Susceptibility of beige mice to Mycobacterium avium: role of neutrophils.

The beige mutation in C57BL/6 mice has been shown to increase the susceptibility to infection by Mycobacterium avium. In this study, we confirmed those results and showed that the effect of the beige mutation was most obvious after infection with a strain of lower virulence than with a highly virulent isolate of M. avium. The dissemination of M. avium from the gut was observed with both C57BL/6 and beige mice but was faster in the latter. The expression of gamma interferon (IFN-gamma) and the priming for tumor necrosis factor production during an in vivo infection were similar between beige and immunocompetent C57BL/6 mice. IFN-gamma produced during the infection of beige mice was protective in the spleen, and the administration of recombinant IFN-gamma restored the resistance in the spleen to levels similar to those found in control mice. There were no histological differences between wild-type and beige mice with respect to granuloma formation in the liver. The increased susceptibility of beige mice to M. avium as manifested in the liver was reduced by transfusing neutrophils from wild-type C57BL/6 mice. Likewise, depletion of neutrophils from C57BL/6 mice rendered them as susceptible to M. avium infection of the liver as beige mice. Our results point to the participation of neutrophils in the defect of beige mice in addition to other defects. Furthermore, these results show that neutrophils play a significant role in the defense mechanisms against mycobacterial infections and that beige animals may be a useful model for study of the role of neutrophils in mycobacteriosis.     

Intranasal infection of beige mice with Mycobacterium avium complex: role of neutrophils and natural killer cells.

Beige mice show increased susceptibility to intranasal infection with organisms of the Mycobacterium avium complex (MAC) compared with their immunocompetent congenics, C57BL/6 mice. This increased susceptibility was clear 2 weeks postinfection, before the activation of the specific immune response. T lymphocytes from 4-week infected beige mice, cultured in vitro, produced amounts of gamma interferon similar to those found in cells from C57BL/6 mice. Macrophage activation, as judged by NO production and lysis of the macrophage target P815, occurred in the lungs of beige mice. Despite the inability of bone marrow-derived NK cells from beige mice to lyse NK-susceptible YAC-1 cells, their gamma interferon production was normal. Monoclonal antibody to NK1.1 was used to deplete C57BL/10 mice of lytic activity against YAC-1 cells without exacerbating infection between 2 and 6 weeks of observation, making it unlikely that any deficiency in NK cells was the cause of susceptibility in beige mice. There was a striking influx of neutrophils in the lungs of beige mice compared with C57BL/6. More than half of the MAC organisms appeared associated with the neutrophils of beige mice, while in C57BL/6 mice, most MAC organisms were associated with cells of macrophage/monocyte morphology. Injection of monoclonal antibody specific for neutrophils failed to eliminate those cells from the lungs of beige mice. However, in C57BL/6 mice, neutrophil numbers were reduced by 95% without exacerbating the infection. We conclude that, although neutrophils are not essential to the relative resistance of C57BL/6 mice, the known deficiencies in both neutrophils and macrophages account for the susceptibility of beige mice.     

Chemokine production and leukocyte recruitment to the lungs of Paracoccidioides brasiliensis-infected mice is modulated by interferon-gamma

Chemokines and chemokine receptors play a role in cell recruitment during granulomatous inflammatory reactions. Here, we evaluated the expression of chemokines and chemokine receptors and their regulation by IFN-gamma in the course of Paracoccidioides brasiliensis (Pb) infection in mice. We found an association between KC and MIP-1alpha (CCL3) production and neutrophil infiltration in the lungs of Pb-infected mice during the early acute phase of infection. High levels of RANTES/CCL5, MCP-1/CCL2, IP-10/CXCL10, and Mig/CXCL9 simultaneously with mononuclear cell infiltration in the lungs was found. In the absence of IFN-gamma (GKO mice) we observed increased production of KC and MIP-1alpha and chronic neutrophilia. Moreover, we found a change in the chemokine receptor profiles expressed by wild-type (WT) versus GKO animals. Increased expression of CXCR3 and CCR5, and low levels of CCR3 and CCR4 were observed in the lungs of Pb-infected WT mice, whereas the opposite effect was observed in the lungs of GKO mice. Consistent with these results, infected cells from WT mice preferentially migrated in response to IP-10 (CXCR3 ligand), while those from GKO mice migrated in response to eotaxin/CCL11 (CCR3 ligand). These results suggest that IFN-gamma modulates the expression of chemokines and chemokine receptors as well as the kind of cells that infiltrate the lungs of Pb-infected mice.     

