Abstract 1665: A comprehensive platform for the screening and assessment of IRAK4-targeting PROTACs

Interleukin-1 (IL-1) receptor-associated kinase (IRAK) is phosphorylated after it is recruited to the receptor, subsequently ubiquitinated, and eventually degraded upon IL-1 stimulation. Although a point mutation changing lysine 134 to arginine (K134R) in IRAK abolished IL-1-induced IRAK ubiquitination and degradation, mutations of serines and threonines adjacent to lysine 134 to alanines ((S/T)A (131-144)) reduced IL-1-induced IRAK phosphorylation and abolished IRAK ubiquitination. Through the study of these IRAK modification mutants, we uncovered two parallel IL-1-mediated signaling pathways for NFkappaB activation, TAK1-dependent and MEKK3-dependent, respectively. These two pathways bifurcate at the level of IRAK modification. The TAK1-dependent pathway leads to IKKalpha/beta phosphorylation and IKKbeta activation, resulting in classical NFkappaB activation through IkappaBalpha phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKKgamma phosphorylation and IKKalpha activation, resulting in NFkappaB activation through IkappaBalpha phosphorylation and subsequent dissociation from NFkappaB but without IkappaBalpha degradation. These results provide significant insight to our further understanding of NFkappaB activation pathways.

Two parallel interleukin-1 (IL-1)-mediated signaling pathways have been uncovered for IL-1R-TLR-mediated NFkappaB activation: TAK1-dependent and MEKK3-dependent pathways, respectively. The TAK1-dependent pathway leads to IKKalpha/beta phosphorylation and IKKbeta activation, resulting in classic NFkappaB activation through IkappaBalpha phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKKgamma phosphorylation and IKKalpha activation, resulting in NFkappaB activation through dissociation of phosphorylated IkappaBalpha from NFkappaB without IkappaBalpha degradation. IL-1 receptor-associated kinase 4 (IRAK4) belongs to the IRAK family of proteins and plays a critical role in IL-1R/TLR-mediated signaling. IRAK4 kinase-inactive mutant failed to mediate the IL-1R-TLR-induced TAK1-dependent NFkappaB activation pathway, but mediated IL-1-induced TAK1-independent NFkappaB activation and retained the ability to activate substantial gene expression, indicating a structural role of IRAK4 in mediating this alternative NFkappaB activation pathway. Deletion analysis of IRAK4 indicates the essential structural role of the IRAK4 death domain in receptor proximal signaling for mediating IL-1R-TLR-induced NFkappaB activation.

