IL-12/IFN-gamma/NO axis plays critical role in development of Th1-mediated experimental autoimmune encephalomyelitis

The importance of the IL-12/IFN-gamma/nitric oxide (NO) axis in the pathogenesis of autoimmune diseases remains controversial. In parallel experiments, we explored the role of the IL-12/IFN-gamma/NO axis in the development of MOG 35-55-induced experimental autoimmune encephalomyelitis (EAE) in mice lacking IL-12, IFN-gamma receptor (IFN-gammaR) and inducible nitric oxide synthase (NOS2), respectively. In comparison with wide-type control mice, IL-12-/-, IFN-gammaR-/- and NOS2-/- mice displayed more severe clinical signs of EAE both in remission and at subsequent relapse. Given the relatively low IFN-gamma production in IL-12-/- mice and the lack of IFN-gamma/IFN-gammaR signaling pathway in IFN-gammaR-/- mice, IL-12-/-, IFN-gammaR-/- and NOS2-/- mice with EAE exhibited low NO production. This correlated negatively with MOG 35-55-induced T cell proliferation. Both ED1-positive macrophages and CD4-positive T cells were increased in spinal cords from IL-12-/-, IFN-gammaR-/- and NOS2-/- compared to control mice. In vitro experiments demonstrate that spleen mononuclear cells from IL-12-/-, IFN-gammaR-/- and NOS2-/-mice with EAE present stronger migration capacity when compared to control mice. These results reveal that the IL-12/IFN-gamma/NO axis plays a critical role in the development of MOG 35-55-induced EAE, possibly over failing NO production.     

Inheritance of immune polarization patterns is linked to resistance versus susceptibility to Cryptococcus neoformans in a mouse model

Genetic background variation between inbred strains accounts for different levels of susceptibility to Cryptococcus neoformans in the mouse infection model. To elucidate the inheritance of immunophenotypic traits and their associations with clearance outcomes during cryptococcal infection, we compared C57BL/6, BALB/c, and their first-generation hybrid, CB6F1 (F1), mice. Mice from each group were infected with C. neoformans (10(4) CFU) and analyzed at weekly intervals over a 6-week period. BALB/c mice progressively cleared the cryptococcal infection in the lungs and showed a Th1-skewed immune response: a Th1-shifted cytokine profile, modest lung pathology, and no significant elevation in the systemic immunoglobulin E (IgE) level. In contrast, C57BL/6 mice developed a chronic infection with a Th2-skewed immune response: a Th2-shifted cytokine profile, pulmonary eosinophilia, severe lung pathology, elevated serum IgE, fungemia, and cryptococcal dissemination in the central nervous system. F1 mice demonstrated intermediate resistance to C. neoformans, with a stronger resemblance to the immunophenotype of the resistant (BALB/c) mice. F1 mice also demonstrated enhanced pulmonary recruitment of lymphocytes, especially CD8(+) T cells, in comparison to both parental strains, suggesting positive heterosis. We conclude that the inheritance of traits responsible for early cytokine induction in the infected lungs and dendritic-cell maturation/activation status in draining nodes is responsible for the intermediate immune response polarization and clearance outcome observed initially in the lungs of F1 mice. The enhanced pulmonary lymphocyte recruitment could be responsible for a gradual shutdown of the undesirable Th2 arm of the immune response and subsequently improved anticryptococcal resistance in F1 mice.     

IL-23 dampens the allergic response to Cryptococcus neoformans through IL-17-independent and -dependent mechanisms

