Luanda Mara da Silva Oliveira

Profile of skin barrier proteins (filaggrin, claudins 1 and 4) and Th1/Th2/Th17 cytokines in adults with atopic dermatitis

Atopic dermatitis (AD) in adults and profile of skin barrier proteins and inflammatory cytokines.

TLR7/TLR8 Activation Restores Defective Cytokine Secretion by Myeloid Dendritic Cells but Not by Plasmacytoid Dendritic Cells in HIV-Infected Pregnant Women and Newborns.

Mother-to-child transmission (MTCT) of HIV-1 has been significantly reduced with the use of antiretroviral therapies, resulting in an increased number of HIV-exposed uninfected infants. The consequences of HIV infection on the innate immune system of both mother-newborn are not well understood. In this study, we analyzed peripheral blood and umbilical cord blood (CB) collected from HIV-1-infected and uninfected pregnant women. We measured TNF-α, IL-10 and IFN-α secretion after the stimulation of the cells with agonists of both extracellular Toll-like receptors (TLRs) (TLR2, TLR4 and TLR5) and intracellular TLRs (TLR7, TLR7/8 and TLR9). Moreover, as an indicator of the innate immune response, we evaluated the responsiveness of myeloid dendritic cells (mDCs) and plasmacytoid DCs (pDCs) to TLRs that are associated with the antiviral response. Our results showed that peripheral blood mononuclear cells (PBMCs) from HIV-1-infected mothers and CB were defective in TNF-α production after activation by TLR2, TLR5, TLR3 and TLR7. However, the TNF-α response was preserved after TLR7/8 (CL097) stimulation, mainly in the neonatal cells. Furthermore, only CL097 activation was able to induce IL-10 and IFN-α secretion in both maternal and CB cells in the infected group. An increase in IFN-α secretion was observed in CL097-treated CB from HIV-infected mothers compared with control mothers. The effectiveness of CL097 stimulation was confirmed by observation of similar mRNA levels of interferon regulatory factor-7 (IRF-7), IFN-α and TNF-α in PBMCs of both groups. The function of both mDCs and pDCs was markedly compromised in the HIV-infected group, and although TLR7/TLR8 activation overcame the impairment in TNF-α secretion by mDCs, such stimulation was unable to reverse the dysfunctional type I IFN response by pDCs in the HIV-infected samples. Our findings highlight the dysfunction of innate immunity in HIV-infected mother-newborn pairs. The activation of the TLR7/8 pathway could function as an adjuvant to improve maternal-neonatal innate immunity.

Distinct natural killer cells in HIV-exposed seronegative subjects with effector cytotoxic CD56(dim) and CD56(bright) cells and memory-like CD57⁺NKG2C⁺CD56(dim) cells.

Background:Innate immunity, including natural killer (NK) cells, may play a significant role in maintaining natural resistance to infection in highly HIV-exposed seronegative (HESN) subjects. The differences between NK-cell subsets, regarding their activating/maturing marker expression and their memory markers, in HESN subjects are not fully defined. Methods:We have conducted an analysis of the activating/memory markers and intracellular CD107a and interferon &ggr; (IFN-&ggr;) expression in NK-cell subsets from HESN and HIV-infected and healthy subjects. Results:HESN individuals showed an increased expression of activating markers, such as NKG2D in CD56bright and CD56dim NK cells, and an increased frequency of CD56brightCD127+ and fully mature CD56dimCD57+ NK cells compared with HIV-infected patients and healthy control subjects. Of note, HESN individuals showed an increased frequency of memory CD56dimCD57+ NK cells, and this is known to be expanded on cytomegalovirus infection, as evidenced by their high rate of cytomegalovirus seropositivity. Simultaneous expression of the CD94, NKG2A, NKG2C, and NKG2D receptors on CD56bright NK cells was detected in HESN subjects, whereas in the HIV-1 group, the expression of these 4 receptors was enhanced in CD56dim NK cells. It was also found that CD56bright and CD56dim NK cells in HESN subjects showed increased CD107a and/or IFN-&ggr; expression. Conclusions:The NK cells from HESN individuals presented a unique activation profile, with increased expression of NKG2D, CD107a, and IFN-&ggr; and “memory” CD57+CD56dim NK cells. The complex network of functional NK-cell activities in HESN individuals may be exploited for long-term protection through vaccination.

