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Flow-cytometric analysis of cytokine production in human paracoccidioidomycosis
www.elsevier.com/locate/issn/10434666 Cytokine 35 (2006) 207–216
Flow-cytometric analysis of cytokine production in human paracoccidioidomycosis Ronei Luciano Mamoni, Maria Heloisa Souza Lima Blotta
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Department of Clinical Pathology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), P.O. Box 6111, 13083-970 Campinas, SP, Brazil Received 9 November 2005; received in revised form 17 July 2006; accepted 27 August 2006
Abstract Human infection with Paracoccidioides brasiliensis may result in three major outcomes: paracoccidioidomycosis-infection (PI), which is observed in healthy carriers living in endemic areas and the adult form (AF) and juvenile form (JF) of the disease. In this study we proposed to examine the intracellular expression of IFN-c, TNF-a, IL-2, IL-10, IL-12, CXCL8, CXCL9 and CXCL10 by peripheral blood mononuclear cells (PBMC) of patients with the JF and AF of the disease, as well as of PI individuals stimulated with PMA plus ionomycin, LPS or anti-CD3 plus anti-CD28, by flow cytometry. The results showed that PI individuals present a higher percentage of cells producing IFN-c, TNF-a, IL-2, CXCL9 and CXCL10 when compared to AF and JF patients. IFN-c was predominantly detected in CD3+CD8+ T cells, whereas IL-2 and TNF-a were mainly expressed in CD3+CD4+ cells. Monocytes of PI individuals also presented higher expression of CD80 and lower expression of CD86 when compared to JF and AF patients, and higher expression of HLA-DR, only when compared to JF patients. These results indicate that the differential production of cytokines and chemokines, as well as the expression of co-stimulatory molecules involved in antigen presentation, may influence the outcome of PCM infection. 2006 Elsevier Ltd. All rights reserved. Keywords: Paracoccidioidomycosis; Flow-cytometry; Cytokines
1. Introduction Paracoccidioidomycosis (PCM) is a deep mycosis restricted to Latin America, most prevalent in rural areas among low-income individuals. This disease is caused by Paracoccidioides brasiliensis, a fungus that undergoes thermal dimorphism, developing as mycelium at room temperature and as yeast like cells at 37 C [1]. The infection probably occurs by the inhalation of fungal particles, which usually leads to an asymptomatic infection [1]. PCM presents a broad range of clinical and immunological manifestations, varying from benign and localized to severe and disseminated forms. According to current classification PCM may be divided in three major groups: the asymptomatic infection observed in healthy individuals
*
Corresponding author. Fax: +55 19 3521 9434. E-mail address: [email protected] (M. H. S. L. Blotta).
1043-4666/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2006.08.005
who live in endemic areas and are positive for the paracoccidioidin cutaneous test (PCM-infection [PI]) and two clinical forms of the disease: the adult (AF) and juvenile (JF) forms. The AF almost always affects adult males, who show a high frequency of pulmonary, skin and visceral involvement, whereas the JF equally affects young patients of both sexes and is characterized by systemic lymph node involvement, hepatosplenomegaly and bone marrow dysfunction [2]. Several studies on the immune responses developed by patients with PCM demonstrated that the mild forms of the disease (unifocal AF) were associated with the production of low levels of antibodies and positive delayed-type hypersensitivity (DTH) reactions, whereas the severe disseminated forms (JF or multifocal AF) were associated with high levels of antibodies and energy in DTH reactions [3–5]. More recent data have shown that effective defense against P. brasiliensis depends strictly upon Th1 cells and acquired resistance is governed by
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T cell- and macrophage-activating cytokines, whereas a Th2 pattern has been associated with susceptibility and more severe disease [3–6]. Patients presenting the disseminated forms of the disease produce higher levels of type 2 cytokines (IL-4, IL-5, IL-10 and TGF-b1) and antibodies (IgE, IgG4, and IgA) in addition to eosinophilia and impaired IFN-c secretion [4–9]. On the other hand, PI individuals present a typical Th1 pattern of cytokines with the production of IFN-c and TNF-a and basal levels of IL-4, IL-5 and IL-10 [5,6]. Among the cytokines associated with resistance to PCM, TNF-a and IFN-c play prominent functions, predominantly activating the oxidative burst in macrophages and maintaining the structural architecture of granuloma [10–14]. In addition, these cytokines are important in stimulating antigen presenting cells to upregulate the expression of molecules like the major histocompatibility complex (MHC) of class I and II and the co-stimulatory molecules CD80 (B7.1) and CD86 (B7.2) [15–17]. Several factors may contribute to the development of a Th1 or a Th2 response against an infection, but there is no consensus on this subject in PCM. Host and pathogen genetic background, virulence and the fungal load in the organism [1,18], as well as environmental factors influencing the host immune response, such as alcoholism, tobacco smoking and malnutrition, are candidates for consideration [19–21]. The development of Th1 and Th2 responses depend on the type of cytokine encountered in the milieu in which the differentiation process occurs. A Th1 cell will be generated in presence of IL-12, whereas a Th2 cell will be generated in IL-4 rich environment [22,23]. In this study, the production of several cytokines and chemokines in cells obtained from individuals presenting PCM-infection or PCM-disease (AF or the JF) were analyzed by flow-cytometry, in response to non-specific stimuli. We found that PI individuals exhibit higher numbers of CD8+IFN-c+, CD4+IL-2+/TNF-a+ as well as CD3+CXCL9+/CXCL10+/CXCR3+ cells as compared to AF and JF patients. In addition, a very low number of cells expressing IL-10 were detected in PI individuals showing that the resistance to disease observed in this group may be associated with the capacity to mount a predominant Th1 response. 2. Material and methods 2.1. Subjects The sample consisted of 38 subjects: 23 patients with newly diagnosed PCM and 15 healthy paracoccidioidin-reactive (PI) individuals living in an endemic area for PCM (Campinas, SP, Brazil). The diagnosis of PCM was established by fungus detection in clinical specimens and serology (immunodiffusion test). The patients were grouped according to clinical form: 8 with the juvenile acute form (JF) and 15 with the multifocal chronic adult form (AF)
of the disease. This research was approved by the Medical School Ethics Committee of the State University of Campinas (UNICAMP) and informed consent was obtained from each participant. 2.2. Determination of cytokine and chemokine production by flow cytometry The production of cytokines and chemokines was determined by intracellular staining using a flow-cytometric technique. Whole blood samples and PBMC obtained by the centrifugation of whole blood on a Ficoll-Hypaque gradient (Amersham Biosciences) were used. The cells were stimulated using different protocols in order to obtain the best results for each parameter analyzed. Whole blood samples were stimulated for 6 h with PMA (50 ng/mL—Sigma, St. Louis, USA) plus ionomycin (250 ng/mL—Sigma). PBMC were stimulated for 6 or 24 h with LPS (10 lg/mL—Sigma), or with antibodies anti-CD3 plus anti-CD28 (1 lg/mL each—R&D Systems, Minneapolis, USA) for 24 h. All samples were incubated during the last 6 h with Brefeldin A (1 lg/ mL—Sigma). Whole blood samples were first incubated with FACS Lysing Solution (BD Biosciences, San Jose, CA, USA) for 10 min at room temperature in order to lyse red blood cells. For the detection of surface molecules, the cells were resuspended at 1 · 107 cells/mL in wash buffer (PBS-sodium azide (2 mM)-BSA (1%)) and 20 lL of this suspension was added to 96 wells plates containing a mixture of monoclonal antibodies (antiCD3-CyChrome; anti-CD8-FITC or PE; anti-CD4 and anti-CD68-FITC; anti-CD80 and anti-CD86-PE, all from BD Biosciences, and anti-CXCR3- FITC, from R & D Systems) or the respective isotype controls and incubated for 30 min at room temperature. The cells were then washed, fixed with paraformaldehyde and permeabilized with a solution of saponin (0.5%) for 10 min at room temperature. After that, the cells were incubated with the monoclonal antibodies against intracellular molecules for 30 min at room temperature (anti-IFN-c-FITC or PE; anti-TNF-a, anti-IL2, anti-CXCL8, anti-IL10 and anti-IL12-PE, all from BD Biosciences, and anti-CXCL9 and anti-CXCL10-FITC both from R&D Systems). After incubation the cells were washed once with permeabilization buffer and once with wash buffer and fixed again with 250 lL of a 2% paraformaldehyde solution. FITC, PE and CyCh labeled control antibodies, as well as control unstimulated cells, were also included in all experiments. The samples were analyzed on a FACScalibur flow cytometer using CellQuest software (BD Biosciences), with selection of the lymphocyte and monocyte/ macrophage regions (gates). In all cases at least 10,000 gated events were acquired. As PMA has a down regulatory effect on CD4 expression [24], the determination of CD4+ cells was inferred by differential gatering in a flow cytometer, considering CD3+ minus CD3+CD8+ T cells.
