Through Toll-like Receptors Signaling Research Articles

Abstract B014: TLR signaling coordinates SAA-dependent liver metastasis in pancreatic cancer

Abstract Metastasis remains the primary cause of mortality in cancer. This is especially true in pancreatic ductal adenocarcinoma (PDAC) - where the liver is the most common site of metastasis and the 5-year overall survival rate for these patients has remained <3% for the past 20 years. Accumulating evidence shows a critical role for a niche microenvironment in the liver, termed the pro-metastatic niche, that supports metastatic seeding and outgrowth. Our previous work demonstrated that hepatocytes orchestrate the formation of this pro-metastatic niche during early tumorigenesis through the release of Serum Amyloid A1 and 2 (SAA). However, the mechanisms by which SAA directs formation of a pro-metastatic niche in the liver, and the impact of this niche on the fate of disseminating tumor cells (DTCs) seeding the liver, remain ill-defined. To begin to identify the cellular determinants of niche formation, we first analyzed the expression of SAA receptors within the normal liver and found predominant expression of receptors on the resident F4/80+ macrophages. This is in line with current published work as SAA is well characterized to control liver biology through toll-like receptors (TLRs) expressed by liver resident immune cells and hepatic stellate cells. Consistent with this, we also found that SAA is a specific, potent agonist of TLR2 in vitro. In tumor bearing mice we then found increased expression of TLR2 transcripts as well as an accumulation of TLR2+ inflammatory monocytes within the niche. Together, these findings suggested that SAA might form a pro-metastatic niche in the liver withough a TLR2-dependent mechanism. To test this, we next examined the livers of TLR2-/- tumor bearing mice and found a decrease in the expression of surrogate markers of the pro-metastatic niche. We also sought to determine a role for TLR2 in metastatic outgrowth. Using TLR2-/- mice in a model of PDAC with direct metastasis to the liver, we found significantly decreased metastatic burden and outgrowth in the liver. Analysis of micro-metastatic lesions in TLR2-/- tumor bearing mice also revealed that DTCs demonstrated molecular markers of cellular dormancy. Altogether, our findings suggest the evolution of an inflammatory mechanism within the niche wherein SAA may continue to signal through TLR2+ inflammatory monocytes, which then act to coordinate metastatic outgrowth. Future work aims to further elucidate the role of TLR2+ resident macrophages and monocytes in the formation of a pro-metastatic niche in the liver and in regulating metastatic outgrowth. Citation Format: Jesse Lee, Meredith L. Stone, Hana Choi, Gregory L. Beatty. TLR signaling coordinates SAA-dependent liver metastasis in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B014.

TLR or NOD receptor signaling skews monocyte fate decision via distinct mechanisms driven by mTOR and miR-155

Monocytes are rapidly recruited to inflamed tissues where they differentiate into monocyte-derived macrophages (mo-mac) or dendritic cells (mo-DC). At infection sites, monocytes encounter a broad range of microbial motifs. How pathogen recognition impacts monocyte fate decision is unclear. Here, we show, using an in vitro model allowing the simultaneous differentiation of human mo-mac and mo-DC, that viruses promote mo-mac while Mycobacteria favor mo-DC differentiation. Mechanistically, we found that pathogen sensing through toll-like receptor (TLR) ligands increases mo-mac differentiation via mTORC1. By contrast, nucleotide-binding oligomerization domain (NOD) ligands favor mo-DC through the induction of TNF-α secretion and miR-155 expression. We confirmed these results in vivo, in mouse skin and by analyzing transcriptomic data from human individuals. Overall, our findings allow a better understanding of the molecular control of monocyte differentiation and of monocyte plasticity upon pathogen sensing.

Dynamics of Toll-like Receptors Signaling in Skeletal Muscle Atrophy.

Skeletal muscle atrophy has been characterized as a state of uncontrolled inflammation and oxidative stress that escalates protein catabolism. Recent advancement supports impinging signaling molecules in the muscle fibers controlled through toll-like receptors (TLR). Activated TLR signaling pathways have been identified as inhibitors of muscle mass and provoke the settings for muscle atrophy. Among them, mainly TLR2 and TLR4 manifest their presence to exacerbate the release of the pro-inflammatory cytokine to deform the synchronized muscle programming. The present review enlightens the TLR signaling mediated muscle loss and the interplay between inflammation and skeletal muscle growth.

