IRAK1 Signaling Research Articles

Acanthoic acid protectsagainst ethanol-induced liver injury: Possible role of AMPK activation and IRAK4 inhibition

The aim of this study was to investigate the effects of acanthoic acid (AA) on the regulation of inflammatory response, lipid accumulation, and fibrosis via AMPK- IRAK4 signaling against chronic alcohol consumption in mice. Ethanol-induced liver injury was induced in male mice by Lieber-DeCarli diet for 28d. And mice in AA groups were gavaged with AA (20 or 40mg/kg) for 28d. AA treatment significantly decreased serum AST and TG, hepatic TG levels, serum ethanol and LPS levels compared with chronic ethanol administration. AA ameliorated histological changes, lipid droplets, hepatic fibrosis, and inflammation induced by ethanol. AA significantly increased the expressions of p-LKB1, p-AMPK, and SIRT1 caused by chronic ethanol administration, and attenuated the increasing protein expressions of IRAK1 and IRAK4.siRNA against AMPKα1 blocked AMPKα1 and increased IRAK4 protein expressions, compared with control-siRNA-transfected group, while AA treatment significantly decreased IRAK4 expressions compared with AMPKα1-siRNA-transfected group. AMPK-siRNA also blocked the decreased effect of AA on inflammatory factors. AA decreased over-expression of IRAK4 and inflammation under ethanol plus LPS challenge. AA recruited LKB1-AMPK phosphorylation and activated SIRT1 to regulate alcoholic liver injury, especially, inhibited IRAK1/4 signaling pathway to regulate lipid metabolism, hepatic fibrosis and inflammation caused by alcohol consumption.

Pacritinib Targets IRAK1 and Shows Synergy with HDAC and BET Inhibitors in Acute Myeloid Leukemia

