Assessment of Allergen-Responsive Regulatory T Cells in Experimental Asthma Induced in Different Mouse Strains.

BACKGROUND: Regulatory T cells (Tregs) are important in regulating responses to innocuous antigens, such as allergens, by controlling the Th2 response, a mechanism that appears to be compromised in atopic asthmatic individuals. Different isogenic mouse strains also have distinct immunological responses and susceptibility to the experimental protocols used to develop lung allergic inflammation. In this work, we investigated the differences in the frequency of Treg cell subtypes among A/J, BALB/c, and C57BL/6, under normal conditions and following induction of allergic asthma with ovalbumin (OVA). METHODS: Subcutaneous sensitization followed by 4 consecutive intranasal OVA challenges induced asthma characteristic changes such as airway hyperreactivity, inflammation, and production of Th2 cytokines (IL-4, IL-13, IL-5, and IL-33) in the lungs of only A/J and BALB/c but not C57BL/6 strain and evaluated by invasive whole-body plethysmography, flow cytometry, and ELISA, respectively. RESULTS: A/J strain naturally showed a higher frequency of CD4+IL-10+ T cells in the lungs of naïve mice compared to the other strains, accompanied by higher frequencies of CD4+IL-4+ T cells. C57BL/6 mice did not develop lung inflammation and presented higher frequency of CD4+CD25+Foxp3+ Treg cells in the bronchoalveolar lavage fluid (BALF) after the allergen challenge. In in vitro settings, allergen-specific stimulation of mediastinal LN (mLN) cells from OVA-challenged animals induced higher frequency of CD4+IL-10+ Treg cells from A/J strain and CD4+CD25+Foxp3+ from C57BL/6. CONCLUSIONS: The observed differences in the frequencies of Treg cell subtypes associated with the susceptibility of the animals to experimental asthma suggest that CD4+CD25+Foxp3+ and IL-10-producing CD4+ Treg cells may play different roles in asthma control. Similar to asthmatic individuals, the lack of an efficient regulatory response and susceptibility to the development of experimental asthma in A/J mice further suggests that this strain could be preferably chosen in experimental models of allergic asthma.

Th17 Cells in Viral Infections-Friend or Foe?

Th17 cells are recognized as indispensable in inducing protective immunity against bacteria and fungi, as they promote the integrity of mucosal epithelial barriers. It is believed that Th17 cells also play a central role in the induction of autoimmune diseases. Recent advances have evaluated Th17 effector functions during viral infections, including their critical role in the production and induction of pro-inflammatory cytokines and in the recruitment and activation of other immune cells. Thus, Th17 is involved in the induction both of pathogenicity and immunoprotective mechanisms seen in the host's immune response against viruses. However, certain Th17 cells can also modulate immune responses, since they can secrete immunosuppressive factors, such as IL-10; these cells are called non-pathogenic Th17 cells. Here, we present a brief review of Th17 cells and highlight their involvement in some virus infections. We cover these notions by highlighting the role of Th17 cells in regulating the protective and pathogenic immune response in the context of viral infections. In addition, we will be describing myocarditis and multiple sclerosis as examples of immune diseases triggered by viral infections, in which we will discuss further the roles of Th17 cells in the induction of tissue damage.

Microneme Proteins 1 and 4 From Toxoplasma gondii Induce IL-10 Production by Macrophages Through TLR4 Endocytosis.

