TLR Stimulation Research Articles

MTDH Promotes Intestinal Inflammation by Positively Regulating TLR Signalling.

Macrophages in the intestinal mucosa can rapidly engage Toll-like receptor [TLR]-mediated inflammatory responses to protect against pathogen invasion, but these same innate immune responses can also drive the induction of colitis. Our previous research revealed that metadherin [MTDH] is overexpressed in multiple cancers and plays vital roles in tumour progression. However, the role of MTDH in intestinal inflammation is largely unknown. In this study, we found the MTDH expression in colonic lamina propria [CLP] macrophages was positively correlated with inflammatory colitis severity. MTDH-/- mice were protected against the symptoms of dextran sodium sulphate [DSS]-induced colitis; however, adoptive transfer of MTDH wild-type [WT] monocytes partially restored the susceptibility of MTDH-/- mice to DSS-induced colitis. TLR stimulation was sufficient to induce the expression of MTDH, whereas the absence of MTDH was sufficient to suppress TLR-induced production of inflammatory cytokines by macrophages. From a mechanistic perspective, MTDH recruited TRAF6 to TAK1, leading to TRAF6-mediated TAK1 K63 ubiquitination and phosphorylation, ultimately facilitating TLR-induced NF-κB and MAPK signalling. Taken together, our results indicate that MTDH contributes to colitis development by promoting TLR-induced pro-inflammatory cytokine production in CLP macrophages and might represent a potential therapeutic approach for intestine inflammation intervention.

CD200R1 and CD200R1L expression is regulated during B cell development in swine and modulates the Ig production in response to the TLR7 ligand imiquimoid.

The CD200R family comprises a group of paired receptors that can modulate the activation of immune cells. They are expressed both on myeloid cells and lymphocyte subsets. Here we report that the expression of these receptors on porcine B cells is tightly regulated, being mainly expressed on mature cells. The expression of the inhibitory receptors CD200R1 and/or its splicing variant CD200R1X2, either in combination or not with the activating receptor CD200R1L, is upregulated in sIgM+ effector/memory cells, and tends to decline thereafter as these cells progress to plasmablasts or switch the Ig isotype. sIgM+ naïve and primed cells only express, by contrast, the CD200R1X2 receptor. B-1 like cells also express CD200R1 isoforms, either alone or in combination with CD200R1L. Treatment of peripheral blood mononuclear cells with a monoclonal antibody specific for inhibitory receptors, enhances the IgM and IgG production induced by TLR7 stimulation suggesting a modulatory role of B cell functions of these receptors.

IgE-activated mast cells enhance TLR4-mediated antigen-specific CD4+ T cell responses

Mast cells are potent mediators of allergy and asthma, yet their role in regulating adaptive immunity remains ambiguous. On the surface of mast cells, the crosslinking of IgE bound to FcεRI by a specific antigen recognized by that IgE triggers the release of immune mediators such as histamine and cytokines capable of activating other immune cells; however, little is known about the mast cell contribution to the induction of endogenous, antigen-specific CD4+ T cells. Here we examined the effects of specific mast cell activation in vivo on the initiation of an antigen-specific CD4+ T cell response. While CD4+ T cells were not enhanced by FcεRI stimulation alone, their activation was synergistically enhanced when FcεRI activation was combined with TLR4 stimulation. This enhanced activation was dependent on global TLR4 stimulation but appeared to be less dependent on mast cell expressed TLR4. This study provides important new evidence to support the role of mast cells as mediators of the antigen-specific adaptive immune response.

Long noncoding RNA MIR4435-2HG enhances metabolic function of myeloid dendritic cells from HIV-1 elite controllers.