Background: The bone marrow microenvironment modulates response of acute myeloid leukemia (AML) to therapy. Thus necessitating the identification of microenvironment-driven signaling mechanisms that contribute to AML pathogenesis. We recently showed that AML growth depends on IL-1 signaling in a majority of cases that include a spectrum of genetic aberrations. IL-1 signals through the serine/threonine kinase IL-1 receptor-associated kinase (IRAK1), a central mediator of immune and inflammatory responses that was recently suggested as a potential therapeutic target in leukemia. Pacritinib is currently under development as a small-molecule JAK2 inhibitor for myelofibrosis; recently a kinome screen of 429 kinases showed that pacritinib also inhibits IRAK1 activity (IC50 = 13.6 nM). Here we provide further evidence to support targeting of IRAK1 by pacritinib as a therapeutic strategy in AML, and show synergistic activity with histone deacetylase (HDAC) or bromodomain (BET) inhibitors to overcome drug resistance across genetic subtypes.Methods: We used inducible shRNA to reduce IRAK1 levels in primary AML cells. To validate the selectivity of pacritinib for IRAK1, we performed targeted mutagenesis on key interacting residues. We compared the efficacy of pacritinib, an established dual FLT3/JAK2 inhibitor, to that of the FLT3 inhibitor quizartinib and the JAK2 inhibitor ruxolitinib in 12 cell lines and 46 primary AML samples with various genetic lesions. We tested the synergy of pacritinib in combination with various drugs, including HDAC, BET, and BTK inhibitors, and BH3 mimetic. Equimolar concentrations of both agents were tested at a dose gradient of 1.0-5000 nM. Synergy and combination index were calculated using Calcusyn software. The effects of drug treatments were evaluated on cell viability, survival, differentiation, and downstream signaling.Results: Genetic knockdown of IRAK1 reduced the viability of AML cell lines and primary AML cells by 70% and reduced p38 and ERK1/2 kinase activity. Molecular docking simulation showed that pacritinib forms favorable interactions with D298 and S295 residues in the IRAK1 kinase domain. IRAK1D298K and IRAK1S295D conferred significant pacritinib resistance compared to native IRAK1, confirming the selective binding and on-target inhibitory effect of pacritinib. Primary AML samples and cell lines showed sensitivity to pacritinib (median IC50 = 90 nm, range = 2.0-1000 nM, p<0.004), with a higher response rate (44/46 or 96%, or IC50 < 1000 nM) than that observed with ruxolitinib (15%) or quizartinib (80%). With regard to IC90 < 1000 nM, a more stringent measure of efficacy, a higher percentage of primary AML samples (33%) showed sensitivity to pacritinib than that for quizartinib (13%, p<0.0001). Mechanistic studies showed that pacritinib inhibits the activity of downstream effectors in the IL-1 pathway, including IRAK1 and p38, in addition to inhibiting the secretion of various inflammatory cytokines. Consistent with previous studies, pacritinib inhibited FLT3 and JAK2/JAK3 activity in FLT3- or JAK-dependent AML cells, respectively. Additionally, our data suggest that pacritinib reduced the viability of AML primary samples and cell lines harboring a variety of genetic abnormalities, including TET2, MLL, ASXL1, NPM1, CEBPA, and RAS mutations, by inhibiting IRAK1 activity. To identify potential combination therapies, we combined pacritinib with agents from several classes and found synergy with the HDAC inhibitor panobinostat and the BET inhibitor JQ1 with a combination index <1. Specifically, treatment with 50 nM pacritinib or 50 nM JQ1 alone reduced the viability of primary AML cells to 50%, while the combined treatment reduced viability to ~25%. Similarly, 50 nM pacritinib or 20 nM panobinostat alone reduced the viability of AML cells to 50%, while the combined treatment reduced viability to ~10%. Pacritinib blocked cell growth, while HDAC or BET inhibitors promoted differentiation of AML cells and reduced numbers of early progenitors. The combinations blocked not only IRAK1 and ERK activity, but also c-Myc.Conclusions: IRAK1 is a potential therapeutic target in AML across genetic subtypes. Pacritinib, in addition to suppressing JAK2 and FLT3 signaling, specifically blocks IRAK1 signaling in AML and synergizes with HDAC or BET inhibitors to block growth and promote differentiation, establishing its potential clinical utility. DisclosuresDruker:Agios: Honoraria; Ambit BioSciences: Consultancy; ARIAD: Patents & Royalties, Research Funding; Array: Patents & Royalties; AstraZeneca: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Other: travel, accommodations, expenses ; BMS: Research Funding; CTI: Equity Ownership; Curis: Patents & Royalties; Cylene: Consultancy, Equity Ownership; D3 Oncology Solutions: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses ; Lorus: Consultancy, Equity Ownership; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Novartis: Research Funding; Oncotide Pharmaceuticals: Research Funding; Pfizer: Patents & Royalties; Roche: Consultancy. Singer:CTI BioPharma Corp.: Employment, Equity Ownership, Other: Leadership . Agarwal:CTI BioPharma Corp: Research Funding.

Lack of IL-1 Receptor-Associated Kinase-4 Leads to Defective Th1 Cell Responses and Renders Mice Susceptible to Mycobacterial Infection.

The Toll-like and IL-1 family receptors play critical roles in innate and adaptive immunity against intracellular pathogens. Although previous data demonstrated the importance of TLRs and IL-1R signaling events for the establishment of an effective immune response to mycobacteria, the possible function of the adaptor molecule IL-1R-associated kinase (IRAK)-4 against this pathogen has not been addressed. In this study, we determined the role of IRAK-4 in signaling pathways responsible for controlling mycobacterial infections. This kinase is important for the production of IL-12 and TNF-α by macrophages and dendritic cells exposed to mycobacteria. Moreover, Mycobacterium bovis-infected IRAK-4-knockout macrophages displayed impaired MAPK and NF-κB activation. IL-1β secretion and caspase-1 activation were also dependent on IRAK-4 signaling. Mice lacking IRAK-4 showed increased M. bovis burden in spleen, liver, and lungs and smaller liver granulomas during 60 d of infection compared with wild-type mice. Furthermore, 80% of IRAK-4(-/-) mice succumbed to virulent M. tuberculosis within 100 d following low-dose infection. This increased susceptibility to mycobacteria correlated with reduced IFN-γ/TNF-α recall responses by splenocytes, as well as fewer IL-12p70-producing APCs. Additionally, we observed that IRAK-4 is also important for the production of IFN-γ by CD4(+) T cells from infected mice. Finally, THP-1 cells treated with an IRAK-4 inhibitor and exposed to M. bovis showed reduced TNF-α and IL-12, suggesting that the results found in mice can be extended to humans. In summary, these data demonstrate that IRAK-4 is essential for innate and adaptive immunity and necessary for efficient control of mycobacterial infections.

