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The interleukin (IL)-36 cytokines are a sub-family of the IL-1 family which are becoming increasingly implicated in the pathogenesis of inflammatory diseases and malignancies. Initial studies of IL-36 signalling in tumorigenesis identified an immune-mediated anti-tumorigenic function for these cytokines. However, more recent studies have shown IL-36 cytokines also contribute to the pathogenesis of lung and colorectal cancer (CRC).The aim of this study was to investigate IL-36 expression in CRC using transcriptomic datasets and software such as several R packages, Cytoscape, GEO2R and AnalyzeR. Validation of results was completed by qRT-PCR on both cell lines and a patient cohort. Cellular proliferation was assessed by flow cytometry and resazurin reduction.We demonstrate that IL-36 gene expression increases with CRC development. Decreased tumoral IL-36 receptor expression was shown to be associated with improved patient outcome. Our differential gene expression analysis revealed a novel role for the IL-36/IL-17/IL-23 axis, with these findings validated using patient-derived samples and cell lines. IL-36γ, together with either IL-17a or IL-22, was able to synergistically induce different genes involved in the IL-17/IL-23 axis in CRC cells and additively induce colon cancer cell proliferation.Collectively, this data support a pro-tumorigenic role for IL-36 signalling in colon cancer, with the IL-17/IL-23 axis influential in IL-36-mediated colon tumorigenesis.

DNA sensor pathways can initiate inflammasome, cell death and type I interferon (IFN) signalling in immune-mediated inflammatory diseases (IMIDs); including type I interferonopathies. We investigated the involvement of these pathways in the pathogenesis of ulcerative colitis (UC); by analysing expression of DNA sensor, inflammasome, and type I IFN biomarker genes in colonic mucosal biopsy tissue from control (n=31), inactive UC (n=31), active UC (n=33) and a UC single cell RNA-Seq dataset. The effects of type I IFN (IFN-β), IFN-γ and TNF-α on gene expression, cytokine production and cell death were investigated in human colonic organoids. In organoids treated with cytokines alone, or in combination with NLRP3, caspase or JAK inhibitors, cell death was measured, and supernatants were assayed for IL-1β/IL-18/CXCL10. The expression of DNA sensor pathway genes - PYHIN family members (AIM2, IFI16, MNDA, PYHIN1), as well as ZBP1, cGAS and DDX41 were increased in active UC and expressed in a cell type restricted pattern. Inflammasome genes (CASP1, IL1B, IL18), type I IFN inducers (STING, TBK1, IRF3), IFNB1 and type I IFN biomarker genes (OAS2, IFIT2, MX2) were also increased in active UC. Co-treatment of organoids with IFN-β or IFN-γ and TNFα increased expression of IFI16, ZBP1, CASP1, cGAS and STING, induced cell death and IL-1β/IL-18 secretion. This inflammatory cell death was blocked by the JAK inhibitor tofacitinib but not by inflammasome or caspase inhibitors. Increased type I IFN activity may drive elevated expression of DNA sensor genes and JAK-dependent but inflammasome-independent inflammatory cell death of colonic epithelial cells in UC.

The IL-36 family of cytokines were first identified in 2000 based on their sequence homology to IL-1 cytokines. Over subsequent years, the ability of these cytokines to either agonise or antagonise an IL-1R homologue, now known as the IL-36 Receptor (IL-36R), was identified and these cytokines went through several cycles of renaming with the current nomenclature being proposed in 2010. Despite being identified over 20 years ago, it is only during the last decade that the function of these cytokines in health and disease has really begun to be appreciated, with both homeostatic functions in wound healing and response to infection, as well as pathological functions now ascribed. In the disease context, over activation of IL-36 has now been associated with many inflammatory diseases including Psoriasis and inflammatory bowel diseases, with roles in cancer also now being investigated. This review summarises the current knowledge of IL-36 biology, its role in inflammatory diseases and focuses on an emerging role for IL-36 in cancer.

