Roderick A. McDonald

Urokinase is required for the pulmonary inflammatory response to Cryptococcus neoformans. A murine transgenic model.

Urokinase (uPA) is hypothesized to provide proteolytic activity enabling inflammatory cells to traverse tissues during recruitment, and it is implicated as a cytokine modulator. Definitive evaluation of these hypotheses in vivo has previously been impossible because uPA could not completely and irreversibly be eliminated. This limitation has been overcome through the development of uPA-deficient transgenic mice (uPA-/-). Using these mice, we evaluated the importance of uPA in the pulmonary inflammatory response to Cryptococcus neoformans (strain 52D). C. neoformans was inoculated into uPA-/- and control mice (uPA+/+), and cell recruitment to the lungs was quantitated. The number of CFU in lung, spleen and brain was determined to assess clearance, and survival curves were generated. By day 21 after inoculation, uPA-/- mice had markedly fewer pulmonary inflammatory (CD45+), CD4+, and CD11b/CD18+ cells compared with uPA+/+ controls (P<0.0007); pulmonary CFUs in the uPA-/- mice continued to increase, whereas CFUs diminished in uPA+/+ mice(P<0.005). In survival studies, only 3/19 uPA+/+ mice died, whereas 15/19 uPA-/- mice died (p<0.001). We have demonstrated that uPA is required for a pulmonary inflammatory response to C. neoformans. Lack of uPA results in inadequate cellular recruitment, uncontrolled infection, and death.

Role of IFN-γ in Regulating T2 Immunity and the Development of Alternatively Activated Macrophages during Allergic Bronchopulmonary Mycosis

Pulmonary Cryptococcus neoformans infection of C57BL/6 mice is an established model of a chronic pulmonary fungal infection accompanied by an “allergic” response (T2) to the infection, i.e., a model of an allergic bronchopulmonary mycosis. Our objective was to determine whether IFN-γ plays a role in regulating the pulmonary T2 immune response in C. neoformans-infected C57BL/6 mice. Long-term pulmonary fungistasis was lost in IFN-γ knockout (KO) mice, resulting in an increased pulmonary burden of fungi at wk 3. IFN-γ was required for the early influx of leukocytes into the lungs but was not required later in the infection. By wk 3, eosinophil and macrophage numbers were elevated in the absence of IFN-γ. The inducible NO synthase to arginase ratio was lower in the lungs of IFN-γ KO mice and the macrophages had increased numbers of intracellular cryptococci and YM1 crystals, indicative of alternatively activated macrophages in these mice. There was evidence of pulmonary fibrosis in both wild-type and IFN-γ KO mice by 5 wk postinfection. IFN-γ production was not required for the development of T2 cytokine (IL-4, IL-5, IL-13) producing cells in the lungs and lung-associated lymph nodes or induction of an IgE response. At a number of time points, T2 cytokine production was enhanced in IFN-γ KO mice. Thus, in the absence of IFN-γ, C57BL/6 mice develop an augmented allergic response to C. neoformans, including enhanced generation of alternatively activated macrophages, which is accompanied by a switch from a chronic to a progressive pulmonary cryptococcal infection.

Induction of Interleukin-12 and Gamma Interferon Requires Tumor Necrosis Factor Alpha for Protective T1-Cell-Mediated Immunity to Pulmonary Cryptococcus neoformans Infection

ABSTRACT 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-α) augments the development of pulmonary T1 immunity to C. neoformans infection. TNF-α 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-α-treated mice at any time point examined during the afferent phase of the response (days 0 to 7). However, neutralization of TNF-α prevented the initiation of pulmonary clearance during the efferent phase of the response (day 14). Administration of anti-TNF-α monoclonal antibody (day 0) diminished the lung levels of TNF-α, interleukin-12 (IL-12), and gamma interferon (IFN-γ) induced by C. neoformans at day 7 postinfection. Neutralization of TNF-α (day 0) also altered the IFN-γ/IL-4 ratio in the lung-associated lymph nodes at day 7 following C. neoformans infection. Anti-TNF-α-treated mice developed a pulmonary eosinophilia at day 14 postinfection. Consistent with the pulmonary eosinophilia, anti-TNF-α-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-γ in response to the infection. These findings demonstrate that afferent-phase TNF-α production is essential for the induction of IL-12 and IFN-γ and neutralization of early TNF-α results in a T2 shift of the T1/T2 balance of antifungal immunity.

Effect of cytokine interplay on macrophage polarization during chronic pulmonary infection with Cryptococcus neoformans.

