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

Swimming exercise modifies oxidative stress in skeletal and cardiac muscles of diabetic rats

Introduction: Oxidative stress is a key factor leading to the deterioration of diabetes. Oxidative stress exacerbates diabetes and induction of the activity of the antioxidant system may be required to prevent this effect. Objetive: The aim of the present study was to evaluate the redox state in the skeletal and cardiac muscles in a diabetes rat model subjected to swimming exercise for 4 weeks. Methods: Wistar rats were divided into four groups: untrained control (C), trained control (T), untrained alloxan-induced diabetes (D), and trained alloxan-induced diabetes (TD). The redox state of the skeletal and cardiac muscles was assessed by analyzing TBARS, -SH groups, H2O2 production, and SOD and catalase activity. The total number of cardiomyocytes and the total area of collagen fibers in the cardiac muscle were measured by histomorphometry. Results: In the Soleus muscles, the TD group showed increased H2O2 levels and catalase activity compared to the T group, and SOD activity compared to the D group. Regarding the red gastrocnemius, the TD group presented higher SOD and lower catalase activities than the D group. Regarding the cardiac muscle, the TD group presented lower TBARS and higher levels of -SH groups and catalase activity than the D group. Swimming exercise decreased hyperglycemia and reduced pathology, as evidenced by the reduced number of cardiomyocytes and the area of collagen fibers. Conclusion: Swimming exercise in diabetic rats controlled hyperglycemia and oxidative damage, and the reduced fibrosis in the cardiac muscle of diabetic rats

The role of CD28 deficiency, a T cell costimulatory molecule, in pulmonary Paracoccidioidomycosis.

Como a imunoproteção na Paracoccidioidomicose (PCM) é principalmente mediada por células T, investigamos o impacto da deficiência de CD28, molécula co-estimulatória de linfócitos T, na gravidade da infecção primária e secundária pelo Paracoccidioides brasiliensis. Quando comparados a camundongos C57Bl/6 normais (WT), camundongos CD28-deficientes (CD28KO) apresentaram infecção mais grave associada à produção diminuída de anticorpos e citocinas. Além disso, a pré-imunização de animais deficientes e normais resultou em imunoproteção equivalente. Inesperadamente, a sobrevida de animais CD28KO foi significativamente maior que a dos WT, apesar da sua elevada carga fúngica tecidual. Em conclusão, nosso trabalho mostrou que a deficiência de CD28 resulta em PCM mais grave, porém não letal, associada a resposta imune deficiente. Além disso, verificamos que carga fúngica elevada, na ausência da imunidade adaptativa efetora, não ocasiona diminuição de sobrevida, revelando que a resposta imune na PCM pode tanto proteger como ser deletéria aos hospedeiros.As immunoprotection in Paracoccidioidomycosis (PCM) is mainly mediated by T cells, and CD28 is a costimulatory molecule for T lymphocytes, we investigated the influence of CD28 deficiency in primary and secondary PCM. Compared with normal C57BL/6 mice, CD28-deficient (CD28KO) mice developed a more severe infection associated with impaired antibody and cytokine production. In addition, CD28KO and WT mice previously immunized by the s.c. route developed equivalent immunoprotection when challenged by the pulmonary route. Interestingly, CD28KO mice presented increased mean survival time despite their elevated fungal loads in the lungs. In conclusion, our work showed, for the first time, that CD28-deficiency results in more severe, but not overwhelming, PCM. Furthermore, in the absence of effector adaptative immunity, elevated fungal loads do not cause lethal infections, revealing the protective and deleterious effects of immune responses to Paracoccidioides brasiliensis infected hosts

The role of regulatory T cells in the pulmonary paracoccidioidomycosis of susceptible and resistant mice to Paracoccidioides brasiliensis.

