Arjunarishta alleviates experimental colitis via suppressing proinflammatory cytokine expression,modulating gut microbiota and enhancing antioxidant effect

Molecular Biology Reports - Traditional ayurvedic medicine, Arjunarishta (AA) is used to treat several inflammatory conditions including dysentery associated with blood. The formulation is a...     

Characterization of proinflammatory cytokine production and CD14 expression by murine alveolar macrophage cell lines

Summary Alveolar macrophages, which play a central role in lung defense, produce cytokines that help orchestrate local inflammatory responses. In sepsis and other pathological conditions, bacterial lipopolysaccharide endotoxin can induce alveolar macrophages (AM) to release proinflammatory cytokines, including tumor necrosis factor-alpha, interleukin-1, and interleukin-6. Studying the mechanisms that control alveolar macrophage cytokine production may lead to better therapies for conditions involving inflammatory lung injury. We and others have noted significant differences between alveolar macrophages and peritoneal macrophages, but large numbers of human or murine alveolar macrophages are rarely available for detailed mechanistic studies. We have obtained three murine alveolar macrophage cell lines (AMJ2C8, AMJ2C11, and AMJ2C20) and have begun to characterize their cytokine responses to proinflammatory stimuli. We measured the effects of endotoxin, interferon gamma, and the combination of the two on production of tumor necrosis factor, interleukin-1 beta, and interleukin-6 in each line. We also studied the expression of the endotoxin receptor CD14 by these cells, and investigated the effect of serum on their endotoxin responsiveness. We show here that all three of the cell lines responded in a manner comparable to that of primary murine alveolar macrophages. Observed variations between these lines may reflect the documented heterogeneity seen in populations of primary alveolar macrophages. These cell lines should expand the repertoire of tools available to investigators studying regulation of murine alveolar macrophage responses.     

An IFN-gamma-independent proinflammatory role of IL-18 in murine streptococcal cell wall arthritis

IL-18 is a member of the IL-1 family of proteins that exerts proinflammatory effects. It was formally known as IFN-gamma-inducing factor and is a pivotal cytokine for the development of Th1 responses. Apart from Th1 immune-stimulatory activity, IL-18 induces the production of proinflammatory cytokines such as TNF-alpha and IL-1 in vitro. The goal was to investigate the role of endogenous IL-18 in murine streptococcal cell wall (SCW)-induced arthritis. Furthermore, we investigated whether IL-18 neutralization had an impact on local TNF and IL-1 production. C57BL/6, BALB/c, and IFN-gamma-deficient mice were injected with 2 mg of rabbit anti-murine IL-18 Abs shortly before induction of arthritis by intra-articular injection of 25 microg of SCW fragments into the right knee joint. Suppression of joint swelling was noted on days 1 and 2 of SCW arthritis after blockade of endogenous IL-18. Analysis of local cytokine concentrations showed that IL-18, TNF-alpha, and IL-1ss levels were decreased. Severe inhibition of chondrocyte proteoglycan synthesis was seen in the vehicle-treated control animals, whereas a reversal of the inhibition of chondrocyte proteoglycan synthesis was found in the anti-IL-18-exposed animals. Blockade of endogenous IL-18 in IFN-gamma-deficient mice showed results similar to those found in wild-type animals, identifying a role for IL-18 that is IFN-gamma independent. The present study indicates that IL-18 is a proinflammatory cytokine during the onset of murine SCW arthritis, and this inflammatory role of IL-18 is IFN-gamma independent.     

The apolipoprotein E-mimetic peptide COG112 inhibits NF-kappaB signaling, proinflammatory cytokine expression, and disease activity in murine models of colitis

