealed. TLR-9 expression in anti-U1-70k-negative, anti-C negative and anti-SmB-negative MCTD patients was higher than in MCTD-positive patients. We observed negative correlations between serum IFN-α levels and TLR-7 expression and C3 and C4 levels in SLE patients. In SLE patients we observed that with increased IFN-γ, TLR-3 and TLR-8 expression increased the value of C3 and C4. Our results confirmed that the endosomal TLR-IFN pathway seems to be more important in SLE than in MCTD or SSc, and that IFN-α and IFN-ß may be possible biomarkers for SLE.

Prognostic factors for survival of herpes simplex virus-associated hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) occurs in neonates with disseminated infection of herpes simplex virus (HSV). Little has been reported on the control of rapid HLH progression. We studied the cytokine profile and genetic basis of two index cases with divergent outcomes after early treatment of type 2 HSV infection. One survivor had fever and elevated serum levels of tumor necrosis factor (TNF)-α, interleukin-6 (IL-6), interferon (IFN)-ß, and IFN-γ at diagnosis. The other neonate had no fever or TNF-α production, but significant IL-6 or IFN responses during the treatment course, and died 19 days after birth. Among 16 reported cases of neonatal HSV-HLH including index cases, eight deceased neonates experienced significantly less fever at presentation (p = 0.028), lower platelet counts (p = 0.019), and lower ratios of soluble IL-2 receptor (sIL-2R) to ferritin levels (p = 0.044) than eight survivors. The 100-day overall survival rates were significantly higher in patients with fever (p = 0.004), > 100 × 109/L of platelet counts (p = 0.035) or > 20 of sIL-2R/ferritin ratio at diagnosis (p = 0.004). The first febrile and cytokine responses to HSV infection predict the early outcome of neonatal HSV-HLH.

Novel nucleotide variants in SLA-DOB and CD4 are associated with immune traits in pregnant sows.

Reinforcing the immunity of pregnant sows can not only improve their own health condition but also increase the survival rate and healthy status of their piglets. This study aims to find single-nucleotide polymorphism (SNP) and molecular markers that are associated with the immune traits of pregnant sows. SLA-DOB and CD4 were selected as candidate genes, and blood samples were randomly collected from pregnant Landrace sows and used to detect T-lymphocyte subsets, interferon alpha, interleukin 6, Toll-like receptor 3, serum antibody immunoglobulin G, and porcine reproductive and respiratory syndrome virus-specific antibody. Then, association analyses were conducted for the polymorphic sites of candidate genes with immune traits. We found 12 mutations in the two genes and conducted an association study with eight of them. Our results indicated that among the eight mutations, SNP1, SNP2, and SNP3 of the SLA-DOB gene and Ins9, SNP10, and SNP11 in the CD4 gene are newly discovered mutations. Except for SNP1, SNP3, and SNP11, the other five SNPs are associated with at least one immune trait tested. Especially, SNP2 and Ins9 are significantly associated with at least one of the T-lymphocyte subgroups and at least one antibody. These novel mutations have potential important effects on the polymorphic loci of the above immune traits in pregnant sows. The results suggest that the SLA-DOB and CD4 genes and their genetic mutations can be considered as important candidate genes and mutations for the immunity of pregnant sows.

Decreased TLR3 in Hyperplastic Adipose Tissue, Blood and Inflamed Adipocytes is Related to Metabolic Inflammation.

