Enhanced innate immune responsiveness to pulmonary Cryptococcus neoformans infection is associated with resistance to progressive infection

Genetically regulated mechanisms of host defense against Cryptococcus neoformans infection are not well understood. In this study, pulmonary infection with the moderately virulent C. neoformans strain 24067 was used to compare the host resistance phenotype of C57BL/6J with that of inbred mouse strain SJL/J. At 7 days or later after infection, C57BL/6J mice exhibited a significantly greater fungal burden in the lungs than SJL/J mice. Characterization of the pulmonary innate immune response at 3 h after cryptococcal infection revealed that resistant SJL/J mice exhibited significantly higher neutrophilia, with elevated levels of inflammatory cytokine tumor necrosis factor alpha (TNF-α) and keratinocyte-derived chemokine (KC)/CXCL1 in the airways, as well as increased whole-lung mRNA expression of chemokines KC/CXCL1, MIP-1α/CCL3, MIP-1β/CCL4, MIP-2/CXCL2, and MCP-1/CCL2 and cytokines interleukin 1β (IL-1β) and IL-1Ra. At 7 and 14 days after infection, SJL/J mice maintained significantly higher levels of TNF-α and KC/CXCL1 in the airways and exhibited a Th1 response characterized by elevated levels of lung gamma interferon (IFN-γ) and IL-12/IL-23p40, while C57BL/6J mice exhibited Th2 immunity as defined by eosinophilia and IL-4 production. Alveolar and resident peritoneal macrophages from SJL/J mice also secreted significantly greater amounts of TNF-α and KC/CXCL1 following in vitro stimulation with C. neoformans. Intracellular signaling analysis demonstrated that TNF-α and KC/CXCL1 production was regulated by NF-κB and phosphatidylinositol 3 kinase in both strains; however, SJL/J macrophages exhibited heightened and prolonged activation in response to C. neoformans infection compared to that of C57BL/6J. Taken together, these data demonstrate that an enhanced innate immune response against pulmonary C. neoformans infection in SJL/J mice is associated with natural resistance to progressive infection.     

Interleukin-17A Enhances Host Defense against Cryptococcal Lung Infection through Effects Mediated by Leukocyte Recruitment,Activation, and Gamma Interferon Production

Infection of C57BL/6 mice with the moderately virulent Cryptococcus neoformans strain 52D models the complex adaptive immune response observed in HIV-negative patients with persistent fungal lung infections. In this model, Th1 and Th2 responses evolve over time, yet the contribution of interleukin-17A (IL-17A) to antifungal host defense is unknown. In this study, we show that fungal lung infection promoted an increase in Th17 T cells that persisted to 8 weeks postinfection. Our comparison of fungal lung infection in wild-type mice and IL-17A-deficient mice (IL-17A^−/− mice; C57BL/6 genetic background) demonstrated that late fungal clearance was impaired in the absence of IL-17A. This finding was associated with reduced intracellular containment of the organism within lung macrophages and deficits in the accumulation of total lung leukocytes, including specific reductions in CD11c⁺ CD11b⁺ myeloid cells (dendritic cells and exudate macrophages), B cells, and CD8⁺ T cells, and a nonsignificant trend in the reduction of lung neutrophils. Although IL-17A did not alter the total number of CD4 T cells, decreases in the total number of CD4 T cells and CD8 T cells expressing gamma interferon (IFN-γ) were observed in IL-17A^−/− mice. Lastly, expression of major histocompatibility complex class II (MHC-II) and the costimulatory molecules CD80 and CD86 on CD11c⁺ CD11b⁺ myeloid cells was diminished in IL-17A^−/− mice. Collectively, these data indicate that IL-17A enhances host defenses against a moderately virulent strain of C. neoformans through effects on leukocyte recruitment, IFN-γ production by CD4 and CD8 T cells, and the activation of lung myeloid cells.     

