Autocrine production of IL-10 mediates defective IL-12 production and NF-kappa B activation in tumor-associated macrophages

IL-12 is a central cytokine in the activation of inflammation and immunity and in the generation of Th1-type responses. Tumor-associated macrophages (TAM) from mouse and human tumors showed defective production of IL-12. Defective IL-12 production was associated with lack of p50/p65 NF-kappa B activation. TAM produced increased amounts of the immunosuppressive cytokine IL-10. Abs against IL-10 restored the defective capacity of TAM to produce IL-12. Our data suggest that during tumor growth an IL-10-dependent pathway of diversion of macrophage function can be activated into the tumor microenvironment and results in the promotion of the IL-10+ IL-12- phenotype of TAM. Blocking IL-10, as well as other immunosuppressive cytokines present in the tumor microenvironment, such as TGF-beta, may complement therapeutic strategies aimed at activating type I antitumor immune responses.     

SHIP negatively regulates IgE + antigen-induced IL-6 production in mast cells by inhibiting NF-kappa B activity

We demonstrate in this study that IgE + Ag-induced proinflammatory cytokine production is substantially higher in Src homology-2-containing inositol 5'-phosphatase (SHIP)(-/-) than in SHIP(+/+) bone marrow-derived mast cells (BMMCs). Focusing on IL-6, we found that the repression of IL-6 mRNA and protein production in SHIP(+/+) BMMCs requires the enzymatic activity of SHIP, because SHIP(-/-) BMMCs expressing wild-type, but not phosphatase-deficient (D675G), SHIP revert the IgE + Ag-induced increase in IL-6 mRNA and protein down to levels seen in SHIP(+/+) BMMCs. Comparing the activation of various signaling pathways to determine which ones might be responsible for the elevated IL-6 production in SHIP(-/-) BMMCs, we found the phosphatidylinositol 3-kinase/protein kinase B (PKB), extracellular signal-related kinase (Erk), p38, c-Jun N-terminal kinase, and protein kinase C (PKC) pathways are all elevated in IgE + Ag-induced SHIP(-/-) cells. Moreover, inhibitor studies suggested that all these pathways play an essential role in IL-6 production. Looking downstream, we found that IgE + Ag-induced IL-6 production is dependent on the activity of NF-kappa B and that I kappa B phosphorylation/degradation and NF-kappa B translocation, DNA binding and transactivation are much higher in SHIP(-/-) BMMCs. Interestingly, using various pathway inhibitors, it appears that the phosphatidylinositol 3-kinase/PKB and PKC pathways elevate IL-6 mRNA synthesis, at least in part, by enhancing the phosphorylation of I kappa B and NF-kappa B DNA binding while the Erk and p38 pathways enhance IL-6 mRNA synthesis by increasing the transactivation potential of NF-kappa B. Taken together, our data are consistent with a model in which SHIP negatively regulates NF-kappa B activity and IL-6 synthesis by reducing IgE + Ag-induced phosphatidylinositol-3,4,5-trisphosphate levels and thus PKB, PKC, Erk, and p38 activation.     

Phosphorylation of Bcl10 negatively regulates T-cell receptor-mediated NF-kappaB activation

Bcl10 (B-cell lymphoma 10) is an adaptor protein comprised of an N-terminal caspase recruitment domain and a C-terminal serine/threonine-rich domain. Bcl10 plays a critical role in antigen receptor-mediated NF-kappaB activation and lymphocyte development and functions. Our current study has discovered that T-cell activation induced monophosphorylation and biphosphorylation of Bcl10 and has identified S138 within Bcl10 as one of the T-cell receptor-induced phosphorylation sites. Alteration of S138 to an alanine residue impaired T-cell activation-induced ubiquitination and subsequent degradation of Bcl10, ultimately resulting in prolongation of TCR-mediated NF-kappaB activation and enhancement of interleukin-2 production. Taken together, our findings demonstrate that phosphorylation of Bcl10 at S138 down-regulates Bcl10 protein levels and thus negatively regulates T-cell receptor-mediated NF-kappaB activation.     