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2.3. Statistical analyses The results were analyzed by SigmaStat v1.0 software (Jandel Co). The differences among the groups were analyzed with the ANOVA test. The differences between stimuli were compared with the t test for paired samples. Data were considered significant when a p value equal or lower than 0.05 was obtained. 3. Results 3.1. Production of Th1 cytokines is higher in PI individuals than in AF or JF patients Individuals of the PI group were observed presenting a higher percentage of cells expressing IFN-c when compared to the AF or JF group, and AF patients also expressed more IFN-c when compared to JF patients (Fig. 1). Interestingly, IFN-c expression was mainly observed in CD3+CD8+ cells, indicating an important role of this cell subset in IFN-c production. As lymphocytes from PI individuals present an intense proliferative response to P. brasiliensis antigen [5], when
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compared to the AF or JF patients, IL-2 production was examined next. The percentage of cells expressing IL-2 was highest in the PI group, as for IFN-c, however, unlike IFN-c production, IL-2 was predominantly detected in CD3+CD4+ cells (Fig. 2). In addition to IFN-c, TNF-a has a key role in PCM, participating in granuloma formation as well as in macrophage activation. Several cell types including lymphocytes and monocytes are able to produce TNF-a. A higher percentage of TNF+CD4+ cells was detected in PI individuals in relation to AF or JF patients (Fig. 3B), however, similar percentages of TNF+CD68+ were detected in the PI and AF groups in both unstimulated and LPS stimulated cells (Fig. 3C). 3.2. CXC chemokines and CXCR3 are predominantly expressed in cells from PI individuals as compared to AF or JF patients It is well known that IFN-c is a potent inducer of chemokines CXCL10 and CXCL9 by macrophages [25]. We then determined whether CXCL9 and CXCL10 production was upregulated in PBMC among PI individuals. For this
Fig. 1. IFN-c production by PBMC from PCM-infection (PI) individuals and patients with the adult (AF) or juvenile form of PCM (JF) after stimulation with PMA (50 ng/mL) plus ionomycin (250 ng/mL) for 6 h, in the presence of brefeldin A (1 lg/mL). (A) Representative dot-plots of CD3+ or CD8+ cells expressing IFN-c. Percentages of positive cells in each quadrant are indicated at the top. (B) Percentages of CD3+IFN-c+, CD3+CD8+IFN- c+ and (CD3+IFN-c+) minus (CD3+CD8+IFN- c+) cells. The cells were gated in lymphocyte region according to their SSC and FSC characteristics. Data are expressed as mean ± SEM. Statistics: ANOVA test, *p < 0.05.
Fig. 2. IL-2 production by PBMC from PCM-infection (PI) individuals and patients with the adult (AF) or juvenile form of PCM (JF) after stimulation with PMA (50 ng/mL) plus ionomycin (250 ng/mL) for 6 h, in the presence of brefeldin A (1 lg/mL). (A) Representative dot-plots of CD3+ or CD8+ cells expressing IL-2. Percentages of positive cells in each quadrant are indicated at the top. (B) Percentages of CD3+IL-2+, CD3+CD8+IL-2+ and (CD3+IL2+) minus (CD3+CD8+IL-2+) cells. The cells were gated in lymphocyte region according to their SSC and FSC characteristics. Data are expressed as mean ± SEM. Statistics: ANOVA test, *p < 0.05.
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Fig. 3. TNF-a production by PBMC from PCM-infection (PI) individuals and patients with the adult (AF) or juvenile form of PCM (JF) after stimulation with PMA (50 ng/mL) plus ionomycin (250 ng/mL) for 6 h, in the presence of brefeldin A (1 lg/mL). (A) Representative dot-plots of CD3+, CD8+ or CD68+ cells expressing TNF-a. Percentages of positive cells in each quadrant are indicated at the top. (B) Percentages of CD3+TNF-a+, CD3+CD8+TNF- a+ and (CD3+TNF-a+) minus (CD3+CD8+TNF- a+) cells. (C) Percentages of CD68+TNF-a+ unstimulated or stimulated with LPS. The cells were gated in lymphocyte or monocyte region according to their SSC and FSC characteristics. Data are expressed as mean ± SEM. Statistics: ANOVA test, *p < 0.05. Note the different scales.
set of experiments PBMC were cultured in the presence of anti-CD3 plus anti-CD28 monoclonal antibodies, for 24 h, in the presence of brefeldin A for the last 6 h.
According to the IFN-c data, the percentage of CXCL9 and CXCL10/CD3+CD4+ cells was higher in the PI group as compared to both AF and JF patients. In contrast, the
Fig. 4. Expression of chemokines (CXCL9, CXCL10 and CXCL8) and CXCR3 in lymphocytes (CD3+ cells) or monocytes (CD68+ cells) from PCMinfection (PI) individuals and patients with the adult (AF) or juvenile form of PCM (JF). Percentage of CD3+ or CD68+ cells expressing CXCL9 (A) or CXCL10 (B) after stimulation with anti-CD3 plus anti-CD28 monoclonal antibodies (1 lg/mL of each for 24 h), in the presence of brefeldin A (1 lg/mL) for the last 6 h. (C) Expression of the chemokine receptor CXCR3 in lymphocytes (CD3+) unstimulated or stimulated with anti-CD3 plus anti-CD28 for 24 h. (D) Percentages of CXCL8 positive monocytes (CD68+ cells) unstimulated or stimulated with LPS for 6 h. The cells were gated in lymphocyte or monocyte region according to their SSC and FSC characteristics. Data are expressed as mean ± SEM. Statistics: ANOVA test, *p < 0.05. Note the different scales.
