IL-36 expression is increased in NSCLC with IL-36 stimulation of lung cancer cells promoting a pro-tumorigenic phenotype.

The IL-36 cytokines are a recently described subset of the IL-1 family of cytokines, and have been shown to play a role in the pathogenesis of respiratory diseases such as asthma and COPD. Given the common aetiological links between COPD and lung cancer development, as well as the involvement of other IL-1 family members in lung tumorigenesis, the aim of this work was to investigate the role of IL-36 cytokines in the pathogenesis of lung cancer. In this study we demonstrate that expression of IL-36 cytokines and receptor mRNA and protein are significantly increased in lung cancer tissue compared to adjacent non-tumour tissue. In vitro assays showed that stimulation of two lung cancer cell lines, SKMES-1 human squamous cell and LLC murine lung cancer, with IL-36R agonists resulted in increased cellular migration and proliferation. All IL-36 cytokines induced the expression of pro-inflammatory chemokines in both lung cancer cell lines with synergistic effects identified upon co-stimulation of cells with IL-17, IL-22 and TNFα. Furthermore, we report that IL-36 cytokines induce protein expression of the immune checkpoint inhibitor protein PD-L1 on lung cancer cells. Taken together, this data indicates that targeting IL-36R signalling may be a useful targeted therapy for lung cancer patients with IL-36R+ cancer cells.

Transcriptomic and functional analyses reveal a tumour-promoting role for the IL-36 receptor in colon cancer and crosstalk between IL-36 signalling and the IL-17/ IL-23 axis.

BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Defining optimal parameters to maximize the effect of electrochemotherapy on lung cancer cells whilst preserving the integrity of immune cells.

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.

DNA sensor-associated type I interferon signaling is increased in ulcerative colitis and induces JAK-dependent inflammatory cell death in colonic organoids.

DNA sensor pathways can initiate inflammasome, cell death, and type I interferon (IFN) signaling 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 analyzing the 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-ß), 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 NLR family pyrin domain-containing 3 (NLRP3), caspase, or JAK inhibitors, cell death was measured, and supernatants were assayed for IL-1ß/IL-18/CXCL10. The expression of DNA sensor pathway genes-PYHIN family members [absent in melanoma 2 (AIM2), IFI16, myeloid cell nuclear differentiation antigen (MNDA), and pyrin and HIN domain family member 1 (PYHIN1)- as well as Z-DNA-binding protein 1 (ZBP1), cyclic GMP-AMP synthase (cGAS), and DDX41 was increased in active UC and expressed in a cell type-restricted pattern. Inflammasome genes (CASP1, IL1B, and IL18), type I IFN inducers [stimulator of interferon response cGAMP interactor 1 (STING), TBK1, and IRF3), IFNB1, and type I IFN biomarker genes (OAS2, IFIT2, and MX2) were also increased in active UC. Cotreatment of organoids with IFN-ß or IFN-γ in combination with TNFα increased expression of IFI16, ZBP1, CASP1, cGAS, and STING induced cell death and IL-1ß/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.NEW & NOTEWORTHY This study found that patients with active UC have significantly increased colonic gene expression of cytosolic DNA sensor, inflammasome, STING, and type I IFN signaling pathways. The type I IFN, IFN-ß, in combination with TNF-α induced JAK-dependent but NLRP3 and inflammasome-independent inflammatory cell death of colonic organoids. This novel inflammatory cell death phenotype is relevant to UC immunopathology and may partially explain the efficacy of the JAKinibs tofacitinib and upadacitinib in patients with UC.

Electrochemotherapy and immune interactions; A boost to the system?

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).

IL-36 signalling enhances a pro-tumorigenic phenotype in colon cancer cells with cancer cell growth restricted by administration of the IL-36R antagonist.

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.

Electroporation of suspension cell lines - A proposed assay set for optimizations.

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.

IL-36 cytokines in inflammatory and malignant diseases: not the new kid on the block anymore.

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.

IL-1 Family Members in Cancer; Two Sides to Every Story.

The IL-1 family of cytokines currently comprises of seven ligands with pro-inflammatory activity (IL-1α and IL-1ß, IL-18, IL-33, IL-36α, IL-36ß, 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.

Engagement of Fas differentially regulates the production of LPS-induced proinflammatory cytokines and type I interferons.

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 (IFNß) production. This differential effect involves the Fas adaptor FADD because while LPS-induced IL-6 production increased in FADD-/- murine embryonic fibroblasts, LPS-induced IFNß production was significantly reduced in these cells. Overexpression of a dominant negative form of FADD (FADD-DD) inhibits LPS-induced IFNß 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 IFNß luciferase. Moreover, FADD-DD inhibits TRIF-, TRAM-, IKKε-, TBK-1- and TRAF3-induced IFNß 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/IFNß 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.

Wounds that heal and wounds that don't - The role of the IL-33/ST2 pathway in tissue repair and tumorigenesis.

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.

An antitumorigenic role for the IL-33 receptor, ST2L, in colon cancer.

