Function Of Toll-like Receptors Research Articles

Toll-like receptors landscape in Takifugu fasciatus: Dynamic responses to Aeromonas hydrophila, poly I:C, and Ichthyophthirius multifiliis challenge

Toll like receptors (TLRs) are essential for host defense, recognizing pathogen-associated molecular patterns (PAMPs) from infectious agents. However, limited information is available on TLRs in Takifugu fasciatus, a model species for comparative genomics and aquaculture. This study identified twelve T. fasciatus TLR genes, mapping them across nine chromosomes. Phylogenetic analysis revealed that the TfTLRs share orthologs from five vertebrate subfamilies, excluding the TLR4 subfamily. Gene synteny analysis confirmed the conservation of neighboring genes of TfTLRs within Neoteleostei, validating our annotation of TfTLRs. RT-PCR analysis showed ubiquitous expression of all TfTLRs across nine tissues, underscoring their role in immune homeostasis. Furthermore, RT-qPCR analysis showed significant expression changes in TfTLR2, TfTLR3, TfTLR5m, TfTLR5s and TfTLR8 in response to Aeromonas hydrophilia infection in vivo, while TfTLR3, TfTLR5m, TfTLR22, and TfTLR23 were responsive to poly I:C both in vivo and in vitro. In contrast, no significant TfTLRs expression changes were observed upon Ichthyophthirius multifiliis infection, suggesting their distinct roles in pathogens recognition. These findings deepen our understanding of TLR function in teleosts, providing valuable insights for future immune research in fish species.

Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling

The discovery of Toll-like receptors (TLRs) represented a significant breakthrough that paved the way for the study of host-pathogen interactions in innate immunity. However, there are still major gaps in understanding TLR function, especially regarding the early dynamics of downstream TLR pathways. Here, we present a label-free optical biosensor-based assay as a method for detecting TLR activation in a native and label-free environment and defining the dynamics of TLR pathway activation. This technology is sufficiently sensitive to detect TLR signaling and readily discriminates between different TLR signaling pathways. We define pharmacological modulators of cell surface and endosomal TLRs and downstream signaling molecules and uncover TLR signaling signatures, including potential biased receptor signaling. These findings highlight that optical biosensor assays complement traditional assays that use a single endpoint and have the potential to facilitate the future design of selective drugs targeting TLRs and their downstream effector cascades.

The Toll-like receptor 4 antagonist TAK-242 in combination with sodium hyaluronate alleviates postoperative abdominal adhesion in a mouse model

Postoperative abdominal adhesion is one of the most common complications after abdominal surgery. The Toll-like receptor 4 (TLR4) signaling pathway is one of the most common inflammation-related pathways, and it has been demonstrated that TLR4 is highly expressed in adhesive tissues; however, the function of TLR4 in adhesion formation has not yet been studied. In the present study, the expression of TLR4 was first detected by immunohistochemical (IHC) and double-immunofluorescence staining in 40 mice, which were randomly divided into four groups, and sacrificed at 1, 3, 5 and 7 days after surgery. Subsequently, another 40 mice were randomly divided into five groups; with the exception of the sham group, the other groups were modeled and treated with saline that contained DMSO, sodium hyaluronate (HA), TAK-242 or TAK-242 + HA (applied to damaged peritoneal wounds). A total of 7 days after surgery, the mice were sacrificed and specimens were collected. Inflammation was detected by hematoxylin and eosin staining, and ELISA of transforming growth factor- β1 (TGF-β1) and interleukin-6 (IL-6); collagen deposition was examined by Masson staining and IHC staining of α-SMA; and reactive oxygen species (ROS) were detected by ROS staining and malondialdehyde (MDA) assay. The results revealed that TLR4 was highly expressed in the adhesive tissues at 3, 5 and 7 days after surgery. In addition, TAK-242 + HA treatment could reduce abdominal adhesion formation, exhibiting lower Nair’s score and inflammation scores, lower TGF-β1 and IL-6 levels, and lower collagen thickness and α-SMA levels compared with those in the control group. In addition, the TAK-242 + HA group had lower levels of ROS and MDA compared with those in the control group. The present study revealed that TLR4 was highly expressed in the process of adhesion formation and its inhibitor, TAK-242, combined with HA, could reduce adhesion formation by reducing inflammation and ROS, and alleviating collagen deposition.