The protozoan parasite Toxoplasma gondii modulates host cell responses to favor its success in the early stage of infections by secreting proteins from its apical organelles. Some of these proteins, including microneme proteins (MICs) 1 and 4, trigger pro-inflammatory host cell responses. The lectins MIC1 and MIC4 interact with N-linked glycans on TLR2 and TLR4, activating NF-κB and producing IL-12, TNF-α, and IL-6. Interestingly, MIC1 and MIC4 also trigger secretion of the anti-inflammatory cytokine IL-10 through mechanisms as yet unknown. Herein, we show that the ability of these MICs to induce macrophages to produce IL-10 depends on TLR4 internalization from the cell surface. Macrophages subjected to blockade of endocytosis by Dynasore continued to release TNF-α, but failed to produce IL-10, in response to MIC1 or MIC4 exposure. Similarly, IL-10 was not produced by Dynasore-conditioned T. gondii-infected macrophages. Furthermore, MIC1- or MIC4-stimulated macrophages gained transient tolerance to LPS. We report a previously undiscovered mechanism by which well-defined T. gondii components inhibit a host inflammatory response.

Recombinant Lactococcus lactis Carrying IL-4 and IL-10 Coding Vectors Protects against Type 1 Diabetes in NOD Mice and Attenuates Insulitis in the STZ-Induced Model.

Type 1 diabetes (T1D) is an autoimmune disease that culminates in beta cell destruction in the pancreas and, subsequently, deficiency in insulin production. Cytokines play a crucial role in the development of diabetes, orchestrating the recruitment and action of immune cells, to not only destroy insulin-producing cells but also preserve them. Therefore, the aim of this study was to investigate the effect of orally administered Lactococcus lactis MG1363 FnBPA+ strains carrying plasmids encoding IL-4 and IL-10 in the streptozotocin- (STZ-) induced diabetes model and in nonobese diabetic (NOD) mice. The STZ-induced mice that were treated with combined bacterial strains carrying plasmids encoding IL-4 and IL-10 showed lower incidence of diabetes and more preserved pancreatic islets than the mice that received the individual bacterial strains. Combined administration of L. lactis MG1363 FnBPA+ (pValac::dts::IL-4) and L. lactis MG1363 FnBPA+ (pValac::IL-10) resulted in protection against diabetes in NOD mice. It was shown that the combined treatment with recombinant bacterial by oral route prevented hyperglycemia and reduced the pancreatic islets-destruction in NOD mice. In addition, increased levels of IL-4 and IL-10 in serum and pancreatic tissue revealed a systemic effect of the treatment and also favored an anti-inflammatory microenvironment. Reduced concentrations of IL-12 in pancreas were essential to the regulation of inflammation, resulting in no incidence of diabetes in treated NOD mice. Normal levels of intestinal sIgA after long-term treatment with the L. lactis strains carrying plasmids encoding IL-4 and IL-10 indicate the development of oral tolerance and corroborate the use of this potent tool of mucosal delivery. For the first time, L. lactis MG1363 FnBPA+ strains carrying eukaryotic expression vectors encoding IL-4 and IL-10 are tested in STZ-induced and NOD mouse models. Therefore, our study demonstrates this innovative strategy provides immunomodulatory potential for further investigations in T1D and other autoimmune diseases.

High- and moderate-intensity training modify LPS-induced ex-vivo interleukin-10 production in obese men in response to an acute exercise bout.

