mRNA Expression Of Anti-inflammatory Cytokines Research Articles

Molecular characterization of an interleukin-4/13B homolog in grass carp (Ctenopharyngodon idella) and its role in fish against Aeromonas hydrophila infection

Mammalian interleukin 4 (IL-4) and interleukin 13 (IL-13) molecules are anti-inflammatory cytokines mediating the alternative activation of macrophages. However, the role of fish IL-4/13 homologs in monocytes/macrophages (MO/MФ) polarization remains unclear. In this study, we have functionally identified an IL-4/13B homolog in grass carp (Ctenopharyngodon idella), which is termed as CiIL-4/13B. Multiple alignment showed that CiIL-4/13B shared the typical characteristics and structure with other known fish IL-4/13. Phylogenetic analysis showed that CiIL-4/13B is evolutionarily closely related to zebrafish (Danio rerio) and common carp (Cyprinus carpio) IL-4/13B. CiIL-4/13B mRNA was constitutively expressed in tissues and peripheral blood lymphocytes (PBLs) examined, with its highest expression seen in PBLs. Following Aeromonas hydrophila infection, CiIL-4/13B mRNA expression was upregulated. Recombinant CiIL-4/13B (rCiIL-4/13B) was overexpressed in Escherichia coli and purified for a functional study. Using prepared anti-rCiIL-4/13B antiserum, Western blot analysis showed that native CiIL-4/13B in grass carp plasma is N-glycosylated. Intraperitoneal injection of bioactive rCiIL-4/13B significantly increased the survival rate of grass carp against A. hydrophila, and decreased the tissue bacterial load, with a higher dose having better effects. Bioactive rCiIL-4/13B treatment decreased nitrite production and mRNA expression of proinflammatory cytokines (IL-1β and TNF-α), while it increased arginase activity and mRNA expression of anti-inflammatory cytokines (TGF-β and IL-10). The phagocytosis by grass carp MO/MФ had no significant changes by the 8 h treatment of bioactive rCiIL-4/13B compared to that of the negative control, while it was significantly inhibited by the 24 h treatment of bioactive rCiIL-4/13B. The inhibitory effect of rCiIL-4/13B on MO/MФ phagocytosis may be a consequence of MO/MФ proliferation. In summary, our results suggest that CiIL-4/13B plays a protective effect in grass carp against A. hydrophila by inducing alternatively activated MO/MФ.

Screening and Characterization of Lactobacillus casei MCL Strain Exhibiting Immunomodulation Activity

As an appraisal for the application of a new starter culture, more than 200 lactic acid bacteria strains were isolated from raw milk and healthy human feces. The strains showing excellent growth and acid production ability in 10% skim milk media were selected and identified as Lactobacillus casei based on the results of their API carbohydrate fermentation patterns, as well as 16S rDNA sequence analysis. To assess the effect of L. casei strains on irritable bowel disease (IBD), the inhibitory effect of the selected strains against the nitric oxide (NO) production of lipopolysaccharide (LPS)-stimulated RAW 264.7 cells was measured. Among the tested L. casei strains, L. casei MCL was observed to have the greatest NO inhibitory activity. Additionally, L. casei MCL was found to inhibit mRNA expression of pro-inflammatory cytokines (interleukin-<TEX>$1{\beta}$</TEX>, IL-6, TNF-<TEX>${\alpha}$</TEX>), as well as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) involved in pathophysiologic processes such as inflammation. The mRNA expression of anti-inflammatory cytokines, including IL-10 and transforming growth factor-<TEX>$1{\beta}$</TEX> (TGF-<TEX>${\beta}$</TEX>) of L. casei MCL, was confirmed using quantitative real-time PCR. In conclusion, L. casei MCL showed decreases in the expression of pro-inflammatory cytokines and up-regulated expression of the anti-inflammatory cytokine.