The cytokines IL-23 and IL-17 have been implicated in resistance to cryptococcal disease, but it is not clear whether IL-23-mediated production of IL-17 promotes fungal containment following pulmonary challenge with Cryptococcus neoformans. We used mice lacking IL-23 (IL-23p19(-/-)) or IL-17RA (IL-17RA(-/-)), and wild type (WT) C57BL/6 mice to examine the IL-23/IL-17 axis after intranasal infection with the C. neoformans strain 52D. The absence of IL-23 or IL-17RA had no effect on pulmonary or brain fungal burden at 1 or 6 weeks after infection. However, survival of IL-23p19(-/-) mice was reduced compared to IL-17RA(-/-) mice. IL-I7 production by CD4 T cells and natural killer T (NKT) cells was impaired in IL-23p19(-/-) lungs, but was not completely abolished. Both IL-23p19(-/-) and IL-17RA(-/-) mice exhibited impaired neutrophil recruitment, increased serum levels of IgE and IgG2b, and increased deposition of YM1/YM2 crystals in the lung, but only IL-23p19(-/-) mice developed persistent lung eosinophilia. Although survival of IL-17RA(-/-) and WT mice was similar after 17 weeks of infection, only surviving IL-17RA(-/-) mice exhibited cryptococcal dissemination to the blood. These data demonstrate that IL-23 dampens the allergic response to cryptococcal infection through IL-17-independent suppression of eosinophil recruitment and IL-17-dependent regulation of antibody production and crystal deposition. Furthermore, IL-23, and to a lesser extent IL-17, contribute to disease resistance.     

Induction of interleukin-12 and gamma interferon requires tumor necrosis factor alpha for protective T1-cell-mediated immunity to pulmonary Cryptococcus neoformans infection

The development of T1-cell-mediated immunity is required to clear a pulmonary Cryptococcus neoformans infection. The objective of these studies was to determine the mechanism by which tumor necrosis factor alpha (TNF-alpha) augments the development of pulmonary T1 immunity to C. neoformans infection. TNF-alpha expression was detected in lavage sample cells at days 2, 3, and 7 following C. neoformans infection. The numbers of CFU in the lung were not different between control and anti-TNF-alpha-treated mice at any time point examined during the afferent phase of the response (days 0 to 7). However, neutralization of TNF-alpha prevented the initiation of pulmonary clearance during the efferent phase of the response (day 14). Administration of anti-TNF-alpha monoclonal antibody (day 0) diminished the lung levels of TNF-alpha, interleukin-12 (IL-12), and gamma interferon (IFN-gamma) induced by C. neoformans at day 7 postinfection. Neutralization of TNF-alpha (day 0) also altered the IFN-gamma/IL-4 ratio in the lung-associated lymph nodes at day 7 following C. neoformans infection. Anti-TNF-alpha-treated mice developed a pulmonary eosinophilia at day 14 postinfection. Consistent with the pulmonary eosinophilia, anti-TNF-alpha-treated mice exhibited elevated serum immunoglobulin E and inhibition of the anticryptococcal delayed-type hypersensitivity response, indicating a shift toward a T2 response. Neutralization of IL-12 also prevented lung leukocyte production of IFN-gamma in response to the infection. These findings demonstrate that afferent-phase TNF-alpha production is essential for the induction of IL-12 and IFN-gamma and neutralization of early TNF-alpha results in a T2 shift of the T1/T2 balance of antifungal immunity.     

Aberrant contraction of antigen-specific CD4 T cells after infection in the absence of gamma interferon or its receptor

Several lines of evidence from different model systems suggest that gamma interferon (IFN-gamma) is an important regulator of T-cell contraction after antigen (Ag)-driven expansion. To specifically investigate the role of IFN-gamma in regulating the contraction of Ag-specific CD4 T cells, we infected IFN-gamma-/- and IFN-gammaR1-/- mice with attenuated Listeria monocytogenes and monitored the numbers of Ag-specific CD4 T cells during the expansion, contraction, and memory phases of the immune response to infection. In the absence of IFN-gamma or the ligand-binding portion of its receptor, Ag-specific CD4 T cells exhibited normal expansion in numbers, but in both strains of deficient mice there was very little decrease in the number of Ag-specific CD4 T cells even at time points later than day 90 after infection. This significant delay in contraction was not due to prolonged infection, since mice treated with antibiotics to conclusively eliminate infection exhibited the same defect in contraction. In addition to altering the number of Ag-specific CD4 T cells, the absence of IFN-gamma signaling also changed the phenotype of cells generated after infection. IFN-gammaR1-/- Ag-specific CD4 T cells reacquired expression of CD127 more quickly than wild-type cells, and more IFN-gammaR1-/- CD4 T cells were capable of producing both IFN-gamma and interleukin 2 following Ag stimulation. From these data we conclude that IFN-gamma regulates the contraction, phenotype, and function of Ag-specific CD4 T cells generated after infection.     

