The role of MAPK in Kupffer cell toll-like receptor (TLR) 2-, TLR4-, and TLR9-mediated signaling following trauma-hemorrhage

Severe injury deranges immune function and increases the risk of sepsis and multiple organ failure. Kupffer cells play a major role in mediating posttraumatic immune responses, in part via different Toll-like receptors (TLR). Although mitogen-activated protein kinases (MAPK) are key elements in the TLR signaling pathway, it remains unclear whether the activation of different MAPK are TLR specific. Male C3H/HeN mice underwent midline laparotomy (i.e., soft tissue injury), hemorrhagic shock (MAP approximately 35 mm Hg for 90 min), and resuscitation. Kupffer cells were isolated 2 h thereafter, lysed and immunoblotted with antibodies to p38, ERK1/2, or JNK proteins. In addition, cells were preincubated with specific inhibitors of p38, ERK1/2, or JNK MAPK followed by stimulation with the TLR2 agonist, zymosan; the TLR4 agonist, LPS; or the TLR9 agonist, CpG DNA. Cytokine (TNF-alpha, interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and KC) production was determined by cytometric bead array after 24 h in culture. MAPK activity as well as TNF-alpha, MCP-1, and KC production by Kupffer cells were significantly increased following trauma-hemorrhage. TLR4 activation by LPS stimulation increased the levels of all measured cytokines. CpG-stimulated TLR9 signaling increased TNF-alpha and IL-6 levels; however, it had no effect on chemokine production. Selective MAPK inhibition demonstrated that chemokine production was mediated via p38 and JNK MAPK activation in TLR2, -4, and -9 signaling. In contrast, TNF-alpha and IL-6 production was differentially regulated by MAPK depending on the TLR pathway stimulated. Thus, Kupffer cell TLR signaling employs different MAPK pathways in eliciting cytokine and chemokine responses following trauma-hemorrhage.     

The tyrosine kinase BMX is an essential mediator of inflammatory arthritis in a kinase-independent manner

Inflammatory cytokines like TNF play a central role in autoimmune disorders such as rheumatoid arthritis. We identified the tyrosine kinase bone marrow kinase on chromosome X (BMX) as an essential component of a shared inflammatory signaling pathway. Transient depletion of BMX strongly reduced secretion of IL-8 in cell lines and primary human cells stimulated by TNF, IL-1β, or TLR agonists. BMX was required for phosphorylation of p38 MAPK and JNK, as well as activation of NF-κB. The following epistasis analysis indicated that BMX acts downstream of or at the same level as the complex TGF-β activated kinase 1 (TAK1)-TAK1 binding protein. At the cellular level, regulation of the IL-8 promoter required the pleckstrin homology domain of BMX, which could be replaced by an ectopic myristylation signal, indicating a requirement for BMX membrane association. In addition, activation of the IL-8 promoter by in vitro BMX overexpression required its catalytic activity. Genetic ablation of BMX conferred protection in the mouse arthritis model of passive K/BxN serum transfer, confirming that BMX is an essential mediator of inflammation in vivo. However, genetic replacement with a catalytically inactive BMX allele was not protective in the same arthritis animal model. We conclude that BMX is an essential component of inflammatory cytokine signaling and that catalytic, as well as noncatalytic functions of BMX are involved.     

Tollip regulates proinflammatory responses to interleukin-1 and lipopolysaccharide

Activation of interleukin-1 (IL-1) receptor (IL-1R), Toll-like receptor 2 (TLR2), and TLR4 triggers NF-kappaB and mitogen-activated protein kinase (MAPK)-dependent signaling, thereby initiating immune responses. Tollip has been implicated as a negative regulator of NF-kappaB signaling triggered by these receptors in in vitro studies. Here, deficient mice were used to determine the physiological contribution of Tollip to immunity. NF-kappaB, as well as MAPK, signaling appeared normal in Tollip-deficient cells stimulated with IL-1beta or the TLR4 ligand lipopolysaccharide (LPS). Similarly, IL-1beta- and TLR-driven activation of dendritic cells and lymphocytes was indistinguishable from wild-type cells. In contrast, the production of the proinflammatory cytokines, IL-6 and tumor necrosis factor alpha was significantly reduced after IL-1beta and LPS treatment at low doses but not at lethal doses of LPS. Tollip therefore controls the magnitude of inflammatory cytokine production in response to IL-1beta and LPS.     

