IL-12 protects against coxsackievirus B3-induced myocarditis by increasing IFN-gamma and macrophage and neutrophil populations in the heart

Th1-type immune responses, mediated by IL-12-induced IFN-gamma, are believed to exacerbate certain autoimmune diseases. We recently found that signaling via IL-12Rbeta1 increases coxsackievirus B3 (CVB3)-induced myocarditis. In this study, we examined the role of IL-12 on the development of CVB3-induced myocarditis using mice deficient in IL-12p35 that lack IL-12p70. We found that IL-12 deficiency did not prevent myocarditis, but viral replication was significantly increased. Although there were no changes in the total percentage of inflammatory cells in IL-12-deficient hearts compared with wild-type BALB/c controls by FACS analysis, macrophage and neutrophil populations were decreased. This decrease corresponded to reduced TNF-alpha and IFN-gamma levels in the heart, suggesting that macrophage and/or neutrophil populations may be a primary source of TNF-alpha and IFN-gamma during acute CVB3 myocarditis. Increased viral replication in IL-12-deficient mice was not mediated by reduced TNFRp55 signaling, because viral replication was unaltered in TNFRp55-deficient mice. However, STAT4 or IFN-gamma deficiency resulted in significantly increased viral replication and significantly reduced TNF-alpha and IFN-gamma levels in the heart, similar to IL-12 deficiency, indicating that the IL-12/STAT4 pathway of IFN-gamma production is important in limiting CVB3 replication. Furthermore, STAT4 or IFN-gamma deficiency also increased chronic CVB3 myocarditis, indicating that therapeutic strategies aimed at reducing Th1-mediated autoimmune diseases may exacerbate common viral infections such as CVB3 and increase chronic inflammatory heart disease.     

T cells contribute to disease severity during coxsackievirus B4 infection

By using a model of coxsackievirus B4-induced disease, the question of whether tissue damage is due to the virus or to immune-mediated mechanisms was addressed. Both viral replication and T-cell function were implicated in contributing to the severity of disease. Three stages (I to III) of disease, which correspond to periods of high viral titers, low viral titers, and no infectious virus, have been identified. Stage I disease is considered to be primarily the result of viral replication. Immunopathological mechanisms appear to contribute to the severity of stage II and III disease. To investigate the role of T cells in contributing to the severity of disease, viral infection in CD8 knockout (ko) mice and CD4 ko mice was analyzed. CD8 T-cell responses appear to be beneficial during early, viral disease but detrimental in later disease when viral titers are diminishing. CD4 ko mice, unlike the parental strain, survived infection. Viral replication was lower in the CD4 ko mice. Was survival due to decreased viral replication or to the lack of T-helper-cell function? To investigate further the role of T helper cells in contributing to tissue damage, viral infection in two additional ko strains (interleukin-4 [IL-4] ko and gamma interferon ko strains) was examined. A clear correlation between viral replication and the outcome of infection was not observed. The absence of IL-4, which may influence T-helper-cell subset development, was advantageous during early viral disease but deleterious in later disease. The results suggest that T-cell-mediated immunity is both beneficial and detrimental during coxsackievirus B4 infection.