Abstract 4798: Efficacy and safety of highly selective novel IRAK4 inhibitors for treatment of ABC-DLBCL

Abstract Interleukin-1 receptor associated kinases (IRAKs) are serine/threonine protein kinases belonging to the tyrosine-like kinase (TLK) family. IRAKs function as mediators of Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling pathways and play an important role in innate immune signaling. TLR/IL-1R stimulation leads to recruitment of MYD88, an adaptor molecule, to the activated receptor complex, which then complexes with IRAK4 and activates IRAK1. TRAF6 is then activated by IRAK1 leading to NFkB activation. Recent studies have reported the occurrence of gain of function oncogenic mutation (L265P) in MYD88 in ∼30% of activated B cell diffuse large B-cell lymphoma(ABC DLBCL) and ∼90% of Waldenstrom's macroglobulinemia (WM) leading to constitutive activation of IRAK4 and NFkB pathway. Among the DLBCL subtypes (GCB, ABC DLBCL and PMBL), ABC DLBCL is the most refractory. Inhibition of constitutive IRAK4 signalling can be used as a therapeutic strategy to treat ABC DLBCL Small molecule inhibitors of IRAK4 were synthesized based on hits originating from Aurigene's compound library. Structure guided drug design approach was used to further improve the potency. Lead compounds demonstrated moderate to very high selectivity towardsIRAK4 (S35 score of 0.03) when screened against a large panel of 329 kinases. Aurigene's lead compounds have excellent PK profile and good oral bioavailability in mice, leading to good in-vivo activity in TLR4 induced cytokine release model. Selected lead compounds were tested in a OCI-Ly3 xenograft model, which has a MYD88(L265P) mutation leading to constitutive activation of IRAK4 signaling. An advanced lead compound has demonstrated excellent efficacy in OCI-Ly3 model, with tumor stasis at low doses and tumor regression at higher doses. The compound is well tolerated and has a good therapeutic window as determined in a 14 day rodent toxicity study. In summary, a selective IRAK4 inhibitor has been identified with excellent efficacy and good safety profile. Citation Format: Wesley Roy Balasubramanian, Venkateshwar Rao Gummadi, Kavitha Nellore, Subhendu Mukherjee, Sivapriya Marappan, Aravind Basavaraju, Bharathi Raja Ainan, Girish Daginakatte, Sreevalsam Gopinath, Sanjeev Giri, Thomas Antony, Shekar Chelur, Susanta Samajdar, Chetan Pandit, Murali Ramachandra. Efficacy and safety of highly selective novel IRAK4 inhibitors for treatment of ABC-DLBCL. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4798.

Abstract 3338: Pacritinib reduces human myeloid leukemia stem cell maintance in a defined niche