The aim of this study was to evaluate the inflammatory (peripheral and lipopolysaccharide (LPS)-stimulated released from whole blood) and metabolic (glucose and insulin) profile of inactive obese men in response to two isoenergetic models of aerobic exercise training (~300  kcal each exercise session). Twenty-two participants (28.7 ± 1.6 years; BMI = 34.4 ± 0.1 kg/m2) were randomized into two groups: I) HIIT: high-intensity interval training (10× 1 bout: 1 min - 100% Maximal Aerobic Velocity) or II) MICT: moderate-intensity continuous training (65% Maximal Aerobic Velocity; kcal equal to HIIT). Both groups trained three times per week for 6-weeks. Fasting blood samples were collected before and 0, 30, and 60 min after exercise during the first and last training sessions for evaluation of: I) MIP-1ɑ, insulin, glucose, visceral and subcutaneous fat depots, oral glucose tolerance test, and homeostatic model assessment of insulin resistance (HOMA-IR) index; II) Peripheral (TNF-α, IL-6, and IL-10) and LPS-stimulated release of TNF-α and IL-10 were analyzed before, 0, and 60 min after sessions. IL-6 concentration remained elevated up to 60-min after the acute exercise session (p < 0.001), and IL-10 concentration was higher after 30 and 60-min (p = 0.001) compared to rest, independent of training period and protocol. AUC of IL-10 presented effect of type of training (p = 0.023) with MICT group showed significantly higher values than the HIIT. The ex-vivo assay showed higher IL-10 secretion in response to LPS immediately (p = 0.003) after both acute MICT and HIIT exercise sessions, independent of training period. Fifteen subjects presented decreased HOMA-IR after 6-weeks and seven presented an increase in this index. When we excluded the two least responsive subjects, it was possible to observe a decrease in HOMA-IR (p = 0.020) after training. Taken together, our results suggest that both HIIT and MICT (with same energy expenditure) promote similar effects on HOMA-IR and led to elevations in IL-10 production in LPS-stimulated whole blood, suggesting that leukocytes had an enhanced ability to secrete anti-inflammatory cytokines after the exercise bout.

IL-10 c.-592C>A (rs1800872) polymorphism is associated with cervical cancer.

PURPOSE: Interleukin-10 (IL-10) is an immunoregulatory cytokine and its cervical and serum concentrations have been associated with a poor prognosis of cervical cancer. The rs1800872 polymorphism (c.-592C>A) in the promotor region of the IL-10 gene affects the production and expression of IL-10 and thus is able to determine the immune response profile in the cervix. Therefore, the aim of this work is to state the association between IL-10 c.-592C>A polymorphism and cervical cancer. METHODS: Genomic DNA was extracted from patient's peripheral blood and tumor biopsy. Socio-demographic, sexual behavior and reproductive characteristics data were collected using a questionnaire. RESULTS: Co-dominant model in logistic binary regression adjusted for confounders, showed that patients presenting with C/A genotype had 2.15 times more chances for developing cervical cancer (OR 2.15; CI95% 1.02-4.56). The dominant model, C/A + A/A, was also independently associated with 2.71 times more chances for cervical cancer development when compared to control patients (OR 2.71; CI95% 1.05-4.47). CONCLUSION: Our study analyses show the association between cervical cancer and IL-10 c.-592C>A polymorphism, demonstrating that the allele A presence was independently associated with higher risks of cervical cancer development.

Proinflammatory and regulatory mechanisms in allergic contact dermatitis caused by methylchloroisothiazolinone and methylisothiazolinone.

BACKGROUND: Methylchloroisothiazolinone (MCI) and methylisothiazolinone (MI) are the cause of an increasing number of contact allergies. Understanding the mechanisms by which MCI/MI induces proinflammatory and regulatory factors production is necessary to understand the outcome of allergic contact dermatitis (ACD). OBJECTIVES: To evaluate the dysfunction of proinflammatory cytokines and regulatory factors in the positive MCI/MI patch test at the transcriptional and protein expression levels. Moreover, to analyse the cytokines production induced by MI in peripheral blood mononuclear cells (PBMCs). MATERIALS AND METHODS: The selected patients had positive MCI/MI patch test results. The expression of proinflammatory factors was evaluated by q-PCR and immunochemistry at 48 hours of positive MCI/MI patch test. The MCI/MI- or MI- induced secretion of IL-1ß, TNF and IL-6 by PBMC was analysed by flow cytometry. RESULTS: The results showed a decreased TLR4 expression with upregulated IL6, FOXP3, IL10 and TGFß mRNA expression as assessed by q-PCR at the site of the MCI/MI skin reaction. We detected increased protein levels of TLR4, FOXP3 and IL-10 in the dermis layer in the ACD reaction by immunocitochemistry. Moreover, MCI/MI induced proinflammatory cytokine production by PBMC through the NF-κB signalling pathway. CONCLUSION: Considering the altered innate immune response triggered by MCI/MI sensitization, these findings indicate that the regulatory process at the induction phase of ACD is a crucial mechanism. Given the increase in occupational and domestic exposure to MCI/MI, the underlying immunological mechanisms should be understood.