Ethyl Pyruvate Downregulates Tumor Necrosis Factor Alpha and Interleukin (IL)-6 and Upregulates IL-10 in Lipopolysaccharide-Stimulated Canine Peripheral Blood Mononuclear Cells

We investigated the anti-inflammatory effects of ethyl pyruvate (EP) on LPS-stimulated canine PBMCs in vitro. We found that EP treatment inhibited the mRNA expressions of proinflammatory cytokines (TNF-α and IL-6), but induced mRNA expression of anti-inflammatory cytokines (IL-10). ELISA measurements revealed that EP also effectively downregulates the LPS-induced increase in proinflammatory cytokine release, while upregulating anti-inflammatory cytokine release. These data indicate that EP could be an effective anti-inflammatory agent in dogs.

Inflammation and Foveolar Hyperplasia Are Reduced by Supplemental Dietary Glutamine during Helicobacter pylori Infection in Mice1–3

We recently showed that L-Gln protects cultured gastric cells from ammonia-induced cell death and predicted that Gln may also protect during Helicobacter pylori infection in vivo. Thus, the aim of this study was to test whether supplemental dietary Gln protects against H. pylori-associated pathology. For this, C57BL/6 mice were fed a purified diet consisting of 20.3% protein (1.9% Gln), 66% carbohydrate, and 5% fat or 25.3% protein (5% supplemental L-Gln; 6.9% total Gln), 61% carbohydrate, and 5% fat. After a 2-wk prefeeding period, mice were divided into sham-(uninfected) or H. pylori-infected groups. Body weight and food consumption were recorded weekly. Tissue histopathology, H. pylori colonization, serum IgG, and pro- and antiinflammatory cytokine mRNA expression were determined at 6, 12, and 20 wk postinfection (wkPI). Inflammation, antiinflammatory cytokine, and interleukin-1β mRNA expression were significantly greater at 6 wkPI in H. pylori-infected mice fed supplemental Gln compared with those fed the control diet. At 20 wkPI, however, inflammation and foveolar hyperplasia were significantly lower in H. pylori-infected mice fed supplemental Gln compared with those fed the control diet. Body weight gain, food consumption, H. pylori colonization, and serum IgG did not differ in H. pylori-infected mice fed supplemental Gln compared with the control diet. Our data demonstrate that H. pylori-infected mice fed supplemental dietary Gln have reduced H. pylori-associated pathology in vivo that is accompanied by beneficial changes in the immune response to H. pylori early in infection. Thus, Gln supplementation may be an alternative therapy for reducing H. pylori-associated pathology.

The immunomodulatory effect of lactulose on Enterococcus faecium fed preruminant calves1

Prebiotics and probiotics could represent an effective alternative to the use of synthetic antibiotics in nutrition. The mechanisms by which prebiotics affect the immune system have not yet been investigated in detail. Most effects have been attributed to increases in the innate and acquired immune responses. This study was conducted to elucidate the long-term effects of orally administered lactulose on the immune response in the intestinal tract of probiotic-fed calves. Preruminant calves were randomized to 3 feeding groups: milk replacer containing 1) no lactulose, 2) 1% lactulose, or 3) 3% lactulose. All 3 milk replacers contained 10(9) cfu Enterococcus faecium/kg. Messenger RNA expression of different cell activation markers, pro- and antiinflammatory cytokines, and IgA Fc receptor was investigated in the ileum, mesenterial lymph node, spleen, and white blood cells. A significantly greater number of blood lymphocytes were detected in the 3% lactulose group (P = 0.02) than in the control group. The expression results in male calves indicated that the transcription of IgA Fc receptor in the ileal mucosa of the 1% lactulose treatment group increased significantly (P = 0.04) and also tended to increase in the 3% lactulose group (P = 0.07). Furthermore, decreases in IL-10 and interferon-gamma mRNA expression were observed in the ileum (P = 0.04). The CD4-presenting lymphocytes were decreased significantly in the ileum (P = 0.04) and mesenteric lymph node (P = 0.01), whereas CD8-presenting lymphocytes were increased in the blood (P = 0.03) of females. Other proinflammatory cytokines (IL-1beta, IL-8, and tumor necrosis factor-alpha) and antiinflammatory cytokines (transforming growth factor-beta1) did not show significant differences in mRNA expression among treatment groups. The results indicate that additional lactulose feeding had an immunomodulatory effect on the composition of T-cell subsets in different immune compartments and had minor effects on pro- and antiinflammatory cytokine mRNA expression.

