Aeroallergens Exacerbate Histoplasma capsulatum Infection

Abstract

Allergens such as house dust mites (HDM) and papain induce strong Th2 responses, including elevated IL-4, IL-5 and IL-13 and marked eosinophilia in the airways. Histoplasma capsulatum (H. capsulatum) is a dimorphic fungal pathogen that induces a strong Th1 response marked by IFNγ and TNFα production, leading to rapid clearance in non-immunocompromised hosts. Th1 responses are generally dominant and overwhelm the Th2 response when stimuli for both are present, though there are instances when Th2 stimuli downregulate a Th1 response. We determined if the Th2 response to allergens prevents the host from mounting a Th1 response to H. capsulatum in vivo. C57BL/6 mice exposed to HDM or papain and infected with H. capsulatum exhibited a dominant Th2 response early, characterized by enhanced eosinophilia and elevated Th2 cytokines in lungs. These mice manifested exacerbated fungal burdens, suggesting that animals skewed towards a Th2 response by an allergen are less able to clear the H. capsulatum infection despite an intact Th1 response. On the other hand, secondary infection is not exacerbated by allergen exposure, indicating the memory response may suppress the Th2 response to HDM and quickly clear the infection. In conclusion, an in vivo skewing towards Th2 by allergens exacerbates fungal infection, even though there is a concurrent and unimpaired Th1 response to H. capsulatum.

Introduction

Asthma is a common chronic disease of the airways characterized by episodes of reversible airflow obstruction. In the United States there has been a steady increase in the prevalence of asthma, especially among children and adult women (1). Sensitivities to allergic stimuli such as house dust mites (HDM), air pollutants or fungal spores induce Th2 responses marked by production of IL-4 and IL-13, influx of eosinophils into the lung and airway hyperreactivity (AHR). Mouse models of allergic asthma often utilize HDM. The active component of HDM is Der p 1(2), which has been shown to disrupt the tight junctions between epithelial cells, contributing to allergic disorders (3). Papain is structurally similar to Der p 1 and can be used to induce a strong allergic response in model systems (4).

Inhalation of environmental fungal spores is known to produce an allergic response. In fact, admission to intensive care for asthma is strongly associated with skin test reactivity to one or more fungi (5, 6). Alternaria species are the most common fungi to induce an allergic response, along with Cladosporium and Coprinus (7–9). Among severe asthmatics, sensitivity to fungi ranges from 25% to >70% (10). The soil-based fungus, Histoplasma capsulatum (H. capsulatum), however, does not induce an allergic phenotype. It is a dimorphic fungus found worldwide and in the U.S. is most prominent in the Ohio and Mississippi River Valleys. Exposure to H. capsulatum spores is common and most individuals are able to clear an infection readily; yet there are approximately 25,000 life-threatening infections in the U.S. every year, chiefly in immunocompromised patients such as those with AIDS (11). Infection with H. capsulatum induces a strong Th1 response highlighted by production of TNFα and IFNγ, and this is critical to fungal clearance. Conversely, in models with a Th2 skewed response there is a significantly worse infection (12, 13).

While Th1 and Th2 responses are not mutually exclusive, one generally governs. The initial cytokine environment is significant in establishing the development of Th1 or Th2 responses and in most cases of competing Th1 and Th2 stimuli, the former are dominant. The Th1 cytokine IL-12 induces IFNγ and thereby prevents development of a Th2 response (14). Additionally, a strong skewing of the cytokine environment towards Th2 in filarial patients in vivo does not influence the Th1 type immune response to purified protein derivative of Mycobacterium tuberculosis or the ability of macrophages to limit M. tuberculosis growth (15, 16). In vitro, when Th1 and Th2 stimuli are added to dendritic cell (DC) cultures simultaneously, only IFNγ-producing cells are generated (14). On the other hand, there is also evidence that Th2 conditioning by helminth extracts downregulates Th1 responses in mice and suppresses DC function (14, 17). Differentiated Th1 cells fail to express IL-4 even when they are re-stimulated under Th2 conditions, although partially differentiated cells retain their capacity to produce IL-4 (18). Conversely, even stably committed Th2 cells are plastic and can switch to produce IFNγ upon stimulation with a Th1 inducer (19).