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percentage of CXCL9 and CXCL10 positive monocytes was significantly higher in the PI and AF groups than in JF patients (Fig. 4A and B). CXCL9 and CXCL10 share the same receptor, CXCR3, expressed predominantly in Th1 lymphocytes. Higher amounts of CXCR3+CD3+ positive cells were detected in PI individuals in both unstimulated or anti-CD3 plus anti-CD28 stimulated cells (Fig. 4C), in agreement with chemokine production. In a different way, the analysis of CXCL8 expression in monocytes showed an elevated number of positive cells among AF patients in relation to PI and JF groups (Fig. 4D). 3.3. Elevated number of IL-12 expressing monocytes is detected in AF patients, while IL-10 is predominantly expressed in patients’ cells when compared to PI group Looking for cytokines involved in T cell differentiation we next examined the expression of IL-4 and IL-12 among the groups. Unfortunately we were not able to detect intracellular IL-4 by flow cytometry. However, the analysis of IL-12 producing cells showed a significantly elevated
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number of positive cells among AF patients, in accordance with previous data from our group [5]. IL-12 was detected in both unstimulated and LPS stimulated CD68+ cells (Fig. 5A). Considering the lack of reactivity for IL-4, we then decided to evaluate IL-10 production, since it also presents a negative control upon the Th1 response [26]. In the present study monocytes expressing IL-10 were mainly detected in patients with PCM (AF and JF) as compared to PI individuals. Moreover, after LPS stimulation it was possible to distinguish between AF and JF patients, with a higher percentage of CD68+IL-10+ cells in the JF group (Fig. 5B). 3.4. Expression of the co-stimulatory molecules distinguishes cells from PI, AF and JF individuals MHC class I and II, in addition to co-stimulatory molecules, are essential to the development of an effective immune response, providing the first and second signals to fully activate T cells. In order to address this aspect of the immune response in human PCM, the expression of HLA-DR (MHC-class II), CD80 and CD86 was assessed in monocytes of PI, AF and JF individuals. CD80 and CD86 were found to be differentially expressed in the cells of the groups analyzed. CD80 expression was higher in monocytes of AF patients in relation to PI individuals when analyzing the unstimulated cells, but after stimulation with LPS for 6 h, there was an upregulation of this molecule in the PI group (Fig. 6A). On the other hand, CD86 was expressed in higher levels in both the AF and JF groups when compared to the PI group in both unstimulated and LPS stimulated monocytes (CD68+ cells) (Fig. 6B). Interestingly, the increased percentage of cells expressing CD80 after stimulation in PI group (0.66 ± 0.20 vs. 20.25 ± 4.17) was related to a significant decrease in CD86 expression in the same cells (84.41 ± 1.19 vs. 71.07 ± 3.58—p < 0.05). This modulation was not observed in the AF and JF patients cells, which presented a slight but not statistically significant decrease in CD86 expression (data not shown). In relation to HLA-DR, in spite of the high expression in monocytes of all groups, a significantly higher percentage of positive cells were detected in the PI group, when compared to AF or JF patients (Fig. 6C). 4. Discussion
Fig. 5. Expression of IL-12 and IL-10 in monocytes (CD68 cells) from PCM-infection (PI) individuals and patients with the adult (AF) or juvenile form of PCM (JF). Percentages of CD68+ cells expressing IL-12 (A) or IL-10 (B) after stimulation with LPS (10 lg/mL) for 24 h, in the presence of brefeldin A (1 lg/mL) in the last 6 h. The cells were gated in monocyte region according to their SSC and FSC characteristics. Data are expressed as mean ± SEM. Statistics: ANOVA test, *p < 0.05. Note the different scales.
In a previous study we showed an early and high IFN-c and TNF-a mRNA expression in cells from PI individuals [6]. The data presented here confirm the higher production of these cytokines in PI individuals after stimuli in comparison with patients with PCM. It was verified that PI individuals are able to produce high amounts of Th1 cytokines, like IFN-c, following stimulation. IFN-c is the main macrophage-activating cytokine, which initiates and drives the Th1 cell response and directs the assembly of tissue granuloma in addition to TNF-a [27]. Once these
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Fig. 6. Expression of CD80 (B7.1), CD86 (B7.2) and HLA-DR in monocytes (CD68+ cells) from PCM-infection (PI) individuals and patients with the adult (AF) or juvenile form of PCM (JF). Percentages of CD68+ cells expressing CD80 (A), CD86 (B) and HLA-DR (C) in cells unstimulated or after stimulation with LPS (10 lg/mL) for 24 h. The cells were gated in monocyte region according to their SSC and FSC characteristics. Data are expressed as mean ± SEM. Statistics: ANOVA test, *p < 0.05. Note the different scales.
cytokines are important to the activation of the oxidative metabolism of macrophages and consequently to the killing of the P. brasiliensis [10–12], the prompt and high IFN-c and TNF-a production may explain the resistance of PI individual. Moreover the analysis of the expression of surface markers showed that IFN-c expressing cells were mainly CD3+CD8+. The importance of the CD8 subset in
response to P. brasiliensis infection was recently shown in the murine model of PCM, in which depletion of CD8+ T lymphocytes resulted in the enhancement of disease severity and dissemination in both susceptible and resistant mice [28]. In addition, patients with pulmonary PCM showed large quantities of CD8+ T cells in bronchoalveolar lavage (BAL), which positively correlated with the production of CCL3 [29], a chemokine known to promote chemotaxis of lymphocytes, selectively recruiting CD8+ T cells [30,31]. CD8+ T cells mediated immune responses are based on cytokine production, mainly IFN-c, as showed here, or cytotoxicity [32]. In PCM, TNF-a has also been implicated in the formation of compact granuloma, which accounts for the resistance to infection [12,27]. In the present study, we showed that TNF-a was expressed at different levels according to the cell type analyzed. CD3+TNF-a+ cells were predominant in the PI group, while in CD68+ cells (monocytes) TNF-a expression was similar in AF patients and in PI individuals in both unstimulated or LPS stimulated cells. TNF-a is a cytokine associated with resistance to several infectious diseases. In the experimental model of histoplasmosis the neutralization of TNF-a results in the increase of fungal dissemination and mortality [33]. Herring et al. [34] observed that the neutralization of TNF-a in the initial phase of the infection with Cryptococcus neoformans diminished IL-12 production and hence IFN-c secretion and, in addition, increased the number of eosinophils in peripheral blood. In a recent study we observed that TNF-a mRNA was earlier and higher expressed in cells of PI individuals as compared to JF and AF patients [6]. The elevated production of TNF-a may be important to increase the Th1 response in PI individuals, while the low TNF-a levels exhibited by JF patients probably contribute to the high susceptibility to infection and may be related to eosinophilia observed in this group [4,5,7]. Besides IFN-c and TNF-a production, cells from the PI individuals showed a higher expression of CD4+IL-2+ cells, when compared to patients with both clinical forms of PCM. IL-2 is produced by lymphocytes, mainly Th1 cells with an essential role in the expansion and survival of specific lymphocytes [35,36], as well as, a crucial participation in DTH responses. In a previous paper we reported that PI individuals presented an intense proliferative response against P. brasiliensis antigen correlated with DTH responses [5], so the high production of IL-2 by PI lymphocytes may explain these data. Chemokine and chemokine receptors, in addition to IFN-c and TNF-a, play an important role in granulomatous inflammatory reactions. The higher expression of chemokine CXCL10 and CXCL9 agrees with the detection of IFN-c positive cells in PI individuals. Moreover the production of such chemokines presented a differential pattern of expression depending on the cell type. CD3+ cells from PI group presented higher expression of