Upregulation of Innate Antiviral Restricting Factor Expression in the Cord Blood and Decidual Tissue of HIV-Infected Mothers

Programs for the prevention of mother-to-child transmission of HIV have reduced the transmission rate of perinatal HIV infection and have thereby increased the number of HIV-exposed uninfected (HEU) infants. Natural immunity to HIV-1 infection in both mothers and newborns needs to be further explored. In this study, we compared the expression of antiviral restricting factors in HIV-infected pregnant mothers treated with antiretroviral therapy (ART) in pregnancy (n=23) and in cord blood (CB) (n=16), placental tissues (n=10-13) and colostrum (n=5-6) samples and compared them to expression in samples from uninfected (UN) pregnant mothers (n=21). Mononuclear cells (MNCs) were prepared from maternal and CB samples following deliveries by cesarean section. Maternal (decidua) and fetal (chorionic villus) placental tissues were obtained, and colostrum was collected 24 h after delivery. The mRNA and protein expression levels of antiviral factors were then evaluated. We observed a significant increase in the mRNA expression levels of antiviral factors in MNCs from HIV-infected mothers and CB, including the apolipoprotein B mRNA-editing enzyme 3G (A3G), A3F, tripartite motif family-5α (TRIM-5α), TRIM-22, myxovirus resistance protein A (MxA), stimulator of interferon (IFN) genes (STING) and IFN-β, compared with the levels detected in uninfected (UN) mother-CB pairs. Moreover, A3G transcript and protein levels and α-defensin transcript levels were decreased in the decidua of HIV-infected mothers. Decreased TRIM-5α protein levels in the villi and increased STING mRNA expression in both placental tissues were also observed in HIV-infected mothers compared with uninfected (UN) mothers. Additionally, colostrum cells from infected mothers showed increased tetherin and IFN-β mRNA levels and CXCL9 protein levels. The data presented here indicate that antiviral restricting factor expression can be induced in utero in HIV-infected mothers. Future studies are warranted to determine whether this upregulation of antiviral factors during the perinatal period has a protective effect against HIV-1 infection.

Increased frequency of circulating Tc22/Th22 cells and polyfunctional CD38 − T cells in HIV-exposed uninfected subjects

Some individuals are resistant to HIV-1 infection despite repeated exposure to the virus, suggesting the presence of a complex antiviral response. Innate factors like IL-22 exert gut mucosal protection and polyfunctional T cells have been associated with low progression in HIV infection; therefore, we evaluated the frequencies of CD4+ and CD8+ T cell-secreting cytokines, including Tc22/Th22 cells and polyfunctional T cells in HIV-1-exposed uninfected individuals (EUs), their HIV-1-infected partners and healthy controls. EUs exhibited an increased frequency of p15 Gag CD4+ IL-22+ secreting T cells, whereas HIV-infected partners demonstrated a high frequency of CD4+ IL-17+ T cells in response to p24. Similar responses of Th22 and Tc22 cells to Gag peptides and Staphylococcal enterotoxin B (SEB) stimulation were detected in the serodiscordant couples. However, polyfunctionality in HIV subjects was associated with an HIV Gag response of CD38+ T cells, whereas polyfunctionality for EUs was induced upon SEB stimulation by CD38- T cells. EUs demonstrated the presence of Tc22/Th22 cells and polyfunctional CD38- T cells with a low activation profile. These data suggest that SEB-induced polyfunctional CD4+ and CD8+ T cells together with Tc22/Th22 cells in EU individuals can provide an immunological advantage in the response to pathogens such as HIV-1.