In this paper we evaluated a system stimulated with the Th2-promoting HDM and Th1-promoting H. capsulatum. Mice treated with HDM and infected with H. capsulatum mount concurrent Th1 and Th2 responses with the Th2 response to the HDM remaining strong initially in the presence of a mounting Th1 response to H. capsulatum before the lung environment switches fully to Th1. However, animals with a memory response to H. capsulatum are unable to mount a Th2 response to HDM during a secondary infection suggesting the order of exposure and the generation of memory responses is very important to determining the outcome.

Methods

Mice

Male WT C57BL/6, and BALB/c mice were obtained from Jackson Labs (Bar Harbor, ME). IL-33^−/− mice were house bred. Animals were housed in isolator cages and were maintained by the Department of Laboratory Animal Medicine, 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.

Allergen administration, preparation of H. capsulatum and infection

HDM (Greer Laboratories, Lenoir, NC), papain (carica papaya, EMD Millipore Corp., Billerica, MA), or HDM combined with papain were administered intranasally to mice anesthetized with isoflurane. On Day 0 they were sensitized with 100 ug of allergen then they were challenged on days 7–11 with the same dosage (Supplemental Fig. 1). Mice were rested two days before infection with H. capsulatum.

Preparation of H. capsulatum and infection of mice

H. capsulatum yeast strain G217B was grown for 72 hours at 37° C as previously described (12). Naïve or allergen sensitized mice were infected intranasally with 2×10⁶ yeast cells in approximately 20 μL of HBSS (HyClone, Logan, UT). Mice receiving allergen received 100 μg a day throughout the course of infection.

Organ culture

Lungs were homogenized in 5 mL sterile HBSS and serially diluted before being plated upon Mycosel-agar (Becton-Dickinson, Franklin Lakes, NJ) plates containing 5% sheep blood agar and 5% glucose. Plates were incubated for 8 days at 30°.

Survival

Mice were sensitized and challenged with allergen and infected as described above. When mice appeared moribund they were euthanized.

RNA isolation, cDNA synthesis and quantitative real-time reverse transcription PCR

Total RNA from whole lungs of mice was isolated using TRIzol (Invitrogen, Carlsbad, CA). Oligo(dT)-primed cDNA was prepared by using the reverse transcriptase system (Promega, Madison, WI) according to the manufacturer’s instructions. Quantitative real-time PCR for analysis of gene transcription was performed using TaqMan™ master mixture and primers obtained from Applied Biosystems (Foster City, CA). Samples were analyzed with ABI Prism 7500 (Applied Biosystems). In each experiment, the hypoxanthine phosphoribosyl transferase housekeeping gene was used as an internal control, and samples are compared to uninfected WT controls. The conditions used for amplification were 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 15s and 60 °C for 1 min.