Effects of hydroxytyrosol‐20 on carrageenan‐induced acute inflammation and hyperalgesia in rats

Hydroxytyrosol (HT) is a simple phenol compound extracted from olive leaves. The content of HT in the studied preparation was about 20%, and the preparation was called hydroxytyrosol-20 (HT-20). HT has antioxidant and antiinflammatory activities. There has been no report so far on the efficacy of HT-20 in carrageenan-induced acute inflammation and hyperalgesia in rats. Therefore, the aim of this study was to assess the inhibitory role of HT-20 on carrageenan-induced swelling and hyperalgesia of rat paw. Paw inflammation was assessed by the increase in paw volume and hyperalgesia. The rat paws were cut out under ether anesthesia at 270 min after administration of carrageenan. The tissue of the right paw was isolated separately from the individual rat. The levels of the tumor necrosis factor-alpha (TNF-alpha), interleukin 1beta (IL-1beta) and interleukin 10 (IL-10) mRNA in the tissue were estimated by reverse transcription-polymerase chain reaction (RT-PCR). The results showed that the paw pressure thresholds of rats orally administered HT-20 significantly increased at 210, 240 and 270 min after administration of carrageenan, compared with corresponding basal paw pressure thresholds; the degree of swelling of the right hind paw showed a statistically significant reduction, compared with rats in the carrageenan-treated control. In this model, HT-20 appears to decrease pro-inflammatory cytokines IL-1beta and TNF-alpha and not to increase the antiinflammatory cytokine mRNA expression of IL-10.

Immunological and pathological changes in the placenta during infection with Listeria monocytogenes in pregnant guinea pigs

Exposure to Listeria monocytogenes during pregnancy can result in spontaneous abortion and stillbirths; however, the mechanisms are unknown. Our objective was to determine the effects of infection on specific inflammatory and anti-inflammatory cytokine mRNA expression and apoptosis in the placenta after infection with L. monocytogenes. Pregnant guinea pigs were treated on gestation day (gd) 35 with 10 8 colony forming units L. monocytogenes and sacrificed on gd 37, 41, 44, or 55. At gd 41, IFN-γ and IL-2 mRNA expression was significantly decreased in placentas from treated dams (0.0012-fold and 0.131-fold, respectively). At gd 55, TNF-α mRNA expression was significantly decreased (0.19-fold), while IFN-γ mRNA expression was significantly increased (32-fold), and apoptosis was detected in 100% of placentas from treated dams. In conclusion, inflammatory cytokine mRNA expression is altered and apoptosis is increased in the placenta after treatment with L. monocytogenes, and these changes may contribute to fetal death.

Liposomal IGF-1 gene transfer modulates pro- and anti-inflammatory cytokine mRNA expression in the burn wound.

