Analysis of the expression of toll-like receptors 2 and 4 and cytokine production during experimental Leishmania chagasi infection

Toll-like receptors (TLRs) recognise pathogen-derived molecules and influence immunity to control parasite infections. This study aimed to evaluate the mRNA expression of TLRs 2 and 4, the expression and production of the cytokines interleukin (IL)-12, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL-17, IL-10 and transforming growth factor (TGF)-β and the production of nitric oxide (NO) in the spleen of mice infected with Leishmania chagasi. It also aimed to evaluate any correlations between mRNA expression TLR2 and 4 and cytokines and NO production. Infection resulted in increased TLR2-4, IL-17, TNF-α and TGF-β mRNA expression during early infection, with decreased expression during late infection correlating with parasite load. IFN-γ and IL-12 mRNA expression decreased at the peak of parasitism. IL-10 mRNA expression increased throughout the entire time period analysed. Although TGF-β, TNF-α and IL-17 were highly produced during the initial phase of infection, IFN-γ and IL-12 exhibited high production during the final phase of infection. IL-10 and NO showed increased production throughout the evaluated time period. In the acute phase of infection, there was a positive correlation between TLR2-4, TNF-α, IL-17, NO, IL-10 and TGF-β expression and parasite load. During the chronic phase of infection, there was a positive correlation between TLR2-4, TNF-α, IL-17 and TGF-β expression and parasite load. Our data suggest that infection by L. chagasi resulted in modulation of TLRs 2 and 4 and cytokines.     

Macrophage colony-stimulating factor (CSF-1) induces pro-inflammatory gene expression and enhances antimicrobial responses of goldfish (Carassius auratus L.) macrophages

We report on the regulation of pro-inflammatory functions of goldfish macrophages and induction of gene expression by recombinant goldfish CSF-1 (rgCSF-1). Recombinant goldfish TNFα-2 (rg TNFα-2), rgIFNγ but not rgTGFβ induced time-dependent increase of CSF-1 expression in macrophages. Treatment of goldfish macrophages with rgCSF-1 increased expression of several immune genes including CXCL-8 (= IL-8), CCL-1, TNFα-1, TNFα-2, IL-1β-1, IL-1β-2, IL-12-p35, IL-12-p40, IFN, IL-10 and iNOS A and B. The rgCSF-1 treatment did not significantly alter the mRNA levels of TGFβ and NRAMP in macrophages up to 48 h post treatment. However, at 72 h post treatment, the expression of TGFβ increased whereas that of NRAMP decreased. The treatment of macrophages with rgCSF-1 enhanced their respiratory burst and nitric oxide responses that were abrogated after addition of soluble CSF-1 receptor (sCSF-1R) to cell cultures. Macrophages exhibited a concentration-dependent chemotactic response toward rgCSF-1 as well as an increase in phagocytic activity that was abrogated after addition of sCSF-1R to cell cultures. Our results indicate that in addition to being an important growth factor of goldfish macrophages, rgCSF-1 also plays a central role in the regulation of their pro-inflammatory responses.     

Francisella noatunensis in Atlantic cod (Gadus morhua L.); waterborne transmission and immune responses

This is the first report that confirms waterborne transmission of francisellosis in Atlantic cod. To investigate the transmission of disease, particle reduced water was transferred from a tank with intraperitoneally infected cod to a tank with healthy cod. Waterborne transmission of Francisella noatunensis was confirmed in the effluent group using immunohistochemistry and real-time quantitative PCR (RT-qPCR). The bacteria were located inside the accumulated macrophage-like cells. Specific and high antibody responses against live and inactivated bacteria were observed. Oil adjuvant had no effect on the antibody responses against inactivated F.?noatunensis compared to saline formulation. The antigen epitope was a 20-25?kDa component of F.?noatunensis suggested to be lipopolysaccharide detected by Western blot, Sypro Ruby and Silver staining. Systemic immune reactions were investigated by measuring the expression of IFN-γ, IL-1β and IL-10 genes with RT-qPCR. After i.p. injection of live bacteria, a significant up-regulation of IFN-γ and IL-1β expression was observed from 15 to 60 days post infection in spleen and head kidney. In intestine, IFN-γ was significantly up-regulated after 30 days whereas rectum showed no significant differences in expression. Elevated expression of IL-10 was observed in all the organs tested but was only significantly up-regulated at 60 days post infection in intestine from i.p. infected fish. For the cohabitant group, IL-1β and IFN-γ was up-regulated in spleen whereas intestine and rectum showed a down-regulation after 60 days. IL-10 was up-regulated in intestine of cohabitant fish from day 30 to day 60. These results indicate that F.?noatunensis infection provokes both specific antibody responses and long term inflammatory responses in cod. The present study provides new knowledge about infection routes and shows that both humoral and cellular defence mechanisms are triggered by F.?noatunensis in cod.     