Atorvastatin inhibits IL-17A, TNF, IL-6, and IL-10 in PBMC cultures from patients with severe rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint damage, and it may present with comorbidities at the systemic level. The Th1/Th2/Th17 CD4+ lymphocyte imbalance produces inflammatory cytokines, which begin to act, injuring joint tissue. Atorvastatin is a cholesterol- lowering drug with a range of biological effects including anti-inflammatory potential. Patients with rheumatoid arthritis who used statins exhibited clinical improvement. However, the mechanism is not fully understood. Therefore, we aimed to evaluate the RA immunomodulatory activity of atorvastatin. METHODS: Peripheral blood mononuclear cells (PBMCs) of RA patients and healthy donors were exposed to atorvastatin in different concentrations following a cytotoxicity assay. Th1, Th2, and Th17 cytokines profiles were evaluated in the culture supernatant by cytometric bead array (CBA). Data were analyzed using the Wilcoxon test, and differences were considered significant when p < 0.05. RESULTS: Atorvastatin showed no toxicity at the tested doses in RA PBMC cultures, and at 10µM, it showed the most significant results, reducing IL-17A (p = 0.002), TNF (p = 0.002), and IL-6 (p = 0.008) supernatant levels. The outcomes also revealed that only patients with more severe disease activity and those sensitive to corticoid treatments were responders to atorvastatin in vitro. CONCLUSION: These findings suggest the potential immunomodulatory action of atorvastatin as a mechanism in rheumatoid arthritis treatment.

Photoactivated resveratrol controls intradermal infection by Staphylococcus aureus in mice: a pilot study.

Staphylococcus aureus is one of the main causative agent of infections acquired in both community and hospital environment. In this context, photodynamic therapy (PDT) consists in using a photosensitizer that, activated by light, evokes the formation of reactive oxygen species (ROS), which lead to the death of microorganisms due to oxidative damage; it is useful tool since this action, harmful to pathogens, does not significantly injure human cells. In view of this, this work proposes a more in-depth study on the use of resveratrol (RSV) as a possible photosensitizer. It was observed, in the intradermal infection model in animals' ear dermis, that photoactivated resveratrol promotes an increase in myeloperoxidase expression with reduced bacterial load in the draining lymph node. Besides that, the draining lymph node of the animals treated with photoactivated RSV controls inflammation through IL-10 production. These are pioneers data and this work being a pilot study; then, other works must be conducted with the objective of elucidate the photoactivated resveratrol mechanism of action.

IgG from atopic dermatitis patients induces non-atopic infant thymic invariant natural killer T (iNKT) cells to produce IL-4, IL-17, and IL-10.

BACKGROUND: Atopic dermatitis (AD) pathogenesis still needs to be elucidated, but invariant natural killer T (iNKT) cell involvement was already described by several groups. Our group has demonstrated that IgG antibodies purified from AD patients can modulate cytokine production by thymic T cells. Here we aimed to investigate if IgG from AD patients can modulate infant non-atopic thymic iNKT cells cytokine production in order to collaborate with the elucidation of AD development in infancy. METHODS: Thymic tissues were obtained from children from non-atopic mothers, and IgG was purified from AD patients diagnosed as moderate or severe and, as controls, from subjects clinically classified as non-atopic individuals. PBMCs from non-atopic individuals were also used in this study. RESULTS: Our results demonstrated that IgG from AD patients could induce non-atopic children thymic iNKT cells to produce higher levels of intracellular IL-4, IL-10, and IL-17 when compared to all control conditions. No effect was observed in non-atopic adults peripheral iNKT. We also observed that IgG from AD patients induces an increase in the expression of CD4 and Rorγt transcription factor in non-atopic children thymic iNKT cells compared to the condition of all controls. CONCLUSIONS: These observations suggest that IgG from AD patients can induce a cytokine profile by thymic iNKT cells from non-atopic infants compatible with the observations in AD development, which can collaborate with the elucidation of AD pathogenesis.