The group B coxsackieviruses, comprising six serotypes (B1 to B6), have been implicated in a variety of diseases such as pancreatitis, type I insulin-dependent diabetes mellitus, myocarditis, and myositis (16, 24, 25, 30). The broad spectrum of diseases associated with the group B viruses reflects the existence of strains, with various degrees of virulence, within a serotype. Although there is a great deal of information on the biochemical, biophysical, and genetic characteristics of the picornaviruses, the mechanisms by which these RNA viruses cause disease are poorly understood. An ongoing question that remains to be resolved is whether tissue damage is due solely to the virus, to immunopathological mechanisms, or to a combination of both. Evidence supporting an immunopathological mechanism during coxsackievirus B3 (CVB3) infection implicates different effector cells such as CD8 T cells (10), CD4 T cells (1, 10), autoantibody-producing B cells (19, 31), and natural killer cells (9). In addition, the type of T-helper-cell response is critical in determining pathogenicity in a myocarditis model (11).To study the intricate virus-host relationship, we have developed a mouse model of CVB4-induced disease. Using two serologically indistinguishable variants of the B4 serotype, CB4-P and CB4-V, we have shown that the development of mild versus severe disease is dependent on the infecting viral strain (3). The molecular determinants of virulence of CB4-V have been identified. A threonine residue at position 129 of VP1 is a major determinant of virulence (2). An arginine residue at position 16 of VP4 also influences virulence but to a lesser extent than Thr-129 of VP1 (26).Regardless of the host’s genetic background, the CB4-P variant induces a transient inflammation of the pancreas (pancreatitis) which is followed by repair of the damaged tissues. However, the CB4-V variant induces a severe pancreatitis that can progress to chronic disease, which results in extensive and irreversible destruction of the pancreas. CB4-V infection is also lethal in some strains of mice. The outcome of infection, in B10 strains, is determined by a locus within the major histocompatibility complex (MHC) (23). During CB4-V infection, pancreatic tissue damage is probably due to a combination of mechanisms, including viral cytolysis, autodigestion by pancreatic enzymes, and immunopathology.In this study, we examined the role of the immune system in contributing to disease during infection with the virulent variant, CB4-V. The approach involved analyzing viral infection in immunologically deficient, knockout (ko) strains of mice. We showed the following: (i) CD8 T-cell responses can be beneficial during early, viral disease but detrimental during later disease; (ii) CD4 T cells contribute to the severity of disease during viral infection; (iii) the absence of interleukin-4 (IL-4) is advantageous during early viral disease but deleterious in later disease; and (iv) the outcome of viral infection can be altered by depletion of specific cellular subsets and by neutralization of specific cytokines.     

IL-12 receptor beta 1 and Toll-like receptor 4 increase IL-1 beta- and IL-18-associated myocarditis and coxsackievirus replication

Th1-type immune responses, mediated by IL-12-induced IFN-gamma, protect the host from most viral infections. To investigate the role of IL-12 and IFN-gamma on the development of Coxsackievirus B3 (CB3)-induced myocarditis, we examined the level of inflammation, viral replication, and cytokine production in IL-12Rbeta1- and IFN-gamma-deficient mice following CB3 infection. We report that IL-12Rbeta1 deficiency results in decreased viral replication and inflammation in the heart, while IFN-gamma deficiency exacerbates CB3 replication. Importantly, decreased IL-1beta and IL-18 levels in IL-12Rbeta1-deficient hearts correlated directly with decreased myocardial inflammation. Because IL-1beta and IL-18 were associated with myocardial inflammation, we examined the effect of TLR4 deficiency on CB3 infection and myocarditis. We found that TLR4-deficient mice also had significantly reduced levels of myocarditis, viral replication, and IL-1beta/IL-18, just as we had observed in IL-12Rbeta1-deficient mice. This is the first report that TLR4 influences CB3 replication. These results show that IL-12Rbeta1 and TLR4 exacerbate CB3 infection and myocarditis while IFN-gamma protects against viral replication. The remarkable similarities between the effects of IL-12Rbeta1 and TLR4 suggest that these receptors share common downstream pathways that directly influence IL-1beta and IL-18 production, and confirm that IL-1beta and IL-18 play a significant role in the pathogenesis of CB3-induced myocarditis. These findings have important implications not only for the pathogenesis of myocarditis, but for other autoimmune diseases triggered by viral infections.     

