Eosinophils subvert host resistance to an intracellular pathogen by instigating non-protective IL-4 in CCR2-/- mice.

Eosinophils contribute to type II immune responses in helminth infections and allergic diseases; however, their influence on intracellular pathogens is less clear. We previously reported that CCR2-/- mice exposed to the intracellular fungal pathogen Histoplasma capsulatum exhibit dampened immunity caused by an early exaggerated interleukin (IL)-4 response. We sought to identify the cellular source promulgating IL-4 in infected mutant animals. Eosinophils were the principal instigators of non-protective IL-4 and depleting this granulocyte population improved fungal clearance in CCR2-/- animals. The deleterious impact of eosinophilia on mycosis was also recapitulated in transgenic animals overexpressing eosinophils. Mechanistic examination of IL-4 induction revealed that phagocytosis of H. capsulatum via the pattern recognition receptor complement receptor (CR) 3 triggered the heightened IL-4 response in murine eosinophils. This phenomenon was conserved in human eosinophils; exposure of cells to the fungal pathogen elicited a robust IL-4 response. Thus, our findings elucidate a detrimental attribute of eosinophil biology in fungal infections that could potentially trigger a collapse in host defenses by instigating type II immunity.

Type II cytokines impair host defense against an intracellular fungal pathogen by amplifying macrophage generation of IL-33.

Interleukin (IL)-4 subverts protective immunity to multiple intracellular pathogens, including the fungus Histoplasma capsulatum. Previously, we reported that H. capsulatum-challenged CCR2(-/-) mice manifest elevated pulmonary fungal burden owing to exaggerated IL-4. Paradoxical to our anticipation in IL-33 driving IL-4, we discovered that the latter prompted IL-33 in mutant mice. In infected CCR2(-/-) animals, amplified IL-33 succeeded the heightened IL-4 response and inhibition of IL-4 signaling decreased IL-33. Moreover, macrophages, but not epithelial cells or dendritic cells, from these mice expressed higher IL-33 in comparison with controls. Dissection of mechanisms that promulgated IL-33 revealed type-II cytokines and H. capsulatum synergistically elicited an IL-33 response in macrophages via signal transducer and activator of transcription factor 6/interferon-regulatory factor-4 and Dectin-1 pathways, respectively. Neutralizing IL-33 in CCR2(-/-) animals, but not controls, enhanced their resistance to histoplasmosis. Thus we describe a previously unrecognized role for IL-4 in instigating IL-33 in macrophages. Furthermore, in the presence of intracellular fungal pathogens, the type-II cytokine-driven IL-33 response impairs immunity.

V beta 6+ T cells are obligatory for vaccine-induced immunity to Histoplasma capsulatum.

We examined TCR usage to a protective fragment of heat shock protein 60 from the fungus, Histoplasma capsulatum. Nearly 90% of T cell clones from C57BL/6 mice vaccinated with this protein were Vbeta6+; the remainder were Vbeta14+. Amino acid motifs of the CDR3 region from Vbeta6+ cells were predominantly IxGGG, IGG, or SxxGG, whereas it was uniformly SFSGG for Vbeta14+ clones. Short term T cell lines from Vbeta6+-depleted mice failed to recognize Ag, and no T cell clones could be generated. To determine whether Vbeta6+ cells were functionally important, we eliminated them during vaccination. Depletion of Vbeta6+ cells abrogated protection in vivo and upon adoptive transfer of cells into TCR alphabeta(-/-) mice. Transfer of a Vbeta6+, but not a Vbeta14+, clone into TCR alphabeta(-/-) mice prolonged survival. Cytokine generation by Ag-stimulated splenocytes from immunized mice depleted of Vbeta6+ cells was similar to that of controls. The efficacy of the Vbeta6+ clone was associated with elevated production of IFN-gamma, TNF-alpha, and GM-CSF compared with that of the Vbeta14+ clone. More Vbeta6+ cells were present in lungs and spleens of TCR alphabeta(-/-) on day 3 postinfection compared with Vbeta14+ cells. Thus, a single Vbeta family was essential for vaccine-induced immunity. Moreover, the mechanism by which Vbeta6+ contributed to protective immunity differed between unfractionated splenocytes and T cell clones.