Polyfunctional natural killer cells with a low activation profile in response to Toll-like receptor 3 activation in HIV-1-exposed seronegative subjects

Natural killer (NK) cells are the main mediator of the cytotoxic response in innate immunity and may be involved in resistance to HIV-1 infection in exposed seronegative (ESN) individuals. Toll-like receptor (TLR) signalling is crucial for NK cell activation. Here, we investigated the polyfunctional NK cell response to TLR3 activation in serodiscordant couples. ESN subjects showed increased IFN-γ and CD107a expression in both NK subsets, CD56bright and CD56dim cells, in response to stimulation with a TLR3 agonist, while expression was impaired in the HIV-1-infected partners. TLR3-induced expression of IFN-γ, TNF and CD107a by polyfunctional CD56bright NK cells was more pronounced in ESN individuals than that in healthy controls. Activated NK cells, as determined by CD38 expression, were increased only in the HIV-1-infected partners, with reduced IFN-γ and CD107a expression. Moreover, CD38+ NK cells of the HIV-1-infected partners were associated with increased expression of inhibitory molecules, such as NKG2A, PD-1 and Tim-3, while NK cells from ESN subjects showed decreased NKG2A expression. Altogether, these findings indicate that NK cells of ESN individuals were highly responsive to TLR3 activation and had a polyfunctional NK cell phenotype, while the impaired TLR3 response in HIV-1-infected partners was associated with an inhibitory/exhaustion NK cell phenotype.

Antiviral factors and type I/III interferon expression associated with regulatory factors in the oral epithelial cells from HIV-1-serodiscordant couples

Individuals who remain HIV-seronegative despite repeated unprotected exposure to the virus are defined as exposed seronegative (ESN) individuals. Innate and adaptive immunity, as well as genetic factors, provide ESNs with important advantages that allow for low infection susceptibility. The majority of HIV-1-infected individuals undergo antiretroviral therapy, which can decrease the level of HIV-1 exposure in ESNs. We analyzed type I interferon (IFN)-related antiviral and regulatory factors in peripheral blood mononuclear cells (PBMCs) and oral epithelial cells from serodiscordant couples. Our findings revealed that ESNs did not induce the expression of antiviral factors (APOBEC-3G, TRIM5-α, SAMDH1, STING, TBk1) or regulatory factors (Trex, Foxo3, Socs3, IL-10) in PBMCs, unlike their HIV-1-infected partners. In contrast, ESNs upregulated APOBEC-3G and type I/III IFNs (IFNs-α,-β/-λ) in oral mucosal epithelial cells similar to their HIV-infected partners. The serodiscordant groups exhibited an increased expression of type I IFN-induced regulators, such as Trex and Foxo3, in oral epithelial cells. TLR7, TLR8 and TLR9 were expressed in oral epithelial cells of both ESNs and HIV-1-infected subjects. These findings revealed evidence of antiviral factors, type I/III interferon and regulatory factor expression only in the oral mucosal compartment of ESNs, while HIV-1-infected partners systemically and oral mucosal expressed the antiviral profile.

Dendritic cells primed with a chimeric plasmid containing HIV-1-gag associated with lysosomal-associated protein-1 (LAMP/gag) is a potential therapeutic vaccine against HIV

