Carbohydrate-rich high-molecular-mass antigens are strongly recognized during experimental Histoplasma capsulatum infection

Introduction: During histoplasmosis, Histoplasma capsulatum soluble antigens (CFAg) can be naturally released by yeast cells. Because CFAg can be specifically targeted during infection, in the present study we investigated CFAg release in experimental murine histoplasmosis, and evaluated the host humoral immune response against high-molecular-mass antigens (hMMAg. >150 kDa), the more immunogenic CFAg fraction. Methods: Mice were infected with 2.2x104 H. capsulatum IMT/HC128 yeast cells. The soluble CFAg, IgG anti-CFAg, IgG anti-hMMAg, and IgG-hMMAg circulating immune complexes (CIC) levels were determined by enzymelinked immunosorbent assay, at days 0, 7, 14, and 28 post-infection. Results: We observed a progressive increase in circulating levels of CFAg, IgG anti-CFAg, IgG anti-hMMAg, and IgG-hMMAg CIC after H. capsulatum infection. The hMMAg showed a high percentage of carbohydrates and at least two main immunogenic components. Conclusions: We verified for the first time that hMMAg from H. capsulatum IMT/HC128 strain induce humoral immune response and lead to CIC formation during experimental histoplasmosis.

Histoplasmosis is a systemic fungal disease caused by the dimorphic fungus Histoplasma capsulatum var.capsulatum.The fungal cells grow in mycelial form at room temperature, with the conversion to yeast form occurring at 37ºC.Histoplasma capsulatum has a worldwide distribution throughout the subtropical and tropical zones 1,2 .When airborne mycelia (microconidia and mycelial fragments) are inhaled, they convert to yeast form in the host lungs 3 .Most cases are asymptomatically or self-limited to the lungs, due to an effective host defense linked to the cellular immune response.However, fungal replication can be reactivated from latent foci if the integrity of the host's immune system is impaired 1,[4][5][6] .
Experimental histoplasmosis allows a better understanding of the disease in humans.Several features of the murine model are similar to those in humans, such as the pathology and the nature of the immune response to H. capsulatum antigens 6 .
During histoplasmosis, H. capsulatum cellfree antigens (CFAg) can be naturally released by the yeast cells.CFAg preparation has several positive characteristics, such as simple and quick preparation, and does not require sophisticated equipment.It is frequently used for diagnosis of paracoccidioidomycosis (PCM), a systemic mycosis caused by the fungus Paracoccidioides brasiliensis 7 .Moreover, CFAg can be used to evaluate cell immunity and to induce protection in a murine model of histoplasmosis 8 .
The presence of soluble antigens is frequently observed in human histoplasmosis, often in association with immunosuppression [9][10][11][12][13][14][15] .The study of these CFAg components is important if we consider that they are easily released components and therefore have the potential to interact with host components.

Tristão FSM et al -Carbohydrate-rich hMMAg recognition during experimental histoplasmosis
A previous study by our group showed the in vitro hemagglutinin activity and syngeneic hemophagocy tosis induction by H. capsulatum soluble antigens, and suggested that high-molecularmass components (>150 kDa) could be responsible for the interaction with red blood cells 16 .
In the present study, we investigated the release of CFAg and highmolecular-mass antigens (hMMAg) during experimental infection with the H. capsulatum IMT/HC128 strain.Our results show for the first time evidence that H. capsulatum CFAg or hMMAg are released during experimental infection and induce humoral immune response and formation of circulating immune complexes (CIC).Moreover, our preliminary data of partial hMMAg characterization indicate that these antigens are carbohydrate-rich with at least two main immunogenic components.

Animals
Male Swiss mice, 60 days old, obtained from the State University of Londrina Animal Facilities, were maintained under specific pathogenfree conditions in micro-isolator cages in the animal housing facility of the Center of Biological Sciences, State University of Londrina, Brazil.The animals were supplied with sterilized food and water ad libitum.Groups of 7-8 mice were used for each period of infection.

Fungal strain and cell-free antigen preparation
The H. capsulatum IMT/HC128 strain (kindly provided by Dr. V.R.B. Castillo, IMT/HC, Lima, Peru) was cultured at 35ºC in Sabouraud agar (Micromed, Rio de Janeiro, Brazil) enriched with 5% goat serum and maintained by subculturing at 5-day intervals.CFAg was obtained as previously described by P. brasiliensis yeast cells 7 .Briefly, the fungal growth was collected by gently scraping the surface and was suspended in 1ml of phosphate buffered saline (PBS, pH 7.2), mixed for 30s on a Vortex-mixer and immediately centrifuged at 10.000g for 60s.Next, the supernatant was mixed to phenylmethylsulfonyl fluoride (PMSF; Sigma, St. Louis, MO; 2.5mM) to reduce the activity of fungal natural proteases that may be liberated during antigen preparation.The supernatant was subsequently frozen and stored at -80ºC.Protein concentration was determined using the Folin-phenol method 17 with bovine serum albumin as a standard.