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CXCL9 and CXCL10 than AF or JF groups, while the expression of these chemokines were similar and elevated in monocytes of AF and PI groups. CXCL10 and CXCL9 are IFN-c inducible and very effectively attract activated T lymphocytes [25]. Both chemokines signal through a common receptor, CXCR3, expressed by memory (CD45RO+) T cells, preferentially of the Th1 subset, and by natural killer cells, but not by monocytes and neutrophils [25,37–40]. Accordingly, CXCR3 was expressed in higher levels in T cells from the PI group than in JF or AF patients. Souto et al. [41] showed that IFN-c modulates the expression of chemokine and chemokine receptors and determines the cells that infiltrate the lungs of mice infected with P. brasiliensis. In this model, mice presented high levels of CCL5, CCL2, CXCL10 and CXCL9, simultaneously with mononuclear cell infiltration in the lungs. The predominance of such chemokines and the expression of their receptor could contribute to the preferential mobilization of Th1 cells in PI individuals, leading to a more effective response against P. brasiliensis infection. In this study we showed that unstimulated CD68+ cells from AF patients express higher levels of CXCL8 expression as compared to the other groups. CXCL8 contains a conserved tripeptide motif glutamic acid–leucine–arginine (ELR) at the N-terminal of the protein, which enables it to attract neutrophils [42,43]. Neutrophils participate in the inflammatory response to P. brasiliensis through the release of oxygen-derived products and endogenous peroxidase located in cytoplasmic granules, killing both ingested and extracellular yeast cells [44,45]. Another study of our group also showed higher levels of CXCL8 in sera of patients with the AF as compared to the JF of PCM [4]. Furthermore, neutrophilic abscesses are a common finding in tissues infected with P. brasiliensis [46]. Elevated production of CXCL9, CXCL10 and CCL8, as well as TNF-a, by monocytes of AF group could contribute to contain fungal dissemination but, as describe for the murine model of PCM, a strong activation of innate immunity can trigger mechanisms that suppress cell-mediated immune response. It is well known that, in terms of clinical presentation, the AF of PCM is very heterogeneous, ranging from isolated lesions in the respiratory tract to widely disseminated forms, what suggests a gradation of the immune response. In our study the majority of AF patients presented the multifocal form of the disease and about 90% showed pulmonary involvement. As previously described for TNF-a and NO [47], the concentration of IL-12 produced during P. brasiliensis infection might be crucial for the development of resistance or susceptibility. In this context, our finding of high numbers of IL-12+ monocytes in AF patients may account for an initial effective immune response, followed by an exacerbated inflammatory reaction. Alveolar macrophages from patients with pulmonary PCM express high levels of adhesion and co-stimulatory molecules
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and produce large amounts of IL-12, in addition to IL6, TNF-a and MIP-1a [29]. Moreover, early treatment with recombinant IL-12 resulted in a less disseminated disease in mice infected with P. brasiliensis, but induced an increased pulmonary mononuclear cell inflammation [13]. Recent studies had demonstrated that susceptible mice respond to the P. brasiliensis infection with an initial exacerbated innate inflammatory response in the lungs, producing high amounts of pro-inflammatory cytokines as IL-12 and high NO expression, whereas resistant mice present a more controlled response, with mild production of IL-10 and TGF-b1, and low levels of pro-inflammatory cytokines [48,49]. Considering these results, it was proposed that an exacerbated innate immune response at the early phase of the disease might lead to an ineffective T cell response in the later phases of the infection, which contributes to the dissemination of the disease. Similarly to that, it was demonstrated that the overexpression of IL-12, in lungs of mice infected with Mycobacterium tuberculosis, diminished the capacity of migration of leukocytes (macrophages, lymphocytes and neutrophils) to this organ, hence leading to an increased susceptibility in later phases of the infection, although that overexpression does not influence the production of IFN-c [50]. Unfortunately we were not able to detect intracellular IL-4, although in previous studies performed by our group a higher expression of IL-4 (mRNA and protein) was found in JF patients as compared to AF and PI group [5,6]. Using different approaches we have shown IL-4 in supernatants of PBMC from JF patients cultured in the presence of P. brasiliensis antigen [5], as well as early expression (6 h) of IL-4 mRNA by JF cells [6]. Here we analyzed the expression of IL-4 by flow cytometry in cells stimulated for 24 h with anti-CD3 plus anti-CD28 mAbs, and a possible explanation for the absence of IL-4 could be the fact that the blocking of protein secretion (Brefeldin A) was made only in the last 6 h of cell culture, when IL-4 could no longer be produced. In experimental models IL-4 production is not clearly linked to susceptibility since antigen stimulated lymph node cells produce IL-10 but not IL-4 during the initial 8 weeks of infection [51]. In addition, the role of IL-4 in P. brasiliensis infection apparently depends on the genetic background of the host, leading to susceptibility in C57BL/6 mice, but being important to the resistance in B.10 lines [52]. In this study a higher number of IL-10 positive monocytes (CD68+ cells) were detected in both, AF and JF patients, whereas only a basal number of cells were found in PI individuals. These results confirm our previous data, in which cells from AF or JF patients stimulated in vitro with P. brasiliensis antigen produced higher levels of IL-10 when compared to PI individuals [5], suggesting that, in addition to IL-4, IL-10 may also contribute to susceptibility in human PCM. IL-10 is an important mediator of the inhibition of nitric oxide (NO) diminishing the expression of the inducible nitric oxide synthase (iNOS), an enzyme induced by IFN-c and TNF-a [53]. Although its
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role in human infection is unknown, it was demonstrated that NO has a fungicidal role in P. brasiliensis infection in mice [27,47]. The expression of surface molecules such as CD80 (B7.1), CD86 (B7.2) and HLA-DR (MHC class II), was also different among the groups. Although the low expression in unstimulated cells, CD80 was upregulated in the cells of the 3 groups analyzed, notably in PI individuals, after LPS stimulation. As mentioned above, the increased percentage of cells expressing CD80 after stimulation in PI group was related to a significant decrease in CD86 expression in the same cells. This fact, not observed in the other groups, may reflect a particular manner of costimulation, differently influencing the immune response. Several studies have shown that the co-stimulation of cells through CD80 or CD86 might be important to the development of a Th1 or a Th2 response [15,54]. The expression of CD80 has been associated with a predominant Th1 response with the activation of CD8+ cells [55,56], while co-stimulation through CD86 would be involved in the production of IL-4 and, consequently, in the Th2 response [56–58]. The expression of HLA-DR in cells of PI individuals was also significantly higher in both unstimulated and LPSstimulated cells when compared to the JF group. Th2 cytokines, such as IL-4 and IL-10, induce the downregulation of CD86, while IFN-c increases the expression of CD80 and CD86, as well as HLA-DR. Since the differentiation of T cells is dependent, among other factors, on the cytokine present in the milieu where the immune response takes place, the co-stimulatory molecules involved in the antigen presentation and the chemokines implicated in differential homing of cells, these data support the implication of a Th1 immune response in the resistance profile exhibited by PI individuals. In summary, the present study confirmed our previous findings, which associated individuals with PCM infection (PI) with the Th1 pole of the immune response characterized by an elevated number of T cells expressing IFN-c, IL-2, TNF-a, CXCL9/CXCL10/CCR3 and low number of IL-10 positive cells. Although the intracellular expression of immune mediators have not demonstrated a clear Th2 polarization in the JF of the disease, low production of pro-inflammatory cytokines and chemokines and high number of IL-10+ cells, in addition to low expression of co-stimulatory and HLA-DR molecules characterized this group of patients. On the other hand, a high number of monocytes expressing IL-12 and TNF-a and chemokines such as CXCL9, CXCL10 and CXCL8 probably enables AF patients to better control the fungal dissemination, resulting in moderate forms of the disease. However, in analogy to the animal model of PCM, we may suppose that a misbalance favoring a strong pro-inflammatory response rather than a protective one is detrimental to the development of an effective cellular immune response, which leads to severe forms of the disease.