Acute liver failure is regulated by MYC- and microbiome-dependent programs.

Acute liver failure (ALF) is a fulminant complication of multiple etiologies, characterized by rapid hepatic destruction, multi-organ failure and mortality. ALF treatment is mainly limited to supportive care and liver transplantation. Here we utilize the acetaminophen (APAP) and thioacetamide (TAA) ALF models in characterizing 56,527 single-cell transcriptomes to define the mouse ALF cellular atlas. We demonstrate that unique, previously uncharacterized stellate cell, endothelial cell, Kupffer cell, monocyte and neutrophil subsets, and their intricate intercellular crosstalk, drive ALF. We unravel a common MYC-dependent transcriptional program orchestrating stellate, endothelial and Kupffer cell activation during ALF, which is regulated by the gut microbiome through Toll-like receptor (TLR) signaling. Pharmacological inhibition of MYC, upstream TLR signaling checkpoints or microbiome depletion suppress this cell-specific, MYC-dependent program, thereby attenuating ALF. In humans, we demonstrate upregulated hepatic MYC expression in ALF transplant recipients compared to healthy donors. Collectively we demonstrate that detailed cellular/genetic decoding may enable pathway-specific ALF therapeutic intervention.

Regulation of GLI3 expression by TLR4 signaling

Abstract While acute inflammation is an important host response to infections, chronic inflammation has emerged as a mechanism driving many diseases including autoimmune and malignant diseases. Monocytes are a subset of immune cells that secrete inflammatory cytokines in response to infections through toll-like receptor (TLR) signaling. The cross-talk between TLR signaling and other pathways has been shown to be important for downstream effector functions. We identify a novel role for the transcription factor GLI3, a negative regulator of Hedgehog signaling, in mediating TLR4-induced inflammation. Stimulation of monocytes with LPS increases GLI3 mRNA and protein expression. This occurred in a dose- and time-dependent manner. Investigation of the signaling mechanism suggests that LPS induced GLI3 occurs through the adaptor protein TRIF (but not MyD88). Targeting of transcription factors NFkb, AP-1 and IRF3 by pharmaceutical inhibitors suggests that TLR4-mediated GLI3 is regulated by IRF3. Transfection of our monocyte cell lines with shRNA targeting GLI3 suggests a regulatory role of transcription factor GLI3 in mRNA expression of essential cytokines involved in inflammation including IL-6 and CCL2. We generated conditional GLI3 knockout in mice myeloid cells using LysM-cre mediated recombination. IFA-elicited macrophages from mice with conditional GLI3 knockout driven by LysM-cre stimulated with LPS show reduced IL-6 and CCL2 secretion compared to macrophages from wild-type littermates. Taken together, these data suggest that GLI3 plays a role in innate immunity by regulating TLR4-induced inflammation.

Divergent roles for TLR7 and TLR9 in modulating the NFκB pathway in B cells

Abstract BCR signal strength plays a key role in the fate decision of B cells with strong BCR signals promoting plasma cell differentiation and weaker BCR signals driving germinal center formation. Signals through toll-like receptors (TLRs) also influence B cell differentiation. It is still unclear how the BCR and TLR signaling cascades interface to modulate B cell responses and differentiation. To address this question, we used a BCR sdTg mouse model, AM14, in which all mature B cells have a naïve follicular phenotype. The AM14 BCR recognizes IgG2a-bound immune complexes (ICs) and B cell activation depends on TLR7 and/or TLR9. ICs formed using the auto-antibody (autoAb) PL2-3 can activate AM14 B cells through either BCR/TLR9 or BCR/TLR7, allowing us to use TLR knockout lines to dissect signaling pathways unique to each TLR. Using this system, we reported earlier that BCR/TLR7-activated AM14 B cells differentiate into short-lived plasmablasts while plasma cell development does not occur in BCR/TLR9-activated AM14 B cells. Intriguingly, we now show that BCR/TLR9 activation of AM14 B cells leads to sustained activation of the NFκB pathway. BCR/TLR7-activated AM14 B cells are unable to sustain the initial NFκB response and fail to express the non-classical IκB member, IκBζ. IκBζ is known to be important in regulating cytokine responses in T cells and macrophages. However, its role in B cells is not well defined. We find that several IκBζ regulated cytokines are expressed after BCR/TLR9 dependent signaling but not after BCR/TLR7 dependent signaling. Our results point to divergent roles for TLR7 and TLR9 in modulating the NFκB signaling cascades, leading to differential expression of NFκB target genes which may influence B cell fate decisions.