Recombinant glycoprotein 63 (Gp63) of Trypanosoma carassii suppresses antimicrobial responses of goldfish (Carassius auratus L.) monocytes and macrophages

We previously reported that proteins secreted by Trypanosoma carassii play a role in evasion of fish host immune responses. To further understand how these parasites survive in the host, we cloned and expressed T. carassii glycoprotein 63 (Tcagp63), and generated a rabbit polyclonal antibody to the recombinant protein (rTcagp63). Tcagp63 was similar to gp63 of other trypanosomes and grouped with Trypanosoma cruzi and Trypanosoma brucei gp63 in phylogenetic analysis. We showed that rTcagp63 down-regulated Aeromonas salmonicida and recombinant goldfish TNFα2-induced production of reactive oxygen and nitrogen intermediates. Macrophages treated with rTcagp63 also exhibited significant reduction in the expression of inducible nitric oxide synthase (iNOS)-A, TNFα-1 and TNFα-2. Recombinant Tcagp63 bound to and was internalised by goldfish macrophages. The Tcagp63 may act by altering the signalling events important in downstream monocyte/macrophage antimicrobial and other cytokine-induced functions. We believe that this is the first report on downregulation of antimicrobial responses by trypanosome gp63.     

TNF blockade aggravates experimental chronic Chagas disease cardiomyopathy

Chronic Chagas disease cardiomyopathy (CCC), caused by Trypanosoma cruzi, is an inflammatory dilated cardiomyopathy associated with increased circulating levels of TNF-alpha. We investigate whether TNF blockade with Etanercept during the chronic phase of T. cruzi infection could attenuate experimental CCC development. The effect of Etanercept was evaluated after 11 months of T. cruzi infection on survival, parasitism, left ventricular function, intensity of myocarditis, fibrosis, and left ventricular mRNA expression of cytokines and TNF-alpha-induced genes. Left ventricular function was significantly reduced in treated animals as compared to infected untreated animals. Blood and cardiac parasitism as well as survival rate were not altered with Etanercept treatment. Inflammatory infiltrates were located predominantly in the subendocardic region in treated animals, whereas in untreated animals inflammation was scattered throughout the myocardium. Left ventricular mRNA IL-10 expression was significantly higher, and iNOS, significantly lower in treated than in untreated animals. mRNA expression of TNF-alpha, IFN-gamma, TGF-beta, A20 and ANP was similar in both groups. Our results suggest that TNF-alpha/LT-alpha blockade with Etanercept enhances left ventricular dysfunction in T. cruzi-induced chronic cardiomyopathy and the absence of TNF signaling may be deleterious to the failing heart in Chagas disease cardiomyopathy.     

