Paracoccidioides brasilinsis-Induced Migration of Dendritic Cells and Subsequent T-Cell Activation in the Lung-Draining Lymph Nodes

Paracoccidioidomycosis is a mycotic disease caused by a dimorphic fungus, Paracoccidioides brasiliensis (Pb), that starts with inhalation of the fungus; thus, lung cells such as DC are part of the first line of defense against this microorganism. Migration of DC to the lymph nodes is the first step in initiating T cell responses. The mechanisms involved in resistance to Pb infection are poorly understood, but it is likely that DC play a pivotal role in the induction of effector T cells that control Pb infection. In this study, we showed that after Pb Infection, an important modification of lung DC receptor expression occurred. We observed an increased expression of CCR7 and CD103 on lung DC after infection, as well as MHC-II. After Pb infection, bone marrow-derived DC as well lung DC, migrate to lymph nodes. Migration of lung DC could represent an important mechanism of pathogenesis during PCM infection. In resume our data showed that Pb induced DC migration. Furthermore, we demonstrated that bone marrow-derived DC stimulated by Pb migrate to the lymph nodes and activate a T helper (Th) response. To the best of our knowledge, this is the first reported data showing that Pb induces migration of DC and activate a T helper (Th) response.


Introduction
Paracoccidioidomycosis (PCM) is a mycotic disease caused by a dimorphic fungus, Paracoccidioides brasiliensis (Pb), which initiates a deep mycosis that primarily attacks lung tissue. PCM is the most prevalent deep mycosis in Latin America. Epidemiological and experimental evidence suggests that natural infection is initiated after inhalation of the conidia produced by the mycelial form of the fungus [1]. Clinical forms of PCM vary from a localized and benign disease to a progressive and potentially lethal systemic infection [2]. The broad spectrum of clinical forms is a result of the influence of several factors on disease severity including host response and virulence of the infecting agent [3]. The acute or severe form of the disease is associated with deficient cell immunity, high antibodies (Abs) levels, and preferential secretion of type 2 cytokines [4].
Dendritic cells (DC) are antigen-presenting cells that act as sentinels in peripheral tissues, constantly sampling the antigens in their environment. Lung DC play a pivotal role in infections caused by airborne pathogens such as Mycobacterium tuberculosis, Aspergillus fumigatus and Cryptococcus neoformans [5] [6][7] [8]. The DC population lining the lungs is key to the initiation of T cell responses after pulmonary challenge [9]. DC remain quiescent until activated, at which point they migrate to the draining lymph nodes (DLN) [10], present antigen, and initiate T cell activation [11].
The mechanisms involved in resistance to Pb infection are poorly understood, but it is likely that DC play a pivotal role in the induction of effector T cells that control Pb infection. For example, the production of IFN-c and IL-10, both effector T cell products, appears to be involved in the resolution and dissemination of PCM in both mice and humans [12] [13].
Because DC are the most effective antigen presenting cells (APC) for inducing cell-mediated immune responses, it is important to investigate lung DC and their potential for lymph node migration and immune response initiation. In this study, we analyzed the role that DC played in modulating Pb infection. To the best of our knowledge, this is the first reported data showing that Pb induces migration of DC. Furthermore, we demonstrated that bone marrow-derived DC stimulated by Pb migrate to the lymph nodes and activate a T-cell response.

Ethics Statement
The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Sao Paulo in 02/2009 (Permit Number: 154).

Animals
For this study, we used 8 to 12-week-old BALB/c female mice obtained from the specific pathogen free facility of the University of Sao Paulo and kept in the animal room, with constant temperature and humidity with cycle of dark/light, conformed to institutional guidelines for animal care and welfare.

P. brasiliensis strains
The yeast form of the highly virulent P. brasiliensis strain 18 was grown in Sabouraud-agar. The strain used was recovered from animals before the experiments. A suspension of fungi was prepered with sterile PBS and yeast cells were adjusted to 1610 5 cells in 50 ml, based on hemocytometer counts. Viability was determined with Janus Green B vital dye (Merck) and was always higher than 90%.