Um mecanismo de tolerância periférica estudado é mediado por células T reguladoras (Tregs) que expressa o marcador CD25 e o fator de transcrição FoxP3. Estudamos o papel das Tregs nos fenômenos de imunossupressão na resistência e susceptibilidade genética dos hospedeiros contra o fungo. Animais A/J e B10.A foram depletados de células CD25 e outro grupo recebeu Ig de rato. Na 2ª semana de infecção, a depleção de células CD25 resultou em doença menos grave em ambas as linhagens com intenso afluxo de linfócitos T e macrófagos somente nos animais A/J. Na 10ª semana, a depleção de células CD25 também resultou em doença menos grave com aumento dos níveis de citocinas em ambas as linhagens. Neste período, entre os grupos controle, somente os animais A/J apresentaram aumento de linfócitos T e macrófagos. Em conclusão, os animais A/J apresentaram aumento da ativação da resposta imune enquanto que animais B10.A não desenvolveram eficiente ativação da resposta adaptativa mas que podem ter apresentado uma imunidade inata mais eficiente no controle do crescimento fúngico.A mechanism for studying peripheral tolerance is mediated by regulatory T cells (Tregs) expressing the marker CD25 and the transcription factor FoxP3. We studied the role of Tregs in the mechanism of immunosuppression in genetic susceptibility and resistance of host against the fungus. A/J and B10.A mice were depleted of CD25 and control group received rat Ig. In the second week post infection, the depletion of CD25 cells resulted in low severity of disease in both mouse strains, with an intense influx of T lymphocytes and macrophages only in A/J. At 10 weeks, the depletion of CD25 also resulted in low severity disease with increased levels of cytokines in both mouse strains. Moreover, between the control group, only A/J mice showed an increase of T lymphocytes and macrophages. In conclusion, A/J mice showed increased activation of the immune response, whereas B10.A mice that had not developed efficient activation of the adaptive immune response, but that probably had an innate immunity that was more effective in controlling fungal growth

TLR2 Is a Negative Regulator of Th17 Cells and Tissue Pathology in a Pulmonary Model of Fungal Infection

To study the role of TLR2 in a experimental model of chronic pulmonary infection, TLR2-deficient and wild-type mice were intratracheally infected with Paracoccidioides brasiliensis, a primary fungal pathogen. Compared with control, TLR2(-/-) mice developed a less severe pulmonary infection and decreased NO synthesis. Equivalent results were detected with in vitro-infected macrophages. Unexpectedly, despite the differences in fungal loads both mouse strains showed equivalent survival times and severe pulmonary inflammatory reactions. Studies on lung-infiltrating leukocytes of TLR2(-/-) mice demonstrated an increased presence of polymorphonuclear neutrophils that control fungal loads but were associated with diminished numbers of activated CD4(+) and CD8(+) T lymphocytes. TLR2 deficiency leads to minor differences in the levels of pulmonary type 1 and type 2 cytokines, but results in increased production of KC, a CXC chemokine involved in neutrophils chemotaxis, as well as TGF-beta, IL-6, IL-23, and IL-17 skewing T cell immunity to a Th17 pattern. In addition, the preferential Th17 immunity of TLR2(-/-) mice was associated with impaired expansion of regulatory CD4(+)CD25(+)FoxP3(+) T cells. This is the first study to show that TLR2 activation controls innate and adaptive immunity to P. brasiliensis infection. TLR2 deficiency results in increased Th17 immunity associated with diminished expansion of regulatory T cells and increased lung pathology due to unrestrained inflammatory reactions. The Journal of Immunology, 2009, 183: 1279-1290.Fundacao de Amparo a PesquisaConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CNPq Conselho Nacional de Pesquisa

CD28 Exerts Protective and Detrimental Effects in a Pulmonary Model of Paracoccidioidomycosis▿

T-cell immunity has been claimed as the main immunoprotective mechanism against Paracoccidioides brasiliensis infection, the most important fungal infection in Latin America. As the initial events that control T-cell activation in paracoccidioidomycosis (PCM) are not well established, we decided to investigate the role of CD28, an important costimulatory molecule for the activation of effector and regulatory T cells, in the immunity against this pulmonary pathogen. Using CD28-deficient (CD28−/−) and normal wild-type (WT) C57BL/6 mice, we were able to demonstrate that CD28 costimulation determines in pulmonary paracoccidioidomycosis an early immunoprotection but a late deleterious effect associated with impaired immunity and uncontrolled fungal growth. Up to week 10 postinfection, CD28−/− mice presented increased pulmonary and hepatic fungal loads allied with diminished production of antibodies and pro- and anti-inflammatory cytokines besides impaired activation and migration of effector and regulatory T (Treg) cells to the lungs. Unexpectedly, CD28-sufficient mice progressively lost the control of fungal growth, resulting in an increased mortality associated with persistent presence of Treg cells, deactivation of inflammatory macrophages and T cells, prevalent presence of anti-inflammatory cytokines, elevated fungal burdens, and extensive hepatic lesions. As a whole, our findings suggest that CD28 is required for the early protective T-cell responses to P. brasiliensis infection, but it also induces the expansion of regulatory circuits that lately impair adaptive immunity, allowing uncontrolled fungal growth and overwhelming infection, which leads to precocious mortality of mice

