A novel IRAK4/PIM1 inhibitor ameliorates rheumatoid arthritis and lymphoid malignancy by blocking the TLR/MYD88-mediated NF-κB pathway

Interleukin-1 receptor-associated kinase 4 (IRAK4) is a pivotal enzyme in the Toll-like receptor (TLR)/MYD88 dependent signaling pathway, which is highly activated in rheumatoid arthritis tissues and activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL). Inflammatory responses followed by IRAK4 activation promote B-cell proliferation and aggressiveness of lymphoma. Moreover, proviral integration site for Moloney murine leukemia virus 1 (PIM1) functions as an anti-apoptotic kinase in propagation of ABC-DLBCL with ibrutinib resistance. We developed a dual IRAK4/PIM1 inhibitor KIC-0101 that potently suppresses the NF-κB pathway and proinflammatory cytokine induction in vitro and in vivo. In rheumatoid arthritis mouse models, treatment with KIC-0101 significantly ameliorated cartilage damage and inflammation. KIC-0101 inhibited the nuclear translocation of NF-κB and activation of JAK/STAT pathway in ABC-DLBCLs. In addition, KIC-0101 exhibited an anti-tumor effect on ibrutinib-resistant cells by synergistic dual suppression of TLR/MYD88-mediated NF-κB pathway and PIM1 kinase. Our results suggest that KIC-0101 is a promising drug candidate for autoimmune diseases and ibrutinib-resistant B-cell lymphomas.

Effect of Celastrol Based on IRAK4/ERK/p38 Signaling Pathway on Proliferation and Apoptosis of Multiple Myeloma Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of celastrol on the proliferation and apoptosis of human multiple myeloma (MM) cell lines, reveal the relationship between IRAK4/ERK/p38 signaling pathway and celastrol regulating the proliferation and apoptosis of H929 and ARP-1 cells, and explore whether celastrol combined with bortezomib has synergistic effect. @*METHODS@#CCK-8 method was used to detect the viability of MM cell lines H929 and ARP-1 treated by different concentrations of celastrol, bortezomib, and their combination, and the synergistic effect was determined by Kim's formula. The apoptosis rate of H929 cells and necrosis rate of ARP-1 were detected by Annexin V/PI method. The expression of key proteins and apoptosis proteins in IRAK4/ERK/p38 signaling pathway were detected by Western blot. @*RESULTS@#Celastrol could significantly inhibit the proliferation of H929 and ARP-1 cells (r=0.9018, r=0.9244) and induce apoptosis in a time-dependent manner. Compared with the control group, celastrol could significantly up-regulate the expression of PARP and cleaved caspase-3 while down-regulate the expression of p-IRAK4, p-ERK, and p-p38 in H929 and ARP-1 cells. Celastrol and bortezomib alone inhibited the proliferation of H929 and ARP-1 cells. Compared with celastrol and bortezomib alone, their combination had lower cell survival rate and higher apoptosis rate (P<0.05). @*CONCLUSION@#Celastrol can inhibit the proliferation and promote the apoptosis of H929 and ARP-1 cells, which may be related to inhibiting the phosphorylation of IRAK4 and blocking the activation of IRAK4/ERK/p38 signaling pathway. Celastrol combined with bortezomib has synergistic effect, which can more effectively inhibit the proliferation and induce apoptosis of H929 and ARP-1 cells.

Oral carriage of staphylococcus aureus in people with different body mass index / Colonización oral por staphylococcus aureus en personas con diferente índice de masa corporal

