Gr-1+ cells play an essential role in an experimental model of disseminated histoplasmosis

Microbes and Infection 9 (2007) 1393e1401www.elsevier.com/locate/micinf

Original article

Gr-1þ cells play an essential role in an experimental modelof disseminated histoplasmosis

Anderson Sa-Nunes a,1, Alexandra I. Medeiros a, Carlos A. Sorgi a, Edson G. Soares b,Claudia M.L. Maffei c, Celio L. Silva d, Lucia H. Faccioli a,*

a Departamento de Analises Clınicas, Toxicologicas e Bromatologicas, Faculdade de Ciencias Farmaceuticas de Ribeir~ao Preto,Universidade de S~ao Paulo, Av. do Cafe s/no, Ribeir~ao Preto, S~ao Paulo 14040-903, Brazil

b Departamento de Patologia, Faculdade de Medicina de Ribeir~ao Preto, Universidade de S~ao Paulo, Av. Bandeirantes 3900,

Ribeir~ao Preto, S~ao Paulo 14049-900, Brazilc Departamento de Biologia Celular e Molecular e Bioagentes Patogenicos, Faculdade de Medicina de Ribeir~ao Preto,

Universidade de S~ao Paulo, Av. Bandeirantes 3900, Ribeir~ao Preto, S~ao Paulo 14049-900, Brazild Centro de Pesquisas em Tuberculose, Faculdade de Medicina de Ribeir~ao Preto, Instituto do Milenio Rede-TB,

Universidade de S~ao Paulo, Av. Bandeirantes 3900, Ribeir~ao Preto, S~ao Paulo 14049-900, Brazil

Received 30 June 2006; accepted 6 October 2006

Available online 12 December 2006

Abstract

Recent studies have shown the participation of Gr-1þ cells in many types of infections; however, the role played by these cells in the immuneresponse to fungal pathogens is controversial. In this study we determined whether Gr-1þ cells are involved in the protective immune response insystemic Histoplasma capsulatum infection. Depletion of Gr-1þ cells using the monoclonal antibody (MAb) RB6-8C5 increased histoplasmosisseverity and inhibited the subsequent development of a protective immune response. In addition to the increased fungal burden in lungs andspleens, the Th1 response was found to be unbalanced in these mice and the suppression of the cellular immune response seemed to be asso-ciated with increased nitric oxide production. Taken together, these results indicate that Gr-1þ cell depletion at the beginning of infection allowsyeast multiplication and increases mice mortality. This study improves the understanding of the role of Gr-1þ cells on the protective immunity inhistoplasmosis.� 2006 Elsevier Masson SAS. All rights reserved.

Keywords: Histoplasma capsulatum; Gr-1þ cells; MAb RB6-8C5; Immune response; Cytokines; Nitric oxide

1. Introduction

The magnitude of exposure and immune status of the hostinfluence clinical manifestations of histoplasmosis, a highlyprevalent fungal disease in Central and North America [1].Severe symptomatic disseminated histoplasmosis occurs pri-marily in immunocompromised individuals such as HIV-infected

* Corresponding author. Tel.: þ55 16 602 4303; fax: þ55 16 633 1936.

E-mail address: faccioli@fcfrp.usp.br (L.H. Faccioli).1 Current address: Section of Vector Biology, Laboratory of Malaria and

Vector Research, National Institute of Allergy and Infectious Diseases,

National Institutes of Health, 12735 Twinbrook Parkway, Building Twinbrook

III, Room 2E-28, Rockville, MD, USA.

1286-4579/$ - see front matter � 2006 Elsevier Masson SAS. All rights reserve

doi:10.1016/j.micinf.2006.10.007

patients, receiving chemotherapy for cancer or under immuno-suppressant treatment for transplantation [2]. To mimic thissystemic disease, the experimental model of intravenous yeastinfection has been typically employed [3e5].

More than 10 years ago, the monoclonal antibody (MAb)RB6-8C5 that recognizes the granulocyte-differentiationantigen (Gr-1) was shown to be highly efficient at neutrophildepletion [6]. In mice, the use of MAb RB6-8C5 has beencorrelated with increased susceptibility to a wide range ofmicrobes, including viruses: herpes simplex virus [7] and Juninvirus [8]; bacteria: Mycobacterium tuberculosis [9], Legionellapneumophila [10] and Listeria monocytogenes [11]; parasites:Leishmania major [12], Toxoplasma gondii [13] and numerousothers. However, the role played by Gr-1þ cells in the immune

d.