Bruton's tyrosine kinase is required for TLR2 and TLR4-induced TNF, but not IL-6, production

Bruton's tyrosine kinase (Btk), the gene mutated in the human immunodeficiency X-linked agammaglobulinemia, is activated by LPS and is required for LPS-induced TNF production. In this study, we have investigated the role of Btk both in signaling via another TLR (TLR2) and in the production of other proinflammatory cytokines such as IL-1beta, IL-6, and IL-8. Our data show that in X-linked agammaglobulinemia PBMCs, stimulation with TLR4 (LPS) or TLR2 (N-palmitoyl-S-[2, 3-bis(palmitoyloxy)-(2R)-propyl]-(R)-cysteine) ligands produces significantly less TNF and IL-1beta than in normal controls. In contrast, a lack of Btk has no impact on the production of IL-6, IL-8, or the anti-inflammatory cytokine, IL-10. Our previous data suggested that Btk lies within a p38-dependent pathway that stabilizes TNF mRNA. Accordingly, TaqMan quantitative PCR analysis of actinomycin D time courses presented in this work shows that overexpression of Btk is able to stabilize TNF, but not IL-6 mRNA. Furthermore, using the p38 inhibitor SB203580, we show that the TLR4-induced production of TNF, but not IL-6, requires the activity of p38 MAPK. These data provide evidence for a common requirement for Btk in TLR2- and TLR4-mediated induction of two important proinflammatory cytokines, TNF and IL-1beta, and reveal important differences in the TLR-mediated signals required for the production of IL-6, IL-8, and IL-10.     

TNFα and IL-1β are mediated by both TLR4 and Nod1 pathways in the cultured HAPI cells stimulated by LPS

A growing body of evidence recently suggests that glial cell activation plays an important role in several neurodegenerative diseases and neuropathic pain. Microglia in the central nervous system express toll-like receptor 4 (TLR4) that is traditionally accepted as the primary receptor of lipopolysaccharide (LPS). LPS activates TLR4 signaling pathways to induce the production of proinflammatory molecules. In the present studies, we verified the LPS signaling pathways using cultured highly aggressively proliferating immortalized (HAPI) microglial cells. We found that HAPI cells treated with LPS upregulated the expression of TLR4, phospho-JNK (pJNK) and phospho-NF-κB (pNF-κB), TNFα and IL-1β. Silencing TLR4 with siRNA reduced the expression of pJNK, TNFα and IL-1β, but not pNF-κB in the cells. Inhibition of JNK with SP600125 (a JNK inhibitor) decreased the expression of TNFα and IL-1β. Unexpectedly, we found that inhibition of Nod1 with ML130 significantly reduced the expression of pNF-κB. Inhibition of NF-κB also reduced the expression of TNFα and IL-1β. Nod1 ligand, DAP induced the upregulation of pNF-κB which was blocked by Nod1 inhibitor. These data indicate that LPS-induced pJNK is TLR4-dependent, and that pNF-κB is Nod1-dependent in HAPI cells treated with LPS. Either TLR4-JNK or Nod1-NF-κB pathways is involved in the expression of TNFα and IL-1β.     

Myxoma virus lacking the pyrin-like protein M013 is sensed in human myeloid cells by both NLRP3 and multiple Toll-like receptors, which independently activate the inflammasome and NF-κB innate response pathways