Inhibition of ubiquitin-specific processing proteases 10 and 13 modulates IL-10 and TNF-α production in response to LPS in murine macrophages

Abstract Innate immune response is activated by pathogen-associated molecular patterns (PAMPs) through Toll-like receptors (TLRs). To maintain immune homeostasis, TLR signaling molecules are regulated by a dynamic interplay of various ubiquitination (Ub) and deubiquitylation (DUB) events. Specifically, Lys-63 (K63)-linked ubiquitination on TRAF6 is involved in phosphorylation and activation of downstream MAPKs and IkB kinases. While certain types of ubiquitin modifications lead to activation and stability of proteins, other types cause increased proteasomal degradation and hence deactivation. DUBs are a class of the family of proteases that remove ubiquitin or ubiquitin-like proteins from a variety of protein-substrates. The role of DUBs in regulating the TLR pathway has not been well studied. Through RNA sequencing, we identified that LPS activation of bone marrow derived murine macrophages (BMDMs) leads to significant increase of two DUBs: ubiquitin-specific protease (USP) 10 and 13. To further study their role, we used Spautin-1, an inhibitor of USP10 and USP13. Spautin-1 modulated the LPS-mediated cytokine production and led to upregulation of IL-10, reduction of TNF-α, and had no effect on IL-6 production. However, we found no changes in expression of TRAF-6 or downstream MAPKs, including ERK, JNK, and P38. Alternative pathways were also studied with no effect in pSTAT-1, pSTAT-3, or A20 expression. Altered expression of pro-inflammatory mediators in the presence of Spautin-1 may have clinical importance associated with processes such as acute or chronic inflammatory processes.

Biglycan is a new high-affinity ligand for CD14 in macrophages

Sterile inflammation is a therapeutic target in many diseases where it represents an important initiator of disease progression. However, the detailed mechanisms underlying its evolution and biological relevance are not yet completely elucidated. Biglycan, a prototype extracellular matrix-derived damage-associated molecular pattern, mediates sterile inflammation in macrophages through Toll-like receptor (TLR) 2 and/or TLR4-dependent signaling pathways. Here we discovered that soluble biglycan is a novel high-affinity ligand for CD14, a well-known GPI-anchored co-receptor for TLRs. CD14 is required for all biglycan-mediated TLR2/4 dependent inflammatory signaling pathways in macrophages. By binding to CD14 and choosing different TLR signaling branches, biglycan induced TNF-α and CCL2 via TLR2/4, HSP70 through TLR2, and CCL5 via TLR4. Mechanistically, biglycan evoked phosphorylation and subsequent nuclear translocation of p38, p44/42, and NF-κB, and these effects were due to a specific, high-affinity interaction between biglycan protein core and CD14. Finally, we provide proof-of-principle for the requirement of CD14, by transiently overexpressing biglycan in a mouse model of renal ischemia/reperfusion injury performed in Cd14−/− mice. Lack of Cd14 prevented biglycan-mediated cytokine expression, recruitment of macrophages, M1 macrophage polarization as well as mitigated the tubular damage and serum creatinine levels, thereby improving renal function. Thus, CD14 inhibition could lead to the reduction in the activation of biglycan-TLR2/4 signaling pathways and could be a novel therapeutic approach in inflammatory kidney diseases.

Thymoquinone alleviates the experimentally induced Alzheimer's disease inflammation by modulation of TLRs signaling.

