Prostaglandin E2 suppresses lipopolysaccharide-stimulated IFN-beta production

Macrophages activate the production of cytokines and chemokines in response to LPS through signaling cascades downstream from TLR4. Lipid mediators such as PGE(2), which are produced during inflammatory responses, have been shown to suppress MyD88-dependent gene expression upon TLR4 activation in macrophages. The study reported here investigated the effect of PGE(2) on TLR3- and TLR4-dependent, MyD88-independent gene expression in murine J774A.1 macrophages, as well as the molecular mechanism underlying such an effect. We demonstrate that PGE(2) strongly suppresses LPS-induced IFN-beta production at the mRNA and protein levels. Poly (I:C)-induced IFN-beta and LPS-induced CCL5 production were also suppressed by PGE(2). The inhibitory effect of PGE(2) on LPS-induced IFN-beta expression is mediated through PGE(2) receptor subtypes EP(2) and EP(4), and mimicked by the cAMP analog 8-Br-cAMP as well as by the adenylyl cyclase activator forskolin. The downstream effector molecule responsible for the cAMP-induced suppressive effect is exchange protein directly activated by cAMP (Epac) but not protein kinase A. Moreover, data demonstrate that Epac-mediated signaling proceeds through PI3K, Akt, and GSK3beta. In contrast, PGE(2) inhibits LPS-induced TNF-alpha production in these cells through a distinct pathway requiring protein kinase A activity and independent of Epac/PI3K/Akt. In vivo, administration of a cyclooxygenase inhibitor before LPS injection resulted in enhanced serum IFN-beta concentration in mice. Collectively, data demonstrate that PGE(2) is a negative regulator for IFN-beta production in activated macrophages and during endotoxemia.     

Signaling events leading to peroxiredoxin 5 up-regulation in immunostimulated macrophages

Peroxiredoxins (PRXs) are thiol peroxidases associated with many cellular functions including proliferation, cell cycle, apoptosis, and differentiation. There is also increasing evidence that these ubiquitous antioxidant enzymes control H₂O₂ signaling in eukaryotes. Here, we provide evidence that the LPS/TLR4 and the Th1 cytokine IFN-γ pathways induce expression of PRX5, a potent peroxide and peroxynitrite reductase, in primary macrophages. Furthermore, deletion of TRIF, MyD88, or type I IFN receptor revealed that the LPS/TLR4-dependent increase in PRX5 expression is mediated by a TRIF-dependent/IFN-β-independent pathway. IFN-γ-dependent induction of the PRX5 gene was markedly reduced in MyD88^−/− and TNF^−/− macrophages. Moreover, addition of exogenous TNF allowed the recovery of full PRX5 expression in both MyD88^−/− and TNF^−/− cells stimulated with IFN-γ, suggesting that basal TNF produced in an MyD88-dependent manner contributes to PRX5 induction. Downstream of the TLR pathways, we have explored the role of MAPK activation and found that p38 and JNK mainly contribute to PRX5 up-regulation in immunostimulated macrophages. Expression of PRX5 is thus responsive to innate immunity signals, and we propose that PRX5 is an additional host defense weapon of activated macrophages.     

TLR2, TLR4 and the MyD88 Signaling Are Crucial For the In Vivo Generation and the Longevity of Long-Lived Antibody-Secreting Cells