Electrochemotherapy (ECT), the application of an electric impulse to deliver chemotherapy drugs into cells, has been in clinical trials since the early 1990s and has been used for a variety of different malignancies including melanoma and sarcoma. A standard operating procedure for the use of ECT in clinical settings has been established since 2006. ECT is very effective in reducing the local tumour burden via T-cell dependent killing of the cancer cells; however abscopal effects are not consistently observed. Currently little is known or understood about how ECT affects the immune cell population within the treated tumour and how these changes could impact the immune response. In this manuscript, we will review the current knowledge on ECT in the context of its interactions with the immune system and discuss how the gained knowledge could be harnessed to develop a potent ECT-immune co-treatment combination (Electroimmunotherapy).

Electrochemotherapy (ECT) is becoming an established therapy for melanoma and is under investigation for application in additional cancer types. One potential cancer type that may benefit from ECT is lung cancer as lung cancer treatments remain unable to deliver long-lasting treatment responses. Given the importance of the immune system in lung cancer, here we have also examined the impact of ECT on immune populations. The impact of electroporation and ECT on three human lung cancer cell lines (A549, H460, SK-MES 1), one murine cell line (LLC) and murine T cells, dendritic cells and macrophages was examined. The viability, metabolic activity and recovery potential post-treatment of all cell types was determined to evaluate the potential utility of ECT as a lung cancer treatment. Our findings demonstrate that cisplatin at 11 µM would be the suggested drug of choice when using ECT for lung cancer treatment. Our study also shows that T cells are not impacted by any tested condition, whilst dendritic cells and macrophages are significantly negatively impacted by electric field strengths surpassing 800 V/cm in vitro. Therefore, current ECT protocols (using 1000 V/cm in vivo) might need to adapted to improve viability of the immune population, thus improving therapy outcomes.

The IL-36 cytokines are a recently described subset of the IL-1 family of cytokines, shown to play a role in the pathogenesis of intestinal diseases such as Inflammatory Bowel Disease (IBD). Given the link between IBD and colitis –associated cancer, as well as the involvement of other IL-1 family members in intestinal tumorigenesis, the aim of this work was to investigate whether IL-36 cytokines play a role in the pathogenesis of colon cancer. Whilst research to date has focused on the role of IL-36 family members in augmenting the immune response to induce tumour rejection, very little remains known about IL-36R signalling in tumour cells in this context. In this study we demonstrate that expression of IL-36 family member mRNA and protein are significantly increased in colorectal cancer tissue compared to adjacent non-tumour. In vitro assays showed stimulation of colon cancer cell lines with IL-36R agonists resulted in the activation of the pro-tumorigenic phenotypes of increased cellular migration, invasion and proliferation in both 2D and 3D models. In addition, the IL-36 cytokines induced strong expression of pro-inflammatory chemokines in both human and murine cell lines. Intraperitoneal injection of IL-36Ra significantly reduced tumour burden using the subcutaneous CT26 tumour model in syngeneic Balb/mice, and this was associated with a decrease in Ki-67 expression by tumour cells in the IL-36Ra- treated group relative to untreated, suggesting the inhibition of the pro-proliferative signalling of IL-36 agonists resulted in the decreased tumour size. Moreover, colon cancer cells lacking the IL-36R also showed reduced tumour growth and reduced Ki-67 expression in vivo. Taken together, this data suggests that targeting IL-36R signalling may be a useful targeted therapy for colorectal cancer patients with IL-36R+ tumour cells.