Protection against cryptococcosis by using a murine gamma interferon-producing Cryptococcus neoformans strain

We evaluated cell-mediated immune (CMI) responses in mice given a pulmonary infection with a Cryptococcus neoformans strain engineered to produce the Th1-type cytokine gamma interferon (IFN-gamma). Mice given a pulmonary infection with an IFN-gamma-producing C. neoformans strain were able to resolve the primary infection and demonstrated complete (100%) protection against a second pulmonary challenge with a pathogenic C. neoformans strain. Pulmonary cytokine analyses showed that Th1-type/proinflammatory cytokine and chemokine expression were significantly higher and Th2-type cytokine expression was significantly lower in mice infected with the IFN-gamma-producing C. neoformans strain compared to wild-type-infected mice. This increased pulmonary Th1-type cytokine expression was also associated with significantly lower pulmonary fungal burden and significantly higher pulmonary leukocyte and T-lymphocyte recruitment in mice infected with the IFN-gamma-producing C. neoformans strain compared to wild-type-infected mice. Our results demonstrate that pulmonary infection of mice with a C. neoformans strain expressing IFN-gamma results in the stimulation of local Th1-type anti-cryptococcal CMI responses and the development of protective host immunity against future pulmonary cryptococcal infections. The use of fungi engineered to produce host cytokines is a novel method to study immune responses to infection and may be useful in developing vaccine strategies in humans.     

Antibody-mediated protection in murine Cryptococcus neoformans infection is associated with pleotrophic effects on cytokine and leukocyte responses

Cryptococcus neoformans, an encapsulated yeast, is a common cause of life-threatening meningoencephalitis in immunosuppressed patients. We previously observed that administration of a monoclonal antibody (MAb) to the capsular polysaccharide to mice with pulmonary infection prolonged survival and enhanced granulomatous inflammation without reducing lung CFU. To understand the mechanism of MAb action, we studied leukocyte recruitment and cytokine profiles in lungs of A/JCr mice. B lymphocytes were the predominant cell type in lung infiltrates, comprising 15 to 30% of the leukocytes. Despite alterations in histological appearance, fluorescence-activated cell sorter analysis revealed no significant difference in total numbers of lung leukocytes in MAb-treated mice and controls. Differences in the immune response to C. neoformans between MAb-treated mice and controls included (i) an increase in the percentage of granulocytes among lung leukocytes on day 14, (ii) higher macrophage surface expression of CD86 on day 28, (iii) larger amounts of IL-10 in lung homogenates at day 7, (iv) a trend toward smaller amounts of gamma interferon mRNA and protein on day 7, and (v) a smaller increase in the levels of interleukin-4 mRNA and protein on day 7. Hence, the immune responses to C. neoformans infection in the presence and absence of specific antibody were qualitatively similar, and antibody administration was associated with several subtle quantitative differences in immune response parameters that could translate into enhanced survival. MAb may function partly by down-regulating the inflammatory response and reducing host damage. Our findings demonstrate unexpected complexity in the interaction between specific MAb and other components of the host immune response.     

Interleukin-12 can directly induce T-helper 1 responses in interferon-γ (IFN-γ) receptor-deficient mice, but requires IFN-γ signalling to downregulate T-helper 2 responses

An in vivo model of pulmonary granuloma formation around embolized schistosome eggs was investigated as an environment in which to analyse a role for interleukin-12 (IL-12) in the differentiation of T-helper 1 (Th1) and Th2 subsets. Specifically, mice deficient for the interferon-gamma receptor (IFN-gammaR-/-) were used to determine the role for IL-12 in the absence of IFN-gamma-mediated signalling. We show that recombinant IL-12 administered to IFN-gammaR-/- mice caused the up-regulation of mRNA for IFN-gamma in lung tissue, and the secretion of abundant IFN-gamma by in vitro-cultured lymph node cells in response to egg antigens. This indicates that IL-12 can act independently of IFN-gamma to induce the development of Th1 cells. Administration of rIL-12 to wild-type mice markedly reduced the secretion of Th2-associated cytokines, IL-4 and IL-5. However, these cytokines were not dramatically reduced in IFN-gammaR-/- mice treated with IL-12. We conclude that inhibition of these cytokines by IL-12 is primarily dependent upon effective IFN-gamma signalling, although abrogation of T-cell derived IL-10 appeared to be dependent upon IL-12. We also show that increases in mRNA for the beta2 subunit of the IL-12 receptor and the p40 subunit of IL-12 after rIL-12 treatment were lower in IFN-gammaR-/- mice, compared to wild-type mice, indicating that their expression was primarily dependent upon IFN-gamma with only a minor role for IL-12.     