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Flow-cytometric analysis of cytokine production in human paracoccidioidomycosis Ronei Luciano Mamoni, Maria Heloisa Souza Lima Blotta
*
Department of Clinical Pathology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), P.O. Box 6111, 13083-970 Campinas, SP, Brazil Received 9 November 2005; received in revised form 17 July 2006; accepted 27 August 2006
Abstract Human infection with Paracoccidioides brasiliensis may result in three major outcomes: paracoccidioidomycosis-infection (PI), which is observed in healthy carriers living in endemic areas and the adult form (AF) and juvenile form (JF) of the disease. In this study we proposed to examine the intracellular expression of IFN-c, TNF-a, IL-2, IL-10, IL-12, CXCL8, CXCL9 and CXCL10 by peripheral blood mononuclear cells (PBMC) of patients with the JF and AF of the disease, as well as of PI individuals stimulated with PMA plus ionomycin, LPS or anti-CD3 plus anti-CD28, by flow cytometry. The results showed that PI individuals present a higher percentage of cells producing IFN-c, TNF-a, IL-2, CXCL9 and CXCL10 when compared to AF and JF patients. IFN-c was predominantly detected in CD3+CD8+ T cells, whereas IL-2 and TNF-a were mainly expressed in CD3+CD4+ cells. Monocytes of PI individuals also presented higher expression of CD80 and lower expression of CD86 when compared to JF and AF patients, and higher expression of HLA-DR, only when compared to JF patients. These results indicate that the differential production of cytokines and chemokines, as well as the expression of co-stimulatory molecules involved in antigen presentation, may influence the outcome of PCM infection. 2006 Elsevier Ltd. All rights reserved. Keywords: Paracoccidioidomycosis; Flow-cytometry; Cytokines
1. Introduction Paracoccidioidomycosis (PCM) is a deep mycosis restricted to Latin America, most prevalent in rural areas among low-income individuals. This disease is caused by Paracoccidioides brasiliensis, a fungus that undergoes thermal dimorphism, developing as mycelium at room temperature and as yeast like cells at 37 C [1]. The infection probably occurs by the inhalation of fungal particles, which usually leads to an asymptomatic infection [1]. PCM presents a broad range of clinical and immunological manifestations, varying from benign and localized to severe and disseminated forms. According to current classification PCM may be divided in three major groups: the asymptomatic infection observed in healthy individuals
*
Corresponding author. Fax: +55 19 3521 9434. E-mail address: [email protected] (M. H. S. L. Blotta).
1043-4666/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2006.08.005
who live in endemic areas and are positive for the paracoccidioidin cutaneous test (PCM-infection [PI]) and two clinical forms of the disease: the adult (AF) and juvenile (JF) forms. The AF almost always affects adult males, who show a high frequency of pulmonary, skin and visceral involvement, whereas the JF equally affects young patients of both sexes and is characterized by systemic lymph node involvement, hepatosplenomegaly and bone marrow dysfunction [2]. Several studies on the immune responses developed by patients with PCM demonstrated that the mild forms of the disease (unifocal AF) were associated with the production of low levels of antibodies and positive delayed-type hypersensitivity (DTH) reactions, whereas the severe disseminated forms (JF or multifocal AF) were associated with high levels of antibodies and energy in DTH reactions [3–5]. More recent data have shown that effective defense against P. brasiliensis depends strictly upon Th1 cells and acquired resistance is governed by
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T cell- and macrophage-activating cytokines, whereas a Th2 pattern has been associated with susceptibility and more severe disease [3–6]. Patients presenting the disseminated forms of the disease produce higher levels of type 2 cytokines (IL-4, IL-5, IL-10 and TGF-b1) and antibodies (IgE, IgG4, and IgA) in addition to eosinophilia and impaired IFN-c secretion [4–9]. On the other hand, PI individuals present a typical Th1 pattern of cytokines with the production of IFN-c and TNF-a and basal levels of IL-4, IL-5 and IL-10 [5,6]. Among the cytokines associated with resistance to PCM, TNF-a and IFN-c play prominent functions, predominantly activating the oxidative burst in macrophages and maintaining the structural architecture of granuloma [10–14]. In addition, these cytokines are important in stimulating antigen presenting cells to upregulate the expression of molecules like the major histocompatibility complex (MHC) of class I and II and the co-stimulatory molecules CD80 (B7.1) and CD86 (B7.2) [15–17]. Several factors may contribute to the development of a Th1 or a Th2 response against an infection, but there is no consensus on this subject in PCM. Host and pathogen genetic background, virulence and the fungal load in the organism [1,18], as well as environmental factors influencing the host immune response, such as alcoholism, tobacco smoking and malnutrition, are candidates for consideration [19–21]. The development of Th1 and Th2 responses depend on the type of cytokine encountered in the milieu in which the differentiation process occurs. A Th1 cell will be generated in presence of IL-12, whereas a Th2 cell will be generated in IL-4 rich environment [22,23]. In this study, the production of several cytokines and chemokines in cells obtained from individuals presenting PCM-infection or PCM-disease (AF or the JF) were analyzed by flow-cytometry, in response to non-specific stimuli. We found that PI individuals exhibit higher numbers of CD8+IFN-c+, CD4+IL-2+/TNF-a+ as well as CD3+CXCL9+/CXCL10+/CXCR3+ cells as compared to AF and JF patients. In addition, a very low number of cells expressing IL-10 were detected in PI individuals showing that the resistance to disease observed in this group may be associated with the capacity to mount a predominant Th1 response. 2. Material and methods 2.1. Subjects The sample consisted of 38 subjects: 23 patients with newly diagnosed PCM and 15 healthy paracoccidioidin-reactive (PI) individuals living in an endemic area for PCM (Campinas, SP, Brazil). The diagnosis of PCM was established by fungus detection in clinical specimens and serology (immunodiffusion test). The patients were grouped according to clinical form: 8 with the juvenile acute form (JF) and 15 with the multifocal chronic adult form (AF)
of the disease. This research was approved by the Medical School Ethics Committee of the State University of Campinas (UNICAMP) and informed consent was obtained from each participant. 2.2. Determination of cytokine and chemokine production by flow cytometry The production of cytokines and chemokines was determined by intracellular staining using a flow-cytometric technique. Whole blood samples and PBMC obtained by the centrifugation of whole blood on a Ficoll-Hypaque gradient (Amersham Biosciences) were used. The cells were stimulated using different protocols in order to obtain the best results for each parameter analyzed. Whole blood samples were stimulated for 6 h with PMA (50 ng/mL—Sigma, St. Louis, USA) plus ionomycin (250 ng/mL—Sigma). PBMC were stimulated for 6 or 24 h with LPS (10 lg/mL—Sigma), or with antibodies anti-CD3 plus anti-CD28 (1 lg/mL each—R&D Systems, Minneapolis, USA) for 24 h. All samples were incubated during the last 6 h with Brefeldin A (1 lg/ mL—Sigma). Whole blood samples were first incubated with FACS Lysing Solution (BD Biosciences, San Jose, CA, USA) for 10 min at room temperature in order to lyse red blood cells. For the detection of surface molecules, the cells were resuspended at 1 · 107 cells/mL in wash buffer (PBS-sodium azide (2 mM)-BSA (1%)) and 20 lL of this suspension was added to 96 wells plates containing a mixture of monoclonal antibodies (antiCD3-CyChrome; anti-CD8-FITC or PE; anti-CD4 and anti-CD68-FITC; anti-CD80 and anti-CD86-PE, all from BD Biosciences, and anti-CXCR3- FITC, from R & D Systems) or the respective isotype controls and incubated for 30 min at room temperature. The cells were then washed, fixed with paraformaldehyde and permeabilized with a solution of saponin (0.5%) for 10 min at room temperature. After that, the cells were incubated with the monoclonal antibodies against intracellular molecules for 30 min at room temperature (anti-IFN-c-FITC or PE; anti-TNF-a, anti-IL2, anti-CXCL8, anti-IL10 and anti-IL12-PE, all from BD Biosciences, and anti-CXCL9 and anti-CXCL10-FITC both from R&D Systems). After incubation the cells were washed once with permeabilization buffer and once with wash buffer and fixed again with 250 lL of a 2% paraformaldehyde solution. FITC, PE and CyCh labeled control antibodies, as well as control unstimulated cells, were also included in all experiments. The samples were analyzed on a FACScalibur flow cytometer using CellQuest software (BD Biosciences), with selection of the lymphocyte and monocyte/ macrophage regions (gates). In all cases at least 10,000 gated events were acquired. As PMA has a down regulatory effect on CD4 expression [24], the determination of CD4+ cells was inferred by differential gatering in a flow cytometer, considering CD3+ minus CD3+CD8+ T cells.
