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The Journal of Immunology, 2006, 177: 1763-1771.
Copyright © 2006 by The American Association of Immunologists

B Cells and CD4–CD8– T Cells Are Key Regulators of the Severity of Reactivation Histoplasmosis1

Holly L. Allen and George S. Deepe, Jr.2

Division of Infectious Diseases, Veterans Affairs Hospital, University of Cincinnati College of Medicine, Cincinnati, OH 45267


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The fungus, Histoplasma capsulatum, produces a persistent infection. Reactivation histoplasmosis is largely a result of impaired immunity, but the perturbations associated with escape of the fungus from host defenses remain ill-defined. We analyzed a murine model of reactivation to elucidate the host defects that permit reactivation. C57BL/6 mice were infected intranasally and, 42 days later, they were depleted of CD4+ and CD8+ cells. Elimination of these cells, but not either alone, produced a persistent infection over several weeks. Neutralization of IFN-{gamma}, TNF-{alpha}, or both did not induce reactivation. Endogenous IL-10 exacerbated reactivation. Depletion of T cells in B cell–/– mice induced a markedly higher burden in organs when compared with wild type. However, the infection remained persistent. Elimination of CD4+ cells alone or neutralization of cytokines increased the fungal load. The persistent infection was not dependent on {gamma}{delta} T cells or NK cells. Elimination of Thy-1.2+ cells in mice given mAb to CD4 and CD8 transformed reactivation into a progressive, lethal infection in B cell–/– and wild-type mice, but the tempo of progression was accelerated in the former. The data reveal the complex control by the host to prevent reactivation of this fungus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Upon disruption of the soil, microconidia of the fungus, Histoplasma capsulatum, become airborne and are inhaled coincidentally. The particles settle into terminal bronchioles and alveoli and convert into the pathogenic yeast phase. Subsequently, yeasts disseminate to visceral and lymphoid organs. This form evokes granuloma formation and virtually all of the clinicopathological manifestations of histoplasmosis (1). Infection with H. capsulatum produces a broad spectrum of disease, but the vast majority of exposed individuals recover without sequelae. The organism establishes a persistent state within visceral and lymphoid tissues (2, 3). If those who harbor dormant organisms develop an immunosuppressive condition, they become at-risk for reactivation. The AIDS epidemic before antiretroviral therapy was associated with marked increases in cases of disseminated histoplasmosis, much of it a result of reactivation from latent foci (2, 3, 4, 5). Pharmacologic agents to combat malignancy, autoimmunity, aggressive inflammatory diseases, or organ graft rejection inhibit expression of cellular immunity, and consequently, there is an increase in transforming a latent infection to a clinically active one.

Reactivation of microbes is a widely recognized clinical event (6, 7, 8). This form of infection occurs in the absence of known immunosuppression; in many cases, the organism emerges from its dormant state to cause disease in a host whose immune system, particularly the adaptive arm, is impaired. The underlying immunological mechanisms that prevent reactivation and the perturbations that lead to reactivation are not fully understood in histoplasmosis. We report the development of chronic and progressive murine models of reactivation. Functional T and B cells are necessary to prevent reactivation, and the cytokines, IL-10, IFN-{gamma}, and TNF-{alpha}, are important in controlling the severity of reactivation disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6, µMT (Igh-6tm1Cgn), IL-10–/– (Il10tm1Cgn), and {gamma}{delta} TCR–/– (Tcrdtm1Mom) mice were purchased from The Jackson Laboratory. Athymic nude mice were purchased from National Cancer Institute and used to produce ascites. Animals were housed in isolator cages and were maintained by the Department of Laboratory Animal Medicine (University of Cincinnati), which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. All animal experiments were performed in accordance with the Animal Welfare Act guidelines of the National Institutes of Health, and all protocols were approved by the Institutional Animal Care and Use Committee of the University of Cincinnati.

Preparation of H. capsulatum and infection of mice

H. capsulatum yeast (strain G217B) was prepared as described previously (9). To produce infection in naive mice, animals were inoculated intranasally (i.n.)3 with 2 x 106 H. capsulatum yeast cells in a 30-µl volume of HBSS.