Protective efficacy of H antigen from Histoplasma capsulatum in a murine model of pulmonary histoplasmosis.

We previously reported that immunization with H antigen from Histoplasma capsulatum did not protect mice against an intravenous challenge with yeasts. Here, we investigated the utility of H antigen to protect mice in a model of pulmonary histoplasmosis. Mice immunized with H antigen and challenged intranasally 4 weeks postvaccination were protected against sublethal and lethal challenges with H. capsulatum yeasts. If the challenge was performed 3 months after vaccination, there was a reduction in fungal burden following sublethal challenge and a modest delay in mortality in mice given a lethal inoculum. Vaccination was associated with production of gamma interferon, granulocyte-macrophage colony-stimulating factor, interleukin-4, and interleukin-10 by splenocytes. Vaccination with H antigen was not accompanied by a major expansion of CD4(+) or CD8(+) cells in spleens of mice. These results demonstrate that H antigen may be useful as a protective immunogen against pulmonary exposure to H. capsulatum.

V beta 6+ and V beta 4+ T cells exert cooperative activity in clearance of secondary infection with Histoplasma capsulatum.

We previously studied the lung Vbeta TCR repertoire of C57BL/6 mice during primary infection with the pathogen Histoplasma capsulatum. We observed a consistent oligoclonal expansion of Vbeta4(+) T cells during the peak of infection and early stages of resolution. The Vbeta4(+) family played a role in protective immunity against the fungus. Depletion of this subpopulation of T cells hindered optimal clearance of infection from tissues. In this report we analyze the flux of the Vbeta TCR repertoire in the lungs of C57BL/6 mice with reinfection histoplasmosis. We observed a significant increase in Vbeta6(+) T cells on days 7, 10, and 14, the peak and early resolution phases of infection. This skewing was preceded by an increased number of memory T cells within Vbeta6(+) cells. The VDJ sequences of Vbeta6 chains were oligoclonal during the early stages of the infection, suggesting that the expansion was driven by a small number of Ags. More than 96% of the expanded Vbeta6(+) cells were CD4(+). Depletion of Vbeta6(+) T cells but not Vbeta4(+) T cells induced a modest but significant delay in fungal clearance. Simultaneous depletion of Vbeta4(+) and Vbeta6(+) T cells induced a more pronounced impairment of host resistance. These studies illustrate the dynamic interactions between Vbeta families in the response to microbial challenge.

Molecular cloning, characterization and expression of the heat shock protein 60 gene from the human pathogenic fungus Paracoccidioides brasiliensis.

A gene encoding the heat shock protein (HSP) 60 from Paracoccidioides brasiliensis (Pb) was cloned and characterized. The hsp60 gene is composed of three exons divided by two introns. Structural analysis of the promoter detected canonical sequences characteristic of regulatory regions from eukaryotic genes. The deduced amino acid sequence of the Pb hsp60 gene and the respective cloned cDNA consists of 592 residues highly homologous to other fungal HSP60 proteins. The hsp60 gene is present as a single copy in the genome, as shown by Southern blot analysis. The HSP60 protein was isolated from Pb yeast cellular extracts. N-terminal amino acid sequencing of HSP60 confirmed that the cloned hsp60 gene correlated to the predicted protein in Pb. HSP60 expression appeared to be regulated during form transition in Pb, as different levels of expression were detected in in vitro labeling of cells and northern blot analysis. The complete coding region of Pb hsp60 was fused with plasmid pGEX-4T-3 and expressed in Escherichia coli as a glutathione S-transferase-tagged recombinant protein. The protein reacted with a mouse monoclonal antibody raised to a human recombinant HSP60. Western immunoblot experiments demonstrated that the recombinant protein and the native HSP60 were recognized by sera from humans with paracoccidioidomycosis (PCM).