Toll-Like Receptor 2 Deficiency Exacerbates Dextran Sodium Sulfate-Induced Intestinal Injury through Marinifilaceae-Dependent Attenuation of Cell Cycle Signaling

Background: Ulcerative colitis (UC) is an intestinal disorder marked by chronic, recurring inflammation, yet its underlying mechanisms have not been fully elucidated. Methods: The current research dealt with examining the biological impacts of toll-like receptor 2 (TLR2) on dextran sulfate sodium (DSS)-triggered inflammation in the intestines of wild-type (WT) and TLR2-knockout (TLR2-KO) colitis mouse models. To elucidate the protective function of TLR2 in DSS-triggered colitis, RNA-sequencing (RNA-Seq) was carried out to compare the global gene expression data in the gut of WT and TLR2-KO mice. Further, 16S rRNA gene sequencing revealed notable variations in gut microbiota composition between WT and TLR2-KO colitis mice. Results: It was revealed that TLR2-KO mice exhibited increased susceptibility to DSS-triggered colitis. RNA-Seq results demonstrated that cell cycle pathway-related genes were notably downregulated in TLR2-KO colitis mice (enrichment score = 30, p < 0.001). 16S rRNA gene sequencing revealed that in comparison to the WT colitis mice, the relative abundance of Marinifilacea (p = 0.006), Rikenellacea (p = 0.005), Desulfovibrionaceae (p = 0.045), Tannerellaceae (p = 0.038), Ruminococcaceae (p = 0.003), Clostridia (p = 0.027), and Mycoplasmataceae (p = 0.0009) was significantly increased at the family level in the gut of TLR2-KO colitis mice. In addition, microbiome diversity-transcriptome collaboration analysis highlighted that the relative abundance of Marinifilaceae was negatively linked to the expression of cell cycle signaling-related genes (p values were all less than 0.001). Conclusion: Based on these findings, we concluded that TLR2-KO exacerbates DSS-triggered intestinal injury by mitigating cell cycle signaling in a Marinifilaceae-dependent manner.

Toll-like Receptor Expression in Pelodiscus sinensis Reveals Differential Responses after Aeromonas hydrophila Infection.

Background: Toll-like receptor (TLR), as an important pattern recognition receptor, is a bridge between non-specific immunity and specific immunity, and plays a vital role in the disease resistance of aquatic animals. However, the function of TLR in Pelodiscus sinensis is still unclear. Methods and Results: The sequence characteristics and homology of three TLRs (PsTLR2, PsTLR3 and PsTLR5) were determined in this investigation. Their annotation and orthologies were supported by phylogenetic analysis, functional domain prediction, and sequence similarity analysis. qPCR showed that the identified TLRs were expressed in all tissues, among the high expression of PsTLR5 in the brain and liver and the high expression of PsTLR2 and PsTLR3 in the liver. PsTLR2 mRNA expression increased 6.7-fold in the liver 12 h after Aeromonas hydrophila infection, while the mRNA expression of PsTLR3 was down-regulated by 0.29 times in liver and 0.31 times in spleen. The mRNA expression of PsTLR5 was significantly up-regulated in four immune tissues, and it was up-regulated by 122.8 times in the spleen after 72 h infection. Finally, the recombinant proteins of extracellular LRR domains of these three TLRs were obtained by prokaryotic expression technology, and the binding tests were performed to discover their ability of binding pathogenic microorganisms. Microbial binding test showed that rPsTLR2, rPsTLR3 and rPsTLR5 can combine A. hydrophila, Edwardsiella tarda, Vibrio parahaemolyticus, Staphylococcus aureus, Streptococcus agalactiae and Candida albicans, while rPsTLR3 can bind A. hydrophila, E. tarda, V. parahaemolyticus and C. albicans. Conclusions: Our findings suggested that TLRs may be crucial to turtles' innate immune response against microbes.

Toll-Like Receptor 8 is Expressed in Monocytes in Contrast to Plasmacytoid Dendritic Cells and Mediates Aberrant Interleukin-10 Responses in Patients With Systemic Sclerosis.

