Ana Paula Campanelli

CD4+CD25+ T Cells in Skin Lesions of Patients with Cutaneous Leishmaniasis Exhibit Phenotypic and Functional Characteristics of Natural Regulatory T Cells

Endogenous regulatory T (Treg) cells are involved in the control of infections, including Leishmania infection in mice. Leishmania viannia braziliensis is the main etiologic agent of cutaneous leishmaniasis (CL) in Brazil, and it is also responsible for the more severe mucocutaneous form. Here, we investigated the possible involvement of Treg cells in the control of the immune response in human skin lesions caused by L. viannia braziliensis infection. We show that functional Treg cells can be found in skin lesions of patients with CL. These cells express phenotypic markers of Treg cells--such as CD25, cytotoxic T lymphocyte-associated antigen 4, Foxp3, and glucocorticoid-induced tumor necrosis factor receptor--and are able to produce large amounts of interleukin-10 and transforming growth factor- beta . Furthermore, CD4+CD25+ T cells derived from the skin lesions of 4 of 6 patients with CL significantly suppressed in vitro the phytohemagglutinin-induced proliferative T cell responses of allogeneic peripheral-blood mononuclear cells (PBMCs) from healthy control subjects at a ratio of 1 Treg cell to 10 allogeneic PBMCs. These findings suggest that functional Treg cells accumulate at sites of Leishmania infection in humans and possibly contribute to the local control of effector T cell functions.

Systemic and Local Characterization of Regulatory T Cells in a Chronic Fungal Infection in Humans

The long-term persistence of pathogens in a host is a hallmark of certain infectious diseases, including schistosomiasis, leishmaniasis, and paracoccidioidomycosis (PCM). Natural regulatory T (Treg) cells are involved in control of the immune responses, including response to pathogens. Because CTLA-4 is constitutively expressed in Treg cells and it acts as a negative regulator of T cell activation in patients with PCM, here we investigated the involvement of Treg cells in the control of systemic and local immune response in patients with PCM. We found that the leukocyte subsets were similar in patients and controls, except for CD11c+CD1a+ cells. However, a higher frequency of CD4+CD25+ T cells expressing CTLA-4, glucorticoid-inducible TNFR, membrane-bound TGF-β, and forkhead-box 3 were observed in PBMC of patients. In accordance, these cells exhibited stronger suppressive activity when compared with those from controls (94.0 vs 67.5% of inhibition of allogeneic T cell proliferation). In addition, the data showed that CD4+CD25+ T cells expressing CTLA-4+, glucocorticoid-inducible TNFR positive, CD103+, CD45RO+, membrane-bound TGF-β, forkhead-box 3 positive, and the chemokines receptors CCR4 and CCR5 accumulate in the Paracoccidioides brasiliensis-induced lesions. Indeed, the secreted CCL17 and CCL22, both associated with the migration of Treg cells to peripheral tissues, were also detected in the biopsies. Moreover, the CD4+CD25+ T cell derived from lesions, most of them TGF-β+, also exhibited functional activity in vitro. Altogether, these data provide the first evidence that Treg cells play a role in controlling local and systemic immune response in patients with a fungal-induced granulomatous disease advancing our understanding about the immune regulation in human chronic diseases.