Mouse infection
Mice were challenged with an intratracheal inoculation of 1610 5 yeast cells of the virulent Pb strain 18. After 12 or 24 hrs, the lungs and lymph nodes were removed prior to DC analysis.

Analysis of lung and lymph node DC phenotype after Pb infection
The effects of Pb on surface molecule expression in lung DC were investigated after intra-tracheal (i.t.) infection at 12 and 24 h. Cells were isolated from lungs as previously described [14]. After perfusing the pulmonary vasculature with 5 ml of PBS containing 100 U/ml heparin, the lungs were minced and incubated for 90 min at 37uC in digestion buffer containing 0.7 mg/ml collagenase IV (Sigma-Aldrich-St. Louis-USA) and 30 mg/ml type IV bovine pancreatic DNase I (Sigma-Aldrich-St. Louis-USA). Large particulate matter was removed by passing the cell suspension through a small loose nylon wool plug. The mediastinal and axillary lymph nodes were removed and disrupted, and the cells were analyzed. DC phenotype was determined by flow cytometry using a FACSCantoII (Becton Dickinson). In order to determine the expression of class II MHC co-stimulatory and adhesion molecules, we used labeled Mabs against mouse PE-CD11c (HL3) PECy5-CD11c (HL3), FITC-CD11c (HL3), PE-MHC-II (M5-114.15.2), PE-CD80 (16-10A1), FITC-CD86 (GL1), PE-CCR7 (4B12), APC-DC-SIGN (LWC06), and APC-CD103 (2E7) (All antibodies were obtained from BD Biosciences, San Jose, CA). FlowJo was used for analysis of flow cytometry data. To distinguish autofluorescent cells from cells expressing low levels of individual surface markers, we established upper thresholds for autofluorescence by staining samples with fluorescence-minus-one (FMO) control stain sets [15]. In these sets, a reagent for a channel of interest is omitted.
Bone marrow-derived DCs were generated according to described methods [16]. Femurs and tibias were flushed with 3-5 ml of PBS in 1% BSA. Bone marrow cells were differentiated into DC by culturing in RPMI supplemented with 10% Fetal Calf Serum (FCS), 10 mg/ml gentamicin, and recombinant cytokine GM-CSF (50 ng/ml) for 7 days. On days 3 and 5, the nonadherent cells (granulocytes and lymphocytes) were removed, and the media was replaced with fresh media and growth factor. On day 7, the nonadherent cells were removed and analyzed by FACS using DC cell surface markers. The bone marrow-derived DCs expressed MHC class II, CD80, CD40, CD11b and CD11c (data not shown).

Analysis of in vivo DC migration
In order to analyze DC migration capability following Pb interaction, we first used Bone-marrow derived dendritic cell (BM-DC) marked with CFSE. BM-DCs were labeled for 20 min at 37uC with 5 mM carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR). Following incubation, labeling was stopped by the addition of RPMI/10% FBS, and the cells were washed 3 times with RPMI at room temperature. To reduce the amount of unbound CFSE in cell suspensions, the cells were incubated at 37uC for 5 min after the second wash and prior to the third wash. The CFSE-labeled DCs were incubated with Pb (1:1) for 4 hrs and injected intratracheally (1610 5 DC in 50 ml PBS). After 12 and 24 hrs, the mediastinal and axillary lymph nodes were removed. Cells were isolated and analyzed by flow cytometry as described above. As a control, we used CFSE-labeled BM-DC without incubation with Pb.
CD4 T-cell activation after Pb-pulsed DC administration To determine the type of T-cell activation occurring after administration of Pb-pulsed DC, BM-DC (1610 5 ) were incubated with Pb (1:1) for 4 hrs and injected intratracheally. After 5 days, the mediastinal and axillary lymph nodes were removed and disrupted, and the cells were analyzed. After surface staining for CD4, the lymph node cells were fixed, permeabilized, and stained by anti-IL-10, anti-IL-4 and anti-IFN-c Abs (BD). The cells were analyzed by cytometry as described above.

