Lower cytokine secretion ex vivo by natural killer T cells in HIV-infected individuals is associated with higher CD161 expression

Objective: Natural killer T (NKT) cells are efficiently targeted by HIV and severely reduced in numbers in the circulation of infected individuals. The functional capacity of the remaining NKT cells in HIV-infected individuals is poorly characterized. This study measured NKT cell cytokine production directly ex vivo and compared these responses with both the disease status and NKT subset distribution of individual patients. Methods: NKT cell frequencies, subsets, and ex-vivo effector functions were measured in the peripheral blood mononuclear cells of HIV-infected patients and healthy controls by flow cytometry. We measured cytokines from NKT cells after stimulation with either a-galactosyl ceramide-loaded CD1d dimers (DimerX-alpha GalCer) or phorbol myristate acetate and ionomycin. Results: The frequencies of NKT cells secreting interferon-gamma and tumor necrosis factor-alpha were significantly lower in HIV-infected patients than healthy controls after DimerX-alpha GalCer treatment, but responses were similar after treatment with phorbol myristate acetate and ionomycin. The magnitude of the interferon-gamma response to DimerX-alpha GalCer correlated inversely with the number of years of infection. Both interferon-gamma and tumor necrosis factor-alpha production in response to DimerX-alpha GalCer correlated inversely with CD161 expression. Conclusion: The ex-vivo Th1 responses of circulating NKT cells to CD1d-glycolipid complexes are impaired in HIV-infected patients. NKT cell functions may be progressively lost over time in HIV infection, and CD161 is implicated in the regulation of NKT cell responsiveness. (C) 2009 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins

Novel humanized anti-CD3 antibodies induce a predominantly immunoregulatory profile in human peripheral blood mononuclear cells

Strategies to minimize the immunogenicity and toxicity of murine anti-CD3 antibodies (e.g. OKT3) are of special interest for organ transplantation and for the treatment of autoimmune diseases. In the present work, we have developed two humanized anti-CD3 antibodies. These molecules were shown to bind to human CD3, though less efficiently, and display less mitogenic activity than CKT3. These results prompted us to investigate whether this reduced mitogenic potential was associated with the development of anti-inflammatory properties. Indeed, in peripheral blood mononuclear cells (PBMCs), the humanized antibody versions induced a predominantly anti-inflammatory cytokine profile, in contrast with the pro-inflammatory profile induced by OKT3. Neither OKT3 nor the humanized versions induced the expression of IL-4, IL-2 or TGF-beta. Both humanized antibodies induced significantly lower production of IFN-gamma and IL-5 and slightly higher production of IL-10 than OKT3. This immunomodulatory profile was most evident by the 80-fold higher ratio of IL-10/IFN-gamma production in PBMCs cultured in the presence of the humanized antibodies, compared to those stimulated with CKT3. Furthermore, these humanized anti-CD3 antibodies induced a late FOXP3 gene expression while OKT3 led to a more transient expression of FOXP3. Taken our results, we suggest that these humanized anti-CD3 antibodies may promote the development of T cells with immunoregulatory activity. (C) 2009 Elsevier B.V. All rights reserved.

In vivo electrochemical characterization and inflammatory response of multiwalled carbon nanotube-based electrodes in rat hippocampus

Stimulating neural electrodes are required to deliver charge to an environment that presents itself as hostile. The electrodes need to maintain their electrical characteristics (charge and impedance) in vivo for a proper functioning of neural prostheses. Here we design implantable multi-walled carbon nanotubes coating for stainless steel substrate electrodes, targeted at wide frequency stimulation of deep brain structures. In well-controlled, low-frequency stimulation acute experiments, we show that multi-walled carbon nanotube electrodes maintain their charge storage capacity (CSC) and impedance in vivo. The difference in average CSCs (n = 4) between the in vivo (1.111 mC cm(-2)) and in vitro (1.008 mC cm(-2)) model was statistically insignificant (p > 0.05 or P-value = 0.715, two tailed). We also report on the transcription levels of the pro-inflammatory cytokine IL-1 beta and TLR2 receptor as an immediate response to low-frequency stimulation using RT-PCR. We show here that the IL-1 beta is part of the inflammatory response to low-frequency stimulation, but TLR2 is not significantly increased in stimulated tissue when compared to controls. The early stages of neuroinflammation due to mechanical and electrical trauma induced by implants can be better understood by detection of pro-inflammatory molecules rather than by histological studies. Tracking of such quantitative response profits from better analysis methods over several temporal and spatial scales. Our results concerning the evaluation of such inflammatory molecules revealed that transcripts for the cytokine IL-1 beta are upregulated in response to low-frequency stimulation, whereas no modulation was observed for TLR2. This result indicates that the early response of the brain to mechanical trauma and low-frequency stimulation activates the IL-1 beta signaling cascade but not that of TLR2.