Neutrophil depletion exacerbates experimental Chagas' disease in BALB/c, but protects C57BL/6 mice through modulating the Th1/Th2 dichotomy in different directions

To elucidate the roles of neutrophils in experimental Chagas' disease, we depleted the peripheral neutrophils in BALB/c and C57BL/6 mice with a monoclonal antibody 1 day before Trypanosoma cruzi infection. Neutrophil depletion in BALB/ c mice resulted in exacerbation of the disease and decreased expression of mRNA for Th1 cytokines, including IL-2 and IFN-gamma, IL-12p40 and TNF-alpha in their spleens after the infection, while a Th2 cytokine, IL-10, increased especially 1 day after infection. Neutrophils from infected BALB / c mice expressed mRNA for IL-12p40, IFN-gamma, TNF-alpha and Th1 chemoattractive chemokines, monokine induced by IFN-gamma (MIG) and macrophage inflammatory protein-1alpha (MIP-1alpha ). In contrast, in C57BL/6 mice neutrophil depletion induced resistance to the disease and enhanced the expression of the above Th1 cytokines, although IL-10 mRNA in neutrophil-depleted C57BL/6 mice was also higher than in control mice. Neutrophils from C57BL/6 mice did not express IL-12p40, IFN-gamma and MIG but expressed TNF-alpha, MIP-1alpha and IL-10. Therefore, neutrophils may play opposite roles in these two strains of mice with respect to protection versus exacerbation of T. cruzi infection, possibly through modulating the Th1/Th2 dichotomy in different directions.     

Transgenic mice expressing human interleukin-10 in the antigen-presenting cell compartment show increased susceptibility to infection with Mycobacterium avium associated with decreased macrophage effector function and apoptosis

Interleukin-10 (IL-10) is thought to play an important role in the regulation of microbial immunity. While T-cell-derived IL-10 has been shown to suppress cell-mediated immunity, there has been debate as to whether antigen presenting cell (APC)-derived cytokine can perform the same function in vivo. To assess the influence of APC-produced IL-10 on host resistance to mycobacterial infection, transgenic mice expressing human IL-10 under the control of the major histocompatibility complex class II promoter (hu10Tg) were infected with Mycobacterium avium, and bacterial burdens and immune responses were compared with those observed in wild-type (wt) animals. Hu10Tg mice harbored substantially higher numbers of M. avium and succumbed 16 to 18 weeks postinfection. The granulomas in infected hu10Tg mice showed marked increases in both acid-fast bacilli and host macrophages. In addition, these animals displayed a dramatic increase in hepatic fibrosis. The increased susceptibility of the hu10Tg mice to M. avium infection is independent of T-cell-produced endogenous murine IL-10, since bacterial burdens in mice derived by crossing hu10Tg mice with murine IL-10-deficient mice were not significantly different from those in hu10Tg mice. Importantly, gamma interferon (IFN-gamma) responses were not decreased in the infected transgenic animals from those in wt animals, suggesting the normal development of Th1 effector cells. In contrast, mycobacterium-induced macrophage apoptosis as well as production of TNF, nitric oxide, and IL-12p40 were strongly inhibited in hu10Tg mice. Together, these data indicate that APC-derived IL-10 can exert a major inhibitory effect on control of mycobacterial infection by a mechanism involving the suppression of macrophage effector function and apoptosis.     

The relative impact of bacterial virulence and host genetic background on cytokine expression during Mycobacterium avium infection of mice.

Resistance to Mycobacterium avium depends on both genetically encoded macrophage functions and acquired T-cell immunity. Cytokines may play a role in either type of resistance. We studied the expression of interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma) in naturally susceptible BALB/c (Bcgs) and naturally resistant C.D2 (Bcgr) congenic mice infected with two strains of M. avium (one highly virulent and another of low virulence). We observed that cytokine expression patterns correlated better with the virulence of the micro-organism than with the genetic background of the host. The control of the infection by the low virulence strain in either mouse strain was associated with an increased expression of IFN-gamma and IL-2. Only Bcgs mice infected with a virulent strain of M. avium were unable to restrict bacterial growth. An increased expression of IL-4, early during infection, was detected in the course of the latter infection but played no role in determining the susceptibility to infection. Neutralization of IFN-gamma or IL-2 with specific monoclonal antibodies led to an exacerbation of the infection in Bcgr mice by the two strains of M. avium and in Bcgs mice infected with the low virulence strain of M. avium.     

