Mahboob Hossain Qureshi

IL-18 protects mice against pulmonary and disseminated infection with Cryptococcus neoformans by inducing IFN-gamma production.

We examined the effects of a newly identified cytokine, IL-18, originally designated as IFN-gamma-inducing factor, in a mouse model of pulmonary and disseminated infection with a highly virulent strain of Cryptococcus neoformans. Administration of murine rIL-18 enhanced elimination of live micro-organisms from the lungs, prevented fungal dissemination to the brain, reduced the level of serum cryptococcal capsular polysaccharide Ags, and increased the survival rate of infected mice. Histologic examination of lung sections of infected and PBS-treated mice showed a poor cellular inflammatory reaction and a large number of multiplying C. neoformans yeast cells in alveolar spaces. In contrast, massive infiltration of inflammatory cells, consisting mainly of mononuclear cells, characterized sections of lungs of infected animals treated with IL-18. Treatment with IL-18 also increased the level of serum IFN-gamma. In addition, the protective effect of IL-18 on cryptococcal infection was abrogated by administration of neutralizing anti-IFN-gamma mAb. Finally, we examined the effect of neutralizing anti-IL-18 Ab on cryptococcal infection to define the physiologic role of this cytokine in host defense using another weakly virulent strain of C. neoformans, which induced the expression of IL-18 mRNA in the infected lungs. Administration of this Ab exacerbated the infection, as shown by the increased lung burden. Our results indicate that IL-18 plays an important role in host defense against infection with C. neoformans.

IL-18 contributes to host resistance against infection with Cryptococcus neoformans in mice with defective IL-12 synthesis through induction of IFN-γ production by NK cells.

The aim of this study was to examine the contribution of IL-18 in host defense against infection caused by Cryptococcus neoformans in mice with defective IL-12 production. Experiments were conducted in mice with a targeted disruption of the gene for IL-12p40 subunit (IL-12p40−/− mice). In these mice, host resistance was impaired, as shown by increased number of organisms in both lungs and brains, compared with control mice. Serum IFN-γ was still detected in these mice at a considerable level (20–30% of that in control mice). The host resistance was moderately impaired in IL-12p40−/− mice compared with IFN-γ−/− mice. Neutralizing anti-IFN-γ mAb further increased the lung burdens of organisms. In addition, treatment with neutralizing anti-IL-18 Ab almost completely abrogated the production of IFN-γ and also impaired the host resistance. Host resistance in IL-12p40−/− IL-18−/− mice was more profoundly impaired than in IL-12p40−/− mice. Administration of IL-12 as well as IL-18 increased the serum levels of IFN-γ and significantly restored the reduced host resistance. Spleen cells obtained from infected IL-12p40−/− mice did not produce any IFN-γ upon restimulation with the same organisms, while those from infected and IL-12-treated mice produced IFN-γ. In contrast, IL-18 did not show such effect. Finally, depletion of NK cells by anti-asialo GM1 Ab mostly abrogated the residual production of IFN-γ in IL-12p40−/− mice. Our results indicate that IL-18 contributes to host resistance to cryptococcal infection through the induction of IFN-γ production by NK cells, but not through the development of Th1 cells, under the condition in which IL-12 synthesis is deficient.

Combined effects of IL-12 and IL-18 on the clinical course and local cytokine production in murine pulmonary infection with Cryptococcus neoformans