The myxoma virus (MYXV)-encoded pyrin domain-containing protein M013 coregulates inflammatory responses mediated by both the inflammasome and the NF-κB pathways. Infection of human THP-1 monocytic cells with a MYXV construct deleted for the M013 gene (vMyxM013-KO), but not the parental MYXV, activates both the inflammasome and NF-κB pathways and induces a spectrum of proinflammatory cytokines and chemokines, like interleukin-1β (IL-1β), tumor necrosis factor (TNF), IL-6, and monocyte chemoattractant protein 1. Here, we report that vMyxM013-KO virus-mediated activation of inflammasomes and secretion of IL-1β are dependent on the adaptor protein ASC, caspase-1, and NLRP3 receptor. However, vMyxM013-KO virus-mediated activation of NF-κB signaling, which induces TNF secretion, was independent of ASC, caspase-1, and either the NLRP3 or AIM2 inflammasome receptors. We also report that early synthesis of pro-IL-1β in response to vMyxM013-KO infection is dependent upon the components of the inflammasome complex. Activation of the NLRP3 inflammasome and secretion of IL-1β was also dependent on the release of cathepsin B and production of reactive oxygen species (ROS). By using small interfering RNA screening, we further demonstrated that, among the RIG-I-like receptors (RLRs) and Toll-like receptors (TLRs), only TLR2, TLR6, TLR7, and TLR9 contribute to the NF-κB-dependent secretion of TNF and the inflammasome-dependent secretion of IL-1β in response to vMyxM013-KO virus infection. Additionally, we demonstrate that early triggering of the mitogen-activated protein kinase pathway by vMyxM013-KO virus infection of THP-1 cells plays a critical common upstream role in the coordinate induction of both NF-κB and inflammasome pathways. We conclude that an additional cellular sensor(s)/receptor(s) in addition to the known RLRs/TLRs plays a role in the M013 knockout virus-induced activation of NF-κB pathway signaling, but the activation of inflammasomes entirely depends on sensing by the NLRP3 receptor in response to vMyxM013-KO infection of human myeloid cells.     

TGFβ Activated Kinase 1 (TAK1) at the Crossroad of B Cell Receptor and Toll-Like Receptor 9 Signaling Pathways in Human B Cells

B cell development and activation are regulated by combined signals mediated by the B cell receptor (BCR), receptors for the B-cell activating factor of the tumor necrosis factor family (BAFF-R) and the innate receptor, Toll-like receptor 9 (TLR9). However, the underlying mechanisms by which these signals cooperate in human B cells remain unclear. Our aim was to elucidate the key signaling molecules at the crossroads of BCR, BAFF-R and TLR9 mediated pathways and to follow the functional consequences of costimulation.Therefore we stimulated purified human B cells by combinations of anti-Ig, B-cell activating factor of the tumor necrosis factor family (BAFF) and the TLR9 agonist, CpG oligodeoxynucleotide. Phosphorylation status of various signaling molecules, B cell proliferation, cytokine secretion, plasma blast generation and the frequency of IgG producing cells were investigated. We have found that BCR induced signals cooperate with BAFF-R- and TLR9-mediated signals at different levels of cell activation. BCR and BAFF- as well as TLR9 and BAFF-mediated signals cooperate at NFκB activation, while BCR and TLR9 synergistically costimulate mitogen activated protein kinases (MAPKs), ERK, JNK and p38. We show here for the first time that the MAP3K7 (TGF beta activated kinase, TAK1) is responsible for the synergistic costimulation of B cells by BCR and TLR9, resulting in an enhanced cell proliferation, plasma blast generation, cytokine and antibody production. Specific inhibitor of TAK1 as well as knocking down TAK1 by siRNA abrogates the synergistic signals. We conclude that TAK1 is a key regulator of receptor crosstalk between BCR and TLR9, thus plays a critical role in B cell development and activation.     

Involvement of TLR4/type I IL-1 receptor signaling in the induction of inflammatory mediators and cell death induced by ethanol in cultured astrocytes

Activated astroglial cells are implicated in neuropathogenesis of many infectious and inflammatory diseases of the brain. A number of inflammatory mediators and cytokines have been proposed to play a key role in glial cell-related brain damage. Cytokine production seems to be initiated by signaling through TLR4/type I IL-1R (IL-1RI) in response to their ligands, LPS and IL-1beta, playing vital roles in innate host defense against infections, inflammation, injury, and stress. We have shown that glial cells are stimulated by ethanol, up-regulating cytokines and inflammatory mediators associated with TLR4 and IL-1RI signaling pathways in brain, suggesting that ethanol may contribute to brain damage via inflammation. We explore the possibility that ethanol, in the absence of LPS or IL-1beta, triggers signaling pathways and inflammatory mediators through TLR4 and/or IL-1RI activation in astrocytes. We show in this study that ethanol, at physiologically relevant concentrations, is capable of inducing rapid phosphorylation within 10 min of IL-1R-associated kinase, ERK1/2, stress-activated protein kinase/JNK, and p38 MAPK in astrocytes. Then an activation of NF-kappaB and AP-1 occurs after 30 min of ethanol treatment along with an up-regulation of inducible NO synthase and cyclooxygenase-2 expression. Finally, we note an increase in cell death after 3 h of treatment. Furthermore, by using either anti-TLR4- or anti-IL-1RI-neutralizing Abs, before and during ethanol treatment, we inhibit ethanol-induced signaling events, including NF-kappaB and AP-1 activation, inducible NO synthase, and cyclooxygenase-2 up-regulation and astrocyte death. In summary, these findings indicate that both TLR4 and IL-1RI activation occur upon ethanol treatment, and suggest that signaling through these receptors mediates ethanol-induced inflammatory events in astrocytes and brain.     