Inflammatory bowel disease (IBD), consisting of Crohn's disease and ulcerative colitis, is a source of substantial morbidity and remains difficult to treat. New strategies for beneficial anti-inflammatory therapies would be highly desirable. Apolipoprotein (apo) E has immunomodulatory effects and synthetically derived apoE-mimetic peptides are beneficial in models of sepsis and neuroinflammation. We have reported that the antennapedia-linked apoE-mimetic peptide COG112 inhibits the inflammatory response to the colitis-inducing pathogen Citrobacter rodentium in vitro by inhibiting NF-κB activation. We now determined the effect of COG112 in mouse models of colitis. Using C. rodentium as an infection model, and dextran sulfate sodium (DSS) as an injury model, mice were treated with COG112 by intraperitoneal injection. With C. rodentium, COG112 improved the clinical parameters of survival, body weight, colon weight, and histologic injury. With DSS, COG112 ameliorated the loss of body weight, reduction in colon length, and histologic injury, whether administered concurrently with induction of colitis, during induction plus recovery, or only during the recovery phase of disease. In both colitis models, COG112 inhibited colon tissue inducible nitric-oxide synthase (iNOS), KC, TNF-α, IFN-γ, and IL-17 mRNA expression, and reduced nuclear translocation of NF-κB, as determined by immunoblot and immunofluorescence confocal microscopy. IκB kinase (IKK) activity was also reduced, which is necessary for activation of the canonical NF-κB pathway. Isolated colonic epithelial cells exhibited marked attenuation of expression of iNOS and the CXC chemokines KC and MIP-2. These studies indicate that apoE-mimetic peptides such as COG112 are novel potential therapies for IBD.     

Acute ethanol administration profoundly alters poly I:C-induced cytokine expression in mice by a mechanism that is not dependent on corticosterone

Polyinosinic polycytidylic acid (poly I:C) is an analog of double stranded RNA, which is a common replication intermediate for many viruses. It acts through a toll-like receptor (TLR3) to induce a group of cytokines that can mediate host resistance to viruses and some cancers. The effect of ethanol (EtOH) on induction of this set of cytokines has not been determined. Mice were treated with a single dose of EtOH (by gavage) at the same time as poly I:C was administered (intraperitoneally), and cytokine mRNA expression was measured by RNAse protection assay. Concentrations of IFN-alpha, IL-10, and IL-12 in the serum were measured by ELISA. A single dose of EtOH suppressed induction of mRNA for IFN-alpha, IFN-beta, IFN-gamma, IL-6, IL-9, IL-12, and IL-15. The concentrations of IFN-alpha and IL-12 in the serum were also decreased. In contrast, IL-10 was minimally induced by poly I:C alone, but it was substantially induced by poly I:C plus EtOH. Dose response and time course studies demonstrated that significant alterations of IFN-alpha, IL-10, and IL-12 expression occurred at dosages as low as 4 g/kg (a dosage previously shown to produce blood EtOH concentrations of approximately 0.2%) and that alterations persisted at least 4-6 hr after administration of EtOH. The glucocorticoid synthesis inhibitor, aminoglutethimide, diminished corticosterone levels to normal, but did not block the effects of EtOH on cytokine expression. These results demonstrate that EtOH affects the expression of poly I:C-induced cytokines and that this action is not mediated by corticosterone. These results plus previously published findings are consistent with the idea that EtOH may be a generalized suppressor of toll-like receptor signaling.     

IL-20, an anti-angiogenic cytokine that inhibits COX-2 expression

COX-2 overexpression and subsequent PGE(2) production are frequently associated with non-small cell lung cancer and are implicated in tumor-mediated angiogenesis. Here, we report for the first time that IL-20 downregulates COX-2 and PGE(2) in human bronchial epithelial and endothelial cells. Flow cytometry analysis suggests that IL-20-dependent inhibition of COX-2/PGE(2) occurs through the IL-22R1/IL-20R2 dimers. In addition, we report that IL-20 exerts anti-angiogenic effects, inhibiting experimental angiogenesis. IL-20-mediated inhibition of PMA-induced angiogenesis occurs through the COX-2 regulatory pathway. Altogether our findings revealed that IL-20 is a negative modulator of COX-2/PGE(2) and inhibits angiogenesis.     

Suppression of proinflammatory cytokine expression by herpes simplex virus type 1