Abstract Introduction Pacritinib, a potent clinical small molecule inhibitor of JAK2, it also suppresses signaling through wild-type and mutant FLT3, IRAK1, and CSF1R. Pacritinib does not cause marrow suppression and has demonstrated single agent activity preclinically in myelofibrosis and other myeloid neoplasms including AML and CMML. Stromal protection was not observed. However, the capacity of pacritinib to eradicate therapy resistant leukemia stem cells (LSC), residing in the bone marrow niche, had not been examined. Thus, we investigated the impact of pacritinib alone or in combination with standard of care therapy on primary blast crisis chronic myeloid leukemia (BC CML), myelofibrosis (MF) and AML LSC survival and self-renewal in a stem cell supportive niche. Methods Genetically engineered mouse bone marrow fibroblasts producing human SCF, IL3 and G-CSF were used as stromal monolayers to support LSC survival and self-renewal. Human primary CD34+ cells were selected from BC CML (n = 5), MF (n = 5) and relapsed AML (n = 4) before and after clinical treatment with azacitidine. As a control, CD34+ cells from age matched normal bone marrow (a-NBM, n = 4) were used for the co-culture. Survival and self-renewal of the cells were investigated by colony forming and replating assays. Pacritinib was used at concentrations ranging from 10 to 50 nM alone and in combination with 1 nM dasatinib. Results Pacritinib alone induced dose-dependent inhibition of self-renewal in a-NBM, AML, MF and CML-BC, with the optimal concentration of 20nM leading to IC50 diversity in the response between normal and leukemia progenitors. AML and MF responded uniformly and inhibition reached 50% at 10nM concentration. BC CML cells were more divergent: 40% demonstrated &amp;gt;50% inhibition, in another 40% it was 20-50% and in 20% inhibition was &amp;lt;20%. Combined treatment with the low dose of dasatinib and pacritinib doses of 10 or 20 nM resulted in a significant (p&amp;lt;0.001, Anova) difference in self-renewal of all BC CML cells, raising a possibility of an additive/synergistic mechanisms. AML cells collected before and after clinical treatment with azacitidine uniformly showed a significant decrease in self-renewal starting with 10 nM pacritinib alone and combined treatment with dasatinib did not enhance the inhibition, suggesting a prospect of using pacritinib as a single agent in the treatment of relapsed AML. Conclusions Together these data indicate that possibly through inhibition of CSF1 and IRAK1 signaling in addition to suppression of JAK2, even in the presence of a LSC supportive niche, readily clinically achievable low nM concentrations of pacritinib alone are effective in reducing self-renewal of MF and relapsed AML. However, a combination of dasatinib and pacritinib is required to eliminate self-renewing LSC in BC CML with minimal toxicity toward normal progenitors. Targeting niche-dependent signaling could represent a robust avenue for treatment of refractory myeloid leukemia. Citation Format: Larisa Balaian, Anna Kulijian, Edward D. Ball, Catriona H.M Jamieson. Pacritinib reduces human myeloid leukemia stem cell maintance in a defined niche. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3338.

IRAK1 Drives Intestinal Inflammation by Promoting the Generation of Effector Th Cells with Optimal Gut-Homing Capacity.

IL-1R-associated kinase (IRAK) 1 is an important component of the IL-1R and TLR signaling pathways, which influence Th cell differentiation. In this study, we show that IRAK1 promotes Th17 development by mediating IL-1β-induced upregulation of IL-23R and subsequent STAT3 phosphorylation, thus enabling sustained IL-17 production. Moreover, we show that IRAK1 signaling fosters Th1 differentiation by mediating T-bet induction and counteracts regulatory T cell generation. Cotransfer experiments revealed that Irak1-deficient CD4(+) T cells have a cell-intrinsic defect in generating Th1 and Th17 cells under inflammatory conditions in spleen, mesenteric lymph nodes, and colon tissue. Furthermore, IRAK1 expression in T cells was shown to be essential for T cell accumulation in the inflamed intestine and mesenteric lymph nodes. Transcriptome analysis ex vivo revealed that IRAK1 promotes T cell activation and induction of gut-homing molecules in a cell-intrinsic manner. Accordingly, Irak1-deficient T cells failed to upregulate surface expression of α4β7 integrin after transfer into Rag1(-/-) mice, and their ability to induce colitis was greatly impaired. Lack of IRAK1 in recipient mice provided additional protection from colitis. Therefore, IRAK1 plays an important role in intestinal inflammation by mediating T cell activation, differentiation, and accumulation in the gut. Thus, IRAK1 is a promising novel target for therapy of inflammatory bowel diseases.