Background & Aims: The IL-1 subfamily, IL-36, is increasingly being implicated as a potent cytokine family involved in chronic inflammatory conditions such as psoriasis and IBD. Recent work has also suggested IL-36 cytokines may act as a potential adjuvant in immunotherapy regimens to augment the anti-cancer immune response. Given the pluripotent nature of IL-1 cytokines and the relationship between inflammation and tumorigenesis, we investigated the effects of IL-36 signalling in colorectal cancer (CRC). Methods: IL-36 family expression in CRC progression was investigated by the NCBI GEO transcriptomic database, and then by immunohistochemistry (IHC) and qRT-PCR. Pro-tumorigenic properties such as cancer cell migration, invasion and proliferation were investigated in 2D and 3D models. In vivo models were performed using Balb-c mice which were subcutaneously inoculated with CT26 cells and treated with IP injections of recombinant IL-36R agonists (IL-36α, β, γ) or antagonist (IL-36RN). Tumour tissues were excised and studied by flow cytometry and IHC analysis. Additionally, CRISPR-Cas9 mediated IL-36R KO CT26 cells were generated and subcutaneously injected into Balb-c mice, with changes in tumour growth and immune cell infiltrate investigated. Results: IL-36R expression was shown to significantly increase with stage of disease in the adenoma-carcinoma sequence, including distant metastases. Additionally, expression of all family members was increased in tumour tissue relative to adjacent normal tissue in a separate colon cancer cohort. In vitro IL-36R signalling on cancer cells augmented a pro-tumorigenic phenotype by induction of pro-tumorigenic cytokines/chemokine production, increased cellular migration, invasion and proliferation of tumour cells. In vivo inhibition of IL-36R signalling by both administration of recombinant IL-36RN and through deletion of the IL-36R gene in CT26 cells, resulted in a significant reduction in tumour growth. This reduction was associated with a significant decrease in tumour cell proliferation, as well as an increase in CD8+ T cell infiltrate in IL-36R KO groups. Of note, administration of IL-36R agonists also resulted in reduced tumour growth, albeit to a lesser extent, associated with an increase in CD4+ and CD8+ T cell infiltration. Conclusions: This data indicates that IL-36 family members, similar to other IL-1 family members, have dual functions in colon cancer. In addition, this data suggests that targeting IL-36R signalling may be a useful targeted therapy for CRC patients with IL-36R+ tumour cells. Citation Format: Kevin J. Baker, Charlotte O'Donnell, Micheal O'Riordain, Elizabeth Brint, Maura Bendix, Aileen Houston. IL-36 signalling enhances a pro-tumorigenic phenotype in colon cancer cells with cancer cell growth restricted by administration of the IL-36R antagonist [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1324.

To make in vitro single cell electroporation protocols more comparable between various cancer types and groups, we propose a set of assays to test a range of electric field strengths at the start of any new project to determine the optimal electric field strength for a given cell line. While testing a range of electric field strengths, we kept the other ESOPE parameters constant (8 pulses, 100 µs pulse duration, 1 Hz pulse frequency). Basic assays were employed to measure short-term viability, effectiveness of treatment, metabolic activity, and recovery potential post-treatment to determine the optimal field strength for a particular cell line. Six cancer cell lines were tested, three of human (A549, A375 and Pan02) and three murine (LLC, B16F10 and MIA-PACA2). Our findings demonstrate that the optimal electroporation setting while keeping with all other ESOPE parameters are 800 V/cm for A549 and Pan02, 700 V/cm for A375, Mia-PACA2, and B16F10, and 1300 V/cm for LLC. Having an agreed upon set of assays to determine each cell lines optimal electric field strength should allow an improve translation of findings between cell lines for in vitro work from various groups and potentially improve translation into the clinic.

The IL-1 family of cytokines currently comprises of seven ligands with pro-inflammatory activity (IL-1α and IL-1β, IL-18, IL-33, IL-36α, IL-36β, IL-36γ) as well as two ligands with anti-inflammatory activity (IL-37, IL-38). These cytokines are known to play a key role in modulating both the innate and adaptive immunes response, with dysregulation linked to a variety of autoimmune and inflammatory diseases. Given the increasing appreciation of the link between inflammation and cancer, the role of several members of this family in the pathogenesis of cancer has been extensively investigated. In this review, we highlight both the pro- and anti-tumorigenic effects identified for almost all members of this family, and explore potential underlying mechanisms accounting for these divergent effects. Such dual functions need to be carefully assessed when developing therapeutic intervention strategies targeting these cytokines in cancer.

A role for the IL-36 family of cytokines has been identified in the pathogenesis of psoriasis. Although significant mechanistic overlap can exist between psoriasis and inflammatory bowel disease (IBD), to date there have been no reports investigating the IL-36 family in gastrointestinal inflammation. Here we demonstrate that expression levels of IL-36α are specifically elevated in the colonic mucosa of ulcerative colitis patients. This elevated expression is mirrored in the inflamed colonic mucosa of mice, wherein IL-36 receptor deficiency confirmed this pathway as a mediator of mucosal inflammation. Il36r-/- mice exhibited reduced disease severity in an acute DSS-induced model of colitis in association with decreased innate inflammatory cell infiltration to the colon lamina propria. Consistent with these data, infection with the enteropathogenic bacteria Citrobacter rodentium, resulted in reduced innate inflammatory cell recruitment and increased bacterial colonization in the colons of il36r-/- mice. Il36r-/- mice also exhibited altered T helper cell responses in this model, with enhanced Th17 and reduced Th1 responses, demonstrating that IL-36R signaling also regulates intestinal mucosal T-cell responses. These data identify a novel role for IL-36 signaling in colonic inflammation and indicate that the IL-36R pathway may represent a novel target for therapeutic intervention in IBD.