Oral administration of EPA-rich oil impairs collagen reorganization due to elevated production of IL-10 during skin wound healing in mice.

Wound healing is an essential process for organism survival. Some fatty acids have been described as modulators of wound healing. However, the role of omega-3 fatty acids is unclear. In the present work, we investigate the effects of oral administration of eicosapentaenoic acid (EPA)-rich oil on wound healing in mice. After 4 weeks of EPA-rich oil supplementation (2 g/kg of body weight), mice had increased serum concentrations of EPA (20:5ω-3) (6-fold) and docosahexaenoic acid (DHA; 22:6ω-3) (33%) in relation to control mice. Omega-3 fatty acids were also incorporated into skin in the EPA fed mice. The wound healing process was delayed at the 3rd and 7th days after wounding in mice that received EPA-rich oil when compared to control mice but there was no effect on the total time required for wound closure. Collagen reorganization, that impacts the quality of the wound tissue, was impaired after EPA-rich oil supplementation. These effects were associated with an increase of M2 macrophages (twice in relation to control animals) and interleukin-10 (IL-10) concentrations in tissue in the initial stages of wound healing. In the absence of IL-10 (IL-10-/- mice), wound closure and organization of collagen were normalized even when EPA was fed, supporting that the deleterious effects of EPA-rich oil supplementation were due to the excessive production of IL-10. In conclusion, oral administration of EPA-rich oil impairs the quality of wound healing without affecting the wound closure time likely due to an elevation of the anti-inflammatory cytokine IL-10.

T-Cell Immunophenotyping and Cytokine Production Analysis in Patients with Chagas Disease 4 Years after Benznidazole Treatment.

The major problem with Chagas disease is evolution of the chronic indeterminate form to a progressive cardiac disease. Treatment diminishes parasitemia but not clinical progression, and the immunological features involved are unclear. Here, we studied the clinical course and the immune response in patients with chronic-phase Chagas disease at 48 months after benznidazole treatment. Progression to the cardiac form of Chagas disease or its aggravation was associated with higher in vitro antigen-specific production of interferon gamma (IFN-γ) in patients with cardiac Chagas disease than in patients with the indeterminate form. Predominance of IFN-γ production over interleukin-10 (IL-10) production in antigen-specific cultures was associated with cardiac involvement. Significantly higher numbers of antigen-specific T helper 1 cells (T-Bet+ IFN-γ+) and a significantly higher IFN-γ+/IL-10+ ratio were observed in patients with cardiac Chagas disease than in patients with the indeterminate form. Cardiac damage was associated with higher numbers of T helper cells than cytotoxic T lymphocytes producing IFN-γ. Patients with cardiac Chagas disease had predominant CD25- and CD25low T regulatory (Treg) subpopulations, whereas patients with the indeterminate form manifested a higher relative mean percentage of CD25high Treg subpopulations. These findings suggest that at 48 months after benznidazole treatment, the disease can worsen or progress to the cardiac form. The progression may be related to increased IFN-γ production (mostly from CD4+ T cells) relative to IL-10 production and increased Treg percentages. Patients with the indeterminate form of Chagas disease show a more balanced ratio of proinflammatory and anti-inflammatory cytokines.

Leishmania braziliensis Infection Enhances Toll-Like Receptors 2 and 4 Expression and Triggers TNF-α and IL-10 Production in Human Cutaneous Leishmaniasis.