Flow cytometry

Lungs were homogenized with 10 mg collagenase and 200 units of DNase I for 30 minutes at 37° C in a 5% CO² incubator before being filtered and then fixed in 4% paraformaldehyde. The phenotype of cells was determined by incubating lung homogenates with the indicated antibodies and CD16/32 to limit non-specific binding. Cells were stained at 4° C for 30 minutes in PBS containing 1% BSA and 0.01% sodium azide. For intracellular staining, mice were given 250 μg brefeldin A IP. After 6 hours lungs were collected and processed as above (20). Cells were stained with combinations of the following antibodies: PE-Cy7-conjugated CD11c, AlexaFluor 700-conjugated Ly6G and Ly6C, PE-conjugated CD11b, APC-conjugated CD4, APC-Cy7-conjugated Ly6C and CD4, Horizon PE-CF594-conjugated SiglecF, Horizon V500-conjugated MHCII and CD8, APC-conjugated CD3, PE-conjugated IFNγ, and PE-conjugated IL-4 from BD Biosciences (San Diego, CA). efluor 450-conjugated CD11b and CD45, FITC-conjugated CD45 from eBioscience (Thermo Fisher Scientific, San Diego, CA. Pacific Blue-conjugated CD11c and PE-conjugated CD64 from Biolegend (San Diego, CA). PE-conjugated ST2 from R&D Systems (Minneapolis, MN). Cells were washed and resuspended in PBS containing 1% BSA and 0.01% sodium azide. Appropriate isotype controls were performed in parallel. Data were acquired using a LSRII (BD Biosciences) flow cytometer in the FACS core at Cincinnati Children’s Hospital and were analyzed using FCS Express 6.02 (DeNovo Software, Glendale, CA). See Supplemental Figure.2 for gating strategy and naïve controls. Eosinophils were defined as CD45⁺CD11b⁺CD11c^-SiglecF⁺. Alveolar macrophages were defined as CD45⁺CD11b⁺CD11c^-SiglecF⁺CD64⁺. Neutrophils were defined as CD45⁺CD11b⁺Ly6G⁺. Inflammatory monocytes were defined as CD45⁺CD11c^-CD11b⁺Ly6C^hi. Monocyte-derived DCs were defined as CD45⁺CD11c⁺CD11b⁺CD64⁺. T cells were defined as CD45⁺CD3⁺ and CD4⁺ or CD8⁺

Measurement of cytokines and chemokines by ELISA

Eotaxin-1, IL-4, IL-12 and IFNγ protein concentration was quantified in lung homogenates by using ELISA kits from R&D systems.

Histologic analysis

Lungs were inflated with 3 ml formalin before paraffin-embedded sections were stained by PAS and H&E at the Histology core at Cincinnati Children’s Hospital. Images were acquired with on a Zeiss Imager A.2 with an AxioCam MRc5. Images were analyzed using AxioVs40×60 V 4.9.1.0.

Statistics

Statistics were performed using the Student’s t-test or ANOVA with Boneferroni’s correction. P value of <0.05 was considered statistically significant. For all graphs, *P = 0.01–0.05, ** P = 0.005–0.01, and *** P = <0.005.

Results

HDM exacerbates H. capsulatum infection

C57BL/6 mice were sensitized to HDM before being infected with H. capsulatum to skew the animals towards a Th2 response, and throughout the course of infection mice were given HDM daily to maintain a Th2 response. Mice exposed to HDM had significantly elevated fungal burdens at late intervals during infection compared with infected controls (Fig. 1A). This finding was also true for the more Th2-skewed BALB/c mouse background (Supplemental Fig. 3A). Though the fungal burden plateaued around day 7, animals treated with HDM began to die by D13, and by D16 50% had died (Fig. 1B).

Figure 1:

Pretreatment with HDM exacerbates fungal infection. A) Pulmonary fungal burden represented as log₁₀ CFU in control or HDM-treated mice at day 3, 5, 7, and 10 p.i. B) Survival curve for control and HDM-treated mice infected with 2×10⁶ CFU. C-G) Cell populations presented as percentage of CD45⁺ leukocytes in lung homogenates from HDM treated, infected, and HDM-treated and infected mice at days 3, 5, 7, and 10 p.i. H) Percentage of CD45⁺CD4⁺ T cells that are IFNγ⁺ or IL-4⁺ in uninfected mice and at day 7 p.i. Data are represented as mean ± SEM, 2–3 independent experiments, n= 8–12 mice.