1394 A. Sa-Nunes et al. / Microbes and Infection 9 (2007) 1393e1401

response to fungal pathogens is conflicting. In some studies, ithas been shown that depletion of Gr-1þ cells increased suscep-tibility to Candida albicans [14], Aspergillus fumigatus [15]and Paracoccidioides brasiliensis [16]. In contrast, the oppo-site effect was observed in Cryptococcus neoformans infection[17]. Since a number of studies have demonstrated that MAbRB6-8C5 also reacts against subsets of eosinophils [18], den-dritic cells [19] and T CD8þ lymphocytes [20], it is possiblethat most of these reports had been equivocally discussed con-sidering only neutrophil depletion.

In a previous study, it was reported that mortality in Gr-1þ

depleted mice infected with Histoplasma capsulatum washigher than in non-depleted mice [4]. In the present study,we have investigated the role of Gr-1þ cells on an experimentalmodel of systemic disseminated histoplasmosis by analyzingthe effect of a single dose of MAb RB6-8C5 in the host’sdefense to H. capsulatum infection.

2. Materials and methods

2.1. Mice and infection

Six-week-old male C57BL/6 mice were obtained from theanimal facilities of the Faculdade de Ciencias Farmaceuticasde Ribeir~ao Preto, Universidade de S~ao Paulo. Mice were in-oculated intravenously with 106 H. capsulatum yeast cells in0.1 ml of sterile PBS and control mice received i.v. PBSonly. All experiments were approved by and conducted inaccordance with guidelines established by the Universidadede S~ao Paulo Animal Care Committee.

2.2. Preparation of H. capsulatum

The H. capsulatum strain was isolated from a patient at theUniversity Hospital from Faculdade de Medicina de Ribeir~aoPreto, Universidade de S~ao Paulo. Live mycelia were obtainedby fungal culture at 25 �C on Sabouraud dextrose agar (Difco,Detroit, MI, USA). H. capsulatum yeast cells were grownat 37 �C for 36 h in Ham’s F12 medium (Gibco-BRL, GrandIsland, NY). Yeast cells were washed three times and sus-pended in PBS [21].

2.3. Treatment of mice with MAb RB6-8C5

The hybridoma secreting the MAb RB6-8C5 was a giftfrom Dr. M. Mariano (Departamento de Microbiologia, Imu-nologia e Parasitologia, UNIFESP, S~ao Paulo, Brazil). Anti-bodies were obtained from the culture supernatant ofrespective hybridomas and purified using a protein G-agarosecolumn (Gibco-BRL, Grand Island, NY). Twenty-four hoursbefore H. capsulatum infection, mice were intraperitoneallyinjected with 250 mg of MAb RB6-8C5 (namely ‘‘depletedmice’’) or rat IgG (namely ‘‘non-depleted mice’’) in 0.5 mlof PBS. Uninfected mice were used for comparison in someexperiments.

2.4. Blood cell counts

At various time points post-infection, blood samples werecollected by cardiac puncture. Total cell counts were per-formed using diluting solution in a Neubauer chamber anddifferential counts were obtained using Rosenfeld-stainedsmears [22].

2.5. Cytokine measurement

At 7 and 15 days post-infection, splenocytes from unin-fected mice and H. capsulatum-infected (depleted or non-depleted) mice were suspended at a concentration of 5� 105

cells in 0.1 ml of complete medium (RPMI 1640 supple-mented with 10% fetal bovine serum, 100 U/ml penicillin,100 mg/ml streptomycin, 2 mM L-glutamine, and 0.05 mM 2-mercaptoethanol) in 96-well plates. Cells were incubated at37 �C with the same volume of medium or 5 mg/well ofH. capsulatum cell-free antigens (CFAgs) in medium, preparedas previously described [23]. After 72 h incubation, the con-centrations of IL-4, IL-10 and IFN-g were determined inculture supernatants. At the same infection time points, thesemice had their left lungs removed, homogenized in 2 ml ofRPMI 1640 and centrifuged at 1500� g. The supernatantswere filter-sterilized and levels of IL-4, IL-10, IFN-g andTNF-a were determined. Blood samples were centrifugedand serum concentrations of IL-4, IL-10 and IFN-g were de-termined. Cytokines were measured by ELISA using commer-cially available antibodies with reference standard curves(Pharmingen, San Diego, CA).

