Transient neutralization of tumor necrosis factor alpha can produce a chronic fungal infection in an immunocompetent host: potential role of immature dendritic cells

The mechanisms underlying induction of immune dysregulation and chronic fungal infection by a transient tumor necrosis factor alpha (TNF-alpha) deficiency remain to be defined. The objective of our studies was to determine the potential contribution of neutropenia and immature dendritic cells to the immune deviation. Administration of an anti-TNF-alpha monoclonal antibody at day 0 neutralized TNF-alpha only during the first week of a pulmonary Cryptococcus neoformans infection. Transient neutralization of TNF-alpha resulted in transient depression of interleukin-12 (IL-12), monocyte chemotactic protein 1 (MCP-1), and gamma interferon (IFN-gamma) production but permanently impaired long-term clearance of the infection from the lungs even after the levels of these cytokines increased and a vigorous inflammatory response developed. Early neutrophil recruitment was defective in the absence of TNF-alpha. However, as demonstrated by neutrophil depletion studies, this did not account for the decrease in IL-12 and IFN-gamma levels and did not play a role in establishing chronic pulmonary cryptococcal infection. Transient TNF-alpha neutralization also produced a deficiency in CD11c(+) MHC II(+) cells and IL-12 in the lymph nodes, potentially implicating a defect in mature dendritic cell trafficking. Transfer of cryptococcal antigen-pulsed immature dendritic cells into naive mice prior to intratracheal challenge resulted in the development of a nonprotective immune response to C. neoformans that was similar to that observed in anti-TNF-alpha-treated mice (increased IL-4, IL-5, and IL-10 levels, pulmonary eosinophilia, and decreased clearance). Thus, stimulation of an antifungal response by immature dendritic cells can result in an immune deviation similar to that produced by transient TNF-alpha deficiency, identifying a new mechanism by which a chronic fungal infection can occur in an immunocompetent host.     

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.     

Stimulation via Toll-like receptor 9 reduces Cryptococcus neoformans-induced pulmonary inflammation in an IL-12-dependent manner

Cytosine-phosphate-guanosine-containing oligodeoxynucleotides (CpG ODN) are important vaccine adjuvants that promote Th1-type immune responses. Cryptococcus neoformans is a serious human pathogen that replicates in the lung but may disseminate systemically leading to meningitis, particularly in immunocompromised individuals. Immunization of susceptible C57BL/6 mice with CpG ODN deviates the immune response from a Th2- toward a Th1-type response following infection with C. neoformans. CpG also induces IL-12, TNF, MCP-1 and macrophage nitric oxide production. CD4(+) and CD8(+) T cells producing IFN-gamma increase in frequency, while those producing IL-5 decrease. More importantly, pulmonary eosinophilia is significantly reduced, an effect that depends on IL-12 and CD8(+) T cells but not NK cells. CpG treatment also reduces the burden of C. neoformans in the lung, an effect that is IL-12-, NK cell- and T cell-independent and probably reflects a direct effect of CpG on pathogen opsonization or an enhancement of macrophage antimicrobial activity. An equivalent beneficial effect is also observed when CpG ODN treatment is delivered during established cryptococcal disease. This is the first study documenting that promotion of lung TLR9 signaling using synthetic agonists enhances host defense. Activation of innate immunity has clear therapeutic potential and may even be beneficial in patients with acquired immune deficiency.     

Role of tumor necrosis factor and gamma interferon in acquired resistance to Cryptococcus neoformans in the central nervous system of mice.