Systemic sclerosis (SSc) is a severe rheumatic disease causing fibrotic tissue rearrangement. Aberrant toll-like receptor (TLR) 8 transcripts in plasmacytoid dendritic cells (pDCs) were recently linked to SSc pathogenesis, which is at least partially mediated by increased type I interferon (IFN-I) responses. Here, we addressed the functional role of TLR8 signaling in different immune cell subsets of patients with SSc. Monocytes, conventional dendritic cells (cDCs), and pDCs from the blood and skin of patients with SSc were analyzed for TLR8 protein expression. To assess TLR function, cytokine responses upon TLR7 and TLR8 stimulation were studied. To identify relevant alterations specific for patients with SSc (n = 16), patients with primary Sjögren disease (pSS; n = 10) and healthy controls (HCs; n = 13) were included into the study. In all individuals, TLR8 was expressed in monocytes and cDCs but not in pDCs. The TLR8 expression levels were overall similar in patients with SSc and pSS and HCs. Additionally, in all study participants, TLR8 stimulation of pDCs did not induce IFN-I expression. In contrast, monocytes from patients with SSc revealed increased interleukin (IL)-10 responses upon TLR8 (patients with SSc vs HCs, P = 0.0126) and TLR7/8 stimulation (patients with SSc vs HCs, P = 0.0170). TLR8 protein is not expressed in pDCs of patients with SSc. Accordingly, they do not respond to TLR8 stimulation. In contrast, monocytes of patients with SSc respond to TLR8 stimulation with increased IL-10 responses. Therefore, TLR8 signaling in monocytes participates in SSc pathogenesis by conferring aberrant IL-10 expression.

Toll-like receptors and integrins crosstalk.

Immune system recognizes invading microbes at both pathogen and antigen levels. Toll-like receptors (TLRs) play a key role in the first-line defense against pathogens. Major functions of TLRs include cytokine and chemokine production. TLRs share common downstream signaling pathways with other receptors. The crosstalk revolving around TLRs is rather significant and complex, underscoring the intricate nature of immune system. The profiles of produced cytokines and chemokines via TLRs can be affected by other receptors. Integrins are critical heterodimeric adhesion molecules expressed on many different cells. There are studies describing synergetic or inhibitory interplay between TLRs and integrins. Thus, we reviewed the crosstalk between TLRs and integrins. Understanding the nature of the crosstalk could allow us to modulate TLR functions via integrins.

Toll-like receptors (TLR-2 and TLR-4): putative role in the uterine inflammatory response in cows – A mini review

Metritis and endometritis are the most prevalent uterine inflammatory diseases in bovines and cause infertility and high economic loss to farmers. Many studies have been conducted to investigate the mechanism of pathology, immune response and therapeutics associated with these diseases. This review focuses mainly on the immune system present in the uterus and on the involvement of Toll-like receptors. This study aimed to provide a deep understanding of the structure, function and expression pathways of Toll-like receptors (TLR-2 and TLR-4) in the treatment of pathogenic microorganisms invading the uterus and their significance in inflammatory conditions such as metritis and clinical and subclinical endometritis. The genetic associations of TLRs with diseases and some therapeutic interventions are also briefly discussed. As such, TLRs-2 and 4 play a critical role in providing a uterine defense mechanism against foreign substances (embryos, sperm, and pathogenic bacteria). In the present era, the use of TLR agonists is being unfurled for treatment, and further exploration and study of this topic in the future are needed.

Motor, Cognitive, and Behavioral Impairment in TLR3 and TLR9 Deficient Male Mice: Insights into the Non-Immunological Roles of Toll-Like Receptors

