Interleukins 17 and 23 influence the host response to Histoplasma capsulatum.

Host defenses against Histoplasma capsulatum require the action of several cytokines. Here, we explored the influence of interleukin (IL)-17 and IL-23 on immunity to H. capsulatum infection in mice. In lungs, synthesis of IL-17 was up-regulated during acute infection, and the cells producing it were predominantly CD3(+). Neutralization of IL-17A blunted fungal clearance but did not promote progressive infection. Decreased inflammatory cell recruitment and increased levels of IL-6 and IL-10 were associated with impaired clearance. To determine whether the elevated cytokine levels were important in the action of IL-17A, IL-6(-/-) or IL-10(-/-) mice were treated with anti-IL-17A; neutralization of IL-17A did not alter fungal burden in either group of knockout mice. We explored the relationship between IL-17 and IL-23 because they have been reported to form a regulatory network. IL-23 transcription and protein level were increased in the lungs of infected mice. Mice producing IL-23 in the absence of IL-12 manifested prolonged survival that was IL-17 dependent. Thus, IL-17 is requisite for the generation of optimal inflammatory and protective responses. Generation of functional IL-17(+) cells is dependent on IL-6 and IL-10. Our findings also establish the existence a regulatory IL-17/IL-23 axis in histoplasmosis.

Histoplasma capsulatum manifests preferential invasion of phagocytic subpopulations in murine lungs.

Numerous in vitro studies have demonstrated that Histoplasma capsulatum is engulfed by the diverse populations of phagocytic cells including monocytes/macrophages (Mphi), immature dendritic cells (DC), and neutrophils. The in vivo distribution of H. capsulatum has yet to be examined following an intrapulmonary challenge. To accomplish this goal, we engineered GFP into two genetically dissimilar strains of H. capsulatum, G217B and 186R. C57BL/6 mice were infected with each of these strains, and we analyzed the distribution of this fungus in the three major phagocytic populations on successive days. Yeast cells were found in all three populations of cells from Days 1 through 7. Proportionally, DC dominated at Day 1, whereas the majority of yeast cells was detected in neutrophils thereafter. Yeast cells were present in inflammatory and resident Mphi on Day 3, but on Day 7, they were chiefly in inflammatory Mphi. Yeast cells were predominantly in a CD11c(+intermediate/high), F4/80(-), CD11b(+), Ly-6C(+), CD205(+) DC population. Neutralization of TNF-alpha or IFN-gamma produced a significant redistribution of yeast cells. These results reveal the complex nature of intracellular residence of this fungus. Moreover, the findings demonstrate that there is a skewing in the subpopulations of cells that are infected, especially DC.

TNF-alpha antagonism generates a population of antigen-specific CD4+CD25+ T cells that inhibit protective immunity in murine histoplasmosis.

In both humans and mice, treatment with TNF-alpha antagonists is associated with serious infectious complications including disseminated histoplasmosis. The mechanisms by which inhibition of endogenous TNF-alpha alter protective immunity remain obscure. Herein, we tested the possibility that neutralization of this cytokine triggered the emergence of T cells that dampen immunity. The lungs of mice given mAb to TNF-alpha contained a higher proportion and number of CD4+CD25+ cells than controls. This elevation was not observed in IFN-gamma- or GM-CSF-deficient mice or in those given a high inoculum. Phenotypic analysis revealed that these cells lacked many of the characteristics of natural regulatory T cells, including Foxp3. CD4+CD25+ cells from TNF-alpha-neutralized mice suppressed Ag-specific, but not nonspecific, responses in vitro. Elimination of CD25+ cells in vivo restored protective immunity in mice given mAb to TNF-alpha and adoptive transfer of CD4+CD25+ cells inhibited immunity. In vitro and in vivo, the suppressive effect was reversed by mAb to IL-10. Thus, neutralization of TNF-alpha is associated with the induction of a population of regulatory T cells that alter protective immunity in an Ag-specific manner to Histoplasma capsulatum.

Expression of hygromycin phosphotransferase alters virulence of Histoplasma capsulatum.