We reported recently that interleukin (IL)‐12 and IL‐18 synergistically increased the fungicidal activity of mouse peritoneal exudate cells against Cryptococcus neoformans by inducing the production of interferon (IFN)‐γ by natural killer (NK) cells. To confirm these findings in vivo, we examined the effect of combined treatment using these two cytokines on the course of experimentally induced pulmonary and disseminated cryptococcosis in mice. IL‐12 andIL‐18 were used at subtherapeutic doses (0.005 and 2 μg/mouse/day, respectively). A single administration of either cytokine was not effective in protecting mice against the infection, while combined treatment significantly prolonged survival time of infected mice and reduced the lung and brain loads of organisms. These protective effects were associated with elevated IFN‐γ and reduced IL‐4 levels in bronchoalveolar lavage fluid. Finally, depletion of NK and γ δ T cells, but not of CD4+ T cells, by administration of specific antibodies, significantly reduced the production of IFN‐γ in lungs by IL‐12/IL‐18 treatment during the 7 days of infection. Our results demonstrated that IL‐12 and IL‐18 protected mice against cryptococcal infection in a synergistic manner by enhancing the local production of IFN‐γ by NK and γ δ T cells in the early phase of infection and by suppressing the production of IL‐4 in lungs.

Resveratrol attenuates hepatic steatosis in high-fat fed mice by decreasing lipogenesis and inflammation

OBJECTIVE Resveratrol (RSV) is the most studied natural compound that activates sirtuins, which produce beneficial metabolic effects on lipid and glucose metabolism. The aim of the present study was to investigate the role of resveratrol in preventing nonalcoholic fatty liver disease (NAFLD) and expression of liver inflammatory markers in mice treated with a high-fat diet. METHODS AND PROCEDURES Eighteen male mice were divided into three groups and fed for 60 d with a standard diet (ST), high-fat diet (HFD), or high-fat diet plus resveratrol (HFD + RSV, 30 mg/kg/d). Body weight, food intake, and serum total cholesterol, triacylglycerol, insulin, alanine transaminase (ALT), and aspartate aminotransferase (AST) were evaluated. Liver histology was analyzed. Expression of ACC, PPAR-γ, ChREBP, SREBP-1 c, CPT-1, tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), NF-κB, interleukin 1 β (IL-1 β), and SIRT1 were evaluated by quantitative real-time reverse transcriptase PCR (qRT-PCR). RESULTS The major finding of the present study was that RSV reduced body fat, total cholesterol, triacylglycerol, transaminases, and insulin plasma level. These results were accompanied with a significant reduction in TNF-α, IL-6, and NF-κB mRNA expression in the liver. Analyses of liver adipogenesis related genes indicated that ACC, PPAR-γ, and SREBP-1 mRNA expression were significantly suppressed in HFD + RSV mice. In addition, we observed increased expression of SIRT1 in the HFD + RSV group. CONCLUSIONS We observed that treatment with resveratrol improved lipid metabolism, and decreased NAFLD and pro-inflammatory profile in liver of mice with obesity-inducible diets. These data suggest an important clinical application of RSV in preventing liver diseases.

Contribution of tumour necrosis factor-alpha (TNF-α) in host defence mechanism against Cryptococcus neoformans

We investigated the role of TNF‐α in the host defence mechanism against infection with a virulent strain of Cryptococcus neoformans. Administration of exogenous recombinant human TNF‐α significantly prolonged the survival time of mice infected by intratracheal instillation of the organism. Surprisingly, neutralizing MoAb to murine TNF‐α did not shorten their survival time, a finding inconsistent with previous results. To investigate the cause of this inconsistency, we examined the production of TNF‐α in the lungs of infected mice. During the course of cryptococcosis, there was little or no generation of TNF‐α mRNA in the lung. This might be partly due to a direct inhibitory action of the fungal microorganism on TNF‐α production by macrophages. In vitro production of TNF‐α by murine interferon‐gamma (IFN‐γ)‐ and lipopolysaccharide (LPS)‐stimulated macrophages was strongly inhibited by co‐culturing with the whole yeast cells. In contrast, administration of recombinant murine IL‐12 markedly induced TNF‐α production and the neutralizing anti‐TNF‐α MoAb strongly blocked IL‐12‐induced protection of mice against cryptococcal infection. These results indicate that endogenously synthesized TNF‐α has the potential to contribute to the elimination of C. neoformans and partly mediates the protective effect of IL‐12.