BACKGROUND/AIMS: Obesity is characterized by the immune activation that eventually dampens insulin sensitivity and changes metabolism. This study explores the impact of different inflammatory/ anti-inflammatory paradigms on the expression of toll-like receptors (TLR) found in adipocyte cultures, adipose tissue, and blood. METHODS: We evaluated by real time PCR the impact of acute surgery stress in vivo (adipose tissue) and macrophages (MCM) in vitro (adipocytes). Weight loss was chosen as an anti-inflammatory model, so TLR were analyzed in fat samples collected before and after bariatric surgery-induced weight loss. Associations with inflammatory and metabolic parameters were analyzed in non-obese and obese subjects, in parallel with gene expression measures taken in blood and isolated adipocytes/ stromal-vascular cells (SVC). Treatments with an agonist of TLR3 were conducted in human adipocyte cultures under normal conditions and upon conditions that simulated the chronic low-grade inflammatory state of obesity. RESULTS: Surgery stress raised TLR1 and TLR8 in subcutaneous (SAT), and TLR2 in SAT and visceral (VAT) adipose tissue, while decreasing VAT TLR3 and TLR4. MCM led to increased TLR2 and diminished TLR3, TLR4, and TLR5 expressions in human adipocytes. The anti-inflammatory impact of weight loss was concomitant with decreased TLR1, TLR3, and TLR8 in SAT. Cross-sectional associations confirmed increased V/ SAT TLR1 and TLR8, and decreased TLR3 in obese patients, as compared with non-obese subjects. As expected, TLR were predominant in SVC and adipocyte precursor cells, even though expression of all of them but TLR8 (very low levels) was also found in ex vivo isolated and in vitro differentiated adipocytes. Among SVC, CD14+ macrophages showed increased TLR1, TLR2, and TLR7, but decreased TLR3 mRNA. The opposite patterns shown for TLR2 and TLR3 in V/ SAT, SVC, and inflamed adipocytes were observed in blood as well, being TLR3 more likely linked to lymphocyte instead of neutrophil counts. On the other hand, decreased TLR3 in adipocytes challenged with MCM dampened lipogenesis and the inflammatory response to Poly(I:C). CONCLUSION: Functional variations in the expression of TLR found in blood and hypertrophied fat depots, namely decreased TLR3 in lymphocytes and inflamed adipocytes, are linked to metabolic inflammation.

TLR2 and TLR3 expression as a biomarker for the risk of doxorubicin-induced heart failure.

Doxorubicin (Dox) is limited in its use because of its adverse effect of inducing irreversible heart dysfunction. Innate immune factors, including toll-like receptors (TLRs), play important roles in most cardiac diseases and doxorubicin-induced cardiotoxicity. In this study, subjects were divided into the following groups: healthy controls (n = 62), HF group (n = 60), Dox group (n = 82), and Dox-HF group (n = 32). Expressions of TLR mRNAs in peripheral blood mononuclear cells were detected by RT-PCR. Western blotting was used to quantify protein expressions of Peripheral blood mononuclear cells (PBMCs) TLRs and their downstream signal proteins. The release of inflammatory factors was detected by ELISA. Results indicated that TLR2 was increased and TLR3 was decreased between the control group and Dox group, and between the Dox group and Dox-HF group. Serum inflammatory factors were comparable between the HF group, the Dox group, and the Dox-HF group. This study suggested that TLR2 and TLR3 are up- and down-regulated, respectively, in doxorubicin-treated patients who develop heart dysfunctions. This may suggest a predictive role for TLR2-TLR3 imbalance in doxorubicin-induced heart failure.

Analysis of host Toll-like receptor 3 and RIG-I-like receptor gene expression in patients with abdominal aortic aneurysm.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is a vascular disease relatively common in the elderly population. Although some events that contribute to the development and progression of AAA are known, there are limited data examining the association of Toll-like receptor 3 (TLR3) and RIG-I-like receptor expression with the pathogenesis of AAAs. In this study, we investigated the gene and protein expression of TLR3 and RIG-I-like receptors (RIG-I and MDA5) in aortic wall and blood of AAA patients and examined the relationship between their expression and immune response. METHODS: Total RNA was extracted from aortic wall tissues and blood samples collected from 20 patients with AAA and blood samples of 17 healthy volunteers without aortic aneurysm. To evaluate the DDX58 (RIG-I), IFIH1 (MDA5), and TLR3 gene expression level, quantitative real-time polymerase chain reaction was used. Extracellular cytokine and pattern recognition receptor levels were quantified by enzyme-linked immunosorbent assays. RESULTS: TLR3, RIG-I, and MDA5 were constitutively expressed in both aortic tissues and blood samples from AAA patients and healthy volunteers. In patients with AAA, higher TLR3 expression in aortic tissues than in blood was found (P = .004). The DDX58 messenger RNA expression was higher in blood of patients with AAA compared with healthy subjects (P = .021). A significantly higher level of plasma interleukin 4 was noticed in patients with AAA than in healthy individuals (P = .008). CONCLUSIONS: This study suggests that RIG-I and TLR3 seem to be important factors in the pathogenesis of AAA.