2.6. Organ culture for H. capsulatum

To recover H. capsulatum, the lungs from each mouse wereaseptically excised, washed in PBS, minced with scissors, andenzymatically digested for 30 min in 15 ml of RPMI 1640with 1 mg/ml of liberase Cl (Roche Molecular Biochemicals,Indianapolis, IN). The cell suspension and tissue fragmentswere further dispersed by drawing up and down through thebore of a 10 ml syringe and were centrifuged. Spleens frominfected mice were aseptically removed as described above.Cells from both organs were suspended in media containingantibiotics and 0.2 ml of serial dilutions (10-fold) were addedon BHI-agar-blood plates. After incubation at 37 �C for 21days, colony-forming units (CFU) were counted and expressedper spleen and per gram of lung� SEM. The detection limitwas 5� 102 CFU.

2.7. Induction and measurement of cellular immuneresponse in vivo (DTH reaction) and in vitro (spleen cellproliferation assay)

H. capsulatum-infected (depleted and non-depleted) micewere submitted to intradermal challenge with an optimal inoc-ulum of 5 mg of CFAgs in a volume of 0.05 ml at 7 and 15days post-infection. Footpad swelling was measured with amicrometer (Mitutoyo Sul Americana, Suzano, SP, Brazil).

1395A. Sa-Nunes et al. / Microbes and Infection 9 (2007) 1393e1401

The delayed-type hypersensitivity (DTH) reaction was ex-pressed as the mean� SEM of the difference (in millimeters)between footpad thickness measured immediately before and24 h after antigen challenge. As a negative control, DTH reac-tion to CFAgs was measured in uninfected mice [23].

At 7 and 15 days post-infection, spleen cells were prepared asdescribed above and incubated with medium or stimulated withCFAgs (5 mg/well) or Concanavalin A (1 mg/ml) [23]. Prolifer-ation of spleen cells was measured after 72 h by adding 0.5 mCi/well of [3H]-thymidine (Amersham, Bucks, UK) in the last 18 hincubation. Cells were harvested and processed for scintillationcounting. Results are expressed as the stimulation index whichrepresents the proliferation induced by the stimuli divided bythe proliferation observed in the presence of medium.

2.8. Nitrite production by spleen cells andin lung homogenates

Nitrite (NO2�) concentration in supernatants of spleen cells

and in lung homogenates was measured by Griess reaction,employing a method previously described [24]. Briefly,100 ml of the samples were incubated at room temperaturewith an equal volume of the Griess reagent. The NO2

� concen-tration was determined using a standard curve of 1e200 mMNaNO2. The data are presented as micromoles of NO2

� inthe culture supernatant (mean� SEM).

2.9. Statistics

Statistical variations were determined by analysis of vari-ance (ANOVA) and Student’s t-test. The log-rank test wasused for statistical analysis of mortality. Values of P< 0.05were considered significant.

3. Results

3.1. Blood cell counts in depleted and non-depleted mice

No differences in blood cell counts were observed betweenuninfected mice receiving rat IgG or PBS (vehicle) in the pe-riod analyzed (data not shown). Fig. 1A shows that systemicH. capsulatum infection in non-depleted mice induced neutro-philia at the first day post-infection which returned to thecontrol level (uninfected mice) at 3 and 5 days post-infectionand significantly decreased at 7 days post-infection. RB6-8C5treatment decreased blood neutrophils by 80e90% in bothuninfected or infected mice at 1 and 3 days post-infection(P< 0.01). In the infected depleted group, neutrophil numbersreturned to the control levels at 5 days post-infection andalso decreased in the infected non-depleted group at 7 dayspost-infection.

H. capsulatum infection did not affect blood mononuclearcells until 5 days post-infection; however, a significant reduc-tion in these cells at 7 days post-infection was observed(P< 0.01). Mononuclear cells from both uninfected andinfected mice were affected by RB6-8C5 treatment at 1 daypost-infection in comparison with uninfected non-depleted

mice. However, these cells remain diminished at 3 dayspost-infection only in the infected group. At 5 and 7 dayspost-infection, infected depleted mice had the same mononu-clear cell profile in blood as the infected non-depleted mice.As observed for neutrophils, the uninfected depleted groupdid not show changes in blood mononuclear cell numbers at7 days post-infection when compared with the uninfectednon-depleted group (Fig. 1B).