Restriction of HIV-1 replication in elite controllers (ECs) is frequently attributed to T cell-mediated immune responses, while the specific contribution of innate immune cells is less clear. Here, we demonstrate an upregulation of the host long noncoding RNA (lncRNA) MIR4435-2HG in primary myeloid dendritic cells (mDCs) from ECs. Elevated expression of this lncRNA in mDCs was associated with a distinct immunometabolic profile, characterized by increased oxidative phosphorylation and glycolysis activities in response to TLR3 stimulation. Using functional assays, we show that MIR4435-2HG directly influenced the metabolic state of mDCs, likely through epigenetic mechanisms involving H3K27ac enrichment at an intronic enhancer in the RPTOR gene locus, the main component of the mammalian target of rapamycin complex 1 (mTORC1). Together, these results suggest a role of MIR4435-2HG for enhancing immunometabolic activities of mDCs in ECs through targeted epigenetic modifications of a member of the mTOR signaling pathway.

Capsule-dependent metabolic immunosuppression of host cells by Francisella tularensis

Abstract Francisella tularensis subsp. tularensis (Ftt) is a highly pathogenic, intracellular bacterium capable of replicating while avoiding detection by the host immune system. An O-antigen polysaccharide capsule has been identified as an important virulence factor involved in suppressing the immune response. Previous studies have shown that Ftt capsule limits the metabolic shift from oxidative phosphorylation to glycolysis needed for activation of host defense mechanisms. The specific mechanism by which Ftt capsule alters host metabolism is not fully understood but involves improvement of mitochondrial function. In this study, central metabolism in primary cell culture was mapped using targeted metabolomics following TLR stimulation and capsule treatment. Consistent with previous findings, capsule stimulated TCA cycle activity to increase mitochondrial respiration. Ftt capsule treatment did not inhibit increased glycolytic flux associated with TLR-induced metabolic activation. However, capsule limited accumulation of lactate and depletion of acetyl-CoA pools associated with TLR-driven uncoupling of glycolysis and the TCA cycle. These data point to capsule-dependent manipulation of pyruvate dehydrogenase, the enzyme responsible for converting pyruvate made during glycolysis into acetyl-CoA for utilization within the TCA-cycle. Together, these results demonstrate a novel mechanism by which a bacterial capsule can limit inflammation by maintaining coupling of central metabolic pathways.

Mechanisms of TRAF3 Regulation of TLR Signaling in B Lymphocytes

Abstract TRAF3 is an adaptor protein that binds to receptors of the TNFR superfamily and exerts multiple regulatory functions in signaling pathways. However, functions are cell type and context dependent. B cell TRAF3 inhibits homeostatic NFκB2 activation, CD40/BAFFR signaling and other pro-survival signaling pathways; restraint of survival is a B cell unique TRAF3 function. Loss of function mutations of human TRAF3 are detected in B cell lymphoma and multiple myeloma. In addition to homeostatic survival, we found that TRAF3 inhibits B cell TLR signaling, a pathway implicated in B cell lymphomagenesis and autoimmunity. Numerous reports describe TLR function in myeloid cells, but specific TLR signaling pathways in B cells are under-studied. B cell TRAF3 inhibits TLR-mediated cytokine and Ab production, and Ig class switching. Our <underline>working hypothesis</underline> is that B cell TRAF3 regulates the expression, recruitment and/or post-translational modification of TLR signaling proteins. Preliminary data show that B cell TRAF3 associates with signaling proteins MyD88 and IRAK1, and the kinase Syk following TLR stimulation. We are currently investigating how this association normally restrains the functions of these proteins in promoting TLR signals to B cells. We expect that information gained will provide new insights into the molecular mechanisms contributing to the regulation of B cells by TRAF3 in TLR signaling, potentially informing selection and development of more targeted therapies for B cell malignancies and autoimmunity.

Integrin αvβ3 limits TLR signaling in plasmacytoid dendritic cells to prevent autoimmunity.