The innate immune response is linked to the adaptive immune response through several families of the pattern recognition receptors, among which Toll-like receptors (TLRs) are the most studied. Triggering of the pattern recognition receptors results in production of cytokines and chemokines and enhancement of both innate and adaptive immune responses. TLR signaling pathways share similar elements with interleukin-1 receptor (IL-1R)/IL-18R signaling pathways. Activation of the receptors after ligand binding leads to the recruitment of the Toll/IL-1R domain–containing adaptor molecule, myeloid differentiation factor 88, to the TLRs (1). Thereafter, several kinases such as IL-1R–associated kinase 4 (IRAK-4) and IRAK-1, as well as tumor necrosis factor receptor–associated factor 6, are recruited to the receptor complex (Figure 1). IRAK-4 is activated, and after hyperphosphorylation of IRAK-1, the receptor complex is further activated by transforming growth factor –activated kinase 1–dependent and –independent pathways, leading finally to activation of the downstream mediators NFB and activator protein 1 (2,3). An important factor in the activation of this inflammatory pathway is IRAK-4. IRAK-4 is a member of a family of protein kinases that also contains IRAK-1, IRAK-2, and IRAK-M. It has been demonstrated that IRAK-4 is crucial for the signaling pathway of the IL-1 and IL-18 receptors. In addition, IRAK-4 is needed for activation of nearly all of the TLRs, with the exception of TLR-3. Using IRAK-4 kinase-inactive knockin mice, it has been shown that IRAK-4 is important for IL-1–, lipopolysaccharide (LPS; TLR-4)–, or R848 (TLR-7)– induced cytokine and chemokine production (4–6). Interestingly, TLR-4 signaling is only partly disrupted in IRAK-4 kinase-inactive knockin mice, since cells from these mice respond to LPS exposure with subnormal cytokine production (7). The partial TLR-4 responsiveness is mediated by a secondary pathway involving the TRIF adaptor molecule (Figure 1). However, IRAK-4 kinase-inactive knockin mice are resistant to LPSor CpG-induced lethal shock (5). The role of active IRAK-4 has not been extensively studied in animal models of complex diseases. Very recently, it was shown that IRAK-4 is crucial in the development of vascular inflammation. IRAK-4 kinaseinactive knockin mice backcrossed in apolipoprotein E–knockout mice were almost completely protected against atherosclerosis (8). However, until now very little has been known about the effect of IRAK-4 in models of arthritis. In this issue of Arthritis & Rheumatism, KoziczakHolbro and colleagues demonstrate for the first time that IRAK-4 kinase-inactive knockin mice are completely protected against the development of antibodyinduced arthritis (K/BxN mouse model). Compared with wild-type (WT) mice, IRAK-4 kinase-inactive knockin mice did not have joint swelling, cartilage damage, or bone erosions (9). It has been previously shown that this particular animal model of arthritis is highly IL-1 dependent, since IL-1–deficient mice were extremely resistant to the K/BxN model of experimental arthritis (10). In line with these results, Koziczak-Holbro and colleagues confirmed the critical role of IRAK-4 in the IL-1R signaling pathway (9). Interestingly, however, the authors also showed that IRAK-4 kinase activity was not necessary for trafficking of T cells, B cells, and macrophages to the site of inflammation, whereas IRAK-4 was crucial for residential stromal cells to recruit inflammatory cells to the joint. These data identify IRAK-4 as a pivotal upstream kinase and potential therapeutic target to modulate synoviocyte activation in rheumatoid arthritis (RA). Furthermore, Koziczak-Holbro and colleagues Leo A. B. Joosten, PhD, Mihai G. Netea, MD, PhD: Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands. Address correspondence and reprint requests to Leo A. B. Joosten, PhD, Department of Medicine (463), Radboud University Nijmegen Medical Centre, Geert Grooteplein zuid 8, 6525 GA Nijmegen, The Netherlands. E-mail: l.joosten@aig.umcn.nl. Submitted for publication February 3, 2009; accepted in revised form February 25, 2009.

BackgroundThe aim of this study was to assess the effects of interleukin-1 (IL-1) receptor associated kinase (IRAK) inhibitors on intestinal injury induced by necrotizing enterocolitis (NEC) in neonatal rats and its regulation on the intestinal Toll-like receptor (TLR) inflammatory signaling pathway.Material/MethodsThe neonatal rat models of NEC were established though hypoxia-cold stimulation. All rats were divided into 3 groups: an NEC model group (NEC group), an IRAK inhibitor group (IRAKI group), and a normal control group (NC group). At 72 h after the models were established, intestinal tissues were collected for histopathological examination, enzyme-linked immunosorbent assay (ELISA), Western blotting, and immunohistochemistry.ResultsAfter IRAK inhibitor intervention, the symptoms of NEC in neonatal rats were alleviated, and the degree of weight loss was reduced. In the IRAK group, the intestinal pathology of neonatal rats was improved, pathological score was decreased, and the incidence rate of NEC was significantly reduced. The levels of tumor necrosis factor-alpha (TNF-α), IL-1β, and IL-6 in the IRAK group were significantly decreased compared with those in the NEC group. There were no significant differences in IRAK1 and IRAK4 protein expression levels between the IRAK group and the NEC group. The phosphorylated IRAK1 and IRAK4 in the IRAK group were significantly decreased. Nuclear factor-kappa B (NF-κB) level of intestinal tissues in the IRAK group was reduced compared with that in the NEC group.ConclusionsIRAK inhibitors can inhibit the inflammatory response of the NEC model, reduce the release of pro-inflammatory cytokines, and alleviate the damage to intestinal tissues by inhibiting conduction of the TLR signaling pathway.