Although naive C.B-17 and BALB/cBy mice die of meningoencephalitis within 5 weeks of intravenous infection with an opportunistic strain of Cryptococcus neoformans, immunized mice express an acquired, CD4+ T-cell-dependent immunity and survive an intravenous infection. Infusion of lymphocytes from immune mice into severe combined immunodeficiency (SCID) mice renders these mice more resistant to cryptococcal brain infection than uninfused controls. We have investigated the role of gamma interferon (IFN-gamma) and tumor necrosis factor (TNF) in acquired resistance to C. neoformans. Neutralization of either IFN-gamma or TNF impaired resistance of immune BALB/cBy or C.B-17 mice to cryptococci. At 10 days postinfection, there were approximately 10 times as many yeast cells in the brains of mice treated with either anticytokine antibody as in the brains of mice treated with control antibody. Simultaneous neutralization of IFN-gamma and TNF further exacerbated infection. Neutralization of IFN-gamma or TNF also impaired resistance in immune lymphocyte-infused SCID mice, resulting in significantly higher yeast burdens in brains of cytokine-neutralized mice than in brains of controls. Concurrent neutralization of IFN-gamma and TNF rendered SCID recipients of immune cells equivalent to uninfused SCID mice with respect both to brain yeast burdens at 10 days and to survival. Anti-TNF treatment alone also curtailed survival. Histological examination of the brains of cytokine-neutralized mice revealed deficiencies in ability to focus inflammatory cells at brain lesions. These data demonstrate that both IFN-gamma and TNF are important mediators of acquired resistance to cryptococcal meningoencephalitis.     

Roles of interleukin-12 and gamma interferon in murine Chlamydia pneumoniae infection

BALB/c and strain 129 mice infected intranasally with Chlamydia pneumoniae displayed a moderate-to-severe inflammation in the lungs and produced interleukin-12 (IL-12), gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and IL-10, with peak levels on days 1 to 3 postinfection (p.i.), returning to basal levels by day 16 p.i. Anti-IL-12 treatment resulted in less-severe pathological changes but higher bacterial titers on days 3 and 7 p.i. By day 16 p.i., the inflammatory responses of control antibody-treated mice subsided. The bacterial titers of both anti-IL-12- and control antibody-treated mice decreased within 3 weeks to marginally detectable levels. Anti-IL-12 treatment significantly reduced lung IFN-gamma production and in vitro spleen cell IFN-gamma production in response to either C. pneumoniae or concanavalin A. In gamma-irradiated infected mice, cytokine production was delayed, and this delay correlated with high bacterial titers in the lungs. Following C. pneumoniae infection, 129 mice lacking the IFN-gamma receptor alpha chain gene (G129 mice) produced similar IL-12 levels and exhibited similarly severe pathological changes but had higher bacterial titers than 129 mice. However, by day 45 p.i., bacterial titers became undetectable in both wild-type 129 and G129 mice. Thus, during C. pneumoniae lung infection, IL-12, more than IFN-gamma, plays a role in pulmonary-cell infiltration. IFN-gamma and IL-12, acting mostly through its induction of IFN-gamma and Th1 responses, play an important role in controlling acute C. pneumoniae infection in the lungs, but eventually all mice control the infection to undetectable levels by IL-12- and IFN-gamma-independent mechanisms.     

Interleukin-12 enhances murine survival against acute toxoplasmosis.

Protective immunity against Toxoplasma gondii is mediated by the host cellular immune response. Interleukin-12 (IL-12), a recently described cytokine that stimulates NK cells to produce gamma interferon (IFN-gamma), is able to enhance host protection against this parasite in SCID mice. Administration of IL-12 to A/J mice significantly increased survival over that of control mice when IL-12 was delivered early in the course of acute infection. If it was administered at day 3 or thereafter, there was no observed difference in mortality between treated and control mice. Antibody depletion of IL-12 increased susceptibility to infection, as measured by mortality, only when the IL-12 was administered before day 3 postinfection. Mice treated with IL-12 at day 0 postinfection exhibited a significant rise above the control in both IL-2 and IFN-gamma production. Once infection has been established in the host (3 days), administration of exogenous IL-12 is unable to alter parasite-induced downregulation of IFN-gamma production. Thus, IL-12 appears to play an important, but transitory, role in protection against acute infection with T. gondii in the normal murine host.     