Abstract SLE is a chronic, systemic autoimmune disease affecting millions of people worldwide. SLE is caused by a breakdown of tolerance to self-derived nucleic acids (NA) and characterized by the presence of autoantibodies to DNA/RNA. Plasmacytoid dendritic cells (pDCs) are highly tuned to respond to NA and express a range of receptors for RNA and DNA, including TLRs 7 and 9. Following TLR engagement, pDCs produce high levels of type I interferon (IFN-I), which promotes loss of tolerance, implicating pDCs in development of autoimmunity. However, relatively little is known about how innate immune signaling is regulated in pDCs, and particularly how they distinguish self-derived from foreign NA. TLRs 7 and 9 are both localized in endosomal compartments, and their NA ligands must be internalized and processed before they can bind. One source of these NA is dying cells and loss of their receptors is associated with SLE. We studied the role of an apoptotic cell receptor, Integrin αvβ3, in TLR signaling of pDCs and found that αvβ3 limits IFN-I production. αvβ3-knockout pDCs show increased TLR signaling and produce more IFN-I and inflammatory cytokines when stimulated in culture. In mice without αvβ3, there is a systemic increase in circulating ISGs and IFN-I after TLR7 stimulation. This corresponds with an increase in pDC activation and a decrease in marginal zone B cells, indicating elevated IFN-I production. These data indicate αvβ3 has a pDC-intrinsic role in limiting TLR signaling, which we propose is through activation of non-canonical autophagy. Based on these data, we hypothesize that co-receptors involved in capture and internalization of nucleic acids provide contextual ‘cues’ to regulate TLR signaling and prevent autoimmunity.

Sphingosine‐1‐Phosphate Contributes to TLR9‐Induced TNF‐α Release in Lung Tumor Cells

Sphingosine-1-phosphate (S1P) is a membrane-derived bioactive phospholipid involved in many lung physiological and pathological processes. Higher levels of S1P have been registered in a broad range of respiratory diseases, including inflammatory disorders and cancer. The aim of our study was to understand the role of the S1P in healthy versus tumor cells after Toll-Like Receptors (TLRs) activation, well-known modulators of sphingolipid metabolism. We found that S1P was involved in TLR9-induced TNF-α release in that the inhibition of both ceramidase and sphingosine kinase I/II (SPHK I/II) significantly reduced the levels of TNF-α after TLR9 triggering in lung adenocarcinoma cells. These results were not observed in healthy fibroblasts, implying that this pathway was mainly involved in pathological conditions. Moreover, the activation of TLR4 by means of LPS did not have similar effects as in the case of CpG-stimulated TLR9. Importantly, the activation of TLR9 induced S1P generation and allowed it to interact on the outside receptors S1P1 and S1P3 via the efflux through its membrane transporter SPNS2. Indeed, both the blockade of S1P3 and the transporter SPNS2 significantly reduced the activity of S1P on TNF-α release from lung adenocarcinoma cells. To note, although CpG induced higher levels of TNF-α than exogenous S1P, we found that TLR9 stimulation was involved in S1P synthesis. In conclusion, our study identified a novel inflammatory pathway in that TLR9 increases a pro-inflammatory cytokine, such as TNF-α, via the induction of a ceramide/S1P imbalance in favor of S1P. Therefore, these data will shed more light on the role of the higher levels of S1P during inflammatory conditions.

IL-17 receptor A signaling on B cells promotes B-cell responses to TLR7 and enhances autoreactive germinal center B-cell development

Abstract IL-17 receptor A (IL-17RA) expression has been identified in B cells and macrophages. There was low expression of IL-17RA on T cells. Surprisingly, we previously showed that IL-17RA signaling through NF-κB p65/p50 has a complex effect on both B and T-cell localization and germinal center (GC) architecture, making it difficult to dissect the specific effects of IL-17RA on B cells. To isolate the effects of the absence of IL-17RA specifically on B cells, we have generated a B-cell specific IL-17RA knockout (Il17rafloxed/floxed x Cd19.Cre) BXD2 mice. Flow cytometry confirmed the loss of IL-17RA expression in only CD19+ B cells but not CD3+ T cells or CD11b+ macrophages. There was a 2-fold decreased spontaneous GC B cell population in Il17rafloxed/floxed x Cd19.Cre BXD2, compared to Il17rawt/floxed x Cd19.Cre BXD2 mice. Interestingly, the abnormal activation and expansion of T-follicular helper cell (Tfh) phenotype in BXD2-Il17ra−/− mice was abrogated in Il17rafloxed/floxed x Cd19.Cre BXD2 mice. In the BXD2-Il17ra−/− mice, B cells were anergic to TLR7 stimulation and exhibited a strong stimulus-specific transcription repressor p50/p50 homodimer and a lower NF-κB phospho-p65. To demonstrate that this anergy is due specifically to IL-17RA on B cells but not other cells, we have identified that there was a lower TLR7-induced CD69 in vitro in Il17rafloxed/floxed x Cd19.Cre BXD2 mice. Taken together, our results suggest that IL-17RA signaling directly regulates B cell autoimmune phenotype in the BXD2 mice through an enhancement of B-cell responses stimulated by the NF-κB signaling pathway. A deficiency of such signal enforces B cell anergy and prevents autoreactive GC formation.