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2.3. Statistical analyses The results were analyzed by SigmaStat v1.0 software (Jandel Co). The differences among the groups were analyzed with the ANOVA test. The differences between stimuli were compared with the t test for paired samples. Data were considered significant when a p value equal or lower than 0.05 was obtained. 3. Results 3.1. Production of Th1 cytokines is higher in PI individuals than in AF or JF patients Individuals of the PI group were observed presenting a higher percentage of cells expressing IFN-c when compared to the AF or JF group, and AF patients also expressed more IFN-c when compared to JF patients (Fig. 1). Interestingly, IFN-c expression was mainly observed in CD3+CD8+ cells, indicating an important role of this cell subset in IFN-c production. As lymphocytes from PI individuals present an intense proliferative response to P. brasiliensis antigen [5], when
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compared to the AF or JF patients, IL-2 production was examined next. The percentage of cells expressing IL-2 was highest in the PI group, as for IFN-c, however, unlike IFN-c production, IL-2 was predominantly detected in CD3+CD4+ cells (Fig. 2). In addition to IFN-c, TNF-a has a key role in PCM, participating in granuloma formation as well as in macrophage activation. Several cell types including lymphocytes and monocytes are able to produce TNF-a. A higher percentage of TNF+CD4+ cells was detected in PI individuals in relation to AF or JF patients (Fig. 3B), however, similar percentages of TNF+CD68+ were detected in the PI and AF groups in both unstimulated and LPS stimulated cells (Fig. 3C). 3.2. CXC chemokines and CXCR3 are predominantly expressed in cells from PI individuals as compared to AF or JF patients It is well known that IFN-c is a potent inducer of chemokines CXCL10 and CXCL9 by macrophages [25]. We then determined whether CXCL9 and CXCL10 production was upregulated in PBMC among PI individuals. For this
Fig. 1. IFN-c production by PBMC from PCM-infection (PI) individuals and patients with the adult (AF) or juvenile form of PCM (JF) after stimulation with PMA (50 ng/mL) plus ionomycin (250 ng/mL) for 6 h, in the presence of brefeldin A (1 lg/mL). (A) Representative dot-plots of CD3+ or CD8+ cells expressing IFN-c. Percentages of positive cells in each quadrant are indicated at the top. (B) Percentages of CD3+IFN-c+, CD3+CD8+IFN- c+ and (CD3+IFN-c+) minus (CD3+CD8+IFN- c+) cells. The cells were gated in lymphocyte region according to their SSC and FSC characteristics. Data are expressed as mean ± SEM. Statistics: ANOVA test, *p < 0.05.
Fig. 2. IL-2 production by PBMC from PCM-infection (PI) individuals and patients with the adult (AF) or juvenile form of PCM (JF) after stimulation with PMA (50 ng/mL) plus ionomycin (250 ng/mL) for 6 h, in the presence of brefeldin A (1 lg/mL). (A) Representative dot-plots of CD3+ or CD8+ cells expressing IL-2. Percentages of positive cells in each quadrant are indicated at the top. (B) Percentages of CD3+IL-2+, CD3+CD8+IL-2+ and (CD3+IL2+) minus (CD3+CD8+IL-2+) cells. The cells were gated in lymphocyte region according to their SSC and FSC characteristics. Data are expressed as mean ± SEM. Statistics: ANOVA test, *p < 0.05.
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Fig. 3. TNF-a production by PBMC from PCM-infection (PI) individuals and patients with the adult (AF) or juvenile form of PCM (JF) after stimulation with PMA (50 ng/mL) plus ionomycin (250 ng/mL) for 6 h, in the presence of brefeldin A (1 lg/mL). (A) Representative dot-plots of CD3+, CD8+ or CD68+ cells expressing TNF-a. Percentages of positive cells in each quadrant are indicated at the top. (B) Percentages of CD3+TNF-a+, CD3+CD8+TNF- a+ and (CD3+TNF-a+) minus (CD3+CD8+TNF- a+) cells. (C) Percentages of CD68+TNF-a+ unstimulated or stimulated with LPS. The cells were gated in lymphocyte or monocyte region according to their SSC and FSC characteristics. Data are expressed as mean ± SEM. Statistics: ANOVA test, *p < 0.05. Note the different scales.
set of experiments PBMC were cultured in the presence of anti-CD3 plus anti-CD28 monoclonal antibodies, for 24 h, in the presence of brefeldin A for the last 6 h.
According to the IFN-c data, the percentage of CXCL9 and CXCL10/CD3+CD4+ cells was higher in the PI group as compared to both AF and JF patients. In contrast, the
Fig. 4. Expression of chemokines (CXCL9, CXCL10 and CXCL8) and CXCR3 in lymphocytes (CD3+ cells) or monocytes (CD68+ cells) from PCMinfection (PI) individuals and patients with the adult (AF) or juvenile form of PCM (JF). Percentage of CD3+ or CD68+ cells expressing CXCL9 (A) or CXCL10 (B) after stimulation with anti-CD3 plus anti-CD28 monoclonal antibodies (1 lg/mL of each for 24 h), in the presence of brefeldin A (1 lg/mL) for the last 6 h. (C) Expression of the chemokine receptor CXCR3 in lymphocytes (CD3+) unstimulated or stimulated with anti-CD3 plus anti-CD28 for 24 h. (D) Percentages of CXCL8 positive monocytes (CD68+ cells) unstimulated or stimulated with LPS for 6 h. The cells were gated in lymphocyte or monocyte region according to their SSC and FSC characteristics. Data are expressed as mean ± SEM. Statistics: ANOVA test, *p < 0.05. Note the different scales.