Regulatory T cells attenuate experimental periodontitis progression in mice

AIMS The aim of this study was to identify the presence and characterize the function of regulatory T cells (Tregs) in experimental periodontitis in mice. MATERIAL AND METHODS C57Bl/6 mice infected with Actinobacillus actinomycetemcomitans, treated or not with anti-glucocorticoid-inducible tumour necrosis factor receptor (anti-GITR) to inhibit Tregs function, were analysed regarding inflammatory cell and Tregs influx, alveolar bone loss and cytokine expression/production (analysed by real-time polymerase chain reaction and ELISA) throughout experimental periodontitis. RESULTS A. actinomycetemcomitans inoculation in mice resulted in periodontal disease characterized by marked alveolar bone loss and an influx of inflammatory cells. Flow cytometry evaluation of inflammatory cells demonstrated an increased number of CD4(+)CD25(+) and CD4(+)FOXp3(+) cells, characterizing the presence of Tregs in the periodontal environment in a late stage after infection. Tregs-associated cytokines interleukin-10 (IL-10), cytotoxic T lymphocyte-associated molecule 4 (CTLA-4) and transforming growth factor-beta (TGF-beta) were found to be expressed/produced in a kinetics that resembles Tregs migration. Treatment with anti-GITR, which inhibits Tregs function, showed increased alveolar bone loss and inflammatory cell migration. A reduction in IL-10, CTLA-4 and TGF-beta levels was also observed, while interferon-gamma, tumour necrosis factor-alpha and receptor activator for nuclear factor kappaB ligand levels were increased. However, bacterial load and C-reactive protein serum did not show any differences. CONCLUSION Taken together, our results showed that the presence of Treg cells attenuates the severity of experimental periodontitis without impairment in the control of infection.

The broad effects of the functional IL-10 promoter-592 polymorphism : modulation of IL-10, TIMP-3, and OPG expression and their association with periodontal disease outcome

Periodontal diseases are infectious diseases, in which periodontopathogens trigger chronic inflammatory and immune responses that lead to tissue destruction. It occurs through the generation of metalloproteinases and the activation of bone resorption mechanisms. Anti‐inflammatory cytokines such as IL‐10 seem to attenuate periodontal tissue destruction through the induction of tissue inhibitors of metalloproteinases (TIMPs) and the inhibitor of osteoclastogenesis osteoprotegerin (OPG). A high individual variation in levels of IL‐10 mRNA is verified in periodontitis patients, which is possibly determined by genetic polymorphisms. In this study, the IL‐10 promoter ‐592C/A single nucleotide polymorphism (SNP), which is associated with a decrease in IL‐10 production, was analyzed by RFLP in 116 chronic periodontitis (CP) patients and 173 control (C) subjects, and the IL‐10, TIMPs, and OPG mRNA expression levels in diseased gingival tissues were determined by real‐time‐PCR. The IL‐10‐592 SNP CA (P=0.0012/OR=2.4/CI:1.4‐4.1), AA (P=0.0458/OR=2.3/CI:1.1‐4.9), and CA+AA (P=0.0006/OR=2.4/CI:1.4‐3.4) genotypes and the allele A (P=0.0036/OR=1.7/CI:1.2‐2.4) were found to be significantly more prevalent in the CP group when compared with control subjects. Both CA and AA genotypes were associated with lower levels of IL‐10, TIMP‐3, and OPG mRNA expression in diseased periodontal tissues and were also associated with disease severity as mean pocket depth. Taken together, the results presented here demonstrate that IL10‐592 SNP is functional in CP, being associated with lower levels of IL‐10 mRNA expression, which is supposed to consequently decrease the expression of the downstream genes TIMP‐3 and OPG, and influence periodontal disease outcome.

Differential Patterns of Receptor Activator of Nuclear Factor Kappa B Ligand/Osteoprotegerin Expression in Human Periapical Granulomas: Possible Association with Progressive or Stable Nature of the Lesions

Receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) are expressed in apical periodontitis, suggesting a role for these molecules during lesion development. However, the profiles of RANKL/OPG expression in periapical lesions remain unknown. In this study we investigated the patterns of RANKL and OPG mRNA expression by real-time polymerase chain reaction in human periapical granulomas (N = 44) and compared them with sites presenting characteristic bone resorbing activity: healthy (n = 14) and orthodontically stretched and compressed periodontal ligament (n = 26), healthy gingiva (n = 24), chronic gingivitis (n = 32), and chronic periodontitis (n = 34) samples. Both RANKL and OPG mRNA expression was higher in periapical granulomas when compared with healthy periodontal ligament. Distinct patterns of RANKL and OPG expression ratio were found in the granulomas and in different physiologic and pathologic conditions, with characteristic bone resorption activity potentially being indicative of the stable or progressive nature of the lesions. Lesions with radiographic image smaller than 5 mm showed higher RANKL/OPG expression than images greater than 5 mm. Periapical granulomas presented heterogeneous patterns of RANKL and OPG expression, ranging from samples with RANKL/OPG ratio similar to that seen in sites with minimal or absent bone resorption to samples with RANKL/OPG expression pattern comparable with active bone resorption sites.