Toll-like receptor 3 (TLR-3), TLR-4 and CD80 expression in peripheral blood mononuclear cells and urinary CD80 levels in children with idiopathic nephrotic syndrome.

BACKGROUND: The aims of this study were (1) to detect toll-like receptor (TLR)-3, TLR-4 and CD80 expression in peripheral blood mononuclear cells (PBMCs) and estimate urinary CD80 levels in children with idiopathic nephrotic syndrome and (2) to investigate the utility of these markers to differentiate between biopsy-proven minimal change disease (MCD) and focal segmental glomeruloscelerosis (FSGS). METHODS: The study included 70 patients with idiopathic nephrotic syndrome (NS), of whom 40 had steroid-sensitive NS (SSNS; 25 with active NS, 15 in remission) and 30 had steroid-resistant NS (SRNS) patients, and 23 healthy controls. TLR-3, TLR- 4 and CD80 mRNA expression levels in PBMCs were determined and the urinary CD80 level estimated. RESULTS: Median TLR-3, TLR-4 and CD80 mRNA expression levels were higher in patients with active SSNS than in those with SRNS, and the latter patient group also had significantly lower expression levels than the controls. The expression levels of these markers were associated with reductions in remission. Patients with biopsy-proven MCD had higher median expression levels of these markers than those with FSGS, but the differences were not statistically significant. Median urinary CD80/creatinine values were significantly higher in patients with SSNS and SRNS than in the controls and steroid-sensitive patients in remission (p < 0.001). CD80 levels were also significantly higher in patients with MCD than in those with FSGS (p = 0.002). A cut-off level of >914.5 ng/g had a sensitivity of 86.6%, specificity 71.4% and area under the curve of 0.828 (95% confidence interval 0.678-0.978, p = 0.002) for the diagnosis of MCD. CONCLUSIONS: Increased expressions of TLR-3, TLR-4 and CD80 mRNA and the level of urinary CD80/creatinine could be useful markers to differentiate patients of SSNS in relapse from those with SRNS. Further these markers can also distinguish biopsy proven MCD from FSGS in SRNS patients.

Low expression of Toll-like receptors in peripheral blood mononuclear cells of pediatric patients with acute lymphoblastic leukemia.

Cancer is the second most common cause of death among children aged 1-14 years. Leukemia accounts for one-third of all childhood cancers, 78% of which is acute lymphoblastic leukemia (ALL). The development of cancer has been associated with malignant cells that express low levels of immunogenic molecules, which facilitates their escape from the antineoplastic immune response. It is thought that it may be possible to rescue the antineoplastic immune response through the activation of recognition receptors, such as Toll-like receptors (TLRs), which activate the innate immune system. TLRs are type I membrane glycoproteins expressed mainly in immune system cells such as monocytes, neutrophils, macrophages, dendritic cells, T, B and natural killer cells. The aim of the present study was to evaluate the expression of TLR1, TLR3, TLR4, TLR7 and TLR9 in peripheral blood mononuclear cells (PBMCs) in patients with ALL and prior to any treatment. PBMCs were obtained from 50 pediatric patients diagnosed with ALL and from 20 children attending the ophthalmology and orthopedics services. The mean fluorescence intensity was obtained by analysis of immunofluorescence. We found lower expression levels of TLR1, TLR3, TLR4, TLR7 and TLR9 in PBMCs from patients with ALL compared with those from control patients. We also observed that the PBMCs from patients with Pre-B and B ALL had lower TLR4 expression than controls and patients with Pro-B, Pre-B, B and T ALL had lower TLR7 expression than controls. The present study is the first to demonstrate reduced expression of TLRs in PBMCs from pediatric patients with ALL. This finding is of great relevance and may partly explain the reduction in the antineoplastic immune response in patients with ALL.

Expression of Toll­like receptors 3 and 9 in Egyptian systemic lupus erythematosus patients.