A BALB/c mouse model shows that liver involvement in dengue disease is immune-mediated

In the present study, BALB/c mice were used to develop a model for the hepatic injury associated to dengue infection. Histological analysis after subcutaneous inoculation with a low viral dose of dengue-2 virus showed Kupffer cell hyperplasia and an increased inflammatory cellular infiltrate next to the bile ducts on days 5, 7 and 14 post-inoculation, mainly characterized by the presence of mononuclear cells. The liver mRNA transcription level of IL-1 beta was highest on the 5th day post-infection (p.i.) and decreased by the 21st day, TNF-alpha showed a peak of mRNA transcription after 14 days p.i. coinciding with the regression of cellular infiltrates and elevated expression of TGF-beta mRNA. Serum AST and ALT levels were slightly elevated at 7 and 14 days post-infection. Dengue-2 RNA levels were undetectable in the liver on any of the days following inoculation. Our observations suggest that, as it is true for humans, the animals undergo a transient and slight liver inflammation, probably due to local cytokine production and cellular infiltration in the liver. (C) 2010 Elsevier Inc. All rights reserved.

IL-17 Receptor Signaling Is Required to Control Polymicrobial Sepsis

Sepsis is a systemic inflammatory response resulting from the inability of the host to contain the infection locally. Previously, we demonstrated that during severe sepsis there is a marked failure of neutrophil migration to the infection site, which contributes to dissemination of infection, resulting in high mortality. IL-17 plays an important role in neutrophil recruitment. Herein, we investigated the role of IL-17R signaling in polymicrobial sepsis induced by cecal ligation and puncture (CLP). It was observed that IL-17R-deficient mice, subjected to CLP-induced non-severe sepsis, show reduced neutrophil recruitment into the peritoneal cavity, spread of infection, and increased systemic inflammatory response as compared with C57BL/6 littermates. As a consequence, the mice showed an increased mortality rate. The ability of IL-17 to induce neutrophil migration was demonstrated in vivo and in vitro. Beside its role in neutrophil recruitment to the infection focus, IL-17 enhanced the microbicidal activity of the migrating neutrophils by a mechanism dependent on NO. Therefore, IL-17 plays a critical role in host protection during polymicrobial sepsis. The Journal of Immunology, 2009, 182: 7846-7854.

Role of cytokines (TNF-alpha, IL-1 beta and KC) in the pathogenesis of CPT-11-induced intestinal mucositis in mice: effect of pentoxifylline and thalidomide