Role of granulocyte macrophage colony-stimulating factor during gram-negative lung infection with Pseudomonas aeruginosa

Granulocyte macrophage colony-stimulating factor (GM-CSF) stimulates survival, proliferation, differentiation, and function of myeloid cells. Recently, GM-CSF has been shown to be important for normal pulmonary homeostasis. We report that GM-CSF is induced in lung leukocytes during infection with Gram-negative bacteria. Therefore, we postulated that deficiencies in GM-CSF would increase susceptibility to Gram-negative infection in vivo. After an intratracheal inoculum with Pseudomonas aeruginosa, GM-CSF-/- mice show decreased survival compared with wild-type mice. GM-CSF-/- mice show increased lung, spleen, and blood bacterial CFU. GM-CSF-/- mice are defective in the production of cysteinyl leukotrienes, prostaglandin E2, macrophage inflammatory protein, and keratinocyte-derived chemokine in lung leukocytes postinfection. Despite these defects, inflammatory cell recruitment is not diminished at 6 or 24 h postinfection, and the functional activity of polymorphonuclear leukocytes from the lung and peritoneum against P. aeruginosa is enhanced in GM-CSF-/- mice. In contrast, alveolar macrophage (AM) phagocytosis, killing, and H2O2 production are defective in GM-CSF-/- mice. Although the absence of GM-CSF has profound effects on AMs, peritoneal macrophages seem to have normal bactericidal activities in GM-CSF-/- mice. Defects in AM function may be related to diminished levels of IFN-gamma and TNF-alpha postinfection. Thus, GM-CSF-/- mice are more susceptible to lung infection with P. aeruginosa as a result of impaired AM function.     

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.     

Murine gammaherpesvirus-68 infection causes multi-organ fibrosis and alters leukocyte trafficking in interferon-gamma receptor knockout mice

Murine gammaherpesvirus-68 (MHV-68) infection in interferon-gamma receptor knockout mice (IFN-gammaR(-)/(-)) results in splenic fibrosis and excessive loss of splenocytes. In our present study we found that MHV-68 infection in IFN-gammaR(-)/(-) mice also resulted in fibrosis and atrophy of the mediastinal lymph nodes, interstitial pulmonary fibrosis and fibrotic changes in the liver. Atrophy and cellular depletion of the spleen in IFN-gammaR(-)/(-) was not the result of increased cell death. The loss of splenocytes in IFN-gammaR(-)/(-) mice, which was most evident on day 23 after infection, correlated with an increase in the number of leukocytes in peripheral blood. At the peak of leukocytosis, on day 23 after infection, peripheral blood cells from infected IFN-gammaR(-)/(-) mice were unable to traffic through the fibrosed spleens of IFN-gammaR(-)/(-) mice but were able to enter the spleens of wild-type mice. This indicates that leukocytosis was in part the result of emigration of cells from the spleen and their subsequent exclusion of re-entry at the height of fibrosis. Significant cytokine and chemokine changes were observed in spleens of IFN-gammaR(-)/(-) mice. IFN-gamma, tumor necrosis factor-alpha (TNF-alpha ), TNF-beta, interleukin-1beta (IL-1beta), transforming growth factor-beta1 (TGF-beta1), lymphotactin, and MIP-1beta were elevated on day 14 after infection whereas chemokines IP-10 and MIG were significantly reduced. These changes suggest a role for dysregulated cytokines and chemokines in severe organ-specific fibrosis with implications for immune-mediated fibrotic disorders.     

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.     

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.     