Susceptibility to Progressive Cryptococcus neoformans Pulmonary Infection Is Regulated by Loci on Mouse Chromosomes 1 and 9

Genetic factors that regulate the pathogenesis of pneumonia caused by the fungus Cryptococcus neoformans are poorly understood. Through a phenotypic strain survey we observed that inbred C3H/HeN mice develop a significantly greater lung fungal burden than mice of the resistant CBA/J strain 4 weeks following intratracheal infection with C. neoformans ATCC 24067. The aim of the present study was to characterize the inflammatory response of C3H/HeN mice following C. neoformans pulmonary infection and to identify genetic loci that regulate host defense. Following cryptococcal infection, C3H/HeN mice demonstrated a Th2 immune response with heightened airway and tissue eosinophilia, goblet cell metaplasia, and significantly higher lung interleukin-5 (IL-5) and IL-13 protein expression relative to CBA/J mice. Conversely, CBA/J mice exhibited greater airway and tissue neutrophilia that was associated with significantly higher pulmonary expression of gamma interferon, CXCL10, and IL-17 proteins than C3H/HeN mice. Using the fungal burden at 4 weeks postinfection as a phenotype, genome-wide quantitative trait locus (QTL) analysis among 435 segregating (C3H/HeN × CBA/J)F2 (C3HCBAF2) hybrids identified two significant QTLs on chromosomes 1 (Cnes4) and 9 (Cnes5) that control susceptibility to cryptococcal pneumonia in an additive manner. Susceptible C3H/HeN mice carry a resistance allele at Cnes4 and a susceptibility allele at Cnes5. These studies reveal additional genetic complexity of the host response to C. neoformans that is associated with divergent patterns of pulmonary inflammation.     

Neither Dectin-2 nor the Mannose Receptor Is Required for Resistance to Coccidioides immitis in Mice

We investigated the roles of the mannose receptor (MR) and Dectin-2 in resistance to pulmonary coccidioidomycosis in C57BL/6 (B6) mice and in the interaction of myeloid cells with spherules, using B6 mice with targeted mutations in Mrc1 and Clec4n. Spherules are the tissue form of Coccidioides, and we determined that the MR on bone marrow-derived dendritic cells (BMDC) was important for recognition of spherules (formalin-killed spherules [FKS]) and for secretion of interleukin 10 (IL-10) and proinflammatory cytokines in response to FKS by both elicited macrophages and BMDC. Infected MR knockout (KO) mice produced more IL-10 in their lungs than did B6 mice, and MR KO mice also made more protective Th-17 cytokines. In contrast to the MR, Dectin-2 was not required for recognition of FKS by BMDC or for the production of cytokines by BMDC in response to FKS. However, Dectin-2 KO was required for stimulation of elicited peritoneal macrophages. Despite that, lung cytokine levels were not significantly different in Dectin-2 KO mice and B6 mice 14 days after infection, except for IL-1β, which was higher in Dectin-2 KO lungs. Although both Dectin-2^−/− and MR^−/− myeloid cells had reduced proinflammatory cytokine responses to FKS in vitro, neither MR nor Dectin-2 deficiency reduced the resistance of B6 mice to pulmonary coccidioidomycosis.     

DAP12 Inhibits Pulmonary Immune Responses to Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen that is inhaled into the lungs and can lead to life-threatening meningoencephalitis in immunocompromised patients. Currently, the molecular mechanisms that regulate the mammalian immune response to respiratory cryptococcal challenge remain poorly defined. DAP12, a signaling adapter for multiple pattern recognition receptors in myeloid and natural killer (NK) cells, has been shown to play both activating and inhibitory roles during lung infections by different bacteria and fungi. In this study, we demonstrate that DAP12 plays an important inhibitory role in the immune response to C. neoformans. Infectious outcomes in DAP12^−/− mice, including survival and lung fungal burden, are significantly improved compared to those in C57BL/6 wild-type (WT) mice. We find that eosinophils and macrophages are decreased while NK cells are increased in the lungs of infected DAP12^−/− mice. In contrast to WT NK cells, DAP12^−/− NK cells are able to repress C. neoformans growth in vitro. Additionally, DAP12^−/− macrophages are more highly activated than WT macrophages, with increased production of tumor necrosis factor (TNF) and CCL5/RANTES and more efficient uptake and killing of C. neoformans. These findings suggest that DAP12 acts as a brake on the pulmonary immune response to C. neoformans by promoting pulmonary eosinophilia and by inhibiting the activation and antifungal activities of effector cells, including NK cells and macrophages.     