Toll-like receptors (TLRs) play a critical role in initiating the innate immune response to infection or injury. Recent studies have uncovered their intriguing functions as moonlighting proteins involved in various biological processes, including development, learning, and memory. However, the specific functions of individual TLRs are still largely unknown. We investigated the effects of TLR3 and TLR9 receptor deficiency on motor, cognitive, and behavioral functions during development using genetically modified male mice of different ages. We evaluated the motor coordination, anxiety-like behavior, spatial learning, and working memory of male mice lacking the TLR3 and TLR9 genes at different ages (two, four, six, and eight months) using the rotarod, open field, water maze, and T-maze tests. We observed that the deletion of either TLR3 or TLR9 resulted in impaired motor performance. Furthermore, young TLR3-deficient mice exhibited reduced anxiety-like behavior and spatial learning deficits; however, their working memory was unaffected. In contrast, young TLR9-knockout mice showed hyperactivity and a tendency toward decreased working memory. These findings provide valuable insights into the broader roles of the TLR system beyond the innate immune response, revealing its involvement in pathways associated with the central nervous system. Importantly, our results establish a strong association between the endosomal receptors TLR3 and TLR9 and the performance of motor, cognitive, and behavioral tasks that change over time. This study contributes to the growing body of research on the multifaceted functions of TLRs and enhances our understanding of their participation in non-immune-related processes.

TRAF3 regulation of proximal TLR signaling in B cells.

Toll-like receptors are pattern recognition receptors that bridge the innate and adaptive immune responses and are critical for host defense. Most studies of Toll-like receptors have focused upon their roles in myeloid cells. B lymphocytes express most Toll-like receptors and are responsive to Toll-like receptor ligands, yet Toll-like receptor-mediated signaling in B cells is relatively understudied. This is an important knowledge gap, as Toll-like receptor functions can be cell type specific. In striking contrast to myeloid cells, TRAF3 inhibits TLR-mediated functions in B cells. TRAF3-deficient B cells display enhanced IRF3 and NFκB activation, cytokine production, immunoglobulin isotype switching, and antibody production in response to Toll-like receptors 3, 4, 7, and 9. Here, we address the question of how TRAF3 impacts initial B-cell Toll-like receptor signals to regulate downstream activation. We found that TRAF3 in B cells associated with proximal Toll-like receptor 4 and 7 signaling proteins, including MyD88, TRAF6, and the tyrosine kinase Syk. In the absence of TRAF3, TRAF6 showed a greater association with several Toll-like receptor signaling proteins, suggesting that TRAF3 may inhibit TRAF6 access to Toll-like receptor signaling complexes and thus early Toll-like receptor signaling. In addition, our results highlight a key role for Syk in Toll-like receptor signaling in B cells. In the absence of TRAF3, Syk activation was enhanced in response to ligands for Toll-like receptors 4 and 7, and Syk inhibition reduced downstream Toll-like receptor-mediated NFκB activation and proinflammatory cytokine production. This study reveals multiple mechanisms by which TRAF3 serves as a key negative regulator of early Toll-like receptor signaling events in B cells.

UNC93B1 variants underlie TLR7-dependent autoimmunity.

UNC93B1 is critical for trafficking and function of nucleic acid-sensing Toll-like receptors (TLRs) TLR3, TLR7, TLR8, and TLR9, which are essential for antiviral immunity. Overactive TLR7 signaling induced by recognition of self-nucleic acids has been implicated in systemic lupus erythematosus (SLE). Here, we report UNC93B1 variants (E92G and R336L) in four patients with early-onset SLE. Patient cells or mouse macrophages carrying the UNC93B1 variants produced high amounts of TNF-α and IL-6 and upon stimulation with TLR7/TLR8 agonist, but not with TLR3 or TLR9 agonists. E92G causes UNC93B1 protein instability and reduced interaction with TLR7, leading to selective TLR7 hyperactivation with constitutive type I IFN signaling. Thus, UNC93B1 regulates TLR subtype-specific mechanisms of ligand recognition. Our findings establish a pivotal role for UNC93B1 in TLR7-dependent autoimmunity and highlight the therapeutic potential of targeting TLR7 in SLE.

Effects of histone acetyltransferase (HAT) and histone deacetylase (HDAC) inhibitors on proliferative, differentiative, and regenerative functions of Toll-like receptor 2 (TLR-2)-stimulated human dental pulp cells (hDPCs).

