Paula A. Velilla

Functional Regulatory T Cells Accumulate in Aged Hosts and Promote Chronic Infectious Disease Reactivation

Declines in immune function are well described in the elderly and are considered to contribute significantly to the disease burden in this population. Regulatory T cells (Tregs), a CD4+ T cell subset usually characterized by high CD25 expression, control the intensity of immune responses both in rodents and humans. However, because CD25 expression does not define all Tregs, especially in aged hosts, we characterized Tregs by the expression of FOXP3, a transcription factor crucial for Treg differentiation and function. The proportion of FOXP3+CD4+ Tregs increased in the blood of the elderly and the lymphoid tissues of aged mice. The expression of functional markers, such as CTLA-4 and GITR, was either preserved or increased on FOXP3+ Tregs from aged hosts, depending on the tissue analyzed. In vitro depletion of peripheral Tregs from elderly humans improves effector T cell responses in most subjects. Importantly, Tregs from old FoxP3-GFP knock-in mice were suppressive, exhibiting a higher level of suppression per cell than young Tregs. The increased proportion of Tregs in aged mice was associated with the spontaneous reactivation of chronic Leishmania major infection in old mice, likely because old Tregs efficiently suppressed the production of IFN-γ by effector T cells. Finally, in vivo depletion of Tregs in old mice attenuated disease severity. Accumulation of functional Tregs in aged hosts could therefore play an important role in the frequent reactivation of chronic infections that occurs in aging. Manipulation of Treg numbers and/or activity may be envisioned to enhance the control of infectious diseases in this fragile population.

Immunological characterization of compensatory anti-inflammatory response syndrome in patients with severe sepsis: a longitudinal study*.

Objectives:To perform a complete immunological characterization of compensatory anti-inflammatory response syndrome in patients with sepsis and to explore the relationship between these changes and clinical outcomes of 28-day mortality and secondary infections. Design:Prospective single-center study conducted between April 2011 and December 2012. Setting:ICUs from Hospital Universitario San Vicente Fundación at Medellin, Colombia. Patients:One hundred forty-eight patients with severe sepsis. Interventions:None. Measurements and Main Results:At days 0, 1, 3, 5, 10, and 28, we determined the expression of HLA-DR in monocytes and the apoptosis and the proliferation index in T lymphocytes, as well as the levels of tumor necrosis factor-&agr;, interleukin-6, interleukin-1&bgr;, interleukin-10, and transforming growth factor-&bgr; in both plasma and cell culture supernatants of peripheral blood mononuclear cells. The mean percentage of HLA-DR+ was 60.7 at enrollment and increased by 0.9% (95% CI, 0.7–1.2%) per day. The mean percentage of CD4 T cells and CD8 T cells AV+/7-AAD– at enrollment was 37.2% and 20.4%, respectively, but it diminished at a rate of –0.5% (95% CI, –0.7% to –0.3%) and –0.3% (95% CI, –0.4% to –0.2%) per day, respectively. Plasma levels of interleukin-6 and interleukin-10 were 290 and 166 pg/mL and decreased at a rate of –7.8 pg/mL (95% CI, –9.5 to –6.1 pg/mL) and –4 pg/mL (95% CI, –5.1 to –2.8 pg/mL) per day, respectively. After controlling for confounders, only sustained plasma levels of interleukin-6 increase the risk of death (hazard ratio 1.003; 95% CI, 1.001–1.006). Conclusions:We found no evidence to support a two-phase model of sepsis pathophysiology. However, immunological variables did behave in a mixed and time-dependent manner. Further studies should evaluate changes over time of interleukin-6 plasma levels as a prognostic biomarker for critically ill patients.

HIV-Induced T-Cell Activation/Exhaustion in Rectal Mucosa Is Controlled Only Partially by Antiretroviral Treatment

Peripheral blood T-cells from untreated HIV-1-infected patients exhibit reduced immune responses, usually associated with a hyperactivated/exhausted phenotype compared to HAART treated patients. However, it is not clear whether HAART ameliorates this altered phenotype of T-cells in the gastrointestinal-associated lymphoid tissue (GALT), the main site for viral replication. Here, we compared T-cells from peripheral blood and GALT of two groups of chronically HIV-1-infected patients: untreated patients with active viral replication, and patients on suppressive HAART. We characterized the T-cell phenotype by measuring PD-1, CTLA-4, HLA-DR, CD25, Foxp3 and granzyme A expression by flow cytometry; mRNA expression of T-bet, GATA-3, ROR-γt and Foxp3, and was also evaluated in peripheral blood mononuclear cells and rectal lymphoid cells. In HIV-1+ patients, the frequency of PD-1+ and CTLA-4+ T-cells (both CD4+ and CD8+ T cells) was higher in the GALT than in the blood. The expression of PD-1 by T-cells from GALT was higher in HIV-1-infected subjects with active viral replication compared to controls. Moreover, the expression per cell of PD-1 and CTLA-4 in CD4+ T-cells from blood and GALT was positively correlated with viral load. HAART treatment decreased the expression of CTLA-4 in CD8+ T cells from blood and GALT to levels similar as those observed in controls. Frequency of Granzyme A+ CD8+ T-cells in both tissues was low in the untreated group, compared to controls and HAART-treated patients. Finally, a switch towards Treg polarization was found in untreated patients, in both tissues. Together, these findings suggest that chronic HIV-1 infection results in an activated/exhausted T-cell phenotype, despite T-cell polarization towards a regulatory profile; these alterations are more pronounced in the GALT compared to peripheral blood, and are only partiality modulated by HAART.