Synergistic Blockade Of Activated B Cell-Like DLBCL Proliferation With a Selective Inhibitor Of IRAK4 In Combination With Inhibition Of The B-Cell Receptor Signaling Network

Toll-Like Receptor (TLR) and IL-1 signaling is mediated by the adaptor protein MyD88 through IRAK4 activation. TLR and IL-1 family ligands activate NFkB through this pathway and stimulate proliferation and cell survival, as well as induce cytokine and chemokine production that can amplify tumor cell survival. The gain-of-function L265P mutation in MyD88 occurs in ∼30% of patients with activated B-cell like diffuse large B-cell lymphoma (ABC-DLBCL) and ∼90% of Waldenström's macroglobulinemia. Therefore, inhibition of IRAK4 may be therapeutically relevant in hematologic malignancies containing MyD88 mutations. Recent clinical results with kinase inhibitors strongly support a role for signaling through the B-cell receptor (BCR) pathway in the progression of hematological malignancies including ABC-DLBCL. We were interested to understand the potential utility of selective IRAK4 inhibitors in combination with inhibition of the BCR signaling networks. We have reported previously the identification and characterization of potent and selective IRAK4 inhibitors that are effective in blocking inflammatory signaling in immune cells and demonstrate efficacy in vivo in models of autoimmune disease. ND-2158, a potent (Ki of 1.2 nM) and highly selective IRAK4 inhibitor has been shown to be effective in reducing the proliferation of ABC-DLBCL cell lines. ND-2158 does not decrease cell viability for other cell lines that lack the MyD88 mutation including a germinal center-like DLBCL cell line, BJAB, suggesting that the anti-proliferative effects in ABC-DLBCL cells relate in part to the activating MyD88 mutation. Complete cross-over dose-response proliferation studies of the ABC-DLBCL cell line, OCI-LY10, were conducted using ND-2158 in combination with blockade of key BCR signaling network nodes, using inhibitors of either Btk (ibrutinib), PI3Kdelta (GS-1101), or Syk (P505-15). Isobologram analysis using the Chou-Talalay method revealed that ND-2158 was able to synergistically block cell proliferation in combination with ibrutinib, P505-15, or GS-1101. Interestingly, we find that blockade of SYK, PI3Kdelta, or BTK signaling enhances the potency of ND-2158 in ABC-DLBCL cells. The IC50 values observed in this context are comparable to the potency of ND-2158 when used as a single agent to inhibit inflammatory signaling in immune cells that are not dependent on BCR signaling. The cell proliferation blockade IC50for ND-2158 shifted from an average value of ∼7 μM to 0.19, 0.05, or 0.15 μM, when combined with the IC50 concentrations of the inhibitors of BTK, PI3Kdelta or SYK kinases, respectively. These results suggest that inhibition of both BCR signaling pathways that are amplified in ABC-DLBCL, and IRAK4 signaling activated through MyD88 mutations, are required for a more complete blockade of ABC-DLBCL proliferation. Moreover, we explored ND-2158 combination with lenalidomide, known to be synergistic with BCR and NFkB pathway inhibitors. In contrast to combinations with BCR signaling inhibition, studies with lenalidomide failed to demonstrate an additive or synergistic activity when combined with IRAK4 inhibition in ABC-DLBCL cell lines. Therefore, we conclude that IRAK4 activation, as well as aberrant BCR signaling, are likely to contribute to the proliferative capacity of ABC-DLBCL. We propose that combinatorial therapeutic approaches, including inhibition of IRAK4, may provide benefit for patients with ABC-DLBCL. Disclosures:Chaudhary:Nimbus Discovery Inc.: Employment. Off Label Use: Exploratory inhibitor of IRAK4 for research purposes. Wood:Nimbus Discovery Inc.: Employment. Romero:Nimbus Discovery Inc.: Consultancy, Equity Ownership. Robinson:Schrodinger Inc. Consultant to Nimbus Discovery Inc.: Consultancy. Greenwood:Schrodinger Inc. Consultant to Nimbus Discovery Inc.: Consultancy. Shelley:Schrodinger Inc. Consultant to Nimbus Discovery Inc.: Consultancy. Morin:Nimbus Discovery Inc.: Consultancy. Kapeller:Nimbus Discovery Inc.: Employment. Westlin:Nimbus Discovery Inc.: Employment, Equity Ownership.