CCR5-Dependent Regulatory T Cell Migration Mediates Fungal Survival and Severe Immunosuppression

Paracoccidioidomycosis, a debilitating pulmonary mycosis, is caused by the dimorphic fungus Paracoccidioides brasiliensis. The infection results in the formation of granulomas containing viable yeast cells that are the fungal sources for disease reactivation. Because CD4+CD25+ regulatory T cells (Tregs) are in the lesions of patients with paracoccidioidomycosis, the migration of Treg cells is dependent on the axis chemokine-chemokine receptors, and CCR5 ligands are produced in P. brasiliensis-induced lesions, we investigated the role of CCR5 in the control of the infection. The results showed that CCR5−/− mice are more efficient in controlling fungal growth and dissemination and exhibited smaller granulomas than wild-type (WT) mice. In the absence of CCR5, the percentage of CD4+CD25+ T cells expressing Foxp3, glucocorticoid-induced TNFR (GITR), CD103, CD45low, and CTLA-4 in the granulomas was significantly decreased. Interestingly, P. brasiliensis infection resulted in an absence of T cell proliferation in response to Con A in WT but not CCR5−/− mice that was abrogated by anti-CTLA-4 mAb and anti-GITR mAb. Moreover, the adoptive transfer of CD4+CD25+ but not CD4+CD25− T cells from infected WT to infected CCR5−/− mice resulted in a significant increase in fungal load. Overall, CCR5 is a key receptor for the migration of Treg cells to the site of P. brasiliensis infection, leading to down-modulation of effector immune response and the long-term presence of the fungus in the granulomas. Thus, a tight control of Treg cell migration to the granulomatous lesions could be an important mechanism for avoiding exacerbation and reactivation of the disease.

Chemokine Production and Leukocyte Recruitment to the Lungs of Paracoccidioides brasiliensis-Infected Mice Is Modulated by Interferon-γ

Chemokines and chemokine receptors play a role in cell recruitment during granulomatous inflammatory reactions. Here, we evaluated the expression of chemokines and chemokine receptors and their regulation by IFN-gamma in the course of Paracoccidioides brasiliensis (Pb) infection in mice. We found an association between KC and MIP-1alpha (CCL3) production and neutrophil infiltration in the lungs of Pb-infected mice during the early acute phase of infection. High levels of RANTES/CCL5, MCP-1/CCL2, IP-10/CXCL10, and Mig/CXCL9 simultaneously with mononuclear cell infiltration in the lungs was found. In the absence of IFN-gamma (GKO mice) we observed increased production of KC and MIP-1alpha and chronic neutrophilia. Moreover, we found a change in the chemokine receptor profiles expressed by wild-type (WT) versus GKO animals. Increased expression of CXCR3 and CCR5, and low levels of CCR3 and CCR4 were observed in the lungs of Pb-infected WT mice, whereas the opposite effect was observed in the lungs of GKO mice. Consistent with these results, infected cells from WT mice preferentially migrated in response to IP-10 (CXCR3 ligand), while those from GKO mice migrated in response to eotaxin/CCL11 (CCR3 ligand). These results suggest that IFN-gamma modulates the expression of chemokines and chemokine receptors as well as the kind of cells that infiltrate the lungs of Pb-infected mice.

