Anti-CD25 Treatment Depletes Treg Cells and Decreases Disease Severity in Susceptible and Resistant Mice Infected with Paracoccidioides brasiliensis

Regulatory T (Treg) cells are fundamental in the control of immunity and excessive tissue pathology. In paracoccidioidomycosis, an endemic mycosis of Latin America, the immunoregulatory mechanisms that control the progressive and regressive forms of this infection are poorly known. Due to its modulatory activity on Treg cells, we investigated the effects of anti-CD25 treatment over the course of pulmonary infection in resistant (A/J) and susceptible (B10.A) mice infected with Paracoccidioides brasiliensis. We verified that the resistant A/J mice developed higher numbers and more potent Treg cells than susceptible B10.A mice. Compared to B10.A cells, the CD4(+)CD25(+)Foxp3(+) Treg cells of A/J mice expressed higher levels of CD25, CTLA4, GITR, Foxp3, LAP and intracellular IL-10 and TGF-beta. In both resistant and susceptible mice, anti-CD25 treatment decreased the CD4(+)CD25(+)Foxp3(+) Treg cell number, impaired indoleamine 2,3-dioxygenase expression and resulted in decreased fungal loads in the lungs, liver and spleen. In A/J mice, anti-CD25 treatment led to an early increase in T cell immunity, demonstrated by the augmented influx of activated CD4(+) and CD8(+) T cells, macrophages and dendritic cells to the lungs. At a later phase, the mild infection was associated with decreased inflammatory reactions and increased Th1/Th2/Th17 cytokine production. In B10.A mice, anti-CD25 treatment did not alter the inflammatory reactions but increased the fungicidal mechanisms and late secretion of Th1/Th2/Th17 cytokines. Importantly, in both mouse strains, the early depletion of CD25(+) cells resulted in less severe tissue pathology and abolished the enhanced mortality observed in susceptible mice. In conclusion, this study is the first to demonstrate that anti-CD25 treatment is beneficial to the progressive and regressive forms of paracoccidioidomycosis, potentially due to the anti-CD25-mediated reduction of Treg cells, as these cells have suppressive effects on the early T cell response in resistant mice and the clearance mechanisms of fungal cells in susceptible mice.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Pesquisas (CNPq)Conselho Nacional de Pesquisas (CNPq

TLR-4 Cooperates with Dectin-1 and Mannose Receptor to Expand Th17 and Tc17 Cells Induced by Paracoccidioides brasiliensis Stimulated Dendritic Cells

The concomitant use of diverse Pattern Recognition Receptors (PRRs) by innate immune cells can result in synergistic or inhibitory activities that profoundly influence anti-microbial immunity. Dectin-1 and the Mannose Receptor (MR) are C-type Lectin Receptors (CLRs) previously reported to cooperate with Toll Like Receptors (TLRs) signaling in the initial inflammatory response and in the induction of adaptive Th17 and Tc17 immunity mediated by CD4+ and CD8+ T cells, respectively. The protective immunity against paracoccidioidomycosis, the most prevalent fungal infection of Latin America, was previously shown to be influenced by these T cell subsets motivating us to study the contribution of TLRs, Dectin-1 and MR to the development of Th17/Tc17 immunity. First, curdlan a specific Dectin-1 agonist was used to characterize the influence of this receptor in the proliferative response and Th17/Tc17 differentiation of naïve lymphocytes induced by Paracoccidioides brasiliensis activated dendritic cells (DCs) from C57BL/6 mice. Then, WT, Dectin-1-/-, TLR-2-/- and TLR-4-/- DCs treated or untreated with anti-Dectin-1 and anti-MR antibodies were used to investigate the contribution of these receptors in lymphocyte activation and differentiation. We verified that curdlan induces an enhanced lymphocyte proliferation and development of IL-17 producing CD4+ and CD8+ T cells. In addition, treatment of WT, TLR-2-/- and TLR-4-/- DCs by anti-Dectin-1 antibodies or antigen presentation by Dectin-1-/- DCs led to decreased lymphoproliferation and impaired Th17 and Tc17 expansion. These responses were also inhibited by anti-MR treatment of DCs, but a synergistic action on Th17/Tc17 differentiation was mediated by TLR-4 and MR. Taken together, our results indicate that diverse TLRs and CLRs are involved in the induction of lymphocyte proliferation and Th17/Tc17 differentiation mediated by P. brasiliensis activated DCs, but a synergist action was restricted to Dectin-1, TLR-4 and MR

Expression of Dectin-1 and Enhanced Activation of NALP3 Inflammasome Are Associated with Resistance to Paracoccidioidomycosis