The Role of Proprotein Convertases in the Regulation of the Function of Immune Cells in the Oncoimmune Response.

Proprotein convertases (PC) are a family of 9 serine proteases involved in the processing of cellular pro-proteins. They trigger the activation, inactivation or functional changes of many hormones, neuropeptides, growth factors and receptors. Therefore, these enzymes are essential for cellular homeostasis in health and disease. Nine PC subtilisin/kexin genes (PCSK1 to PCSK9) encoding for PC1/3, PC2, furin, PC4, PC5/6, PACE4, PC7, SKI-1/S1P and PCSK9 are known. The expression of PC1/3, PC2, PC5/6, Furin and PC7 in lymphoid organs such as lymph nodes, thymus and spleen has suggested a role for these enzymes in immunity. In fact, knock-out of Furin in T cells was associated with high secretion of pro-inflammatory cytokines and autoantibody production in mice. This suggested a key role for this enzyme in immune tolerance. Moreover, Furin through its proteolytic activity, regulates the suppressive functions of Treg and thus prevents chronic inflammation and autoimmune diseases. In macrophages, Furin is also involved in the regulation of their inflammatory phenotype. Similarly, PC1/3 inhibition combined with TLR4 stimulation triggers the activation of the NF-κB signaling pathway with an increased secretion of pro-inflammatory cytokines. Factors secreted by PC1/3 KD macrophages stimulated with LPS exert a chemoattractive effect on naive auxiliary T lymphocytes (Th0) and anti-tumoral activities. The link between TLR and PCs is thus very important in inflammatory response regulation. Furin regulates TL7 and TLR8 processing and trafficking whereas PC1/3 controls TLR4 and TLR9 trafficking. Since PC1/3 and Furin are key regulators of both the innate and adaptive immune responses their inhibition may play a major role in oncoimmune therapy. The role of PCs in the oncoimmune response and therapeutic strategies based on PCs inhibition are proposed in the present review.

Identification of TLR2 Signalling Mechanisms Which Contribute to Barrett's and Oesophageal Adenocarcinoma Disease Progression.

Simple SummaryOesophageal adenocarcinoma (EAC) is a common type of oesophageal cancer with a rapidly rising incidence. Risk factors such as reflux, smoking, obesity and Barrett’s oesophagus cause chronic irritation and inflammation in the oesophagus. A receptor that causes inflammation, called Toll-like receptor 2 (TLR2), is expressed at higher levels in oesophageal cells from patients with Barrett’s and EAC, compared to disease-free patients. This study aimed to identify mechanisms involved in TLR2-mediated inflammation in oesophageal cells; and to assess whether TLR2 represents a therapeutic target to limit EAC development. Findings reveal that TLR2 activation in Barrett’s organoids and oesophageal cancer cells amplifies inflammation and promotes cancer development by causing the secretion of several inflammatory factors, most notably the nuclear protein, HMGB1. We demonstrate that TLR2 neutralisation efficiently blocks the inflammatory effects of TLR2 in these systems, revealing the therapeutic potential of TLR2 targeting to limit oesophageal disease and cancer progression.Chronic inflammation plays an important role in the pathogenesis of oesophageal adenocarcinoma (EAC) and its only known precursor, Barrett’s oesophagus (BE). Recent studies have shown that oesophageal TLR2 levels increase from normal epithelium towards EAC. TLR2 signalling is therefore likely to be important during EAC development and progression, which requires an inflammatory microenvironment. Here, we show that, in response to TLR2 stimulation, BE organoids and early-stage EAC cells secrete pro-inflammatory cytokines and chemokines which recruit macrophages to the tumour site. Factors secreted from TLR2-stimulated EAC cells are shown to subsequently activate TLR2 on naïve macrophages, priming them for inflammasome activation and inducing their differentiation to an M2/TAM-like phenotype. We identify the endogenous TLR2 ligand, HMGB1, as the factor secreted from EAC cells responsible for the observed TLR2-mediated effects on macrophages. Our results indicate that HMGB1 signalling between EAC cells and macrophages creates an inflammatory tumour microenvironment to facilitate EAC progression. In addition to identifying HMGB1 as a potential target for early-stage EAC treatment, our data suggest that blocking TLR2 signalling represents a mechanism to limit HMGB1 release, inflammatory cell infiltration and inflammation during EAC progression.