Neutropenia alters lung cytokine production in mice and reduces their susceptibility to pulmonary cryptococcosis

Neutrophils are generally considered to contribute to host defense through their potent microbicidal activity. However, there is accumulating evidence that neutrophils also have an important regulatory role in establishing the balance of Th1 and Th2 responses. This study investigated the role of neutrophils in defense against pulmonary Cryptococcus neoformans infection using neutrophil-depleted BALB/c mice generated by administering mAb RB6-8C5. Neutropenic mice with pulmonary infection survived significantly longer than control mice, but there was no difference between groups infected intravenously. On day 1 of infection, neutropenic mice had significantly smaller fungal burdens than control mice. On day 7, neutropenic mice had significantly higher lung concentrations of IL-10, TNF-alpha, IL-4, and IL-12 than control mice, but there was no difference in IFN-gamma and MCP-1 levels. Neutrophils influenced the outcome of cryptococcal infection in mice through mechanisms that did not involve a reduction in early fungal burden. The absence of neutrophils in lung tissue during the initial stages of infection appeared to alter the inflammatory response in a manner that was subsequently beneficial to the host. Higher levels of Th1- and Th2-associated cytokines in neutropenic mice could have simultaneously promoted a strong cellular response while reducing inflammatory damage to the lung. Our results support the emerging concept that neutrophils play an important function in modulating the development of the immune response.     

Sex-determined resistance to Toxoplasma gondii is associated with temporal differences in cytokine production.

Examination of a wide range of inbred mice of diverse genetic backgrounds and major histocompatibility complex haplotypes revealed a dramatic difference in the susceptibilities of males and females to Toxoplasma gondii infection. Female mice were found to be more susceptible to acute infection, as determined by higher mortality levels, than male mice, while those female mice surviving to have chronic infections harbored more cysts in their brains than did surviving males. This phenomenon was therefore investigated in greater depth immunologically in the BALB/K mouse, a strain showing moderate susceptibility to infection with T. gondii. Plasma tumor necrosis factor alpha (TNF-alpha) levels were elevated in both male and female BALB/K mice on days 8 and 10 postinfection, but not thereafter, with males producing significantly higher levels than females. However, it was not until day 12 postinfection that the first deaths occurred, and these were among female mice, indicating that TNF-alpha production was not responsible for mortality. In vitro examination of T. gondii-specific T-cell proliferative responses from day 15 postinfection onwards revealed significantly higher stimulation indices in male mice than in their female counterparts. This difference was most apparent in splenocyte cultures initiated at day 15 postinfection, where complete suppression of proliferation was noted in the splenocytes from female mice but not from male mice. Analysis of tissue culture supernatants from these cultures revealed distinct differences in the kinetics of production as well as the quantities of gamma interferon (IFN-gamma) and interleukin 10 (IL-10) produced. Spleen cells from male mice produced higher levels of IFN-gamma in the early stages of infection than those from female mice. IFN-gamma levels were highest in the supernatants from male splenocyte cultures initiated at day 15 postinfection. Similar levels of IFN-gamma were not obtained from the supernatants of female splenocyte cultures until day 22 postinfection. IL-10 production, on the other hand, peaked at maximal levels in the cell cultures from both sexes initiated at day 22 postinfection. These results suggest that, in male mice, a rapid response to infection with high levels of TNF-alpha and IFN-gamma helps to control parasite multiplication, after which IL-10 production may be important in down regulating these potentially harmful inflammatory mediators. The failure of female mice to respond quickly in terms of T-cell proliferation and IFN-gamma production compared with their male counterparts may account for their poor survival rates and higher cyst burdens.     

Immune responses to Yersinia enterocolitica in susceptible BALB/c and resistant C57BL/6 mice: an essential role for gamma interferon.