The Escherichia coli hygromycin phosphotransferase (hph) gene, which confers hygromycin resistance, is commonly used as a dominant selectable marker in genetically modified bacteria, fungi, plants, insects, and mammalian cells. Expression of the hph gene has rarely been reported to induce effects other than those expected. Hygromycin B is the most common dominant selectable marker used in the molecular manipulation of Histoplasma capsulatum in the generation of knockout strains of H. capsulatum or as a marker in mutant strains. hph-expressing organisms appear to have no defect in long-term in vitro growth and survival and have been successfully used to exploit host-parasite interaction in short-term cell culture systems and animal experiments. We introduced the hph gene as a selectable marker together with the gene encoding green fluorescent protein into wild-type strains of H. capsulatum. Infection of mice with hph-expressing H. capsulatum yeast cells at sublethal doses resulted in lethality. The lethality was not attributable to the site of integration of the hph construct into the genomes or to the method of integration and was not H. capsulatum strain related. Death of mice was not caused by altered cytokine profiles or an overwhelming fungal burden. The lethality was dependent on the kinase activity of hygromycin phosphotransferase. These results should raise awareness of the potential detrimental effects of the hph gene.

Lung-restricted macrophage activation in the pearl mouse model of Hermansky-Pudlak syndrome.

Pulmonary inflammation, abnormalities in alveolar type II cell and macrophage morphology, and pulmonary fibrosis are features of Hermansky-Pudlak Syndrome (HPS). We used the naturally occurring "pearl" HPS2 mouse model to investigate the mechanisms of lung inflammation observed in HPS. Although baseline bronchoalveolar lavage (BAL) cell counts and differentials were similar in pearl and strain-matched wild-type (WT) mice, elevated levels of proinflammatory (MIP1gamma) and counterregulatory (IL-12p40, soluble TNFr1/2) factors, but not TNF-alpha, were detected in BAL from pearl mice. After intranasal LPS challenge, BAL levels of TNF-alpha, MIP1alpha, KC, and MCP-1 were 2- to 3-fold greater in pearl than WT mice. At baseline, cultured pearl alveolar macrophages (AMs) had markedly increased production of inflammatory cytokines. Furthermore, pearl AMs had exaggerated TNF-alpha responses to TLR4, TLR2, and TLR3 ligands, as well as increased IFN-gamma/LPS-induced NO production. After 24 h in culture, pearl AM LPS responses reverted to WT levels, and pearl AMs were appropriately refractory to continuous LPS exposure. In contrast, cultured pearl peritoneal macrophages and peripheral blood monocytes did not produce TNF-alpha at baseline and had LPS responses which were no different from WT controls. Exposure of WT AMs to heat- and protease-labile components of pearl BAL, but not WT BAL, resulted in robust TNF-alpha secretion. Similar abnormalities were identified in AMs and BAL from another HPS model, pale ear HPS1 mice. We conclude that the lungs of HPS mice exhibit hyperresponsiveness to LPS and constitutive and organ-specific macrophage activation.

T cells require tumor necrosis factor-alpha to provide protective immunity in mice infected with Histoplasma capsulatum.

We examined whether neutralization of tumor necrosis factor (TNF)-alpha after intranasal exposure of mice to Histoplasma capsulatum was necessary for control of primary or secondary infection. All mice given monoclonal antibody to TNF-alpha on the day of infection or on day 3 after infection died. When antibody was administered on day 5 after infection, 60% of mice with primary infection died, whereas none with secondary infection did. Antibody treatment on day 7 after infection produced a transiently higher fungal burden. Because optimal clearance required TNF-alpha after the onset of infection, we hypothesized that it may regulate T cell function. Lung CD3+ cells were the dominant population of TNF-alpha-producing cells (approximately 40%-70%). Neutralization of this cytokine decreased the number of memory T cells but not the number of activated, proliferating, or interferon-gamma-producing cells. T cells from infected, TNF-alpha-neutralized mice failed to protect T cell-deficient mice. The absence of TNF-alpha induces a defect in T cell-mediated protection.

Immune defects in 28-kDa proteasome activator gamma-deficient mice.