Recombinant murine granulocyte-macrophage colony-stimulating factor modulates the course of pulmonary histoplasmosis in immunocompetent and immunodeficient mice.

Several endogenous cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF), are necessary for eliminating Histoplasma capsulatum from tissues. In this study, we explored the efficacy of recombinant murine GM-CSF in the treatment of pulmonary histoplasmosis. This cytokine significantly reduced fungal burden in a dose-dependent manner. Pretreatment did not consistently produce a better result than treatment started after infection. The biological effectiveness of GM-CSF was not associated with modulation of lung cytokine production or alteration in lung inflammation, but it directly activated a nonadherent lung cell population to exert anti-Histoplasma activity. GM-CSF improved survival of T-cell-depleted mice exposed to H. capsulatum. When combined with a suboptimal amount of amphotericin B, GM-CSF enhanced survival of normal or T-cell-depleted mice given a lethal challenge. These results suggest that this cytokine may be useful as an adjunctive treatment for histoplasmosis.

Immune response to early and late Histoplasma capsulatum infections.

Knowledge of the host response to the intracellular pathogenic fungus Histoplasma capsulatum has increased dramatically. Information has accumulated regarding the cellular and molecular determinants that lead to resolution of both primary and secondary infection. The significance of cytokines and other endogenous soluble mediators to the protective immune response have been analyzed. Moreover, work concerning the relative importance of T cell subsets to protective immunity has been initiated.

Regulation of infection with Histoplasma capsulatum by TNFR1 and -2.

The concerted action of several cytokines is necessary for resolution of both primary and secondary infection with Histoplasma capsulatum. Among the soluble factors that contribute to tissue sterilization, TNF-alpha stands as a central mediator of protective immunity to this fungus. In this study, we explored the regulation of protective immunity by TNFR1 and -2. In primary pulmonary infection, both TNFR1-/- and -2-/- mice manifested a high mortality after infection with H. capsulatum, although TNFR1-/- mice were more susceptible than TNFR2 -/- mice. Overwhelming infection in the former was associated with a pronounced decrement in the number of inflammatory cells in the lungs and elevated IFN-gamma and TNF-alpha levels in the lungs. In contrast, IFN-gamma levels were markedly decreased in TNFR2-/- mice, and treatment with this cytokine restored protective immunity. Lung macrophages from both groups of knockout mice released substantial amounts of NO. Upon secondary infection, TNFR2-/- mice survived rechallenge and cleared infection as efficiently as C57BL/6 animals. In contrast, mice given mAb to TNFR1 succumbed to reexposure, and the high mortality was accompanied by a significant increase in fungal burden in the lungs. Both IL-4 and IL-10 were elevated in the lungs of these mice. The results demonstrate the pivotal influence of TNFR1 and -2 in controlling primary infection and highlight the differences between these receptors for regulation reexposure histoplasmosis.

Applying in vivo expression technology (IVET) to the fungal pathogen Histoplasma capsulatum.

Understanding how pathogens survive within the host cell is of paramount importance in the development of vaccines and therapeutic agents. This task has been particularly daunting in the study of fungal pathogens due to the lack of easily manipulated genetic systems. In recent years several molecular genetic reporter systems have been developed to identify genes expressed during the infection process and potential virulence determinants. The development of one method in particular, in vivo expression technology (IVET), has led to the discovery of several genes from various bacterial pathogens necessary for survival during infection. The recent development of molecular genetic tools for Histoplasma capsulatum has enabled us to adapt the IVET technology for this pathogenic fungus utilizing the URA5 gene, which is essential for H. capsulatum survival in mice and in cultured macrophages, as a reporter of in vivo gene expression. We report the first successful application of IVET screening of a fungal pathogen for genes expressed exclusively during infection.

