Regulatory T-Cell Dynamics in Cutaneous and Mucocutaneous Leishmaniasis due to Leishmania braziliensis.

To evaluate the dynamics of regulatory T cells (Tregs) during tegumentary leishmaniasis, we assessed peripheral blood and biopsies from 54 patients. Patients with cutaneous leishmaniasis (CL) had a decreased proportion of Tregs in the peripheral blood, but the proportion was higher in the biopsies of lesions. During treatment of CL, circulating Tregs increased reaching normal proportions, whereas antigen-specific interferon-γ responses diminished. By contrast, circulating Tregs from mucosal leishmaniasis patients failed to normalize during treatment. C-C chemokine receptor type 5 was expressed on a large proportion of Tregs at the site of infection. These results demonstrate increased Tregs at the site of infection, possibly homing from the peripheral circulation.

Microparticles derived from obese adipose tissue elicit a pro-inflammatory phenotype of CD16+, CCR5+ and TLR8+ monocytes.

Macrophage infiltration into adipose tissue (AT) is a hallmark of the chronic inflammatory response in obesity and is supported by an intense monocyte migration towards AT. Although it has been detected an increased proportion of circulating CD16+ monocyte subsets in obese subjects, the mechanisms underlying this effect and the contribution of these cells to the inflamed profile of obese AT are still poorly understood. We investigated whether factors secreted by human obese omental AT could polarize monocytes to CD16+ enriched phenotype, and how these changes could modify their migratory capacity towards adipose tissue itself. We show that explants of human obese omental AT, obtained during bariatric surgery, released higher levels of MIP1-α, TNFα, leptin and also VEGF, together with increasing amounts of microparticles (MP), when compared to explants of lean subcutaneous AT. A higher content of circulating MP derived from preadipocytes and leukocytes was also detected in plasma of obese subjects. Conditioned media or MP released from obese omental AT increased CD16 and CCR5 expression on CD14+CD16- monocytes and augmented their migratory capacity towards the conditioned media from obese omental AT, itself. This effect was inhibited when MIP1-α was neutralized. Additionally, we demonstrate that MP derived from obese omental AT carry and transfer TLR8 to monocytes, thus triggering an increase in CD16 expression in those cells. Our data shows a positive feedback loop between blood monocytes and obese omental AT, which releases chemotactic mediators and TLR8-enriched MP, thus inducing an up-regulation of CD16+ monocytes, favoring leukocyte infiltration in the obese omental AT.

Variability of the dendritic cell response triggered by different serotypes of Aggregatibacter actinomycetemcomitans or Porphyromonas gingivalis is toll-like receptor 2 (TLR2) or TLR4 dependent.

BACKGROUND: Different serotypes of Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis have been shown to induce differential dendritic cell (DC) responses. This study investigates whether cytokine and CC-chemokine receptor (CCR) production by DCs stimulated with different serotypes of A. actinomycetemcomitans or P. gingivalis is Toll-like receptor 2 (TLR2) and/or TLR4 dependent. METHODS: DCs were obtained from healthy individuals and primed at a multiplicity of infection (MOI) of 10(2) with different A. actinomycetemcomitans or P. gingivalis serotypes in the presence or absence of anti-TLR2 or anti-TLR4 blocking antibodies. TLR2 and TLR4 expression, CCR5 and CCR6 expression, and interleukin (IL)-1ß, IL-10, IL-12, and IL-23 expression and secretion were quantified by flow cytometry, real-time reverse-transcription polymerase chain reaction, and enzyme-linked immunosorbent assay. RESULTS: When DCs were stimulated with serotype b of A. actinomycetemcomitans or serotype K1 of P. gingivalis, higher levels of TLR2 or TLR4, respectively, were detected compared to DCs stimulated with the other serotypes. Similarly, higher levels of cytokines and CCRs were detected in serotype b- or serotype K1-primed DCs compared to the others, and these increased levels positively correlated with levels of TLR2 or TLR4. When TLR2 signaling was blocked using a specific anti-TLR2 monoclonal antibody, serotype b-induced cytokine and CCR expression was inhibited; when TLR4 signaling was blocked, serotype K1-induced response was inhibited. CONCLUSIONS: These results demonstrate that the variability of secretion of cytokines and expression of CCRs detected in DCs stimulated with different serotypes of A. actinomycetemcomitans or P. gingivalis is TLR2 or TLR4 dependent, respectively.

CD4+ T-cell activation impairs serogroup C Neisseria meningitis vaccine response in HIV-infected children.