IL-1 receptor-associated kinase (IRAK) is phosphorylated, ubiquitinated, and degraded upon interleukin-1 (IL-1) stimulation. In this study, we showed that IRAK can be ubiquitinated through both Lys-48- and Lys-63-linked polyubiquitin chains upon IL-1 induction. Pellino 3b is the RING-like motif ubiquitin protein ligase that promotes the Lys-63-linked polyubiquitination on IRAK. Pellino 3b-mediated Lys-63-linked IRAK polyubiquitination competed with Lys-48-linked IRAK polyubiquitination for the same ubiquitination site, Lys-134 of IRAK, thereby blocking IL-1-induced IRAK degradation. Importantly, the negative impact of Pellino 3b on IL-1-induced IRAK degradation correlated with the inhibitory effect of Pellino 3b on the IL-1-induced TAK1-dependent pathway, suggesting that a positive role of IRAK degradation in IL-1 induced TAK1 activation. Taken together, our results suggest that Pellino 3b acts as a negative regulator for IL-1 signaling by regulating IRAK degradation through its ubiquitin protein ligase activity.

Most members of the Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) families transduce signals via a canonical pathway involving the MyD88 adapter and the interleukin-1 receptor-associated kinase (IRAK) complex. This complex contains four molecules, including at least two (IRAK-1 and IRAK-4) active kinases. In mice and humans, deficiencies of IRAK-4 or MyD88 abolish most TLR (except for TLR3 and some TLR4) and IL-1R signaling in both leukocytes and fibroblasts. TLR and IL-1R responses are weak but not abolished in mice lacking IRAK-1, whereas the role of IRAK-1 in humans remains unclear. We describe here a boy with X-linked MECP2 deficiency-related syndrome due to a large de novo Xq28 chromosomal deletion encompassing both MECP2 and IRAK1 Like many boys with MECP2 null mutations, this child died very early, at the age of 7 mo. Unlike most IRAK-4- or MyD88-deficient patients, he did not suffer from invasive bacterial diseases during his short life. The IRAK-1 protein was completely absent from the patient's fibroblasts, which responded very poorly to all TLR2/6 (PAM2CSK4, LTA, FSL-1), TLR1/2 (PAM3CSK4), and TLR4 (LPS, MPLA) agonists tested but had almost unimpaired responses to IL-1β. By contrast, the patient's peripheral blood mononuclear cells responded normally to all TLR1/2, TLR2/6, TLR4, TLR7, and TLR8 (R848) agonists tested, and to IL-1β. The death of this child precluded long-term evaluations of the clinical consequences of inherited IRAK-1 deficiency. However, these findings suggest that human IRAK-1 is essential downstream from TLRs but not IL-1Rs in fibroblasts, whereas it plays a redundant role downstream from both TLRs and IL-1Rs in leukocytes.

ABSTRACTSystemic lupus erythematosus (SLE) is a typical autoimmune disease. Genome-wide analyses have revealed that interleukin-1 receptor-associated kinase 1 (IRAK1) is associated with susceptibility to SLE. Our previous study investigated the role of IRAK1 in nuclear factor-κB (NF-κB)-related pathways in a mouse model of lupus. In this study, we aimed to further explore the etiological role of IRAK1. The gene expression and phosphorylation of IRAK1 in CD4+ T cells from lupus patients and healthy controls were examined by quantitative reverse transcription-polymerase chain reaction and western blotting, respectively. The percentage of circulating Th17 cells and plasma IL-17A levels were evaluated by flow cytometry and enzyme-linked immunosorbent assay, respectively. The influence of IRAK1 suppression on Th17 development was assessed using an IRAK1 inhibitor and small interfering RNA. We found that IRAK1 transcript levels in CD4+ T cells were significantly upregulated in SLE patients in comparison to controls and were positively correlated with disease activity. In vitro experiments showed that lupus CD4+ T cells had more pronounced IRAK1 phosphorylation at threonine-209 upon IL-1β stimulation than did control cells. Moreover, IRAK1 expression was positively associated with Th17/IL-17A in patients. When naïve CD4+ T cells were polarized toward the Th17 subset, IRAK1 inhibition significantly repressed IL-17A production and the gene expression of Th17 markers, namely, retinoic acid receptor-related orphan receptor c, IL-23 receptor and IL-17A. In summary, IRAK1 is overexpressed and hyperactivated in CD4+ T cells from SLE patients. IRAK1 inhibition attenuates Th17 differentiation in the context of human SLE, suggesting a therapeutic opportunity.