TNF receptor-associated factor-2 is involved in both IL-1 beta and TNF-alpha signaling cascades leading to NF-kappa B activation and IL-8 expression in human intestinal epithelial cells

Cytokine signaling involves the participation of many adaptor proteins, including the docking protein TNF receptor-associated factor-2 (TRAF-2), which is believed to transmit the TNF-alpha signal through both the I kappa B/NF-kappa B and c-Jun N-terminal kinase (JNK)/stress-related protein kinase (SAPK) pathways. The physiological role of TRAF proteins in cytokine signaling in intestinal epithelial cells (IEC) is unknown. We characterized the effect of a dominant-negative TRAF-2 delivered by an adenoviral vector (Ad5dnTRAF-2) on the cytokine signaling cascade in several IEC and also investigated whether inhibiting the TRAF-2-transmitting signal blocked TNF-alpha-induced NF-kappa B and IL-8 gene expression. A high efficacy and level of Ad5dnTRAF-2 gene transfer were obtained in IEC using a multiplicity of infection of 50. Ad5dnTRAF-2 expression prevented TNF-alpha-induced, but not IL-1 beta-induced, I kappa B alpha degradation and NF-kappa B activation in NIH-3T3 and IEC-6 cells. TNF-alpha-induced JNK activation was also inhibited in Ad5dnTRAF-2-infected HT-29 cells. Induction of IL-8 gene expression by TNF-alpha was partially inhibited in Ad5dnTRAF-2-transfected HT-29, but not in control Ad5LacZ-infected, cells. Surprisingly, IL-1 beta-mediated IL-8 gene expression was also inhibited in HT-29 cells as measured by Northern blot and ELISA. We concluded that TRAF-2 is partially involved in TNF-alpha-mediated signaling through I kappa B/NF-kappa B in IEC. In addition, our data suggest that TRAF-2 is involved in IL-1 beta signaling in HT-29 cells. Manipulation of cytokine signaling pathways represents a new approach for inhibiting proinflammatory gene expression in IEC.     

Coinfection with Schistosoma mansoni is associated with decreased HIV-specific cytolysis and increased IL-10 production

Impaired virus-specific immune responses have previously been observed with Schistosoma mansoni coinfection. We characterized Gag-specific responses in HIV-1-positive Ugandans with and without S. mansoni coinfection. We observed no significant difference in the frequency of IFN-gamma CD8+ T cells between the two groups. Interestingly, expression of CD107, a marker for cytolytic activity, was significantly lower in volunteers with S. mansoni coinfection compared with those with HIV-1 infection alone (p = 0.002). In contrast, the frequency of IL-10-positive Gag-specific CD8+ T cell responses was higher in volunteers with S. mansoni coinfection (p = 0.004). Analysis of human CMV-specific CD8+ T cell responses in the same individuals failed to reveal a similar pattern of altered CD107 and IL-10 expression. Our results suggest that S. mansoni coinfection is associated with decreased Gag-specific CD8+ cytolytic T cell responses and increased number of Gag-specific IL-10 positive CD8+ T cells. Our findings may have important implications toward the implementation of HIV preventive and therapeutic programs in Africa.     

Morphine reciprocally regulates IL-10 and IL-12 production by monocyte-derived human dendritic cells and enhances T cell activation

We evaluated the effect of morphine on human dendritic cells (DCs). Interestingly, immature DCs were found to express all 3 (mu, kappa, delta) opioid receptors on the cell surface. Chronic morphine treatment (10(-8) to 10(-12) M) during the development of DCs from monocytes augmented LPS-induced upregulation of HLA-DR, CD86, CD80, and CD83 and increased the T cell stimulatory capacity of DCs, which could be inhibited by naloxone, an opioid receptor antagonist. The change in surface phenotype was paralleled by a p38 MAPK-dependent decrease in IL-10 and increase in IL-12 secretion. Our data indicate that morphine exerts an immunostimulatory effect by modulating LPS-induced DC maturation.     