OBJECTIVE: To investigate the influence of CD4 T-cell activation and regulatory populations in HIV-infected children antibody response to vaccination with a conjugate C polysaccharide vaccine. DESIGN: CD4 T-cell activation was evaluated by expression of CD38, HLA-DR and CCR5 molecules. Regulatory CD4 T cells (TReg) were characterized as FoxP3CD127CD25 and inducer T cells (TInd) as CD4FoxP3CD25CD39. METHODS: All patients (n = 36) were HIV-vertically infected, aged 2-17 years-old and were vaccinated with one vaccine injection. Blood samples were obtained before and after immunization to determine bactericidal antibody titers (SBA), CD4 T-cell activation and frequency of TReg and TInd subsets (multiparametric flow cytometry). RESULTS: Children not-responding (n = 18) to MenC vaccine expressed higher frequency of activated CD4 T cells (HLA-DRCD38CCR5) than responders (n = 18), both before and after vaccination (P < 0.05). A significant higher frequency of TReg was detected in responders compared with nonresponders (P = 0.0001). We also detected an inverse correlation between CD4DRCD38CCR5 (P = 0.01) or CD4DRCD38 (P = 0.02) T cells and TReg cell frequency after vaccination. CD4 T-cell activation negatively correlated (P = 0.006) with postvaccination SBA titers but a positive correlation (P = 0.0001) was detected between TReg cells and SBA. TReg and TInd subsets were inversely correlated (P = 0.04). CONCLUSION: Our findings suggest that higher CD4 T-cell activation leads to poor vaccine response in children living with HIV, which may be associated with a TReg/TInd disequilibrium.

Paradoxical role of CD16+CCR2+CCR5+ monocytes in tuberculosis: efficient APC in pleural effusion but also mark disease severity in blood.

The role of CD16(-) and CD16(+) Mo subsets in human TB remains unknown. Our aim was to characterize Mo subsets from TB patients and to assess whether the inflammatory milieu from TB pleurisy modulate their phenotype and recruitment. We found an expansion of peripheral CD16(+) Mo that correlated with disease severity and with TNF-α plasma levels. Circulating Mo from TB patients are activated, showing a higher CD14, CD16, and CD11b expression and Mtb binding than HS. Both subsets coexpressed CCR2/CCR5, showing a potential ability to migrate to the inflammatory site. In tuberculous PF, the CD16(+) subset was the main Mo/MΦ population, accumulation that can be favored by the induction of CD16 expression in CD16(-) Mo triggered by soluble factors found in this inflammatory milieu. CD16(+) Mo in PF were characterized by a high density of receptors for Mtb recognition (DC-SIGN, MR, CD11b) and for lipid-antigens presentation (CD1b), allowing them to induce a successful, specific T cell proliferation response. Hence, in tuberculous PF, CD16(+) Mo constitute the main APC population; whereas in PB, their predominance is associated with the severity of pulmonary TB, suggesting a paradoxical role of the CD16(+) Mo subset that depends on the cellular localization.

The dual role of p55 tumour necrosis factor-alpha receptor in Actinobacillus actinomycetemcomitans-induced experimental periodontitis: host protection and tissue destruction.

Inflammatory immune reactions in response to periodontopathogens are thought to protect the host against infection, but may trigger periodontal destruction. Thus, we examined the mechanisms by which the proinflammatory cytokine tumour necrosis factor (TNF)-alpha modulates the outcome of Actinobacillus actinomycetemcomitans-induced periodontal disease in mice. Our results showed that TNF-alpha receptor p55-deficient mice [p55TNF-knock-out (KO)] developed a less severe periodontitis in response to A. actinomycetemcomitans infection, characterized by significantly less alveolar bone loss and inflammatory reaction. Real-time polymerase chain reaction (PCR) demonstrated that levels of chemokines (CXCL1, 3 and 10; CCL3 and 5) and their receptors (CXCR2 and 3, CCR5) were lower in p55TNF-KO mice, as were matrix metalloproteinase (MMP)-1, 2 and 9 and receptor activator of nuclear factor kB ligand (RANKL) mRNA levels. However, the absence of the TNF-alpha p55 results in an impairment of protective immunity to A. actinomycetemcomitans infection, characterized by increased bacterial load and higher levels of C-reactive protein during the course of disease. Such impaired host response may be the result of the reduced chemoattraction of lymphocytes, neutrophils and macrophages, and reduced inducible nitric oxide synthase expression (iNOS) and myeloperoxidase (MPO) production in periodontal tissues of p55 TNF-KO mice. Our results demonstrate the mechanisms involved determining periodontal disease severity by TNF-alpha receptor p55, and its role in providing immune protection to A. actinomycetemcomitans periodontal infection.