Organ culture for H. capsulatum

Recovery of H. capsulatum was performed as described elsewhere (9). Fungal burden was expressed as mean log10 CFU per whole organ ± SEM. The limit of detection was 102 CFU.

Cytokine measurement

Lungs from infected mice were excised and homogenized in 10 ml of HBSS. The homogenates were centrifuged at 1500 x g, filter sterilized, and was stored at –70°C until assayed. IFN-{gamma} levels were assessed by a commercially available ELISA kit (Endogen).

Monoclonal Abs

The following mAb were used: rat anti-mouse CD4 (clone GK1.5), rat anti-mouse CD8 (clone 2.43), rat anti-mouse Thy1.2 (clone 30-H12), rat anti-mouse TNF-{alpha} (clone XT-22.1), rat anti-mouse IFN-{gamma} (clone XMG1.2), rat anti-mouse GM-CSF (clone MP1–22E9), rat anti-mouse IL-10 (clone JES5A), and rat anti-mouse NK1.1 (clone PK136). mAb to CD4, CD8, Thy-1.2, TNF-{alpha}, and IFN-{gamma} were purchased from the National Cell Culture Center. mAb to GMCSF, IL-10, and NK1.1 were purified from ascites. The concentration of rat IgG was assessed by ELISA after protein G purification. The amount was calculated by linear regression from a rat IgG standard curve. Anti-asialo GM Ab was purchased from Wako Chemicals.

Treatment of mice with depleting and/or neutralizing mAb

Mice were injected i.p. with mAb beginning at 42 days postinfection and treatment was continued weekly until the end of the experiment. Either 100 µg (anti-CD4, anti-CD8) or 1 mg (anti-Thy1.2, anti-TNF-{alpha}, anti-IFN-{gamma}, anti-GMCSF, anti-IL-10) was administered. To deplete NK cells, mice were treated with 1 mg of anti-NK1.1 and 50 µg of anti-asialo-GM each wk. Control animals received rat IgG.

Treatment with aminoguanidine

Mice were injected twice daily i.p. with 100 µg of aminoguanidine (Sigma-Aldrich) dissolved in 500 µl of HBSS or an equal volume of HBSS. In exploratory studies, this dose of aminoguanidine reduced NO production by >90% in alveolar macrophages from mice infected with 2 x 106 H. capsulatum (G. S. Deepe, unpublished observations). This dose also has been shown to induce sufficient inhibition of NO in vivo (10).

Flow cytometry

The efficacy of cell depletion was confirmed by flow cytometry. The following Abs were purchased from BD Biosciences: CD3-PE, CD4-FITC, CD8-PerCP, and Thy1.2-allophycocyanin. Spleens were teased apart with the frosted ends of two glass slides and washed HBSS. A total of 2 x 106 cells were incubated with 0.5 µg of Ab in staining buffer (1% BSA in PBS) for 10 min at 4°C. The cells were washed in staining buffer and fluorescence was measured using a FACSCalibur flow cytometer (BD Pharmingen).

Statistical analyses

ANOVA was used to compare groups. A value of p < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Reactivation is dependent on the loss of T cells

C57BL/6 mice infected with 2 x 106 H. capsulatum yeast cells i.n. reduce the fungal burden below the detection limits of 102 CFU/organ by 28 days (9, 11). Although fungi are not isolated beyond that day, we reasoned that they may persist at <102 for a subsequent period of time, and that we could induce reactivation. A dysfunction or loss of T cells is often associated with reactivation histoplasmosis (5, 12, 13). We infected mice with the above inoculum and at day 42 postinfection, we commenced depletion of CD4+ and CD8+ cells; treatment continued every 7 days. A control group of mice received an equivalent amount of rat IgG. Mice were sacrificed at serial intervals after initiation of reactivation (Fig. 1); spleens and lungs were assessed for fungal burden. No fungi were detected in lungs and spleens of mice that received rat IgG throughout the duration of study. Elimination of CD4+ and CD8+ cells was associated with recovery of yeasts at each of the days postreactivation in both lungs and spleens. At day 14, CFU in spleens were similar to that of lungs but exceeded those in lungs at each of the days thereafter (Fig. 2, A and B). The number of CFU in lungs gradually declined. In spleens, CFU peaked at day 28 postreactivation and remained constant.