This in vitro study aimed to modify TLR-2-mediated effects on the paracrine, proliferative, and differentiation potentials of human dental pulp-derived cells using histone acetyltransferase (HAT) and histone deacetylase (HDAC) inhibitors. Cell viability was assessed using the XTT assay. Cells were either treated with 10μg/ml Pam3CSK4 only, or pre-treated with valproic acid (VPA) (3mM), trichostatin A (TSA) (3μM), and MG-149 (3μM) for a total of 4h and 24h. Control groups included unstimulated cells and cells incubated with inhibitors solvents only. Transcript levels for NANOG, OCT3-4, FGF-1 and 2, NGF, VEGF, COL-1A1, TLR-2, hβD-2 and 3, BMP-2, DSPP, and ALP were assessed through qPCR. After 24h, TSA pre-treatment significantly upregulated the defensins and maintained the elevated pro-inflammatory cytokines, but significantly reduced healing and differentiation genes. VPA significantly upregulated the pro-inflammatory cytokine levels, while MG-149 significantly downregulated them. Pluripotency genes were not significantly affected by any regimen. At the attempted concentrations, TSA upregulated the defensins gene expression levels, and MG-149 exerted a remarkable anti-inflammatory effect; therefore, they could favorably impact the immunological profile of hDPCs. Targeting hDPC nuclear function could be a promising option in the scope of the biological management of inflammatory pulp diseases.

Toll-like receptor expression and functional behavior in platelets from patients with systemic lupus erythematosus

BackgroundMultiple blood cell abnormalities participate in the development of inflammation in systemic lupus erythematosus (SLE). Although platelets have been suggested as one of these contributors through the release of their content during activation, there are limited specific data about their role as immune players in SLE. Materials and MethodsThirteen SLE patients were included. Flow cytometry was used to measure Toll-like receptors (TLR) 2, 4, and 9 in resting platelets, platelet-activation markers (PAC-1 binding, P-selectin, CD63, and CD40 ligand -L) and platelet-leukocyte aggregates before and after specific TLR stimulation. Soluble CD40L and von Willebrand factor (vWf) release from stimulated platelets was measured using ELISA. ResultsIn resting conditions, SLE platelets showed normal expression levels of TLR 2, 4 and 9. Platelet surface activation markers, soluble CD40L, and vWf release were normal at baseline and after TLR stimulation. Platelet-monocyte aggregates were elevated in resting conditions in SLE samples and showed only a marginal increase after TLR stimulation, while baseline and stimulated platelet-neutrophil and platelet-lymphocyte aggregates were normal. C-reactive protein levels positively correlated with platelet-monocyte aggregates both at baseline and after stimulation with the TLR-2 agonist PAM3CSK4, suggesting these complexes could reflect the inflammatory activity in SLE. In our cohort, 12 of 13 patients received treatment with hydroxychloroquine (HCQ), a known inhibitor of endosomal activity and a potential inhibitor of platelet activation. The fact that SLE platelets showed an adequate response to TLR agonists suggests that, despite this treatment, they retain the ability to respond to the increased levels of damage-associated molecular patterns (DAMPs), which represent known TLR ligands, present in the circulation of SLE patients. Interestingly, elevated plasma levels of high mobility group box 1 (HMGB1), a classical DAMP, correlated with vWf release from TLR-stimulated platelets, suggesting that HMGB1 may also be released by platelets, thereby creating a positive feedback loop for platelet activation that contributes to inflammation. ConclusionOur study demonstrates normal platelet TLR expression and function together with increased circulating platelet-monocyte aggregates. In addition, a direct correlation was observed between plasma HMGB1 levels and platelet vWf release following TLR2 stimulation. This platelet behavior in a group of patients undergoing HCQ treatment suggests that platelets could play a role in the inflammatory state of SLE.