Diabetes acceleration or prevention by a coxsackievirus B4 infection: critical requirements for both interleukin-4 and gamma interferon

Type 1 diabetes acceleration in nonobese diabetic (NOD) mice through coxsackievirus B4 (CVB4) infection requires a preexisting critical mass of autoreactive T cells in pancreatic islets, and in the absence of this insulitic threshold, CVB4 infection leads to long-term disease protection. To understand this acceleration and protection process, we challenged 8- and 12-week-old NOD mice containing a disruption in interleukin-4 (IL-4) or gamma interferon (IFN-gamma) genes (NOD IL-4-/- and NOD IFN-gamma-/-, respectively) with a diabetogenic, pancreatropic Edwards strain of CVB4. The elimination of IL-4 did not alter the rate of insulitis or diabetes development in NOD mice, while the elimination of IFN-gamma delayed these events several weeks. CVB4 infection in 8-week-old mice only significantly accelerated the onset of diabetes in a subset of standard, but not IL-4- or IFN-gamma-deficient, NOD mice. Long-term diabetes protection was established in standard NOD mice as well as in the NOD IFN-gamma-/- mice that did not rapidly develop disease following CVB4 infection at 8 weeks of age. When mice were infected at 12 weeks of age, the onset of diabetes was accelerated in NOD IL-4-/- mice, while neither acceleration nor long-term protection was elicited in NOD IFN-gamma-/- mice. No differences were observed in the kinetics of CVB4 clearance in pancreases from NOD, NOD IL-4-/-, and NOD IFN-gamma-/- mice. Collectively, these results suggest that at the insulitis threshold at which CVB4 infection can first accelerate the onset of diabetes in NOD mice, IL-4 as well as IFN-gamma contributes to this pathogenic process. The protective mechanism against diabetes elicited in NOD mice infected with CVB4 prior to the development of a critical threshold level of insulitis requires neither IL-4 nor IFN-gamma.     

Cutting edge: IL-12 is required for the maintenance of IFN-gamma production in T cells mediating chronic resistance to the intracellular pathogen, Toxoplasma gondii

IL-12 is required for the development of IFN-gamma-dependent resistance to intracellular pathogens but is not thought to play a major role in its maintenance. To directly assess the requirement for continuous IL-12 signaling in long-term cell-mediated immunity, recombinant cytokine was transiently administered to IL-12 p40-deficient mice during the first 2 wk of infection with the intracellular pathogen Toxoplasma gondii. As expected, these animals survived the acute phase and established chronic infections. However, 4-6 wk after IL-12 withdrawal, the mice exhibited increased brain cyst burdens and succumbed to toxoplasmic encephalitis. Reactivation was associated with a loss of T-dependent IFN-gamma production without a concomitant increase in Th2 cytokine expression. Importantly, parasite Ag-induced IFN-gamma synthesis by purified T cells from these animals could be restored by in vitro exposure to IL-12. These results argue that endogenous IL-12 is required for the long-term maintenance of IFN-gamma-dependent resistance against intracellular pathogens.     

Expression of immunoregulatory cytokines by recombinant coxsackievirus B3 variants confers protection against virus-caused myocarditis

Clinical and laboratory investigations have demonstrated the involvement of viruses and bacteria as potential causative agents in cardiovascular disease and have specifically found coxsackievirus B3 (CVB3) to be a leading cause. Experimental data indicate that cytokines are involved in controlling CVB3 replication. Therefore, recombinant CVB3 (CVB3rec) variants expressing the T-helper-1 (T(H)1)-specific gamma interferon (IFN-gamma) or the T(H)2-specific interleukin-10 (IL-10) as well as the control virus CVB3(muIL-10), which produce only biologically inactive IL-10, were established. Coding regions of murine cytokines were cloned into the 5' end of the CVB3 wild type (CVB3wt) open reading frame and were supplied with an artificial viral 3Cpro-specific Q-G cleavage site. Correct processing releases active cytokines, and the concentration of IFN-gamma and IL-10 was analyzed by enzyme-linked immunosorbent assay and bioassays. In mice, CVB3wt was detectable in pancreas and heart tissue, causing massive destruction of the exocrine pancreas as well as myocardial inflammation and heart cell lysis. Most of the CVB3wt-infected mice revealed virus-associated symptoms, and some died within 28 days postinfection. In contrast, CVB3rec variants were present only in the pancreas of infected mice, causing local inflammation with subsequent healing. Four weeks after the first infection, surviving mice were challenged with the lethal CVB3H3 variant, causing casualties in the CVB3wt- and CVB3(muIL-10)-infected groups, whereas almost none of the CVB3(IFN-gamma)- and CVB3(IL-10)-infected mice died and no pathological disorders were detectable. This study demonstrates that expression of immunoregulatory cytokines during CVB3 replication simultaneously protects mice against a lethal disease and prevents virus-caused tissue destruction.     