Figure 1
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  		FIGURE 1. Regimen of assessing reactivation. Mice were infected with 2 x 106 yeast i.n. and at the designated times, they were treated with mAb to cells or to cytokines. Arrows indicate the days at which treatment with mAb was given. Thick arrows represent days in which mice were sacrificed and fungal burden was assessed.

 

Figure 2
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  		FIGURE 2. Depletion of CD4+ and CD8+ T cells induces reactivation histoplasmosis in C57BL/6 mice. C57BL/6 mice were infected with 2 x 106 yeast cells i.n. Six weeks later, mice were treated with mAb i.p. weekly until the end of the experiment where fungal burden was assessed in the lungs (A and C) and spleens (B and D). A and B, Mice were treated with both anti-CD4 and anti-CD8 and fungal burden was assessed at 14, 28, 42, and 56 days postreactivation. A and B, Mice were given anti-CD4 or anti-CD8 alone or in combination and fungal burden was assessed at 14 and 56 days postreactivation. Data represent mean ± SEM of at least six animals per group.

 
We assessed the contribution of individual T cell subsets to reactivation. Mice were depleted of CD4+, CD8+, or CD4+ and CD8+ cells and assayed on days 14 and 56 postreactivation. The absence of CD4+ or CD8+ cells alone did not induce reactivation (Fig. 2, C and D).

A concern of using mAb to deplete cells is that the host mounts a response to foreign protein (in this case rat IgG), and the efficacy of depletion is attenuated. To address this issue, we analyzed the proportion of CD4+ and CD8+ cells in mice given rat IgG or mAb to CD4 and CD8 at 56 days postreactivation. Continued administration of mAb to CD4 and CD8 did not blunt the efficacy of cell depletion (Table I).


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  		Table I. Percentages of CD4+ and CD8+ cells in the spleen in reactivation histoplasmosisa

 
Reactivation in B cell–/– mice

Clinically, reactivation histoplasmosis may manifest as a chronic or acute infection. The former is insidious and may be slowly progressive over time whereas the latter is rapidly progressive and often associated with hemologic abnormalities, high fever, and a sepsis-like syndrome (14). The model we developed mimicked a chronic persistent reactivation rather than a rapidly progressive illness. To determine whether the other major lymphocyte population, B cells, shaped the profile of reactivation, we infected B cell–/– and wild-type mice with 2 x 106 yeasts i.n. and at 42 days postinfection, we initiated treatment with mAb to CD4 or CD8 or both. Of note, the course of primary and secondary histoplasmosis in B cell–/– mice is nearly identical with that in wild-type (12). Elimination of T cells in B cell–/– mice produced reactivation in lungs and spleens (Fig. 3, A and B). The fungal burden was significantly higher in B cell–/– mice as compared with wild type although the pattern of reactivation was similar (Fig. 3, A and B).


Figure 3
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  		FIGURE 3. Reactivation histoplasmosis in B cell–/– mice. B cell–/– (µMT) and wild-type (C57BL/6) mice were infected with 2 x 106 yeast cells i.n. Six weeks later, mice were treated with mAb i.p. weekly until the end of the experiment where fungal burden was assessed in the lungs (A and C) and spleens (B and D). A and B, Mice were treated with both anti-CD4 and anti-CD8 and fungal burden was assessed at 14, 28, 42, and 56 days postreactivation. C and D, Mice were given anti-CD4 or anti-CD8 alone or in combination and fungal burden was assessed at 14 and 56 days postreactivation. Data represent mean ± SEM of at least eight animals per group. A and B, ***, p < 0.001 when compared with wild-type values at the same time point. C and D, ***, p < 0.001, when compared with day 14.