Introduction Irinotecan (CPT-11) is an inhibitor of DNA topoisomerase I and is clinically effective against several cancers. A major toxic effect of CPT-11 is delayed diarrhea; however, the exact mechanism by which the drug induces diarrhea has not been established. Purpose Elucidate the mechanisms of induction of delayed diarrhea and determine the effects of the cytokine production inhibitor pentoxifylline (PTX) and thalidomide (TLD) in the experimental model of intestinal mucositis, induced by CPT-11. Materials and methods Intestinal mucositis was induced in male Swiss mice by intraperitoneal administration of CPT-11 (75 mg/kg) daily for 4 days. Animals received subcutaneous PTX (1.7, 5 and 15 mg/kg) or TLD (15, 30, 60 mg/kg) or 0.5 ml of saline daily for 5 and 7 days, starting 1 day before the first CPT-11 injection. The incidence of delayed diarrhea was monitored by scores and the animals were sacrificed on the 5th and 7th experimental day for histological analysis, immunohistochemistry for TNF-alpha and assay of myeloperoxidase (MPO) activity, tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and KC ELISA. Results CPT-11 caused significant diarrhea, histopathological alterations (inflammatory cell infiltration, loss of crypt architecture and villus shortening) and increased intestinal tissue MPO activity, TNF-alpha, IL-1 beta and KC level and TNF-alpha immuno-staining. PTX inhibited delayed diarrhea of mice submitted to intestinal mucositis and reduced histopathological damage, intestinal MPO activity, tissue level of TNF-alpha, IL-1 beta and KC and TNF-alpha immuno-staining. TLD significantly reduced the lesions induced by CPT-11 in intestinal mucosa, decreased MPO activity, TNF-alpha tissue level and TNF-alpha immuno-staining, but did not reduce the severity of diarrhea. Conclusion These results suggest an important role of TNF-alpha, IL-1 beta and KC in the pathogenesis of intestinal mucositis induced by CPT-11.

Down-regulation of expression of osteoblast and osteocyte markers in periodontal tissues associated with the spontaneous alveolar bone loss of interleukin-10 knockout mice

The aim of this study was to unravel the mechanisms by which interleukin (IL)-10, a potent pleiotropic cytokine, modulates alveolar bone homeostasis in C57BL/6 wild-type (WT) and IL-10 knockout (IL-10 KO) mice, evaluated at 8, 24, and 48 wk of age. Interleukin-10 KO mice presented significant alveolar bone loss when compared with WT mice, and this was not associated with changes in leukocyte counts or bacterial load. The levels of expression of messenger RNA (mRNA) for tumor necrosis factor-alpha (TNF-alpha), IL-1 beta, IL-6, transforming growth factor-beta (TGF-beta), receptor activator of nuclear factor kappa B ligand (RANKL), osteoprotegerin (OPG), and matrix metalloproteinase 13 (MMP13) were similar between both strains, whereas a significant decrease of tissue inhibitor of metalloproteinase 1 (TIMP1) mRNA expression was found at 48 wk in IL-10 KO mice. The osteoblast markers core binding factor alpha1 (CBFA1) and type I collagen (COL-I) were expressed at similar levels in both strains, whereas the levels of alkaline phosphatase (ALP) and osteocalcin (OCN), and those of the osteocyte markers phosphate-regulating gene endopeptidases (PHEX) and dentin matrix protein 1 (DMP1) were significantly lower in IL-10 KO mice. Our results demonstrate that the alveolar bone loss in the absence of IL-10 was associated with a reduced expression of osteoblast and osteocyte markers, an effect independent of microbial, inflammatory or bone-resorptive pathways.

Experimental Chemotherapy in Paracoccidioidomycosis Using Ruthenium NO Donor

Paracoccidioidomycosis (PCM) is a granulomatous disease caused by a dimorphic fungus, Paracoccidioides brasiliensis (Pb). To determine the influence of nitric oxide (NO) on this disease, we tested cis-[Ru(bpy)2(NO)SO3](PF6), ruthenium nitrosyl, which releases NO when activated by biological reducing agents, in BALB/c mice infected intravenously with Pb 18 isolate. In a previous study by our group, the fungicidal activity of ruthenium nitrosyl was evaluated in a mouse model of acute PCM, by measuring the immune cellular response (DTH), histopathological characteristics of the granulomatous lesions (and numbers), cytokines, and NO production. We found that cis-[Ru(bpy)2(NO)SO3](PF6)-treated mice were more resistant to infection, since they exhibited higher survival when compared with the control group. Furthermore, we observed a decreased influx of inflammatory cells in the lung and liver tissue of treated mice, possibly because of a minor reduction in fungal cell numbers. Moreover, an increased production of IL-10 and a decrease in TNF-alpha levels were detected in lung tissues of infected mice treated with cis-[Ru(bpy)2(NO)SO3](PF6). Immunohistochemistry showed that there was no difference in the number of VEGF- expressing cells. The animals treated with cis-[Ru(bpy)2(NO)SO3](PF6) showed high NO levels at 40 days after infection. These results show that NO is effectively involved in the mechanism that regulates the immune response in lung of Pb-infected mice. These data suggest that NO is a resistance factor during paracoccidioidomycosis by controlling fungal proliferation, influencing cytokine production, and consequently moderating the development of a strong inflammatory response.