Intranasal interleukin-12 treatment for protection against respiratory infection with the Francisella tularensis live vaccine strain

Francisella tularensis is a gram-negative intracellular bacterium that can induce lethal respiratory infection in humans and rodents. However, little is known about the role of innate or adaptive immunity in protection from respiratory tularemia. In the present study, the role of interleukin-12 (IL-12) in inducing protective immunity in the lungs against intranasal infection of mice with the live vaccine strain (LVS) of F. tularensis was investigated. It was found that gamma interferon (IFN-gamma) and IL-12 were strictly required for protection, since mice deficient in IFN-gamma, IL-12 p35, or IL-12 p40 all succumbed to LVS doses that were sublethal for wild-type mice. Furthermore, exogenous IL-12 treatment 24 h before intranasal infection with a lethal dose of LVS (10,000 CFU) significantly decreased bacterial loads in the lungs, livers, and spleens of wild-type BALB/c and C57BL/6 mice and allowed the animals to survive infection; such protection was not observed in IFN-gamma-deficient mice. The resistance induced by IL-12 to LVS infection was still observed in NK cell-deficient beige mice but not in CD8-/- mice. These results demonstrate that exogenous IL-12 delivered intranasally can prevent respiratory tularemia through a mechanism that is at least partially dependent upon the expression of IFN-gamma and CD8 T cells.     

Cytokine and chemokine transcription profile during Mycoplasma pulmonis infection in susceptible and resistant strains of mice: macrophage inflammatory protein 1beta (CCL4) and monocyte chemoattractant protein 2 (CCL8) and accumulation of CCR5+ Th cells

The progression of murine mycoplasma pneumonia is dependent on T cells and other immune cells. The role of cytokines in immunity are complex, and identifying the network of cytokines produced after infection of mice is essential in dissecting the key cytokine cascades involved mycoplasma disease pathogenesis. In the present study, mRNA expression of 143 different cytokines, chemokines, or receptors were evaluated in lung tissues from both susceptible (BALB/c and C3H/HeN) and resistant (C57BL/6) mice after Mycoplasma pulmonis infection. To accomplish this, membrane-based cDNA microarrays were used to monitor changes mRNA expression in lungs. There was a clear association with disease susceptibility and development of cytokine mRNA expression. In addition to proinflammatory cytokines, mRNA expression of an anti-inflammatory cytokine, interleukin-10, increased with disease severity, suggesting an attempt to moderate the severity of the inflammatory response. Furthermore, it is clear that an array of chemokines produced in susceptible mice could contribute to the recruitment and maintenance of inflammatory cells at the site of disease. In support of this, there was an increase in macrophage inflammatory protein 1beta (MIP-1beta; CCL4) and monocyte chemoattractant protein 2 (MCP-2; CCL8) mRNA levels from mycoplasma-infected mice and a corresponding accumulation of CD4+ Th cells expressing the MIP-1beta/MCP-2 receptor, CCR5, in the lungs of mice. Furthermore, MIP-1beta- and MCP-2-producing cells and CD4+ T cells were found to be in close association in pulmonary lesions. Thus, there was a significant cytokine response associated with disease pathogenesis, and these studies provide important leads and insights into ongoing cytokine- and chemokine-mediated processes in this persistent inflammatory disease.     

Mouse Strain-Dependent Chemokine Regulation of the Genital Tract T Helper Cell Type 1 Immune Response

Vaginal infection with the mouse pneumonitis agent of Chlamydia trachomatis (MoPn) produces shorter courses of infection in C57BL/6 and BALB/c mice than in C3H/HeN mice, while C57BL/6 mice are more resistant to oviduct pathology. A robust Th1 response is extremely important in host defense against chlamydia. In this study we examined gamma interferon (IFN-gamma), interleukin 10 (IL-10), and the T-cell-regulatory chemokines macrophage inflammatory protein-1alpha (MIP-1alpha) and monocyte chemoattractant protein-1 (MCP-1) to determine if differences in these responses were associated with the differential courses of infection seen in these three strains of mice. Increased and prolonged IFN-gamma responses and lower IL-10 responses were observed in the C57BL/6 strain compared to BALB/c and C3H. Examination of genital tract chemokines revealed a marked predominance of MIP-1alpha over MCP-1 only in the C57 strain. Thus, a pattern of high MIP-1alpha and low MCP-1 levels during the first week of infection is associated with an increased Th1 response and a shorter, more benign chlamydial infection. Inhibition of the MCP-1 response in C3H mice increased their later T-cell production of IFN-gamma but decreased their early IFN-gamma response and had no effect on the course or outcome of infection. Inhibition of MCP-1 is not beneficial in chlamydial infection because of its pleiotropic effects.     