Inhibition of Toll-like Receptor 4 (TLR4) Promotes Erythroid and Myeloid Differentiation in Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML)

Introduction: The hallmark of Myelodysplastic Syndrome (MDS) is a block in differentiation of hematopoietic precursors resulting in single or multilineage cytopenias. Evaluation of regulatory pathways that are inappropriately overactive and lead to a blockade in erythroid and myeloid differentiation is an important process in determining future therapeutic targets for the treatment of MDS. The Toll-like receptor (TLR) pathways play an important role in the innate immune response and are also highly expressed on hematopoietic stem cells. Aberrant overactivation of these pathways can stimulate inflammation and cytokine production in the marrow microenvironment. Also, while the overactivation of interleukin-1 receptor-associated kinase 4 (IRAK4), tumor necrosis factor receptor associated factor 6 (TRAF6) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways has been seen in Myelodysplastic Syndrome and Acute Myeloid Leukemia (AML), the upstream signals that drive their activation are not fully identified. Results: We investigated the expression of all TLRs in a large database of MDS CD34+ marrow samples and age-matched controls. Our findings revealed that TLR6 and TLR4 were the most significantly overexpressed receptors in MDS samples (FDR<0.001). Furthermore, we observed that the TLR4 cofactor lymphocyte antigen 96 (LY96), also known as myeloid differentiation factor 2 (MD-2), was also overexpressed in MDS samples when compared to controls. Furthermore, the S100A8 and S100A9 proteins are ligands for TLR4 and were found to be positively associated with severity of anemia in the cohort of MDS samples. These data provided the rationale for determining the functional role of the TLR4 pathway in MDS/AML models. To obtain a potent and specific antibody against TLR4, we inoculated rabbits with recombinant human TLR4 and obtained high affinity antibodies that neutralized TLR4 function at low nano-molar range of doses. The TLR4 polyclonal antibody was able to inhibit lipopolysaccharide (LPS)-induced NF-κb activation in leukemic THP-1 cells. The TLR4 antibody also led to a decrease in proliferative phosphorylated extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase in leukemic cells. We then treated samples from MDS/AML patients (n=14) with the TLR4 antibody (1 nM dosing) and assessed for erythroid and myeloid growth and differentiation. Fluorescence activated cell sorting (FACS) analysis for erythroid differentiation showed increased expression of either CD71, CD235a or both after treatment with the TLR4 antibody. FACS analysis for myeloid differentiation showed increased expression of either CD11b, CD14 or both after treatment with the TLR4 antibody. This effect was more pronounced in the intermediate/high risk group than in the low-risk group of patients. Conclusion: Inhibition of the TLR4 pathway with a potent TLR4 antibody promotes erythroid and myeloid maturation and relieves the differentiation block on these lineages in MDS patient samples in the in vitro setting. Our preclinical data supports further evaluation of TLR4 inhibition as a therapeutic target in patients with MDS, as anemia and neutropenia are hallmarks of the disease.

Molecular characteristics and therapeutic implications of Toll-like receptor signaling pathway in melanoma

Melanoma is a malignant tumor of melanocytes and is often considered immunogenic cancer. Toll-like receptor-related genes are expressed differently in most types of cancer, depending on the immune microenvironment inside cancer, and the key function of Toll-like receptors (TLRs) for melanoma has not been fully elucidated. Based on multi-omics data from TCGA and GEO databases, we first performed pan-cancer analysis on TLR, including CNV, SNV, and mRNA changes in TLR-related genes in multiple human cancers, as well as patient prognosis characterization. Then, we divided melanoma patients into three subgroups (clusters 1, 2, and 3) according to the expression of the TLR pathway, and explored the correlation between TLR pathway and melanoma prognosis, immune infiltration, metabolic reprogramming, and oncogene expression characteristics. Finally, through univariate Cox regression analysis and LASSO algorithm, we selected six TLR-related genes to construct a survival prognostic model, divided melanoma patients into the training set, internal validation set 1, internal validation set 2, and external validation set for multiple validations, and discussed the correlation between model genes and clinical features of melanoma patients. In conclusion, we constructed a prognostic survival model based on TLR-related genes that precisely and independently demonstrated the potential to assess the prognosis and immune traits of melanoma patients, which is critical for patients’ survival.

The Protective Role of TLR4 in Intestinal Epithelial Cells through the Regulation of the Gut Microbiota in DSS-Induced Colitis in Mice.