3.2. Mortality and fungal recovery

All H. capsulatum-infected depleted mice died (17.6� 3.2days post-infection), whereas only 20% of the non-depletedinfected mice died during the period of observation(P< 0.01; Fig. 2A). At 7 days post-infection, CFU were foundto be 0.63 log10 higher in the spleens of depleted mice than inthose of non-depleted mice (P< 0.05; Fig. 2B), but CFU from

Fig. 1. Blood cell counts from uninfected and H. capsulatum-infected mice de-

pleted or non-depleted of Gr-1þ cells. Mice received 250 mg of MAb RB6-8C5

or rat IgG intraperitoneally and 24 h later were infected intravenously with 106

viable yeasts of H. capsulatum (Hc). At the indicated time points, blood sam-

ples were collected and neutrophil (A) and mononuclear cell (B) counts were

obtained as described in Section 2. *P< 0.05 versus uninfected; #P< 0.05

versus rat IgGþHc (n¼ 5).

1396 A. Sa-Nunes et al. / Microbes and Infection 9 (2007) 1393e1401

lungs did not differ among these groups at 7 days post-infection(Fig. 2C). At 15 days post-infection, the spleens and lungsof depleted mice contained 2.59 (Fig. 2B) and 1.72 log10

(Fig. 2C) more CFU, respectively, than did those of non-depleted mice (P< 0.05).

3.3. Cytokines in serum, lung homogenates and spleencell culture supernatants

On the first day post-infection, levels of IL-4 were 2-foldhigher in the serum of the infected depleted group

Fig. 2. Effect of Gr-1þ cells depletion on survival and fungal recovery of lungs

and spleens from H. capsulatum-infected mice. Mice received 250 mg of MAb

RB6-8C5 or rat IgG intraperitoneally and 24 h later were infected intravenously

with 106 viable yeasts of H. capsulatum (Hc). Survival rates were observed for

90 days (A). At 7 and 15 days post-infection, lungs and spleens were removed

and CFUs in the organs were determined in non-depleted (white bars) and de-

pleted (black bars) mice (B, C). *P< 0.01, by log-rank test in the mortality as-

say; *P< 0.05 versus rat IgGþHc group (n¼ 5e10).

(P< 0.05) than in the infected non-depleted group or unin-fected group. No differences were observed in IL-4 levels at3 and 5 days post-infection. However, at 7 days post-infectionthe serum of both infected groups had 2-fold more IL-4(P< 0.05) than serum of the uninfected group (Fig. 3A). Incontrast, serum IL-10 levels on the first day post-infectionwere 2-fold higher (P< 0.05) in non-depleted mice in compar-ison with depleted mice or uninfected mice. No differenceswere observed at 3 days post-infection among the groups. At5 days post-infection, serum of infected depleted mice hadsignificantly (P< 0.05) more IL-10 than that from uninfectedor infected non-depleted mice. On the other hand, IL-10 levels

Fig. 3. Cytokine levels in serum of H. capsulatum-infected mice depleted or

non-depleted of Gr-1þ cells. Mice received 250 mg of RB6-8C5 or rat IgG in-

traperitoneally and 24 h later were infected intravenously with 106 viable

yeasts of H. capsulatum (Hc). Uninfected mice were included as a control

(dotted line). At 1, 3, 5 and 7 days post-infection, mice were bled, serum

was collected and cytokine levels were determined by ELISA. *P< 0.05 ver-

sus uninfected; #P< 0.05 versus rat IgGþHc (n¼ 5e8 per group).

1397A. Sa-Nunes et al. / Microbes and Infection 9 (2007) 1393e1401

decreased in the infected non-depleted group when comparedwith uninfected or infected depleted mice at 7 days post-infection (P< 0.05; Fig. 3B). No differences in levels ofserum IFN-g were observed in uninfected mice and both in-fected groups at 1 and 3 days post-infection. At days 5 and7 post-infection a similar increase in the production of thiscytokine (more than 800%) was seen in the serum of bothinfected groups in comparison with serum of the uninfectedgroup (Fig. 3C). However, an earlier and stronger increase inthe ratio between IFN-g and IL-10 and between IFN-g andIL-4 was observed in infected non-depleted mice suggestinga more intense Th1 polarization in this group than in theinfected depleted group.