Dectin-1 is a pattern recognition receptor (PRR) that recognizes β-glucans and plays a major role in the immunity against fungal pathogens. Paracoccidioides brasiliensis, the causative agent of paracoccidioidomycosis, has a sugar-rich cell wall mainly composed of mannans and glucans. To investigate the role of dectin-1 in the innate immunity of resistant (A/J) and susceptible (B10.A) mice to P. brasiliensis infection, we evaluated the role of curdlan (a dectin-1 agonist) and laminarin (a dectin-1 antagonist) in the activation of macrophages from both mouse strains. We verified that curdlan has a negligible role in the activation of B10.A macrophages but enhances the phagocytic and fungicidal abilities of A/J macrophages. Curdlan up-regulated the expression of costimulatory molecules and PRRs in A/J macrophages that express elevated levels of dectin-1, but not in B10.A cells. In addition, curdlan treatment inhibited arginase-1 and enhanced NO-synthase mRNA expression in infected A/J macrophages but had not effect in B10.A cells. In contrast, laminarin reinforced the respective M2/M1 profiles of infected A/J and B10.A macrophages. Following curdlan treatment, A/J macrophages showed significantly higher Syk kinase phosphorylation and expression of intracellular pro-IL-1β . These findings led us to investigate if the NRLP3 inflammasome was differently activated in A/J and B10.A cells. Indeed, compared with B10.A cells A/J macrophages showed an increased expression of NALP3, ASC and IL-1β mRNA. They also showed elevated caspase-1 activity and secreted high levels of mature IL-β and IL-18 after curdlan treatment and P. brasiliensis infection. Our data demonstrate that soluble and particulate β-glucans exert opposed modulatory activities on macrophages of diverse genetic patterns. Moreover, the synergistic action of dectin-1 and NALP3 inflammasome were for the first time associated with the innate response of resistant hosts to P. brasiliensis infection

Loss- and Gain-of-Function Approaches Indicate a Dual Role Exerted by Regulatory T Cells in Pulmonary Paracoccidioidomycosis.

Paracoccidioidomycosis (PCM), is a pulmonary fungal disease whose severity depends on the adequate development of T cell immunity. Although regulatory T (Treg) cells were shown to control immunity against PCM, deleterious or protective effects were described in different experimental settings. To clarify the function of Treg cells in pulmonary PCM, loss-and gain-of-function approaches were performed with Foxp3GFP knock-in mice and immunodeficient Rag1-/- mice, respectively, which were intratracheally infected with 106 yeast cells. The activity of Foxp3-expressing Treg cells in pulmonary PCM was determined in Foxp3GFP transgenic mice. First, it was verified that natural Treg cells migrate to the lungs of infected mice, where they become activated. Depletion of Treg cells led to reduced fungal load, diminished pathogen dissemination and increased Th1/Th2/Th17 immunity. Further, adoptive transfer of diverse T cell subsets to Rag1-/- mice subsequently infected by the pulmonary route demonstrated that isolated CD4+Foxp3+ Treg cells were able to confer some degree of immunoprotection and that CD4+Foxp3- T cells alone reduced fungal growth and enhanced T cell immunity, but induced vigorous inflammatory reactions in the lungs. Nevertheless, transfer of Treg cells combined with CD4+Foxp3- T cells generated more efficient and balanced immune Th1/Th2/Th17 responses able to limit pathogen growth and excessive tissue inflammation, leading to regressive disease and increased survival rates. Altogether, these loss- and gain-of-function approaches allow us to clearly demonstrate the dual role of Treg cells in pulmonary PCM, their deleterious effects by impairing T cell immunity and pathogen eradication, and their protective role by suppressing exacerbated tissue inflammation

Indoleamine 2,3-dioxygenase controls fungal loads and immunity in Paracoccidioidomicosis but is more important to susceptible than resistant hosts.

Paracoccidioidomycosis, a primary fungal infection restricted to Latin America, is acquired by inhalation of fungal particles. The immunoregulatory mechanisms that control the severe and mild forms of paracoccidioidomycosis are still unclear. Indoleamine 2,3-dioxygenase (IDO), an IFN-γ induced enzyme that catalyzes tryptophan metabolism, can control host-pathogen interaction by inhibiting pathogen growth, T cell immunity and tissue inflammation.In this study, we investigated the role of IDO in pulmonary paracoccidioidomycosis of susceptible and resistant mice. IDO was blocked by 1-methyl-dl-tryptophan (1MT), and fungal infection studied in vitro and in vivo. Paracoccidioides brasiliensis infection was more severe in 1MT treated than untreated macrophages of resistant and susceptible mice, concurrently with decreased production of kynurenines and IDO mRNA. Similar results were observed in the pulmonary infection. Independent of the host genetic pattern, IDO inhibition reduced fungal clearance but enhanced T cell immunity. The early IDO inhibition resulted in increased differentiation of dendritic and Th17 cells, accompanied by reduced responses of Th1 and Treg cells. Despite these equivalent biological effects, only in susceptible mice the temporary IDO blockade caused sustained fungal growth, increased tissue pathology and mortality rates. In contrast, resistant mice were able to recover the transitory IDO blockade by the late control of fungal burdens without enhanced tissue pathology.Our studies demonstrate for the first time that in pulmonary paracoccidioidomycosis, IDO is an important immunoregulatory enzyme that promotes fungal clearance and inhibits T cell immunity and inflammation, with prominent importance to susceptible hosts. In fact, only in the susceptible background IDO inhibition resulted in uncontrolled tissue pathology and mortality rates. Our findings open new perspectives to understand the immunopathology of paracoccidioidomycosis, and suggest that an insufficient IDO activity could be associated with the severe cases of human PCM characterized by inefficient fungal clearance and excessive inflammation

Anti-CD25 treatment increases the influx of inflammatory cells to the lungs of resistant but not susceptible mice to <i>P. brasiliensis</i> infection.