Curcumin Blocks Interleukin-1 (IL-1) Signaling by Inhibiting the Recruitment of the IL-1 Receptor–Associated Kinase IRAK in Murine Thymoma EL-4 Cells

Treatment of several autoimmune diseases and types of cancer has been an intense area of research over the past two decades. Many signaling pathways that regulate innate and/or adaptive immunity, as well as those that induce overexpression or mutation of protein kinases, have been targeted for drug discovery. One of the serine/threonine kinases, Interleukin-1 Receptor Associated Kinase 4 (IRAK4) regulates signaling through various Toll-like receptors (TLRs) and interleukin-1 receptor (IL1R). It controls diverse cellular processes including inflammation, apoptosis, and cellular differentiation. MyD88 gain-of-function mutations or overexpression of IRAK4 has been implicated in various types of malignancies such as Waldenström macroglobulinemia, B cell lymphoma, colorectal cancer, pancreatic ductal adenocarcinoma, breast cancer, etc. Moreover, over activation of IRAK4 is also associated with several autoimmune diseases. The significant role of IRAK4 makes it an interesting target for the discovery and development of potent small molecule inhibitors. A few potent IRAK4 inhibitors such as PF-06650833, RA9 and BAY1834845 have recently entered phase I/II clinical trial studies. Nevertheless, there is still a need of selective inhibitors for the treatment of cancer and various autoimmune diseases. A great need for the same intrigued us to perform molecular modeling studies on 4,6-diaminonicotinamide derivatives as IRAK4 inhibitors. We performed molecular docking and dynamics simulation of 50 ns for one of the most active compounds of the dataset. We also carried out MM-PBSA binding free energy calculation to identify the active site residues, interactions of which are contributing to the total binding energy. The final 50 ns conformation of the most active compound was selected to perform dataset alignment in a 3D-QSAR study. Generated RF-CoMFA (q2 = 0.751, ONC = 4, r2 = 0.911) model revealed reasonable statistical results. Overall results of molecular dynamics simulation, MM-PBSA binding free energy calculation and RF-CoMFA model revealed important active site residues of IRAK4 and necessary structural properties of ligand to design more potent IRAK4 inhibitors. We designed few IRAK4 inhibitors based on these results, which possessed higher activity (predicted pIC50) than the most active compounds of the dataset selected for this study. Moreover, ADMET properties of these inhibitors revealed promising results and need to be validated using experimental studies.

Pathologic activation of the Toll-like receptor (TLR) pathway underlies various human disorders such as autoimmune diseases, chronic inflammatory diseases and lymphoid malignancies. Current therapy of these diseases relies on immunosuppressive or chemotherapeutic agents, but more effective therapies tailored to disease-causing mechanisms are required. The IL-1 receptor-associated kinase 4 (IRAK4), is critical to TLR signaling and is recruited to TLRs by the adapter protein MyD88. Gain-of-function MYD88 mutations are activated by IRAK4 kinase in several mature B cell malignancies, including activated B-cell-like diffuse large B cell lymphoma (ABC DLBCL). Development of selective IRAK4 kinase inhibitors has been confounded by the challenging structure of IRAK4 catalytic domain. Using structure-based drug design methodologies, we identified potent and selective IRAK4 kinase inhibitors. ABC DLBCL cell lines that specifically harbor activating MYD88 mutations are killed by these inhibitors, both in vitro and in mouse xenograft models. Gene expression profiling revealed that IRAK4 kinase inhibitors downregulated prosurvival NF-κB signatures, and cytokine analysis showed a decrease in the production of inflammatory cytokines IL-6 and IL-10. Our findings open new possibilities for the therapy of malignant diseases that rely upon IRAK4 kinase. Citation Format: Priscilla N. Kelly, Divya Chaudhary, Ryan M. Young, Art Shaffer, Shaughnessy Robinson, Donna L. Romero, Rosana Kapeller, Louis M. Staudt. Highly potent and selective interleukin-1 receptor-associated kinase 4 inhibitors for the therapy of lymphoid malignancies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-112. doi:10.1158/1538-7445.AM2014-LB-112