At 7 and 15 days post-infection, no differences in IL-4content in lung homogenates were observed between theuninfected group and from both infected groups (Fig. 4A).At 7 days post-infection, infected non-depleted mice presentedsignificantly more IL-10 in lung than did uninfected or in-fected depleted mice (P< 0.05). At 15 days post-infection,more IL-10 was detected in the lung homogenate from in-fected depleted mice than in that from infected non-depletedmice (P< 0.05; Fig. 4B). Similar IFN-g and TNF-a kineticswere observed in lung homogenates from both infectedgroups, in which levels of these cytokines were significantlyhigher than in those from the uninfected group. However, at15 days post-infection, levels of these cytokines in lung ho-mogenates from infected depleted mice were much higherthan in those from infected non-depleted mice (P< 0.05;Fig. 4C and D).

At 7 and 15 days post-infection, no significant differencesin IL-4 production by spleen cells incubated in medium alonewere observed between mice from the uninfected group tothose in either of the infected groups, depleted and non-depleted (Fig. 5A and B). Only a slight increase in IL-4production was observed in the cell culture from non-depletedanimals stimulated with CFAgs at 7 days post-infection(Fig. 5A). Also at day 7 post-infection, spleen cells fromboth infected groups incubated in medium alone producedlevels of IL-10 similar to those observed in spleen cellsfrom the uninfected group. However, when stimulated withCFAgs, production of IL-10 by these cells was significantlyhigher only in the infected depleted mice (P< 0.05;Fig. 5C). At 15 days post-infection, spleen cells from infecteddepleted animals produced more IL-10 than did those fromanimals in the other groups, in which levels of this cytokinewere virtually undetectable (Fig. 5D). In spleen cells from un-infected mice, levels of IFN-g were also barely detectable. Onthe other hand, at 7 and 15 days post-infection, spleen cellsfrom both groups of infected mice produced high amountsof IFN-g (P< 0.05). However, when stimulated with CFAgs,only spleen cells from the infected non-depleted group pro-duced more IFN-g (P< 0.05; Fig. 5E and F).

3.4. Cellular immune response in vivo and in vitro

To evaluate whether depletion of Gr-1þ cells affected thedevelopment of in vivo cellular immune response, acquisitionof DTH reactivity was determined. In both infected groups,

Fig. 4. IL-4, IL-10, IFN-g and TNF-a production in lung tissue of uninfected (control) and H. capsulatum-infected mice, depleted (RB6-8C5þHc) or non-

depleted (rat IgGþHc) of Gr-1þ cells. Mice received 250 mg of MAb RB6-8C5 or rat IgG and, 24 h later, were infected intravenously with 106 yeast cells of

H. capsulatum. At 7 and 15 days post-infection, lungs were removed, homogenized and centrifuged. Cytokine levels in the supernatants were determined by

ELISA. *P< 0.05 versus uninfected; #P< 0.05 versus rat IgGþHc (n¼ 5).

1398 A. Sa-Nunes et al. / Microbes and Infection 9 (2007) 1393e1401

Fig. 5. IL-4, IL-10 and IFN-g production by spleen cells of uninfected (control) and H. capsulatum-infected mice, depleted (RB6-8C5þHc) or non-depleted (rat

IgGþHc) of Gr-1þ cells. Mice received 250 mg of MAb RB6-8C5 or rat IgG and, 24 h later, were infected intravenously with 106 yeast cells of H. capsulatum. At 7

and 15 days post-infection, spleen cells were obtained and incubated for 72 h with medium or stimulated with 5 mg/well of H. capsulatum CFAgs. Cytokine levels

were determined by ELISA, and NO2� was determined by Griess reaction. *P< 0.05 versus uninfected group; #P< 0.05 versus same group incubated with CFAgs

or RB6-8C5 + Hc group incubated with medium; ND, not determined (n¼ 5).

DTH reaction was significantly pronounced at 7 days post-infection (P< 0.05; Fig. 6A). Nevertheless, decreased DTHreaction was found in depleted mice in comparison withnon-depleted mice at 15 days post-infection (P< 0.05;Fig. 6B).