IFN-gamma regulates Fas ligand expression in human CD4+ T lymphocytes and controls their anti-mycobacterial cytotoxic functions

Fas and Fas Ligand (FasL) expression, activation-induced cell death (AICD) and mycobacterial antigen-specific cytotoxicity of peripheral T cells from patients with complete inherited IFN-gamma receptor 1 binding chain deficiency (IFN-gammaR1-/-) were investigated. Fas was equally expressed in both normal and deficient T lymphoblasts and they underwent apoptosis when stimulated with agonist anti-Fas mAb. By contrast, T lymphoblasts and CD4+ T cell clones (TCC) from deficient patients displayed a reduced surface FasL expression and resistance to AICD. CD8+ TCC from healthy and deficient patients displayed similar high level of FasL and susceptibility to AICD. In Jurkat CD4+ T cells competent to transduce IFN-gamma signaling, IFN-gamma induced surface FasL export and their Fas-dependent apoptosis. Effector T cells generated from a patient with a dominant negative mutation of IFN-gammaR1 (IFN-gammaR1DN) following stimulation with mycobacterial antigens were unable to kill MHC class II-matched, mycobacterial antigen-pulsed macrophages. Normal Fas expression in T cells and FasL in CD8+ cells may account for the absence of autoimmune disorders in these patients. Conversely, defective FasL expression on IFN-gammaR1DN CD4+ T cells impairs their cytotoxic functions and highlights a novel role for IFN-gamma signaling in the control of mycobacterial infection in humans.     

Evaluation of host immune responses to pulmonary cryptococcosis using a temperature-sensitive C. neoformans calcineurin A mutant strain

Cryptococcus neoformans is an opportunistic fungal pathogen that threatens individuals with impaired cell-mediated immunity (CMI). Presently, there are no standardized vaccines available to prevent cryptococcal infections and conventional anti-fungal drug therapy does not induce host immune reactivity and thus cannot efficiently resolve C. neoformans infections in immunocompromised individuals. The present study was designed to characterize pulmonary immune responses following infection with an avirulent temperature-sensitive (ts) mutant, calcineurin A1 (cna1) compared to the pathogenic C. neoformans strain H99 and its potential to induce protective anti-cryptococcal immunity. Host CMI responses in cna1-inoculated mice were observed to be dose-dependent, and comprise increases in pulmonary macrophages and CD4(+) T lymphocytes. However, cytokine analysis demonstrated a mixed pulmonary cytokine response (increases in IL-4, and MCP-1) with no induction of IFN-gamma. Also, pre-immunization with the ts cna1 mutant did not result in protection from a subsequent secondary pulmonary infection with the pathogenic C. neoformans strain H99. Taken together, these results suggest that host pulmonary CMI responses to the ts cna1 mutant that is eventually eliminated from the host without the induction of IFN-gamma appear to be dose-dependent, diverse, and require further stimulation to induce C. neoformans-specific Th1-type cytokine responses to resolve subsequent experimental pulmonary cryptococcal infections.     

Role of granulocyte macrophage colony-stimulating factor in host defense against pulmonary Cryptococcus neoformans infection during murine allergic bronchopulmonary mycosis

We investigated the role of granulocyte macrophage colony-stimulating factor (GM-CSF) in host defense in a murine model of pulmonary cryptococcosis induced by intratracheal inoculation of Cryptococcus neoformans. Pulmonary C. neoformans infection of C57BL/6 mice is an established model of an allergic bronchopulmonary mycosis. Our objective was to determine whether GM-CSF regulates the pulmonary Th2 immune response in C. neoformans-infected C57BL/6 mice. Long-term pulmonary fungistasis was lost in GM-CSF knockout (GM(-/-)) mice, resulting in increased pulmonary burden of fungi between weeks 3 and 5. GM-CSF was required for the early influx of macrophages and CD4 and CD8 T cells into the lungs but was not required later in the infection. Lack of GM-CSF also resulted in reduced eosinophil recruitment and delayed recruitment of mononuclear cells into the airspace. Macrophages from GM(+/+) mice showed numerous hallmarks of alternatively activated macrophages: higher numbers of intracellular cryptococci, YM1 crystals, and induction of CCL17. These hallmarks are absent in macrophages from GM(-/-) mice. Mucus-producing goblet cells were abundantly present within the bronchial epithelial layer in GM(+/+) mice but not in GM(-/-) mice at week 5 after infection. Production of both Th1 and Th2 cytokines was impaired in the absence of GM-CSF, consistent with both reduced C. neoformans clearance and absence of allergic lung pathology.     