Mouse strain differences in the chemokine response to acute lung infection with a murine gammaherpesvirus

Numerous mouse strain-based differences in the immune response and in susceptibility to numerous pathogens have been described, but it is not known if these differences extend to chemokine responses to viral infection of the lungs. To define mouse strain-based differences in the host chemokine response and susceptibility to infection with murine gammaherpesvirus-68 (MHV-68), we compared the induced chemokine response to MHV-68 infection in the lungs of BALB/c and C57BL/6 mice at 1-15 days post-infection. CC and CXC chemokines were induced in both BALB/c and C57BL/6 following infection but the level of chemokine induction was significantly higher in the BALB/c mice for all chemokines measured. In addition, interferon-gamma (IFN-gamma) was also induced to a significantly higher level in the lungs of BALB/c infected mice compared to C57BL/6 mice. Interestingly, viral gene expression was lower in the lungs of C57BL/6 mice during the acute phase of replication. Titers of infectious virus were also greater in BALB/c lungs, although they did not achieve statistical significance. In contrast, latent viral load in the spleen, as measured by quantitative real-time PCR, did not significantly differ between mouse strains, suggesting that the establishment of latency is not affected by the amount of virus present during acute infection. This data suggests that robust chemokine response and expression of IFN-gamma in the lungs of infected BALB/c mice does not correlate with increased resistance to infection. In addition, the significant differences in chemokine responses observed will be important factors to consider in future studies of viral pathogenesis using mouse models.     

Endogenous Inhibition of Antimycobacterial Immunity by IL-10 Varies between Mycobacterial Species

Interleukin (IL)-10 is an immunoregulatory cytokine that inhibits both Th1-like T cell responses and macrophage activation. Deficiency of IL-10 has been associated with increased Th1-like CD4+ T-cell responses and increased clearance of some intracellular pathogens, however, its role in mycobacterial infections is controversial. In order to examine the effects of mycobacterial virulence on the outcome of infection we compared infection with Mycobacterium avium and virulent Mycobacterium tuberculosis in C57Bl/6 IL-10-/- mice. M. avium infection in IL-10-/- mice resulted in sustained increases in interferon (IFN)-gamma-secreting T-cell responses and was associated with the increased clearance of M. avium from the liver and lung. By contrast, M. tuberculosis infection in IL-10-/- mice led to a transient increase in IFN-gamma T-cell responses at 4 weeks postinfection, with reduced bacterial burden in the lungs. This was not sustained so that by 8 weeks there was no difference to wild-type (WT) mice. In vitro infection of IL-10-/- macrophages with M. avium, but not M. tuberculosis, led to an increased IL-12 production. Therefore, endogenous IL-10 exerts a significant inhibition on specific IFN-gamma T-cell responses to M. avium infection, however, this effect is short lived during the M. tuberculosis infection, and fails to influence the long-term course of infection.     

L-tryptophan-L-kynurenine pathway metabolism accelerated by Toxoplasma gondii infection is abolished in gamma interferon-gene-deficient mice: cross-regulation between inducible nitric oxide synthase and indoleamine-2,3-dioxygenase.