The in vitro lymphoproliferative response from spleen cellswas also evaluated and the stimulation index of proliferationinduced by Concanavalin A (polyclonal activation) is shownin Fig. 6C. At 7 days post-infection, spleen cells from infecteddepleted mice showed a decreased proliferation in comparisonwith that from infected non-depleted mice (0.67� 0.20 versus1.52� 0.26, respectively; P< 0.05). However, both infectedgroups showed a strongly inhibited proliferation in compari-son with uninfected control mice (15.42� 5.55; P< 0.01).At 15 days post-infection, the suppression in the proliferativeresponse of spleen cells from both infected groups of mice waspartially reversed. However, the proliferation of spleen cellsfrom infected non-depleted mice was significantly higherthan that from infected depleted mice (11.54� 1.71 versus3.88� 0.30, respectively; P< 0.05). As observed at 7 dayspost-infection, proliferation of spleen cells from the uninfectedgroup at 15 days post-infection was higher than that from in-fected groups (17.56� 1.93; P< 0.01). No proliferative

response was observed when CFAgs were used as a stimulusin spleen cell cultures from either uninfected or infectedgroups in both evaluated period (data not shown).

3.5. NO2� production

No differences were observed in NO2� concentration from

the lungs of uninfected and infected groups at 7 days post-infection. However, NO2

� content in the lungs of the infecteddepleted group increased 3-fold over those seen in uninfectedand infected non-depleted mice at 15 days post-infection(P< 0.01; Fig. 6D).

In spleen cells from uninfected mice, NO2� levels were

barely detectable but spleen cells from both infected groupsproduced NO2

� when incubated in medium alone. However,spleen cells from infected depleted mice produced signifi-cantly more NO2

� in this basal condition than did spleen cellsfrom infected non-depleted mice (P< 0.05). On the otherhand, spleen cells from infected non-depleted mice increasedNO2� production when stimulated with CFAgs, although under

the same conditions, this did not occur in spleen cells frominfected depleted animals (Fig. 6E and F).

1399A. Sa-Nunes et al. / Microbes and Infection 9 (2007) 1393e1401

Fig. 6. Cellular immune response and NO2� levels from uninfected (control) and H. capsulatum-infected mice, depleted (RB6-8C5þHc) or non-depleted (rat

IgGþHc) of Gr-1þ cells. Mice received 250 mg of MAb RB6-8C5 or rat IgG and, 24 h later, were infected intravenously with 106 yeast cells of H. capsulatum.

At 7 and 15 days post-infection DTH response was evaluated (A, B), spleen cell proliferation was determined (C) and NO2� was determined in lung homogenates

(D) and spleen cell culture supernatants (E, F) as described in Section 2. *P< 0.05 versus uninfected; #P< 0.05 versus rat IgGþHc or indicated group (n¼ 5).

4. Discussion

A previous study demonstrated that depletion of Gr-1þ

cells at the time of an i.v. H. capsulatum infection resultedin accelerated mortality of mice. However, the role of thesecells in the increased susceptibility was not investigated [4].In this work, the same previously reported mortality usinga single dose of MAb RB6-8C5 was observed (Fig. 2A). Anal-ysis of blood smears of depleted groups during the first weekof infection confirmed a profound decrease in the number ofneutrophils until day 3 post-infection when compared withthe uninfected control group (Fig. 1A). In fact, it is wellknown that during microbial infection, neutrophils contribute

to host protection by promoting phagocytosis and activatingoxidative mechanisms, as well as by stimulating productionof immunoregulatory cytokines and chemokines [25]. How-ever, increasing pieces of evidence have demonstrated thatother Gr-1þ cells more than neutrophils could be responsiblefor the effects observed by the depletion using this MAb. Atleast the mononuclear population in the blood of infectedmice was also affected by the treatment with MAb RB6-8C5, although to a lesser extent than neutrophils (Fig. 1B).When this MAb was used in uninfected mice, a significant de-crease of mononuclear cells in the blood at 1 day post-infec-tion was observed, suggesting that treatment but notinfection was responsible for this decrease.