Kinetics of cytokine profile during intraperitoneal inoculation of Japanese encephalitis virus in BALB/c mice model

Infection with Japanese encephalitis virus (JEV) is mostly asymptomatic/subclinical in 90% of the individuals. Host immune response during subclinical JEV infection is poorly understood. We assessed iNOS, IFN-gamma, TNF-alpha, IL-10 and IL-4 production in spleen, brain and sera of intraperitoneally challenged BALB/c mice by RT-PCR and ELISA along with brain histopathology at different days post inoculation (d.p.i.). In spleen of virus infected mice, expression of all cytokines including iNOS mRNA were upregulated till 5d.p.i. followed by decline. At 5d.p.i., IL-10 expression outcompeted TNF-alpha, IFN-gamma and IL-4. However, in the virus infected mice sera, IL-4 production predominated over TNF-alpha and IL-10 at 5d.p.i. Conversely, cytokines expression and iNOS mRNA remained unchanged in the brain of virus infected mice from 1 to 7d.p.i. A significant increase in the cytokine expression was observed at 11d.p.i. (P<0.05) in virus infected mice brain, with the predominance of IL-10 along with the presence of meningeal inflammation and viral RNA by histology and RT-PCR, respectively. We report a biased pattern of cytokine production in sera, brain and spleen of mice intraperitoneally challenged with JEV. IL-10 exerts neuroprotective function during JEV and regulates deleterious effects of proinflammatory cytokines; however, its mechanism needs further investigation.     

The hamster as a model of human visceral leishmaniasis: progressive disease and impaired generation of nitric oxide in the face of a prominent Th1-like cytokine response

Active human visceral leishmaniasis (VL) is characterized by a progressive increase in visceral parasite burden, cachexia, massive splenomegaly, and hypergammaglobulinemia. In contrast, mice infected with Leishmania donovani, the most commonly studied model of VL, do not develop overt, progressive disease. Furthermore, mice control Leishmania infection through the generation of NO, an effector mechanism that does not have a clear role in human macrophage antimicrobial function. Remarkably, infection of the Syrian hamster (Mesocricetus auratus) with L. donovani reproduced the clinicopathological features of human VL, and investigation into the mechanisms of disease in the hamster revealed striking differences from the murine model. Uncontrolled parasite replication in the hamster liver, spleen, and bone marrow occurred despite a strong Th1-like cytokine (IL-2, IFN-gamma, and TNF/lymphotoxin) response in these organs, suggesting impairment of macrophage effector function. Indeed, throughout the course of infection, inducible NO synthase (iNOS, NOS2) mRNA or enzyme activity in liver or spleen tissue was not detected. In contrast, NOS2 mRNA and enzyme activity was readily detected in the spleens of infected mice. The impaired hamster NOS2 expression could not be explained by an absence of the NOS2 gene, overproduction of IL-4, defective TNF/lymphotoxin production (a potent second signal for NOS2 induction), or early dominant production of the deactivating cytokines IL-10 and TGF-beta. Thus, although a Th1-like cytokine response was prominent, the major antileishmanial effector mechanism that is responsible for control of infection in mice was absent throughout the course of progressive VL in the hamster.     

Interferon-gamma production and host protective response against Mycobacterium tuberculosis in mice lacking both IL-12p40 and IL-18

Interferon (IFN)-gamma plays an essential role in host defense against infection with Mycobacterium tuberculosis, and its synthesis is critically regulated by interleukin (IL)-12, IL-18 and the recently identified IL-23. The present study was designed to determine the roles of these cytokines in IFN-gamma-mediated host defenses against M. tuberculosis. For this purpose, we compared host protective responses in IL-12p40 and IL-18 double-knockout (DKO) mice (which lacked both IL-12/IL-18 and also IL-23) and IFN-gamma gene-disrupted (GKO) mice. DKO mice were more resistant to the infection than GKO mice, as indicated by their extended survival and reduced live colony numbers in spleen, liver and lung. IFN-gamma was detected by ELISA in liver and lung homogenates, but not in spleen and serum, and in all organs by RT-PCR in DKO mice at comparable or reduced levels to those in wild-type mice. IFN-gamma production was reduced by depletion of CD4+ T cells, but not of natural killer (NK), NKT, gammadeltaT and dendritic cells. Neutralization of IFN-gamma or TNF-alpha by specific monoclonal antibodies (mAbs) significantly shortened the survival time of the infected DKO mice. Furthermore, anti-TNF-alpha mAb partially attenuated IFN-gamma synthesis in the liver of these mice. Finally, the expression level of inducible nitric oxide synthase (iNOS) mRNA in the spleen, liver and lung was considerable in DKO mice but only marginal or undetected in GKO mice. Our results indicate the presence of IL-12-, IL-18- and IL-23-independent host protective responses against mycobacterial infection mediated by IFN-gamma, which was secreted from helper T cells.     