L-Tryptophan degradation by indoleamine 2,3-dioxygenase (IDO) might have an important role in gamma interferon (IFN-gamma)-induced antimicrobial effects. In the present study, the effects of Toxoplasma gondii infection on IDO were investigated by using wild-type and IFN-gamma-gene-deficient (knockout) (IFN-gamma KO) mice. In wild-type C57BL/6J mice, enzyme activities and mRNA levels for IDO in both lungs and brain were markedly increased and lung L-tryptophan concentrations were dramatically decreased following T. gondii infection. In contrast, these metabolic changes did not occur in T. gondii-infected IFN-gamma KO mice or in uninfected IFN-gamma KO mice. The levels of inducible nitric oxide synthase (iNOS) induction in infected IFN-gamma KO mice were high in lungs and low in brain compared to those in infected wild-type mice. The extent of increased mRNA expression of T. gondii surface antigen gene 2 (SAG2) induced in lungs and brain by T. gondii infection was significantly enhanced in IFN-gamma KO mice compared to wild-type mice on day 7 postinfection. Treatment with N-nitro-L-arginine methyl ester, an iNOS inhibitor, increased the levels of SAG2 mRNA in brain but not in lungs and of plasma L-kynurenine after T. gondii infection. This in vivo study provides evidence that L-tryptophan depletion caused by T. gondii is directly mediated by IFN-gamma in the lungs, where iNOS is not induced by IFN-gamma. This study suggests that there is an antitoxoplasma mechanism of cross-regulation between iNOS and IDO and that the expression of the main antiparasite effector mechanisms for iNOS and/or IDO may vary among tissues.     

Regulatory Effects of Macrophage Inflammatory Protein 1α/CCL3 on the Development of Immunity to Cryptococcus neoformans Depend on Expression of Early Inflammatory Cytokines

Macrophage inflammatory protein 1alpha (MIP-1alpha)/CCL3 prevents the development of eosinophilic pneumonia (EP) driven by a nonprotective T2-type immunity during infection with a highly virulent strain of Cryptococcus neoformans. The present study evaluated the interaction of MIP-1alpha with other innate immune system cytokines by comparing the immune responses that followed pulmonary infections with high- (C. neoformans 145A) and low (C. neoformans 52D)-virulence strains. In contrast to what was found for C. neoformans 145A infection, lack of MIP-1alpha in C. neoformans 52D infection did not cause the development of EP. C. neoformans 52D induced tumor necrosis factor alpha (TNF-alpha), gamma interferon (IFN-gamma), and MCP-1 in the lungs of infected wild-type (WT) and MIP-1alpha knockout (KO) mice by day 7 postinfection. Both WT and MIP-1alpha KO mice subsequently cleared this infection. Thus, the robust expression of early inflammatory cytokines in C. neoformans 52D-infected mice promoted the development of protective immunity even in the absence of MIP-1alpha. Alternatively, C. neoformans 145A-infected WT and MIP-1alpha KO mice had diminished TNF-alpha, IFN-gamma, and macrophage chemoattractant protein 1 (MCP-1) responses, indicating that virulent C. neoformans 145A evaded early innate host defenses. However C. neoformans 145A-infected WT mice had an early induction of MIP-1alpha and subsequently did not develop EP. In contrast, C. neoformans 145A-infected MIP-1alpha KO mice developed EP and had increased C. neoformans dissemination into the brain by day 35. We conclude that, in the absence of other innate immune response effector molecules, MIP-1alpha is crucial to prevent the development of EP and to control C. neoformans dissemination to the brain.     

Differential chemokine response of murine macrophages stimulated with cytokines and infected with Listeria monocytogenes

During inflammatory processes the infected macrophage is a rich source of chemokines which induce infiltration of leukocytes to the site of infection. We investigated the regulation of chemokine production by murine macrophages in response to infection with the intracellular bacterial pathogen, Listeria monocytogenes. As a source of quiescent macrophages, murine bone marrow-derived macrophages (BMM) cultured under serum-free conditions were used. With RT-PCR, we detected induction of RNA message for the chemokines macrophage inflammatory protein (MIP)-2, KC, MIP-1alpha, MIP-1beta, IFN-gamma-inducible protein- 10 and RANTES in L. monocytogenes-infected macrophages. Accordingly, ELISA-detectable MIP-1alpha, MIP-2 and KC protein was induced by infection with L. monocytogenes. In contrast, L. monocytogenes infection of BMM alone failed to induce considerable expression of monocyte chemoattractant protein (MCP)-1 at the mRNA or protein level, but co-treatment with IFN-gamma was necessary. Release of infection- triggered MIP-2, MIP-1alpha and KC was negatively regulated by IFN- gamma. Similarly, IL-4 stimulated MCP-1 release by infected macrophages but reduced production of MIP-1alpha, MIP-2 and KC. IL-10 turned out to be a general deactivator in terms of macrophage chemokine production. IL-13 had no effect on MIP-1alpha, MIP-2 and KC production by infected BMM, but slightly reduced MCP-1 release. By using IFN-gamma and IL-4 gene deletion mutant mice, in vivo regulation of these chemokines by IL- 4 and IFN-gamma in listeriosis was studied. In summary, our results show that chemokines are produced by macrophages infected with L. monocytogenes, and that chemokine release is differentially regulated by the macrophage modulators IFN-gamma, IL-4, IL-10 and IL-13.      