The essential role of IFN-γ in the control of lethal Aggregatibacter actinomycetemcomitans infection in mice

Inflammatory immune reactions in response to periodontopathogens trigger periodontal destruction, but their role to protect the host against infection remains unknown. Thus, we examined the mechanisms by which IFN-gamma modulates the outcome of Aggregatibacter actinomycetemcomitans-induced periodontal disease in mice. Our results showed that IFN-gamma deficient mice developed less severe periodontitis in response to A. actinomycetemcomitans infection, characterized by significant lower alveolar bone loss and inflammatory reaction. However, the absence of IFN-gamma results in increased bacterial load in periodontal tissues and higher acute phase reaction, followed by a disseminated bacterial infection and mice death during the course of the disease. Such impaired host response was found to be associated with a reduction in the levels of inflammatory cytokines and chemokines and in the number of GR1+, F4/80+, CD4+ and CD8+ leukocytes in the diseased periodontium of IFN-gamma deficient mice. In addition, the levels of both antimicrobial mediators myeloperoxidase and inducible nitric oxide synthase were also found to be reduced in IFN-KO mice. Our results demonstrate for the first time that a periodontal infection may be lethal in an immunocompromised host. In addition, the mechanisms involved in IFN-gamma mediated cell migration to diseased periodontal tissues, and its essential role to control A. actinomycetemcomitans infection were clarified.

An Interleukin-1β (IL-1β) Single-Nucleotide Polymorphism at Position 3954 and Red Complex Periodontopathogens Independently and Additively Modulate the Levels of IL-1β in Diseased Periodontal Tissues

ABSTRACT Inflammatory cytokines such as interleukin-1β (IL-1β) are involved in the pathogenesis of periodontal diseases. A high individual variation in the levels of IL-1β mRNA has been verified, which is possibly determined by genetic polymorphisms and/or by the presence of periodontopathogens such as Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and Aggregatibacter actinomycetemcomitans. In this study, we investigated the role of an IL-1β promoter single-nucleotide polymorphism at position 3954 [IL-1β(3954) SNP] and the presence of the periodontopathogens in the determination of the IL-1β levels in the periodontal tissues of nonsmoking chronic periodontitis (CP) patients (n = 117) and control (C) subjects (n = 175) and the possible correlations with the clinical parameters of the disease. IL-1β(3954) SNP was investigated by restriction fragment length polymorphism, while the IL-1β levels and the presence of the periodontopathogens were determined by real-time PCR. Similar frequencies of IL-1β(3954) SNP were found in the C and CP groups, in spite of a trend toward a higher incidence of T alleles in the CP group. The IL-1β(3954) SNP CT and TT genotypes, as well as P. gingivalis, T. forsythia, and T. denticola, were associated with higher IL-1β levels and with higher values of the clinical parameters of disease severity. Concomitant analyses demonstrate that IL-1β(3954) and the red complex periodontopathogens were found to independently and additively modulate the levels of IL-1β in periodontal tissues. Similarly, the concurrent presence of both factors was associated with increased scores of disease severity. IL-1β(3954) genotypes and red complex periodontopathogens, individually and additively, modulate the levels of IL-1β in the diseased tissues of nonsmoking CP patients and, consequently, are potentially involved in the determination of the disease outcome.

Confocal laser scanning microscopy is appropriate to detect viability of Enterococcus faecalis in infected dentin.

The purpose of this study was to explore the potential of confocal laser scanning microscopy (CLSM) for in situ identification of live and dead Enterococcus faecalis in infected dentin. Eight cylindrical dentin specimens were infected with Enterococcus faecalis in BHI for 21 days. After the experimental period, the specimens were stained with fluorescein diacetate (FDA) and propidium iodide (PI) or acridine orange (0.01%) and analyzed by CLSM. Two noninfected dentin specimens were used as negative controls. CLSM analysis shows that the discrimination between viable (green) and dead (red) bacteria in infected dentinal tubules could be observed after staining with FDA/PI. Acridine orange was able to show metabolic activity of the E. faecalis cells inside the dentinal tubules showed by its red fluorescence. The viability of bacteria in infected dentin can be determined in situ by CLSM. FDA/PI and acridine orange are useful for this technique.