This study was undertaken to gain better insights into the role of TLRs and MyD88 in the development and differentiation of memory B cells, especially of ASC, during the Th2 polarized memory response induced by Natterins. Our in vivo findings demonstrated that the anaphylactic IgG1 production is dependent on TLR2 and MyD88 signaling, and that TLR4 acts as adjuvant accelerating the synthesis of high affinity-IgE. Also, TLR4 (MyD88-independent) modulated the migration of innate-like B cells (B1a and B2) out of the peritoneal cavity, and the emigration from the spleen of B1b and B2 cells. TLR4 (MyD88-independent) modulated the emigration from the spleen of Bmem as well as ASC B220^pos. TLR2 triggered to the egress from the peritoneum of Bmem (MyD88-dependent) and ASC B220^pos (MyD88-independent). We showed that TLR4 regulates the degree of expansion of Bmem in the peritoneum (MyD88-dependent) and in BM (MyD88-independent) as well as of ASC B220^neg in the spleen (MyD88-independent). TLR2 regulated the intensity of the expansion of Bmem (MyD88-independent) and ASC B220^pos (MyD88-dependent) in BM. Finally, TLR4 signals sustained the longevity of ASC B220^pos (MyD88-independent) and ASC B220^neg into the peritoneum (MyD88-dependent) and TLR2 MyD88-dependent signaling supported the persistence of B2 cells in BM, Bmem in the spleen and ASC B220^neg in peritoneum and BM. Terminally differentiated ASC B220^neg required the cooperation of both signals through TLR2/TLR4 via MyD88 for longevity in peritoneum, whereas Bmem required only TLR2/MyD88 to stay in spleen, and ASC B220^pos rested in peritoneum dependent on TLR4 signaling. Our data sustain that earlier events on memory B cells differentiation induced in secondary immune response against Natterins, after secondary lymph organs influx and egress, may be the key to determining peripheral localization of innate-like B cells and memory B cells as ASC B220^pos and ASC B220^neg.     

TIRAP mediates endotoxin-induced NF-kappaB activation and apoptosis in endothelial cells

Bacterial lipopolysaccharide (LPS) initiates multiple signaling events in vascular endothelial cells that can result in activation and/or cell death. LPS-induced activation of endothelial cells elicits a wide array of vascular endothelial responses, many of which are dependent on NF-kappaB activation. Several of the signaling molecules that mediate LPS-induced NF-kappaB activation, including Tlr-4, MyD88, and IRAK-1, have been similarly reported to mediate LPS pro-apoptotic signaling. Recently, a new signaling molecule, TIRAP, has been identified that mediates LPS-induced NF-kappaB signaling in monocytes and macrophages. Using a TIRAP dominant negative construct, we have identified a role for TIRAP in mediating LPS-induced NF-kappaB activation and apoptosis in human endothelial cells. These data identify TIRAP as a dual functioning signaling molecule and suggest the presence of a MyD88-independent LPS signaling pathway in human endothelial cells.     

Effects of myeloid differentiation primary response gene 88 (MyD88) activation on Helicobacter infection in vivo and induction of a Th17 response

Background:  Helicobacter pylori is a spiral‐shaped Gram‐negative microaerophilic bacterium associated with a number of gastrointestinal disorders, including gastritis, peptic ulcers, and gastric cancer. Several studies have implicated a Th17 response as a key to protective immunity against Helicobacter. Materials and Methods:  Wild type (WT) and MyD88‐deficient (MyD88^?/?) mice in the C57BL/6 background were infected with H. felis for 6 and 25 weeks and colonization density and host response evaluated. Real‐time PCR was used to determine the expression of cytokines and antimicrobial peptides in the gastric tissue of mice. Results:  mRNA expression levels of the Th17 cytokines interleukin‐17A (IL‐17A) and IL‐22 were markedly up‐regulated in WT compared with MyD88^?/? mice both at 6 and at 25 weeks in response to infection with H. felis, indicating that induction of Th17 responses depends on MyD88 signaling. Furthermore, reduction in the expression of Th17‐dependent intestinal antimicrobial peptide lipocalin‐2 was linked with increased bacterial burden in the absence of MyD88 signaling. Conclusion:  We provide evidence showing that MyD88‐dependent signaling is required for the host to induce a Th17 response for the control of Helicobacter infection.     