CFSE labeling of migrating DC
To detect migrating lung DC, mice were lightly anesthetized and intratracheally administered 1610 5 Pb diluted in PBS and 50 ml 8 mM CFSE (Fluka, Buchs, Switzerland) diluted in PBS. This method is based on the labeling of intracellular proteins and provides stable labels for at least 8 weeks in non-dividing lymphocytes. This labeling procedure has frequently been used to stain cells in the lungs and has allowed the tracking of respiratory DC migration. After 12 and 24 hrs, the mediastinal and axillary lymph nodes were removed. Cells were isolated and analyzed by flow cytometry as described above. Mice administered only CFSE were used as controls.
DC transport yeast form of Pb to draining lymph nodes Labeling of yeast with FITC. Live yeast were suspended in 0.1 M carbonate buffer (pH 9.3) at 2610 8 /ml and added to 200 ml of FITC in DMSO, as described [17]. After incubation for 2 h at room temperature while being protected from light, the suspensions were diluted and washed twice in PBS (pH 7.2) to remove all detectable free-FITC as determined by fluorescence measurement of the supernatants when compared to PBS alone. The yeast pellets were then counted and diluted in PBS to the desired concentration. FITC labeling did not affect the viability of cells. After labeling, 1610 6 Pb were injected intratracheally. After 12 h, the lymph node cells were removed and analyzed by cytometry as described above. Animals injected with FITC only were used as controls.

Statistics
Statistical comparisons were made by analysis of variance (ANOVA) and by Tukey-Kramer test. All values were reported as the mean +/2 standard error of the means.

Pb infection alters the number and phenotype of lung DCs
To study the role of Pb infection on lung DC, we infected BALB/c mice intratracheally (i.t.) with the yeast form of Pb. At 12 and 24 hrs after infection, we analyzed the number and phenotypes of lung DC by flow cytometry. We showed an increased in the number of MHC-II + /CD11c + cells (DC) after   Figure 1A). In addition, an increase of DC expression of CD103, MHC-II, and CCR7 when compared with the controls (PBS) was observed ( Figure 1B). No significant differences were observed in the expression of CD80, CD86 and DC-SIGN (data not shown).
In vivo DC Migration to the lung-draining lymph nodes.
Naïve T cell priming occurs in lymph nodes draining from the site of infection. To study the capability of DC to migrate to the lung-draining mediastinal lymph nodes, we labeled BM-DC with CFSE, incubated with Pb and administered the solution intratracheally to mice. Our results showed an increase of CD11+/CFSE+ in the lymph nodes 12 hrs after administration of fungus, but after 24 hrs the number decreased to control levels ( Figure 2B). Only CD11+CFSE+ cells incubated with Pb were found in the lymph nodes ( Figure 2A). These cells also expressed mature phenotypic markers (MHC-II high ). These results demonstrate that BM-DC, in the presence of Pb, migrate to draining lymph nodes.
In addition, we analyzed the capacity of lung DC to migrate to the lung-draining mediastinal lymph nodes. Mice were injected (i.t.) with CFSE and Pb. This procedure enabled us to track DC migrating from the lungs to the mediastinal lymph nodes during Pb infection. In addition, flow cytometry allowed for the separation of migrating DC and resident DC taken from the lymph nodes. CFSE treatment alone did not induce significant DC migration as demonstrated by the minimal number of CFSE-labeled cells in the mediastinal lymph nodes after infection. After intratracheal infection with Pb, the CFSE-labeled cells observed in the mediastinal lymph nodes were predominantly CD11c cells ( Figure 3A). It was observed CFSE/CD11c+ cell at 12 hrs after Pb infection, and after 24 hrs these cells decreased to control levels ( Figure 3B). These results show that lung DC have the capability to migrate to the draining lymph nodes in the presence of Pb.

DC transport yeast of Pb from the airways to the thoracic lymph nodes
Recently, our group demonstrated that lung DC phagocytose Pb yeast in vivo [18]. Moreover, in this study, we have shown that lung DC migrate to the lymph nodes. Here, we address the question of whether pulmonary DC, after phagocytosis of the fungus, transport Pb yeast from the airways to the lymph nodes. To this end, FITC-labeled yeast cells were i.t. injected, and the FITCpositive DC were enumerated in lymph nodes by FACS analysis. Our results showed that CD11c + FITC + cells appeared in the thoracic lymph nodes 12 hrs after infection. These results indicate that pulmonary DC transport Pb yeast to the draining lymph nodes (Figure 4).