Candida albicans suppresses nitric oxide (NO) production by interferon-gamma (IFN-γ) and lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages

We examined the in vitro effect of Candida albicans on NO production by macrophages. Candida albicans suppressed not only NO production but also expression of inducible NO synthase (iNOS) mRNA by murine IFN‐γ and bacterial LPS‐stimulated peritoneal macrophages. The suppression was not associated with inhibition but rather stimulation of IL‐1β production. This effect was observed when more than 1 × 103/ml of Candida albicans were added to macrophage cultures (1 × 106 cells/ml) and reached a maximal level at 1 × 106/ml. The NO inhibitory effect of Candida albicans was mediated predominantly by as yet unidentified soluble factor(s) and to a lesser extent by direct contact. In addition, heat‐ or paraformaldehyde‐killed Candida albicans did not show this inhibitory activity. Culture supernatant of Candida albicans also inhibited NO production by activated macrophages in a dose‐dependent manner, and increased IL‐1β production. Finally, the inhibitory effect was not mediated by IL‐10 and transforming growth factor‐beta (TGF‐β), since neutralizing antibodies to these cytokines did not influence Candida albicans‐induced reduction in macrophage NO production. Our results suggest that Candida albicans may evade host defence mechanism(s) through a soluble factor‐mediated suppression of NO production by stimulated macrophages, and that the effect is independent of production of immunosuppressive cytokines such as IL‐10 and TGF‐β.

IL-10 Modulates Host Responses and Lung Damage Induced by Pneumocystis carinii Infection

Host responses to Pneumocystis carinii infection mediate impairment of pulmonary function and contribute to the pathogenesis of pneumonia. IL-10 is known to inhibit inflammation and reduce the severity of pathology caused by a number of infectious organisms. In the present studies, IL-10-deficient (IL-10 knockout (KO)) mice were infected with P. carinii to determine whether the severity of pathogenesis and the efficiency of clearance of the organisms could be altered in the absence of IL-10. The clearance kinetics of P. carinii from IL-10 KO mice was significantly enhanced compared with that of wild-type (WT) mice. This corresponded to a more intense CD4+ and CD8+ T cell response as well as an earlier neutrophil response in the lungs of IL-10 KO mice. Furthermore, IL-12, IL-18, and IFN-γ were found in the bronchoalveolar lavage fluids at earlier time points in IL-10 KO mice suggesting that alveolar macrophages were activated earlier than in WT mice. However, when CD4+ cells were depleted from P. carinii-infected IL-10 KO mice, the ability to enhance clearance was lost. Furthermore, CD4-depleted IL-10 KO mice had significantly more lung injury than CD4-depleted WT mice even though the intensity of the inflammatory responses was similar. This was characterized by increased vascular leakage, decreased oxygenation, and decreased arterial pH. These data indicate that IL-10 down-regulates the immune response to P. carinii in WT mice; however, in the absence of CD4+ T cells, IL-10 plays a critical role in controlling lung damage independent of modulating the inflammatory response.

Neonatal T Cells in an Adult Lung Environment Are Competent to Resolve Pneumocystis carinii Pneumonia

Initiation of the pulmonary inflammatory response to Pneumocystis carinii is delayed by 3 wk in mice infected as neonates compared with adults. There was no difference in the proliferative response of draining lymph node T cells from mice infected as neonates compared with adults when stimulated in vitro with either Con A or anti-CD3 mAb. However, TNF-α and IFN-γ mRNA expression in the lungs of P. carinii-infected neonates was significantly lower than in adults indicating a lack of appropriate activation signaling in the local environment. This may have been due to active suppression because TGF-β mRNA expression was significantly elevated in neonatal lungs compared with adults. To determine whether T cells from 10-day-old mice would effect resolution of P. carinii if harbored in an adult lung environment, cells were adoptively transferred to SCID mice with established P. carinii infections. There was no difference in the kinetics of T cell migration into the lungs or of clearance of P. carinii organisms when SCID mice were reconstituted with splenocytes from young mice as compared with adult mice. Furthermore, splenocytes from young mice stimulated both TNF-α and IFN-γ mRNA expression to levels that were similar to that in the lungs of SCID mice reconstituted with adult cells. These data indicate that neonatal lymphocytes are competent to resolve P. carinii infection when harbored in an adult lung environment, suggesting that the neonatal lung environment, and not the T cells, is ineffective at responding to P. carinii infection.