MYD88 L265P Promotes Survival of Waldenstrom's Macroglobulinemia Cells by Activation of Bruton's Tyrosine Kinase

Abstract 897 Background:MYD88 is an adapter molecule which promotes Toll-Like Receptor and IL-1 Receptor signaling through the IRAK 1/4/TRAF6/NF-κB pathway. MYD88 L265P is a recently described somatic mutation that is widely expressed (>90%) in patients with Waldenstrom’s Macroglobulinemia (Treon et al, NEJM 2012). The signaling pathway by which MYD88 L265P promotes WM growth and survival remains to be fully clarified. We therefore sought to identify pathway(s) responsible for MYD88 L265P pro-survival signaling. Patients and Methods:Primary WM-LPCs were isolated from bone marrow biopsy specimens from WM patients. Normal CD19+ B-cells were isolated from the peripheral blood of healthy donors. Western blot analyses was performed using total and phospho-specific antibodies in the MYD88 L265P expressing WM cell lines BCWM.1 and MCWL-1 following MYD88 knockdown by lentiviral transduction, and/or use of MYD88 or IRAK signal inhibitors. WM cells were also treated with the BTK inhibitor PCI-32765, in the presence or absence of an IRAK1/4 inhibitor. Cell survival was evaluated by Annexin V/PI staining and/or AlamarBlue® or ATPLite® assays after treatments with inhibitors. Synergistic interactions were assessed with CalcuSyn software. Results:Lentiviral knockdown of MYD88 and/or use of an inhibitor of MYD88 homo-dimerization led to decreased survival of MYD88 L265P expressing BCWM.1 and MWCL-1 cells, as well as primary WM LPCs expressing MYD88 L265P. In contrast minimal killing was observed in MYD88 Wild Type (WT) expressing Ramos, MM.1S, or healthy donor CD19+ selected B-cells. Phospho-specific western blot analysis showed decreased IRAK1, NF-kB and STAT3 signaling following inhibition of MYD88 signaling in L265P expressing WM cells. Conversely, use of an IRAK1/4 kinase inhibitor showed reduced tumor cell killing, NF-kB and STAT3 signaling. We therefore performed co-immunopreciptation experiments to identify other MYD88 binding partners in L265P expressing WM cells. These studies identified Bruton's Tyrosine Kinase (BTK) in complex with MYD88, with MYD88 showing more robust binding to phospho-BTK in L265P expressing WM cells. Lentiviral knockdown of MYD88 and/or use of an inhibitor of MYD88 homodimerization led to decreased BTK phosphorylation. Moreover, overexpression by lentiviral transduction of MYD88 L265P showed more robust BTK phosphorylation versus WM cells transduced with MYD88 WT. Lastly, the use of a BTK-inhibitor (PCI-32765) blocked NF-kB and STAT3 signaling, as well as induced WM tumor cell killing that was synergistically enhanced by the presence of an IRAK1/4 kinase inhibitor. Conclusion:MYD88 L265P promotes survival of WM cells by activation of BTK. These studies provide the framework for the investigation of BTK inhibitors in WM, as single agents and in combination with other MYD88 pathway inhibitors. Disclosures:Treon:Onyx: Research Funding; Celgene: Research Funding; Pharmacyclics: Research Funding; Cephalon: Consultancy; Avila: Consultancy.