Interleukin-1 receptor (IL1R)-associated kinase 4 (IRAK4) is a central regulator of innate immune signaling, controlling IL1R and Toll-like receptor (TLR)-mediated responses and containing both scaffolding and kinase activities. Humans deficient in IRAK4 activity have autosomal recessive primary immune deficiency (PID). Here, we characterized the molecular mechanism of dysfunction of two IRAK4 PID variants, G298D and the compound variant R12C (R12C/R391H/T458I). Using these variants and the kinase-inactive D329A variant to delineate the contributions of IRAK4's scaffolding and kinase activities to IL1R signaling, we found that the G298D variant is kinase-inactive and expressed at extremely low levels, acting functionally as a null mutation. The R12C compound variant possessed WT kinase activity, but could not interact with myeloid differentiation primary response 88 (MyD88) and IRAK1, causing impairment of IL-1-induced signaling and cytokine production. Quantitation of IL-1 signaling in IRAK4-deficient cells complemented with either WT or the R12C or D329A variant indicated that the loss of MyD88 interaction had a greater impact on IL-1-induced signaling and cytokine expression than the loss of IRAK4 kinase activity. Importantly, kinase-inactive IRAK4 exhibited a greater association with MyD88 and a weaker association with IRAK1 in IRAK4-deficient cells expressing kinase-inactive IRAK4 and in primary cells treated with a selective IRAK4 inhibitor. Loss of IRAK4 kinase activity only partially inhibited IL-1-induced cytokine and NF-κB signaling. Therefore, the IRAK4-MyD88 scaffolding function is essential for IL-1 signaling, but IRAK4 kinase activity can control IL-1 signal strength by modulating the association of IRAK4, MyD88, and IRAK1.

Background:AML is an aggressive hematopoietic malignancy that arises from a population of aberrant hematopoietic stem cells in the bone marrow (BM). Advances in understanding the molecular basis of AML has led to the development of new targeted therapies. CA‐4948 is a novel, oral IRAK4 kinase inhibitor with additional inhibitory activity against wild‐type (wt) and mutated FLT3 kinase. IRAK4 (Interleukin‐1 Receptor Associated Kinase 4) is required for interleukin 1 receptor (IL‐1R) and toll‐like receptor (TLR) innate immune pathway signaling, pathways that are frequently over activated in AML and myelodysplastic syndromes (MDS). For example, AML patients have increased IL‐1R agonist (IL‐1ß) levels that promote the survival of AML cells and IL‐1R KO represses AML cell growth in vitro and in vivo (Carey et al 2017). Dysregulation of the FLT3 signaling pathway is a well validated driver of AML. Constitutively activating mutations in FLT3 that comprise the ITD or the tyrosine kinase domain (KD) are frequently acquired late in AML disease and are poor prognostic factors with high relapse rates. FLT3 kinase inhibitors targeting FLT3‐ITD or ITD/KD double mutations show high remission rates; however, multiple resistance mechanisms have been reported in both nonclinical models and AML patients. CA‐4948 has both IRAK4 and FLT3 inhibitory activity, which may impart benefit to FLT3‐wt and FLT3‐mutant AML patients.Aims:Evaluate the ability of a novel IRAK4/FLT3 inhibitor, CA‐4948, to block IRAK4 and FLT3 in FLT3‐wt and FLT3‐mutant AML in vitro and in vivo.Methods:CA‐4948's kinome profile was assessed against 378 kinases and 9 FLT3 mutant variants (DiscoverX). CA‐4948's ability to inhibit TLR/IL‐1R or FLT3 signaling pathways was evaluated using NF‐kB reporter, cytokine production, or western blot. The growth inhibitory and pro‐apoptotic activity of CA‐4948 was tested in vitro against AML cell lines by viability assay or flow cytometry. For AML FLT3‐wt in vivo efficacy, THP‐1 cells were tail‐vein injected into mice and animal survival and degree of AML cell engraftment in BM were monitored in CA‐4948, FLT3i, or vehicle treated mice. For AML FLT3 mutant in vivo efficacy, subcutaneous MV4–11 and MOLM‐14 FLT3‐ITD and MOLM‐14 double FLT3‐ITD/KD tumor models were treated with CA‐4948 or other FLT3i.Results:CA‐4948 exhibited 23 nM (Kd) binding affinity for IRAK4, >500‐fold selectivity against IRAK1, and high binding affinity for FLT3‐wt, ‐ITD, ‐ITD/D835 V, and ‐ITD/F691L at 8–31 nM (Kd). CA‐4948 blocked cellular TLR and IL‐1R‐stimulated signaling from 150–220 nM (IC50). Consistent with CA‐4948's additional FLT3 inhibitory activity, CA‐4948 induced apoptosis and exhibited cytotoxic activity against AML FLT3 mutant lines in vitro at 58–200 nM (IC50). In FLT3‐wt in vivo studies, CA‐4948 blocked THP‐1 bone marrow engraftment, while FLT3i quizartinib (Q) and midostaurin (M) had no inhibitory effect. In AML FLT3‐ITD tumors, CA‐4948 induced tumor regression equivalent to Q and M. CA‐4948 also induced tumor regression in FLT3‐ITD/D835Y tumors similar to M and showed greater efficacy in FLT3‐ITD/F691L tumors as compared to Q.Summary/Conclusion:These results demonstrate that targeting the IL‐1R/TLR signaling pathway with IRAK4 inhibitor CA‐4849 may be an effective therapeutic strategy in FLT3‐wt AML. Furthermore, CA‐4948's additional FLT3 inhibitory activity may repress the emergence of FLT3‐mutant AML clones. CA‐4948's dual IRAK4/FLT3 inhibitory activity merits further clinical investigation in FLT3‐wt and FLT3‐mutant AML/MDS.