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percentage of CXCL9 and CXCL10 positive monocytes was significantly higher in the PI and AF groups than in JF patients (Fig. 4A and B). CXCL9 and CXCL10 share the same receptor, CXCR3, expressed predominantly in Th1 lymphocytes. Higher amounts of CXCR3+CD3+ positive cells were detected in PI individuals in both unstimulated or anti-CD3 plus anti-CD28 stimulated cells (Fig. 4C), in agreement with chemokine production. In a different way, the analysis of CXCL8 expression in monocytes showed an elevated number of positive cells among AF patients in relation to PI and JF groups (Fig. 4D). 3.3. Elevated number of IL-12 expressing monocytes is detected in AF patients, while IL-10 is predominantly expressed in patients’ cells when compared to PI group Looking for cytokines involved in T cell differentiation we next examined the expression of IL-4 and IL-12 among the groups. Unfortunately we were not able to detect intracellular IL-4 by flow cytometry. However, the analysis of IL-12 producing cells showed a significantly elevated
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number of positive cells among AF patients, in accordance with previous data from our group [5]. IL-12 was detected in both unstimulated and LPS stimulated CD68+ cells (Fig. 5A). Considering the lack of reactivity for IL-4, we then decided to evaluate IL-10 production, since it also presents a negative control upon the Th1 response [26]. In the present study monocytes expressing IL-10 were mainly detected in patients with PCM (AF and JF) as compared to PI individuals. Moreover, after LPS stimulation it was possible to distinguish between AF and JF patients, with a higher percentage of CD68+IL-10+ cells in the JF group (Fig. 5B). 3.4. Expression of the co-stimulatory molecules distinguishes cells from PI, AF and JF individuals MHC class I and II, in addition to co-stimulatory molecules, are essential to the development of an effective immune response, providing the first and second signals to fully activate T cells. In order to address this aspect of the immune response in human PCM, the expression of HLA-DR (MHC-class II), CD80 and CD86 was assessed in monocytes of PI, AF and JF individuals. CD80 and CD86 were found to be differentially expressed in the cells of the groups analyzed. CD80 expression was higher in monocytes of AF patients in relation to PI individuals when analyzing the unstimulated cells, but after stimulation with LPS for 6 h, there was an upregulation of this molecule in the PI group (Fig. 6A). On the other hand, CD86 was expressed in higher levels in both the AF and JF groups when compared to the PI group in both unstimulated and LPS stimulated monocytes (CD68+ cells) (Fig. 6B). Interestingly, the increased percentage of cells expressing CD80 after stimulation in PI group (0.66 ± 0.20 vs. 20.25 ± 4.17) was related to a significant decrease in CD86 expression in the same cells (84.41 ± 1.19 vs. 71.07 ± 3.58—p < 0.05). This modulation was not observed in the AF and JF patients cells, which presented a slight but not statistically significant decrease in CD86 expression (data not shown). In relation to HLA-DR, in spite of the high expression in monocytes of all groups, a significantly higher percentage of positive cells were detected in the PI group, when compared to AF or JF patients (Fig. 6C). 4. Discussion
Fig. 5. Expression of IL-12 and IL-10 in monocytes (CD68 cells) from PCM-infection (PI) individuals and patients with the adult (AF) or juvenile form of PCM (JF). Percentages of CD68+ cells expressing IL-12 (A) or IL-10 (B) after stimulation with LPS (10 lg/mL) for 24 h, in the presence of brefeldin A (1 lg/mL) in the last 6 h. The cells were gated in monocyte region according to their SSC and FSC characteristics. Data are expressed as mean ± SEM. Statistics: ANOVA test, *p < 0.05. Note the different scales.
In a previous study we showed an early and high IFN-c and TNF-a mRNA expression in cells from PI individuals [6]. The data presented here confirm the higher production of these cytokines in PI individuals after stimuli in comparison with patients with PCM. It was verified that PI individuals are able to produce high amounts of Th1 cytokines, like IFN-c, following stimulation. IFN-c is the main macrophage-activating cytokine, which initiates and drives the Th1 cell response and directs the assembly of tissue granuloma in addition to TNF-a [27]. Once these
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Fig. 6. Expression of CD80 (B7.1), CD86 (B7.2) and HLA-DR in monocytes (CD68+ cells) from PCM-infection (PI) individuals and patients with the adult (AF) or juvenile form of PCM (JF). Percentages of CD68+ cells expressing CD80 (A), CD86 (B) and HLA-DR (C) in cells unstimulated or after stimulation with LPS (10 lg/mL) for 24 h. The cells were gated in monocyte region according to their SSC and FSC characteristics. Data are expressed as mean ± SEM. Statistics: ANOVA test, *p < 0.05. Note the different scales.
cytokines are important to the activation of the oxidative metabolism of macrophages and consequently to the killing of the P. brasiliensis [10–12], the prompt and high IFN-c and TNF-a production may explain the resistance of PI individual. Moreover the analysis of the expression of surface markers showed that IFN-c expressing cells were mainly CD3+CD8+. The importance of the CD8 subset in
response to P. brasiliensis infection was recently shown in the murine model of PCM, in which depletion of CD8+ T lymphocytes resulted in the enhancement of disease severity and dissemination in both susceptible and resistant mice [28]. In addition, patients with pulmonary PCM showed large quantities of CD8+ T cells in bronchoalveolar lavage (BAL), which positively correlated with the production of CCL3 [29], a chemokine known to promote chemotaxis of lymphocytes, selectively recruiting CD8+ T cells [30,31]. CD8+ T cells mediated immune responses are based on cytokine production, mainly IFN-c, as showed here, or cytotoxicity [32]. In PCM, TNF-a has also been implicated in the formation of compact granuloma, which accounts for the resistance to infection [12,27]. In the present study, we showed that TNF-a was expressed at different levels according to the cell type analyzed. CD3+TNF-a+ cells were predominant in the PI group, while in CD68+ cells (monocytes) TNF-a expression was similar in AF patients and in PI individuals in both unstimulated or LPS stimulated cells. TNF-a is a cytokine associated with resistance to several infectious diseases. In the experimental model of histoplasmosis the neutralization of TNF-a results in the increase of fungal dissemination and mortality [33]. Herring et al. [34] observed that the neutralization of TNF-a in the initial phase of the infection with Cryptococcus neoformans diminished IL-12 production and hence IFN-c secretion and, in addition, increased the number of eosinophils in peripheral blood. In a recent study we observed that TNF-a mRNA was earlier and higher expressed in cells of PI individuals as compared to JF and AF patients [6]. The elevated production of TNF-a may be important to increase the Th1 response in PI individuals, while the low TNF-a levels exhibited by JF patients probably contribute to the high susceptibility to infection and may be related to eosinophilia observed in this group [4,5,7]. Besides IFN-c and TNF-a production, cells from the PI individuals showed a higher expression of CD4+IL-2+ cells, when compared to patients with both clinical forms of PCM. IL-2 is produced by lymphocytes, mainly Th1 cells with an essential role in the expansion and survival of specific lymphocytes [35,36], as well as, a crucial participation in DTH responses. In a previous paper we reported that PI individuals presented an intense proliferative response against P. brasiliensis antigen correlated with DTH responses [5], so the high production of IL-2 by PI lymphocytes may explain these data. Chemokine and chemokine receptors, in addition to IFN-c and TNF-a, play an important role in granulomatous inflammatory reactions. The higher expression of chemokine CXCL10 and CXCL9 agrees with the detection of IFN-c positive cells in PI individuals. Moreover the production of such chemokines presented a differential pattern of expression depending on the cell type. CD3+ cells from PI group presented higher expression of