Susceptibility of mice to infection with Yersinia enterocolitica has been shown to be related to neither the Ity locus encoding for resistance to Salmonella typhimurium and other pathogens nor the H-2 locus. Recent studies in our laboratory have demonstrated that T-cell-mediated immune responses are required for overcoming primary Yersinia infection. In the present study, we investigated the course of infection with Y. enterocolitica and the resulting immune responses in Yersinia-susceptible BALB/c and Yersinia-resistant C57BL/6 mice. In the early phase of infection, the clearance of the pathogen was comparable in both strains of mice, suggesting similar mechanisms of innate resistance. Splenic T cells from Yersinia-infected C57BL/6 mice exhibited marked proliferative responses and produced gamma interferon (IFN-gamma) upon exposure to heat-killed yersiniae. By contrast, the Yersinia-specific T-cell response in BALB/c mice was weak, and IFN-gamma production could not be detected before day 21 postinfection. T cells isolated from C57BL/6 mice 7 days after infection mediated immunity to Y. enterocolitica but those from BALB/c mice did not, while at 21 days postinfection T cells from both strains mediated protection. Neutralization of IFN-gamma abrogated resistance to yersiniae in C57BL/6 mice but to a far smaller extent in BALB/c mice. Administration of recombinant IFN-gamma or anti-interleukin-4 antibodies rendered BALB/c mice resistant to yersiniae, whereas this treatment did not significantly affect the course of the infection in C57BL/6 mice. These results indicate that the cellular immune response, in particular the production of IFN-gamma by Yersinia-specific T cells, is associated with resistance of mice to Y. enterocolitica.     

The gamma interferon receptor is required for the protective pulmonary inflammatory response to Cryptococcus neoformans

Mice with a null deletion mutation in the gamma interferon (IFN-gamma) receptor gene were used to study the role of IFN-gamma responsiveness during experimental pulmonary cryptococcosis. Cryptococcus neoformans was inoculated intratracheally into mice lacking the IFN-gamma receptor gene (IFN-gammaR-/-) and into control mice (IFN-gammaR+/+). The numbers of CFU in lung, spleen, and brain were determined to assess clearance; cytokines produced by lung leukocytes were measured, and survival curves were generated. In the present study, we demonstrate the following points. (i) IFN-gammaR-/- mice are markedly more susceptible to C. neoformans infection than IFN-gammaR+/+ mice. (ii) In the absence of IFN-gamma signaling, pulmonary CFU continue to increase over the course of infection, and the infection disseminates to the brain. (iii) In the absence of IFN-gamma receptor, recruitment of inflammatory cells in response to pulmonary cryptococcal infection is not impaired. (iv) At week 5 postinfection, IFN-gammaR-/- mice have recruited greater numbers of leukocytes into their lungs, with neutrophils, eosinophils, and lymphocytes accounting for this cellular increase. (v) IFN-gamma signaling is required for the development of a T1 over a T2 immune response in the lung following cryptococcal infection. These results indicate that in the absence of IFN- gamma responsiveness, even though the recruitment of pulmonary inflammatory cells is not impaired and the secretion of IFN-gamma is not affected, IFN-gammaR-/- mice do not have the ability to resolve the cryptococcal infection. In conclusion, our data suggest that proper functional IFN-gamma signaling, possibly through a mechanism which inhibits the potentially disease-promoting T2 response, is required for mice to confine the cryptococcal infection.     

CpG oligodeoxynucleotides promote the host protective response against infection with Cryptococcus neoformans through induction of interferon-gamma production by CD4+ T cells