Human cardiac fibroblasts produce pro-inflammatory cytokines upon TLRs and RLRs stimulation

Heart inflammation is one of the major causes of heart damage that leads to dilated cardiomyopathy and often progresses to end-stage heart failure. In the present study, we aimed to assess whether human cardiac cells could release immune mediators upon stimulation of Toll-like receptors (TLRs) and Retinoic acid-inducible gene (RIG)-I-like receptors (RLRs).Commercially available human cardiac fibroblasts and an immortalized human cardiomyocyte cell line were stimulated in vitro with TLR2, TLR3, and TLR4 agonists. In addition, cytosolic RLRs were activated in cardiac cells after transfection of polyinosinic-polycytidylic acid (PolyIC). Upon stimulation of TLR3, TLR4, MDA5, and RIG-I, but not upon stimulation of TLR2, human cardiac fibroblasts produced high amounts of the pro-inflammatory cytokines IL-6 and IL-8. On the contrary, the immortalized human cardiomyocyte cell line was unresponsive to the tested TLRs agonists. Upon RLRs stimulation, cardiac fibroblasts, and to a lesser extent the cardiomyocyte cell line, induced anti-viral IFN-β expression.These data demonstrate that human cardiac fibroblasts and an immortalized human cardiomyocyte cell line differently respond to various TLRs and RLRs ligands. In particular, human cardiac fibroblasts were able to induce pro-inflammatory and anti-viral cytokines on their own. These aspects will contribute to better understand the immunological function of the different cell populations that make up the cardiac tissue.

Sphingosine-1-Phosphate Receptor Type 4 (S1P4) Is Differentially Regulated in Peritoneal B1 B Cells upon TLR4 Stimulation and Facilitates the Egress of Peritoneal B1a B Cells and Subsequent Accumulation of Splenic IRA B Cells under Inflammatory Conditions.

Background: Gram-negative infections of the peritoneal cavity result in profound modifications of peritoneal B cell populations and induce the migration of peritoneal B cells to distant secondary lymphoid organs. However, mechanisms controlling the egress of peritoneal B cells from the peritoneal cavity and their subsequent trafficking remain incompletely understood. Sphingosine-1-phosphate (S1P)-mediated signaling controls migratory processes in numerous immune cells. The present work investigates the role of S1P-mediated signaling in peritoneal B cell trafficking under inflammatory conditions. Methods: Differential S1P receptor expression after peritoneal B cell activation was assessed semi‑quantitatively using RT-PCR in vitro. The functional implications of differential S1P1 and S1P4 expression were assessed by transwell migration in vitro, by adoptive peritoneal B cell transfer in a model of sterile lipopolysaccharide (LPS)‑induced peritonitis and in the polymicrobial colon ascendens stent peritonitis (CASP) model. Results: The two sphingosine-1-phosphate receptors (S1PRs) expressed in peritoneal B cell subsets S1P1 and S1P4 are differentially regulated upon stimulation with the TLR4 agonist LPS, but not upon PMA/ionomycin or B cell receptor (BCR) crosslinking. S1P4 deficiency affects both the trafficking of activated peritoneal B cells to secondary lymphoid organs and the positioning of these cells within the functional compartments of the targeted organ. S1P4 deficiency in LPS-activated peritoneal B cells results in significantly reduced numbers of splenic innate response activator B cells. Conclusions: The S1P-S1PR system is implicated in the trafficking of LPS-activated peritoneal B cells. Given the protective role of peritoneal B1a B cells in peritoneal sepsis, further experiments to investigate the impact of S1P4-mediated signaling on the severity and mortality of peritoneal sepsis are warranted.