Regulation of scavenger receptor class B type I in hamster liver and Hep3B cells by endotoxin and cytokines

Multiple changes in HDL metabolism occur during infection and inflammation that could potentially impair the antiatherogenic functions of HDL. Scavenger receptor class B type I (SR-BI) promotes cholesterol efflux from peripheral cells and mediates selective uptake of cholesteryl ester into hepatocytes, thereby playing a pivotal role in reverse cholesterol transport. We studied the effect of endotoxin (lipopolysaccharide, LPS) and cytokines [tumor necrosis factor (TNF) and interleukin 1 (IL-1)] on hepatic SR-BI mRNA and protein levels in Syrian hamsters. LPS significantly decreased SR-BI mRNA levels in hamster liver. This effect was rapid and sustained, and was associated with a decrease in hepatic SR-BI protein levels. High cholesterol diet did not change hepatic SR-BI mRNA levels, and LPS was able to decrease SR-BI mRNA levels during high cholesterol feeding. TNF and IL-1 decreased SR-BI mRNA levels in the liver, and the effects of TNF and IL-1 were additive. TNF and IL-1 also decreased SR-BI levels in Hep3B hepatoma cells. More importantly, TNF and IL-1 decreased the uptake of HDL cholesteryl ester into Hep3B cells. In addition, we studied the effect of LPS on SR-BI mRNA in RAW 264.7 cells, a macrophage cell line. LPS rapidly decreased SR-BI mRNA levels in RAW 264.7 cells, but the effect was not sustained and did not lead to a reduction in SR-BI protein levels. Our results suggest that the decrease in hepatic SR-BI levels due to LPS and cytokines during infection and inflammation may decrease selective uptake of cholesteryl ester into the liver and result in impaired reverse cholesterol transport.     

Differential control of IFN-gamma and IL-2 production during Trypanosoma cruzi infection

In murine infection with Trypanosoma cruzi, immune responsiveness to parasite and non-parasite Ag becomes suppressed during the acute phase of infection, and this suppression is known to extend to the production of IL-2. To determine whether suppression of lymphokine production was specific for IL-2, or was a generalized phenomenon involving suppressed production of other lymphokines, we have begun an investigation of the ability of mice to produce of a number of lymphokines during infection, initially addressing this question by studying IFN-gamma production. Supernatants from Con A-stimulated spleen cells from infected resistant (C57B1/6) and susceptible (C3H) mice were assayed for IFN-gamma. Supernatants known to be suppressed with respect to IL-2 production from both mouse strains contained IFN-gamma at or above that of supernatants from normal spleen cells. Samples were assayed in an IFN bioassay to ensure that the IFN-gamma detected by ELISA was biologically active. Thus, suppression during T. cruzi infection does not extend to the production of all lymphokines. The stimulation of IFN-gamma production was confirmed by detection of IFN-gamma mRNA in unstimulated spleen cells from infected animals, and in Con A, Con A + PMA, and in some cases, parasite Ag-stimulated spleen cells from infected animals. IFN-gamma mRNA levels in mitogen-stimulated spleen cells equalled or exceeded those found in similarly stimulated normal cells. In contrast, stimulated spleen cells from infected animals had reduced levels of IL-2 mRNA relative to normal spleen cells. Thus at both the protein and mRNA level, IFN-gamma production is stimulated by T. cruzi infection, whereas IL-2 production is suppressed. Serum IFN-gamma in infected C57B1/6 and C3H mice was detected 8 days after infection, peaked on day 20 of infection, and subsequently fell, but remained detectable at low levels throughout the life of infected mice. Infected animals were depleted of cell populations known to be capable of producing IFN-gamma, and Thy-1+, CD4-, CD8-, NK- cells, and to a lesser degree, CD4+ and CD8+ cells were found to be responsible for the production of IFN-gamma during infection. We also report that IL-2 can induce IFN-gamma production in vitro and in vivo by spleen cells from infected animals, and that IL-2 can synergize with epimastigote or trypomastigote antigen to produce high levels of IFN-gamma comparable to those found in supernatants from mitogen-stimulated cells.     