Differential recruitment of B and T cells in coxsackievirus B4-induced pancreatitis is influenced by a capsid protein.

Two genetically similar variants of coxsackievirus B4, CB4-P and CB4-V, cause distinct disease syndromes in mice. A multidisciplinary approach was used to examine the events occurring in situ. The CB4-P variant induced acute pancreatitis, followed by repair of the exocrine tissues, while the CB4-V variant induced chronic pancreatitis, characterized by extensive destruction of the exocrine tissues. Since CB4-V replicated more efficiently than CB4-P in vivo, the more extensive tissue injury associated with CB4-V infection could be explained as the result of a higher level of viral replication. However, the fact that CB4-V replicated more efficiently in a mouse strain that survives infection than in a strain that succumbs to infection suggests that immune-mediated mechanisms as well as viral cytolysis may contribute to pancreatic tissue injury. To address the role of the immune system in virus-induced pancreatitis, the cell types within the inflammatory infiltrate were analyzed by flow cytometry. B cells (34 to 75%) were the most abundant, followed by T cells (10 to 30%), natural killer cells (4 to 8%), and macrophages (0 to 6%). Recruitment (and perhaps proliferation) of B and T cells to the pancreatic tissues was influenced by viral strain. Differential recruitment of T and B cells may reflect altered antigenic sites between CB4-P and CB4-V. The viral sequence that affected T- and B-cell recruitment was identified as a threonine residue at position 129 of the VP1 capsid protein.     

The tyrosine kinase p56lck is essential in coxsackievirus B3-mediated heart disease

Infections are thought to be important in the pathogenesis of many heart diseases. Coxsackievirus B3 (CVB3) has been linked to chronic dilated cardiomyopathy, a common cause of progressive heart disease, heart failure and sudden death. We show here that the sarcoma (Src) family kinase Lck (p56lck) is required for efficient CVB3 replication in T-cell lines and for viral replication and persistence in vivo. Whereas infection of wild-type mice with human pathogenic CVB3 caused acute and very severe myocarditis, meningitis, hepatitis, pancreatitis and dilated cardiomyopathy, mice lacking the p56lck gene were completely protected from CVB3-induced acute pathogenicity and chronic heart disease. These data identify a previously unknown function of Src family kinases and indicate that p56lck is the essential host factor that controls the replication and pathogenicity of CVB3.     

Therapeutic protective effects of IL-12 combined with soluble IL-4 receptor against established infections of herpes simplex virus type 1 in thermally injured mice.

The effect of combination therapy between IL-12 and soluble IL-4R (sIL-4R) on the established infection of HSV-1 in thermally injured mice (TI mice) was investigated. All of the TI mice infected with lethal amounts of HSV-1 died when IL-12 was given therapeutically at a dose of 500 U/mouse. However, 80% of these mice treated prophylactically with IL-12 survived compared with 0% survival of the same mice treated with saline. The therapeutic administration of IL-12 to TI mice currently infected with HSV-1 caused an 80% survival of these mice when the treatment was combined with sIL-4R. Although IL-12 did not stimulate IFN-gamma production in cultures of splenic T cells from TI mice, IFN-gamma was produced by stimulation with IL-12 when the producer cells were prepared from TI mice that had been treated previously with sIL-4R. After stimulation with anti-CD3 mAb, splenic T cells from TI mice with the established infection of HSV-1 produced IL-4 into their culture fluids. However, IL-4 was not produced by splenic T cells that were prepared from the same infected mice treated with IL-12 and sIL-4R in combination. The results obtained herein indicate that the efficacies of the combination therapy against the established infection of HSV-1 may result from the IFN-gamma production stimulated by IL-12 in TI mice that are treated with sIL-4R for reducing burn-associated type 2 T cell responses.     