In the present study, we elucidated the effect of synthetic CpG-containing oligodeoxynucleotides (ODN) on pulmonary and disseminated infection caused by Cryptococcus neoformans. CDF-1 mice were inoculated intratracheally with a highly virulent strain of this pathogen, which resulted in massive bacterial growth in the lung, dissemination to the brain and death. Administration of CpG-ODN promoted the clearance of C. neoformans in the lungs, decreased their dissemination to brain and prolonged the survival of infected mice. These effects correlated well with the enhanced production of interleukin (IL)-12 and interferon (IFN)-gamma and attenuated secretion of IL-4 in bronchoalveolar lavage fluids (BALF) and promoted development of Th1 cells, as indicated by the increased production of IFN-gamma by paratracheal lymph node cells upon restimulation with cryptococcal antigens. The IFN-gamma synthesis in BALF was inhibited by depletion of CD8(+) and CD4(+) T cells on days 7 and 14 after infection, respectively, but not by depletion of NK and gammadelta T cells. Consistent with these data, intracellular expression of IFN-gamma was detected predominantly in CD8(+) and CD4(+) T cells in the lung on days 7 and 14, respectively. The protective effect of CpG-ODN, as shown by the prolonged survival, was completely and partially inhibited by depletion of CD4(+) or CD8(+) T cells, respectively, but not by depletion of other cells. Finally, TNF-alpha was markedly induced by CpG-ODN, and the protective effect of this agent was strongly inhibited by neutralizing anti-TNF-alpha MoAb. Our results indicate that CpG-ODN alters the Th1-Th2 cytokine balance and promotes host resistance against infection with C. neoformans.     

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)-gamma 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 and IL-18 were used at subtherapeutic doses (0.005 and 2 microg/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-gamma and reduced IL-4 levels in bronchoalveolar lavage fluid. Finally, depletion of NK and gammadelta T cells, but not of CD4+ T cells, by administration of specific antibodies, significantly reduced the production of IFN-gamma 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-gamma by NK and gammadelta T cells in the early phase of infection and by suppressing the production of IL-4 in lungs.     

Reciprocal immunomodulatory effects of gamma interferon and interleukin-4 on filaria-induced airway hyperresponsiveness

Tropical pulmonary eosinophilia (TPE) is a severe asthmatic syndrome of lymphatic filariasis, in which an allergic response is induced to microfilariae (Mf) in the lungs. Previously, in a murine model for TPE, we have demonstrated that recombinant interleukin-12 (IL-12) suppresses pulmonary eosinophilia and airway hyperresponsiveness (AHR) by modulating the T helper (Th) response in the lungs from Th2- to Th1-like, with elevated gamma-interferon (IFN-gamma) production and decreased IL-4 and IL-5 production. The present study examined the immunomodulatory roles of IL-4 and IFN-gamma in filaria-induced AHR and pulmonary inflammation using mice genetically deficient in these cytokines. C57BL/6, IL-4 gene knockout (IL-4(-/-)), and IFN-gamma(-/-) mice were first immunized with soluble Brugia malayi antigens and then inoculated intravenously with 200,000 live Mf. Compared with C57BL/6 mice, IL-4(-/-) mice exhibited significantly reduced AHR, whereas IFN-gamma(-/-) mice had increased AHR. Histopathologically, each mouse strain showed increased cellular infiltration into the lung parenchyma and bronchoalveolar space compared with na?ve animals. However, consistent with changes in AHR, IL-4(-/-) mice had less inflammation than C57BL/6 mice, whereas IFN-gamma(-/-) mice had exacerbated pulmonary inflammation with the loss of pulmonary architecture. Systemically, IL-4(-/-) mice produced significantly higher IFN-gamma levels compared with C57BL/6 mice, whereas IFN-gamma(-/-) mice produced significantly higher IL-4 levels. These data indicate that IL-4 is required for the induction of filaria-induced AHR, whereas IFN-gamma suppresses AHR.     

Enhanced gamma interferon production through activation of Valpha14(+) natural killer T cells by alpha-galactosylceramide in interleukin-18-deficient mice with systemic cryptococcosis