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-beta, 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-beta, 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-beta+, 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.

Differential expression of chemokines and chemokine receptors in inflammatory periapical diseases.

BACKGROUND: Periapical lesions are thought to be the result of a local inflammatory response mediated by inflammatory cell infiltration and production of inflammatory mediators. Although chemokines are strongly implicated in the migration and activation of leukocytes in different inflammatory diseases and experimental models, little is known regarding the expression of chemokines and their receptors in human apical periodontitis. OBJECTIVE AND METHODS: The objective of this study was to determine the expression of chemokines and their receptors by real-time polymerase chain reaction in samples obtained from healthy gingiva, periapical granulomas, and inflammatory periradicular cysts. The inflammatory infiltrate was characterized by immunohistochemistry. RESULTS: Comparing cysts and granulomas, an increase in CD4+ and CD8+ cells was observed in granulomas, despite the similar numbers of CD45RO-positive cells detected in both lesions. The analysis of mRNA expression revealed increased levels of CCR1, CCR2, CCR3, CCR5, CXCR1, and CXCR3 in both types of lesion compared with controls. Cysts exhibited a higher expression of CCR3, CCR5, CXCR1, and CXCR3 compared to granulomas. A significantly higher expression of RANTES, IP-10, and MCP-1 was detected in cysts compared with controls or granulomas. The expression of interleukin-8, MIP-1alpha, and MIP-1beta was not different in the three experimental groups. CONCLUSIONS: The increase in Th1 type (CCR1, CCR5, and CXCR3) and Th2 type (CCR2 and CCR3) receptors in both periapical lesions suggests the concomitant occurrence of Th1 and Th2 responses. Furthermore, the prevalent expression of the receptors CCR3, CCR5, CXCR1, and CXCR3 and of the chemokines RANTES, IP-10, and MCP-1 in cysts may point to a role in the progression of granulomas to cysts.

[CXCR-4 AND CCR-5 expression in normal term human placenta]. / Expresión de receptores de quimiocinas (CXCR-4 y CCR-5) en placenta humana normal a término.

The maternal-fetal interphase has an active Immunitary System (IS) whose mediators -cells, cytokines and chemokines- coordinately act to favour pregnancy normal development. It is not known exactly which of those mediators are present in each placental cellular stratus and what the physiological or potentially pathologic consequences derived from their presence can be. It is known that chemokines recruit cells with regulatory activity towards the deciduous and some of their receptors are coreceptors to infectious agents like HIV, making research of chemokines expression and their receptors in the maternal-fetal interphase of great interest in recent years. In the present study, the CXCR-4 and CCR-5 expression was investigated in 8 samples of normal human placenta obtained from term pregnancies, with low obstetric risk, by using Immunocitochemical techniques (Biotin-Avidin-Peroxidase). The most relevant finding in this study was the demonstration that CXCR-4 and CCR-5 differential expression in trophoblast, stroma and endothelium represents, as far as we know, the first report of the presence of these receptors in all layers of placental tissue. These results help to broaden the knowledge about the expression of chemokines receptors -that act as main coreceptors in the HIV infection- in the maternal-fetal interphase, and this can be a contribution to be taken into account in the vertical transmission study of this infectious agent.

CCR5 plays a critical role in the development of myocarditis and host protection in mice infected with Trypanosoma cruzi.

The pathogenesis of myocarditis during Trypanosoma cruzi infection is poorly understood. We investigated the role played by chemokine receptor 5 (CCR5) in the influx of T cells to the cardiac tissue of T. cruzi-infected mice. mRNA and protein for the CCR5 ligands CCL3, CCL4, and CCL5 were detected in the hearts of infected mice in association with CD4+ and CD8+ T cells. There was a high level of CCR5 expression on CD8+ T cells in the hearts of infected mice. Moreover, CCR5 expression on CD8+ T cells was positively modulated by T. cruzi infection. CCR5-deficient mice infected with T. cruzi experienced a dramatically inhibited migration of T cells to the heart and were also more susceptible to infection. These results suggest that CCR5 and its ligands play a central role in the control of T cell influx in T. cruzi-infected mice. Knowledge of the mechanisms that trigger and control the migration of cells to the heart in patients with Chagas disease may help in the design of drugs that prevent myocarditis and protect against the development of severe disease.

Patterns of chemokines and chemokine receptors expression in different forms of human periodontal disease.