Eosinophil influx to the lung is induced early in response to HDM, and at day 3 there was no difference in eosinophil percentage between animals given HDM and those animals sensitized HDM and infected with H. capsulatum (Fig. 1C). Throughout the course of infection, the animals treated with HDM and infected exhibited significant decreases in eosinophil numbers compared to the HDM alone group. Alveolar macrophages were increased in response to HDM, however, in infected animals this population disappears by day 10 post infection (p.i.) (Fig. 1C). Neutrophils, inflammatory monocytes and monocyte-derived DCs did not vary greatly between conditions, although there were significantly more inflammatory monocytes at day 10 in the HDM-treated and infected group compared with the other two groups (Fig. 1E-G). Additionally, both IFNγ⁺ and IL-4⁺ CD4⁺ T cells were increased in the animals treated with HDM, however upon infection, HDM treated and infected animals showed no differences from infected controls (Fig. 1H).

HDM-induced Th2 response persists throughout infection

The influx of eosinophils associated with HDM seen in Figure 1 corresponded to elevated levels of the eosinophil chemoattractants eotaxin 1 and 2 throughout the course of the experiment in uninfected mice (Fig. 2A&B). In infected animals treated with HDM, the eotaxins were significantly elevated early before declining thereafter (Fig. 2A & B). The canonical Th2 cytokines IL-4 and IL-13 followed a similar profile, suggesting the H. capsulatum infection may suppress the Th2 response late in infection (Fig. 2C & D). Protein analysis of lung homogenate supernatants at day 7 p.i. for eotaxin 1 and IL-4 exhibit a similar pattern to the RNA expression data (Fig. 2E-F).

Figure 2:

HDM induces a Th2 response early but is eventually suppressed by the infection. A-D) Quantitative real-time reverse transcription PCR (qRT-PCR) analysis of Th2 chemokine and cytokine genes in response to HDM, infection, or HDM and infection at days 3, 5, 7, and 10 p.i. E-F) ELISA quantification of Eotaxin 1 and IL-4 protein levels at day 7 p.i. Data are represented as mean ± SEM, n= 8–12 mice.

Th1 response to H. capsulatum is intact

The strong Th2 response to HDM was established before the animals are infected and could therefore suppress the Th1 response to the infection. However, both IL-12 and IFNγ expression (Fig. 3A & B) and protein concentration (Fig. 3C & D) were similar between the infected and the infected and HDM treated groups suggesting an intact Th1 response. Similar trends were observed in the BALB/c background for Th1 and Th2 cytokines (Supplemental Fig. 3B-D).

Figure 3:

HDM induced Th2 response does not suppress H. capsulatum Th1 response. A-B) qRT-PCR analysis of Th1 cytokine genes in response to HDM, infection, or HDM and infection at days 3, 5, 7, and 10 p.i. C-D) ELISA analysis of IL-12 and IFNγ at day 7 p.i. Data are represented as mean ± SEM, 2–3 independent experiments, n= 8–12 mice.

IL-4, but not IL-33, is responsible for HDM phenotype

IL-4 was elevated in response to HDM (Fig. 2C), therefore we ascertained if neutralization of this cytokine would prevent the HDM-associated increase in fungal burden (Supplemental Fig. 1B for method schematic). Animals treated with αIL-4 antibody have significantly reduced fungal burden when exposed to HDM than seen in the IgG control animals (Fig. 4A). Additionally, αIL-4 and HDM treated animals did not exhibit as much eosinophil influx as controls (Fig. 4B). αIL-4 reduced eotaxin 2 while having no effect on IFNγ (Fig. 4C & D). We next asked if IL-33 influenced the exaggerated fungal burden. IL-33^−/− animals sensitized with HDM exhibited elevated fungal burdens similar to WT mice treated with HDM and IL-4 and IFNγ levels were also similar (Supplemental Fig. 4 A and B). Thus, the elevated fungal burdens associated with HDM were not caused by IL-33.