Alzheimer's disease (AD) is characterized by a robust inflammatory response elicited by the accumulation and deposition of amyloid-β (Aβ) within the brain. Aβ induces detrimental inflammatory responses through toll-like receptors (TLRs) signaling pathway. Thymoquinone (TQ), the main active constituent of Nigella sativa oil, has been reported by several previous studies for its potent anti-inflammatory effect. The aim of this study is to elucidate the effect of TQ in improving learning and memory, using a rat model of AD induced by a combination of aluminum chloride (AlCl3) and d-galactose (d-Gal). TQ was administered orally at doses of 10, 20, and 40 mg/kg/day for 14 days after AD induction. Memory functions were assessed using the step through passive avoidance test. Amyloid plaques were shown to be present using hematoxylin and eosin staining. Tumor necrosis factor-alpha (TNF-α) and Interleukin-1beta (IL-1β) levels in brain were assessed via ELISA and profiling TLR-2, TLR-4, myeloid differential factor 88, toll-interleukin-1 receptor domain-containing adapter-inducing interferon-β, interferon regulatory factor 3 (IRF-3), and nuclear factor-κB (NF-κB) expressions via real-time polymerase chain reaction. TQ improved AD rat cognitive decline, decreased Aβ formation and accumulation, significantly decreased TNF-α and IL-1β at all levels of doses and significantly downregulated the expression of TLRs pathway components as well as their downstream effectors NF-κB and IRF-3 mRNAs at all levels of doses ( p < 0.05). We concluded that TQ reduced the inflammation induced by d-Gal/AlCl3 combination. It is therefore reasonable to assign the anti-inflammatory responses to the modulation of TLRs pathway.

Alpha(v) Integrins Engage Autophagy Components to Regulate B cell Immune Response

Abstract Signaling through Toll-like receptors (TLR) has been implicated in activation of immune cell populations such as B lymphocytes. TLR ligands associated with self-antigens can drive increased self-reactive B cell responses however TLR ligands present in pathogenic products or vaccines can also enhance protective antibody responses. Our recent work has identified a novel mechanism by which αv integrins, a family of adhesion molecules and the autophagy proteins limit excessive B cell TLR signaling. Specifically, we have found that αvβ3 integrin regulates TLR signaling (NFKB and IRF7 activation) by directing maturation of TLR containing endosomes, through activation of components of the autophagy pathway (LC3 and Atg5). B cells lacking either αv integrins or autophagy components show enhanced TLR signaling and increased proliferative responses to TLR ligands. Mice with disruption in this pathway develop increased autoantibodies with age and develop accelerated autoimmunity in murine lupus models. Therefore, we propose that αv-mediated regulation of TLR signaling exists to limit excessive B cell responses to self-antigens. In addition, we find that mice in which αv is specifically deleted from B cells mount stronger antibody responses when immunized with exogenous antigens containing TLR-ligand adjuvants. Immune responses to these antigens are characterized by increased expansion of germinal center cells, increased somatic hyper-mutation and higher levels of high-affinity class-switched antibodies. Therefore this αv mediated regulation of TLR signaling not only regulates B cell responses to self-antigens but it is also important for regulating germinal center B cell responses and production of high-affinity antibodies.

Controlling T cell development and function via innate Toll-like receptor signaling for autoimmune therapy

Abstract Innate regulation through Toll-like receptor (TLR) signaling has been shown to be important for promoting T cell subset development and function. However, limited information is known about whether differential TLR signaling can selectively inhibit Th17 and/or Th1 cells, important for controlling excessive inflammation and autoimmune responses. Here we demonstrate that activation of TLR7 signaling in T cells can inhibit both Th17 cell differentiation from naïve T cells and IL-17 production in established Th17 cells. We further report that down-regulation of STAT3 signaling is responsible for TLR7-mediated inhibition of Th17 cells due to induction of SOCS3 and SOCS5. TLR7-mediated suppression of Th17 cells does not require DC involvement. In addition, we show that TLR7 signaling can suppress Th1 cell development and function due to a mechanism different from Th17 cell suppression. Importantly, our complementary in vivo studies demonstrate that treatment with the TLR7 ligand imiquimod can inhibit both Th1 and Th17 cells, resulting in prevention of and immunotherapeutic reduction in experimental autoimmune encephalomyelitis (EAE). These studies identify a new strategy to manipulate Th17/Th1 cells through TLR7 signaling, with important implications for successful immunotherapy against autoimmune and inflammatory diseases.