Toll-like receptor 4 and MYD88-dependent signaling mechanisms of the innate immune system are essential for the response to lipopolysaccharide by epithelial and stromal cells of the bovine endometrium

Infection of the bovine endometrium with Gram-negative bacteria commonly causes uterine disease. Toll-like receptor 4 (TLR4) on cells of the immune system bind Gram-negative bacterial lipopolysaccharide (LPS), stimulating the secretion of the proinflammatory cytokines interleukin 1B (IL1B) and IL6, and the chemokine IL8. Because the endometrium is the first barrier to infection of the uterus, the signaling cascade triggered by LPS and the subsequent expression of inflammatory mediators were investigated in endometrial epithelial and stromal cells, and the key pathways identified using short interfering RNA (siRNA) and biochemical inhibitors. Treatment of endometrial cells with ultrapure LPS stimulated an inflammatory response characterized by increased IL1B, IL6, and IL8 mRNA expression, and IL6 protein accumulation in epithelial cells, and by increased IL1B and IL8 mRNA expression, and IL6 and IL8 protein accumulation in stromal cells. Treatment of endometrial cells with LPS also induced the degradation of IKB and the nuclear translocation of NFKB, as well as rapid phosphorylation of mitogen-activated protein kinase 3/1 (MAPK3/1) and MAPK14. Knockdown of TLR4 or its signaling adaptor molecule, myeloid differentiation factor 88 (MYD88), using siRNA reduced the inflammatory response to LPS in epithelial and stromal cells. Biochemical inhibition of MAPK3/1, but not JNK or MAPK14, reduced LPS-induced IL1B, IL6, and IL8 expression in endometrial cells. In conclusion, epithelial and stromal cells have an intrinsic role in innate immune surveillance in the endometrium, and in the case of LPS this recognition occurs via TLR4- and MYD88-dependent cell signaling pathways.     

Bruton's tyrosine kinase separately regulates NFkappaB p65RelA activation and cytokine interleukin (IL)-10/IL-12 production in TLR9-stimulated B Cells

B lymphocytes express both B cell receptor and Toll-like receptors (TLR). We show here that Bruton's tyrosine kinase (Btk), a critical component in B cell receptor signaling, is also involved in TLR9 signaling in B cells. Stimulation of B cells with TLR9 ligand CpG oligodeoxynucleotide (ODN) leads to transient phosphorylation of Btk, and in the absence of Btk, TLR9-induced proliferation of B cells is impaired. Interestingly, Btk(-/-) B cells secrete significantly more interleukin (IL)-12 but much less IL-10 compared with wild type B cells upon TLR9 stimulation. Immunization of Btk(-/-) mice with CpG ODN also leads to elevated levels of IL-12 in vivo and consequently, a greater -fold increment in the production of Th1 type IgG2b and IgG3 antibodies in these mice compared with wild type controls. The addition of exogenous recombinant IL-10 could suppress IL-12 production by TLR9-activated Btk(-/-) B cells, suggesting that in B cells, Btk negatively regulates IL-12 through the induction of autocrine IL-10 production. TLR9 signaling also leads to the activation of NFkappaB, including the p65RelA subunit in wild type B cells. The lack of Btk signaling affects the activation of NFkappaB and impairs the translocation of the p65RelA subunit to the nucleus of B cells upon TLR9 stimulation. However, p65RelA(-/-) B cells could respond similarly to wild type B cells in terms of IL-10 and IL-12 secretion when stimulated with CpG ODN, suggesting that the defect in NFkappaB p65RelA activation is additional to the impairment in cytokine production in TLR9-activated Btk(-/-) B cells. Thus, Btk plays an important role in TLR9 signaling and acts separately to regulate NFkappaB RelA activation as well as IL-10 and IL-12 production in B cells.     