ABSTRACT The immune response to Cryptococcus neoformans following pulmonary infection of C57BL/6 wild-type (WT) mice results in the development of persistent infection with characteristics of allergic bronchopulmonary mycosis (ABPM). To further clarify the role of Th1/Th2 polarizing cytokines in this model, we performed kinetic analysis of cytokine responses and compared cytokine profiles, pathologies, and macrophage (Mac) polarization status in C. neoformans-infected WT, interleukin-4-deficient (IL-4−/−), and gamma interferon-deficient (IFN-γ−/−) C57BL/6 mice. Results show that cytokine expression in the infected WT mice is not permanently Th2 biased but changes dynamically over time. Using multiple Mac activation markers, we further demonstrate that IL-4 and IFN-γ regulate the polarization state of Macs in this model. A higher IL-4/IFN-γ ratio leads to the development of alternatively activated Macs (aaMacs), whereas a higher IFN-γ/IL-4 ratio leads to the generation of classically activated Macs (caMacs). WT mice that coexpress IL-4 and IFN-γ during fungal infection concurrently display both types of Mac polarization markers. Concurrent stimulation of Macs with IFN-γ and IL-4 results in an upregulation of both sets of markers within the same cells, i.e., formation of an intermediate aaMac/caMac phenotype. These cells express both inducible nitric oxide synthase (important for clearance) and arginase (associated with chronic/progressive infection). Together, our data demonstrate that the interplay between Th1 and Th2 cytokines supports chronic infection, chronic inflammation, and the development of ABPM pathology in C. neoformans-infected lungs. This cytokine interplay modulates Mac differentiation, including generation of an intermediate caMac/aaMac phenotype, which in turn may support chronic “steady-state” fungal infection and the resultant ABPM pathology.

Distinct Roles for IL-4 and IL-10 in Regulating T2 Immunity during Allergic Bronchopulmonary Mycosis

Pulmonary Cryptococcus neoformans infection of C57BL/6 mice is an established model of an allergic bronchopulmonary mycosis that has also been used to test a number of immunomodulatory agents. Our objective was to determine the role of IL-4 and IL-10 in the development/manifestation of the T2 response to C. neoformans in the lungs and lung-associated lymph nodes. In contrast to wild-type (WT) mice, which develop a chronic infection, pulmonary clearance was significantly greater in IL-4 knockout (KO) and IL-10 KO mice but was not due to an up-regulation of a non-T cell effector mechanism. Pulmonary eosinophilia was absent in both IL-4 KO and IL-10 KO mice compared with WT mice. The production of IL-4, IL-5, and IL-13 by lung leukocytes from IL-4 KO and IL-10 KO mice was lower but IFN-γ levels remained the same. TNF-α and IL-12 production by lung leukocytes was up-regulated in IL-10 KO but not IL-4 KO mice. Overall, IL-4 KO mice did not develop the systemic (lung-associated lymph nodes and serum) or local (lungs) T2 responses characteristic of the allergic bronchopulmonary C. neoformans infection. In contrast, the systemic T2 elements of the response remained unaltered in IL-10 KO mice whereas the T2 response in the lungs failed to develop indicating that the action of IL-10 in T cell regulation was distinct from that of IL-4. Thus, although IL-10 has been reported to down-regulate pulmonary T2 responses to isolated fungal Ags, IL-10 can augment pulmonary T2 responses if they occur in the context of fungal infection.

The role of macrophage inflammatory protein-1α/CCL3 in regulation of T cell-mediated immunity to Cryptococcus neoformans infection.

Macrophage inflammatory protein-1α (MIP-1α/CCL3) is a CC chemokine required for optimal recruitment of leukocytes in response to cryptococcal Ags. MIP-1α is expressed in the lungs by day 6 post Cryptococcus neoformans infection and could play a role in the development of cell-mediated immunity. To address this possibility, wild-type (MIP-1α+/+) mice and MIP-1α knockout (MIP-1α−/−) mice were infected intratracheally with a highly virulent strain of C. neoformans (145A). MIP-1α message was detected in the lungs on days 3, 7, and 14 in MIP-1α+/+ mice, but it was undetectable in MIP-1α−/− mice. On day 16, MIP-1α−/− mice had a 7-fold increase in C. neoformans burden in the lungs, but no decrease in pulmonary leukocyte recruitment. MIP-1α+/+ and MIP-1α−/− mice had similar numbers of recruited lymphocytes and monocytes/macrophages. Notably, MIP-1α−/− mice had a significantly greater number of eosinophils. MIP-1α−/− mice had extremely high levels of serum IgE. This switch of immune response to a T2 phenotype was associated with enhanced IL-4 and IL-13 expression in the lungs of MIP-1α−/− mice compared with MIP-1α +/+ mice. Progression of pulmonary cryptococcosis in the presence of nonprotective T2 immunity resulted in profound lung damage in MIP-1α−/− mice (eosinophilic crystal deposition, destruction of lung parenchyma, and pulmonary hemorrhage). Twelve-week survival was dramatically decreased in MIP-1α−/− mice. These studies, together with our previous studies, demonstrate that MIP-1α plays a role in both the afferent (T1/T2 development) and efferent (T1-mediated leukocyte recruitment) phases of cell-mediated immunity to C. neoformans.