Figure 4:

IL-4 is responsible for part, but not all, of the HDM phenotype. A) Day 7 p.i. pulmonary fungal burdens represented as CFUs in control and HDM-treated IgG and αIL-4-treated mice. B) Eosinophil populations presented as percentage of CD45⁺ leukocytes in lung homogenates from HDM treated, and control and HDM-treated infected mice for day 7 p.i. qRT-PCR analysis of whole lung homogenates from IgG and αIL-4-treated mice for eotaxin2 (C) and IFNy (D) following infection and HDM treatment. Data are represented as mean ± SEM, 3 independent experiments, n= 12 mice.

Papain treatment also exacerbates infection

Papain is a protease allergen with similar activity to the active component of HDM, Der P 1 (4). When administered using the protocol established for HDM, papain exacerbated fungal infection more severely than HDM (Fig. 5A). Administration of both to mice produced a similar fungal burden as that of papain alone. Papain also induced slightly more death than HDM (Fig. 5B). Similar to HDM, animals sensitized with papain or papain + HDM (H + P) have an intact Th1 response and produce IL-12b and IFNγ in response to infection, but also displayed elevated levels of Th2 cytokines Arg1, IL-4, eotaxin 1 and eotaxin 2 (Fig. 5C-H). Similar results were seen in BALB/c mice, a more Th2 prone mouse strain (Supplemental Fig. 3).

Figure 5:

Papain similarly exacerbates infection. A) Pulmonary fungal burden represented as log₁₀ CFU in control, HDM-, papain-, or HDM and papain-treated mice at day 7 p.i. B) Survival curve for control infected, HDM treated and infected, and papain treated and infected mice infected with 2×10⁶ CFU. C-D) qRT-PCR analysis of whole lung homogenates for Th1 cytokine genes at day 7 p.i. E-I) qRT-PCR analysis of whole lung homogenates for Th2 cytokine and chemokine genes, significance indicated when compared to control. Data are represented as mean ± SEM, 2–3 independent experiments, n= 8–12 mice.

Mucus accumulates in the airways of the lungs when responding to an allergen challenge (21). Gob-5 production is elevated in response to allergen challenge, but upon infection disappears (Fig. 5I). PAS stained lung histology sections of day 7 show significant mucus staining surrounding the airways of the allergen treated animals (arrows, Fig. 6A). In contrast, massive cell infiltrates are present in the infected and allergen-sensitized animals (Fig. 6 A & B). Papain induced severe inflammation that is further exacerbated by infection and is a possible explanation for the worse infection in the papain treated animals compared to the HDM treated animals.

Figure 6:

Allergens induce mucus accumulation in the airways. Day 7 p.i. histology of PAS (A) and H&E (B) stained lung sections from control mice and mice treated with HDM, papain or HDM and papain, both uninfected and infected. Arrows indicate mucin⁺ airways in the PAS stained sections. Magnification of 25x. Micrographs are a representative of n=4.

Primary infection does not suppress Th2 response to HDM

Mice were given one sensitization dose of allergen before infection and then challenged with allergen during infection. These mice still exhibited increased fungal burdens compared to control animals, despite not being fully skewed towards a Th2 response before being infected (Fig. 7A). In fact, one sensitization dose of papain given a week before infection, with no additional allergen challenges, was enough to significantly increase fungal burden. IFNγ was unchanged between the conditions (Fig. 8B). The mice that were challenged with allergen after infection still had elevated levels of the IL-4, eotaxins eotaxin 1 and 2 and Gob-5 (Fig. 7C-F).

Figure 7:

Primary infection does not overwhelm allergic response. A) Pulmonary fungal burden represented as log₁₀ CFU in control, HDM- and papain-treated mice at 7 days p.i. Allergen treated mice were sensitized to the allergen 7 days before infection. During infection mice were either left unchallenged (S) or challenged (S+C) with daily doses of 100 ug of HDM or papain throughout the course of infection. qRT-PCR analysis of whole lung homogenates for Th1 cytokine genes (B) and Th2 cytokine and chemokine genes (C-F), significance indicated when compared to control infected. Data are represented as mean ± SEM, 2 independent experiments, n= 8 mice.