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CXCL9 and CXCL10 than AF or JF groups, while the expression of these chemokines were similar and elevated in monocytes of AF and PI groups. CXCL10 and CXCL9 are IFN-c inducible and very effectively attract activated T lymphocytes [25]. Both chemokines signal through a common receptor, CXCR3, expressed by memory (CD45RO+) T cells, preferentially of the Th1 subset, and by natural killer cells, but not by monocytes and neutrophils [25,37–40]. Accordingly, CXCR3 was expressed in higher levels in T cells from the PI group than in JF or AF patients. Souto et al. [41] showed that IFN-c modulates the expression of chemokine and chemokine receptors and determines the cells that infiltrate the lungs of mice infected with P. brasiliensis. In this model, mice presented high levels of CCL5, CCL2, CXCL10 and CXCL9, simultaneously with mononuclear cell infiltration in the lungs. The predominance of such chemokines and the expression of their receptor could contribute to the preferential mobilization of Th1 cells in PI individuals, leading to a more effective response against P. brasiliensis infection. In this study we showed that unstimulated CD68+ cells from AF patients express higher levels of CXCL8 expression as compared to the other groups. CXCL8 contains a conserved tripeptide motif glutamic acid–leucine–arginine (ELR) at the N-terminal of the protein, which enables it to attract neutrophils [42,43]. Neutrophils participate in the inflammatory response to P. brasiliensis through the release of oxygen-derived products and endogenous peroxidase located in cytoplasmic granules, killing both ingested and extracellular yeast cells [44,45]. Another study of our group also showed higher levels of CXCL8 in sera of patients with the AF as compared to the JF of PCM [4]. Furthermore, neutrophilic abscesses are a common finding in tissues infected with P. brasiliensis [46]. Elevated production of CXCL9, CXCL10 and CCL8, as well as TNF-a, by monocytes of AF group could contribute to contain fungal dissemination but, as describe for the murine model of PCM, a strong activation of innate immunity can trigger mechanisms that suppress cell-mediated immune response. It is well known that, in terms of clinical presentation, the AF of PCM is very heterogeneous, ranging from isolated lesions in the respiratory tract to widely disseminated forms, what suggests a gradation of the immune response. In our study the majority of AF patients presented the multifocal form of the disease and about 90% showed pulmonary involvement. As previously described for TNF-a and NO [47], the concentration of IL-12 produced during P. brasiliensis infection might be crucial for the development of resistance or susceptibility. In this context, our finding of high numbers of IL-12+ monocytes in AF patients may account for an initial effective immune response, followed by an exacerbated inflammatory reaction. Alveolar macrophages from patients with pulmonary PCM express high levels of adhesion and co-stimulatory molecules
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and produce large amounts of IL-12, in addition to IL6, TNF-a and MIP-1a [29]. Moreover, early treatment with recombinant IL-12 resulted in a less disseminated disease in mice infected with P. brasiliensis, but induced an increased pulmonary mononuclear cell inflammation [13]. Recent studies had demonstrated that susceptible mice respond to the P. brasiliensis infection with an initial exacerbated innate inflammatory response in the lungs, producing high amounts of pro-inflammatory cytokines as IL-12 and high NO expression, whereas resistant mice present a more controlled response, with mild production of IL-10 and TGF-b1, and low levels of pro-inflammatory cytokines [48,49]. Considering these results, it was proposed that an exacerbated innate immune response at the early phase of the disease might lead to an ineffective T cell response in the later phases of the infection, which contributes to the dissemination of the disease. Similarly to that, it was demonstrated that the overexpression of IL-12, in lungs of mice infected with Mycobacterium tuberculosis, diminished the capacity of migration of leukocytes (macrophages, lymphocytes and neutrophils) to this organ, hence leading to an increased susceptibility in later phases of the infection, although that overexpression does not influence the production of IFN-c [50]. Unfortunately we were not able to detect intracellular IL-4, although in previous studies performed by our group a higher expression of IL-4 (mRNA and protein) was found in JF patients as compared to AF and PI group [5,6]. Using different approaches we have shown IL-4 in supernatants of PBMC from JF patients cultured in the presence of P. brasiliensis antigen [5], as well as early expression (6 h) of IL-4 mRNA by JF cells [6]. Here we analyzed the expression of IL-4 by flow cytometry in cells stimulated for 24 h with anti-CD3 plus anti-CD28 mAbs, and a possible explanation for the absence of IL-4 could be the fact that the blocking of protein secretion (Brefeldin A) was made only in the last 6 h of cell culture, when IL-4 could no longer be produced. In experimental models IL-4 production is not clearly linked to susceptibility since antigen stimulated lymph node cells produce IL-10 but not IL-4 during the initial 8 weeks of infection [51]. In addition, the role of IL-4 in P. brasiliensis infection apparently depends on the genetic background of the host, leading to susceptibility in C57BL/6 mice, but being important to the resistance in B.10 lines [52]. In this study a higher number of IL-10 positive monocytes (CD68+ cells) were detected in both, AF and JF patients, whereas only a basal number of cells were found in PI individuals. These results confirm our previous data, in which cells from AF or JF patients stimulated in vitro with P. brasiliensis antigen produced higher levels of IL-10 when compared to PI individuals [5], suggesting that, in addition to IL-4, IL-10 may also contribute to susceptibility in human PCM. IL-10 is an important mediator of the inhibition of nitric oxide (NO) diminishing the expression of the inducible nitric oxide synthase (iNOS), an enzyme induced by IFN-c and TNF-a [53]. Although its
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role in human infection is unknown, it was demonstrated that NO has a fungicidal role in P. brasiliensis infection in mice [27,47]. The expression of surface molecules such as CD80 (B7.1), CD86 (B7.2) and HLA-DR (MHC class II), was also different among the groups. Although the low expression in unstimulated cells, CD80 was upregulated in the cells of the 3 groups analyzed, notably in PI individuals, after LPS stimulation. As mentioned above, the increased percentage of cells expressing CD80 after stimulation in PI group was related to a significant decrease in CD86 expression in the same cells. This fact, not observed in the other groups, may reflect a particular manner of costimulation, differently influencing the immune response. Several studies have shown that the co-stimulation of cells through CD80 or CD86 might be important to the development of a Th1 or a Th2 response [15,54]. The expression of CD80 has been associated with a predominant Th1 response with the activation of CD8+ cells [55,56], while co-stimulation through CD86 would be involved in the production of IL-4 and, consequently, in the Th2 response [56–58]. The expression of HLA-DR in cells of PI individuals was also significantly higher in both unstimulated and LPSstimulated cells when compared to the JF group. Th2 cytokines, such as IL-4 and IL-10, induce the downregulation of CD86, while IFN-c increases the expression of CD80 and CD86, as well as HLA-DR. Since the differentiation of T cells is dependent, among other factors, on the cytokine present in the milieu where the immune response takes place, the co-stimulatory molecules involved in the antigen presentation and the chemokines implicated in differential homing of cells, these data support the implication of a Th1 immune response in the resistance profile exhibited by PI individuals. In summary, the present study confirmed our previous findings, which associated individuals with PCM infection (PI) with the Th1 pole of the immune response characterized by an elevated number of T cells expressing IFN-c, IL-2, TNF-a, CXCL9/CXCL10/CCR3 and low number of IL-10 positive cells. Although the intracellular expression of immune mediators have not demonstrated a clear Th2 polarization in the JF of the disease, low production of pro-inflammatory cytokines and chemokines and high number of IL-10+ cells, in addition to low expression of co-stimulatory and HLA-DR molecules characterized this group of patients. On the other hand, a high number of monocytes expressing IL-12 and TNF-a and chemokines such as CXCL9, CXCL10 and CXCL8 probably enables AF patients to better control the fungal dissemination, resulting in moderate forms of the disease. However, in analogy to the animal model of PCM, we may suppose that a misbalance favoring a strong pro-inflammatory response rather than a protective one is detrimental to the development of an effective cellular immune response, which leads to severe forms of the disease.
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