Fas (CD95) signalling is best known for its role in apoptosis, however, recent reports have shown it to be involved in other cellular responses as well, including inflammation. Fas and its adaptor protein FADD are known to negatively regulate LPS-induced proinflammatory responses, but their role in LPS-induced type I interferon production is unknown. Here, we demonstrate that Fas engagement on macrophages, using an agonistic Fas antibody CH11, augments LPS-induced NF-κB responses, causing increased production of TNFα, IL-8, IL-6 and IL-12. Conversely, costimulation with both LPS and CH11 causes a significant reduction in the level of interferon-beta (IFNβ) production. This differential effect involves the Fas adaptor FADD because while LPS-induced IL-6 production increased in FADD-/- murine embryonic fibroblasts, LPS-induced IFNβ production was significantly reduced in these cells. Overexpression of a dominant negative form of FADD (FADD-DD) inhibits LPS-induced IFNβ luciferase but not LPS-induced NF-κB luciferase. In contrast, overexpression of full-length FADD inhibited LPS-induced NF-κB luciferase activation but was seen to augment LPS-induced IFNβ luciferase. Moreover, FADD-DD inhibits TRIF-, TRAM-, IKKe-, TBK-1- and TRAF3-induced IFNβ luciferase production, with coimmunoprecipitation experiments demonstrating an interaction between FADD and TRIF. These data identify FADD as a novel component of the noncanonical Toll-like receptor 4/IFNβ signalling pathway and demonstrate that both Fas and its adaptor FADD can differentially regulate the production of LPS-induced proinflammatory cytokines and type I interferons.

The pathogenic gram-positive bacteria, Listeria monocytogenes is a facultative infectious intracellular pathogen that causes listeriosis. Effective elimination of infection is dependent upon a functioning innate immune system and activation of inflammatory responses by pathogen recognition receptors (PRRs). In this review, we trace the route of L. monocytogenes invasion as it disseminates from the intestinal epithelium, through the bloodstream of the host, to the liver and spleen. Along this route, we highlight the diverse, region specific, innate defences in place throughout the course of infection. We provide an overview of recent advances in our knowledge of key innate immune defences against L. monocytogenes, focusing on the PRRs in various cell types known to be critical in the detection of this pathogen.

Listeria monocytogenes is a Gram-positive bacterium that can cause septicemia and meningitis. TLRs are central receptors of the innate immune system that drive inflammatory responses to invading microbes such as L. monocytogenes. Although intestinal epithelial cells (IECs) represent the initial point of entry used by L. monocytogenes for infection, the innate immune response to L. monocytogenes in these cells has been poorly characterized to date. The aim of this study was to determine which TLRs are involved in mediating the immune response to L. monocytogenes in IECs. We performed an RNA interference screen of TLRs 1–10 in the HT-29 IEC cell line and observed the most significant reduction in chemokine output following silencing of TLR10. This effect was also observed in the macrophage cell line THP-1. The chemokines CCL20, CCL1, and IL-8 were reduced following knockdown of TLR10. Silencing of TLR10 resulted in increased viability of L. monocytogenes in both HT-29 and THP-1 cells. TLR10 was found to be predominantly expressed intracellularly in epithelia, and activation required viable L. monocytogenes. NF-κB activation was seen to require TLR2 in addition to TLR10. Taken together, these data indicate novel roles for TLR10 in sensing pathogenic infection in both the epithelium and macrophages and have identified L. monocytogenes as a source of ligand for the orphan receptor TLR10.