Induction of IL-12p40 and type 1 immunity by Toxoplasma gondii in the absence of the TLR-MyD88 signaling cascade

Toxoplasma gondii is an orally acquired pathogen that induces strong IFN-γ based immunity conferring protection but that can also be the cause of immunopathology. The response in mice is driven in part by well-characterized MyD88-dependent signaling pathways. Here we focus on induction of less well understood immune responses that do not involve this Toll-like receptor (TLR)/IL-1 family receptor adaptor molecule, in particular as they occur in the intestinal mucosa. Using eYFP-IL-12p40 reporter mice on an MyD88^-/- background, we identified dendritic cells, macrophages, and neutrophils as cellular sources of MyD88-independent IL-12 after peroral T. gondii infection. Infection-induced IL-12 was lower in the absence of MyD88, but was still clearly above noninfected levels. Overall, this carried through to the IFN-γ response, which while generally decreased was still remarkably robust in the absence of MyD88. In the latter mice, IL-12 was strictly required to induce type I immunity. Type 1 and type 3 innate lymphoid cells (ILC), CD4⁺ T cells, and CD8⁺ T cells each contributed to the IFN-γ pool. We report that ILC3 were expanded in infected MyD88^-/- mice relative to their MyD88^+/+ counterparts, suggesting a compensatory response triggered by loss of MyD88. Furthermore, bacterial flagellin and Toxoplasma specific CD4⁺ T cell populations in the lamina propria expanded in response to infection in both WT and KO mice. Finally, we show that My88-independent IL-12 and T cell mediated IFN-γ production require the presence of the intestinal microbiota. Our results identify MyD88-independent intestinal immune pathways induced by T. gondii including myeloid cell derived IL-12 production, downstream type I immunity and IFN-γ production by ILC1, ILC3, and T lymphocytes. Collectively, our data reveal an underlying network of immune responses that do not involve signaling through MyD88.     

Endotoxin-induced maturation of MyD88-deficient dendritic cells

LPS, a major component of the cell wall of Gram-negative bacteria, can induce a variety of biological responses including cytokine production from macrophages, B cell proliferation, and endotoxin shock. All of them were completely abolished in MyD88-deficient mice, indicating the essential role of MyD88 in LPS signaling. However, MyD88-deficient cells still show activation of NF-kappaB and mitogen-activated protein kinase cascades, although the biological significance of this activation is not clear. In this study, we have examined the effects of LPS on dendritic cells (DCs) from wild-type and several mutant mice. LPS-induced cytokine production from DCs was dependent on MyD88. However, LPS could induce functional maturation of MyD88-deficient DCs, including up-regulation of costimulatory molecules and enhancement of APC activity. MyD88-deficient DCs could not mature in response to bacterial DNA, the ligand for Toll-like receptor (TLR)9, indicating that MyD88 is differentially required for TLR family signaling. MyD88-dependent and -independent pathways originate at the intracytoplasmic region of TLR4, because both cytokine induction and functional maturation were abolished in DCs from C3H/HeJ mice carrying the point mutation in the region. Finally, in vivo analysis revealed that MyD88-, but not TLR4-, deficient splenic CD11c(+) DCs could up-regulate their costimulatory molecule expression in response to LPS. Collectively, the present study provides the first evidence that the MyD88-independent pathway downstream of TLR4 can lead to functional DC maturation, which is critical for a link between innate and adaptive immunity.     

Tumor Progression Locus 2 (Tpl2) Kinase Promotes Chemokine Receptor Expression and Macrophage Migration during Acute Inflammation

In autoimmune diseases, the accumulation of activated leukocytes correlates with inflammation and disease progression, and, therefore, the disruption of leukocyte trafficking is an active area of research. The serine/threonine protein kinase Tpl2 (MAP3K8) regulates leukocyte inflammatory responses and is also being investigated for therapeutic inhibition during autoimmunity. Here we addressed the contribution of Tpl2 to the regulation of macrophage chemokine receptor expression and migration in vivo using a mouse model of Tpl2 ablation. LPS stimulation of bone marrow-derived macrophages induced early CCR1 chemokine receptor expression but repressed CCR2 and CCR5 expression. Notably, early induction of CCR1 expression by LPS was dependent upon a signaling pathway involving Tpl2, PI3K, and ERK. On the contrary, Tpl2 was required to maintain the basal expression of CCR2 and CCR5 as well as to stabilize CCR5 mRNA expression. Consistent with impairments in chemokine receptor expression, tpl2^−/− macrophages were defective in trafficking to the peritoneal cavity following thioglycollate-induced inflammation. Overall, this study demonstrates a Tpl2-dependent mechanism for macrophage expression of select chemokine receptors and provides further insight into how Tpl2 inhibition may be used therapeutically to disrupt inflammatory networks in vivo.     