Toll‐like receptor 2 (TLR2)‐TLR9 crosstalk dictates IL‐12 family cytokine production in microglia

Microglia are the resident mononuclear phagocytes of the CNS parenchyma and represent an initial line of defense against invading microorganisms. Microglia utilize Toll-like receptors (TLRs) for pathogen recognition and TLR2 specifically senses conserved motifs of gram-positive bacteria including lipoproteins, lipoteichoic acids, and peptidoglycan (PGN) leading to cytokine/chemokine production. Interestingly, primary microglia derived from TLR2 knockout (KO) mice over-expressed numerous IL-12 family members, including IL-12p40, IL-12p70, and IL-27 in response to intact S. aureus, but not the less structurally complex TLR2 ligands Pam3CSK4 or PGN. The ability of intact bacteria to augment IL-12 family member expression was specific for gram-positive organisms, since numerous gram-negative strains were unable to elicit exaggerated responses in TLR2 KO microglia. Inhibition of SYK or IRAK4 signaling did not impact heightened IL-12 family member production in S. aureus-treated TLR2 KO microglia, whereas PI3K, MAPK, and JNK inhibitors were all capable of restoring exaggerated cytokine expression to wild type levels. Additionally, elevated IL-12 production in TLR2 KO microglia was ablated by a TLR9 antagonist, suggesting that TLR9 drives IL-12 family member production following exposure to intact bacteria that remains unchecked in the absence of TLR2 signaling. Collectively, these findings indicate crosstalk between TLR2 and TLR9 pathways to regulate IL-12 family member production by microglia. The summation of TLR signals must be tightly controlled to ensure the timely cessation and/or fine tuning of cytokine signaling to avoid nonspecific bystander damage due to sustained IL-12 release.

The Schistosoma mansoni Protein Sm16/SmSLP/SmSPO-1 Assembles into a Nine-Subunit Oligomer with Potential To Inhibit Toll-Like Receptor Signaling

The Sm16/SmSLP/SmSPO-1 (Sm16) protein is secreted by the parasite Schistosoma mansoni during skin penetration and has been ascribed immunosuppressive activities. Here we describe the strategy behind the design of a modified Sm16 protein with a decreased aggregation propensity, thus facilitating the expression and purification of an Sm16 protein that is soluble in physiological buffers. The Stokes radii and sedimentation coefficients of recombinant and native proteins indicate that Sm16 is an approximately nine-subunit oligomer. Analysis of truncated Sm16 derivatives showed that both oligomerization and binding to the plasma membrane of human cells depend on multiple C-terminal regions. For analysis of immunomodulatory activities, Sm16 was expressed in Pichia pastoris to facilitate the preparation of a pyrogen/endotoxin-free purified protein. Recombinant Sm16 was found to have no effect on T-lymphocyte activation, cell proliferation, or the basal level of cytokine production by whole human blood or monocytic cells. However, Sm16 exerts potent inhibition of the cytokine response to the Toll-like receptor (TLR) ligands lipopolysaccharide (LPS) and poly(I:C) while being less efficient at inhibiting the response to the TLR ligand peptidoglycan or a synthetic lipopeptide. Since Sm16 specifically inhibits the degradation of the IRAK1 signaling protein in LPS-stimulated monocytes, our findings indicate that inhibition is exerted proximal to the TLR complex.

A Critical Role for the Innate Immune Signaling Molecule IRAK-4 in T Cell Activation

IRAK-4 is a protein kinase that is pivotal in mediating signals for innate immune responses. Here, we report that IRAK-4 signaling is also essential for eliciting adaptive immune responses. Thus, in the absence of IRAK-4, in vivo T cell responses were significantly impaired. Upon T cell receptor stimulation, IRAK-4 is recruited to T cell lipid rafts, where it induces downstream signals, including protein kinase C activation through the association with Zap70. This signaling pathway was found to be required for optimal activation of nuclear factor kappaB. Our findings suggest that T cells use this critical regulator of innate immunity for the development of acquired immunity, suggesting that IRAK-4 may be involved in direct signal cross talk between the two systems.