Development of chronic cardiomyopathy in canine Chagas disease correlates with high IFN-gamma, TNF-alpha, and low IL-10 production during the acute infection phase

When infected with Trypanosoma cruzi, Beagle dogs develop symptoms similar to those of Chagas disease in human beings, and could be an important experimental model for a better understanding of the immunopathogenic mechanisms involved in chronic chagasic infection. This study evaluates IL-10, IFN-gamma and TNF-alpha production in the sera, culture supernatant, heart and cervical lymph nodes and their correlation with cardiomegaly, cardiac inflammation and fibrosis in Beagle dogs infected with T. cruzi. Pathological analysis showed severe splenomegaly, lymphadenopathy and myocarditis in all infected dogs during the acute phase of the disease, with cardiomegaly, inflammation and fibrosis observed in 83% of the animals infected by T. cruzi during the chronic phase. The data indicate that infected animals producing IL-10 in the heart during the chronic phase and showing high IL-10 production in the culture supernatant and serum during the acute phase had lower cardiac alterations (myocarditis, fibrosis and cardiomegaly) than those with high IFN-gamma and TNF-alpha levels. These animals produced low IL-10 levels in the culture supernatant and serum during the acute phase and did not produce IL-10 in the heart during the chronic phase of the disease. Our findings showed that Beagle dogs are a good model for studying the immunopathogenic mechanism of Chagas disease, since they reproduce the clinical and immunological findings described in chagasic patients. The data suggest that the development of the chronic cardiac form of the disease is related to a strong Th1 response during the acute phase of the disease, while the development of the indeterminate form results from a blend of Th1 and Th2 responses soon after infection, suggesting that the acute phase immune response is important for the genesis of chronic cardiac lesions. Crown Copyright (C) 2009 Published by Elsevier B.V. All rights reserved.

Polysaccharide fraction of Agaricus brasiliensis avoids tumor-induced IL-10 production and changes the microenvironment of subcutaneous Ehrlich adenocarcinoma

Subcutaneous Ehrlich tumor-bearing mice were treated with in situ inoculation of a P-glucan-rich extract of Agaricus brasiliensis (ATF), which reduced tumor growth. Histopathological analysis showed that the tumor masses of control mice (Ehr) presented giant tumor cells and many mitotic figures whereas the tumor tissue obtained from ATF-treated animals (Ehr-ATF) presented a lower frequency of both mitotic and giant cells, associated with a higher frequency of apoptotic cells than Ehr. Analysis of the lymphoproliferative activity of spleen cells showed that the treatment had a suppressive rather than a stimulatory effect. Spleen cells of the Ehr group produced higher in vitro levels of IL-10 than normal controls and this occurrence was partially avoided by treatment with ATF. Analysis of cytokine production by tumor-infiltrating cells (ELISpot) showed that ATF induced a higher number of IFN-gamma-producing cells at 7 and 14 days as well as reduction of IL-10-secreting cells at the latter time. Confocal microscopy analysis showed higher intensity of labeling of CD4+ and Mac-3+ cells in ATF-treated mice. Analysis of in situ expression of angiogenic growth factors showed a slight decrease of FGF-2 mRNA in Ehr-ATF animals (7th day) but not of VEGF-A or TGF-beta expression. This fraction could not directly lyse either lymphocytes or tumor cells and we speculate that antitumor effect of ATF could be due to induction of a selective migration of immunocompetent cells from the spleen to the tumor site and to the switch of cytokine production. (C) 2009 Elsevier Inc. All rights reserved.