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

ABSTRACT Mice with a null deletion mutation in the gamma interferon (IFN-γ) receptor gene were used to study the role of IFN-γ responsiveness during experimental pulmonary cryptococcosis. Cryptococcus neoformans was inoculated intratracheally into mice lacking the IFN-γ receptor gene (IFN-γR−/−) and into control mice (IFN-γR+/+). 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-γR−/− mice are markedly more susceptible to C. neoformans infection than IFN-γR+/+ mice. (ii) In the absence of IFN-γ signaling, pulmonary CFU continue to increase over the course of infection, and the infection disseminates to the brain. (iii) In the absence of IFN-γ receptor, recruitment of inflammatory cells in response to pulmonary cryptococcal infection is not impaired. (iv) At week 5 postinfection, IFN-γR−/− mice have recruited greater numbers of leukocytes into their lungs, with neutrophils, eosinophils, and lymphocytes accounting for this cellular increase. (v) IFN-γ 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- γ responsiveness, even though the recruitment of pulmonary inflammatory cells is not impaired and the secretion of IFN-γ is not affected, IFN-γR−/− mice do not have the ability to resolve the cryptococcal infection. In conclusion, our data suggest that proper functional IFN-γ signaling, possibly through a mechanism which inhibits the potentially disease-promoting T2 response, is required for mice to confine the cryptococcal infection.

Generation of antifungal effector CD8+ T cells in the absence of CD4+ T cells during Cryptococcus neoformans infection

Immunity to the opportunistic fungus Cryptococcus neoformans is dependent on cell-mediated immunity. Individuals with defects in cellular immunity, CD4+ T cells in particular, are susceptible to infection with this pathogen. In host defense against a number of pathogens, CD8+ T cell responses are dependent upon CD4+ T cell help. The goal of these studies was to determine whether CD4+ T cells are required for the generation of antifungal CD8+ T cell effectors during pulmonary C. neoformans infection. Using a murine intratracheal infection model, our results demonstrated that CD4+ T cells were not required for the expansion and trafficking of CD8+ T cells to the site of infection. CD4+ T cells were also not required for the generation of IFN-γ-producing CD8+ T cell effectors in the lungs. In CD4− mice, depletion of CD8+ T cells resulted in increased intracellular infection of pulmonary macrophages by C. neoformans, increasing the pulmonary burden of the infection. Neutralization of IFN-γ in CD4−CD8+ mice similarly increased macrophage infection by C. neoformans, thereby blocking the protection provided by CD8+ T cells. Altogether, these data support the hypothesis that effector CD8+ T cell function is independent of CD4+ T cells and that IFN-γ production from CD8+ T cells plays a role in controlling C. neoformans by limiting survival of C. neoformans within macrophages.

Coevolution of TH1, TH2, and TH17 Responses during Repeated Pulmonary Exposure to Aspergillus fumigatus Conidia

ABSTRACT Aspergillus fumigatus, a ubiquitous airborne fungus, can cause invasive infection in immunocompromised individuals but also triggers allergic bronchopulmonary aspergillosis in a subset of otherwise healthy individuals repeatedly exposed to the organism. This study addresses a critical gap in our understanding of the immunoregulation in response to repeated exposure to A. fumigatus conidia. C57BL/6 mice were challenged intranasally with A. fumigatus conidia weekly, and leukocyte composition, activation, and cytokine production were examined after two, four, and eight challenges. Approximately 99% of A. fumigatus conidia were cleared within 24 h after inoculation, and repeated exposure to A. fumigatus conidia did not result in hyphal growth or accumulation of conidia with time. After 2 challenges, there was an early influx of neutrophils and regulatory T (Treg) cells into the lungs but minimal inflammation. Repeated exposure promoted sustained expansion of the draining lymph nodes, while the influx of eosinophils and other myeloid cells into the lungs peaked after four exposures and then decreased despite continued A. fumigatus challenges. Goblet cell metaplasia and low-level fibrosis were evident during the response. Repeated exposure to A. fumigatus conidia induced T cell activation in the lungs and the codevelopment by four exposures of TH1, TH2, and TH17 responses in the lungs, which were maintained through eight exposures. Changes in CD4 T cell polarization or Treg numbers did not account for the reduction in myeloid cell numbers later in the response, suggesting a non-T-cell regulatory pathway involved in dampening inflammation during repeated exposure to A. fumigatus conidia.

Regulatory effects of macrophage inflammatory protein 1α/CCL3 on the development of immunity to Cryptococcus neoformans depend on expression of early inflammatory cytokines

ABSTRACT Macrophage inflammatory protein 1α (MIP-1α)/CCL3 prevents the development of eosinophilic pneumonia (EP) driven by a nonprotective T2-type immunity during infection with a highly virulent strain ofCryptococcus neoformans. The present study evaluated the interaction of MIP-1α 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-1α in C. neoformans 52D infection did not cause the development of EP. C. neoformans 52D induced tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), and MCP-1 in the lungs of infected wild-type (WT) and MIP-1α knockout (KO) mice by day 7 postinfection. Both WT and MIP-1α 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-1α. Alternatively, C. neoformans145A-infected WT and MIP-1α KO mice had diminished TNF-α, IFN-γ, and macrophage chemoattractant protein 1 (MCP-1) responses, indicating that virulent C. neoformans 145A evaded early innate host defenses. However C. neoformans145A-infected WT mice had an early induction of MIP-1α and subsequently did not develop EP. In contrast, C. neoformans 145A-infected MIP-1α 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-1α is crucial to prevent the development of EP and to control C. neoformansdissemination to the brain.