Cutaneous leishmaniasis (CL) caused by infection with Leishmania braziliensis is characterized by an exaggerated inflammatory response that controls the parasite burden, but also contributes to pathology. While myeloid cells are required to eliminate the parasite, recent studies indicate that they may also participate in the inflammatory response driving disease progression. The innate immune response to leishmania is driven in part by the Toll-like receptors (TLRs) TLR2, TLR4, and TLR9. In this study, we used flow cytometric analysis to compare TLR2 and TLR4 expression in monocyte subsets (classical, intermediate, and non-classical) from CL patients and healthy subjects (HS). We also determined if there was an association of either the pro-inflammatory cytokine TNF or the anti-inflammatory cytokine IL-10 with TLR2 or TLR4 expression levels after L. braziliensis infection. In vitro infection with L. braziliensis caused CL monocytes to up-regulate TLR2 and TLR4 expression. We also found that intermediate monocytes expressed the highest levels of TLR2 and TLR4 and that infected monocytes produced more TNF and IL-10 than uninfected monocytes. Finally, while classical and intermediate monocytes were mainly responsible for TNF production, classical monocytes were the main source of IL-10. Collectively, our studies revealed that up-regulated TLR2/4 expression and TNF production by intermediate/inflammatory subsets of monocytes from patients correlates with detrimental outcome of cutaneous leishmaniasis.

Interferon-Beta Treatment Differentially Alters TLR2 and TLR4-Dependent Cytokine Production in Multiple Sclerosis Patients.

OBJECTIVE: Multiple sclerosis (MS) is a multifactorial chronic disease that affects the central nervous system (CNS). Toll-like receptors (TLRs) play a central role in cytokine production after pathogen- and danger-associated molecular patterns (PAMPs and DAMPs) and contribute to CNS damage in MS patients. Here, we evaluated the effects of interferon (IFN)-ß treatment in TLR2 and TLR4-dependent cytokine production and mRNA expression in whole-blood cell cultures from MS patients. METHODS: We evaluated cytokine production by ELISA from whole-blood cell culture supernatants and mRNA expression by real-time polymerase chain reaction in peripheral blood mononuclear cells (PBMCs). RESULTS: In patients treated with IFN-ß, tumor necrosis factor (TNF)-α production after exposure to TLR2 agonist (Pam3Cys) was lower than in healthy controls and untreated MS patients. However, IFN-ß treatment had no significant effect on TNF-α production after TLR4 agonist (LPS) stimulation. On the other hand, interleukin (IL)-10 production was increased in TLR4- but not in TLR2-stimulated whole-blood cell culture from MS patients under IFN-ß treatment when compared to the controls. No differences in TNF-α or IL-10 mRNA expression in PBMCs from healthy controls and untreated or treated MS patients were detected, although PBMCs from treated patients presented higher levels of IL-32γ mRNA than those from controls. CONCLUSIONS: Our data suggest that IFN-ß treatment alters the TLR-dependent immune response of PBMCs from MS patients. This may contribute to the beneficial effects of IFN-ß treatment.

Myeloid-Derived Suppressor Cells Show Different Frequencies in Diabetics and Subjects with Arterial Hypertension.

Type 2 diabetes mellitus (DM2) is strongly associated with other comorbidities such as obesity, atherosclerosis, and hypertension. Obesity is associated with sustained low-grade inflammatory response due to the production of proinflammatory cytokines. This inflammatory process promotes the differentiation of some myeloid cells, including myeloid-derived suppressor cells (MDSCs). In this study, two groups of individuals were included: DM2 patients and non-DM2 individuals with similar characteristics. Immunolabeling of CD15+ CD14- and CD33+ HLA-DR-/low was performed from whole peripheral blood, and samples were analyzed by flow cytometry, and frequencies of MDSCs and the relationship of these with clinical variables, cytokine profile (measured by cytometric bead array), and anthropometric variables were analyzed. The frequency of CD33+ HLA-DR-/low MDSCs (that produce IL-10 and TGF-ß, according to an intracellular detection) is higher in patients with DM2 (P < 0.05), and there is a positive correlation between the frequency of CD15+ CD14- and CD33+ HLA-DR-/low MDSC phenotypes. DM2 patients have an increased concentration of serum IL-5 (P < 0.05). Also, a negative correlation between the frequency of CD15+ CD14- MDSCs and LDL cholesterol was found. Our group of DM2 patients have an increased frequency of mononuclear MDSC CD33+ HLA-DR-/low that produce TGF-ß and IL-10. These cytokines have been associated with immune modulation and reduced T cell responses. DM2 and non-DM2 subjects show a similar cytokine profile, but the DM2 patients have an increased concentration of IL-5.