Syk negatively regulates TLR4-mediated IFNβ and IL-10 production and promotes inflammatory responses in dendritic cells

BackgroundWhile Syk has been shown to associate with TLR4, the immune consequences of Syk–TLR interactions and related molecular mechanisms are unclear.MethodsGain- and loss-of-function approaches were utilized to determine the regulatory function of Syk and elucidate the related molecular mechanisms in TLR4-mediated inflammatory responses. Cytokine production was measured by ELISA and phosphorylation of signaling molecules determined by Western blotting.ResultsSyk deficiency in murine dendritic cells resulted in the enhancement of LPS-induced IFNβ and IL-10 but suppression of pro-inflammatory cytokines (TNFα, IL-6). Deficiency of Syk enhanced the activity of PI3K and elevated the phosphorylation of PI3K and Akt, which in turn, lead to the phospho-inactivation of the downstream, central gatekeeper of the innate response, GSK3β. Inhibition of PI3K or Akt abrogated the ability of Syk deficiency to enhance IFNβ and IL-10 in Syk deficient cells, confirmed by the overexpression of Akt (Myr–Akt) or constitutively active GSK3β (GSK3 S9A). Moreover, neither inhibition of PI3K–Akt signaling nor neutralization of de novo synthesized IFNβ could rescue TNFα and IL-6 production in LPS-stimulated Syk deficient cells. Syk deficiency resulted in decreased phosphorylation of IKKβ and the NF-κB p65 subunit, further suggesting a divergent influence of Syk on pro- and anti-inflammatory TLR responses.ConclusionsSyk negatively regulates TLR4-mediated production of IFNβ and IL-10 and promotes inflammatory responses in dendritic cells through divergent regulation of downstream PI3K–Akt and NF-κB signaling pathways.General significanceSyk may represent a novel target for manipulating the direction or intensity of the innate response, depending on clinical necessity.     

Tyk2 negatively regulates adaptive Th1 immunity by mediating IL-10 signaling and promoting IFN-gamma-dependent IL-10 reactivation

The Jak, Tyk2, is activated in response to IL-12 and IFN-alphabeta and promotes IFN-gamma production by Th1-type CD4 cells. Mice deficient in Tyk2 function have been previously shown to be resistant to autoimmune arthritis and septic shock but are acutely susceptible to opportunistic pathogens such as Toxoplasma gondii. In this study, we show that Tyk2, in addition to mediating the biological effects of IL-12 and IFN-alphabeta, is an important regulator for the signaling and expression of the immunosuppressive cytokine IL-10. In the absence of Tyk2, Ag-reactive CD4 cells exhibit impaired IL-10 synthesis following rechallenge of T. gondii vaccine-primed mice. The impaired IL-10 reactivation leads to unopposed antimicrobial effector mechanisms which results in a paradoxically superior protection of immune Tyk2(-/-) mice against virulent T. gondii challenge. We further demonstrate that Tyk2 indirectly controls CD4 IL-10 reactivation by signaling for maximal IFN-gamma secretion. The unexpected role of IFN-gamma in mediating IL-10 reactivation by Th1 cells provides compelling evidence that conditions driving Th1 responses establish a negative feedback loop, which will ultimately lead to its autoregulation. Thus, Tyk2 can be viewed as a dual-function Jak, mediating both pro and anti-inflammatory cytokine responses.     

Tripartite-motif protein 30 negatively regulates NLRP3 inflammasome activation by modulating reactive oxygen species production

The NLR family, pyrin domain-containing 3 (NLRP3) inflammasome is critical for caspase-1 activation and the proteolytic processing of pro-IL-1β. However, the mechanism that regulates NLRP3 inflammasome activation remains unclear. In this paper, we demonstrate that tripartite-motif protein 30 (TRIM30) negatively regulates NLRP3 inflammasome activation. After stimulation with ATP, an agonist of the NLRP3 inflammasome, knockdown of TRIM30 enhanced caspase-1 activation and increased production of IL-1β in both J774 cells and bone marrow-derived macrophages. Similarly with ATP, knockdown of TRIM30 increased caspase-1 activation and IL-1β production triggered by other NLRP3 inflammasome agonists, including nigericin, monosodium urate, and silica. Production of reactive oxygen species was increased in TRIM30 knockdown cells, and its increase was required for enhanced NLRP3 inflammasome activation, because antioxidant treatment blocked excess IL-1β production. Conversely, overexpression of TRIM30 attenuated reactive oxygen species production and NLRP3 inflammasome activation. Finally, in a crystal-induced NLRP3 inflammasome-dependent peritonitis model, monosodium urate-induced neutrophil flux and IL-1β production was reduced significantly in TRIM30 transgenic mice as compared with that in their nontransgenic littermates. Taken together, our results indicate that TRIM30 is a negative regulator of NLRP3 inflammasome activation and provide insights into the role of TRIM30 in maintaining inflammatory responses.     