IL-33 is a member of the IL-1 family of cytokines. IL-33 is predominantly located within the nucleus of cells where it plays a role in gene regulation. Given the right combination of signals and cellular damage, stored IL-33 is released from the cell where it can interact with its receptor ST2, triggering danger-associated responses and act as a cellular “alarmin”. Whilst IL-33/ST2 signalling has been shown to induce potent pro-inflammatory responses that can be detrimental in certain disease states, a dichotomous, protective role of IL-33 in promoting wound healing has also emerged in multiple tissues types. This review will explore the current literature concerning this homeostatic role of IL-33/ST2 in tissue repair and also review its role in uncontrolled wound responses as seen in both fibrosis and tumorigenesis.

The pathogenesis of irritable bowel syndrome (IBS) is poorly understood. One contributory factor may be low-grade mucosal inflammation, perhaps initiated by the microbiota. Toll-like receptors (TLRs) are a family of pathogen-recognition receptors of the innate immune system. The aim of this study was to evaluate the potential involvement of TLRs in IBS to further understand the involvement of the innate immune system in this complex disorder.The expression of TLRs was investigated in colonic biopsy samples obtained from 26 IBS patients and compared with 19 healthy controls. Protein expression of TLR4, TLR7, and TLR8 was confirmed by immunofluorescence and alterations in the TLR4 protein were confirmed by western blot.Quantitative reverse transcriptase-PCR showed increased levels of TLR4 (P≤0.001) and TLR5 (P=0.0013) and decreased levels of TLR7 (P≤0.001) and TLR8 (P=0.0019) in IBS patients.Our results support the presence of an immune engagement between the microbiota and the host in IBS; an interaction that involves innate immunity and could generate a low-grade inflammatory response. These findings could also offer an additional biomarker of the disease or a disease subset.

Summary The innate immune system is currently seen as the probable initiator of events which culminate in the development of inflammatory bowel disease (IBD) with Toll-like receptors (TLRs) known to be involved in this disease process. Many regulators of TLRs have been described, and dysregulation of these may also be important in the pathogenesis of IBD. The aim of this study was to perform a co-ordinated analysis of the expression levels of both key intestinal TLRs and their inhibitory proteins in the same IBD cohorts, both ulcerative colitis (UC) and Crohn's disease (CD), in order to evaluate the potential roles of these proteins in the pathogenesis of IBD. Of the six TLRs (TLRs 1, 2, 4, 5, 6 and 9) examined, only TLR-4 was increased significantly in IBD, specifically in active UC. In contrast, differential alterations in expression of TLR inhibitory proteins were observed. A20 and suppressor of cytokine signalling 1 (SOCS1) were increased only in active UC while interleukin-1 receptor-associated kinase 1 (IRAK-m) and B cell lymphoma 3 protein (Bcl-3) were increased in both active UC and CD. In contrast, expression of both peroxisome proliferator-activated receptor gamma (PPARγ) and Toll interacting protein (Tollip) was decreased in both active and inactive UC and CD and at both mRNA and protein levels. In addition, expression of both PPARγ and A20 expression was increased by stimulation of a colonic epithelial cell line Caco-2 with both TLR ligands and commensal bacterial strains. These data suggest that IBD may be associated with distinctive changes in TLR-4 and TLR inhibitory proteins, implying that alterations in these may contribute to the pathogenesis of IBD.

T1/ST2 is a member of the interleukin (IL)-1 receptor superfamily, possessing three immunoglobulin domains extracellularly and a Toll/IL1R (TIR) domain intracellularly. The ligand for T1/ST2 is not known. T1/ST2 is expressed on Type 2 T helper (Th2) cells, and its role appears to be in the regulation of Th2 cell function. Here, we have investigated T1/ST2 signal transduction, using either transient overexpression of T1/ST2 or a cross-linking monoclonal antibody to activate cells. We demonstrate that T1/ST2 does not activate the transcription factor NF-κB when overexpressed in murine thymoma EL4 cells, or in the mast cell line P815 treated with the anti-T1/ST2 antibody. However, a chimera comprising the extracellular domain of the type 1 IL-1 receptor and the intracellular domain of T1/ST2 activates NF-κB both by overexpression and in response to IL-1. This artificial activation requires the IL1RAcP recruited via the extracellular portion (IL1R1) of the chimera. T1/ST2 is, however, able to activate the transcription factor activator protein-1 (AP-1), increase phosphorylation of c-Jun, and activate the MAP kinases c-Jun N-terminal kinase (JNK), p42/p44 and p38. Anti-T1/ST2 also induces the selective expression of IL-4 but not IFN-γ in naive T cells. Importantly, this effect is blocked by prior treatment with the JNK inhibitor SP600125 confirming that JNK as a key effector in T1/ST2 signaling. The lack of effect on NF-κB when T1/ST2 is homodimerized identifies T1/ST2 as the first member of the IL-1 receptor superfamily so far studied that is apparently unable to activate NF-κB, consistent with evidence indicating the lack of a role for NF-κB in Th2 cell function.