Dual role of interleukin-4 (IL-4) in pulmonary paracoccidioidomycosis: endogenous IL-4 can induce protection or exacerbation of disease depending on the host genetic pattern

Resistance to paracoccidioidomycosis, the most important endemic mycosis in Latin America, is thought to be primarily mediated by cellular immunity and the production of gamma interferon. To assess the role of interleukin-4 (IL-4), a Th2 cytokine, pulmonary paracoccidioidomycosis in IL-4-depleted susceptible (B10.A) and intermediate (C57BL/6) mice was studied. Two different protocols were used to neutralize endogenous IL-4 in B10.A mice: 1 mg of anti-IL-4 monoclonal antibody (MAb)/week and 8 mg 1 day before intratracheal infection with 10(6) Paracoccidioides brasiliensis yeast cells. Unexpectedly, both protocols enhanced pulmonary infection but did not alter the levels of pulmonary cytokines and specific antibodies. Since in a previous work it was verified that C57BL/6 mice genetically deficient in IL-4 were more resistant to P. brasiliensis infection, we also investigated the effect of IL-4 depletion in this mouse strain. Treatment with the MAb at 1 mg/week led to less severe pulmonary disease associated with impaired synthesis of Th2 cytokines in the lungs and liver of control C57BL/6 mice. Conversely, in IL-4-depleted C57BL/6 mice, increased levels of tumor necrosis factor alpha and IL-12 were found in the lungs and liver, respectively. In addition, higher levels of immunoglobulin G2a (IgG2a) and lower levels of IgG1 antibodies were produced by IL-4-depleted mice than by control mice. Lung pathologic findings were equivalent in IL-4-depleted and untreated B10.A mice. In IL-4-depleted C57BL/6 mice, however, smaller and well-organized granulomas replaced the more extensive lesions that developed in untreated mice. These results clearly showed that IL-4 can have a protective or a disease-promoting effect in pulmonary paracoccidioidomycosis depending on the genetic background of the host.     

Dual Role of Interleukin-10 in Murine Lyme Disease: Regulation of Arthritis Severity and Host Defense

In the murine model of Lyme disease, C3H/He mice exhibit severe arthritis while C57BL/6N mice exhibit mild lesions when infected with Borrelia burgdorferi. Joint tissues from these two strains of mice harbor similar concentrations of B. burgdorferi, suggesting that the difference in disease severity reflects differences in the magnitude of the inflammatory response to B. burgdorferi lipoproteins. Stimulation of bone marrow macrophages from C3H/HeN mice with the B. burgdorferi lipoprotein OspA resulted in higher-level production of the inflammatory mediators tumor necrosis factor alpha, nitric oxide, and interleukin-6 (IL-6) than that of macrophages from C57BL/6N mice. In contrast, macrophages from C57BL/6N mice consistently produced larger amounts of the anti-inflammatory cytokine IL-10 than did C3H/HeN macrophages. Addition of recombinant IL-10 suppressed the production of inflammatory mediators by macrophages from both strains. IL-10 was found to modulate B. burgdorferi-induced inflammation in vivo, since C57BL/6J mice deficient in IL-10 (IL-10-/-) developed more severe arthritis than wild-type C57BL/6J mice. The increase in arthritis severity was associated with a 10-fold decrease in the number of B. burgdorferi organisms present in ankle tissues from IL-10-/- mice. These findings suggest that in C57BL/6 mice, IL-10-dependent regulation of arthritis severity occurs at the expense of effective control of bacterial numbers.     