Background: The association between obesity and the oral microbiome has received great attention. Objective: This study aimed to determine the association of oral Staphylococcus aureus with different body mass index people. Material and Methods: A total of 155 saliva samples were collected. The individuals were grouped into three categories according to their BMI, normal weight, overweight and obese individuals. A loopful of saliva sample was cultured and incubated at 37°C for 24. Staphylococcus aureus isolates were diagnosed by colony characteristics, morphology, and biochemical tests. Results: The oral carriage rate of Staphylococcus aureus was 61.3% (65.1% females and 56.5% males). The Staphylococcus aureus rate was 68% in married and 60% in single people. The differences of oral carriage rates of Staphylococcus aureus in obese (73.6%) and overweight (85.4%) populations was statistically significant (p<0.0001) compared to the rate in normal weight group (34%). Among males, the highest oral carriage rate of Staphylococcus aureus was in overweight individuals (82.6%). Likewise, in females, the highest rate of salivary Staphylococcus aureus was among the overweight group (88.9%). Regarding marital status, in single people, the differences of Staphylococcus aureus in obese (p=0.0003) and overweight (p<0.0001) people was significantly compared to normal weight people. But, in married people, the differences in Staphylococcus aureus rates among all groups were statistically not significant (p=0.0935). Conclusion: Staphylococcus aureus was significantly related to overweight and obese individuals. The human oral Staphylococcus aureus may play a key role in the manifestation of obesity. The oral microbiota could provide a new target for improving the physical well being of humans.

Antecedentes: la asociación entre la obesidad y el microbioma oral ha recibido gran atención. Objetivo: Este estudio tuvo como objetivo determinar la asociación de Staphylococcus aureus oral en personas con diferentes índices de masa corporal. Material y Métodos: Se recolectaron un total de 155 muestras de saliva. Los individuos fueron agrupados en tres categorías según su indice de masa corporal: normopeso, sobrepeso y obesos. Se cultivó un asa de muestra de saliva y se incubó a 37°C durante 24 horas. Los aislamientos de Staphylococcus aureus se identificaron mediante las características de la colonia, la morfología y las pruebas bioquímicas. Resultados:La tasa de colonización oral por Staphylococcus aureus fue del 61,3% (65,1% mujeres y 56,5% hombres). La tasa de colonización por Staphylococcus aureus fue del 68% en casados y del 60% en solteros. Las diferencias de las tasas de portación oral de Staphylococcus aureus en las poblaciones obesas (73,6%) y con sobrepeso (85,4%) fueron estadísticamente significativas (p<0,0001) en comparación con la tasa en el grupo de peso normal (34.0%). Entre los hombres, la tasa más alta de portadores orales de Staphylococcus aureus fue en individuos con sobrepeso (82,6%). En las mujeres, la tasa más alta de Staphylococcus aureus salival se también se presentó en el grupo con sobrepeso (88,9 %). En cuantal estado civil, en solteros, las diferencias de Staphylococcus aureus en obesos (p=0,0003) y con sobrepeso (p<0,0001) fueron significativas compararadas con normopeso. Pero, en personas casadas, las diferencias en las tasas de Staphylococcus aureus entre todos los grupos no fueron estadísticamente significativas (p=0,0935). Conclusion: Staphylococcus aureus salival se relacionó significativamente en individuos con sobrepeso y obesidad. El Staphylococcus aureus oral humano puede jugar un papel clave en la manifestación de la obesidad. La microbiota oral podría proporcionar una nueva diana para mejorar el estado físico de los humanos

Effect of Sishenwan on Toll-like Receptor 4 and IRAK-M Expression in Colonic Tissue of Rats with Ulcerative Colitis of Spleen-kidney Yang Deficiency Type / 中国实验方剂学杂志