Induction of experimental autoimmune encephalomyelitis in C57BL/6 mice deficient in either the chemokine macrophage inflammatory protein-1alpha or its CCR5 receptor

Macrophage inflammatory protein (MIP)-1alpha is a chemokine that is associated with Th1 cytokine responses. Expression and antibody blocking studies have implicated MIP-1alpha in multiple sclerosis (MS) and in experimental autoimmune encephalomyelitis (EAE). We examined the role of MIP-1alpha and its CCR5 receptor in the induction of EAE by immunizing C57BL / 6 mice deficient in either MIP-1alpha or CCR5 with myelin oligodendrocyte glycoprotein (MOG). We found that MIP-1alpha-deficient mice were fully susceptible to MOG-induced EAE. These knockout animals were indistinguishable from wild-type mice in Th1 cytokine gene expression, the kinetics and severity of disease, and infiltration of the central nervous system by lymphocytes, macrophages and granulocytes. RNase protection assays showed comparable accumulation of mRNA for the chemokines interferon-inducible protein-10, RANTES, macrophage chemoattractant protein-1, MIP-1beta, MIP-2, lymphotactin and T cell activation gene-3 during the course of the disease. CCR5-deficient mice were also susceptible to disease induction by MOG. The dispensability of MIP-1alpha and CCR5 for MOG-induced EAE in C57BL / 6 mice supports the idea that differential chemokine expression patterns represent differences in disease mechanism that underlie various models of EAE, and possibly distinct patterns of pathology seen in MS.     

Chemokine regulation of the inflammatory response to a low-dose influenza infection in CCR2-/- mice

Influenza virus infections induce chemokines and cytokines, which regulate the immune response. The chemokine receptor CCR2 plays an important role in macrophage recruitment and in the development of T1 immunity. In the present study, we addressed the role of CCR2 in influenza A virus infection. CCR2 knockout (-/-) mice are protected against influenza A virus infection, despite delayed recruitment of macrophages. We show that low-dose influenza infection of CCR2-/- mice leads to increased neutrophilia between Days 5 and 10 after infection and decreased monocyte/macrophage and CD4(+) T cell recruitment to the lungs between Days 5 and 7 after infection. These changes in leukocyte recruitment did not result from or cause increased viral titers or delayed viral clearance. Neutrophilia in the lungs correlated with increased keratinocyte-derived chemokine (KC) and/or MIP-2 expression in CCR2-/- mice between Days 5 to 10 after infection, although the kinetics of neutrophil recruitment was not altered. MIP-2 mRNA and protein expression was increased three- to fivefold, and KC protein levels were increased two- to threefold in CCR2-/- compared with CCR2 wild-type mice at Day 5 after infection. This preceded the peak neutrophil influx, which occurred 7 days after infection. In vitro studies confirmed that MIP-2 and KC accounted for neutrophil chemotactic activity in the bronchoalveolar lavage. CCR2 deficiency also resulted in increased MIP-1alpha, MIP-1beta, MCP-1, and IFN-inducible protein 10 and decreased RANTES mRNA expression. Furthermore, IL-6 and TNF-alpha cytokine production were elevated after infection. These studies suggest that CCR2 plays a multifactorial role in the development of the immune response to influenza.     

Interleukin-23 (IL-23)-IL-17 cytokine axis in murine Pneumocystis carinii infection