Figure 8:

Secondary H. capsulatum infection suppresses HDM-induced Th2 phenotype. A) Pulmonary fungal burden represented as log₁₀ CFU in control, HDM-treated mice at 7 days post-secondary infection. B) qRT-PCR analysis of whole lung homogenates of Th1 cytokine genes C-E) qRT-PCR analysis of whole lung homogenates of Th2 cytokine genes. F) ELISA analysis of IFNγ, IL-4, IL-5 and IL-13 at day 7 p.i. Data are represented as mean ± SEM, 2 independent experiments, n= 8 mice.

Memory response to H. capsulatum suppresses Th2 response to HDM

During acute infections, HDM causes elevated fungal burdens. To determine what transpires in a secondary infection, mice were infected with H. capsulatum before being allowed to recover and then treated to the same protocol as before to ascertain the effect of a memory response to H. capsulatum (Supplemental Fig. 1C). This memory response resulted in similar fungal burdens between control animals and those treated with HDM (Fig. 8A). IFNγ was also similar between the infected groups (Fig. 8B). Unlike the elevation of Th2 cytokines seen in response to HDM in a primary infection; IL-4, eotaxin 1 and eotaxin 2 were absent during secondary infection in animals treated with HDM. (Fig. 8C-E). However, uninfected age-matched animals did exhibit an elevated Th2 response to HDM. IFNγ protein levels are slightly elevated in infected mice treated with HDM, however all Th2 cytokines are similar between the groups (Fig. 8F).

Discussion

In this study we sought to understand the interplay between a Th2 stimuli and a Th1 stimuli in vivo. HDM establishes a strong Th2 phenotype, with influx of eosinophils to the lung, elevated levels of Th2 cytokines and chemokines and an increase in mucus production. The Th2 response induced by HDM was able to persist through the initial H. capsulatum infection before fading after about a week, with eosinophil and alveolar macrophage influx receding. However, by the time the Th1 response appeared to be suppressing the Th2 response, animals had already incurred so much lung inflammation and fungal burden half of them had died. While in vivo results suggest a quick switch from Th2 to Th1, in vivo is more complicated and appears to allow concurrent responses to form (19).

HDM sensitization creates a distinct Th2 phenotype; however, skewing to a Th2 phenotype does not appear to hinder a Th1 response to the H. capsulatum infection. IL-12b and IFNγ expression are similar between infected controls and those mice first treated with HDM. The expression of these cytokines demonstrates that the established Th2 environment in the lung does not suppress a Th1 response from forming. Peng et al. found mice stimulated with goat anti-mouse IgD antiserum (GαMδ) and then infected with H. capsulatum showed an initial Th2 response that resulted in increased fungal burden that was quickly skewed to Th1 in response to the infection. However, unlike our study, this phenotype was dependent upon IL-4 and IL-10 (22); infected mice treated with HDM or not showed no difference in IL-10 expression levels (Supplemental Fig. 4C). In our study, the response to the infection appears to gradually switch the lung environment from Th2 to Th1 and within a week of infection the animals no longer exhibit an influx of eosinophils in the lung, Th2 cytokines or chemokines or many mucin+ cells. IFNγ is known to block the accumulation of eosinophils in the airway by binding its receptor and possibly inducing apoptosis or recirculation to the lymph nodes and may be a reason for their disappearance (23). Despite the ability to switch the lung environment to one more favorable to resolving infection, initial contact in a Th2 environment is sufficient to result in the death of 50% of the mice pretreated with HDM and 75% of the mice pretreated with papain. This is a more severe phenotype than seen in other model systems; mice do not exhibit a more severe M. tuberculosis infection in a Th2-skewed filarial infection environment (15).