Mice infection
The H. capsulatum IMT/HC128 yeast cells were collected, washed in PBS, and counted in a hemocytometer.The concentration was adjusted to 2.2x10 5 cells/ml.The mice were intravenously infected (i.v.) with 100µl of the fungal preparation described above, corresponding to 2.2x10 4 yeast cells.Control mice received sterile PBS (100µl, i.v.).

Gel filtration chromatography and hMMAg fractions
The CFAg chromatography was performed as previously described 16 , with some modifications.Two milliliters of CFAg (5.7mg/ml) were applied to a Sephadex G75-120 column (1.5 x 22cm, Sigma-Aldrich) buffered with PBS.Eluted samples (1.5ml per tube) were collected in an automatic fraction collector and read into a spectrophotometer (UV/Visible, Ultrospec-2.000;Pharmacia Biotech, Auckland, Northland) at 280nm.For preliminary estimation of molecular mass, dextran blue [exclusion volume up to 150kDa] was applied to the same column.The hMMAg fractions were identified according the absorbance profile, considering the samples that made up the first absorbance peak (fractions 7 to 10).

IgG anti-CFAg and IgG anti-hMMAg preparation
The IgG anti-CFAg was prepared both in New Zealand rabbits and Swiss mice (2-3 animals per group) by subcutaneous injection of CFAg (250μg in rabbits and 75µg in mice) emulsified in Freund's adjuvant (v/v).The first inoculation was performed with complete Freund's adjuvant and the two successive injections with incomplete Freund's adjuvant at intervals of two weeks.In addition, Swiss male mice were inoculated with 50µg hMMAg (pool of fractions 7 to 10, v/v).After bleeding, the hyper immune serum was submitted through a Sepharose-protein G chromatography and the protein concentration was determined using the Folin-phenol method 17 with bovine serum albumin as a standard.

Determination of immune complexes
The IgG-hMMAg circulating immune complexes were detected in immune serum, as previously described 18 , with small modifications.Briefly, 70μl of serum were diluted in PBS (v/v) plus 140μl of polyethylene glycol 5% (PEG 5%) and incubated overnight at 4ºC.After centrifugation (3min, 4ºC, 300g), the resulting precipitate was washed in 500μl of cold 2.5% PEG and centrifuged again.The precipitated immune complexes were dissolved in 100μl of blocking buffer containing 10mM EDTA and 0.02% sodium azide, and then incubated for 30min at 37ºC.To increase the proportion of free antibodies, 40μl of 0.1M HCl-glycine buffer (pH 2.8) were added to each sample and neutralized with 70μl of 1M Tris (pH 9.0).Then, 100μl of each sample were immediately dispensed in duplicate into immunoplates coated with CFAg (600ng, 100µl/well).After blocking (blocking buffer), the technique was processed as described above for specific IgG detection.

Carbohydrates analysis
The carbohydrate analysis of the fractions eluted from Sephadex G75-120 column was performed using the Dubois method as previously described 20 .The results are expressed in optical density (O.D.) at 492nm.

Statistical analysis
Statistical analysis was performed using the parametric Tukey-Kramer test for two-group comparison (GraphPad software).Values of p<0.05 were considered to indicate statistical significance.The results are expressed as the mean ± SEM.

Ethical considerations
The Ethics Committee for Animal Experimentation of our university approved all experimental protocols described in the present study.

IMT/HC128 CFAg are produced and recognized during experimental histoplasmosis
To evaluate whether H. capsulatum IMT/HC128 cell-free antigens are produced after IMT/HC128 infection, we measured CFAg level in the serum from infected mice during experimental histoplasmosis.The antigenemy analysis showed the presence of CFAg in all evaluated time points (Figure 1).The amount of circulating soluble antigens increased during the line of infection (0.100 ± 0.06 at 7 days, 0.13 ± 0.03 at 14 days, and 0.27 ± 0.18 at 28 days p.i.).No CFAg was detected in uninfected mice (Figure 1).
We next investigated the immune recognition of H. capsulatum IMT/HC128 cell-free antigens during experimental histoplasmosis.The specific IgG anti-CFAg was measured in the serum from IMT/ HC128 infected mice.As shown in Figure 2, all the IMT/HC128 infected mice produced IgG anti-CFAg.In addition, uninfected mice showed low IgG anti-CFAg synthesis (0.079 ± 0.01), which increased progressively according the course of infection (0.093 ± 0.025 at 7 days, 0.11 ± 0.04 at 14 days, and 0.21 ± 0.1 at 28 days p.i.).