 
We assessed whether elimination of CD4+ or CD8+ cells alone caused reactivation in B cell–/– mice. Infected animals were administered mAb to CD4 or CD8 or both, and fungal burden was assessed at 14 and 56 days postreactivation. The lungs and spleens of mice depleted of CD4+ cells contained H. capsulatum at 56 days postreactivation, but not 14 days (Fig. 3, C and D). However, the number of CFU in lungs and spleens was not as high as in mice in which both subsets were depleted. Elimination of CD8+ cells did not induce reactivation.

Influence of cytokines that enhance protective immunity in reactivation

A principal effector mechanism in host resistance to H. capsulatum is cytokines. IFN-{gamma}, TNF-{alpha}, and GM-CSF are necessary for protective immunity in primary or secondary histoplasmosis or both (9, 11, 15, 16, 17, 18, 19, 20). To determine whether any or all of these cytokines influenced reactivation, wild-type and B cell–/– mice were infected with 2 x 106 yeast cells i.n. and at 42 days postinfection, they were treated with 1 mg of mAb to IFN-{gamma}, TNF-{alpha}, or both. Although a few CFU were detected in mice treated with these mAb, there was no marked increase in CFU in either lungs or spleens (Table II).


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  		Table II. Fungal burden in C57BL/6 mice treated with neutralizing Ab to TNF-{alpha} and/or IFN-{gamma}a

 
Subsequently, we sought to determine whether neutralization of endogenous IFN-{gamma} and/or TNF-{alpha}would alter the profile of reactivation in T cell-deficient mice. Groups of CD4+ and CD8+-depleted animals were given mAb to IFN-{gamma}, TNF-{alpha}, or both. Neutralization of these cytokines in the absence of T cells did not alter the fungal recovery when compared with T cell-depleted mice whether they were wild type or B cell–/–. Because GM-CSF is critically important in host control of primary infection, we treated T cell-depleted mice with mAb to TNF-{alpha}, IFN-{gamma}, and GM-CSF and assessed CFU in lungs and spleens. Administration of mAb to GM-CSF in conjunction with mAb to IFN-{gamma} and TNF-{alpha} did not enhance the severity of reactivation in T cell-depleted mice (Fig. 4, A and B).


Figure 4
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  		FIGURE 4. Neutralization of Th1 cytokines in combination with T cell depletion modulates reactivation histoplasmosis in C57BL/6 mice. Wild-type (A and B) or B cell–/– (C–E) mice were infected with 2 x 106 yeast cells i.n. Six weeks later, mice were treated with mAb i.p. weekly until the end of the experiment where fungal burden was assessed in the lungs (A, C, and E) and spleens (B, D, and F) at 14 and 56 days postreactivation. All mice were treated with 100 µg of anti-CD4 plus anti-CD8 or 100 µg of anti-CD4 alone (B cell–/– only). Some groups were also treated with 1 mg of anti-TNF-{alpha}, anti-IFN-{gamma}, and/or anti-GMCSF. Data represent mean ± SEM of at least eight animals per group. **, p < 0.01 when compared with controls; ***, p < 0.001 when compared with controls and ###, p < 0.001 when compared with each other.

 
In parallel, we tested the effect of neutralization of these cytokines in B cell–/– mice depleted of either CD4+ and CD8+ cells or CD4+ cells alone. A significant increase in CFU in lungs and spleens of T cell-deficient mice was induced by neutralization of IFN-{gamma} (Fig. 4, C and D). The burden did not differ in IFN-{gamma} and TNF-{alpha}-neutralized mice. In mice lacking only CD4+ cells, reactivation was exacerbated when IFN-{gamma} or IFN-{gamma} and TNF-{alpha} were neutralized. (Fig. 4, E and F).

Role of NO

IFN-{gamma} and TNF-{alpha} may exert a regulatory effect through the induction of NO (9, 20, 21, 22). This soluble mediator is important in reactivation of murine tuberculosis (23, 24). We tested the influence of NO by treatment with aminoguanidine in T cell sufficient and deficient wild-type mice. Daily administration of aminoguanidine to T cell sufficient mice did not cause reactivation whereas this compound enhanced fungal burden in T cell-depleted animals in the spleen (Fig. 5, A and B).