We showed recently that activation of Valpha14(+) natural killer T cells (NKT cells) by alpha-galactosylceramide (alpha-GalCer) resulted in increased gamma interferon (IFN-gamma) production and host resistance to intravenous infection with Cryptococcus neoformans. In other studies, interleukin-18 (IL-18) activated NKT cells in collaboration with IL-12, suggesting the possible contribution of this cytokine to alpha-GalCer-induced IFN-gamma synthesis. Here we examined the role of IL-18 in alpha-GalCer-induced Th1 response by using IL-18KO mice with this infection. In these mice, levels of IFN-gamma in serum and its synthesis in vitro by spleen cells stimulated with live organisms were not reduced, but rather enhanced, compared to those in wild-type (WT) mice, while such production was completely absent in IL-12KO mice. The enhanced production of IFN-gamma correlated with increased IL-12 synthesis but not with reduced production of IL-4, which was rather increased. IFN-gamma synthesis in IL-18KO mice was abolished by neutralizing anti-IL-12 antibody and significantly inhibited by neutralization of endogenous IL-4 with a specific monoclonal antibody. In addition, administration of recombinant IL-4 significantly enhanced the production of IFN-gamma in WT mice. Finally, the enhanced production of IFN-gamma in IL-18KO mice correlated with increased host defense against cryptococcal infection, as indicated by enhancement in alpha-GalCer-related clearance of microorganisms. Our results indicated that in IL-18KO mice, IFN-gamma synthesis was enhanced through overproduction of IL-12 and IL-4 after intravenous infection with C. neoformans and a ligand-specific activation of Valpha14(+) NKT cells.     

In vivo modulation of the murine immune response to Francisella tularensis LVS by administration of anticytokine antibodies.

The role(s) of gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and interleukin-4 (IL-4) in establishment and maintenance of protective immunity to Francisella tularensis LVS in mice (C3H/HeN) was examined by selective removal of these cytokines in vivo with neutralizing antibodies. The 50% lethal dose (LD50) for mice infected intradermally with F. tularensis alone was 136,000 CFU; treatment of mice with anti-IFN-gamma or anti-TNF-alpha at the time of infection significantly reduced (P much less than 0.05) the LD50 to 2 and 5 CFU, respectively. Abrogation of protective immunity, however, was effective only when anti-IFN-gamma or anti-TNF-alpha was administered prior to day 3 postinfection. In contrast, the LD50 for mice treated with anti-IL-4 was repeatedly higher (555,000 CFU) than for controls; this difference, however, was not significant (P greater than 0.05). Thus, IL-4 may be detrimental, while IFN-gamma and TNF-alpha were clearly crucial to the establishment of protective immunity to F. tularensis during a primary infection. The importance of IFN-gamma and TNF-alpha during a secondary immune response to F. tularensis was also investigated. Spleen cells from immune mice passively transfer protective immunity to recipient mice in the absence of confounding antibody-mediated immunity. This passive transfer of immunity, however, was abrogated by treatment of recipient mice with anti-IFN-gamma or anti-TNF-alpha at the time of challenge infection. That anticytokines effectively abrogate protective immunity very early in the course of infection with F. tularensis suggests that T-cell-dependent activation of macrophages for microbicidal activity is unlikely. These T-cell-independent events early in the course of infection may suppress bacterial replication until a T-cell-dependent response ultimately clears the bacteria.     

Cytokine and chemokine expression in the central nervous system associated with protective cell-mediated immunity against Cryptococcus neoformans.

Cryptococcus neoformans is a yeast that causes cryptococcosis, a life-threatening disease that develops following inhalation and dissemination of the organisms. C. neoformans has a predilection for the central nervous system (CNS) and mortality is most frequently associated with meningoencephalitis. Susceptibility to cryptococcosis is increased in patients with deficiencies in cell-mediated immunity (CMI). Because cryptococcal CNS infections are associated with mortality and diagnosis of cryptococcosis is often not made until after dissemination to the CNS, a better understanding of host defense mechanisms against C. neoformans in the CNS is needed to design improved therapies for immunocompromised individuals suffering from cryptococcosis. Using a mouse model, we previously described a protective cell-mediated immune response induced in the periphery that limited the growth of C. neoformans in the CNS. In the current investigation, we examined cytokine and chemokine expression in the CNS to identify factors important in achieving protective immunity. We observed increased expression of IL-1beta, TNF-alpha, IFN-gamma, MCP-1, RANTES, and IP-10 in C. neoformans-infected brains of immune mice compared to control mice suggesting that these cytokines and chemokines are associated with the protective immune response. Furthermore, the Th1-type cytokines TNF-alpha and IFN-gamma, but not the Th2 cytokines IL-4 and IL-5, were secreted at significantly higher levels in C. neoformans-infected brains of immune mice compared to control mice. Our results demonstrate that cytokines and chemokines associated with CMI are produced following infection in the CNS of immunized mice, and the expression of these factors correlates with protection against C. neoformans in the CNS.     