The interleukin-1 (IL-1) receptor colocalizes with focal adhesion complexes (FACs), actin-enriched structures involved in cell adhesion and signaling in fibroblasts and chondrocytes. The colocalization of FACs and IL-1 receptors has been implicated in the restriction of IL-1 signaling transduction to ERK; however, the mechanism of this restriction and the requirement of IL-1 receptor-associated proteins have not been characterized. We determined if the association kinetics of the interleukin-1 receptor-associated kinase (IRAK) colocalizes with FACs and the requirement for IRAK in IL-1-dependent ERK activation. Human gingival fibroblasts were incubated with collagen-coated beads to induce the assembly of FACs at sites of cell-bead contact. Immunoblot analysis of bead-isolated FACs showed a time-dependent assembly of the focal adhesion proteins beta-actin, vinculin, and talin, which was blocked by the actin monomer sequestering toxin latrunculin B. Although no IRAK was isolated with FACs from unstimulated cells, phosphorylated IRAK was transiently associated with FACs isolated from IL-1beta-stimulated fibroblasts. Fibroblasts plated on tissue culture plastic (which permitted the formation of focal adhesions) showed phosphorylation of ERK, JNK, and p38. Cells plated on poly-l-lysine (to prevent the formation of focal adhesions) showed activation only of JNK and p38. ERK activation was partially restored by incubating cells plated on poly-l-lysine with collagen-coated beads before IL-1 stimulation. Cells treated with latrunculin B or swinholide A, which caused a progressive depolymerization of actin filaments, showed a reduction or elimination of IL-1-induced ERK activation, respectively. Fibroblasts electroinjected with a mouse monoclonal anti-IRAK antibody to block the recruitment of IRAK into FACs failed to activate ERK after IL-1 treatment, indicating that FAC-associated IRAK is required for the activation of ERK. These data indicate that the integrity of actin filament arrays and the recruitment of IRAK into focal adhesions are involved in the restriction of IL-1 signaling to ERK.