Figure 5
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  		FIGURE 5. Inhibition of NOS or IL-10 production in vivo modulates reactivation histoplasmosis in C57BL/6. C57BL/6 mice were infected with 2 x 106 yeast cells i.n. Six weeks later, mice were treated with mAb i.p. weekly until the end of the experiment where fungal burden was assessed in the lungs (A, C, and E) and spleens (B, D, and F) at 14 and 56 days postreactivation. A and B, Mice were either treated with 100 µg of anti-CD4 and anti-CD8, aminoguanidine alone (AMG, 100 µg i.p., twice a day) or a combination of mAb and AMG treatment together. C and D, Wild-type mice were treated with anti-CD4 and anti-CD8 with or without 1 mg of anti-IL-10. E and F, Wild-type or IL-10–/– mice were treated with 100 µg of anti-CD4 and CD8 beginning 42 days postinfection. Data represent mean ± SEM of at least eight animals per group. *, 0.05; **, p < 0.01; ***, p < 0.001 when compared with controls. #, No CFU detected.

 
IL-10 and reactivation

IL-10 diminishes the cellular immune response to H. capsulatum and its presence is associated with a higher fungal burden when compared with mice lacking the IL-10 gene. We examined the influence of IL-10 on reactivation. Wild-type mice were depleted of T cells and concomitantly treated with mAb to IL-10 and at 14 and 56 days postreactivation, the lungs and spleens were evaluated for fungal burden. mAb to IL-10 significantly blunted reactivation (Fig. 5, C and D). Because IL-10 neutralization reduced the severity of reactivation, we determined whether this effect was associated with an increase in IFN-{gamma} production. Lung and spleen homogenates from wild-type mice and IL-10-neutralized mice were assessed for IFN-{gamma}. At days 14 and 56 of reactivation, the levels of IFN-{gamma} in recipients of mAb to IL-10 exceeded (p < 0.05) those of controls (Table III).


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  		Table III. IFN-{gamma} levels in lungs and spleens of mice given mAb to IL-10 during reactivationa

 
Because mAb neutralization may not abrogate the presence of endogenous IL-10, we infected wild-type and IL-10–/– mice with 2 x 106 yeasts i.n. and at 42 days postinfection; they were given either rat IgG or mAb to CD4+ and CD8+ cells. H. capsulatum was recovered from wild-type, but not IL-10–/–, mice on days 14 and 56 (Fig. 5, E and F).

Is there another lymphocyte population that prevents fatal reactivation?

Because depletion of CD4+ and CD8+ cells induced a persistent infection in wild-type or B cell–/– mice, we sought to determine whether there was a lymphocyte population that prevented unrestrained growth of the fungus. We infected wild-type and {gamma}{delta} TCR–/– mice and at 42 days postinfection both groups of mice were depleted of CD4+ and CD8+ cells. At day 42, the fungal burden was below the detection limit of 102 CFU. The lack of {gamma}{delta} TCR+ did not transform reactivation from persistent to fatal (Fig. 6, A and B). Elimination of NK cells in T cell-depleted animals did not alter the magnitude of CFU (Fig. 6, C and D).


Figure 6
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  		FIGURE 6. Reactivation in {gamma}{delta} TCR–/– mice and in mice depleted of NK cells. A and B, Wild-type or {gamma}{delta} TCR–/– mice were infected with 2 x 106 yeasts i.n. and at 42 days postinfection, mice were depleted of CD4+ and CD8+ cells. Organs were cultured at days 14 and 56 postreactivation. C and D, Infected mice were depleted of CD4+ and CD8+ cells or both plus NK cells. Data represent mean ± SEM of at least six per group.