The cause of ulcerative colitis (UC) is not yet fully understood. Previous research has pointed towards a potential role for mutations in nucleotide-binding oligomerization domain-containing protein 2 (NOD2) in promoting the onset and progression of inflammatory bowel disease (IBD) by altering the microbiota of the gut. However, the relationship between toll-like receptor 4 (TLR4) and gut microbiota in IBD is not well understood. To shed light on this, the interaction between TLR4 and gut microbiota was studied using a mouse model of IBD. To examine the function of TLR4 signaling in intestinal injury repair, researchers developed Dextran Sulfate Sodium Salt (DSS)-induced colitis and injury models in both wild-type (WT) mice and TLR4 knockout (TLR4-KO) mice. To assess changes in the gut microbiota, 16S rRNA sequencing was conducted on fecal samples from both the TLR4-KO and WT enteritis mouse models. The data obtained depicted a protective function of TLR4 against DSS-induced colitis. The gut microbiota composition was found to vary considerably between the WT and TLR4-KO mice groups as indicated by β-diversity analysis and operational taxonomic units (OTUs) cluster. Statistical analysis of microbial multivariate variables depicted an elevated abundance of Escherichia coli/Shigella, Gammaproteobacteria, Tenerlcutes, Deferribacteres, Enterobacteria, Rikenellaceae, and Proteobacteria in the gut microbiota of TLR4-KO mice, whereas there was a considerable reduction in Bacteroidetes at five different levels of the phylogenetic hierarchy including phylum, class, order, family, and genus in comparison with the WT control. TLR4 may protect intestinal epithelial cells from damage in response to DSS-induced injury by controlling the microbiota in the gut.

How Toll-like receptors influence Parkinson's disease in the microbiome-gut-brain axis.

Recently, a large number of experimenters have found that the pathogenesis of Parkinson's disease may be related to the gut microbiome and proposed the microbiome-gut-brain axis. Studies have shown that Toll-like receptors, especially Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4), are key mediators of gut homeostasis. In addition to their established role in innate immunity throughout the body, research is increasingly showing that the Toll-like receptor 2 and Toll-like receptor 4 signaling pathways shape the development and function of the gut and enteric nervous system. Notably, Toll-like receptor 2 and Toll-like receptor 4 are dysregulated in Parkinson's disease patients and may therefore be identified as the core of early gut dysfunction in Parkinson's disease. To better understand the contribution of Toll-like receptor 2 and Toll-like receptor 4 dysfunction in the gut to early α-synuclein aggregation, we discussed the structural function of Toll-like receptor 2 and Toll-like receptor 4 and signal transduction of Toll-like receptor 2 and Toll-like receptor 4 in Parkinson's disease by reviewing clinical, animal models, and in vitro studies. We also present a conceptual model of the pathogenesis of Parkinson's disease, in which microbial dysbiosis alters the gut barrier as well as the Toll-like receptor 2 and Toll-like receptor 4 signaling pathways, ultimately leading to a positive feedback loop for chronic gut dysfunction, promoting α-synuclein aggregation in the gut and vagus nerve.

Abstract 554: Monocyte Toll-like Receptor 4 (tlr4) Signaling Is Essential For Post Thrombotic Upa Expression

Background: Leukocyte expression of Toll-like receptor 4 (TLR4) increases with transformation to chronic deep venous thrombosis (DVT) & in non-resolving thrombi, but TLR4 function during thrombus clearance is unclear. We hypothesized TLR4 signaling to regulate expression of fibrinolytic factors in monocytes & macrophages (MO/MFs) for VT resolution. Methods: Small interfering RNA (siRNA) knockdown (KD) of TLR4 & disruption of Myd88-dependent/independent pathways occurred in immortalized MFs (RAW264.7), with quantitative real-time PCR (qRT-PCR) for a panel of fibrinolytic factors & immunoblotting for urokinase (uPA). Tlr4-/- mice & wild-type controls underwent IVC stasis/ligation, yielding VT at 2h, 2d, & 8d. Murine/chronic human thrombi underwent immunofluorescent staining for indicated targets & murine thrombi weight-length ratios were found. Splenic & Bone marrow-derived MFs from Tlr4 Cre mice were immunoblotted for uPA. Results: TLR4 silencing in RAW264.7 cells yielded suppression of uPA mRNA (Fig 1B) & protein levels (not shown). TRIF KD yielded decreased uPA mRNA (Fig 1C) & protein levels (not shown), implicating Myd88-independent signaling in MO/MF uPA production. Human (not shown) & murine chronic thrombi staining saw TLR4 co-localization with MF marker CCR2 (Fig 1D). Global knockout (KO) of murine TLR4 did not affect early thrombogenesis (Fig 1E left), with delayed VT resolution seen from 2d (Fig 1E right). Decreased uPA protein levels were observed in Tlr4 KO mice (Fig 1F). Conclusions: TLR4 signaling stimulates in vivo VT resolution. Expression of fibrinolytic factor uPA is enforced in MO/MFs by TLR4-Myd88-independent signaling, a potential target for VT resolution.