The use of systemic IGF-1 has been shown to attenuate the postburn hypermetabolic response and improve burn wound healing. Local IGF-1 gene therapy, however, promotes re-epithelialization in the burn wound without the side-effects associated with systemic delivery. We tested the hypothesis that these beneficial effects are due to changes in local cytokine production. Adult male Sprague-Dawley rats received a 40% total body surface area full-thickness scald burn and randomly received a subcutaneous injection at the burn wound margin of saline or cationic liposomes containing a IGF-1 cDNA construct. Animals were killed at 1, 4, 7 and 10 days after burn trauma. Skin biopsies at the wound border were harvested for total RNA extraction. Cytokine mRNA expression was determined using a multi-probe RNase protection assay. Data are presented as means +/- s.e.m. Statistical analysis used the unpaired t-test or Mann-Whitney test where appropriate. Significance was accepted at P < 0.05. Treatment of the burn wound with liposomal IGF-1-cDNA transfer decreased IL-1beta mRNA levels on day 10 after burn trauma from five-fold burn-induced increases compared with sham-treated rats, to near the control values present in unburned skin samples. Similarly, there was an eight-fold increase in TNF-alpha mRNA expression on postburn day 10 that was abrogated by IGF-1 gene therapy. Local IGF-1 gene transfer attenuates the mRNA expression of the inflammatory cytokines IL-1beta and TNF-alpha in the burn wound. This change may improve burn wound healing by decreasing prolonged local inflammation.

Characterization of acute versus chronic relapsing autoimmune encephalomyelitis in DA rats

This study was undertaken to better understand the role of cytokines in the pathogenesis, especially in the mechanisms of relapse, of experimental autoimmune encephalomyelitis (EAE). For this purpose, we induced acute and chronic relapsing (CR) EAE in DA rats and determined several immunological parameters in rats at various stages of two types of EAE. Histopathological analysis revealed that there was no significant difference in the severity of inflammation in the spinal cord lesions between the two groups. However, demyelination was observed only in rats with CR EAE. Cytokine analysis by competitive PCR demonstrated that levels of TNF-α, IL-6 and IL-12 p40 mRNA in the spinal cord at the first attack of CR EAE were significantly higher than those at the peak stage of acute EAE. The mRNA expression of anti-inflammatory cytokines, IL-10 and TGF-β1, was generally low in both acute EAE and the first attack of CR EAE and upregulated at later stages of CR EAE. These findings suggest that persistent high-level expression of pro-inflammatory cytokines is closely associated with demyelination and relapse of EAE. In contrast, anti-inflammatory cytokines play only a minor role in the relapse.

Differential Expression of Inflammatory Cytokines Parallels Progression of Central Nervous System Pathology in Two Clinically Distinct Models of Multiple Sclerosis

Multiple sclerosis is an immune-mediated demyelinating disease of unknown etiology that presents with either a chronic-progressive or relapsing-remitting clinical course. Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) and relapsing-remitting experimental autoimmune encephalomyelitis (R-EAE) in the SJL/J mouse are both relevant murine CD4+ T cell-mediated demyelinating models that recapitulate the multiple sclerosis disease phenotypes. To determine the cellular and molecular basis for these observed differences in clinical course, we quantitatively analyzed the temporal expression of pro- and antiinflammatory cytokine mRNA expression in the central nervous system (CNS) and the phenotype of the inflammatory mononuclear infiltrates. TMEV-infected SJL/J mice expressed IFN-gamma, TNF-alpha, IL-10, and IL-4 mRNA during the preclinical phase, and their levels continued to increase throughout the duration of the chronic-progressive disease course. These data correlated with the continued presence of both CD4+ T cells and F4/80+ macrophages within the CNS infiltrates. In contrast, SJL/J mice with PLP(139-151)-induced R-EAE displayed a biphasic pattern of CNS expression for the proinflammatory cytokines, IFN-gamma and TNF-alpha, with expression peaking at the height of the acute phase and relapse(s). This pattern correlated with dynamic changes in the CD4+ T cell and F4/80+ macrophage populations during relapsing-remitting disease progression. Interestingly, IL-4 message was undetectable until disease remission(s), demonstrating its potential role in the intrinsic regulation of ongoing disease, whereas IL-10 was continuously expressed, arguing against a regulatory role in either disease. These data suggest that the kinetics of cytokine expression together with the nature of the persistent inflammatory infiltrates are major contributors to the differences in clinical course between TMEV-IDD and R-EAE.