Interferon regulatory factor 1 in mycobacterial infection

In order to understand the role of IRF-1 in the development of murine tuberculosis in vivo, IRF-1 knockout mice were infected with Mycobacterium tuberculosis by placing them in the exposure chamber of an airborne infection apparatus. These knockout mice developed multifocal necrotic lesions in the lung, liver and spleen tissues and died of disseminated tuberculosis within 43 days of infection. Compared with the levels in wild-type mice, the pulmonary inducible NO synthase (iNOS) mRNA expression level was significantly lower, but IL-18 and IL-6 mRNA levels were higher. There was no statistically significant difference in the expression of IFN-gamma and TNF-alpha mRNA between the IRF-1 knockout and wild-type mice. IRF-1 is indirectly responsible for iNOS mRNA expression and plays an important role in the pathogenesis of murine tuberculosis.     

Differential expression of IL-1 and TNF receptors in murine macrophages infected with virulent vs. avirulent Legionella pneumophila

Infection of macrophages from genetically susceptible A/J mice with Legionella pneumophila induces high levels of various cytokines in serum as well as in cultures of spleen or peritoneal cells from the mice. However, modulation of receptor expression for these cytokines during infection has not been studied in detail, even though these receptors on macrophages have a critical role in inflammatory responses during the infection. In the present study, the differential expression of mRNA for TNF and IL-1 receptors as well as receptor antigens during infection of macrophages with virulent vs. avirulent L. pneumophila was investigated. Mouse thioglycollate-elicited peritoneal macrophages showed by RT-PCR constitutive steady-state levels of mRNA for TNF-type I and -type II receptors as well as IL-1 type I receptor. However, IL-1 type II receptor mRNA was not expressed in thioglycollate-elicited macrophages. Infection of macrophages with virulent bacteria caused an upregulation of IL-1 type I and TNF type I receptor mRNA, but had no effect on TNF type II receptor message. Avirulent L. pneumophila infection caused much less induction of these receptor mRNAs. The amount of receptor antigen of IL-1 type I on the surface of macrophages was also increased by infection with virulent L. pneumophila determined by flow cytometric analysis. These results indicate that L. pneumophila infection not only causes induction of various cytokines, but also modulation of certain cytokine receptors, which may regulate the susceptibility to infection.     

IL-17RA Signaling Reduces Inflammation and Mortality during Trypanosoma cruzi Infection by Recruiting Suppressive IL-10-Producing Neutrophils

Members of the IL-17 cytokine family play an important role in protection against pathogens through the induction of different effector mechanisms. We determined that IL-17A, IL-17E and IL-17F are produced during the acute phase of T. cruzi infection. Using IL-17RA knockout (KO) mice, we demonstrate that IL-17RA, the common receptor subunit for many IL-17 family members, is required for host resistance during T. cruzi infection. Furthermore, infected IL-17RA KO mice that lack of response to several IL-17 cytokines showed amplified inflammatory responses with exuberant IFN-γ and TNF production that promoted hepatic damage and mortality. Absence of IL-17RA during T. cruzi infection resulted in reduced CXCL1 and CXCL2 expression in spleen and liver and limited neutrophil recruitment. T. cruzi-stimulated neutrophils secreted IL-10 and showed an IL-10-dependent suppressive phenotype in vitro inhibiting T-cell proliferation and IFN-γ production. Specific depletion of Ly-6G+ neutrophils in vivo during T. cruzi infection raised parasitemia and serum IFN-γ concentration and resulted in increased liver pathology in WT mice and overwhelming wasting disease in IL-17RA KO mice. Adoptively transferred neutrophils were unable to migrate to tissues and to restore resistant phenotype in infected IL-17RA KO mice but migrated to spleen and liver of infected WT mice and downregulated IFN-γ production and increased survival in an IL-10 dependent manner. Our results underscore the role of IL-17RA in the modulation of IFN-γ-mediated inflammatory responses during infections and uncover a previously unrecognized regulatory mechanism that involves the IL-17RA-mediated recruitment of suppressive IL-10-producing neutrophils.     