Objective:To observe the Toll-like receptor 4(TLR4)and its negative regulating factorInterleukin-1 receptor-associated kinase-M(IRAK-M)in colonic mucosa of rats with experimental ulcerative colitis(UC), and to discuss the mechanism of the Chinese medicine Sishenwan. Method:The 90 Wistar rats were randomly divide into six groups, blank group, model group, sulfasalazine group(0.36 g·kg-1), Sishenwan low, medium and high-dose group(2.5, 5, 10 g·kg-1), 15 cases in each group. A rat model of UC was prepared by using a solution of trinitrobenzenesulfonic acid/ethano.The histopathological changes of colon were observed by hematoxylin-eosin (HE) staining. The contents of serum free triiodothyroid acid (FT3), serum free thyroxine (FT4), immunoglobulin (Ig) E and interleukin (IL)-2 were determined by radioimmunoassay. The activity of superoxide dismutase (SOD) in rat serum was determined by xanthine oxidation method. The activity of malondialdehyde (MDA) in serum of rats was determined by thiobarbituric acid (TBA) colorimetry. Result:Compared with blank group, intestinal mucosal injury score of rats in model group was significantly increased (PP3, FT4, IL-2 and SOD were significantly decreased (PPPPPP3, FT4, IL-2, and SOD contents were significantly increased (PPPPPConclusion:The unbalanced expressions of TLR4 and its negative regulating factor IRAK-M are connected with the pathogenesis of UC.Sishenwan can cure UC and control the expression of TLR4 and promote the expression of IRAK-M.

Effects of IRF3 knockdown on the nuclear expression of Irak1bp1 protein in LPS-stimulated Raw 264.7 cells / 上海交通大学学报（医学版）

Objective • To amplify the interferon regulator factor 3 (IRF3) short hairpin RNA (shRNA) virus and investigate the effect of the virus on the nuclear expression of Irak1bp1 protein in lipopolysaccharide (LPS)-stimulated Raw 264.7 cells. Methods • Adenovirus was amplified in HEK293T cells and the virus titer was detected by TCID 50 assay. The Raw 264.7 cells were randomly divided into four groups including adenovirus (-) LPS (-) group, adenovirus (-) LPS (+) group, adenovirus (+) LPS (-) group and adenovirus (+) LPS (+) group. The expression of intracellular IRF3 mRNA was detected by real-time PCR, and the nuclear expression of IRF3 and Irak1bp1 protein were detected by Western blotting. Results • The titer of adenovirus was 2.2×1011 PFU/mL and the best MOI was 300. The expression of IRF3 mRNA and nuclear IRF3 protein in LPS-stimulated Raw 264.7 cells were significantly higher than those of the control group. The cellular constitutive expression of IRF3 at mRNA level and the LPS-induced expression of IRF3 were significantly inhibited after transfection of Raw 264.7 cells with adenovirus strains carrying IRF3 shRNA. However, the nuclear constitutive expression of IRF3 protein was not affected by IRF3 shRNA in the unstimulated state. The expression of nuclear Irak1bp1 protein was significantly higher than that of the control group. The nuclear constitutive expression and the LPS-induced expression of Irak1bp1 protein were not affected by IRF3 shRNA. Conclusion • Transfection of LPS-stimulated Raw 264.7 cells with adenovirus strains carrying IRF3 shRNA could effectively inhibit the expression of IRF3, but not affect the nuclear expression of Irak1bp1 protein.

Effects of IRF3 knockdown on the nuclear expression of Irak1bp1 protein in LPS-stimulated Raw 264.7 cells / 上海交通大学学报（医学版）

Objective·To amplify the interferon regulator factor 3 (IRF3) short hairpin RNA (shRNA) virus and investigate the effect of the virus on the nuclear expression of Irak1bp1 protein in lipopolysaccharide (LPS)-stimulated Raw 264.7 cells. Methods?·?Adenovirus was amplified in HEK293T cells and the virus titer was detected by TCID 50 assay. The Raw 264.7 cells were randomly divided into four groups including adenovirus (-) LPS (-) group, adenovirus (-) LPS (+) group, adenovirus (+) LPS (-) group and adenovirus (+) LPS (+) group. The expression of intracellular IRF3 mRNA was detected by real-time PCR, and the nuclear expression of IRF3 and Irak1bp1 protein were detected by Western blotting. Results?·?The titer of adenovirus was 2.2×1011PFU/mL and the best MOI was 300. The expression of IRF3 mRNA and nuclear IRF3 protein in LPS-stimulated Raw 264.7 cells were significantly higher than those of the control group. The cellular constitutive expression of IRF3 at mRNA level and the LPS-induced expression of IRF3 were significantly inhibited after transfection of Raw 264.7 cells with adenovirus strains carrying IRF3 shRNA. However, the nuclear constitutive expression of IRF3 protein was not affected by IRF3 shRNA in the unstimulated state. The expression of nuclear Irak1bp1 protein was significantly higher than that of the control group. The nuclear constitutive expression and the LPS-induced expression of Irak1bp1 protein were not affected by IRF3 shRNA. Conclusion?·?Transfection of LPS-stimulated Raw 264.7 cells with adenovirus strains carrying IRF3 shRNA could effectively inhibit the expression of IRF3, but not affect the nuclear expression of Irak1bp1 protein.