Ischemia-Reperfusion Lung Injury Is Attenuated in MyD88-Deficient Mice

Ischemia-reperfusion lung injury is a common cause of acute morbidity and mortality in lung transplant recipients and has been associated with subsequent development of bronchiolitis obliterans syndrome. Recognition of endogenous ligands released during cellular injury (damage-associated molecular patterns; DAMPs) by Toll-like receptors (TLRs), especially TLR4, has increasingly been recognized as a mechanism for inflammation resulting from tissue damage. TLR4 is implicated in the pathogenesis of ischemia-reperfusion injury of multiple organs including heart, liver, kidney and lung. Additionally, activation of TLRs other than TLR4 by DAMPs has been identified in tissues other than the lung. Because all known TLRs, with the exception of TLR3, signal via the MyD88 adapter protein, we hypothesized that lung ischemia-reperfusion injury was mediated by MyD88-dependent signaling. To test this hypothesis, we subjected C57BL/6 wildtype, Myd88 ^-/-, and Tlr4 ^-/- mice to 1 hr of left lung warm ischemia followed by 4 hr of reperfusion. We found that Myd88 ^-/- mice had significantly less MCP-1/CCL2 in the left lung following ischemia-reperfusion as compared with wildtype mice. This difference was associated with dramatically reduced lung permeability. Interestingly, Tlr4 ^-/- mice had only partial protection from ischemia-reperfusion as compared to Myd88 ^-/- mice, implicating other MyD88-dependent pathways in lung injury following ischemia-reperfusion. We also found that left lung ischemia-reperfusion caused remote inflammation in the right lung. Finally, using chimeric mice with MyD88 expression restricted to either myeloid or non-myeloid cells, we found that MyD88-dependent signaling in myeloid cells was necessary for ischemia-reperfusion induced lung permeability. We conclude that MyD88-dependent signaling through multiple receptors is important in the pathogenesis of acute lung inflammation and injury following ischemia and reperfusion.     

Lipopolysaccharide-driven Th2 Cytokine Production in Macrophages Is Regulated by Both MyD88 and TRAM