BACKGROUND: Despite the importance of inflammation in cancer, the role of the cytokine IL-33, and its receptor ST2, in colon cancer is unclear. The aim of this study was to investigate the role of IL-33, and its receptor isoforms (ST2 and ST2L), in colon cancer. METHODS: Serum levels of IL-33 and sST2 were determined with ELISA. ST2 and IL-33 expression was detected with quantitative real-time PCR (qRT-PCR), western blotting and immunohistochemistry. ST2 expression in CT26 cells was stably suppressed using ST2-specific shRNA. Cytokine and chemokine gene expression was detected with qRT-PCR. RESULTS: Human colon tumours showed lower expression of ST2L as compared with adjacent non-tumour tissue (P<0.01). Moreover, the higher the tumour grade, the lower the expression of ST2L (P=0.026). Colon cancer cells expressed ST2 and IL-33 in vitro. Functional analyses showed that stimulation of tumour cells with IL-33 induced the expression of chemokine (C-C motif) ligand 2 (CCL2). Knockdown of ST2 in murine colon cancer cells resulted in enhanced tumour growth (P<0.05) in BALB/c mice in vivo. This was associated with a decrease in macrophage infiltration, with IL-33-induced macrophage recruitment reduced by antagonising CCL2 in vitro. CONCLUSION: The IL-33/ST2 signalling axis may have a protective role in colon carcinogenesis.

Engagement of Fas on Macrophages Modulates Poly I:C induced cytokine production with specific enhancement of IP-10.

Viral double-stranded RNA (dsRNA) is recognised by pathogen recognition receptors such as Toll-Like Receptor 3 (TLR3) and retinoic acid inducible gene-I (RIG-I), and results in cytokine and interferon production. Fas, a well characterised death receptor, has recently been shown to play a role in the inflammatory response. In this study we investigated the role of Fas in the anti-viral immune response. Stimulation of Fas on macrophages did not induce significant cytokine production. However, activation of Fas modified the response of macrophages to the viral dsRNA analogue poly I:C. In particular, poly I:C-induced IP-10 production was significantly enhanced. A similar augmentation of IP-10 by Fas was observed following stimulation with both poly A:U and Sendai virus. Fas activation suppressed poly I:C-induced phosphorylation of the MAP kinases p38 and JNK, while overexpression of the Fas adaptor protein, Fas-associated protein with death domain (FADD), activated AP-1 and inhibited poly I:C-induced IP-10 production. Consistent with an inhibitory role for AP-1 in IP-10 production, mutation of the AP-1 binding site on the IP-10 promoter resulted in augmented poly I:C-induced IP-10. These results demonstrate that engagement of the Fas receptor plays a role in modifying the innate immune response to viral RNA.

Intestinal expression of Fas and Fas ligand is upregulated by bacterial signaling through TLR4 and TLR5, with activation of Fas modulating intestinal TLR-mediated inflammation.

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.

Identification of TLR10 as a key mediator of the inflammatory response to Listeria monocytogenes in intestinal epithelial cells and macrophages.

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.

Life in the Fas lane: differential outcomes of Fas signaling.

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.

Differential expression of toll-like receptors in patients with irritable bowel syndrome.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Mal interacts with tumor necrosis factor receptor-associated factor (TRAF)-6 to mediate NF-kappaB activation by toll-like receptor (TLR)-2 and TLR4.

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-kappaB-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-kappaB. These results identify a specific role for Mal in TLR-mediated signaling in regulating NF-kappaB-dependent gene transcription via its interaction with TRAF6.

ST2 is an inhibitor of interleukin 1 receptor and Toll-like receptor 4 signaling and maintains endotoxin tolerance.

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.

Bruton's tyrosine kinase is a Toll/interleukin-1 receptor domain-binding protein that participates in nuclear factor kappaB activation by Toll-like receptor 4.

In this study we have identified members of the Toll-like receptor (TLR) family (namely, TLRs 4, 6, 8, and 9) as proteins to which the intracellular protein tyrosine kinase, Bruton's tyrosine kinase (Btk), binds. Detailed analysis of the interaction between Btk and TLR8 demonstrates that the presence of both Box 2 and 3 motifs in the Toll/interleukin-1 receptor domain was required for the interaction. Furthermore, co-immunoprecipitation experiments revealed that Btk can also interact with key proteins involved in TLR4 signal transduction, namely, MyD88, Mal (MyD88 adapter-like protein), and interleukin-1 receptor-associated kinase-1, but not TRAF-6. The ability of Btk to interact with TLR4 and Mal suggests a role for Btk in lipopolysaccharide (LPS) signal transduction. Stimulation of the human monocytic cell line THP-1 with LPS resulted in an increase in the level of tyrosine phosphorylation of Btk (indicative of activation). The autokinase activity of Btk was also stimulated after LPS stimulation. In addition, a dominant negative form of Btk inhibited TLR4-mediated activation of a nuclear factor kappaB (NFkappaB)-dependent reporter gene in HEK293 cells as well as LPS-induced activation of NFkappaB in the astrocytoma cell line U373 and the monocytic cell line RAW264.7. Further investigation revealed that the Btk-specific inhibitor, LFM-A13, inhibited the activation of NFkappaB by LPS in THP-1 cells. Our findings implicate Btk as a Toll/interleukin-1 receptor domain-binding protein that is important for NFkappaB activation by TLR4.