Immunodeficiency in a female patient with Incontinentia Pigmenti

Incontinentia pigmenti (IP) is a rare X-linked dominant neuroectodermal disorder characterized by ocular, dermatologic, and neurological abnormalities. It is caused by heterozygous mutations in IKBKG (NEMO), which encodes a regulatory factor involved in NF-kB activation. Random and skewed X-inactivation allows for female survival and determines extent of disease. Although immune function is typically normal in IP patients, hypomorphic IKBKG mutations are known to cause immunodeficiency, as occurs in X-linked NEMO deficiency. A female newborn presented with bullous vesicles, peeling skin, alopecia, hearing loss, and tonic-clonic seizures with MRI demonstrating evidence of in utero stroke. She developed intermittent fevers and cellulitis and was diagnosed with IP based on clinical findings. Following discharge, she was readmitted at 1 month of age due to bronchiolitis complicated by pulmonary abscess. Labs demonstrated hyperleukocytosis with hypereosinophilia, normal T, B, and NK lymphocyte enumeration, abnormal lymphocyte proliferation to phytohemagglutinin (PHA) and anti-CD3, and abnormal toll-like receptor (TLR) function. Genetic analysis revealed a heterozygous deletion of exons 4–10 in IKBKG. Labs at 1 year follow up showed normal lymphocyte (T, B, and NK) enumeration and mitogen responses, normal immunoglobulin levels, and normal responses to pneumococcal and tetanus vaccines. While about 70% of IP patients harbor a deletion of exons 4–10 in IKBKG. unlike other hypomorphic NEMO mutations, this mutation is not typically associated with altered immune function. This case highlights that immunodeficiency may be an early clinical manifestation in female patients with IP.

Effects of TcpC’s NADase Activity on Inhibition of Innate Immune Signaling

Abstract The innate immune system is activated by pattern recognition receptors such as Toll-like receptors (TLRs), which differentiate between self and non-self by recognizing distinct molecular structures on pathogens. As this process threatens pathogen survival in the host, bacteria have developed mechanisms to combat this. Uropathogenic Escherichia coli (UPEC) express the Toll/Interleukin-1 Receptor (TIR) domain-containing protein TcpC, which inhibits the function of TLRs by binding to the signaling adapter protein myeloid differentiation factor 88 (MyD88). Bacterial TIR proteins, including TcpC, recently have been found to also exhibit NADase activity. We hypothesize that the ability of TcpC to block TLR signaling and its NADase function are interrelated and that the TcpC EA mutant, which has been shown to have diminished NADase activity will not bind efficiently to MyD88. We performed in vitro translation assays to first produce both WT and EA mutant versions of TcpC that contain both a streptavidin and histidine tag. Then we conducted pull-down experiments to determine if the TcpC EA mutant could successfully bind to streptavidin beads, as well as MyD88, potentially resulting in clustering with the TIR signaling protein and blocking the signaling pathway entirely. While we have successfully produced tagged WT and EA TcpC proteins and pulled down the proteins using streptavidin beads, we are working to optimize conditions to determine if the NADase-deficient TcpC EA mutant is able to bind MyD88, as has been shown previously for WT TcpC. The resulting information will give us a closer look into TcpC’s effect on virulence and the suppression of the innate immune response via TLR and MyD88 related signaling pathways. This research was partially funded by the USM LSAMP program, supported by NSF LSAMP Award #1619676, the Towson OSPR Seed Funding for Grant Seekers, and the Bridges to Doctorate Scholars Program, NIH-R25GM119970 04.