<p>Anti-CD25-treated and untreated A/J and B10.A mice were inoculated i.t. with 1×10⁶<i>P. brasiliensis</i> yeast cells. At weeks 2 and 10 of infection lungs of both mouse strains (n = 6) were excised, minced, and digested enzymatically. Lung cells suspensions were obtained, counted, stained for CD3 (T cells), CD19 (B cells) and GR1 (myeloid cells, including neutrophils and monocytes) by flow cytometry. Anti-CD25 treatment significantly alters the number (A) but not the frequency of inflammatory cells in the lungs (B) of infected mice. At week 2, anti-CD25-treated A/J mice showed increased influx of T cells, B cells and myeloid cells, whereas at week 10 these populations appeared in decreased numbers (C, D). In B10.A mice only GR1⁺ cells appeared in decreased numbers at week 10 of infection (C, D). The data represent the mean ± SEM of the results from 6–8 mice per group and are representative of two independent experiments. * (<i>P</i><0.05), ** (<i>P</i><0.01), and *** (<i>P</i><0.001) compared with IgG-treated mice.</p

Treg cells from resistant mice have a higher suppressive potency than those of susceptible mice.

<p>CFSE-labeled responder CD4⁺CD25⁻ T cells from naïve mice were stimulated by irradiated naïve APCs plus anti-CD3 antibodies and cultured in the presence or absence of several ratios of CD25⁺ T cells obtained from lungs of resistant and susceptible mice at weeks 2 (A) and 10 (B) after infection with 1×10⁶<i>P.brasiliensis</i> yeasts. Cells were cultured for 5 days and the proliferative response of CFSE-labeled cells was measured by flow cytometry. The proliferation index (PI) was calculated as describe in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051071#s2" target="_blank">materials and methods</a> and the percentage of inhibition considered as 100% the PI of APC-stimulated CD4⁺CD25⁻ responder cells in the absence of CD4⁺CD25⁺ cells. The data represent the mean ± SEM of the results from 6 mice per group and are representative of two independent experiments. * (<i>P</i><0.05), ** (<i>P</i><0.01), and *** (<i>P</i><0.001) compared with A/J mice.</p

Depletion of CD25⁺ cells diminishes the inflammatory reactions in the liver of A/J mice and abolishes the hepatic fungal lesions of B10.A mice.

<p>Characterization of leukocyte subsets and activation profile of cells by flow cytometry in the liver infiltrating leucocytes (LIL) from anti-CD25-treated and untreated A/J and B10.A mice inoculated i.t. with 1×10⁶<i>P. brasiliensis</i> yeast cells. At week 10 after infection liver cell suspensions were obtained and stained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051071#s2" target="_blank">Materials and Methods</a>. The acquisition and analysis gates were restricted to macrophages or lymphocytes. A, CD4⁺ T cells; B, CD8⁺ T cells; C- Activated/Treg CD4⁺ T cells. D- Liver macrophages. E- CD4⁺CD25⁺Foxp3⁺ Treg cells. The data represent the mean ± SEM of the results from 5–6 mice per group and are representative of two experiments. * (<i>P</i><0.05), ** (<i>P</i><0.01), and *** (<i>P</i><0.001) compared with IgG controls or B10.A strain. F- Histopathology of liver. Anti-CD25 treated and untreated A/J mice did not show hepatic lesions at week 10 of infection (data not shown). In contrast, control B10.A mice presented extensive hepatic lesions containing large numbers of fungal cells (F, a,b). Anti-CD25 treatment practically abolished the inflammatory lesions (F, c,d) and the fungal loads of B10.A mice. a, c (HE, X 100); b, d (Groccot X 100).</p

Histopathology of pulmonary lesions of anti-CD25-treated and untreated A/J and B10.A mice at week 10 post-infection with 1×10⁶<i>P. brasiliensis</i> yeasts.

<p>IgG-treated (A, B) and anti-CD25-treated (C, D) A/J mice showed equivalent diffuse inflammatory reactions characterized by a small number of yeasts in the presence of elevated number of macrophages, lymphocytes and plasma cells; small portions of lung tissue were preserved, with limited signs of inflammatory cell recruitment. IgG-treated B10.A mice (E, F) presented an elevated number of well-defined, confluent, necrotic, granulomas of various sizes (E) containing an elevated number of fungal cells (arrows in F); these lesions occupy a large area of lung tissue (E, F). Compared with control B10.A mice, anti-CD25-treated B10.A mice showed significantly smaller lesions (G) containing a few number of yeasts (H). A, C, E, G, (HE, X 100); B, D, F, H (Groccot X 100). I- Total area of lung lesions of mice (n = 6) at week 10 after infection. ** (<i>P</i><0.01), and *** (<i>P</i><0.001) compared with IgG-treated controls or the susceptible strain.</p