 
We reasoned that another population of T cells that either escape elimination or did not express CD4 or CD8 might be involved in persistence. A CD4–, CD8– Thy-1.2+ population of cells has been reported to influence infection with Mycobacterium tuberculosis (25). We analyzed mice depleted of CD4+ and CD8+ cells for the presence of a Thy-1.2+, CD4–, CD8– population. In wild-type and B cell–/– mice, treatment with mAb to CD4 and CD8 abolished these cell populations by >97% (Fig. 7, A and C vs E and G). Analysis of the remaining cells demonstrated a small population bearing Thy-1.2 (Fig. 7, F and H). Subsequently, wild-type and B cell–/– mice were infected and, 42 days later, mice were administered mAb to CD4, CD8, with or without mAb to Thy-1.2. Another group received mAb to Thy-1.2 alone which substantially reduced CD4+ and CD8+ by ~80% but did not abolish them. At 14 days postreactivation, CFU in lungs, but not spleens, of wild-type mice that received all three mAb was significantly higher (p < 0.001) than that of mice that received mAb to CD4 and CD8 or mice that received mAb to Thy-1.2 alone (Fig. 8, A and B). Wild-type mice treated with all three mAb became markedly ill between days 45 and 55 of reactivation and were sacrificed on day 56. The burden in their organs was significantly higher than in the other two groups. Interestingly, mice that received only anti-Thy-1.2 did not manifest reactivation at day 56. B cell–/– recipients of the three mAb became moribund between days 25 and 30 of reactivation whereas the other two groups were not. These animals were euthanized and organs were cultured. CFU in lungs and spleens of the moribund animals were strikingly elevated and far exceeded (p < 0.001) that in the other two groups (Fig. 8, C and D).


Figure 7
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  		FIGURE 7. Phenotype of splenocytes harvested from C57BL/6 and µMT mice during reactivation histoplasmosis. Splenocytes from C57BL/6 (A, B, E, and F) and µMT (C, D, G, and H) mice were harvested at 56 days postreactivation. The cells were stained with CD4-FITC, CD8-PerCP, and Thy-1.2-allophycocyanin and analyzed first by CD4-FITC and CD8-PerCP expression (A, C, E, and G). A and C represent wild-type and B cell–/– mice that have not been depleted. B, D, F, and H are gated on the CD4–CD8– population (lower left quadrant) to determine Thy-1.2-allophycocyanin expression of this population. Dot plots show a representative analysis from one of six mice. SSC denotes side scatter.

 

Figure 8
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  		FIGURE 8. A Thy-1.2+ population modulates the severity of reactivation. Infected wild-type (A and B) and B cell–/– (C and D) mice were treated with mAb to CD4 and CD8, mAb to Thy-1.2, or mAb to CD4, CD8 and Thy-1.2 and organs cultured at days 14 and 56 postreactivation. Data represent a mean ± SEM of at least eight mice per group. *, p < 0.05, ***, p < 0.001 when compared with controls.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Reactivation of dormant foci is a major pathogenic feature of numerous microbes, including H. capsulatum. Abundant literature has established that patients from endemic areas may reactivate after they have left an endemic regions (2, 3). Several risk factors have been associated with reactivation in humans including age, receipt of immunosuppressive agents, and AIDS, especially when the CD4+ counts are <100 cells/µl (26, 27, 28). Missing from these risk analyses has been a comprehensive understanding of the immunological disturbances that trigger reactivation.

A crucial feature of this study was the creation of a model of reactivation histoplasmosis that largely mimics the human condition. A hallmark of humans latently infected is that the microbe is not recovered until disease becomes apparent. The finding that H. capsulatum was not recovered before reactivation distinguishes our model from several others with higher prokaryotes or eukaryotes in which organisms are easily detected before reactivation is induced (7, 29, 30, 31). In several models, the microbial burden is relatively large. This circumstance is not present in humans. When the burden is detectable, "reactivation" may be construed as disease exacerbation.

One of those models referred to above-used H. capsulatum albeit with a different fungal strain and B6C3F1 mice. These animals manifested a high burden of infection (>104 to 106 CFU) in lungs and spleens for >3 mo; 50% of them reactivated spontaneously with a fatal outcome (29). In contrast, the fungal burden in C57BL/6 mice before reactivation was <102 CFU/organ and unprovoked reactivation did not transpire during the 98 days of observation.