Delayed Inflammatory Response to Pneumocystis carinii Infection in Neonatal Mice Is Due to an Inadequate Lung Environment

Challenge of neonatal mice with an intranasal inoculation of Pneumocystis carinii results in a subclinical infection that takes 6 wk to resolve, whereas adult mice resolve a comparable challenge within 3 wk. This delayed clearance is due to a delayed inflammatory response in neonatal mice; however, the reason for this delay has been unknown. To determine whether the neonatal lung environment is sufficient to attract immunocompetent lymphocytes into the lungs, an adoptive transfer strategy was employed in which splenocytes from adult BALB/c mice were transferred into P. carinii-infected neonatal or adult SCID mice. All adults, but no pups, resolved their infections by day 37 postreconstitution. Despite reconstitution with adult splenocytes, pups had a negligible lung inflammatory response until day 24, whereas adult mice had activated CD4+ and CD8+ cells in the lung by day 13. The delay in neonates corresponded to delayed kinetics of expression of lung cytokines TNF-α and IFN-γ mRNA and chemokines lymphotactin, RANTES, and macrophage inflammatory protein-1β mRNA. Phagocytic cells from neonatal mice were significantly less efficient than adult cells at migrating to the draining lymph nodes after phagocytosing fluorescent beads. There were fewer dendritic cells and Ia+ myeloid cells in the lungs of P. carinii-infected neonatal mice compared with adults. These data indicate that the lung environment of neonatal mice is insufficient for migration of T cells, due at least in part to inefficient phagocytosis and migration of APCs to the lymph nodes as well as delayed chemokine and TNF-α mRNA expression.

Interferon-gamma (IFN-γ)-dependent protection and synthesis of chemoattractants for mononuclear leucocytes caused by IL-12 in the lungs of mice infected with Cryptococcus neoformans

We have recently demonstrated that IL‐12 induced cellular inflammatory responses consisting mainly of accumulation of mononuclear leucocytes in the lungs of mice infected with Cryptococcus neoformans and protected mice against fulminant infection. We examined the involvement of endogenously synthesized IFN‐γ in such a response by investigating the effects of a neutralizing monoclonal antibody against this cytokine. The latter treatment completely abrogated the positive effects of IL‐12 on survival of infected mice and prevented IL‐12‐induced elimination of microbials from the lungs. Histopathological examination showed that accumulation of mononuclear leucocytes in the infected lungs caused by IL‐12 was clearly inhibited by anti‐IFN‐γ MoAb. We also examined the local production of mononuclear cell‐attracting chemokines such as monocyte chemotactic protein‐1 (MCP‐1), regulated upon activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory protein‐1α (MIP‐1α), MIP‐1β and IFN‐γ‐inducible protein 10 (IP‐10) in the lungs using a reverse transcriptase‐polymerase chain reaction (RT‐PCR) method. We found that these chemokines were not synthesized in the infected lungs, while IL‐12 treatment markedly induced their production. Interestingly, neutralizing anti‐IFN‐γ MoAb strongly suppressed IL‐12‐induced production of these chemokines. Similar results were obtained with MCP‐1 and MIP‐1α when their synthesis was measured at the protein level using respective ELISA kits. Our results indicate that IFN‐γ plays a central role in the protective effects of IL‐12 by inducing mononuclear leucocyte‐attracting chemokines and cellular inflammatory responses.