1400 A. Sa-Nunes et al. / Microbes and Infection 9 (2007) 1393e1401

The initial results on serum cytokine levels suggest a typicalTh1 polarization in H. capsulatum-infected mice. Neverthe-less, depleted mice seem to present a less robust Th1 polariza-tion than non-depleted mice in the beginning of infection(Fig. 3). The cytokine measurement in lungs and spleensshows a more complicated correlation. Except for some differ-ences in IL-10 production, levels of IL-4, IFN-g and TNF-a inlungs (Fig. 4) and IL-4 and IFN-g in spleens (Fig. 5) were verysimilar among both infected groups at 7 days post-infection.On the other hand, the absence of Gr-1þ cells in the beginningof infection seems to exacerbate the fungal proliferation anddissemination, which was confirmed by the CFU recovery(Fig. 2B and C) and by the histopathological analysis of liversand lungs (refer to Supplementary data, Appendices A and B).This explains the overproduction of Th1-related cytokines ob-served in lungs and spleens of the infected depleted mice, butnot in the non-depleted mice at 15 days post-infection (Figs. 4and 5). The production of Th1 cytokines associated with ex-cessive fungal burden in the organs at 15 days post-infectionseemed to be deleterious instead of protective to the miceand it was associated with increased nitric oxide productionin spleen cells and lungs (Fig. 6). Nitric oxide is one of themost important molecules with anti-Histoplasma activity[24], but its overproduction is known to be immunosuppres-sive and anti-proliferative [26]. In fact, the DTH response,which is an important determinant of the protective responsein histoplasmosis [23] was suppressed in infected depletedmice (Fig. 6B), as well as in the in vitro lymphoproliferativeresponse (Fig. 6C). The absence of specific proliferation foundin both infected groups seems to be related with nitric oxideproduction, since the addition of aminoguanidine in the culturerestored the proliferation of spleen cells from H. capsulatum-infected mice [26]. Taken together these previous data andours, we suggest that nitric oxide production affected thecellular immune response in vivo and in vitro.

There are several reports suggesting a role for each Gr-1þ

subpopulations in histoplasmosis. For example, it has beenshown that H. capsulatum mycelia are susceptible to killingby human neutrophils in vitro, although H. capsulatum yeastcells are not [27]. These data are consistent with those fromother studies showing that H. capsulatum resists killing byneutrophils, which have only a fungistatic activity againstyeast cells [28]. The importance of dendritic cells in anti-histoplasma immunity is becoming clearer. It has been demon-strated that human dendritic cells are able to phagocytoseH. capsulatum through the VLA-5 integrin and present fungalantigen to activate CD4þ T cells [29]. CD8þ T cells are notessential but contribute to the optimal clearance of H. capsu-latum from tissues in the systemic model [3]. However, inthe absence of CD4þ T cells, dendritic cells can activateCD8þ T cells by direct uptake of the fungus, or by cross-presentation of apoptotic macrophage-associated fungal anti-gens inducing a protective response [30]. Finally, althougheosinophils have been associated with histoplasmosis, eosino-phil depletion by MAb TRFK-5 did affect either mortality oracquisition of cellular immunity in histoplasmosis (data notshown). Based on these pieces of evidence, we suggest that

the absence of the neutrophil fungistatic activity and thediminished antigen presentation by dendritic cells could explainwhy mice depleted of Gr-1þ cells are more susceptible tosystemic H. capsulatum infection. Unfortunately, we havenot addressed the specific roles played by either neutrophilsor dendritic cells in this work. Finally, Zhou et al. [4] demon-strated that, after the development of an effective acquiredresponse, Gr-1þ cells are not essential for the maintenanceof immunity. Therefore, we did not investigate the impact ofGr-1þ cells’ depletion on secondary infection.

Taken together, these data show that the unbalanced Th1polarization, defective control of fungal proliferation and over-production of nitric oxide associated with immunosuppressionof in vivo and in vitro cellular immune response could explainthe increased susceptibility to histoplasmosis in the absence ofGr-1þ cells. In conclusion, this study provides further informa-tion regarding the role played by Gr-1þ cells in fungal dis-eases, particularly in disseminated histoplasmosis.

Acknowledgements

This study was supported by grants from the Fundac~ao deAmparo a Pesquisa do Estado de S~ao Paulo (FAPESP-95/9691-6 and 00/01079-0), the Conselho Nacional de Desenvol-vimento Cientıfico e Tecnologico, and the Fundac~ao de Amparoao Ensino, Pesquisa e Assistencia do Hospital das Clınicas daFMRP-USP, Brazil. The authors would like to thank Marly deCastro and Ana Maria da Rocha from Faculdade de Medicinade Ribeir~ao Preto for their technical support, and Dr. AndreBafica and Susannah Cleary for their critical reading of themanuscript.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at doi: 10.1016/j.micinf.2006.10.007.

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