Interleukin-12 neutralization alters lung inflammation and leukocyte expression of CD80, CD86, and major histocompatibility complex class II in mice infected with Histoplasma capsulatum.

Histoplasma capsulatum induces a cell-mediated immune response in lungs and lymphoid organs of mammals. Resolution of primary infection in mice depends on interleukin-12 (IL-12), since neutralization of this monokine increases susceptibility to infection. The present study was designed to determine if blockade of IL-12 disrupts the protective immune response by altering the influx of lineage-specific cells into infected lungs and the numbers of cells expressing CD80, CD86, CD119, and major histocompatibility complex class II (MHC II) molecules. In mice given anti-IL-12, there was a 2.5-fold decrease in total numbers of T cells on days 3 to 10 of infection and a 4-fold increase in Mac-1/Gr-1(+) cells on days 7 and 10 compared to infected controls. CD80(+) lung cells from anti-IL-12-treated mice were 2- to 3-fold greater than those from controls on days 7 and 10, whereas the total numbers of CD86(+) cells were 2- to 3-fold less and MHC II(+) cells were 1.5- to 2-fold less on days 3 and 5. Cells expressing CD119 were reduced 1.5-fold on day 5. Treatment with monoclonal antibodies (MAb) to CD80, CD86, or both reduced the fungal burden slightly compared to that in rat immunoglobulin G-treated controls, whereas after IL-12 neutralization, blocking of CD80 reduced the tissue burden by 2. 5-fold and this correlated with a decrease in IL-4. Regardless, mortality was not altered by treatment with MAb to CD80 or CD86. We conclude that (i) IL-12 neutralization alters the nature of the inflammatory response in lungs and the expression of CD80 and CD86 on lineage-specific cells, (ii) the immune response during infection with H. capsulatum is controlled via mechanisms independent of the CD80 and CD86 costimulatory pathways, and (iii) decreased expression of CD86 and MHC II may modulate generation of optimal protective immunity.

Knockout mice as experimental models of virulence.

Infection models with animals whose immune systems have been selectively altered by neutralization of endogenous cytokines or by deletion of a gene have provided a valuable means to study the function of cells or cytokines in the context of complex multidimensional interactions. In particular, knockout mice have allowed a deeper insight into the in vivo performance of antifungal innate and acquired immunity, whose interplay is considered fundamental in the general defense against infections. It is conceivable that such an integrated view of effector and regulatory immune mechanisms operating in opportunistic fungal infections would facilitate the search for cells, cytokines and molecular pathways that are essential to control fungal infectivity or oppose fungus-associated immunopathology.

Neutralization of endogenous granulocyte-macrophage colony-stimulating factor subverts the protective immune response to Histoplasma capsulatum.

We examined the influence of endogenous GM-CSF on the course of primary and secondary pulmonary histoplasmosis. A high proportion (>/=75%) of C57BL/6 mice given mAb to GM-CSF did not survive primary infection, whereas 88-94% of infected controls survived. Analysis of leukocytes revealed significantly fewer CD4+ and CD8+ cells in lungs, but not airways, of anti-GM-CSF-treated mice as compared with infected controls. However, the histopathology was similar between the two groups. Lungs of mice given mAb to GM-CSF manifested depressed levels of TNF-alpha, IFN-gamma, and reactive nitrogen intermediates and elevated levels of IL-4 and IL-10. Administration of mAb to IL-4, to IL-10, or both restored protective immunity in GM-CSF-neutralized mice. In secondary infection, administration of mAb to GM-CSF exacerbated infection but did not alter survival over 30 days. The character of the inflammatory response was similar, and no differences were detected in Th1 or Th2 cytokine production between the two groups. Thus, endogenous GM-CSF is essential for survival in primary but not secondary infection, and blockade perturbs protective immunity. These findings reveal a new mechanism whereby GM-CSF contributes to host protection and demonstrate differences in control of primary and secondary histoplasmosis.