We were able to generate two separate outcomes in mice that have reactivated H. capsulatum. One result was the creation of a chronic persistent infection similar to chronic disseminated histoplasmosis, and the second was a progressive infection similar to the more aggressive infantile form. Depletion of CD4+ and CD8+ cells, but not either population alone, in immunocompetent C57BL/6 mice caused reactivation. Fourteen days after the loss of these cells, fungus was recovered simultaneously in lungs and spleens. During the course of reactivation, CFU in spleens remained constant whereas those in the lungs slowly declined. The reason for the disparate responses between the two organs is not known. The data indicate that lungs do not require optimal numbers of T cells to combat this fungus. This assertion is supported by the pattern of elimination of this fungus in secondary infection. Elimination of CD4+ and CD8+ cells in mice with secondary histoplasmosis was associated with a marked increase in CFU in spleens whereas there was a decrement in lungs (12). In secondary disease, synthesis of TNF-{alpha} by lungs, but not spleens, was one explanation for the differential response. In reactivation disease, this consideration seems unlikely since neutralization of this cytokine did not modulate the course of infection. Elimination of Thy-1.2+ cells in CD4+ and CD8+-depleted mice transformed a persistent infection into progressive and lethal one. A population of Thy-1.2+ cells that were CD4– and CD8– appears to be necessary for the persistence of the fungus. Interestingly, the burden in spleens were higher than in lungs of mice lacking all three cell subsets. This finding suggests that the spleen may be more dependent on this population for control of reactivation than the lung. The population of Thy-1.2+ cells was not {gamma}{delta} TCR+ cells or NKT cells because depletion of these cell populations did not modulate the chronic persistence.

The influence of so called "double-negative" T cells on infections appears to be limited. These cells have been shown to present in human and murine tuberculosis, but their contribution to host control of infection remains modest at best (32, 33). In contrast, this population in our model was an exceptionally prominent cell in preventing reactivation from becoming fatal.

Reactivation was induced in B cell–/– mice. Although the pattern of reactivity was similar to that of B cell+/+ animals, there were several notable differences. First, the elimination of CD4+ cells led to reactivation in B cell–/– mice. Second, the fungal burden was distinctly higher in these mice. This finding contrasts with that of primary and secondary pulmonary histoplasmosis in B cell–/– mice in which the fungal burden in these mice does not differ from that of wild type (12). Third, the response of T cell-depleted B cell–/– mice to neutralization of IFN-{gamma} and TNF-{alpha} differed from that of wild type. These results indicate that B cells and/or their products contribute notably to reactivation. This point has been speculated upon in murine tuberculosis, but no data exist that have addressed this issue (34). The findings in B cell–/– mice may be influenced by the fact that these animals innately manifest an enhanced Th1 response (35). This could account for the enhanced response to neutralization with mAb to IFN-{gamma} and TNF-{alpha}. In addition, these mutant mice exhibit selective expression of IgA which may modulate the response in the airways (36). The involvement of B cells in humans may seem remote in infections that distinctly rely on T cell-mediated immunity. However, B cell dysfunction is a consequence of immunosuppressive agents and is observed in AIDS (37, 38, 39, 40, 41).

Prevention of reactivation in H. capsulatum-exposed wild-type mice only required the presence of CD4+ or CD8+ cells. This finding was not true for B cell–/– mice in which depletion of CD4+ cells alone caused reactivation. These results suggest that in wild-type mice, CD8+ cells cooperate with B cells or their products to obviate reactivation. When B cells were absent, the CD8+ cells did not inhibit reactivation. Thus, the CD8+-B cell axis is a crucial one in limiting the extent of reactivation. However, the absence of B cells did not convert the persistent infection to a fatal one in either CD4+ cell-depleted mice or in animals lacking both subsets.