Distinct pathways for NF-kappa B regulation are associated with aberrant macrophage IL-12 production in lupus- and diabetes-prone mouse strains

One characteristic of mice prone to a variety of autoimmune diseases is the aberrant regulation of cytokine production by macrophages (Mphi), noted in cells isolated well before the onset of disease. Strikingly, the pattern of IL-12 dysregulation, in particular, is consistent with the nature of the autoimmune disease that will develop in each strain, i.e., elevated in mice prone to Th1-mediated organ-specific disease (nonobese diabetic (NOD) and SJL mice) and reduced in lupus-prone strains (MRL/+ and NZB/W). Mechanistically, the abnormal regulation of IL-12 in these strains was found to be strictly associated with novel patterns of Rel binding in vitro to the unique NF-kappaB site in the IL-12 p40 promoter. In this study, we report several new findings related to these Rel-kappaB interactions. Evaluation of the p40 NF-kappaB site in vivo, assessed by chromatin immunoprecipitation, revealed Rel usage patterns similar to those found in vitro using EMSA, with preferential association of the p40 kappaB site with c-Rel in NOD Mphi but with p50 in NZB/W Mphi. Moreover, blocking c-Rel in primary Mphi, using short interfering RNA, selectively blocked IL-12 production and normalized the minimal, residual IL-12 levels. Nuclear extracts from NOD Mphi were characterized by c-Rel hyperphosphorylation, and dephosphorylation of nuclear proteins completely blocked binding to the kappaB site. In contrast, elevated IkappaB appears to be a likely mechanism accounting for the reduced nuclear c-Rel levels noted in NZB/W Mphi. Alterations in NF-kappaB metabolism thus appear to define a pathway regulating intrinsic IL-12 defects in both diabetes- and lupus-prone strains.     

IL-1beta stimulates IL-6 production in cultured skeletal muscle cells through activation of MAP kinase signaling pathway and NF-kappa B

Recent studies suggest that the skeletal muscle may be a significant site of IL-6 production in various conditions, including exercise, inflammation, hypoperfusion, denervation, and local muscle injury. The mediators and molecular mechanisms regulating muscle IL-6 production are poorly understood. We tested the hypothesis that IL-6 production in muscle cells is regulated by IL-1beta and that mitogen-activated protein (MAP) kinase signaling and NF-kappaB activation are involved in IL-1beta-induced IL-6 production. Cultured C2C12 cells, a mouse skeletal muscle cell line, were treated with different concentrations (0.1-2 ng/ml) of IL-1beta in the absence or presence of the p38 MAP kinase inhibitor SB-208350 or the p42/44 inhibitor PD-98059. Protein and gene expression of IL-6 were determined by ELISA and real-time PCR, respectively. NF-kappaB DNA binding activity was determined by electrophoretic mobility shift assay and by transfecting myocytes with a luciferase reporter plasmid containing a promoter construct with multiple repeats of NF-kappaB binding site. Treatment of myotubes with IL-1beta resulted in a dose- and time-dependent increase of IL-6 production accompanied by an approximately 25-fold increase in IL-6 mRNA levels. IL-1beta stimulated NF-kappaB DNA binding activity and gene activation. SB-208350 and PD-98059 inhibited the increase in IL-6 production induced by IL-1beta. The present results support the concept that skeletal muscle is an important site of IL-6 production. In addition, the results suggest the IL-1beta regulates muscle IL-6 production at least in part by activating the MAP kinase pathway and NF-kappaB.     

Polycomb group protein Bmi1 negatively regulates IL-10 expression in activated macrophages

Macrophages exert a wide variety of functions, which necessitate a high level of plasticity on the chromatin level. In the work presented here, we analyzed the role of the polycomb group protein Bmi1 during the acute response of bone marrow derived macrophages (BMDM) to lipopolysaccharide (LPS). Unexpectedly, we observed that Bmi1 was rapidly induced at the protein level and transiently phosphorylated upon LPS treatment. The induction of Bmi1 was dependent on MAP-kinase signaling. LPS treatment of BMDM in the absence of Bmi1 resulted in a pronounced increase in expression of the anti-inflammatory cytokine interleukin-10 (IL-10). Our results identify Bmi1 as a repressor of IL-10 expression during macrophage activation.