A comparative investigation on the role and interaction of EsxA and EsxB in host immune response

Staphylococcus aureus (S. aureus) is a frequent and major cause of bovine mastitis; it poses a tremendous economic burden to dairy industries of numerous countries. Early-secretion antigen-6 secretion system (ESS) has been viewed as an essential virulence and pathogenic factor of S. aureus. EsxA and EsxB are small acidic proteins secreted by ESS and identified as potential T-cell antigens of S. aureus. Unlike those of Mycobacterium tuberculosis (M. tuberculosis), the EsxA and EsxB of S. aureus do not form a dimer. Instead, EsxA dimerizes with itself or EsaC. Therefore, the interaction of EsxA and EsxB remains incompletely understood. In this study, to explore their interactions, EsxA and EsxB were expressed and used for immunization, alone or in combination, of murine infection models. Both components can interact with each other. Through the analysis of the immune response by immunological method, EsxB could significantly enhance the EsxA-specific IgG2a antibody level and increase the proliferation proportion of CD8+ T cells. These results indicate that when vaccinated with EsxA, EsxB can play a critical role in stimulating T helper 1 immunity by activating IgG2a and CD8+ T cells. We further show that vaccination with the combination of EsxA and EsxB resulted in enhanced stimulation of TLR-4 and improved protection against S. aureus. The findings may help us better understand the role of EsxB in the virulence and pathogenesis of S. aureus.

The Relationship Between Androgens and Days per Month of Period Pain, Pelvic Pain, Headache, and TLR4 Responsiveness of Peripheral Blood Mononuclear Cells in Young Women with Dysmenorrhoea.

PurposeWomen bear a disproportionate burden of persistent pain conditions when compared to men. To determine whether the hormonal environment affects the clinical experience of pain, as measured by the days per month of pelvic pain (DPelvicPM), period pain (DPeriodPM), headache (DHeadachePM) or the in vitro EC50 for Interleukin-1β (IL-1β) release following TLR4 stimulation with Lipopolysaccharide from Peripheral Blood Mononuclear Cells (PBMCs). Findings were stratified according to use or non-use of the oral contraceptive pill.Patients and MethodsFifty-six women aged 16–35 years, with minimal or severe dysmenorrhea, and use or non-use of the OC, were enrolled. Blood was collected on two occasions in a single menstrual cycle: Days 1–2 and Days 7–10. Hormonal analysis for testosterone, dihydrotestosterone, dehydroepiandrosterone, Androstenedione, 3α-Androstanediol, 3β-androstanediol, estradiol, estrone, 17α-hydroxyprogesterone, progesterone, cortisol and sex-hormone binding globulin was undertaken using ultra-sensitive Liquid Chromatography Mass–Spectrometry (LC-MS). PBMCs were exposed to lipopolysaccharide (LPS) and the resulting Interleukin-1β output was determined.ResultsNon-users of the OC showed a strongly inverse correlation between a reducing free androgen index (FAI) and increasing DPelvicPM (p=0.0032), DPeriodPM (p=0.013), DHeadachePM (p=0.041). Non-users of the OC showed a significant increase in DPelvicPM (p=0.049) on Days 7–10. Modestly significant associations were found between reduced androgens and potentiated LPS-induced IL-1β (lower EC50).ConclusionThis is the first study to investigate the relationship between the hormonal environment and activation of the immune system in young women with dysmenorrhoea-related pain conditions. Low androgen levels were consistently associated with increased pain. Translational implications for the findings are discussed.