Toll-Like Receptor 4 Signaling Leads to Severe Fungal Infection Associated with Enhanced Proinflammatory Immunity and Impaired Expansion of Regulatory T Cells▿

Toll-like receptors (TLRs) present in innate immune cells recognize pathogen molecular patterns and influence immunity to control the host-parasite interaction. The objective of this study was to characterize the involvement of TLR4 in the innate and adaptive immunity to Paracoccidioides brasiliensis, the most important primary fungal pathogen of Latin America. We compared the responses of C3H/HeJ mice, which are naturally defective in TLR4 signaling, with those of C3H/HePas mice, which express functional receptors, after in vitro and in vivo infection with P. brasiliensis. Unexpectedly, we verified that TLR4-defective macrophages infected in vitro with P. brasiliensis presented decreased fungal loads associated with impaired synthesis of nitric oxide, interleukin-12 (IL-12), and macrophage chemotactic protein 1 (MCP-1). After intratracheal infection with 1 million yeasts, TLR4-defective mice developed reduced fungal burdens and decreased levels of pulmonary nitric oxide, proinflammatory cytokines, and antibodies. TLR4-competent mice produced elevated levels of IL-12 and tumor necrosis factor alpha (TNF-α), besides cytokines of the Th17 pattern, indicating a proinflammatory role for TLR4 signaling. The more severe infection of TLR4-normal mice resulted in increased influx of activated macrophages and T cells to the lungs and progressive control of fungal burdens but impaired expansion of regulatory T cells (Treg cells). In contrast, TLR4-defective mice were not able to clear their diminished fungal burdens totally, a defect associated with deficient activation of T-cell immunity and enhanced development of Treg cells. These divergent patterns of immunity, however, resulted in equivalent mortality rates, indicating that control of elevated fungal growth mediated by vigorous inflammatory reactions is as deleterious to the hosts as low fungal loads inefficiently controlled by limited inflammatory reactions

Intradermal Application of Crotamine Induces Inflammatory and Immunological Changes In Vivo

Crotamine is a single-chain polypeptide with cell-penetrating properties, which is considered a promising molecule for clinical use. Nevertheless, its biosafety data are still scarce. Herein, we assessed the in vivo proinflammatory properties of crotamine, including its local effect and systemic serum parameters. Sixty male Wistar rats were intradermically injected with 200, 400 and 800 µg crotamine and analyzed after 1, 3 and 7 days. Local effect of crotamine was assessed by determination of MPO and NAG activities, NO levels and angiogenesis. Systemic inflammatory response was assessed by determination of IL-10, TNF-α, CRP, NO, TBARS and SH groups. Crotamine induced macrophages and neutrophils chemotaxis as evidenced by the upregulation of both NAG (0.5–0.6 OD/mg) and MPO (0.1–0.2 OD/mg) activities, on the first and third day of analysis, respectively. High levels of NO were observed for all concentrations and time-points. Moreover, 800 μg crotamine resulted in serum NO (64.7 μM) and local tissue NO (58.5 μM) levels higher or equivalent to those recorded for their respective histamine controls (55.7 μM and 59.0 μM). Crotamine also induced a significant angiogenic response compared to histamine. Systemically, crotamine induced a progressive increase in serum CRP levels up to the third day of analysis (22.4–45.8 mg/mL), which was significantly greater than control values. Crotamine (400 μg) also caused an increase in serum TNF-α, in the first day of analysis (1095.4 pg/mL), however a significant increase in IL-10 (122.2 pg/mL) was also recorded for the same time-point, suggesting the induction of an anti-inflammatory effect. Finally, crotamine changed the systemic redox state by inducing gradual increase in serum levels of TBARS (1.0–1.8 μM/mL) and decrease in SH levels (124.7–19.5 μM/mL) throughout the experimental period of analysis. In summary, rats intradermally injected with crotamine presented local and systemic acute inflammatory responses similarly to histamine, which limits crotamine therapeutic use on its original form