IL-4 is not solely responsible for the exacerbated H. capsulatum infection exhibited with HDM pretreatment. α-IL-4 reduced the fungal burden, but not quite to the burden of control mice. Another possible mediator is IL-33. It has been shown to be mediate inflammatory cell influx due to HDM and papain (24–26). Despite multiple reports signifying a key role for IL-33 it does not appear to contribute (Sup Fig. 3), in our system. IL-13 is a possible candidate responsible for mediating the HDM exacerbation of fungal infection, although there are no data regarding the influence of IL-13 on host defenses to this pathogen. It is another canonical Th2 cytokine known to be elevated in response to HDM and induces AHR independent of IgE and eosinophils responding to the IL-4 (27). IL-13 plays an important role in the effector phase of asthma by inducing airway remodeling, AHR and mucus hyperproduction (26). In OVA-induced AHR, IL-4 deficiency was not enough to prevent the onset of Th2 immune responses, but deficiency in IL-13 was (28). HDM-treated mice exhibited elevated IL-13 which persisted through early infection and may have contributed to the elevated fungal burdens in these mice.

While most studies investigating asthma or allergy in mice use BALB/c, the more Th2-inclined biased mouse strain, we used C57BL/6 which is considered to be more Th1 prone (34). Despite this predisposition the C57BL/6 mice responded to HDM similarly to the BALB/c mice, with a robust Th2 response that persisted for almost a week before switching to a Th1 phenotype induced by H. capsulatum infection. BALB/c mice are often chosen for allergy studies because C57BL/6 mice are more apt to become tolerant (35), and in some model systems do not show persistence of AHR or inflammation (36). This does not appear to be the case in our model, as the mice continue to have robust Th2 responses throughout the course of the experiment in uninfected animals.

Infection with H. capsulatum in an established Th2 lung environment exacerbated the fungal burden. Likewise, exposure to HDM or papain in mice with ongoing infection impaired antifungal immunity. These mice were given two days to establish a Th1 environment before being given a Th2 stimulus, and therefore, should have been skewed towards Th1 (14, 18). However, the lungs of these mice exhibited elevated levels of eotaxins 1 and 2 and IL-4, while maintaining IFNγ levels, indicating that the Th2 response was developing despite a pre-existing Th1. This suggests that despite the dogma of a Th1 environment suppressing the development of a Th2 response, the mice were still able to develop a Th2 response, maintaining both Th1 and Th2 concurrently.

In a secondary infection, HDM treatment did not exacerbate infection and there was no Th2 response. These results suggest memory T cells against H. capsulatum and likely Th1-skewed were quickly dominant and able to suppress T cells that may have otherwise been skewed towards Th2. However, passive transfer of Th1 cells before OVA-induced airway inflammation resulted in exacerbated inflammation (37), not the lack of Th2 response we saw in our secondary infection and HDM treated animals. This observation supports the hygiene hypothesis that theorizes that increased cleanliness has led to an increase in asthma prevalence. And while there is debate over the legitimacy of the hypothesis (38), at least for our model it appears that a memory response to H. capsulatum is able to suppress HDM-induced Th2 responses during secondary infection.

Individuals from endemic areas of H. capsulatum will have been exposed early in life and therefore likely have a memory response to Histoplasma. However, non-endemic asthmatics visiting an endemic region may be more susceptible to developing histoplasmosis, as a case study of a young asthmatic who developed pulmonary histoplasmosis after visiting an endemic region shows (39).

In summary, primary H. capsulatum infection is exacerbated when initiated in a Th2 environment. While the host is able to relatively quickly skew the lung towards a Th1 response to fight the infection, it is not always enough to resolve the infection. However, a secondary infection with an intact memory response results in quick clearance and quick suppression of the allergen-induced Th2 phenotype. Susceptible populations from non-endemic regions may wish to exhibit caution if their symptoms are not well controlled.

Abbreviations Used

AHR

(airway hyperreactivity)

DC

(dendritic cell), HDM (house dust mites)

HDM

+ Papain (H + P)

(p.i.)

Post infection

Th1

(Type 1 T helper)

Th2

(Type 2 T helper)