TLRs play an important role in mediating intestinal inflammation and homeostasis. Fas is best studied in terms of its function in apoptosis, but recent studies demonstrate that Fas signaling may mediate additional functions such as inflammation. The role of Fas, and the Fas ligand (FasL), in the intestine is poorly understood. The aim of this study was to evaluate potential cross-talk between TLRs and Fas/FasL system in intestinal epithelial cells (IECs). IECs were stimulated with TLR ligands, and expression of Fas and FasL was investigated. Treatment with TLR4 and TLR5 ligands, but not TLR2 and 9 ligands, increased expression of Fas and FasL in IECs in vitro. Consistent with this finding, expression of intestinal Fas and FasL was reduced in vivo in the epithelium of TLR4 knockout (KO), 5KO, and germ-free mice, but not in TLR2KO mice. Modulating Fas signaling using agonistic anti-Fas augmented TLR4- and TLR5-mediated TNF-α and IL-8 production by IECs. In addition, suppression of Fas in IECs reduced the ability of TLR4 and TLR5 ligands and the intestinal pathogens Salmonella typhimurium and Listeria monocytogenes to induce the expression of IL-8. In conclusion, this study demonstrates that extensive cross-talk in IECs occurs between the Fas and TLR signaling pathways, with the FasL/Fas system playing a role in TLR-mediated inflammatory responses in the intestine.

The recognition of microbial pathogens by the innate immune system involves Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns. Different TLRs recognize different pathogen-associated molecular patterns, with TLR-4 mediating the response to lipopolysaccharide from Gram-negative bacteria. All TLRs have a Toll/IL-1 receptor (TIR) domain, which is responsible for signal transduction. MyD88 is one such protein that contains a TIR domain. It acts as an adapter, being involved in TLR-2, TLR-4 and TLR-9 signalling; however, our understanding of how TLR-4 signals is incomplete. Here we describe a protein, Mal (MyD88-adapter-like), which joins MyD88 as a cytoplasmic TIR-domain-containing protein in the human genome. Mal activates NF-kappaB, Jun amino-terminal kinase and extracellular signal-regulated kinase-1 and -2. Mal can form homodimers and can also form heterodimers with MyD88. Activation of NF-kappaB by Mal requires IRAK-2, but not IRAK, whereas MyD88 requires both IRAKs. Mal associates with IRAK-2 by means of its TIR domain. A dominant negative form of Mal inhibits NF-kappaB, which is activated by TLR-4 or lipopolysaccharide, but it does not inhibit NF-kappaB activation by IL-1RI or IL-18R. Mal associates with TLR-4. Mal is therefore an adapter in TLR-4 signal transduction.

Fas, also known as CD95 or APO-1, is a member of the tumor necrosis factor/nerve growth factor superfamily. Although best characterized in terms of its apoptotic function, recent studies have identified several other cellular responses emanating from Fas. These responses include migration, invasion, inflammation, and proliferation. In this review, we focus on the diverse cellular outcomes of Fas signaling and the molecular switches identified to date that regulate its pro- and anti-apoptotic functions. Such switches occur at different levels of signal transduction, ranging from the receptor through to cross-talk with other signaling pathways. Factors identified to date including other extracellular signals, proteins recruited to the death-inducing signaling complex, and the availability of different intracellular components of signal transduction pathways. The success of therapeutically targeting Fas will require a better understanding of these pathways, as well as the regulatory mechanisms that determine cellular outcome following receptor activation.