Enhanced viral immunoinflammatory lesions in mice lacking IL-23 responses

Herpes simplex virus (HSV) infection of the cornea culminates in an immunopathological lesion (stromal keratitis--SK) that impairs vision. This report shows that HSV infection results in IL-23 up-regulation, but if this response fails to occur, as was noted in p19-/- mice, the severity of lesions, their incidence and the level of viral induced angiogenesis were significantly increased compared to wild-type (WT) animals (p<0.05). The higher disease severity in p19-/- mice appeared to be the consequence of an increased IL-12 response that in turn led to the induction of higher numbers of IFN-gamma producing CD4(+)T cells, the principal orchestrators of SK. Our results indicate that the severity of HSV induced immunopathological lesions may be mainly the consequence of IL-12 driven Th1 T cell reactions rather than the action of IL-17 producing cells controlled by IL-23.     

IL-23 provides a limited mechanism of resistance to acute toxoplasmosis in the absence of IL-12

IL-23 and IL-12 are heterodimeric cytokines which share the p40 subunit, but which have unique second subunits, IL-23p19 and IL-12p35. Since p40 is required for the development of the Th1 type response necessary for resistance to Toxoplasma gondii, studies were performed to assess the role of IL-23 in resistance to this pathogen. Increased levels of IL-23 were detected in mice infected with T. gondii and in vitro stimulation of dendritic cells with this pathogen resulted in increased levels of mRNA for this cytokine. To address the role of IL-23 in resistance to T. gondii, mice lacking the p40 subunit (common to IL-12 and IL-23) and mice that lack IL-12 p35 (specific for IL-12) were infected and their responses were compared. These studies revealed that p40(-/-) mice rapidly succumbed to toxoplasmosis, while p35(-/-) mice displayed enhanced resistance though they eventually succumbed to this infection. In addition, the administration of IL-23 to p40(-/-) mice infected with T. gondii resulted in a decreased parasite burden and enhanced resistance. However, the enhanced resistance of p35(-/-) mice or p40(-/-) mice treated with IL-23 was not associated with increased production of IFN-gamma. When IL-23p19(-/-) mice were infected with T. gondii these mice developed normal T cell responses and controlled parasite replication to the same extent as wild-type mice. Together, these studies indicate that IL-12, not IL-23, plays a dominant role in resistance to toxoplasmosis but, in the absence of IL-12, IL-23 can provide a limited mechanism of resistance to this infection.     

IL-12-independent IFN-gamma production by T cells in experimental Chagas' disease is mediated by IL-18.

IL-12p35-deficient (IL-12p35(-/-)) mice were highly susceptible to Trypanosoma cruzi infection and succumbed during acute infection, demonstrating the crucial importance of endogenous IL-12 in resistance to experimental Chagas' disease. Delayed immune responses were observed in mutant mice, although comparable IFN-gamma and TNF-alpha blood levels as in wild-type mice were detected 2 wk postinfection. In vivo and in vitro analysis demonstrated that T cells, but not NK cells, were recruited to infected organs. Analysis of mice double deficient in the recombinase-activating gene 2 (RAG2) and IL-12p35, as well as studies involving T cell depletion, identified CD4(+) T cells as the cellular source for IL-12-independent IFN-gamma production. IL-18 was induced in IL-12p35(-/-) mice and was responsible for IFN-gamma production, as demonstrated by in vivo IL-18 neutralization studies. In conclusion, evidence is presented for an IL-12-independent IFN-gamma production in experimental Chagas' disease that is T cell and IL-18 dependent.     