Viral immune evasion strategies are important for establishment and maintenance of infections. Many viruses are in possession of mechanisms to counteract the antiviral response raised by the infected host. Here we show that a herpes simplex virus type 1 (HSV-1) mutant lacking functional viral protein 16 (VP16)-a tegument protein promoting viral gene expression-induced significantly higher levels of proinflammatory cytokines than wild-type HSV-1. This was observed in several cell lines and primary murine macrophages, as well as in peritoneal cells harvested from mice infected in vivo. The enhanced ability to stimulate cytokine expression in the absence of VP16 was not mediated directly by VP16 but was dependent on the viral immediate-early genes for infected cell protein 4 (ICP4) and ICP27, which are expressed in a VP16-dependent manner during primary HSV infection. The virus appeared to target cellular factors other than interferon-induced double-stranded RNA-activated protein kinase R (PKR), since the virus mutants remained stronger inducers of cytokines in cells stably expressing a dominant-negative mutant form of PKR. Finally, mRNA stability assay revealed a significantly longer half-life for interleukin-6 mRNA after infection with the VP16 mutant than after infection with the wild-type virus. Thus, HSV is able to suppress expression of proinflammatory cytokines by decreasing the stability of mRNAs, thereby potentially impeding the antiviral host response to infection.     

IL-6 is required for the development of Th1 cell-mediated murine colitis

Proinflammatory cytokines have been demonstrated to play a crucial role in the pathogenesis of Crohn's disease. Among those cytokines, strong expression of IL-6 has been repeatedly demonstrated. To examine the role for IL-6 in the pathogenesis of Crohn's disease, we introduced anti-IL-6R mAb to a murine model of colitis. Colitis was induced in C.B-17-scid mice transferred with CD45RBhigh CD4+ T cells from BALB/c mice. Anti-IL-6R mAb or rat IgG was administered weekly after T cell transfer. ICAM-1 and VCAM-1 expression were analyzed by immunohistochemistry. Colonic cytokine expression was determined by RT-PCR. Mice treated with mAb showed normal growth, whereas controls lost weight. The average colitis score was 0.64 for mAb-treated mice and 1.80 for controls. T cell expansion in treated mice was less remarkable than in the controls. Colonic ICAM-1 and VCAM-1 expression were markedly suppressed by mAb. IFN-gamma, TNF-alpha, and IL-1beta mRNA were reduced by the treatment. The results presented here show a crucial role for IL-6 in the pathogenesis of murine colitis and suggest a therapeutic potential of anti-IL-6R mAb for treatment of human Crohn's disease.     

ZAK: a MAP3Kinase that transduces Shiga toxin- and ricin-induced proinflammatory cytokine expression

Shiga toxins (Stxs) and ricin initiate damage to host cells by cleaving a single adenine residue on the α-sarcin loop of the 28S ribosomal RNA. This molecular insult results in a cascade of intracellular events termed the ribotoxic stress response (RSR). Although Stxs and ricin have been shown to cause the RSR, the mitogen-activated protein kinase kinase kinase (MAP3K) that transduces the signal from intoxicated ribosomes to activate SAPKinases has remained elusive. We show in vitro that DHP-2 (7-[3-fluoro-4-aminophenyl-(4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl))]-quinoline), a zipper sterile-α-motif kinase (ZAK)-specific inhibitor, blocks Stx2/ricin-induced SAPKinase activation. Treatment of cells with DHP-2 also blocks Stx2/ricin-mediated upregulation of the proinflammatory cytokine interleukin-8 and results in a modest but statistically significant improvement in cell viability following Stx2/ricin treatment. Finally we show that siRNA directed against the N-terminus of ZAK diminishes Stx2/Ricin-induced SAPKinase activation. Together, these data demonstrate that a ZAK isoform(s) is the MAP3Kinase that transduces the RSR. Therefore, ZAKα and/or β isoforms may act as potential therapeutic target(s) for treating Stx/ricin-associated illnesses. Furthermore, a small molecule inhibitor like DHP-2 may prove valuable in preventing the Stx/ricin-induced proinflammatory and/or apoptotic effects that are thought to contribute to pathogenesis by Stx-producing Escherichia coli and ricin.     

Role of IL-10 in regulating proinflammatory cytokine release by Kupffer cells following trauma-hemorrhage