As discussed in all chapters in this book, individual Toll-like receptors (TLRs) recognise distinct pathogen associated molecular patterns (PAMPs) that have been evolutionarily conserved in certain classes of microbes including bacteria [1–3], viruses [4–7], fungi [8] and parasites [9]. Since the importance of TLRs in the innate immune response was first realised much work has been carried out to elucidate the series of events triggered by ligand binding to TLRs which eventually result in proinflammatory cytokine production and also the importance of TLRs in the pathogenesis of various disease states. It has become clear that TLRs are, on one hand, essential regulators of both innate and adaptive immune responses and, on the other, have been implicated in the development of autoimmune, chronic inflammatory and infectious diseases. One of the best characterised diseases to be implicated with TLR signalling is sepsis and its most severe form, septic shock, a syndrome associated with bacterial infection. More than 50% of the infections are associated with Gramnegative bacteria [10] and therefore with lipopolysaccharide (LPS) a ligand for TLR4 [1]. Signalling through TLR9 has been implicated in the production of autoantibodies recognising self-DNA, a detrimental state seen in patients with systemic lupus erythematosus (SLE) [11, 12]. People carrying the well studied D299G polymorphism of TLR4 show a reduced risk of carotid artery atherosclerosis [13] and acute coronary events [14]. Patients with mutations in downstream components of the signalling pathway such as NEMO and IRAK4 showed increased risk of bacterial and viral infections [15]. The importance of the link between TLRs and various disease states is evident and most of the aforementioned diseases and others are discussed elsewhere in this book. It now seems possible that most immune and inflammatory diseases will have a TLR component at some level. The immune response needs to constantly strike a balance between activation and inhibition to avoid detrimental inflammatory responses. TLR signalling and functions are therefore, of necessity, under tight regulation. Many of the aforementioned disease states may arise through a lack of regulation of TLR signalling, either

Toll-like receptors (TLRs) are involved in host defence against invading pathogens, functioning as primary sensors of microbial products and activating signalling pathways that induce the expression of immune and pro-inflammatory genes. However, TLRs have also been implicated in several immune-mediated and inflammatory diseases. As the immune system needs to constantly strike a balance between activation and inhibition to avoid detrimental and inappropriate inflammatory responses, TLR signalling must be tightly regulated. Here, we discuss the various negative regulatory mechanisms that have evolved to attenuate TLR signalling to maintain this immunological balance.

Contents: TLRs as bacterial sensors.- Toll-like receptors and rheumatoid arthritis: is there a connection?- Toll-like receptor 9 and systemic autoimmune diseases.- Toll-like receptors and airway disease.- Toll-like receptors and vascular disease.- Toll-like receptors and inflammatory bowel disease.- Toll-like receptor signaling in the liver.- Toll-like receptors as key sensors of viral infection.- IRAK-4: a key kinase involved in toll-like receptor signaling and resistance to bacterial infection.- Endogenous regulation of toll-like receptor signalling.- Tuning of inflammatory cytokines and toll-like receptors by TIR8/SIGIRR, a member of the IL-1 receptor family with unique structure and regulation.- Toll-like receptors as pharmacological targets.

PUBLISHED, The Toll-interleukin-1 receptor domain-containing adapter Mal (MyD88 adapter-like protein) is involved in Toll-like receptor (TLR)-2 and TLR4 signal transduction. However, no studies have yet identified a function for Mal distinct from the related adapter MyD88. In this study, we have identified a putative TRAF6 interaction site in Mal but not in MyD88 and we demonstrate that Mal can be co-immunoprecipitated with TRAF6. Overexpression of MalE190A, which contains a mutation within the TRAF6-binding motif, failed to induce the expression of an NF-?B-dependent reporter gene, p65-mediated transactivation of gene expression, or activation of Jun N-terminal kinase or p42/p44 MAP kinase, which are induced with wild type Mal. MalE190A inhibited TLR2- and TLR4-mediated activation of NF-?B. These results identify a specific role for Mal in TLR-mediated signaling in regulating NF-?B-dependent gene transcription via its interaction with TRAF6.