B cell αv integrins regulate development of autoimmunity in murine lupus models

Abstract B cell activation by self-antigen and signaling through Toll-like receptors (TLR) such as TLR7 and TLR9 has been implicated in the development of autoimmunity. We have recently identified a mechanism by which αvβ3 integrin limit excessive B cell responses to antigens containing TLR ligands, including self-antigens. We have previously reported that mice in which αv integrins are selectively deleted from B cells develop increased levels of autoantibodies to RNA and double stranded DNA. Based on these findings, we hypothesized that deletion of αv integrins would accelerate TLR dependent autoimmunity. To study this further, we crossed mice lacking αv integrins on B cells (αv-CD19 mice) with mice expressing increased levels of TLR7 (TLR7.1tg mice) that develop Lupus-like autoimmunity. We find that the resulting αv-CD19 TLR7tg mice had increased spleen weight in comparison with age-matched TLR7tg littermates. αv-CD19 TLR7tg mice also had increased number of IgG2c+ plasma cells and increased autoantibodies to RNA, all indicative of enhanced B Cell activation and development of autoimmunity. In addition, deletion of αv integrin on B Cells in this model leads to enhanced activation of T Cells. Currently, we are investigating the role of αv integrins on B Cell subpopulations in driving T Cell activation and antigen presentation in this model of autoimmunity. B Cells contribute to autoimmunity by activating T Cells in addition to producing autoantibodies and cytokines. These studies allow us to understand how altered B Cell TLR signaling can lead to enhanced immune activation in autoimmunity and highlight the importance of αv integrins in regulating this process.

αv integrins engage autophagy components to regulate B cell immune response

Abstract Signaling through Toll-like receptors (TLR) such as TLR7 and TLR9 has been implicated in B cell activation by self-antigens and development of autoimmunity. Our recent work based on murine models has identified a novel mechanism by which a family of adhesion molecules called αv integrins and the autophagy pathway limit excessive B cell responses to antigens containing TLR ligands and self-derived antigens. Specifically, we have found that αvβ3 integrin regulates B cell TLR signaling by directing trafficking and maturation of TLR containing endosomes and engaging components of the autophagy pathway. When B cells lack either αv integrins or autophagy components LC3 and Atg5 endosomal trafficking of the TLRs is disrupted and leads to enhanced TLR signaling and increased B cell responses. We have used av conditional knockout mice to determine the consequences of loss of this αvβ3-mediated immune regulation. Mice lacking B cell av show increased antibody responses to antigens containing TLR-ligands and develop increased levels of autoantibodies. We therefore propose that αv-mediated TLR regulation serves to maintain the balance between protective immunity to microbes and potential pathological autoimmune responses. We are currently studying the role of αv signaling and autophagy components in development of class switched, high affinity antibodies in response to antigens in the context of infection with a pathogen or in response to self-antigens.

Integration of innate into adaptive immune responses in ZAP-70–positive chronic lymphocytic leukemia

AbstractThe crucial dependence of chronic lymphocytic leukemia (CLL) cells on signals derived from the B cell receptor (BCR) has encouraged the development of new inhibitors, which interfere with BCR signaling and demonstrate clinical benefits in nearly all patients. In addition, signaling through Toll-like receptor (TLR) 9 of the innate immune system has been shown to further contribute to the activation of CLL cells. However, responses to TLR9 engagement are not uniform, but diametrically opposed with cell death in some patients and cell proliferation in others. We now provide evidence that heterogeneous responses to TLR agonists are related to differences in the ability of CLL cells to activate the BCR-associated kinase Syk. Notably, expression of ZAP-70 appears to be of crucial importance for TLR9-mediated activation of Syk. We show that the activation of Syk provides an antiapoptotic signal, which is independent of Mcl-1, Bcl-2, and Bcl-XL, but related to the degradation of the proapoptotic Bim. Mechanistically, TLR9-mediated antiapoptotic signals in ZAP-70–positive CLL trigger secretion of immunoglobulin M, which then serves as (auto-) antigen for a prosurvival BCR signal. Thus, our data show that single activation of the innate immune receptor TLR9 is sufficient to fully engage BCR signaling in ZAP-70–positive CLL, protecting malignant cells from apoptosis. We conclude that the integration of TLR signaling into an adaptive immune response can further promote survival of CLL cells and may contribute to the unfavorable prognosis of ZAP-70–positive CLL.