Lymph node T-CD4 cell activation by DC
As demonstrated above, DC phagocytose the yeast form of Pb and migrate to the lymph nodes. Given these results, DC were considered likely to contribute to the induction of T cell responses.  To investigate which CD4 T cell subtypes could be activated by DC, animals were injected with Pb or Pb-pulsed BM-DC, and intracellular cytokines from T cells present in the lymph nodes were investigated. Our results showed that animals infected with Pb induced a mixed pattern of CD4 T cell cytokines, compatible with a Th1/Th2 response. However, when Pb-pulsed DC was injected, a Th2 response was observed in the draining lymph node ( Figure 5).

Discussion
PCM starts with inhalation of the fungus; thus, lung cells such as DC are part of the first line of defense against this microorganism. Migration of DC to the lymph nodes is the first step in initiating T cell responses. DC are highly efficient antigenpresenting cells that are central to the induction and regulation of most adaptive immune responses. Their specialized capabilities of acquiring, processing, retaining, and finally presenting peptides on major histocompatibility complex (MHC) molecules are critical properties that account in part for their major role in antigen presentation [19]. Unlike other antigen-presenting cells, DCs are specialized for homing efficiently to the T cell zones of lymphoid organs, allowing for optimal interactions with T lymphocytes. DC migratory capacity distinguishes them from macrophages. Therefore, we postulated that DC, after interaction with Pb, could migrate to the draining lymph nodes and activate CD4 T-cells.
In this study, we showed that after Pb Infection, an important modification of DC receptor expression occurred. We observed an increased expression of CCR7 and CD103 on lung DC after infection, as well as MHC-II. Several studies have shown that pathogen recognition through PRRs activates DC leading to an increase in DC expression of the chemokine receptors CCR7 and CD103 [20][21] [11]. The increased expression enables DC to migrate from the site of infection to the secondary lymphoid tissues. Therefore, these results could indicate that DC are capable of migration after interacting with Pb. Next, we infected mice with Pb-pulsed BM-DC. Our results showed that DC pulsed with Pb migrate to the lymph nodes. CFSE+ DC migrate to the lymph nodes, therein coming into contact with naive T cells [11], and this interaction results in the proliferation of naive T cells. Our results showed that Pb-pulsed BM-DC induce a Th2-like response when compared to infection with only Pb. These results demonstrate the low capability of BM-DC to induce a Th1 response in the presence of Pb. Recently, our group showed that BM-DC, in the presence of Pb, decreased production of IL-12, a cytokine important for the induction of Th1 [22].
However, it is known that BM-DC and lung DC have differences in response, so it was necessary to test the in vivo capacity of lung DC to migrate to lymph nodes following Pb infection. Recently, we showed that lung DC can phagocytose the yeast form of Pb in vivo. The results of this study showed that lung DC migrate and transport the yeast form of Pb to lymph nodes. Migration of lung DC could represent an important mechanism of pathogenesis during PCM infection. The lung DC could act as Trojan horses for Pb and, depending on the circumstances, influence disease susceptibility. However, this hypothesis requires more study. In other model of fungal infection it was demostrated that interactions between DC and Crytococcus neoformans alter DC antigen-presenting functions and modulate resultant T-cell responses in vitro [23]. Moreover, it was shown that after cryptococcal infection in vivo, DC migrate to thoracic lymph nodes and active T-cell to produce cytokine [24] [25]. Conversely, some pathogens are known to use mechanisms to arrest DC migration to protect themselves from the generation of potent adaptive immune responses. Schistosomal parasites produce prostaglandin D2 to inhibit DC migration to lymph nodes after infection through skin [26], and microbial antigens derived from Borrelia garinii, the causative agent of chronic Lyme disease, significantly downregulate CD38 and CCR7 expression in DC, thereby hampering their migratory capability [27]. Elucidating the mechanisms of regulation and coordination of DC migration by pathogen-derived signals requires more study. An understanding of the signals that regulate DC migration could allow for the development of methods to induce efficient T-cell activation that would aid in the control of PCM infection.