ECTV Abolishes the Ability of GM-BM Cells to Stimulate Allogeneic CD4 T Cells in a Mouse Strain-Independent Manner

Ectromelia virus (ECTV) is the etiological agent of mousepox, an acute and systemic disease with high mortality rates in susceptible strains of mice. Resistance and susceptibility to mousepox are triggered by the dichotomous T-helper (Th) immune response generated in infected animals, with strong protective Th1 or nonprotective Th2 profile, respectively. Th1/Th2 balance is influenced by dendritic cells (DCs), which were shown to differ in their ability to polarize naïve CD4⁺ T cells in different mouse strains. Therefore, we have studied the inner-strain differences in the ability of conventional DCs (cDCs), generated from resistant (C57BL/6) and susceptible (BALB/c) mice, to stimulate proliferation and activation of Th cells upon ECTV infection. We found that ECTV infection of GM-CSF-derived bone marrow (GM-BM) cells, composed of cDCs and macrophages, affected initiation of allogeneic CD4⁺ T cells proliferation in a mouse strain-independent manner. Moreover, infected GM-BM cells from both mouse strains failed to induce and even inhibited the production of Th1 (IFN-γ and IL-2), Th2 (IL-4 and IL-10) and Th17 (IL-17A) cytokines by allogeneic CD4⁺ T cells. These results indicate that in in vitro conditions ECTV compromises the ability of cDCs to initiate/polarize adaptive antiviral immune response independently of the host strain resistance/susceptibility to lethal infection.     

Interleukin-10 inhibits Mycobacterium bovis bacillus Calmette�CGu��rin (BCG)-induced macrophage cytotoxicity against bladder cancer cells

The mechanisms underlying bacillus Calmette–Guérin (BCG) immunotherapy of bladder cancer currently remain elusive. Previously, we demonstrated that macrophages were cytotoxic to bladder cancer cells upon BCG stimulation in vitro. However, macrophages from C57BL/6 mice were less potent than those from C3H/HeN mice for the killing of bladder cancer cells. This study was to determine whether interleukin (IL)-10 produced by macrophages in response to BCG is a causative factor for the reduced cytotoxicity in BCG-stimulated C57BL/6 macrophages. Thioglycollate-elicited peritoneal macrophages were prepared and analysed for the BCG induction of cytotoxicity, cytokines and nitric oxide (NO) in vitro. Compared to BCG-stimulated C3H/HeN macrophages, BCG-stimulated C57BL/6 macrophages exhibited reduced killing of bladder cancer MBT-2 cells and MB49 cells. Studies demonstrated further that BCG-stimulated C57BL/6 macrophages produced a high level of IL-10, which correlated with reduced production of tumour necrosis factor (TNF)-α, IL-6 and NO. Neutralizing endogenous IL-10 during BCG stimulation increased C57BL/6 macrophage cytotoxicity against MB49 cells by 3·2-fold, along with increased production of TNF-α by 6·4-fold and NO by 3·6-fold, respectively. Macrophages from C57BL/6 IL-10^−/− mice also exhibited increased killing of MB49 cells and production of TNF-α and NO upon BCG stimulation. In addition, supplementation of exogenous recombinant IL-10 reduced BCG-induced C3H/HeN macrophage cytotoxicity against both MBT-2 cells and MB49 cells in a dose-dependent manner. These results reveal the inhibitory role of IL-10 in BCG-induced macrophage cytotoxicity, suggesting that blockage of IL-10 may potentially enhance the effect of BCG in the treatment of bladder cancer patients.     

Ambiguous Role of Interleukin-12 in Yersinia enterocolitica Infection in Susceptible and Resistant Mouse Strains

Endogenous interleukin-12 (IL-12) mediates protection against Yersinia enterocolitica in C57BL/6 mice by triggering gamma interferon (IFN-γ) production in NK and CD4⁺ T cells. Administration of exogenous IL-12 confers protection against yersiniae in Yersinia-susceptible BALB/c mice but exacerbates yersiniosis in resistant C57BL/6 mice. Therefore, we wanted to dissect the different mechanisms exerted by IL-12 during Yersinia infections by using different models of Yersinia-resistant and -susceptible mice, including resistant C57BL/6 mice, susceptible BALB/c mice, intermediate-susceptible wild-type 129/Sv mice, 129/Sv IFN-γ-receptor-deficient (IFN-γR^−/−) mice and C57BL/6 tumor necrosis factor (TNF) receptor p55 chain-deficient (TNFR p55^−/−) mice. IFN-γR^−/− mice turned out to be highly susceptible to infection by Y. enterocolitica compared with IFN-γR^+/+ mice. Administration of IL-12 was protective in IFN-γR^+/+ mice but not in IFN-γR^−/− mice, suggesting that IFN-γR-induced mechanisms are essential for IL-12-induced resistance against yersiniae. BALB/c mice could be rendered Yersinia resistant by administration of anti-CD4 antibodies or by administration of IL-12. In contrast, C57BL/6 mice could be rendered more resistant by administration of transforming growth factor β (TGF-β). Furthermore, IL-12-triggered toxic effects in C57BL/6 mice were abrogated by coadministration of TGF-β. While administration of IL-12 alone increased TNF-α levels, administration of TGF-β or TGF-β plus IL-12 decreased both TNF-α and IFN-γ levels in Yersinia-infected C57BL/6 mice. Moreover, IL-12 did not induce toxicity in Yersinia-infected TNFR p55^−/− mice, suggesting that TNF-α accounts for IL-12-induced toxicity. Taken together, IL-12 may induce different effector mechanisms in BALB/c and C57BL/6 mice resulting either in protection or exacerbation. These results are important for understanding the critical balance of proinflammatory and regulatory cytokines in bacterial infections which is decisive for beneficial effects of cytokine therapy.     