Cytokine (tumor necrosis factor, IL-6, and IL-8) production by respiratory syncytial virus-infected human alveolar macrophages.

Human alveolar macrophages (AM) are susceptible to infection with respiratory syncytial virus (RSV), but the infection is abortive after the initial cycles of virus replication. We have investigated if RSV infection of AM results in the production of cytokines TNF, IL-6, and IL-8, all of which may modulate inflammatory and immune responses to the virus, as well as may directly protect respiratory epithelial cells against spread of infection. Within 1 h after interaction with RSV, increased mRNA levels were found for all three cytokines. Peak expression of the mRNAs occurred at 3 to 6 h. The virus most effectively induced TNF mRNA expression greater than IL-6 mRNA greater than IL-8 mRNA, as compared to cytokine mRNA expression induced by bacterial endotoxin. Inactivated virus was almost as effective as live virus in inducing and maintaining increased IL-6 and IL-8 mRNA over 16 h, whereas live infectious RSV was necessary for maintaining TNF mRNA expression over the same time. Protein concentrations of the different cytokines in the supernatants of infected AM reflected the increased levels of mRNA in the cells. Despite the high levels of cytokines with possible antiviral activity (TNF and IL-6) in the AM supernatants, neither supernatants nor rTNF when added to bronchial epithelial cells protected them from infection with RSV. However, TNF, IL-1, and RSV, but not IL-6, induced IL-8 and IL-6 mRNA expression by the bronchial epithelial cells suggesting that cytokines produced by RSV-infected AM may be more important in modulating the inflammatory response in infection than directly interfering with virus infection/replication of airway epithelium.     

Immunoregulation by Toxoplasma gondii infection prevents allergic immune responses in mice

Toxoplasma gondii is a ubiquitous intracellular parasite affecting most mammals including humans. In epidemiological studies, infection with T. gondii and allergy development have been postulated to be inversely related. Using a mouse model of birch pollen allergy we investigated whether infection with T. gondii influences allergic immune responses to birch pollen. BALB/c mice were infected with T. gondii oocysts either before or at the end of sensitisation with the major birch pollen allergen Bet v 1 and thereafter aerosol challenged with birch pollen extract. During the acute phase of infection, clinical signs correlated with increased levels of serum TNF-α, IL-6, IFN-γ and anti-Toxoplasma-IgM. In the chronic phase, Toxoplasma-specific serum IgG, brain tissue cysts and high IFN-γ production in spleen cell cultures were detected. Mice infected prior to allergic sensitisation produced significantly less allergen-specific IgE and IgG1, while IgG2a levels were markedly increased. IL-5 levels in spleen cell cultures and bronchoalveolar lavage fluid were significantly reduced, and airway inflammation was prevented in these mice. Notably, in mice infected at the end of the allergic sensitisation process, systemic and local immune responses to the allergen were markedly reduced. T.gondii infection was associated with up-regulation of Toll-like receptor 2 (TLR2), 4, 9 and 11, as well as T-bet (a differentiation factor for Th1 cells) mRNA expression in splenocytes; moreover, enhanced TGF-β, IL-10 and Foxp3 mRNA expression in these cells suggested that regulatory mechanisms were involved in suppression of the allergic immune response. Kinetic studies confirmed the induction of Foxp3⁺CD4⁺CD25⁺ regulatory T cells preferentially during the chronic phase of T. gondii infection. Our data demonstrate that T. gondii exhibits strong immunomodulating properties which lead to prevention of allergic immune responses and thereby support the hygiene hypothesis.