Gram-negative bacterial lipopolysaccharide (LPS) activates macrophages by interacting with Toll-like receptor 4 (TLR4) and triggers the production of various pro-inflammatory Th1 type (type 1) cytokines such as IFNγ, TNFα, and IL8. Though some recent studies cited macrophages as potential sources for Th2 type (type 2) cytokines, little however is known about the intracellular events that lead to LPS-induced type 2 cytokines in macrophages. To understand the mechanisms by which LPS induces type 2 cytokine gene expression, macrophages were stimulated with LPS, and the expression of IL-4 and IL-5 genes were examined. LPS, acting through TLR4, activates both type 1 and type 2 cytokine production both in vitro and in vivo by using macrophages from C3H/HeJ or C3H/HeOuJ mice. Although the baseline level of both TNFα and IL-4 protein was very low, TNFα was released rapidly after stimulation (within 4 h); however, IL-4 was released after 48 h LPS stimulation in secreted form. Silencing of myeloid differentiation protein (MyD88) and TRIF-related adaptor molecule (TRAM), using small interfering RNA abolished IL-4 induction induced by LPS whereas silencing of TRAM has no effect on TNFα induction, thereby indicating that LPS-induced TNFα is MyD88-dependent but IL-4 is required both MyD88 and TRAM. These findings suggest a novel function of LPS and the signaling pathways in the induction of IL-4 gene expression.Pathogen-associated molecular patterns (PAMPs)² such as bacterial LPS are powerful activators of the innate immune system. Exposure to LPS induces an inflammatory reaction in the lung, mediated primarily by an array of inflammatory chemokines and cytokines released by blood monocytes and alveolar macrophages. Mammalian Toll-like receptors (TLRs) are key molecules for recognizing microbial PAMPs and transducing the subsequent inflammatory response (1). LPS is well known to interact with macrophages via TLR4 receptor resulting in cellular activation and synthesis and release of type 1 proinflammatory cytokines such as IFNγ, IL-2, and TNFα (2, 3). These cytokines can further activate monocytes, neutrophils, and lymphocytes, initiating cellular injury and tissue damage (4, 5).Inhaled LPS signaling through TLR4 has also been shown to be necessary to induce type 2 responses to inhaled antigens in a mouse model of allergic asthma (6). IL-4, the prototypic type 2 cytokine, is a pleiotropic cytokine with regulatory effects on B cell growth, T cell growth, and function, immunoglobulin class switching to IgE during the development of immune responses (7). It is also involved in promoting cellular inflammation in the asthmatic lung and contributes to the pathogenesis of allergy and lung remodeling in chronic asthma (8, 9). Different cell types have been reported to produce IL-4 including the well known CD4+ and CD8+ T cells (10, 11), basophils (12), natural killer cells (13), mast cells (14), and eosinophils (15). Pouliot et al. (3) have shown that human alveolar macrophages (AMs) can produce IL-4 in response to PMA and calcium ionophore A23187, and they suggest that AMs might play a crucial role in the type 1/type 2 balance in the lung.LPS-stimulated production of type 1 cytokines such as TNFα and INFγ has been extensively studied in macrophages; however, LPS-stimulated production of type 2 cytokines by macrophages has not yet been well defined. Because the presence of IL-4 at the site of a developing immune response can skew the ultimate cytokine pattern, alveolar macrophage produced IL-4 may be important in the development of allergic airway disease. Indeed, TLR4-defective mice studied using a standard murine model of allergic airway inflammation had an overall decrease in lung inflammatory responses, a dramatic reduction of eosinophils and lymphocytes, and lower circulating levels of OVA-specific IgE (16).The intracellular events following LPS stimulation of TLR4 depends on different sets of Toll/interleukin-1 resistance (TIR) domain containing adaptor molecules. These adaptors provide a structural platform for the recruitment of downstream effector molecules (17, 18). Two distinct responses following engagement of TLR4 with LPS have been described. An early response leading to activation of NF-κB is dependent on MyD88, while a late response utilizes TIR domain-containing adaptor-inducing interferon-γ (TRIF) and TRIF-related adaptor molecule (TRAM) to activate NF-κB (19). While TRIF is common to both TLR3 and TLR4 pathways, TRAM is highly specific for TLR4 (20). The complex signaling network initiated by the interaction of the adaptor and effector proteins ultimately decides the specific pattern of gene expression that is elicited in response to TLR agonists and the particular type of cytokine that is produced determines the recruitment and activation of other immune cells. Therefore, further clarification of the cellular responses following the activation of TLR is crucial and fundamental to our understanding of immune responses.In this report, we show that LPS can stimulate de novo IL-4 gene expression in murine macrophages, both in vitro and in vivo. Utilizing RNA interference we further showed that the induction of IL-4 is both MyD88- and TRAM-dependent (MyD88-independent), while LPS-induced TNFα is strictly dependent on MyD88. These results indicate that LPS induces IL-4 production by macrophages, and provide a new molecular mechanism controlling the regulation of IL-4 prior to the emergence of a polarized adaptive immune response.     