Ectopic expression of gamma interferon in the eye protects transgenic mice from intraocular herpes simplex virus type 1 infections.

Transgenic (rho gamma) mice provide a model for studying the influence of gamma interferon (IFN-gamma) produced in the eye on ocular and cerebral viral infection. To establish this model, we injected BALB/c- and C57BL/6-derived transgenic and nontransgenic mice of different ages intravitreally with herpes simplex virus type 1 (HSV-1) strain F. Eye and brain tissues of these mice were assessed for pathological and immunocytochemical changes. HSV-1 infection induced severe retinitis of the injected eyes and infection of the brain in all mice. In transgenic mice inoculated with HSV-1, the left, nontreated eyes were protected from retinitis, whereas nontransgenic mice developed bilateral retinitis. Additional intravitreal injection of IFN-gamma with the virus protected the noninoculated eyes of nontransgenic mice. Three-week-old nontransgenic mice died from HSV-1 infection, whereas transgenic mice of the same age and nontransgenic mice intravitreally treated with IFN-gamma survived. Ocular IFN-gamma production increased the extent of inflammation in transgenic mice but did not have a significant influence on the growth of HSV-1 until day 3 after inoculation and did not influence the neuroinvasion of this virus. Thus, the effects of IFN-gamma were not caused by an early block of viral replication. Possible mechanisms of IFN-gamma action include activation of the immune response, alteration of the properties of the virus, and direct protection of neurons.     

IL-23 p19 Knockout Mice Exhibit Minimal Defects in Responses to Primary and Secondary Infection with Francisella tularensis LVS

Our laboratory’s investigations into mechanisms of protective immunity against Francisella tularensis Live Vaccine Strain (LVS) have uncovered mediators important in host defense against primary infection, as well as those correlated with successful vaccination. One such potential correlate was IL-12p40, a pleiotropic cytokine that promotes Th1 T cell function as part of IL-12p70. LVS-infected IL-12p40 deficient knockout (KO) mice maintain a chronic infection, but IL-12p35 KO mice clear LVS infection; thus the role that IL-12p40 plays in immunity to LVS is independent of the IL-12p70 heterodimer. IL-12p40 can also partner with IL-23p19 to create the heterodimeric cytokine IL-23. Here, we directly tested the role of IL-23 in LVS resistance, and found IL-23 to be largely dispensable for immunity to LVS following intradermal or intranasal infection. IL-23p19 KO splenocytes were fully competent in controlling intramacrophage LVS replication in an in vitro overlay assay. Further, antibody responses in IL-23p19 KO mice were similar to those of normal wild type mice after LVS infection. IL-23p19 KO mice or normal wild type mice that survived primary LVS infection survived maximal doses of LVS secondary challenge. Thus p40 has a novel role in clearance of LVS infection that is unrelated to either IL-12 or IL-23.     

Using recombinant coxsackievirus B3 to evaluate the induction and protective efficacy of CD8+ T cells during picornavirus infection