IRF1 governs the differential interferon-stimulated gene responses in human monocytes and macrophages by regulating chromatin accessibility

SUMMARYMyeloid lineage cells use TLRs to recognize and respond to diverse microbial ligands. Although unique transcription factors dictate the outcome of specific TLR signaling, whether lineage-specific differences exist to further modulate the quality of TLR-induced inflammation remains unclear. Comprehensive analysis of global gene transcription in human monocytes, monocyte-derived macrophages, and monocyte-derived dendritic cells stimulated with various TLR ligands identifies multiple lineage-specific, TLR-responsive gene programs. Monocytes are hyperresponsive to TLR7/8 stimulation that correlates with the higher expression of the receptors. While macrophages and monocytes express similar levels of TLR4, macrophages, but not monocytes, upregulate interferon-stimulated genes (ISGs) in response to TLR4 stimulation. We find that TLR4 signaling in macrophages uniquely engages transcription factor IRF1, which facilitates the opening of ISG loci for transcription. This study provides a critical mechanistic basis for lineage-specific TLR responses and uncovers IRF1 as a master regulator for the ISG transcriptional program in human macrophages.

TLR7/8 agonist treatment induces an increase in bone marrow resident dendritic cells and hematopoietic progenitor expansion and mobilization

There is accumulating evidence suggesting that toll-like receptor (TLR) signals play an important role in the regulation of hematopoietic stem/progenitor cells (HSPCs). TLR7/8 stimulation induces the myeloid differentiation of normal HSPCs and acute myeloid leukemia cells. However, the in vivo effect of TLR7/8 agonists on hematopoiesis is largely unknown. Here, we show that, similar to TLR4 and TLR2, treatment with the TLR7/8 agonist R848 induces an expansion of phenotypic hematopoietic stem cells (HSCs) with reduced repopulating potential and HSPC mobilization. In contrast to chronic TLR4 stimulation, treatment with R848 for 5 days did not induce a significant increase in myeloid-biased HSCs. Treatment with R848 results in a significant increase in classic dendritic cells (DCs) in the bone marrow, but a decrease in common dendritic cell progenitors and pre-DCs. Phenotypic analysis of DCs revealed that R848 treatment is associated with altered expression of certain chemokines, activation markers, and migratory receptors. Together, these data indicate that systemic administration of a TLR7/8 agonist has unique effects on hematopoiesis, including the expansion of DCs in the bone marrow, that might have clinical relevance to augment responses to certain immunotherapies, such as cancer vaccines and immune checkpoint blockade.

Interaction of TLR4 and TLR8 in the Innate Immune Response against Mycobacterium Tuberculosis.

The interaction and crosstalk of Toll-like receptors (TLRs) is an established pathway in which the innate immune system recognises and fights pathogens. In a single nucleotide polymorphisms (SNP) analysis of an Indian cohort, we found evidence for both TLR4-399T and TRL8-1A conveying increased susceptibility towards tuberculosis (TB) in an interdependent manner, even though there is no established TLR4 ligand present in Mycobacterium tuberculosis (Mtb), which is the causative pathogen of TB. Docking studies revealed that TLR4 and TLR8 can build a heterodimer, allowing interaction with TLR8 ligands. The conformational change of TLR4-399T might impair this interaction. With immunoprecipitation and mass spectrometry, we precipitated TLR4 with TLR8-targeted antibodies, indicating heterodimerisation. Confocal microscopy confirmed a high co-localisation frequency of TLR4 and TLR8 that further increased upon TLR8 stimulation. The heterodimerisation of TLR4 and TLR8 led to an induction of IL12p40, NF-κB, and IRF3. TLR4-399T in interaction with TLR8 induced an increased NF-κB response as compared to TLR4-399C, which was potentially caused by an alteration of subsequent immunological pathways involving type I IFNs. In summary, we present evidence that the heterodimerisation of TLR4 and TLR8 at the endosome is involved in Mtb recognition via TLR8 ligands, such as microbial RNA, which induces a Th1 response. These findings may lead to novel targets for therapeutic interventions and vaccine development regarding TB.