Role of Toll-Like Receptors in Hepatitis C Virus Pathogenesis and Treatment.

Viral infections are rising every day, and viruses appear to be the most dangerous pathogens in the world. Hepatitis C virus (HCV) is accepted as one of the major destructive factors of promoting severe hepatic disorders by infecting more than 180 million individuals throughout the world. Chronic infection caused by HCV poses a serious global health emergency and appears to be a powerful threat to humanity. Almost 20 years have passed since the disclosure of HCV, but even now, treatment preferences remain limited. Humans are born with a rapid and nonspecific mechanism to prevent viral attacks through Toll-like receptors (TLRs), which are evolutionary conserved cellular activator proteins responsible for recognizing specific components present on penetrating microbes and viruses. Recent research efforts in TLR biology suggest that targeting the TLRs and their signaling pathways during HCV infection could contribute to novel therapies against HCV. The mobilization of TLRs boosts antiviral communication and integrates the development of long-lasting acquired immune responses to limit viral pathogenesis. Both activation and suppression of TLRs are necessary for the efficient treatment of HCV. For the proper management and eradication of HCV, novel drugs that target TLRs and their signaling pathway are needed. This review summarizes the role of TLR signaling in HCV infection and treatment.

A Blunted Response to the Anti-Inflammatory Cytokine IL-10 Contributes to Excessive TNF Production in Myeloproliferative Neoplasm

Rationale:Tumor Necrosis Factor-alpha (TNF) is elevated in myeloproliferative neoplasm (MPN) and plays a key role in expansion of the JAK2V617F neoplastic clone. A high TNF environment, as is the case in MPN patients, gives TNF resistant JAK2V617F mutant cells a selective advantage over their TNF sensitive non-mutant counterparts resulting in expansion of the neoplastic clone. To efficiently target TNF production therapeutically it is necessary to identify the mechanism driving this excessive TNF production. TNF is classically produced by monocytes after stimulation through Toll-like receptors (TLR), crucial pattern recognition receptors for microbial products. Because TLR signaling plays an integral role in inflammation and TNF production, we hypothesized that exaggerated signaling of the TLR pathway is the mechanism by which TNF is overproduced in MPN.Results:We compared the response of peripheral blood monocytes from MPN versus normal controls to the TLR agonists R848 (TLR7/8), LPS (TLR4), or zymosan (TLR2). After stimulation with each of these TLR agonists for 24 hours, CD14+ monocytes from MPN patients (n=18) produced increased amounts of TNF (measured by ELISA) as compared to normal controls (n=10) at all concentrations tested (p<0.05). The increased TNF production in MPN monocytes could not be explained by a higher fraction of inflammatory (CD16+ CD14+) monocytes in MPN. We next used phosflow to detect whether MPN patients have abnormal activation of downstream signaling molecules following stimulation with TLR agonists. At early time points (15min) following stimulation, MPN patients (n=6) and normal controls (n=6) phosphorylated p38 and ERK1/2 equally. At later time points (2hrs) MPN patients maintained phosphorylation of p38 and ERK1/2, whereas in normal controls phosphorylation of p38 and ERK1/2 returned to baseline (p<0.05).This suggested that the negative feedback regulation of TLR signaling may be defective in MPN patients resulting in prolonged TNF production after stimulation. To test this, we compared the tempo of TNF production following LPS stimulation in MPN versus normal monocytes using intracellular flow cytometry as well as ELISA. Monocytes from normal controls (n=12) cease production of TNF by 9 hours post LPS stimulation, whereas MPN monocytes (n=12) continue to produce TNF at 18 hours (p<0.05).LPS stimulation induces a negative feedback loop culminating in the production of IL-10, an anti-inflammatory cytokine which serves to dampen TNF production. We found that MPN monocytes produce abundant IL-10 in response to LPS stimulation. Next we measured the ability of IL-10 to dampen TNF production in LPS stimulated monocytes comparing MPN patients versus normal controls. Addition of low concentrations of rhIL-10 (1ng/ml) to LPS stimulated monocytes reduced TNF production by 50% in normal controls (n=8), whereas it reduced TNF production by only 10-20% in MPN (n=5). However, higher concentrations of IL-10 (>10ng/ml) was able to dampen TNF production in LPS stimulated MPN monocytes by 50%.Conclusions:MPN monocytes produce excessive amounts of TNF in response to TLR ligation due to a defect in the negative regulatory feedback loop which normally serves to dampen TNF production following TLR ligation. We have localized this defect to a blunted response to the anti-inflammatory cytokine IL-10. MPN monocytes are less responsive to the anti-inflammatory actions of IL-10 at low concentrations but these effects can be restored by increasing the concentration of IL-10. Together, these data suggest that administration of IL-10 may reduce excessive inflammatory cytokine production in MPN. DisclosuresNo relevant conflicts of interest to declare.