Human CD8 T-cell activation in acute and chronic chikungunya infection.

There is a need for more detailed elucidation of T-cell immunity in chikungunya infection. CD8 T cells are one of main actors against viruses. Here, we analysed CD8+ T lymphocytes from patients in the acute and chronic phases of chikungunya disease (CHIKD). Our results demonstrate that CD8+ T cells expressed higher ex vivo granzyme B, perforin and CD107A expression in patients in the acute phase of CHIKD compared with healthy individuals and higher ex vivo expression of CD69, interleukin-17A, interleukin-10 and CD95 ligand, and co-expression of CD95/CD95 ligand. These results elucidate the importance of these lymphocytes, demonstrating immune mechanisms mediated in human chikungunya infection.

Vaccination with a CD4+ and CD8+ T-cell epitopes-based recombinant chimeric protein derived from Leishmania infantum proteins confers protective immunity against visceral leishmaniasis.

Vaccination seems to be the best approach to control visceral leishmaniasis (VL). Resistance against infection is based on the development of a Th1 immune response characterized by the production of interferons-γ (IFN-γ), interleukin-12 (IL-12), granulocyte-macrophage-colony-stimulating factor (GM-CSF), and tumor necrosis factor-α (TNF-α), among others. A number of antigens have been tested as potential targets against the disease; few of them are able to stimulate human immune cells. In the present study, 1 prediction of MHC class I and II molecules-specific epitopes in the amino acid sequences of 3 Leishmania proteins: 1 hypothetical, prohibitin, and small glutamine-rich tetratricopeptide repeat-containing proteins, was performed using bioinformatics tools, and a T-cell epitopes-based recombinant chimeric protein was constructed, synthetized and purified to be evaluated in invitro and in vivo experiments. The purified protein was tested regarding its immunogenicity in peripheral blood mononuclear cells (PBMCs) from healthy subjects and VL patients, as well as to its immunogenicity and protective efficacy in a murine model against Leishmania infantum infection. Results showed a Th1 response based on high IFN-γ and low IL-10 levels derived from in chimera-stimulated PBMCs in both healthy subjects and VL patients. In addition, chimera and/or saponin-immunized mice presented significantly lower parasite burden in distinct evaluated organs, when compared to the controls, besides higher levels of IFN-γ, IL-2, IL-12, and GM-CSF, and an IgG2a isotype-based humoral response. In addition, the CD4+ and CD8+ T-cell subtypes contributed to IFN-γ production in the protected animals. The results showed the immunogenicity in human cells and the protective efficacy against L. infantum in a murine model, and well indicate that this recombinant chimera can be considered as a promising strategy to be used against human disease.

IgG from atopic dermatitis patients induces IL-17 and IL-10 production in infant intrathymic TCD4 and TCD8 cells.