IL-6 and TNF-alpha production by Kupffer cells is markedly stimulated following trauma-hemorrhage (T-H). Because IL-10 is an anti-inflammatory cytokine, the aim of this study was to determine whether IL-10 regulates Kupffer cell proinflammatory cytokine release following T-H. To study this, we subjected adult male Sprague-Dawley rats to sham operation or T-H. The procedure involved a 5-cm midline laparotomy and approximately 90 min of hemorrhagic shock (35 mmHg), followed by resuscitation with four times the shed blood volume in the form of Ringer's lactate. At 2 h after the end of resuscitation, livers were perfused in vitro and perfusate was collected. In separate studies, Kupffer cells were isolated and incubated with different concentrations of anti-IL-10 MAb. IgG was used as control. After 16 h of incubation, IL-6 and TNF-alpha levels were measured by ELISA. Plasma IL-10 levels increased significantly following T-H. IL-10 levels in the perfusate and IL-10 production by cultured Kupffer cells were also significantly higher in the T-H group. When Kupffer cells were incubated with 10 microg/ml of anti-IL-10 MAb, IL-6 and TNF-alpha production were significantly increased in both sham and T-H groups compared with those not treated with anti-IL-10 MAb. However, these changes were not observed when the cells were incubated with irrelevant (control) IgG. These results indicate that IL-10 production by Kupffer cells early after T-H may play a pivotal role in attenuating the proinflammatory cytokine environment, possibly in an autocrine/paracrine manner.     

High-fat-diet-associated intestinal microbiota exacerbates psoriasis-like inflammation by enhancing systemic γδ T cell IL-17 production

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Gamma delta-T cells are critical for survival and early proinflammatory cytokine gene expression during murine Klebsiella pneumonia

Although cells of the innate inflammatory response, such as macrophages and neutrophils, have been extensively studied in the arena of Gram-negative bacterial pneumonia, a role for T cells remains unknown. To study the role of specific T cell populations in bacterial pneumonia, mice deleted of their TCR beta- and/or delta-chain were intratracheally inoculated with Klebsiella pneumoniae. Gamma delta T cell knockout mice displayed increased mortality at both early and late time points. In contrast, mice specifically lacking only alpha beta-T cells were no more susceptible than wild-type mice. Pulmonary bacterial clearance in gamma delta-T cell knockout mice was unimpaired. Interestingly, these mice displayed increased peripheral blood dissemination. Rapid up-regulation of IFN-gamma and TNF-alpha gene expression, critical during bacterial infections, was markedly impaired in lung and liver tissue from gamma delta-T cell-deficient mice 24 h postinfection. The increased peripheral blood bacterial dissemination correlated with impaired hepatic bacterial clearance following pulmonary infection and increased hepatic injury as measured by plasma aspartate aminotransferase activity. Combined, these data suggest that mice lacking gamma delta-T cells have an impaired ability to resolve disseminated bacterial infections subsequent to the initial pulmonary infection. These data indicate that gamma delta-T cells comprise a critical component of the acute inflammatory response toward extracellular Gram-negative bacterial infections and are vital for the early production of the proinflammatory cytokines IFN-gamma and TNF-alpha.     

IL-12/IL-18-dependent IFN-gamma release by murine dendritic cells.

Dendritic cells (DC) develop in GM-CSF-stimulated cultures from murine bone marrow progenitors in serum-free (or low serum) medium. CD11c(+) myeloid DC from 7-day cultures stimulated with TNF-alpha, IFN-alpha, IFN-gamma, or LPS up-regulated surface expression of CD40 and CD86 costimulator and MHC class II molecules, did not up-regulate the low "spontaneous" release of IL-18, and did not release IFN-gamma. Stimulation of in vitro-generated DC with exogenous IL-12 and IL-18 (but not with IL-4 or LPS plus IL-18) induced IFN-gamma expression and release in 15-20% of the DC (detectable by FACS analyses or ELISA). Endogenous IL-12 p70 produced by DC in response to ligation of CD40 stimulated IFN-gamma release when exogenous IL-18 was supplied. In vivo-generated, splenic CD8alpha(+) and CD8alpha(-) DC (from immunocompetent and immunodeficient H-2(d) and H-2(b) mice) cultured with IL-12 and IL-18 released IFN-gamma. The presence of LPS during the stimulation of DC with IL-18 plus endogenous (CD40 ligation) or exogenous IL-12 did not affect their IFN-gamma release. In contrast, splenic DC pretreated in vitro or in vivo by LPS strikingly down-regulated IFN-gamma release in response to stimulation by IL-18 and (endogenous or exogenous) IL-12. Hence, DC are a source of early IFN-gamma generated in response to a cascade of cytokine- and/or cell-derived signals that can be positively and negatively regulated.     