Design, synthesis and biological evaluation of pyridine-based IRAK4 inhibitors / 中国药科大学学报

Based on the known IRAK4 inhibitors MK-32 and AU-5,we designed and synthesized 12 pyridine-based target compounds by adopting open-ring and hybrid strategies,and combining molecular docking technology.The bioassays determined by radioisotope labeling demonstrated that the target compounds displayed good inhibitory activity against IRAK4.Among them,the IC50 value of 5 compounds was less than 1 μmol/L,suggesting that these compounds may be candidates for further investigation.

Effects of Jiuxieling Granules on Expressions of MyD88 and IRAK1 in Ulcerative Colitis Rats with Spleen-Kidney Yang Deficiency / 中国中医药信息杂志

Objective To observe the effects of Jiuxieling Granules on the expressions of MyD88 and IRAK1 in ulcerative colitis model rats with spleen-kidney yang deficiency; To discuss its mechanism of action. Methods Animal models were established by compound methods. 90 Wistar rats were randomly divided into blank group, model group, positive medicine group, and Jiuxieling Granules high-, medium-, and low-dose groups. Each administration group was given relevant medicine for gavage. RT-qPCR, SP immunohistochemistry and Western blot were used to detect mRNA and proteins of MyD88 and IRAK1 in colon tissues. Results Compared with the blank group, mRNA and proteins of MyD88 and IRAK1 in the model group increased (P<0.01). Compared with the model group, mRNA and proteins of MyD88 and IRAK1 in each administration group decreased (P<0.01), especially in Jiuxieling Granules high-dose group. Conclusion Jiuxieling Granules can reduce the expressions of MyD88 and IRAK1, and then influence the transmission of MyD88 signaling pathway and block the release of downstream inflammatory factors to achieve the result of treating ulcerative colitis with spleen-kidney yang deficiency.

Expression and clinical relevance of IRAK-M in monocytes in patients with systemic lupus erythematosus / 中国免疫学杂志

Objective:To study the expression and clinical relevance of IRAK-M in monocytes in patients with systemic lupus erythematosus. Methods: Real-time quantitative PCR was performed for IRAK-M mRNA measurement and enzyme-linked immunosorbent assay was used for anti-double-stranded DNA antibody ( dsDNA ) and anti-single-stranded DNA antibody ( ssDNA ) . Dynamic scattering turbidimetric immunoassay was applied for complement 3(C3),complement 4(C4) and C-reactive protein(CRP), while Westergren method for erythrocyte sedimentation rate (ESR). Correlation analysis of IRAK-M with SLEDAI,dsDNA,ssDNA,C3, C4,CRP and ESR was computed by Pearson or Spearman. Results:①The result showed that the mRNA expression of IRAK-M in SLE patients was significantly lower than healthy controls (P0. 05). Conclusion: This study indicated that IRAK-M play certain significant roles in the pathogenesis of SLE. We can monitor SLE disease activity and prognosis by quantitative detection of mRNA expression of IRAK-M. Meanwhile,it is very necessary to routinely test and regularly monitor the levels of dsDNA,ssDNA,C3,C4,CRP and ESR in SLE.