Toxoplasma gondii genotype determines MyD88-dependent signaling in infected macrophages

Infection of mouse macrophages with Toxoplasma gondii elicits MAPK activation and IL-12 production, but host cell signaling pathways have not been clearly delineated. Here, we compared macrophage signaling in response to high virulence type I (RH) vs low virulence type II (ME49) strain infection. Tachyzoites of both strains induced p38 MAPK-dependent macrophage IL-12 release, although ME49 elicited 2- to 3-fold more cytokine than RH. IL-12 production was largely restricted to infected cells in each case. RH-induced IL-12 release did not require MyD88, whereas ME49-triggered IL-12 production was substantially dependent on this TLR/IL-1R adaptor molecule. MyD88 was also not required for RH-stimulated p38 MAPK activation, which occurred in the absence of detectable upstream p38 MAPK kinase activity. In contrast, ME49-driven p38 MAPK activation displayed an MyD88-dependent component. This parasite strain also induced MyD88-dependent activation of MKK4, an upstream activator of p38 MAPK. The results suggest that RH triggers MAPK activation and IL-12 production using MyD88-independent signaling, whereas ME49 uses these pathways as well as MyD88-dependent signaling cascades. Differences in host signaling pathways triggered by RH vs ME49 may contribute to the high and low virulence characteristics displayed by these parasite strains.     

Proinflammatory cytokine production in liver regeneration is Myd88-dependent, but independent of Cd14, Tlr2, and Tlr4

TNF and IL-6 are considered to be important to the initiation or priming phase of liver regeneration. However, the signaling pathways that lead to the production of these cytokines after partial hepatectomy (PH) have not been identified. Enteric-derived LPS appears to be important to liver regeneration, possibly by stimulating proinflammatory cytokine production after surgery. To determine whether LPS signaling pathways are involved in the regulation of the proinflammatory cytokines TNF and IL-6 during the priming phase of liver regeneration, we performed PH on mice lacking the TLRs Tlr4 and Tlr2, the LPS coreceptor, Cd14, and Myd88, an adapter protein involved in most TLR and IL-1R pathways. In MyD88 knockout (KO) mice after PH, both liver Tnf mRNA and circulating IL-6 levels were severely depressed compared with heterozygous or wild-type mice. Activation of STAT-3 and three STAT-3 responsive genes, Socs3, Cd14, and serum amyloid A2 were also blocked. In contrast, Tlr4, Tlr2, and Cd14 KO mice showed no deficits in the production of IL-6. Surprisingly, none of these KO mice showed any delay in hepatocyte replication. These data indicate that the LPS receptor TLR4, as well as TLR2 and CD14, do not play roles in regulating cytokine production or DNA replication after PH. In contrast, MyD88-dependent pathways appear to be responsible for TNF, IL-6, and their downstream signaling pathways.     

Kaposi's Sarcoma-Associated Herpesvirus MicroRNAs Target IRAK1 and MYD88, Two Components of the Toll-Like Receptor/Interleukin-1R Signaling Cascade,To Reduce Inflammatory-Cytokine Expression