Coxsackievirus B3 (CVB3) is a common human pathogen that has been associated with serious diseases including myocarditis and pancreatitis. To better understand the effect of cytotoxic T-lymphocyte (CTL) responses in controlling CVB3 infection, we have inserted well-characterized CTL epitopes into the CVB3 genome. Constructs were made by placing the epitope of interest upstream of the open reading frame encoding the CVB3 polyprotein, separated by a poly-glycine linker and an artificial 3Cpro/3CDpro cleavage site. This strategy results in the foreign protein being translated at the amino- terminus of the viral polyprotein, from which it is cleaved prior to viral assembly. In this study, we cloned major histocompatibility complex class I-restricted CTL epitopes from lymphocytic choriomeningitis virus (LCMV) into recombinant CVB3 (rCVB3). In vitro, rCVB3 growth kinetics showed a 1- to 2-h lag period before exponential growth was initiated, and peak titers were approximately 1 log unit lower than for wild-type virus. rCVB3 replicated to high titers in vivo and caused severe pancreatitis but minimal myocarditis. Despite the high virus titers, rCVB3 infection of naive mice failed to induce a strong CD8+ T-cell response to the encoded epitope; this has implications for the proposed role of "cross-priming" during virus infection and for the utility of recombinant picornaviruses as vaccine vectors. In contrast, rCVB3 infection of LCMV-immune mice resulted in direct ex vivo cytotoxic activity against target cells coated with the epitope peptide, demonstrating that the rCVB3-encoded LCMV-specific epitope was expressed and presented in vivo. The preexisting CD8+ memory T cells could limit rCVB replication; compared to naive mice, infection of LCMV-immune mice with rCVB3 resulted in approximately 50-fold-lower virus titers in the heart and approximately 6-fold-lower virus titers in the pancreas. Although the inserted CTL epitope was retained by rCVB3 through several passages in tissue culture, it was lost in an organ-specific manner in vivo; a substantial proportion of viruses from the pancreas retained the insert, compared to only 0 to 1.8% of myocardial viruses. Together, these results show that expression of heterologous viral proteins by recombinant CVB3 provides a useful model for determining the mechanisms underlying the immune response to this viral pathogen.     

Cutting edge: cross-regulation by TLR4 and T cell Ig mucin-3 determines sex differences in inflammatory heart disease

Recent clinical studies have reinforced the importance of sex-related differences in the pathogenesis of cardiovascular diseases, with an increased incidence and mortality in men. Similar to humans, male BALB/c mice infected with coxsackievirus B3 (CVB3) develop more severe inflammation in the heart even though viral replication is no greater than in females. We show that TLR4 and IFN-gamma levels are significantly elevated and regulatory T cell (Treg) populations significantly reduced in the heart of males following CVB3 infection, whereas females have significantly increased T cell Ig mucin (Tim)-3, IL-4 and Treg. Blocking Tim-3 in males significantly increases inflammation and TLR4 expression while reducing Treg. In contrast, defective TLR4 signaling significantly reduces inflammation while increasing Tim-3 expression. Cross-regulation of TLR4 and Tim-3 occurs during the innate and adaptive immune response. This novel mechanism may help explain why inflammatory heart disease is more severe in males.     

IL-4 and IL-10 antagonize IL-12-mediated protection against acute vaccinia virus infection with a limited role of IFN-gamma and nitric oxide synthetase 2

Resistance or susceptibility to most infectious diseases is strongly determined by the balance of type 1 vs type 2 cytokines produced during infection. However, for viruses, this scheme may be applicable only to infections with some cytopathic viruses, where IFN-gamma is considered as mandatory for host defense with little if any participation of type 2 responses. We studied the role of signature Th1 (IL-12, IFN-gamma) and Th2 (IL-4, IL-10) cytokines for immune responses against vaccinia virus (VV). IL-12-/- mice were far more susceptible than IFN-gamma-/- mice, and primary CTL responses against VV were absent in IL-12-/- mice but remained intact in IFN-gamma-/- mice. Both CD4+ and CD8+ T cells from IL-12-/- mice were unimpaired in IFN-gamma production, although CD4+ T cells showed elevated Th2 cytokine responses. Virus replication was impaired in IL-4-/- mice and, even more strikingly, in IL-10-/- mice, which both produced elevated levels of the proinflammatory cytokines IL-1alpha and IL-6. Thus, IL-4 produced by Th2 cells and IL-10 produced by Th2 cells and probably also by macrophages counteract efficient anti-viral host defense. Surprisingly, NO production, which is considered as a major type 1 effector pathway inhibited by type 2 cytokines, appears to play a limited role against VV, because NO sythetase 2-deficient mice did not show increased viral replication. Thus, our results identify a new role for IL-12 in defense beyond the induction of IFN-gamma and show that IL-4 and IL-10 modulate host protective responses to VV.