The decline in number and function of T cells is a hallmark of HIV infection, and preservation or restoration of HIV‐specific cellular immune response is a major goal of AIDS treatment. Dendritic cells (DCs) play a key role in the initiation and maintenance of the immune response, and their use as a vaccine vehicle is a promising strategy for enhancing vaccine efficacy. We evaluated the potential of DC‐mediated immunization with a DNA vaccine consisting of HIV‐1‐p55gag (gag, group‐specific antigen) associated to lysosomal associated protein (LAMP) sequence (LAMP/gag vaccine). Immunization of mice with mouse DCs transfected with LAMP/gag (Lg‐mDCs) stimulated more potent B‐ and T‐cell responses than naked DNA or DCs pulsed with inactivated HIV. Anti‐Gag antibody levels were sustained for at least 3 mo after immunization, and recall T‐cell responses were also strongly detected at this time point. Human DCs transfected with LAMP/gag (Lg‐hDCs) were also activated and able to stimulate greater T‐cell response than native gag‐transfected DCs. Coculture between Lg‐hDCs and T lymphocytes obtained from patients with HIV resulted in upregulation of CD38, CD69, HLA‐DR, and granzyme B by CD4+ and CD8+ T cells, and increased IFN‐γ and TNF‐α production. These results indicate that the use of LAMP/gag‐DC may be an efficient strategy for enhancing immune function in patients with HIV.—Lucas, C. G. D. O., Matassoli, F. L., Peçanha, L. M. T., Santillo, B. T., Oliveira, L. M. D. S., Oshiro, T. M., Marques, E. T. D. A., Jr., Oxenius, A., de Arruda, L. B. Dendritic cells primed with a chimeric plasmid containing HIV‐1‐gag associated with lysosomal‐associated protein‐1 (LAMP/gag) is a potential therapeutic vaccine against HIV. FASEB J. 30, 2970‐2984 (2016). www.fasebj.org

Toll-like receptor agonists partially restore the production of pro-inflammatory cytokines and type I interferon in Sézary syndrome

Sézary syndrome (SS) carries a poor prognosis, and infections represent the most frequent cause of death in SS patients. Toll-like receptors (TLRs) are a family of innate immune receptors that induce protective immune responses against infections. We sought to evaluate the ability of TLR agonists to induce inflammatory cytokine, Th2 cytokine, and type I interferon (IFN-I) production by peripheral blood mononuclear cells (PBMC) of untreated SS patients. We detected impaired IL-6, IL-10 and IL-13 secretion by PBMC induced by the agonists for TLR5, TLR3, TLR7 and TLR9 in SS patients, while it was partially recovered by TLR2/TLR4 and TLR7/8 agonists TNF secretion was restored following stimulation with TLR2/TLR4 agonists. IFN-γ was scarcely produced upon TLR activation in SS cells, albeit TLR 7/8 (CL097) enhanced their secretion at lower levels than the control group. TLR9 agonist efficiently induced IFN-I in SS patients, although this positive regulation was not observed for other cytokines, in direct contrast to the broad activity of CL097. Among the TLR agonists, TLR4 was able to induce pro-inflammatory, IL-10 and Th2 secretion, while TLR7-8 agonist induced the inflammatory cytokines, IFN-I and IFN-γ. These findings reveal a dysfunctional cytokine response upon both extracellular and intracellular TLR activation in SS patients, which was partially restored by TLRs agonists.

Staphylococcus aureus enterotoxins modulate IL-22-secreting cells in adults with atopic dermatitis

Atopic dermatitis (AD) is a chronic inflammatory immune-mediated skin disease characterized by skin colonization by Staphylococcus aureus. Interleukin (IL)-22, in cooperation with IL-17, triggers antimicrobial peptide elaboration and enhances certain immunological responses. In AD, IL-22 is related to epidermal hyperplasia, keratinocyte apoptosis, and inhibition of antimicrobial peptide (AMP) production. We aimed to evaluate the impact of staphylococcal enterotoxins on the Tc22/Th22 induction in the peripheral blood of AD patients and on CD4+/CD8+ T cells expressing IL-22 in AD skin. Our study showed inhibition of the staphylococcal enterotoxins A and B (SEA and SEB) response by Th22 (CD4+IL-22+IL-17A−IFN-γ−) cells in AD patients. In contrast, Tc22 (CD8+IL-22+IL-17A−IFN-γ−) cells were less susceptible to the inhibitory effects of staphylococcal enterotoxins and exhibited an enhanced response to the bacterial stimuli. In AD skin, we detected increased IL-22 transcript expression and T lymphocytes expressing IL-22. Together, our results provide two major findings in response to staphylococcal enterotoxins in adults with AD: dysfunctional CD4+ IL-22 secreting T cells and increased Tc22 cells. Our hypothesis reinforces the relevance of CD8 T cells modulated by staphylococcal enterotoxins as a potential source of IL-22 in adults with AD, which is relevant for the maintenance of immunological imbalance.