Current knowledge states that periodontal diseases are chronic inflammatory reactions raised in response to periodontopathogens. Many cell types and mediators, including Th1 and Th2 lymphocytes, cytokines and chemokines, appear to be involved in the immunopathogenesis of periodontal diseases. Chemokines, a family of chemotactic cytokines, bind to specific receptors and selectively attract different cell subsets to the inflammatory site. They can also interact with classical cytokines and modulate the local immune response. In order to study the role of chemokines in periodontal diseases, we examined the expression of chemokines, chemokine receptors and cytokines by means of reverse transcription-polymerase chain reaction (RT-PCR) techniques. Characteristic patterns of such factors' expression were found in gingival biopsies from patients presenting with aggressive periodontitis and chronic periodontitis. The expression of the chemokines macrophage inflammatory protein-1 alpha (MIP-1alpha) and interferon-gamma inducible protein 10 (IP-10) and of their respective receptors, CCR5 and CXCR3, were more prevalent and higher in aggressive periodontitis, and associated with higher interferon-gamma (IFN-gamma) expression and lower interleukin-10 (IL-10) expression. In contrast, chronic periodontitis patients exhibited a more frequent and higher expression of monocyte chemoattractant protein-1 (MCP-1) and its receptor CCR4, and higher expression of IL-10. It is possible that chemokines, in addition to the classical cytokines, are involved in the immunopathogenesis of periodontal disease, driving the migration and the maintenance of several inflammatory cell types such as polymorphonuclear leukocytes, dendritic cells (DCs), natural killer cells, macrophages, and subsets of lymphocytes in the gingival tissues. These cells are thought to participate in the inflammatory and immune reaction that takes place in periodontal disease, killing pathogens, presenting antigens, and producing cytokines. The selective recruitment of polarized lymphocyte subsets could result in differential cytokine production at the site of response, which is supposed to determine the stable or progressive nature of the lesion. Besides, the role of chemokines as activators and chemoattracts of osteclasts may be involved in the determination of disease severity.

Early inflammatory markers in elicitation of allergic contact dermatitis.

BACKGROUND: Allergic Contact Dermatitis (ACD) is regarded as a T-cell-mediated delayed-type hypersensitivity reaction. We studied the kinetics of the expression of CS-1 fibronectin, thymus and activation-regulated chemokine (CCL17/ TARC) and different chemokine receptors (CR) in skin biopsies from individuals suffering from back problems, with the antigen responsible of their contact dermatitis and an irrelevant antigen. METHODS: Samples were taken at 2, 10, and 48 hours for histological and immunohistochemical studies using monoclonal antibodies against human CS-1 fibronectin, CCL17, CD3, CD68, CD49d, CXCR3, CCR5, and CCR3. RESULTS: At positive antigen stimulated sites there was an early expression of CS-1 fibronectin (2 hours), followed by CCL17 and a later accumulation of alplha4/beta1+ (CD49d), CD3+, CD68+, CXCR3+ and CCR5+ mononuclear cells. At 48 hours, approximately 59 % of infiltrating cells were CXCR3+, 42% CCR5+, and only 14 % CCR3+. CONCLUSIONS: These results showed for the first time a very early expression of CS-1 fibronectin which preceded production of CCL17 in blood endothelial cells (BCEs) from patients' skin with ACD. The role of these molecules in recruitment of monocytes and effector T cells in ACD is discussed.

CCR5 and beta-chemokines in HIV-1 infected children.

The duration from initial infection with HIV-1 to CD4 lymphocyte depletion and progression to AIDS varies among infected individuals. Despite treatment with highly active antiretroviral therapy (HAART), patients still show different stages of disease progression. We examined the role of beta-chemokines and its receptor, CCR5 in HIV-1 infected children in order to define determinants of HIV progression among treated individuals. Population was divided in two groups: Group 1--Long Term Non Progressors (LTNP) includes 10 patients with B1-B2 CDC disease classification and with a less aggressive therapy (only 2 in HAART); Group 2--Rapid Progressors (RP) includes 9 patients with C3 disease classification. All the patients had a CCR5 wild type (wt) genotype indicating that they do not have the 32 base-pair deletion associated with slower progression. There was an increased production of MIP 1-beta in 8/10 LTNP but only in 4/9 Progressors (Paired t-test/Wilcoxon Sign test, p-value < 0.05). The change in the levels of MIP-1 beta after PHA stimulation was statistically significant in both groups. The levels of RANTES increased in LTNP and RP and the change of the levels after mitogen stimulation was statistically significant for both groups included. The production of RANTES and MIP-1 beta in response to stimulation between both groups was not statistically significant. The production of MIP-1 alpha was variable in both groups and the difference in the levels after mitogen stimulation between the groups was not statistically significant. These results suggest that beta-chemokines do not play an important role in HIV-1 progression in children undergoing HAART.