Protein complexes of the 28-kDa proteasome activator (PA28) family activate the proteasome and may alter proteasome cleavage specificity. Initial investigations have demonstrated a role for the IFN-gamma-inducible PA28alpha/beta complex in Ag processing. Although the noninducible and predominantly nuclear PA28gamma complex has been implicated in affecting proteasome-dependent signaling pathways, such as control of the mitotic cell cycle, there is no previous evidence demonstrating a role for this structure in Ag processing. We therefore generated PA28gamma-deficient mice and investigated their immune function. PA28gamma(-/-) mice display a slight reduction in CD8+ T cell numbers and do not effectively clear a pulmonary fungal infection. However, T cell responses in two viral infection models appear normal in both magnitude and the hierarchy of antigenic epitopes recognized. We conclude that PA28gamma(-/-) mice, like PA28alpha(-/-)/beta(-/-) mice, are deficient in the processing of only specific Ags.

Protective and memory immunity to Histoplasma capsulatum in the absence of IL-10.

We determined whether the absence of IL-10 in mice influenced protective and memory immunity to Histoplasma capsulatum. IL-10(-/-) mice cleared primary and secondary infection more rapidly than wild-type controls. Administration of mAb to TNF-alpha or IFN-gamma, but not GM-CSF, abrogated protection in naive IL-10(-/-) mice; mAb to TNF-alpha, but not IFN-gamma or GM-CSF, subverted protective immunity in secondary histoplasmosis. The inflammatory cell composition in IL-10(-/-) mice was altered in those given mAb to IFN-gamma or TNF-alpha. More Gr-1(+) and Mac-3(+) cells were present in lungs of IL-10(-/-) mice given mAb to IFN-gamma, and treatment with mAb to TNF-alpha sharply reduced the number of CD8(+) cells in lungs of IL-10(-/-) mice. We ascertained whether the lack of IL-10 modulated memory T cell generation or the protective function of cells. The percentage of CD3(+), CD44(high), CD62(low), and IFN-gamma(+) cells in IL-10(-/-) mice was higher than that of wild-type at day 7 but not day 21 or 49 after immunization. Fewer splenocytes from immunized IL-10(-/-) mice were required to mediate protection upon adoptive transfer into infected TCR alphabeta(-/-) mice. Hence, deficiency of IL-10 confers a salutary effect on the course of histoplasmosis, and the beneficial effects of IL-10 deficiency require endogenous TNF-alpha and/or IFN-gamma. Memory cell generation was transiently increased in IL-10(-/-) mice, but the protective function conferred by cells from these mice following immunization is strikingly more vigorous than that of wild-type.

Macrophage-independent fungicidal action of the pulmonary collectins.

Histoplasma capsulatum (Hc) is a facultative intracellular fungal pathogen that causes acute and chronic pneumonia. In this study, we investigated the role of the pulmonary collectins, surfactant proteins (SP) A and D, in the clearance of Hc yeast from the lung. Exposure of yeast to either collectin induced a dose-dependent decrease in [3H]leucine incorporation by several strains of Hc. This decrement was attributed to killing of the collectin-exposed yeast since it failed to grow on agar medium. Exposure to SP-A or -D resulted in increased yeast permeability based on a leak of protein from the organism and enhanced access of an impermeant substrate to intracellular alkaline phosphatase. Inbred and outbred SP-A null (-/-) mice were modestly more susceptible to pulmonary infection with Hc than strain and age-matched SP-A (+/+) control mice. The increase in susceptibility was associated with a decrement in the number of CD8+ cells in the lungs of SP-A-/- mice. Neither SP-A nor SP-D inhibited the growth of macrophage-internalized Hc. We conclude that the SP-A and SP-D are antimicrobial proteins that directly inhibit the growth of Hc by increasing permeability of the organism and that Hc gains asylum from collectin-mediated killing by rapid entry into pulmonary macrophages.

Overexpression of interleukin-4 in lungs of mice impairs elimination of Histoplasma capsulatum.