BACKGROUND: Systemic lupus erythematosus (SLE) is a common complex disease characterized by chronic generalized inflammation which may involve several tissues and organs. OBJECTIVE: The aim of this work was to study the expression of Toll-like receptors (TLR) 3 and 9 in SLE patients, and to investigate their relationship to clinical features, disease activity, and damage. PATIENTS AND METHODS: The current study included 24 Egyptian female SLE patients and 15 matched controls. Disease activity was assessed using the SLE Disease Activity Index (SLEDAI) and damage using the Systemic Lupus International Collaborating Clinics (SLICC) index. Expression of TLR3 and TLR9 in B- (CD19-positive) and T-lymphocytes (CD3-positive) was studied using flow cytometry. RESULTS: Patient age ranged between 17 and 42 years (mean 26.17 ± 5.78 years). There was a significant difference between patients and controls regarding TLR3/CD3, TLR3/CD19, TLR9/CD3, and TLR9/CD19 expression (p < 0.0001). There were significant correlations of TLR3/CD3, TLR3/CD19, and TLR9/CD19 with serum creatinine (r = 0.52, p = 0.009; r = 0.504, p = 0.012; and r = 0.58, p = 0.003; respectively) and negative correlations with ALT levels (r = -0.42, p = 0.04; r = -0.49, p = 0.016; and r = -0.472, p = 0.02; respectively). CONCLUSION: The results of the study suggest that TLR3 and TLR9 play a role in the pathogenesis of SLE, and have an impact on organ involvement in this disease. More studies concerning the biology and function of TLRs are required in larger patient cohorts, and may lead to development of a new class of drugs.

TLR3 and TLR4 as potential clinically biomarkers of cardiovascular risk in coronary artery disease (CAD) patients.

Coronary artery disease (CAD), as a lipid-driven and inflammation-driven disease, has threatened thousands of patients' lives. Toll-like receptors, the most characterized innate immune receptors, have recently been demonstrated to play a key role in coronary artery disease, particularly Toll-like receptor (TLR) 3 and TLR4. We examined TLR3, TLR4, and associated inflammatory factors expression in monocytes and their signaling pathway proteins in patients with varying degrees of coronary artery atherosclerosis [group S (single diseased vessel), n = 36; group D (double diseased vessels), n = 36; group T (three diseased vessels), n = 33 compared with controls (n = 35)]. In mononuclear cells, TLR3 mRNA and protein, and IRF-3 were significantly down-regulated as the coronary arteries stenosis number increased. However, TLR4 mRNA and protein, and MyD88 were significantly increased in patients with coronary artery stenosis compared with controls, and were associated with the number of stenoses. In serum, there was significant up-regulation in TNF-α, IL-8, and MCP-1 and obvious down-regulation in INF-ß and IP-10 with severity of CAD. This study demonstrates differential expression of TLR3 and TLR4 at both the mRNA and protein level in both mononuclear cells and downstream serum readouts of patients with CAD compared with the control. The expression of TLR4 and TLR3 closely correlated with the severity of coronary artery disease as reflected by the number of coronary artery stenoses. TLR3 and TLR4 have the potential to be a clinically useful biomarker of cardiovascular risk.

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.

PURPOSE: To investigate Toll-like receptor (TLR) expression and reactivity in patients with the wet form age-related macular degeneration (AMD). METHODS: 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. RESULTS: 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). CONCLUSIONS: Our data suggested that upregulation of TLR2 and TLR3 may be associated with the pathogenesis of wet AMD.

Expression of Toll like receptors 3 & 7 in peripheral blood from patients with chronic hepatitis C virus infection and their correlation with interferon-alpha.

Toll-like receptor 3 (TLR3) and Toll-like receptor 7 (TLR7) are pathogen- recognition receptors that are expressed on innate immune cells. They recognize viral RNA which induces their activation with subsequent increase in IFN-alpha transcription. It has been postulated that HCV may cause down regulation of these receptors as one of immune evading mechanisms that participate in viral persistence. The aim of this work is to investigate the expression of Toll-like receptors 3 (TLR3) and 7 (TLR7) in peripheral blood from patients with chronic hepatitis C infection and correlate their expression to the peripheral blood expression of interferon-alpha (IFN-alpha), viral load and histopathology of the liver. IFN-alpha, TLR3 and TLR7 expression in peripheral blood from patients with chronic hepatitis C infection (n = 30) and from healthy controls (n = 20) were measured by real time polymerase chain reaction. Viral load and Liver biopsy were done for all patients. The results showed lower expression of TLR3 and TLR7 in patients than controls, and levels of expression correlated positively with IFN-alpha expression. No correlation was found between TLR3 and TLR7 and viral load or histopathological staging and grading of the liver tissue. In conclusion, HCV may induce down regulation of TLRs (TLR3 and TLR7) expression on innate immune cells with subsequent decrease in INF-alpha production suggesting that new therapies that aim to increase the expression level of TLRs may help in treatment of HCV infection.