Gamma interferon-independent effects of interleukin-12 on immunity to Salmonella enterica serovar Typhimurium

Interleukin-12 (IL-12) and IL-18 are both central to the induction of gamma interferon (IFN-γ), and various roles for IL-12 and IL-18 in control of intracellular microbial infections have been demonstrated. We used IL-12p40^−/− and IL-18^−/− mice to further investigate the role of IL-12 and IL-18 in control of Salmonella enterica serovar Typhimurium. While C57BL/6 and IL-18^−/− mice were able to resolve attenuated S. enterica serovar Typhimurium infections, the IL-12p40^−/− mice succumbed to a high bacterial burden after 60 days. Using ovalbumin (OVA)-specific T-cell receptor transgenic T cells (OT-II cells), we demonstrated that following oral infection with recombinant S. enterica serovar Typhimurium expressing OVA, the OT-II cells proliferated in the mesenteric lymph nodes of C57BL/6 and IL-18^−/− mice but not in IL-12p40^−/− mice. In addition, we demonstrated by flow cytometry that equivalent or increased numbers of T cells produced IFN-γ in IL-12p40^−/− mice compared with the numbers of T cells that produced IFN-γ in C57BL/6 and IL-18^−/− mice. Finally, we demonstrated that removal of macrophages from S. enterica serovar Typhimurium-infected C57BL/6 and IL-12p40^−/− mice did not affect the bacterial load, suggesting that impaired control of S. enterica serovar Typhimurium infection in the absence of IL-12p40 is not due to reduced macrophage bactericidal activities, while IL-18^−/− mice did rely on the presence of macrophages for control of the infection. Our results suggest that IL-12p40, but not IL-18, is critical to resolution of infections with attenuated S. enterica serovar Typhimurium and that especially the effects of IL-12p40 on proliferative responses of CD4⁺ T cells, but not the ability of these cells to produce IFN-γ, are important in the resolution of infection by this intracellular bacterial pathogen.     

Standardization and performance evaluation of mononuclear cell cytokine secretion assays in a multicenter study

Background

Burkholderia pseudomalleiis the causative agent for melioidosis. For many bacterial infections, cytokine dysregulation is one of the contributing factors to the severe clinical outcomes in the susceptible hosts. The C57BL/6 and BALB/c mice have been established as a differential model of susceptibility in murine melioidosis. In this study, we compared the innate IFN-γ response toB. pseudomalleibetween the C57BL/6 and BALB/c splenocytes and characterized the hyperproduction of IFN-γ in the relatively susceptible BALB/c micein vitro.

Results

Naïve BALB/c splenocytes were found to produce more IFN-γ in response to live bacterial infection compared to C57BL/6 splenocytes. Natural killer cells were found to be the major producers of IFN-γ, while T cells and Gr-1^intermediatecells also contributed to the IFN-γ response. Although anti-Gr-1 depletion substantially reduced the IFN-γ response, this was not due to the contribution of Gr-1^high, Ly-6G expressing neutrophils. We found no differences in the cell types making IFN-γ between BALB/c and C57BL/6 splenocytes. Although IL-12 is essential for the IFN-γ response, BALB/c and C57BL/6 splenocytes made similar amounts of IL-12 after infection. However, BALB/c splenocytes produced higher proinflammatory cytokines such as IL-1β, TNF-α, IL-6, IL-18 than C57BL/6 splenocytes after infection withB. pseudomallei.