Host defense mechanisms against Pneumocystis carinii are not fully understood. Previous work in the murine model has shown that host defense against infection is critically dependent upon host CD4(+) T cells. The recently described Th17 immune response is predominantly a function of effector CD4(+) T cells stimulated by interleukin-23 (IL-23), but whether these cells are required for defense against P. carinii infection is unknown. We tested the hypothesis that P. carinii stimulates the early release of IL-23, leading to increases in IL-17 production and lung effector CD4(+) T-cell population that mediate clearance of infection. In vitro, stimulation of alveolar macrophages with P. carinii induced IL-23, and IL-23p19 mRNA was expressed in lungs of mice infected with this pathogen. To address the role of IL-23 in resistance to P. carinii, IL-23p19-/- and wild-type control C57BL/6 mice were infected and their fungal burdens and cytokine/chemokine responses were compared. IL-23p19-/- mice displayed transient but impaired clearance of infection, which was most apparent 2 weeks after inoculation. In confirmatory studies, the administration of either anti-IL-23p19 or anti-IL-17 neutralizing antibody to wild-type mice infected with P. carinii also caused increases in fungal burdens. IL-17 and the lymphocyte chemokines IP-10, MIG, MIP-1alpha, MIP-1beta, and RANTES were decreased in the lungs of infected IL-23p19-/- mice in comparison to their levels in the lungs of wild-type mice. In IL-23p19-/- mice infected with P. carinii, there were fewer effector CD4(+) T cells in the lung tissue. Collectively, these studies indicate that the IL-23-IL-17 axis participates in host defense against P. carinii.     

Microglia produce IFN-gamma independently from T cells during acute toxoplasmosis in the brain.

We previously reported a requirement of interferon-gamma (IFN-gamma) production by both T cells and cells other than T or natural killer (NK) cells in the brain for prevention of toxoplasmic encephalitis. In the present study, we examined whether microglia, the resident macrophage system in the brain, produce IFN-gamma in response to infection with Toxoplasma gondii in SCID and wild-type BALB/c mice. IFN-gamma was detected in the culture supernatants of microglia purified from the brains of SCID mice that had developed toxoplasmic encephalitis due to reactivation of infection. A significant increase in numbers of IFN-gamma-expressing microglia was also observed by immunostaining for this cytokine in the brains of SCID and BALB/c mice during the acute stage of acquired infection, and those numbers decreased in the later stage of infection in the BALB/c animals. These results indicate that microglia produce IFN-gamma in the presence and absence of T cells in response to reactivated or acute acquired infection in the brain. Because IFN-gamma is the essential effector molecule to control tachyzoites and because this cytokine is a potent inducer of expression of chemokines and MHC antigens important for recruitment and activation of T cells, IFN-gamma production by microglia might play a critical role in the early stage of tachyzoite proliferation in the brain by limiting parasite growth and initiating subsequent T cell immunity.     

Interferon-gamma-dependent mechanisms of mycobacteria-induced pulmonary immunopathology: the role of angiostasis and CXCR3-targeted chemokines for granuloma necrosis

The mechanisms leading to granuloma caseation, a hallmark of tuberculosis (TB) in humans, are poorly understood. Lung histopathology of C57BL/6 (WT) mice 16 weeks after aerosol infection with Mycobacterium avium strain TMC724 is uniquely characterized by centrally necrotizing granulomas, strongly resembling human TB lesions. However, IFN-gamma-deficient (GKO) and IFN-gamma-receptor-deficient (GRKO) mice did not develop granuloma necrosis following M. avium infection. Comparison of differentially expressed genes in infected WT and GKO lungs by DNA microarray and RNase protection assays revealed that the angiostatic chemokines CXCL9-11 were significantly reduced in GKO mice. In contrast, angiogenic mediators such as angiopoietin and vascular endothelial growth factor, and angiogenic chemokines such as CXCL2, CCL3, and CCL4, remained unchanged or were expressed at higher levels than in infected WT mice, suggesting impaired neovascularization of the granuloma as a possible mechanism for caseation in WT mice. Granuloma vascularization was significantly decreased in central, but not peripheral, areas of granulomas of infected WT compared to GKO mice. In contrast to GRKO mice, WT mice showed signs of severe hypoxia in cells immediately surrounding the necrotic core of granulomas as measured immunohistochemically with a reagent detecting pimonidazole adducts. To test the hypothesis that CXCR3, the common receptor for the angiostatic chemokines CXCL9-11, is involved in granuloma caseation, histomorphology was assessed in M. avium-infected mice deficient for CXCR3 (CXCR3-KO). 16 weeks after infection, these mice developed caseating granulomas similar to WT mice. We conclude that IFN-gamma causes a dysbalance between angiostatic and angiogenic mediators and a concomitant reduction in granuloma vascularization, but that CXCR3-targeted chemokines are not sufficient to induce granuloma necrosis in a mouse model of mycobacteria-induced immunopathology.