Involvement of CD14, toll-like receptors 2 and 4, and MyD88 in the host response to the fungal pathogen Cryptococcus neoformans in vivo

The major capsular polysaccharide of Cryptococcus neoformans, glucuronoxylomannan (GXM), is recognized by Toll-like receptor 2 (TLR2), TLR4, and CD14. In these studies, mice deficient in CD14, TLR2, TLR4, and the TLR-associated adaptor protein, MyD88, were utilized to investigate the contribution of TLRs and CD14 to in vivo host defenses against C. neoformans. MyD88(-/-) mice had significantly reduced survival compared with wild-type C57BL/6 mice after intranasal (i.n.) and intravenous (i.v.) infection with live C. neoformans. CD14(-/-) mice had reduced survival when infected i.v., while TLR2(-/-) mice died significantly earlier after i.n. infection. Mortality was similar comparing TLR4 mutant C3H/HeJ mice and control C3H/HeOuJ mice following i.v. or i.n. challenge with C. neoformans. The course of pulmonary cryptococcosis was studied in more detail in the CD14(-/-), TLR2(-/-), and MyD88(-/-) mice. MyD88(-/-) mice infected i.n. had higher numbers of CFU in the lungs as well as higher GXM levels in the sera and lungs 7 days after infection than wild-type mice did. Surprisingly, there were no major differences in the levels of tumor necrosis factor alpha, interleukin-4 (IL-4), IL-10, IL-12p70, or gamma interferon in the lungs of C. neoformans-infected knockout mice compared with wild-type mice. Histopathologic analysis of the lungs on day 7 postinfection revealed minimal inflammation in all mouse groups. These studies demonstrate a major role for MyD88 and relatively minor roles for CD14 and TLR2 in the response to cryptococcal infection, with the decreased survival of MyD88(-/-) mice correlating with increased numbers of lung CFU and serum and lung GXM levels.     

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.     

Interferon-gamma receptor-mediated but not tumor necrosis factor receptor type 1- or type 2-mediated signaling is crucial for the activation of cerebral blood vessel endothelial cells and microglia in murine Toxoplasma encephalitis.

The regulatory role of interferon-gamma receptor (IFN-gammaR)- and tumor necrosis factor receptor (TNFR)-mediated immune reactions for the activation of cerebral endothelial cells, microglia, and astrocytes was evaluated in a model of murine Toxoplasma encephalitis (TE). Brain endothelial cells of wild-type mice reacted in response to Toxoplasma infection with a strong up-regulation of the vascular cell adhesion molecule, the intercellular adhesion molecule (ICAM)-1, and major histocompatibility complex (MHC) class I and II antigens. A similar response was seen in mice genetically deficient for either TNFR1, TNFR2, or both TNFRs, whereas IFN-gammaR-deficient (IFN-gammaR0/0) mice were found to be defective in the up-regulation of these molecules. However, recruitment of leukocytes to the brain and their intracerebral movement were not impaired in IFN-gammaR0/0 mice. In addition, microglia of Toxoplasma gondii-infected IFN-gammaR0/0 mice failed to induce expression of ICAM-1, leukocyte function-associated antigen (LFA)-1, and MHC class I and II antigens, whereas wild-type and TNFR-deficient mice up-regulated these molecules. Moreover, TNF-alpha mRNA production of F4/80(+) microglia/macrophages was impaired in IFN-gammaR0/0 mice, but not in TNFR-deficient mutants. However, induction of interleukin (IL)-1beta, IL-10, IL-12p40, and IL-15 mRNA was independent of IFN-gammaR and TNFR signaling. In conclusion, IFN-gammaR, but not TNFR signaling, is the major pathway for the activation of endothelial cells and microglia in murine TE. These findings differ from observations in other inflammatory central nervous system disorders, indicating specific regulatory mechanisms in this parasitic cerebral infection.