Conclusion

Higher percentages of Gr-1 expressing NK and T cells, poorer ability in controlling bacteria growth, and higher IL-18 could be the factors contributing to IFN-γ hyperproduction in BALB/c mice.     

Dectin-2 Deficiency Promotes Th2 Response and Mucin Production in the Lungs after Pulmonary Infection with Cryptococcus neoformans

Dectin-2 is a C-type lectin receptor that recognizes high mannose polysaccharides. Cryptococcus neoformans, a yeast-form fungal pathogen, is rich in polysaccharides in its cell wall and capsule. In the present study, we analyzed the role of Dectin-2 in the host defense against C. neoformans infection. In Dectin-2 gene-disrupted (knockout) (Dectin-2KO) mice, the clearance of this fungus and the inflammatory response, as shown by histological analysis and accumulation of leukocytes in infected lungs, were comparable to those in wild-type (WT) mice. The production of type 2 helper T (Th2) cytokines in lungs was higher in Dectin-2KO mice than in WT mice after infection, whereas there was no difference in the levels of production of Th1, Th17, and proinflammatory cytokines between these mice. Mucin production was significantly increased in Dectin-2KO mice, and this increase was reversed by administration of anti-interleukin 4 (IL-4) monoclonal antibody (MAb). The levels of expression of β1-defensin, cathelicidin, surfactant protein A (Sp-A), and Sp-D in infected lungs were comparable between these mice. In in vitro experiments, IL-12p40 and tumor necrosis factor alpha (TNF-α) production and expression of CD86 and major histocompatibility complex (MHC) class II by bone marrow-derived dendritic cells and alveolar macrophages were completely abrogated in Dectin-2KO mice. Finally, the disrupted lysates of C. neoformans, but not of whole yeast cells, activated Dectin-2-triggered signaling in an assay with nuclear factor of activated T cells (NFAT)-green fluorescent protein (GFP) reporter cells expressing this receptor. These results suggest that Dectin-2 may oppose the Th2 response and IL-4-dependent mucin production in the lungs after infection with C. neoformans, and it may not be required for the production of Th1, Th17, and proinflammatory cytokines or for clearance of this fungal pathogen.     

Impact of Surfactant Protein D,Interleukin-5, and Eosinophilia on Cryptococcosis

Cryptococcus neoformans is an opportunistic fungal pathogen that initiates infection following inhalation. As a result, the pulmonary immune response provides a first line of defense against C. neoformans. Surfactant protein D (SP-D) is an important regulator of pulmonary immune responses and is typically host protective against bacterial and viral respiratory infections. However, SP-D is not protective against C. neoformans. This is evidenced by previous work from our laboratory demonstrating that SP-D-deficient mice infected with C. neoformans have a lower fungal burden and live longer than wild-type (WT) control animals. We hypothesized that SP-D alters susceptibility to C. neoformans by dysregulating the innate pulmonary immune response following infection. Thus, inflammatory cells and cytokines were compared in the bronchoalveolar lavage fluid from WT and SP-D^−/− mice after C. neoformans infection. Postinfection, mice lacking SP-D have reduced eosinophil infiltration and interleukin-5 (IL-5) in lung lavage fluid. To further explore the interplay of SP-D, eosinophils, and IL-5, mice expressing altered levels of eosinophils and/or IL-5 were infected with C. neoformans to assess the role of these innate immune mediators. IL-5-overexpressing mice have increased pulmonary eosinophilia and are more susceptible to C. neoformans infection than WT mice. Furthermore, susceptibility of SP-D^−/− mice to C. neoformans infection could be restored to the level of WT mice by increasing IL-5 and eosinophils by crossing the IL-5-overexpressing mice with SP-D^−/− mice. Together, these studies support the conclusion that SP-D increases susceptibility to C. neoformans infection by promoting C. neoformans-driven pulmonary IL-5 and eosinophil infiltration.     