The necessity that both subsets of T cells be eliminated to induce reactivation of H. capsulatum in otherwise immunocompetent mice is similar to that observed in experimental toxoplasmosis (30). In contrast, in murine tuberculosis or leishmaniasis, prevention of reactivation can be caused by elimination of CD4+ cells (34, 42). The differences among the models are not explained by the lack of a single cytokine or other soluble mediator. For example, in toxoplasmosis, the principal mediator of reactivation was IFN-{gamma} whereas in tuberculosis, reactivation was independent of this cytokine (30, 34). When B cell–/– mice were examined, reactivation was induced merely by depleting CD4+ cells, thus reactivation in these mice is induced by depleting the same population as that for tuberculosis and cutaneous leishmaniasis. The contribution of B cells to reactivation in these other experimental infections has not been determined. Among the four organisms discussed, B cells appear to be important mediators of resistance only with Toxoplasma gondii (43, 44, 45, 46), yet their contribution to latency or reactivation of tuberculosis, toxoplasmosis, and leishmaniasis remains to be resolved.

Several cytokines are essential for control of primary or secondary histoplasmosis including IFN-{gamma}, GM-CSF, and TNF-{alpha}. Each of these is requisite for control of primary histoplasmosis in mice, and the latter is requisite for control of secondary (9, 11, 19). We observed that neutralization of IFN-{gamma} did impact the fungal burden but only in B cell–/– mice. Although an effect of mAb to TNF-{alpha} was not observed in these mice, when mAb to IFN-{gamma} and TNF-{alpha} were used together, there was an additive or cooperative effect, but only in CD4+ cell-depleted B cell–/– mice. In contrast, neutralization of IFN-{gamma} and TNF-{alpha} in B cell–/– mice that lacked both T cell subsets did not induce an additive effect. The principal difference between these two groups is the presence of CD8+ cells in the former. In the absence of CD4+ and CD8+ cells, neutralization of TNF-{alpha} appeared to interfere with the effect of mAb to IFN-{gamma}. The results suggest that treatment with mAb to TNF-{alpha} induces a mediator that interferes with the effect of mAb to IFN-{gamma}. This supposition is currently under investigation.

Clinically, a small number of humans with inflammatory diseases that are treated with TNF-{alpha} manifest what appears to be reactivation of histoplasmosis (47, 48). Most of these patients if not all of them also are on other disease-modifying drugs including methotrexate, cyclosporine, and/or prednisone. Thus, it was somewhat surprising to us to find that mAb to TNF-{alpha} did not in and of itself alter reactivation. These data suggest that reactivation may occur in recipients of TNF-{alpha} antagonist when IFN-{gamma} levels and B cell function are perturbed by an immunosuppressive agent.

Treatment with aminoguanidine in T cell-deficient animals did not induce a striking alteration in fungal burden, and the modest effect was noted only in the spleen. The absence of a prominent effect of NO mimics that found in reactivation tuberculosis in mice (34). In contrast, neutralization of IL-10 or the absence of this cytokine had a marked effect on the burden in reactivation. Neutralization of this cytokine blunted reactivation whereas no reactivation was induced in IL-10–/– mice. The reduction in burden was accompanied by an increase in IFN-{gamma} in organs. Because T cells were absent, the most likely source of this cytokine is NK cells which are known to generate IFN-{gamma} in histoplasmosis (49) Thus, IL-10 is critical for reactivation as is true for reactivation leishmaniasis (50).

Reactivation of infections continues to plague humans, and therefore efforts to understand the immunological defects that allow the organism to escape its confines are important. The results of this study demonstrate the complex nature of the host control of preventing reactivation. The ability to define the networks that are involved in maintaining a latent state permits the development of rational therapeutics that can obviate the emergence of the pathogen from its niche in visceral and lymphoid tissues.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
G. S. Deepe, Jr. is a consultant with, and recipient of a grant from, Amgen.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants AI-061298, AI-34361, and AI 42747. Back

2 Address correspondence and reprint requests to Dr. George S. Deepe, Jr., Division of Infectious Diseases, Department of Medicine, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0560. E-mail address: george.deepe{at}uc.edu Back

3 Abbreviation used in this paper: i.n., intranasally. Back

Received for publication March 9, 2006. Accepted for publication May 3, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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