Modulatory effect of fatty acids on fungicidal activity, respiratory burst and TNF-alpha and IL-6 production in J774 murine macrophages

The reported effects of different families of fatty acids (FA; SFA, MUFA, n-3 and n-6 PUFA) on human health and the importance of macrophage respiratory burst and cytokine release to immune defence led us to examine the influence of palmitic acid (PA), oleic acid (OA), linoleic acid, arachidonic acid, EPA and DHA on macrophage function. We determined fungicidal activity, reactive oxygen species (ROS) and cytokine production after the treatment of J774 cells with non-toxic concentrations of the FA. PA had a late and discrete stimulating effect on ROS production, which may be associated with the reduced fungicidal activity of the cells after treatment with this FA. OA presented a sustained stimulatory effect on ROS production and increased fungicidal activity of the cells, suggesting that enrichment of diets with OA may be beneficial for pathogen elimination. The effects of PUFA on ROS production were time-and dose-dependently regulated, with no evident differences between n-3 and n-6 PUFA. It was worth noting that most changes induced after stimulation of the cells with lipopolysaccharide were suppressed by the FA. The present results suggest that supplementation of the diet with specific FA, not classes of FA, might enable an improvement in host defence mechanisms or a reduction in adverse immunological reactions.

Molecular Mechanisms by Which Saturated Fatty Acids Modulate TNF-alpha Expression in Mouse Macrophage Lineage

Many macrophage functions are modulated by fatty acids (FAs), including cytokine release, such as tumor necrosis factor-alpha (TNF-alpha). TNF-alpha is of great interest due to its role in the inflammation process observed in several diseases such as rheumatoid arthritis, atherosclerosis, and obesity. However, the mechanisms by which FA effects occur have not been completely elucidated yet. In this study, we used a mouse monocyte lineage (J774 cells) to evaluate the effect of 50 and 100 mu M of saturated (palmitic and stearic acids), monounsaturated (oleic acid) and polyunsaturated (linoleic acid) FAs on TNF-alpha production. Alterations in gene expression, poly(A) tail length and activation of transcription factors were evaluated. Oleic and linoleic acids, usually known as neutral or pro-inflammatory FA, inhibited LPS-induced TNF-alpha secretion by the cells. Saturated FAs were potent inducers of TNF-alpha expression and secretion under basal and inflammatory conditions (in the presence of LPS). Although the effect of the saturated FA was similar, the mechanism involved in each case seem to be distinct, as palmitic acid increased EGR-1 and CREB binding activity and stearic acid increased mRNA poly(A) tail. These results may contribute to the understanding of the molecular mechanisms by which saturated FAs modulate the inflammatory response and may lead to design of associations of dietary and pharmacological strategies to counteract the pathological effects of TNF-alpha.

Reduced maximal oxygen consumption and overproduction of proinflammatory cytokines in athletes

Objective: It was the aim of this study to evaluate whether chronic pain in athletes is related to performance, measured by the maximum oxygen consumption and production of hormones and cytokines. Methods: Fifty-five athletes with a mean age of 31.9 +/- 4.2 years engaged in regular competition and showing no symptoms of acute inflammation, particularly fever, were studied. They were divided into 2 subgroups according to the occurrence of pain. Plasma concentrations of adrenaline, noradrenaline, cortisol, prolactin, growth hormone and dopamine were measured by radioimmunoassay, and the production of the cytokines interleukin (IL)-1, IL-2, IL-4, IL-6, tumor necrosis factor-alpha, interferon-alpha and prostaglandin E-2 by whole-blood culture. Maximal oxygen consumption was determined during an incremental treadmill test. Results: There was no change in the concentration of stress hormones, but the athletes with chronic pain showed a reduction in maximum oxygen consumption (22%) and total consumption at the anaerobic threshold (25%), as well as increased cytokine production. Increases of 2.7-, 8.1-, 1.7- and 3.7-fold were observed for IL-1, IL-2, tumor necrosis factor-alpha and interferon-alpha, respectively. Conclusions: Our data show that athletes with chronic pain have enhanced production of proinflammatory cytokines and lipid mediators and reduced performance in the ergospirometric test. Copyright (c) 2008 S. Karger AG, Basel.