Kaposi''s sarcoma (KS)-associated herpesvirus (KSHV) is the causative agent of KS, an important AIDS-associated malignancy. KSHV expresses at least 18 different mature microRNAs (miRNAs). We identified interleukin-1 receptor (IL-1R)-associated kinase 1 (IRAK1) as a potential target of miR-K12-9 (miR-K9) in an array data set examining changes in cellular gene expression levels in the presence of KSHV miRNAs. Using 3′-untranslated region (3′UTR) luciferase reporter assays, we confirmed that miR-K9 and other miRNAs inhibit IRAK1 expression. In addition, IRAK1 expression is downregulated in cells transfected with miR-K9 and during de novo KSHV infection. IRAK1 is an important component of the Toll-like receptor (TLR)/IL-1R signaling cascade. The downregulation of IRAK1 by miR-K9 resulted in the decreased stimulation of NF-κB activity in endothelial cells treated with IL-1α and in B cells treated with a TLR7/8 agonist. Interestingly, miR-K9 had a greater effect on NF-κB activity than did a small interfering RNA (siRNA) targeting IRAK1 despite the more efficient downregulation of IRAK1 expression with the siRNA. We hypothesized that KSHV miRNAs may also be regulating a second component of the TLR/IL-1R signaling cascade, resulting in a stronger phenotype. Reanalysis of the array data set identified myeloid differentiation primary response protein 88 (MYD88) as an additional potential target. 3′UTR luciferase reporter assays and Western blot analysis confirmed the targeting of MYD88 by miR-K5. The presence of miR-K9 and miR-K5 inhibited the production of IL-6 and IL-8 upon the IL-1α stimulation of endothelial cells. These results demonstrate KSHV-encoded miRNAs regulating the TLR/IL-1R signaling cascade at two distinct points and suggest the importance of these pathways during viral infection.     

IL-17 enhances IL-1beta-mediated CXCL-8 release from human airway smooth muscle cells

Recent studies into the pathogenesis of airway disorders such as asthma have revealed a dynamic role for airway smooth muscle cells in the perpetuation of airway inflammation via secretion of cytokines and chemokines. In this study, we evaluated whether IL-17 could enhance IL-1beta-mediated CXCL-8 release from human airway smooth muscle cells (HASMC) and investigated the upstream and downstream signaling events regulating the induction of CXCL-8. CXCL-8 mRNA and protein induction were assessed by real-time RT-PCR and ELISA from primary HASMC cultures. HASMC transfected with site-mutated activator protein (AP)-1/NF-kappaB CXCL-8 promoter constructs were treated with selective p38, MEK1/2, and phosphatidylinositol 3-kinase (PI3K) inhibitors to determine the importance of MAPK and PI3K signaling pathways as well as AP-1 and NF-kappaB promoter binding sites. We demonstrate IL-17 induced and synergized with IL-1beta to upregulate CXCL-8 mRNA and protein levels. Erk1/2 and p38 modulated IL-17 and IL-1beta CXCL-8 promoter activity; however, IL-1beta also activated the PI3K pathway. The synergistic response mediating CXCL-8 promoter activity was dependent on both MAPK and PI3K signal transduction pathways and required the cooperation of AP-1 and NF-kappaB cis-acting elements upstream of the CXCL-8 gene. Collectively, our observations indicate MAPK and PI3K pathways regulate the synergy of IL-17 and IL-1beta to enhance CXCL-8 promoter activity, mRNA induction, and protein synthesis in HASMC via the cooperative activation of AP-1 and NF-kappaB trans-acting elements.     

Essential role of MAPK phosphatase-1 in the negative control of innate immune responses

TLR-induced innate immunity and inflammation are mediated by signaling cascades leading to activation of the MAPK family of Ser/Thr protein kinases, including p38 MAPK, which controls cytokine release during innate and adoptive immune responses. Failure to terminate such inflammatory reactions may lead to detrimental systemic effects, including septic shock and autoimmunity. In this study, we provide genetic evidence of a critical and nonredundant role of MAPK phosphatase (MKP)-1 in the negative control of MAPK-regulated inflammatory reactions in vivo. MKP-1-/- mice are hyperresponsive to low-dose LPS-induced toxicity and exhibit significantly increased serum TNF-alpha, IL-6, IL-12, MCP-1, IFN-gamma, and IL-10 levels after systemic administration of LPS. Furthermore, absence of MKP-1 increases systemic levels of proinflammatory cytokines and exacerbates disease development in a mouse model of rheumatoid arthritis. When activated through TLR2, TLR3, TLR4, TLR5, and TLR9, bone marrow-derived MKP-1-/- macrophages exhibit increased cytokine production and elevated expression of the differentiation markers B7.2 (CD86) and CD40. MKP-1-deficient macrophages also show enhanced constitutive and TLR-induced activation of p38 MAPK. Based on these findings, we propose that MKP-1 is an essential component of the intracellular homeostasis that controls the threshold and magnitude of p38 MAPK activation in macrophages, and inflammatory conditions accentuate the significance of this regulatory function.