Differential CD86 and CD40 co-stimulatory molecules and cytokine expression pattern induced by Trypanosoma cruzi in APCs from resistant or susceptible mice

Differential aspects of the host immune response generated by Trypanosoma cruzi infection were examined in two different mouse strains, BALB/c (haplotype H2-Kd) which does not overcome the acute phase of the infection and C57BL/6 (haplotype H2-Kb) which survives to the acute phase. After infection an increase in CD3+ T cells was observed in both mouse strains in the peritoneal cavity. However, while the CD3+ T cells from the BALB/c mice showed an increase in the IL-4 cytokine expression level, the same type of cells from the C57BL/6 mice showed an increase in IFN-gamma expression. In addition, only the macrophages from the C57BL/6 mice were activated secreting IL-12 and TNF-alpha and producing, moreover, high levels of nitrites. It was observed that also after parasite infection the expression of macrophage and dendritic cells CD40 and CD86 co-stimulation molecules from the spleen were diminished in BALB/c but not in C57BL/6 mice. In correlation with this observation the macrophages from the spleen of infected BALB/c mice secreted lower concentrations of nitrites than the C57BL/6 mouse cells. Also, the spleen dendritic cells from infected BALB/c mice had a small potential to present alloantigens in contrast to that observed in the infected C57BL/6 mouse cells.     

Pulmonary Infection with an Interferon-γ-Producing Cryptococcus neoformans Strain Results in Classical Macrophage Activation and Protection

Alternative macrophage activation is associated with exacerbated disease in murine models of pulmonary cryptococcosis. The present study evaluated the efficacy of interferon-γ transgene expression by Cryptococcus neoformans strain H99γ in abrogating alternative macrophage activation in infected mice. Macrophage recruitment into the lungs of mice after infection with C. neoformans strain H99γ was comparable with that observed in mice challenged with wild-type C. neoformans. However, pulmonary infection in mice with C. neoformans strain H99γ was associated with reduced pulmonary fungal burden, increased pulmonary Th1-type and interleukin-17 cytokine production, and classical macrophage activation as evidenced by increased inducible nitric oxide synthase expression, histological evidence of enhanced macrophage fungicidal activity, and resolution of inflammation. In contrast, progressive pulmonary infection, enhanced Th2-type cytokine production, and the induction of alternatively activated macrophages expressing arginase-1, found in inflammatory zone 1, Ym1, and macrophage mannose receptor were observed in the lungs of mice infected with wild-type C. neoformans. These alternatively activated macrophages were also shown to harbor highly encapsulated, replicating cryptococci. Our results demonstrate that pulmonary infection with C. neoformans strain H99γ results in the induction of classically activated macrophages and promotes fungal clearance. These studies indicate that phenotype, as opposed to quantity, of infiltrating macrophages correlates with protection against pulmonary C. neoformans infection.Cryptococcus neoformans is an opportunistic fungal pathogen and frequent cause of life-threatening infection in individuals with suppressed cell-mediated immunity.¹ C. neoformans is the most common mycological agent of morbidity and mortality in patients with AIDS with acute mortality rates ranging between 10 and 25% in developed countries worldwide.² Infection is initiated after the inhalation of desiccated basidiospores or yeast into lung alveoli, resulting in asymptomatic disease or mild bronchopneumonia in immunocompetent individuals.¹ However, bronchial infection is severe in immunocompromised patients and often leads to dissemination, resulting in severe meningoencephalitis. As inhalation is the principal route of entry for C. neoformans, clearance from the lungs is largely dependent on the ability of resident alveolar macrophages to degrade the yeast cells, thereby preventing dissemination.Experimental murine models of pulmonary C. neoformans infection suggest that resolution of infection is associated with the induction of Th1-type cytokine responses characterized by the production of interleukin (IL)-2, IL-12, tumor necrosis factor-α, and interferon (IFN)-γ.^{3,4,5,6,7,8,9,10,11} These cytokines, in turn, induce lymphocyte and phagocyte recruitment and activation of anticryptococcal delayed-type hypersensitivity responses. In contrast, uncontrolled fungal growth and exacerbation of pulmonary cryptococcosis is associated with Th2-type cytokine responses and the generation of alternatively activated macrophages (aaMac).^4,11,12 Specifically, aaMac are induced in high IL-4/IL-13 environments and are thought to contribute to pulmonary pathology by a variety of means.^12,13,14,15 First, aaMac up-regulate genes that increase cryptococcal persistence within macrophages, including arginase-1 (Arg1) and the macrophage mannose receptor (CD206). Up-regulation of Arg1 decreases synthesis of fungicidal nitric oxide by competing with inducible nitric oxide synthase (iNOS) for the substrate l-arginine.¹⁶ Increased surface expression of CD206 results in increased phagocytosis but is accompanied by decreased intracellular killing and TNF-α production.^17,18,19,20 Second, aaMac up-regulate proteins implicated in pulmonary pathology, such as chitinase family proteins Ym1, Ym2, and AMCase as well as found in inflammatory zone 1 (FIZZ1) protein.^4,12,13 Pulmonary C. neoformans infection in C57BL/6 mice is characterized by enhanced alternative macrophage activation and disease progression.^4,13 Interestingly, C57BL/6 mice deficient in IFN-γ develop augmented Th2-type cytokine production and the induction of aaMac during pulmonary C. neoformans infection.⁴ Moreover, IL-13 promotes aaMac differentiation, Th2-type cytokine responses, and allergic inflammation during experimental pulmonary cryptococcosis in mice.¹¹ Thus, alternative macrophage activation has a clear role in promoting progressive cryptococcal disease.^4,15,21Experimental pulmonary infection with the wild-type C. neoformans strain H99 results in fatal outcomes associated with overexuberant Th2-type cytokine responses in a variety of mouse models.^12,22 In contrast, experimental pulmonary infection of BALB/c mice with a C. neoformans strain H99 engineered to produce murine IFN-γ (designated H99γ) results in the induction of Th1-type cytokines as well as a significant influx of T cells, granulocytes, and antigen-presenting cells into the lungs.²³ However, the effect of IFN-γ transgene expression by C. neoformans strain H99γ on the macrophage activation profile in infected lungs remains unknown. The objective of these studies was to determine the activation phenotype of macrophages elicited in response to pulmonary infection with C. neoformans strain H99γ compared with that observed in mice infected with the wild-type C. neoformans.     