Histoplasma capsulatum strain variation in both H antigen production and beta-glucosidase activity and overexpression of HAG1 from a telomeric linear plasmid.

The H antigen of the dimorphic fungal pathogen Histoplasma capsulatum was first described over 40 years ago. It is a secreted glycoprotein that is immunogenic during infection. Recent cloning of the H antigen gene (HAG1) indicated sequence homology with genes for fungal beta-glucosidases. To understand the biological role of this immunodominant antigen in H. capsulatum, enzymatic assays were performed to determine whether H. capsulatum contained a beta-glucosidase enzyme activity and whether this activity was encoded by the HAG1 gene. Substrate gels with H. capsulatum culture supernatants revealed beta-glucosidase activity near the predicted mobility of the H antigen. Quantitative microtiter plate assays revealed marked differences in secreted beta-glucosidase activities from three H. capsulatum restriction fragment length polymorphism (RFLP) classes, with RFLP class II strains displaying high levels of enzyme activity, in contrast to the low levels of activity exhibited by class I and III strains. Immunoblotting of culture supernatants with an H antigen-specific antiserum demonstrated differences in H protein expression levels between the H. capsulatum classes, with a correlation between secreted enzyme activity and H protein levels. We took advantage of these class differences to demonstrate multicopy plasmid H gene overexpression by transformation of an HAG1 plasmid into H. capsulatum. Both a class II strain (G217Bura5-23) and a class III strain (G184ASura5-11) transformed with the telomeric overexpression plasmid pMAD401 displayed increased levels of beta-glucosidase enzyme activity and H protein expression compared to the levels in control transformants containing only the single genomic copy of HAG1. This is the first demonstration of telomeric plasmid-mediated protein overexpression in this pathogenic fungus, and the findings support the identification of the H antigen as a beta-glucosidase.

Complex requirements for nascent and memory immunity in pulmonary histoplasmosis.

The presence of functional T cells is often required for successful resolution of infections with intracellular pathogens, yet the mechanisms by which they contribute to elimination of the invading pathogen in primary and secondary immunity are only partly understood. We report that increased mortality of naive alpha/beta TCR+ or CD4+ T cell-depleted mice infected with the fungus Histoplasma capsulatum is associated with impairment of IFN-gamma production. Upon secondary infection, mice concomitantly depleted of CD4+ and CD8+ cells exhibit decreased survival beyond day 25 of rechallenge, whereas elimination of either T cell subset or B cell deficiency does not result in accelerated mortality compared with controls. Remarkably, despite a decrease of H. capsulatum CFU in lungs of CD4+ plus CD8+-deficient mice, a progressive increase in spleen CFU is observed. The ability to control fungus growth in lungs is associated with vigorous TNF-alpha, but not IFN-gamma, production by bronchoalveolar lavage cells. In contrast, spleen cells from CD4+ plus CD8+-deficient mice are unable to produce TNF-alpha. Thus, the cellular and molecular requirements for protective immunity vary between primary and secondary infection. Furthermore, in secondary histoplasmosis, a sharp contrast can be drawn between lungs and spleens in their reliance upon T cells to control fungal replication. The opposing activities of these organs can be ascribed in part to differential production of TNF-alpha.

Molecular cloning, characterization, and expression of the M antigen of Histoplasma capsulatum.