Allergen-induced airway hyperresponsiveness mediated by cyclooxygenase inhibition is not dependent on 5-lipoxygenase or IL-5, but is IL-13 dependent

Cyclooxygenase (COX) inhibition during allergic sensitization and allergen airway challenge results in augmented allergic inflammation. We hypothesized that this increase in allergic inflammation was dependent on increased generation of leukotrienes that results from COX inhibition, as leukotrienes are important proinflammatory mediators of allergic disease. To test this hypothesis, we allergically sensitized and challenged mice deficient in 5-lipoxygenase (5-LO). We found that 5-LO knockout mice that were treated with a COX inhibitor during allergic sensitization and challenge had significantly increased airway hyperresponsiveness (AHR) (p < 0.01) and airway eosinophilia (p < 0.01) compared with 5-LO knockout mice that were treated with vehicle. The proinflammatory cytokines have also been hypothesized to be critical regulators of airway inflammation and AHR. We found that the increase in airway eosinophilia seen with COX inhibition is dependent on IL-5, whereas the increase in AHR is not dependent on this cytokine. In contrast, the COX inhibition-mediated increase in AHR is dependent on IL-13, but airway eosinophilia is not. These results elucidate the pathways by which COX inhibition exerts a critical effect of the pulmonary allergen-induced inflammatory response and confirm that COX products are important regulators of allergic inflammation.     

Constitutive expression of IL-18 binding protein in murine testicular tissues and cells

In the present study, we examined the cellular origin and the expression levels of interleukin-18 binding protein (IL-18 BP), during normal maturation of murine testis. Immunohistochemical staining of testicular tissues from sexually mature mice, shows that testicular germ cells, at different stages of differentiation, express IL-18 BP. Leydig cells/interstitial cells and peritubular cells express higher levels of IL-18 BP, as compared to other testicular cells. The levels of IL-18 BP were similar in testicular tissues and homogenates from sexually immature and mature mice, as examined by western blot, ELISA and real time PCR analysis. Our results demonstrate, for the first time, the expression of IL-18 BP in murine testicular tissues and cells. Together with our previous studies, which showed over-expression of IL-18 in testicular tissues of immature mice as compared with mature mice, these results may indicate a role for IL-18 in testicular development, and also in the regulation of testicular functions under physiological conditions.     

Synergistic effect of IL-2, IL-12, and IL-18 on thymocyte apoptosis and Th1/Th2 cytokine expression

In the periphery, IL-18 synergistically induces the expression of the Th1 cytokine IFN-gamma in the presence of IL-12 and the Th2 cytokines IL-5 and IL-13 in the presence of IL-2. Although the expression of these cytokines has been described in the thymus, their role in thymic development and function remains uncertain. We report here that freshly isolated thymocytes from C57BL/6 and BALB/c mice stimulated in vitro with IL-2-plus-IL-18 or IL-12-plus-IL-18 produce large amounts of IFN-gamma and IL-13. Analysis of the thymic subsets, CD4(-)CD8(-) (DN), CD4(+)CD8(+), CD4(+)CD8(-), and CD4(-)CD8(+) revealed that IL-18 in combination with IL-2 or IL-12 induces IFN-gamma and IL-13 preferentially from DN cells. Moreover, DN2 and DN3 thymocytes contained more IFN-gamma(+) cells than cells in the later stage of maturation. Additionally, IL-18 in combination with IL-2 induces CCR4 (Th2-associated) and CCR5 (Th1-associated) gene expression. In contrast, IL-18-plus-IL-12 specifically induced CCR5 expression. The IL-2-plus-IL-18 or IL-12-plus-IL-18 effect on IFN-gamma and IL-13 expression is dependent on Stat4 and NF-kappaB but independent of Stat6, T-bet, or NFAT. Furthermore, IL-12-plus-IL-18 induces significant thymocyte apoptosis when expressed in vivo or in vitro, and this effect is exacerbated in the absence of IFN-gamma. IL-12-plus-IL-18-stimulated thymocytes can also induce IA-IE expression on cortical and medullary thymic epithelial cells in an IFN-gamma-dependent manner. Thus, the combination of IL-2, IL-12, and IL-18 can induce phenotypic and functional changes in thymocytes that may alter migration, differentiation, and cell death of immature T cells inside the thymus and potentially affect the Th1/Th2 bias in peripheral immune compartments.