Pretreatment with Pam3 CSK4 decreases inflammation in renal tissue from mice with systemic MRSA infection / 中国免疫学杂志

Objective:To observe whether pretreatment with Pam3CSK4,a TLR2 agonist,could decrease the inflammation response in kidney from mice with systemic MRSA infection,and to investigate the mechanism of the attenuation of inflammation with Pam3CSK4 pretreatment. Methods:BALB/c mice were pretreated with Pam3CSK4 (10 μg/100 μl/each mouse) or PBS via tail vein once daily for two consecutive days. All mice were infected with live MRSA (ATCC43300) at 2×107 CFU/each mouse (via tail vein) 24 h after the second treatment. The levels of cytokines in kidney were measured by ELISA and real-time PCR,respectively. The relative expression of TLR2,IRAKs etc. were detected by real-time PCR. Western blot was performed to detect the phosphorylation of NF-κB, the expression of IRAK-M and A20,respectively. Results:The level of TNF-α,IL-6,IL-1β,CCL3 and IFN-γ in renal tissue from mice pretreated with Pam3CSK4 was decreased significantly compared with that from PBS-treated mice,respectively. Pam3CSK4 pretreatment down-regulated the relative expression of TLR2, inhibited the expression of IRAK-1 and the phosphorylation of NF-κB post infection. The expression of IRAK-M,one of the negative regulators in TLRs signaling pathway was increased significantly in renal tissue from Pam3CSK4-treated mice post infection. Conclusion:Pam3CSK4 pretreatment attenuated the inflammation response in kidney from mice with systemic MRSA infection,and these attenuation is related with up-regulation of IRAK-M.

Pretreatment with Pam3 CSK4 decreases inflammation in renal tissue from mice with systemic MRSA infection / 中国免疫学杂志

Objective:To observe whether pretreatment with Pam3CSK4,a TLR2 agonist,could decrease the inflammation response in kidney from mice with systemic MRSA infection,and to investigate the mechanism of the attenuation of inflammation with Pam3CSK4 pretreatment. Methods:BALB/c mice were pretreated with Pam3CSK4 (10 μg/100 μl/each mouse) or PBS via tail vein once daily for two consecutive days. All mice were infected with live MRSA (ATCC43300) at 2×107 CFU/each mouse (via tail vein) 24 h after the second treatment. The levels of cytokines in kidney were measured by ELISA and real-time PCR,respectively. The relative expression of TLR2,IRAKs etc. were detected by real-time PCR. Western blot was performed to detect the phosphorylation of NF-κB, the expression of IRAK-M and A20,respectively. Results:The level of TNF-α,IL-6,IL-1β,CCL3 and IFN-γ in renal tissue from mice pretreated with Pam3CSK4 was decreased significantly compared with that from PBS-treated mice,respectively. Pam3CSK4 pretreatment down-regulated the relative expression of TLR2, inhibited the expression of IRAK-1 and the phosphorylation of NF-κB post infection. The expression of IRAK-M,one of the negative regulators in TLRs signaling pathway was increased significantly in renal tissue from Pam3CSK4-treated mice post infection. Conclusion:Pam3CSK4 pretreatment attenuated the inflammation response in kidney from mice with systemic MRSA infection,and these attenuation is related with up-regulation of IRAK-M.

Suppression of Antimicrobial Defense and Stabilization of STAT3 by IRAK-M Expression in Tumor Cells Promotes Colorectal Carcinogenesis

Different environmental and genetic factors have been attributed to the etiology of colorectal cancer. Dysbiotic gut microbiota is associated with initiation and progression of colon carcinogenesis. Hyperactivation of STAT3 promotes carcinogenesis by upregulating cell proliferation, survival, tumor-induced immunosupression and angiogenesis. IRAK-M is a negative regulator of toll-like receptor signaling and inhibits innate immune response. The cancer cell may exploit this property of IRAK-M and evade host immune surveillance. Recently, it has been found that IRAK-M provide controlled feed back to bacteria involved in colorectal cancer by reducing antibacterial response in mice. Furthermore, IRAK-M increased the stability of STAT3 in tumor cells that support tumor promotion by upregulating cell proliferation and survival. Thus, it is suggested that IRAK-M promotes colitis associated colon cancer by enhancing bacterial colonization and stabilization of STAT3.