Protection against the pathogenic fungus Histoplasma capsulatum requires Th1 cytokines. Since interleukin-4 (IL-4) can inhibit both Th1 cytokine production and activity, we examined the effects of overproduction of IL-4 in the lung on the course of pulmonary histoplasmosis. IL-4 lung transgenic mice manifested a higher fungal burden in their lungs, but not spleens, compared to wild-type infected controls. Despite the higher burden, the transgenic animals were ultimately capable of controlling infection. The adverse effects of IL-4 on H. capsulatum elimination were not observed during the early phase of infection (days 1 to 3) but were maximal at day 7 postinfection, prior to the induction of cell-mediated immunity. Analysis of total body and lung cytokine levels revealed that gamma interferon and tumor necrosis factor alpha production were not inhibited in the presence of excess IL-4. Our results with transgenic mice were supported by additional in vivo studies in which allergen induction of pulmonary IL-4 was associated with delayed clearance of H. capsulatum yeast and increased fungal burden. These findings demonstrate that excess production of endogenous IL-4 modulates protective immunity to H. capsulatum by delaying clearance of the organism but does not prevent the generation of a Th1 response that ultimately controls infection.

Functional properties of the T cell receptor repertoire in responding to the protective domain of heat-shock protein 60 from Histoplasma capsulatum.

Cells expressing the T cell receptor beta-chain variable region (Vbeta) 6 constitute the majority of T cells responding to the protective domain (F3) of heat-shock protein 60 from Histoplasma capsulatum. This subset of cells exhibits a T helper type 1 (Th1) profile and is pivotal in protection. In this study, additional F3-reactive T cell lines were generated, leading to the discovery of a Th2 line. Vbeta usage by clones was more diverse than has been previously recognized. Nearly all Th2 clones expressed Vbeta8.1/8.2, whereas Th1 clones expressed Vbeta11 or Vbeta6. In adoptive transfer studies, only the Vbeta6(+) Th1 clone prolonged survival; all Th2 clones accelerated mortality. The ameliorative effect of the Vbeta6(+) Th1 clone was abrogated by treatment with monoclonal antibody to interferon-gamma. Neutralization of interleukin-4 reversed the shortened survival of mice to which the Vbeta6(+) Th2 clone was administered. Thus, F3-mediated protection is confined to a defined Vbeta population, but exacerbation of disease is mediated by multiple Vbeta families.

Cellular and molecular regulation of vaccination with heat shock protein 60 from Histoplasma capsulatum.

Vaccination with heat shock protein 60 (Hsp60) from Histoplasma capsulatum induces a protective immune response in mice. We explored the cellular and molecular requirements for the efficacy of recombinant Hsp60 in mice. Depletion of CD4(+), but not CD8(+), cells during the inductive phase of vaccination abolished protection, as assessed by survival and by the fungal burden in lungs and spleens. In the expressive phase, the elimination of CD4(+) or CD8(+) cells after immunization did not significantly alter fungal recovery or survival from a lethal challenge. Depletion of both subpopulations after Hsp60 vaccination resulted in a failure to control a lethal infection and a higher fungal burden in lungs and spleens. Cytokine release by spleen cells from mice vaccinated with Hsp60 produced substantially more gamma interferon and interleukin-10 and -12 than that of cells from mice immunized with either H. capsulatum recombinant Hsp70 or bovine serum albumin. The generation of gamma interferon, but not of interleukin-10, was dependent on T cells, in particular CD4(+) cells. Treatment of Hsp60-immunized mice with monoclonal antibody to gamma interferon or interleukin-10 or -12 in the inductive phase of vaccination was accompanied by increased recovery of yeast cells from lungs and spleens and 100% mortality. Likewise, the neutralization of gamma interferon or interleukin-12 abolished the protective effect of Hsp60 in the expressive phase. These results delineate the complexity of the regulatory elements necessary for vaccination against this fungus.

Discordance between T-cell receptor expression and effector function in mice infected with Histoplasma capsulatum.

V beta 10(+) and V beta 14(+) T cells were selectively increased 7 to 14 days following infection in the lungs of naive mice infected with Histoplasma capsulatum. Following secondary challenge of immune mice, V beta 1(+) and V beta 8.1(+) cells were sporadically increased. Elimination of V beta 10(+) and V beta 14(+) cells from naive mice did not alter the course of infection over a period of 21 days. Thus, overexpression of V beta families does not necessarily signify a key role in host defense.