Toll-like receptor signaling is functional in immune cells of the endangered Tasmanian devil

Devil facial tumour disease (DFTD) is a fatally transmissible cancer that threatens the Tasmanian devil population. As Tasmanian devils do not produce an immune response against DFTD cells, an effective vaccine will require a strong adjuvant. Activation of innate immune system cells through toll-like receptors (TLRs) could provide this stimulation. It is unknown whether marsupials, including Tasmanian devils, express functional TLRs. We isolated RNA from peripheral blood mononuclear cells and, with PCR, detected transcripts for TLRs 2, 3, 4, 5, 6, 7, 8, 9, 10 and 13. Stimulation of the mononuclear cells with agonists to these TLRs increased the expression of downstream TLR signaling products (IL1α, IL6, IL12A and IFNβ). Our data provide the first evidence that TLR signaling is functional in the mononuclear cells of the Tasmanian devil. Future DFTD vaccination trials will incorporate TLR agonists to enhance the immune response against DFTD.

Contribution of MyD88-dependent PKD1 to the development of arthritis (THER2P.954)

Abstract Rheumatoid arthritis (RA) may be initiated in part by signaling through Toll-like receptors (TLRs). Disruption of TLR signaling may provide an opportunity to halt the RA process in the earliest stages. We investigated whether protein kinase D1 (PKD1), which is essential for proinflammatory responses mediated by MyD88-dependent TLRs, plays a role in the development of experimental arthritis. The presence of TLR ligands in joint cavities induces development of reactive arthritis that is characterized by accumulation of activated inflammatory cells in the joints. Deletion of PKD1 in myeloid lineage cells prevented TLR-mediated accumulation of inflammatory cells in joints, thus protecting mice from developing reactive arthritis. Arthritis spontaneously occurring in IL-1R antagonist-deficient (IL-1rn-/-) mice was dependent on MyD88, and suppression of PKD1 expression resulted in inhibition of TLR-mediated cytokine production in IL-1rn-/- macrophages. Daily treatment with a PKD inhibitor substantially reduced the incidence and severity of arthritis in IL-1rn-/- mice. In addition, a PKD inhibitor inhibited the synergistic effect of T cell receptor and TLR2 on IL-6 production in splenocytes, and significantly reduced the incidence and severity of collagen-induced arthritis that closely mimics the pathophysiologic process of human RA. This implies that PKD1 might be one of the key factors that modulate proinflammatory responses in RA, and can be a therapeutic target for RA.

Exaggerated Response to Toll-like Receptor Agonist Contributes to Excessive TNF Production in Myeloproliferative Neoplasm