Abstract PO011: Systemic administration of Poly-ICLC promotes T cell tumor infiltration generating antitumor responses

Abstract Background: Recent advances in immunotherapy have revolutionized cancer treatment. Immune checkpoint blockade and adoptive T cell therapy can induce durable responses in some patients. However, the majority of patients fail to respond. Absence of T cell infiltration to the tumor site in solid tumors is considered as one of the major obstacles for effective immunotherapy. Thus, strategies to enhance T cell trafficking and infiltration into the tumor parenchyma is a major research need. Methods: Using a combination of in vivo mouse tumor models and in vitro assays we compared the capacity of various formulations and routes of administration of poly-IC (a dsRNA mimic that functions as a pattern recognition receptor ligand) in enhancing T cell tumor infiltration and generating antitumor responses. Results: Our results showed that poly-ICLC (poly-IC containing poly-lysine and carboxymethylcellulose) enhanced CD8 T cell infiltration into the tumors resulting in the control of tumor growth. These effects relied both on the route of administration as well as on the formulation of poly-IC. Systemic administration (i.v. or i.m.) of poly-ICLC was significantly more effective in inducing CD8 T cell tumor infiltration as compared to intra-tumoral injections. Also, the effects poly-ICLC were substantially more pronounced as compared to unmodified poly-IC. The antitumor effect of poly-ICLC was mediated via MDA-5 and IFN-I, whereas TLR3 stimulation was not required. Interestingly, poly-ICLC stimulated IFN-I responses on tumor vascular endothelial cells (VECs) enhancing the expression of adhesion molecules (VCAM-I), production of IFN-I and T cell recruiting chemokines (CXCL9/CXCL10). Using conditional knockout mice, showed that ablation of IFNab receptors in VECs impaired the antitumor effects of poly-ICLC. IFN-I production upon MDA5 stimulation is required to enhance the secretion of (CXCL9/CXCL10) by VECs indicating that this IFN-I/CXCL9/CXCL10 regulatory axis is crucial for recruiting effector T cells into the tumor parenchyma. The T cell infiltrating effects of poly-ICLC were mainly observed in tumors and not in normal tissues. Conclusions: Systemic administration of poly-ICLC improves T cell infiltration to solid tumors in an MDA5/IFN-I dependent manner. These findings should have a strong impact on improving T cell-based treatments for solid cancers such as adoptive T cell therapy and antitumor vaccines. Citation Format: Hussein Sultan, Juan Wu, Valentyna Fesenkova, Aaron Fan, Diane Addis, Andres Salazar, Esteban Celis. Systemic administration of Poly-ICLC promotes T cell tumor infiltration generating antitumor responses [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO011.

Effects of innate immune receptor stimulation on extracellular \u03b1-synuclein uptake and degradation by brain resident cells

Synucleinopathies are age-related neurological disorders characterized by the progressive deposition of α-synuclein (α-syn) aggregates and include Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). Although cell-to-cell α-syn transmission is thought to play a key role in the spread of α-syn pathology, the detailed mechanism is still unknown. Neuroinflammation is another key pathological feature of synucleinopathies. Previous studies have identified several immune receptors that mediate neuroinflammation in synucleinopathies, such as Toll-like receptor 2 (TLR2). However, the species of α-syn aggregates varies from study to study, and how different α-syn aggregate species interact with innate immune receptors has yet to be addressed. Therefore, we investigated whether innate immune receptors can facilitate the uptake of different species of α-syn aggregates. Here, we examined whether stimulation of TLRs could modulate the cellular uptake and degradation of α-syn fibrils despite a lack of direct interaction. We observed that stimulation of TLR2 in vitro accelerated α-syn fibril uptake in neurons and glia while delaying the degradation of α-syn in neurons and astrocytes. Internalized α-syn was rapidly degraded in microglia regardless of whether TLR2 was stimulated. However, cellular α-syn uptake and degradation kinetics were not altered by TLR4 stimulation. In addition, upregulation of TLR2 expression in a synucleinopathy mouse model increased the density of Lewy-body-like inclusions and induced morphological changes in microglia. Together, these results suggest that cell type-specific modulation of TLR2 may be a multifaceted and promising therapeutic strategy for synucleinopathies; inhibition of neuronal and astroglial TLR2 decreases pathogenic α-syn transmission, but activation of microglial TLR2 enhances microglial extracellular α-syn clearance.