The carbohydrate-rich hMMAg fractions have high antigenic potential
To identify the antigenic fractions present in the CFAg preparation, the IMT/HC128 CFAg was first submitted to a Sephadex G75-120 column.Figure 3 shows the chromatography's Swiss mice were challenged intravenously with 2.2x10 4 yeast forms of H. capsulatum IMT/HC128 strain.The serum was collected at days 0, 7, 14, and 28 p.i., and the CFAg level was evaluated as described in Methods.The results are expressed in optical density (O.D.) at 492nm, and the scale bars represent the mean ± SEM. #, not detected; *p<0.05.Swiss mice were challenged intravenously with 2.2x10 4 yeast forms of H. capsulatum IMT/HC128 strain.The serum was collected at days 0, 7, 14, and 28 p.i., and the IgG anti-CFAg production was investigated as described in Methods.The results are expressed in optical density (O.D.) at 492nm, and the scale bars represent the mean ± SEM. *p<0.05.elution profile (280nm) of the 16 first collected samples.Our data clearly demonstrated a peak, which is shaped by fractions 7 to 13, with highest value at point 8 (0.19).The void volume marker, dextran blue, was eluted in fraction 8 (data not shown), indicating that samples collected in this tube have molecular mass corresponding to >150 kDa.For this reason, the samples that draw the peak (fractions 7, 8, 9, and 10) were collectively named high-molecular-mass antigens (hMMAg).
The carbohydrate analysis of all collected samples showed a high amount of carbohydrate in most of hMMAg fractions (Figure 3).In detriment of fraction 7, which has few carbohydrates (0.18), fractions 8 (1.39), 9 (1.37), and 10 (0.58) are carbohydrate-rich samples, suggesting that hMMAg fractions have high antigenic potential during experimental IMT/HC128 infection.
In fact, when we performed a Dot-Plot assay using hMMAg samples, our results showed that hMMAg fractions are strongly recognized by immune serum (data not shown).Moreover, our western blotting results indicate that all hMMAg fractions showed at least two main distinct bands when incubated with anti-CFAg serum (Figure 4).The first band has ~113.7kDaand the second one, up to 150kDa (~176kDa).

The specific IgG recognition of hMMAg leads to CIC formation
Considering the presence of hMMAg in the serum from IMT/ HC128 infected mice, we next evaluated the host production of IgG anti-hMMAg.Our results showed higher levels of IgG anti-hMMAg in the immune serum, which increased progressively according the evolution of infection (Figure 5, left panel).At 14 (0.096 ± 0.02) and 28 days p.i (0.243 ± 0.025), the amount of circulating IgG anti-hMMAg was significantly increased compared with uninfected serum (0.043 ± 0.015, p<0.05).
To investigate whether binding of IgG anti-hMMAg and hMMAg leads to CIC, we evaluated the serum level of IgG-hMMAg CIC.As shown in Figure 5 (right panel), the CIC level was also elevated at 14 (0.33 ± 0.035) and 28 days p.i. (0.26 ± 0.02) in comparison with control not-infected serum (0.036 ± 0.025, p<0.05).
Taken together, these results demonstrate that IMT/HC128 infection induces the production of IgG anti-hMMAg; this antibody can integrally bind to hMMAg, giving rise to CIC.

FIGURE 1 -
FIGURE 1 -Cell-free antigens are detected in the serum during experimental histoplasmosis.

FIGURE 2 -
FIGURE 2 -Immunoglobulin G anti-cell free antigens are produced during experimental histoplasmosis.

FIGURE 3 -
FIGURE 3 -H.capsulatum cell-free antigens chromatography profile and carbohydrates levels determination.
Swiss mice were challenged intravenously with 2.2x10 4 yeast forms of H. capsulatum IMT/HC128 strain.The serum was collected at days 0, 7, 14, and 28 days p.i..The IgG anti-hMMAg production and the IgG-hMMAg circulating immune complexes were detected as described in Methods.The results are expressed in optical density (O.D.) at 492nm, and the scale bars represent the mean ± SEM. *, p<0.05.

FIGURE 5 -
FIGURE 5 -Immunoglobulin G anti-high-molecular-mass-antigens are produced during experimental histoplasmosis and can give rise to IgG-hMMAg immune complexes.