Objective To explore the effects of bilirubin on myeloid differentiation factor 88(MyD88)and interleukin-1 receptor associated kinase-4(IRAK-4). Methods Seven-day-old Sprague Dawley rats (clean grade), male or female, weighing 12.0 to 15.0 g, were randomly assigned to 6 groups.There were normal saline group(Ⅰ), lipopolysaccharide(LPS) control group (LPS, Ⅱ), 15 mg/kg bilirubin control (free-LPS) group (Ⅲ), 15 mg/kg group (Ⅳa), 30 mg/kg group (Ⅳb) and 50 mg/kg group (Ⅳc), and then subsequently divided into 2 h, 5 h and 24 h subgroups in each groups.Some of the 200 newborn rats died amid the experiment.Finally a total of 144 were involved in the analysis of results, and 8 rats in each subgroups.Newborn Sprague Dawley rats were administered at various doses of bilirubin(15 mg/kg, 30 mg/kg and 50 mg/kg respectively)intravenously; 1 h after injection, the rats were administered LPS intraperitoneally at a dose of 1 mg/kg; MyD88 and IRAK-4 were detected by immunohistochemistry at 2 h, 5 h and 24 h after the injection of bilirubin. Results (1) LPS could stimulate the expression of MyD88 and IRAK-4 in spleen cells (qMyD88 2 h=49.89, qMyD88 5 h=139.54, qIRAK-4 2 h=7.93, qIRAK-4 5 h=24.30, qIRAK-4 24 h=6.97, P 0.05). Effects of medium and high concentration of bilirubin on LPS stimulation MyD88 were inhibitory(qⅣb 2 h=42.87, qⅣc 2 h=51.38, qⅣb 5 h=103.61, qⅣc 5 h=115.44, qⅣb 24 h=1.18, qⅣc 24 h=11.66, P<0.01). (4) Effects of low, medium and high concentration of bilirubin on LPS stimulation IRAK-4 were inhibitory(qⅣa 2 h=9.52, qⅣb 2 h=14.39, qⅣc 2 h=25.55, qⅣa 5 h=38.83, qⅣb 5 h=54.62, qⅣc 5 h=60.51, qⅣa 24 h=2.41, qⅣb 24 h=1.47, qⅣc 24 h=7.61, P<0.01). (5) The inhibition of bilirubin to MyD88 and IRAK-4 was observed at 2 h, strengthened at 5 h, disappeared at 24 h in low-mid concentrations of bilirubin(P<0.01)while still visible at 24 h in high concentration of bilirubin.(6) There was negatively correlation between the expression level of MyD88, IRAK-4 and bilirubin concentration(rsMyD88 2 h =-0.86, rsMyD88 5 h=-0.92, rsMyD88 24 h=-0.53, rsIRAK-4 2 h=-0.82, rsIRAK-4 5 h=-0.86, rsIRAK-4 24 h=-0.57, P<0.01). (7) Under the effect of bilirubin and LPS, there were positively correlation between the expression levels of MyD88 and IRAK-4 of spleen cells(r2 h=0.77, r5 h=0.9, r24 h=0.67, P<0.01). Conclusion Bilirubin could inhibit the expression of MyD88 and IRAK-4.As the concentration of bilirubin increasing, its inhibition is more obvious and prolonged.The mechanism that bilirubin affects immune function of newborn rat may be related to regulation of expression of MyD88 and IRAK-4 at toll-like receptor 4 signal pathway. Key words: Bilirubin; Hyperbilirubinemia; Myeloid differentiation factor 88; Interleukin-1 receptor associated kinase-4; Rat, newborn,

Systemic lupus erythematosus (SLE) is a debilitating autoimmune disease affecting multiple organs in the body, but therapeutic options are still very limited and often come with adverse effects. Increasing evidence has underlined an important role of the Toll-like receptor 7 (TLR-7)/TLR-9/interleukin-1 receptor-associated kinase 1 (IRAK-1)/interferon regulatory factor 7 (IRF-7) pathway in the development and progression of SLE. Notably, the prolyl isomerase Pin1 is an essential regulator of IRAK-1 in TLR-7/TLR-9 signaling, but its role in SLE is unknown. We undertook this study to determine whether Pin1 is activated and plays any role in the development and treatment of SLE. Activation of Pin1 and TLR-7/TLR-9/IRAK-1/IRF-7 signaling was determined in various cell types among peripheral blood mononuclear cells from healthy controls and SLE patients. The effects of Pin1 and TLR signaling on SLE development were determined using validated Pin1 short hairpin RNA (shRNA), Pin1 genetic knockout, and the small-molecule Pin1 inhibitor all-trans-retinoic acid (ATRA) in immune cells and in several strains of lupus-prone mice. We found abnormal activation of Pin1 and its downstream targets IRAK-1 and IRF-7 in SLE patients. Furthermore, inhibition of Pin1 using either validated Pin1 shRNA or ATRA blocked TLR-7-induced activation of IRAK-1 and IRF-7 in SLE patient-derived immune cells. Moreover, in multiple lupus-prone animals, both Pin1 knockout and ATRA strikingly attenuated the expression of autoimmunity, including skin lesions, lymphadenopathy, splenomegaly, glomerulonephritis, proteinuria, and production of anti-double-stranded DNA antibodies and CD4-CD8- T cells, and also prolonged overall survival in MRL/lpr and B6.lpr mice. Pin1 plays a critical role in the development of SLE, and Pin1-targeted therapy offers a promising new strategy for treating SLE.