CD11c+ CD103+ cells of Mycobacterium tuberculosis‐infected C57BL/6 but not of BALB/c mice induce a high frequency of interferon‐γ‐ or interleukin‐17‐producing CD4+ cells

The magnitude of the cellular adaptive immune response is critical for the control of Mycobacterium tuberculosis infection in the chronic phase. In addition, the genetic background is equally important for resistance or susceptibility to tuberculosis. In this study, we addressed whether lung populations of dendritic cells, obtained from genetically different hosts, would play a role in the magnitude and function of CD4⁺ populations generated after M. tuberculosis infection. Thirty days post-infection, C57BL/6 mice, which generate a stronger interferon-γ (IFN-γ)-mediated immune response than BALB/c mice, exhibited a higher number and frequency of lung CD11c⁺ CD11b⁻ CD103⁺ cells compared with BALB/c mice, which exhibited a high frequency of lung CD11c⁺ CD11b⁺ CD103⁻ cells. CD11c⁺ CD11b⁻ CD103⁺ cells, purified from lungs of infected C57BL/6 mice, but not from infected BALB/c mice, induced a higher frequency of IFN-γ-producing or interleukin-17 (IL-17)-producing CD4⁺ cells. Moreover, CD4⁺ cells also arrive at the lung of C57BL/6 mice faster than in BALB/c mice. This pattern of immune response seems to be associated with higher gene expression for CCL4, CCL19, CCL20 and CCR5 in the lungs of infected C57BL/6 mice compared with infected BALB/c mice. The results described here show that the magnitude of IFN-γ-producing or IL-17-producing CD4⁺ cells is dependent on CD11c⁺ CD11b⁻ CD103⁺ cells, and this pattern of immune response is directly associated with the host genetic background. Therefore, differences in the genetic background contribute to the identification of immunological biomarkers that can be used to design human assays to predict progression of M. tuberculosis infection.