Counter-protective role for interleukin-5 during acute Toxoplasma gondii infection

The role of interleukin-5 (IL-5) during Toxoplasma gondii infection was investigated by comparing disease progression in IL-5 gene deficient (IL-5-/-) mice and their wild-type (WT) counterparts on a C57BL/6 background. IL-5-/- mice infected orally with T. gondii were less susceptible to infection than WT mice as demonstrated by reduced mortality rates. Consistent with this data, orally infected IL-5-/- mice had less severe pathological changes in their small intestines than WT mice at 8 days postinfection. At this time, splenocytes and mesenteric lymph node cells derived from IL-5-/- mice produced levels of IL-12, interferon-gamma (IFN-gamma), IL-4, IL-10, and nitric oxide (measured as nitrite) similar to those derived from WT mice when stimulated with Toxoplasma lysate antigen. However, peak serum IL-12 and IFN-gamma levels (at days 6 and 8, respectively) were significantly higher in IL-5-/- mice than in WT mice. In addition, WT mice but not IL-5-/- mice had raised levels of eosinophils in their peripheral blood between days 5 and 8 following infection. Oral administration of N omega-nitro-L-arginine methyl (from day 4 postinfection) increased mortality rates in both IL-5-/- and WT mice, indicating a protective role for nitric oxide during the early stages of oral T. gondii infection. In comparison with oral infection, no difference in mortality was observed between IL-5-/- and WT mice following intraperitoneal infection with T. gondii, with all mice surviving until 35 days postinfection. Similarly, no significant differences were observed in the severity of the meningitis, perivascular cuffing, or number of microglial nodules or parasites in the brains of intraperitoneally infected mice. Together, these results demonstrate a detrimental role for IL-5 during the early stage of oral infection with T. gondii which is associated with increased small-intestine pathology, eosinophilia, and reduced plasma IL-12 and IFN-gamma levels.     

Both IL-12 and IL-18 contribute to small intestinal Th1-type immunopathology following oral infection with Toxoplasma gondii, but IL-12 is dominant over IL-18 in parasite control

Oral infection of C57BL/6 mice with Toxoplasma gondii results in small intestinal Th1-type immunopathology mediated by local production of IFN-gamma, TNF-alpha, and NO. To analyze whether the proinflammatory cytokines IL-12 and IL-18 play a role in the induction of immunopathology, IL-12p35/p40(-/-) and IL-18(-/-) mice were orally infected with T. gondii. Wild-type mice developed massive necrosis in their small intestines and died 7-10 days post infection. Even though IL-12p35/40(-/-) mice did not develop the necrosis they all died between day 9 and 11 after infection. In contrast, 50% of IL-18(-/-) mice died during the acute phase of infection. Compared to wild-type mice, IL-12p35/p40(-/-) but not IL-18(-/-) mice showed significantly higher parasite numbers in their small intestines and significantly higher numbers of parasite-associated inflammatory foci in their livers. IFN-gamma production was similar in infected wild-type and IL-18(-/-) mice but significantly decreased in IL-12p35/p40(-/-) mice. Treatment of mice with anti-IL-12- or anti-IL-18 antibodies after infection prevented the development of intestinal necrosis. These results reveal that both IL-12 and IL-18 play an important role in the development of intestinal immunopathology following oral infection with T. gondii. However, IL-12 is dominant over IL-18 in the host defense against parasite replication. Therefore, neutralization of IL-18 (rather than TNF-alpha, IL-12, and IFN-gamma) may be a safe strategy for the treatment of Th1-associated diseases.