Inflammation and adipose tissue: effects of progressive load training in rats

Introduction: Cytokines (IL-6, IL-10 and TNF-alpha) are increased after exhaustive exercise in the rat retroperitoneal (RPAT) and mesenteric adipose tissue (MEAT) pads. On the other hand, these cytokines show decreased expression in these depots in response to a chronic exercise protocol. However, the effect of exercise with overload combined with a short recovery period on pro-and anti-inflammatory cytokine expression is unknown. In the present study, we investigated the regulation of cytokine production in the adipose tissue of rats after an overtraining-inducing exercise protocol. Methods: Male Wistar rats were divided into four groups: Control (C), Trained (Tr), Overtrained (OT) and recovered overtrained (R). Cytokines (IL-6, TNF-alpha and IL-10) levels and Toll Like Receptor 4 (TLR4), Nuclear Factor kBBp65 (NF-kBp65), Hormone Sensitive Lipase (HSL) and, Perilipin protein expression were assessed in the adipose tissue. Furthermore, we analysed plasma lipid profile, insulin, testosterone, corticosterone and endotoxin levels, and liver triacylglycerol, cytokine content, as well as apolipoprotein B (apoB) and TLR4 expression in the liver. Results: OT and R groups exhibited reduced performance accompanied by lower testosterone and increased corticosterone and endotoxin levels when compared with the control and trained groups. IL-6 and IL-10 protein levels were increased in the adipose tissue of the group allowed to recover, in comparison with all the other studied groups. TLR-4 and NF-kBp65 were increased in this same group when compared with both control and trained groups. The protein expression of HSL was increased and that of Perilipin, decreased in the adipose in R in relation to the control. In addition, we found increased liver and serum TAG, along with reduced apoB protein expression and IL-6 and IL-10 levels in the of R in relation to the control and trained groups. Conclusion: In conclusion, we have shown that increases in pro-inflammatory cytokines in the adipose tissue after an overtraining protocol may be mediated via TLR-4 and NF-kBp65 signalling, leading to an inflammatory state in this tissue.

Evaluation of Experimental Autoimmune Uveitis in Mice Treated with FTY720

PURPOSE. FTY720 (fingolimod) is an immunomodulatory drug capable of preventing T-cell migration to inflammatory sites by binding to and subsequently downregulating the expression of sphingosine-1 phosphate receptor 1 (S1P(1)) leading in turn to T-cell retention in lymphoid organs. Additional effects of FTY720 by increasing functional activity of regulatory T cells have recently been demonstrated, raising the conversion of conventional T cells into regulatory T cells and affecting the sequestration of regulatory T cells in normal mice. In this study, the action of FTY720 in the ocular autoimmune model in mice was investigated. METHODS. Mice were immunized with 161-180 peptide and pertussis toxin and were treated with 1 mg/kg/d FTY720 by gavage (7-21 days postimmunization [dpi]) or left untreated. Spleen cells, harvested 21 dpi, were cultured and assayed for cytokine production. Draining lymph node, spleen, and eye cells 21 dpi were assayed for quantification of T-cell populations. Disease severity was evaluated by histologic examination of the enucleated eyes at 21 and 49 dpi. In addition, anti-IRBP antibodies were analyzed by ELISA. RESULTS. FTY720 was effective in suppressing the experimental autoimmune uveitis score. Although there was a reduction in the number of eye-infiltrating cells, FTY did not prevent Treg accumulation at this site. FTY720 leads to a significant increase of CD4(+)IFN-gamma(+) and CD4(+)Foxp3(+) cell percentages in lymph nodes, suggesting that this site could be the source of Treg cells found in the eye. CONCLUSIONS. The data showed that treatment in vivo with FTY720 was able to suppress EAU in mice. These results are indicative of the possible therapeutic use of FTY720 in ocular autoimmune processes. (Invest Ophthalmol Vis Sci. 2010;51:2568-2574) DOI:10.1167/iovs.09-4769