Association of genetic variants in the IRAK-4 gene with susceptibility to severe sepsis / 世界急诊医学杂志（英文）

BACKGROUND: The association of genetic variation in the IRAK-1 gene with sepsis outcome has been proved. However, few studies have addressed the impact of the IRAK-4 gene variants on sepsis risk. This study aimed to determine whether the polymorphisms in the IRAK-4 gene are associated with susceptibility to and prognosis of severe sepsis in the Chinese Han ethnic population.METHODS: In this case-control study, 192 patients with severe sepsis hospitalized in the emergency department of Zhongshan Hospital from February 2006 to December 2009 and 192 healthy volunteers were enrolled. Exclusion criteria included metastatic tumors, autoimmune diseases, AIDS or treatment with immunosuppressive drugs. This study was approved by the ethical committee of Zhongshan Hospital, Fudan University. Sepsis patients were divided into a survival group (n=124) and a non-survival group (n=68) according to the 30-day mortality. Primer 3 software was used to design PCR and sequencing primers. Genomic DNA was extracted from peripheral blood mononuclear cells. Seven tagSNPs in IRAK-4 were selected according to the data of the Chinese Han population in Beijing from the Hapmap project and genotyped by direct sequencing. The chi-square test was used to evaluate the differences in genotype and allele frequencies between the two groups.RESULTS: The distributions of all tagSNPs were consistent with Hardy-Weinberg equilibrium. The allele and genotype frequencies of rs4251545 (G/A) were significantly different between the severe sepsis and healthy control groups (P=0.015, P=0.035, respectively). Carriers of the rs4251545A had a higher risk for severe sepsis compared with carriers of the rs4251545G (OR=1.69, 95％ CI: 1.10-2.58). The allele and genotype frequencies of all SNPs were not significantly different between the survival group and non-survival group.CONCLUSION: These findings indicate that the variants in IRAK-4 are significantly associated with susceptibility to severe sepsis in the Chinese Han ethnic population.

Polymorphisms in the interleukin-1 receptor-associated kinase 4 gene are associated withsusceptibility to severe sepsis / 中华急诊医学杂志

Objective To investigate the possible association of IRAK4 polymorphisms with susceptibility to and prognosis of severe sepsis.Methods A total of 192 patients hospitalized in emergency department of Zhongshan Hospital from February 2006 to December 2009,and another 192 healthy volunteers were enrolled in this case-control study.Patients were excluded if they had metastatic tumors,autoimmune diseases,AIDS or received immunosuppressive drugs.This study was approved by the ethical committee of Zhongshan Hospital,Fudan University.Sepsis patients were divided into survival group(n =124)and non-survival group(n =68)according to the 30-day mortality.Primer 3 software was used to design the PCR and sequencing primers.Genomic DNA was extracted from peripheral blood mononuclear cells.Seven tagSNPs were selected based on the data of Chinese Han in Beijing from the Hapmap projectand genotyped by direct sequencing.We used x2 analysis to evaluate the significance of differences in genotype and allele frequencies between different groups.Results The distributions of all tagSNPs were consistent with Hardy-Weinberg equilibrium.The allele and genotype frequencies of rs4251545(G/A)were significantly different between severe sepsis and healthy control groups(P =0.015,P =0.035,respectively).Carriers of the rs4251545A had a higher risk for severe sepsis compared with carriers of the rs4251545G(OR =1.69,95％ CI:1.10-2.58).The allele and genotype frequencies of all SNPs were not significantly different between survivor group and non-survivor group.Conclusions These findings indicated that the variants in IRAK4 are significantly associated with severe sepsis susceptibility in the Chinese Han population.