The major diagnostic antigens of Histoplasma capsulatum are the H and M antigens, pluripotent glycoproteins that elicit both humoral and T-cell-mediated immune responses. These antigens may play a role in the pathogenesis of histoplasmosis. M antigen is considered immunodominant because antibodies against it are the first precipitins to arise in acute histoplasmosis and are commonly present during all phases of infection. The biological activity of monomolecular M antigen and its ability to elicit a protective immune response to H. capsulatum are largely unknown. A molecular approach was used to identify the biological nature of M antigen, including its purification from histoplasmin, partial digestion with proteinases, and reverse-phase high-performance liquid chromatography to separate the released peptides. The amino acid sequences of the purified peptides were obtained by Edman degradation, and using degenerate oligonucleotide primers for PCR, a 321-bp fragment of the gene encoding the M antigen was amplified from genomic H. capsulatum DNA. This fragment was used to screen an H. capsulatum genomic DNA library, leading to the isolation, cloning, and sequencing of the full-length gene. The M gene consists of 2, 187-bp DNA encoding a protein of 80,719 Da, which has significant homology to catalases from Aspergillus fumigatus, Aspergillus niger, and Eimericella nidulans. A cDNA was generated by reverse transcription-PCR and cloned into the expression vector pQE40. The identity of the cloned, expressed protein was confirmed by Western blotting. The recombinant fusion protein was immunoreactive with monoclonal antibodies raised against M antigen, with polyclonal mouse anti-M antiserum, and with a serum sample from a patient with histoplasmosis. The gene encoding the major immunodominant M antigen of H. capsulatum is a presumptive catalase, and the recombinant protein retains serodiagnostic activity.

The URA5 gene is necessary for histoplasma capsulatum growth during infection of mouse and human cells.

The Histoplasma capsulatum URA5 gene, which has recently been cloned and disrupted by allelic replacement, encodes orotidine-5'-monophosphate pyrophosphorylase. Inactivation of URA5 by either targeted or UV mutagenesis results in disruption of the pyrimidine biosynthetic pathway and uracil auxotrophy. We examined the effect of uracil auxotrophy due to a ura5 mutation on H. capsulatum virulence in both cell culture and whole-animal models. Uracil auxotrophs of two H. capsulatum restriction fragment length polymorphism classes were found to be avirulent in cultured murine and human cells, as well as in mice. Moreover, virulence could be restored either by supplying a functional URA5 gene in trans or by supplying exogenous uracil during infection in vitro. These experiments demonstrate that the pyrimidine biosynthetic pathway is essential for H. capsulatum growth and virulence.

Simultaneous infection with Blastomyces dermatitidis and Cryptococcus neoformans.

In this report, we present two cases of simultaneous infection with Blastomyces dermatitidis and Cryptococcus neoformans. In both cases, fungi were isolated from the lungs. The clinical manifestations were not suggestive of infection with two fungi. One patient was receiving immunosuppressive therapy and another was otherwise immunocompetent. A review of the literature failed to uncover any reports of co-infection with these two fungal pathogens. We discuss possible mechanisms for the isolation of both pathogens.

Vbeta4(+) T cells promote clearance of infection in murine pulmonary histoplasmosis.

T cells are essential for controlling infection with Histoplasma capsulatum. Because the T cell receptor is vital for transducing the biological activities of these cells, we sought to determine if exposure to this fungus induced an alteration in the Vbeta repertoire in lungs of C57BL/6 mice infected intranasally. Vbeta2(+) cells were elevated on day 3 after infection; Vbeta4(+) cells were higher than controls on days 7, 10, and 14 after infection. Vbeta10(+) cells were increased on days 14 and 21, and Vbeta11(+) exceeded controls only on day 14. We investigated the clonality and function of Vbeta4(+) cells because their expansion transpired during the critical time of infection, that is, when cellular immunity is activated. Sequence analysis demonstrated preferential use of a restricted set of sequences in the complementarity-determining region 3. Elimination of Vbeta4(+) cells from mice impaired their ability to resolve infection. In contrast, depletion of Vbeta7(+) cells, the abundance of which was similar to that of Vbeta4(+), did not alter elimination of the fungus. The identification of clonotypes of Vbeta4(+) cells suggests that a few antigenic determinants may drive proliferation of this subset, which is necessary for optimal clearance.