B1 cells produce nitric oxide in response to a series of toll-like receptor ligands

The effect of a series of toll-like receptor (TLR) ligands on the production of nitric oxide (NO) in mouse B1 cells was examined by using CD5⁺ IgM⁺ WEHI 231 cells. The stimulation with a series of TLR ligands, which were Pam3Csk4 for TLR1/2, poly I:C for TLR3, lipopolysaccharide (LPS) for TLR4, imiquimod for TLR7 and CpG DNA for TLR9, resulted in enhanced NO production via augmented expression of an inducible type of NO synthase (iNOS). LPS was most potent for the enhancement of NO production, followed by poly I:C and Pam3Csk4. Imiquimod and CpG DNA led to slight NO production. The LPS-induced NO production was dependent on MyD88-dependent pathway consisting of nuclear factor (NF)-κB and a series of mitogen-activated protein kinases (MAPKs). Further, it was also dependent on the MyD88-independent pathway consisting of toll-IL-1R domain-containing adaptor-inducing IFN-β (TRIF) and interferon regulatory factor (IRF)-3. Physiologic peritoneal B1 cells also produced NO via the iNOS expression in response to LPS. The immunological significance of TLR ligands-induced NO production in B1 cells is discussed.     

Flavopiridol inhibits lipopolysaccharide-induced TNF-α production through inactivation of nuclear factor-κB and mitogen-activated protein kinases in the MyD88-dependent pathway

Flavopiridol is a cyclin-dependent kinase inhibitor and inhibits the growth of various cancer cells. The effect of flavopiridol on lipopolysaccharide (LPS)-induced proinflammatory mediator production was examined in RAW 264.7 macrophage-like cells. Flavopiridol significantly reduced the production of tumor necrosis factor-α and, to a lesser extent, nitric oxide in LPS-stimulated cells. Flavopiridol inhibited the activation of nuclear factor-κB and IκB kinase in response to LPS. Flavopiridol also inhibited the activation of a series of mitogen-activated protein kinases, such as p38, stress-activated protein kinase/c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 in response to LPS. However, flavopiridol did not alter the expression of tumor necrosis factor receptor-associated factor 6, myeloid differentiation factor 88 (MyD88) or CD14/toll-like receptor (TLR) 4. Flavopiridol inhibited nitric oxide production induced by a MyD88-dependent TLR2 ligand, but not a MyD88-independent TLR3 ligand. Further, flavopiridol did not alter the phosphorylation of interferon regulatory factor 3 in the MyD88-independent pathway. Therefore, it was suggested that flavopiridol exclusively inhibited the activation of nuclear factor-κB and mitogen-activated protein kinases in the MyD88-dependent pathway. Flavopiridol might be useful for the prevention of LPS-induced inflammatory response.     

Tumor Progression Locus 2-dependent Oxidative Burst Drives Phosphorylation of Extracellular Signal-regulated Kinase during TLR3 and 9 Signaling

Signal transduction via NFκB and MAP kinase cascades is a universal response initiated upon pathogen recognition by Toll-like receptors (TLRs). How activation of these divergent signaling pathways is integrated to dictate distinct immune responses to diverse pathogens is still incompletely understood. Herein, contrary to current perception, we demonstrate that a signaling pathway defined by the inhibitor of κB kinase β (IKKβ), MAP3 kinase tumor progression locus 2 (Tpl2/MAP3K8), and MAP kinase ERK is differentially activated by TLRs. TLRs 2, 4, and 7 directly activate this inflammatory axis, inducing immediate ERK phosphorylation and early TNFα secretion. In addition to TLR adaptor proteins, IKKβ-Tpl2-ERK activation by TLR4 is regulated by the TLR4 co-receptor CD14 and the tyrosine kinase Syk. Signals from TLRs 3 and 9 do not initiate early activation of IKKβ-Tpl2-ERK pathway but instead induce delayed, NADPH-oxidase-dependent ERK phosphorylation and TNFα secretion via autocrine reactive oxygen species signaling. Unexpectedly, Tpl2 is an essential regulator of ROS production during TLR signaling. Overall, our study reveals distinct mechanisms activating a common inflammatory signaling cascade and delineates differences in MyD88-dependent signaling between endosomal TLRs 7 and 9. These findings further confirm the importance of Tpl2 in innate host defense mechanisms and also enhance our understanding of how the immune system tailors pathogen-specific gene expression patterns.