Rationale:Tumor Necrosis Factor-alpha (TNF) is elevated in myeloproliferative neoplasm (MPN) and plays a key role in expansion of the JAK2V617F neoplastic clone. A high TNF environment, as is the case in MPN patients, gives TNF resistant JAK2V617F mutant cells a selective advantage over their TNF sensitive non-mutant counterparts resulting in expansion of the neoplastic clone. To efficiently target TNF production therapeutically it is necessary to identify the mechanism driving this excessive TNF production. TNF is classically produced by monocytes after stimulation through Toll-like receptors (TLR), crucial pattern recognition receptors for microbial products. Because TLR signaling plays an integral role in inflammation and TNF production we hypothesized that exaggerated signaling of the TLR pathway is the mechanism by which TNF is overproduced in MPN. To test this hypothesis we quantified TLR responses in monocytes from MPN patients and normal controls.Results:We compared the response of peripheral blood monocytes from MPN versus normal controls to the TLR agonists R848 (TLR7/8), LPS (TLR4), or zymosan (TLR2). After stimulation with each of these TLR agonists for 24 hours, CD14+ monocytes from MPN patients (n=18) produced increased amounts of TNF (measured by ELISA) as compared to normal controls (n=10) at all concentrations tested (p<0.05). The increased TNF production in MPN monocytes could not be explained by a higher fraction of inflammatory (CD16+ CD14+) monocytes in MPN. We next used phosflow to detect whether MPN patients have abnormal activation of downstream signaling molecules following stimulation with TLR agonists. At early time points (15min) following stimulation, MPN patients (n=6) and normal controls (n=6) phosphorylated p38 and ERK1/2 equally. At later time points (2hrs) MPN patients maintained phosphorylation of p38 and ERK1/2, whereas in normal controls phosphorylation of p38 and ERK1/2 returned to baseline (p<0.05). These data suggest that the excessive production of TNF in MPN patients may be due either to persistent activation of signaling following TLR stimulation or failure to repress the activation state of these two proteins.TLR ligation induces a negative feedback loop culminating in the production of IL-10, an anti-inflammatory cytokine which serves to dampen TNF production. We next used intracellular flow cytometry to determine whether the excessive TNF production in MPN is due to a failure to dampen TNF production over time. Monocytes from normal controls (n=12) cease production of TNF by 9 hours post LPS stimulation, whereas MPN monocytes (n=8) continue producing TNF (p<0.05). This continued TNF production by MPN monocytes is coincident with a failure to produce the anti-inflammatory cytokine IL-10 in response to TLR ligation (p<0.05).Conclusions:We find that MPN monocytes produce excessive amounts of TNF in response to TLR ligation. This excessive production of TNF is due to a defect in the negative regulatory feedback loop which normally serves to dampen TNF production. Targeting the TLR pathway therapeutically in MPN may serve to reduce TNF production and neutralize the selective advantage of the JAK2V617F neoplastic clone, ultimately leading to a reduction in the JAK2V617F allele burden. DisclosuresNo relevant conflicts of interest to declare.

Toll-like receptor-triggered calcium mobilization protects mice against bacterial infection through extracellular ATP release.

Extracellular ATP (eATP), released as a "danger signal" by injured or stressed cells, plays an important role in the regulation of immune responses, but the relationship between ATP release and innate immune responses is still uncertain. In this study, we demonstrated that ATP was released through Toll-like receptor (TLR)-associated signaling in both Escherichia coli-infected mice and lipopolysaccharide (LPS)- or Pam3CSK4-treated macrophages. This ATP release could be blocked completely only by N-ethylmaleimide (NEM), not by carbenoxolone (CBX), flufenamic acid (FFA), or probenecid, suggesting the key role of exocytosis in this process. Furthermore, LPS-induced ATP release could also be reduced dramatically through suppressing calcium mobilization by use of U73122, caffeine, and thapsigargin (TG). In addition, the secretion of interleukin-1β (IL-1β) and CCL-2 was enhanced significantly by ATP, in a time- and dose-dependent manner. Meanwhile, macrophage-mediated phagocytosis of bacteria was also promoted significantly by ATP stimulation. Furthermore, extracellular ATP reduced the number of invading bacteria and protected mice from peritonitis by activating purinergic receptors. Mechanistically, phosphorylation of AKT and ERK was overtly increased by ATP in antibacterial immune responses. Accordingly, if we blocked the P2X- and P2Y-associated signaling pathway by using suramin and pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid), tetrasodium salt (PPADS), the ATP-enhanced immune response was restrained significantly. Taken together, our findings reveal an internal relationship between danger signals and TLR signaling in innate immune responses, which suggests a potential therapeutic significance of calcium mobilization-mediated ATP release in infectious diseases.