PUBLISHED, The Toll/interleukin-1 receptor (TIR) domain is conserved in the intracellular regions of Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs) as well as in several cytoplasmic adapter molecules. This domain has crucial roles in signal transduction by these receptors for host immune response. Here we report the crystal structure at 2.3-? resolution of the TIR domain of human IL-1RAPL, the first structure of a TIR domain of the IL-1R superfamily. There are large structural differences between this TIR domain and that of TLR1 and TLR2. Helix ?D in IL-1RAPL is almost perpendicular to its equivalent in TLR1 or TLR2. The BB loop contains a hydrogen bond unique to IL-1RAPL between Thr residues at the 8th and 10th positions. The structural and sequence diversity among these domains may be important for specificity in the signal transduction by these receptors. A dimer of the TIR domain of IL-1RAPL is observed in the crystal, although this domain is monomeric in solution. Residues in the dimer interface are mostly unique to IL-1RAPL, which is consistent with the distinct functional roles of this receptor. Our functional studies show IL-1RAPL can activate JNK but not the ERK or the p38 MAP kinases, whereas its close homolog, TIGIRR, cannot activate JNK. Deletion mutagenesis studies show that the activation of JNK by IL-1RAPL does not depend on the integrity of its TIR domain, suggesting a distinct mechanism of signaling through this receptor., This work was supported by National Institutes of Health (NIH) Grant AI49475 (to L. T.).

The Toll-interleukin 1 receptor (TIR) superfamily, defined by the presence of an intracellular TIR domain, initiates innate immunity through activation of the transcription factor NF-kappa B, leading to the production of proinflammatory cytokines. ST2 is a member of the TIR family that does not activate NF-kappa B and has been suggested as an important effector molecule of T helper type 2 (T(H)2) responses. We show here that the membrane-bound form of ST2 negatively regulated type I interleukin 1 receptor (IL-1RI) and Toll-like receptor 4 (TLR4) but not TLR3 signaling by sequestrating the adaptors MyD88 and Mal. In contrast to wild-type mice, ST2-deficient mice failed to develop endotoxin tolerance. Thus, these results provide a molecular explanation for the function of ST2 in T(H)2 responses, as inhibition of TLRs promotes a T(H)2 response, and also identify ST2 as a key regulator of endotoxin tolerance.

Intestinal epithelial cells (IECs) represent the initial point of invasion for the pathogen Listeria monocytogenes however; the role of epithelial pattern recognition receptors (PRRs) engagement and subsequent inflammatory cascades remains poorly characterised. The aim of our study was to determine which PRRs are involved in initiating the immune response to L. monocytogenes in IECs. We performed a PRR siRNA library screen of 50 PRRs in the HT-29 IEC prior to infection with L. monocytogenes and then measured immune gene expression by qRT-PCR. Silencing of TLR1, TLR2, TLR10 and NLRP2 resulted in reductions in CCL20 and IL8 induction following infection, while silencing of NLRP7 and NLRP10 increased expression levels. Silencing of TLR10 gave the most significant effect and subsequent stable lentivirus knockdown of TLR10 in HT-29s yielded similar results upon infection. Similarly, silencing of TLR10 in macrophage-like THP-1 cells resulted in markedly reduced cytokine production in response to L. monocytogenes infection. Moreover, TLR10 silencing resulted in greater viability of L. monocytogenes in both THP-1 macrophages and HT-29 epithelia. Using an NF-κB reporter HEK cell line we found that TLR10 requires TLR2 for efficient NF-κB activation following L. monocytogenes infection. These data indicate a previously unknown role for TLR10 in sensing of pathogenic infection and moreover have potentially identified L. monocytogenes as a specific ligand for the orphan receptor TLR10.

G A A b st ra ct s reported to be reduced in UC. SERT knockout (KO), therefore, provides a gain-of-function model to test the role of 5-HT in the pathophysiology of intestinal inflammation. Dextran sodium sulfate (DSS; 3%) was included in the drinking water of wild-type C57/Bl6 mice and their transgenic SERT KO littermates for 7 days to induce colitis. The severity of colitis in SERT KO mice was significantly greater than that in their WT littermates. Body weight reduction, clinical scores, neutrophil infiltration, and mucosal ulcerations were all significantly greater in SERT KO animals; moreover, 50 % of transcripts encoding 70 prominent proinflammatory pathway-related genes (cytokines, interleukins, chemokines and their receptors), assayed by RT-PCR microarray, were also significantly more abundant in the SERT KO mice. Although these observations must still be confirmed in other models of inflammation and in a loss-of-function paradigm, these data are consistent with the hypothesis that 5-HT contributes to the severity of inflammation of the colon. The downregulation of SERT in UC may thus increase the severity of the condition as well as the abnormal intestinal motility and visceral hypersensitivity that accompany it.