INTRODUCTION: Our group recently demonstrated that IgG modulates αßT cell cytokine production during the maturation process in the human thymus. The effects of this modulation are IgG repertoire dependent and can exert a systemic and long-term impact. OBJECTIVE: To investigate whether IgG from atopic dermatitis (AD) patients can modulate cytokine production of infant intrathymic TCD4 and TCD8 cells in vitro. METHODS: Thymic tissues were obtained from newborn children from nonatopic mothers, and thymocytes were cultured for 6 days with purified IgG from AD patients or with intravenous immunoglobulin (IVIG) or mock conditions as controls. Cells were gated as double positive T cells (TDP- CD4+ CD8+ ), TCD4 cells (CD4+ CD8- ), or TCD8 cells (CD4- CD8+ ), and intracellular levels of IL-17A, IFN-γ, TNF-α, IL-4, IL-10, and TGF-ß were evaluated by flow cytometry. RESULTS: Compared to mock and IVIG culture conditions, IgG of AD individuals induced in vitro intracellular production of IL-17 and IL-10 by intrathymic TDP, TCD4, and TCD8 cells of infants. TGF-ß was also detected at a higher frequency in response to AD IgG in TDP and TCD8 cells compared to mock and IVIG cultured conditions. An opposite effect was detected upon IFN-γ production in TCD4 cells, such that AD IgG reduced IFN-γ production compared to production under mock conditions but not under IVIG conditions. CONCLUSION: IgG of AD patients can stimulate cytokine production in infant thymocytes and thus resembles the peripheral profile observed in adults. These findings suggest a novel mechanism that can contribute to AD pathogenesis.

Cytotoxic Effects and Production of Cytokines Induced by the Endophytic Paenibacillus polymyxa RNC-D In Vitro.

BACKGROUND: Prominent among all the organisms that have a potential value for the production of new medicines, are endophytes, fungi and bacteria that live inside plants without harming them. In this study, a total lyophilized extract (TLE) of Paenibacillus polymyxa RNC-D was used. The P. polymyxa lineages are known for their capacity to segregate a large number of extracellular enzymes and bioactive substances. METHODS: The TLE of Paenibacillus polymyxa RNC-D was tested in cell viability assays for cytotoxicity and cytokine production in BALB/3T3 and J774A.1 cell lineages. RESULTS: A 50% mortality rate of fibroblasts (BALB/3T3) was observed in the 1.171±0.161 mg/mL and 0.956±0.112 mg/mL doses after 48 and 72 hours, respectively, as well as a 50% mortality rate of macrophage cells (J774A.1) in the 0.994±0.170 mg/mL and 0.945±0.280 mg/mL doses after 48 and 72 hours, respectively. The ≈1 mg/mL concentration significantly affected the kinetic of growth in all the measured periods. The extract induced apoptosis and necrosis 24 hours after the ≈1 mg/mL concentration in both tested lineages. The treatment with the ≈1 mg/mL concentration led to the production of TNF-α and IFN-γ cytokines in 24 hours. IL-12 and IL-10 began to be detected as a result of the treatment with 0.1 mg/mL. However, with the 0.5 mg/mL dose in 24 hours, a significant reduction in IL-10 was observed. CONCLUSION: Our data suggest that the TLE of P. polymyxa RNC-D modulated the production of cytokines with different patterns of immune response in a dose-dependent way.

Long-term cell culture isolated from lesions of mice infected with Leishmania amazonensis: a new approach to study mononuclear phagocyte subpopulations during the infection.

Leishmanioses are neglected diseases and the parasite Leishmania survives and proliferates within mononuclear phagocytes, particularly macrophages. In vitro studies of the immunology and cell biology of leishmaniosis are performed in murine peritoneum and bone marrow macrophages and immortalized cell lines despite the normal and injured tissue-specific heterogeneity of macrophages. In this work, we established an ex vivo methodology to culture lesional cells from BALB/c mice infected with Leishmania amazonensis. The cells were successfully isolated from footpad skin lesions and those exhibiting macrophage morphology were maintained in long-term culture (12 days), while the small number of lymphocytes, polymorphonuclear and unidentified cells died after 1 day of culture. The frequency of infected cells decreased over 2 days. Most lesional cells cultivated ex vivo were myeloid CD11b+ CD14+ F4/80+ CD68+ cells. Low levels of IFN-γ and IL-4, IL-10 production and low arginase and phagocytic activities were detected in ex vivo lesional cell cultures. The ex vivo model developed in this study open perspectives for studying the biology of leishmanial lesions in cellular subpopulations and at the single-cell level.