Update: biomarkers for idiopathic inflammatory myopathies.

PURPOSE OF REVIEW: Establishing diagnoses and distinguishing active disease from chronic injury remain significant clinical challenges in idiopathic inflammatory myopathies (IIM). Recent 'discovery' approaches utilizing novel genomic and proteomic techniques have revealed candidate molecular biomarkers to augment clinical and classical histological data. RECENT FINDINGS: Whole blood and serum Type 1 interferons (IFN-1) and IFN-1 inducible genes are gaining traction as disease biomarkers in IIM. IFNß is emerging as a disease activity marker specifically for dermatomyositis. Recently, molecules associated with innate immune-cell function, including TLR-3, high mobility group box (HMGB)-1, B7 Homolog 1, S100A4, and resistin have been detected in tissues of dermatomyositis patients. Serum Interleukin-17 (IL-17) and IL-23 correlate with active disease in early IIM. Antibodies recognizing the Survival Motor Neuron complex have been newly identified in a subset of patients with polymyositis. Protein aggregates are potential disease activity sensors for inclusion body myositis. Skin and lung harbor potential biomarkers for IIM. SUMMARY: Recent advances in understanding the pathogenesis of IIM have led to discovery of molecules that are candidate biomarkers of disease activity. Type 1 interferon and myeloid-cell signatures are leading candidate markers for use in IIM activity monitoring.

Increase of toll-like receptor 4 but decrease of interleukin-8 mRNA expression among ischemic stroke patients under aspirin treatment.

OBJECTIVES: Toll-like receptors (TLRs) are molecules conserved in evolution for detecting pathogen invasions and tissue damage and are involved in atherogenesis. This study explores the mRNA expression of TLRs and their probable role in further disease occurrence among ischemic stroke patients. DESIGN AND METHODS: A total of 89 ischemic stroke patients and 166 controls were recruited for this study. Total RNA was extracted and mRNA was reverse-transcribed to cDNA and was analyzed for TLRs and interleukin 8 (IL8). RESULTS: The TLR4 mRNA expression level is significantly higher in the stroke group. Conversely, IL-8 mRNA levels decreased significantly in the patient group. CONCLUSION: Our results suggest that TLR4 overexpression in mRNA levels is observed in stroke patients, which might account for the probable inflammatory injury before or after stroke. A reduction of IL-8 expression could result from the downregulatory effects of aspirin.

Toll-like receptor expression and responsiveness are increased in viraemic HIV-1 infection.

OBJECTIVES: Toll-like receptors (TLR) are important in pathogen recognition and may play a role in HIV disease. We evaluated the effect of chronic untreated and treated HIV-1 infection on systemic TLR expression and TLR signalling. METHODS: Two hundred HIV-infected and uninfected women from a Kenya cohort participated in the studies. TLR1 to TLR10 messenger RNA expression was determined by quantitative reverse transcriptase polymerase chain reaction in peripheral blood mononuclear cells (PBMC). TLR ligand responsiveness was determined in or using ex-vivo PBMC by cytokine production in culture supernatants. RESULTS: Chronic, untreated HIV-1 infection was significantly associated with increased mRNA expression of TLR6, TLR7, and TLR8 and when analysis was limited to those with advanced disease (CD4 cell count < 200 cells/ml) TLR2, TLR3, and TLR4 were additionally elevated. TLR expression correlated with the plasma HIV-RNA load, which was significant for TLR6 and TLR7. In vitro HIV single-stranded RNA alone could enhance TLR mRNA expression. PBMC of HIV-infected subjects also demonstrated profoundly increased proinflammatory responsiveness to TLR ligands, suggesting sensitization of TLR signalling in HIV. Finally, viral suppression by HAART was associated with a normalization of TLR levels. CONCLUSION: Together, these data indicate that chronic viraemic HIV-1 is associated with increased TLR expression and responsiveness, which may perpetuate innate immune dysfunction and activation that underlies HIV pathogenesis, and thus reveal potential new targets for therapy.