Phenotypical characterization of regulatory T cells in humans and rodents.

Regulatory T cells (Tregs) constitute a fascinating subpopulation of CD4+ T cells due to their ability to limit the immune response against self and non‐self antigens. Murine models and antibodies directed against surface and intracellular molecules have allowed elucidation of the mechanisms that govern their development and function. However, these markers used to their classification lack of specificity, as they can be expressed by activated T cells. Similarly, there are slight differences between animal models, in steady state and pathological conditions, anatomical localization and strategy of analysis by flow cytometry. Here, we revised the most common markers utilized for Treg typification by flow cytometry such as CD25, forkhead box protein 3 (FoxP3) and CD127, along with our data obtained in different body compartments of humans, mice and rats. Furthermore, we revised and determined the expression of other molecules important for the phenotypical characterization of Treg cells. We draw attention to the drawbacks of those markers used in chronic states of inflammation. However, until a specific marker for the identification of Tregs is discovered, the best combination of markers will depend upon the tissue or the degree of inflammation from which Tregs derive.

Statins increase the frequency of circulating CD4+ FOXP3+ regulatory T cells in healthy individuals.

Statins have been shown to modulate the number and the suppressive function of CD4+FOXP3+ T cells (Treg) in inflammatory conditions. However, it is not well established whether statin could also affect Treg in absence of inflammation. To address this question, eighteen normocholesterolemic male subjects were treated with lovastatin or atorvastatin daily for 45 days. The frequency and phenotype of circulating Treg were evaluated at days 0, 7, 30, and 45. mRNA levels of FOXP3, IDO, TGF-β, and IL-10 were measured in CD4+ T cells. We found that both statins significantly increased Treg frequency and FOXP3 mRNA levels at day 30. At day 45, Treg numbers returned to baseline values; however, TGF-β and FOXP3 mRNA levels remained high, accompanied by increased percentages of CTLA-4- and GITR-expressing Treg. Treg Ki-67 expression was decreased upon statin treatment. Treg frequency positively correlated with plasma levels of high-density lipoprotein cholesterol (HDL-c), suggesting a role for HDL-c in Treg homeostasis. Therefore, statins appear to have inflammation-independent immune-modulatory effects. Thus, the increase in Treg cells frequency likely contributes to immunomodulatory effect of statins, even in healthy individuals.

Presence of HIV-1 DNA in spermatozoa from HIV-positive patients: changes in the semen parameters.

Although very inefficient, sexual transmission of HIV-1 is responsible for more than 80% of infections worldwide. Yet, the presence of HIV in spermatozoa has been a matter of debate. The aim of this study was to evaluate the presence of HIV nucleic acids and the distribution of mannose receptors in sperm cells, and to determine the semen parameters and cytokine levels in ejaculates from HIV-positive patients. The presence of non-seminal cells in purified sperm was revealed by light microscopy, flow cytometry and RT-PCR. HIV nucleic acids were evaluated by nested PCR; the distributions of mannose receptors on the surface of the sperm and cytokine levels in ejaculates were determined by fluorescence microscopy and flow cytometry respectively. Sperm characteristics were determined by conventional methods. HIV DNA was detected in 69.2% of purified sperm from HIV-positive men; in contrast all purified sperm were negative for HIV RNA. The distribution of mannose receptors and cytokine levels in HIV-1-positive men were similar to uninfected individuals. Using the Principal Component Analysis (PCA) method, it was possible to determine that semen parameters of HIV-positive men exhibit different distributions compared to HIV-negative individuals. Finally, these results indicate that viral DNA is present in purified sperm from HIV-positive men and that HIV infection of spermatozoa could be associated with lower seminal parameters as demonstrated by the PCA method. The similar distribution of mannose receptors between infected and uninfected individuals suggests that sperm cells from infected individuals interact normally with oocytes.

Incomplete Normalization of Regulatory T-Cell Frequency in the Gut Mucosa of Colombian HIV-Infected Patients Receiving Long-Term Antiretroviral Treatment

Introduction To evaluate the effect of late initiation of HAART and poor immune reconstitution on the frequency of regulatory T-cells (Treg) in the peripheral blood and gut of HIV-infected patients, we studied Colombian HIV-infected patients who had been on suppressive HAART for at least one year. They had undetectable viremia but were either immunological responders (HIR); (CD4 counts >500 cells/µl) or non-immunological responders (NIR); (CD4 T-cell count <300 cells/µl). Untreated HIV-infected patients and uninfected controls from the same region were also evaluated. Methods Frequency and phenotype of regulatory T-cells (Treg) were analyzed in gut biopsies and blood samples. The functional effect of Treg depletion on CMV and HIV responses was determined. Markers of immune activation and circulating LPS levels were quantified. Results Untreated patients exhibited high Treg frequency in PBMC and gut, and their Treg express high levels of CTLA-4 and PD-1. Although HAART significantly decreased mucosal Treg frequency, it did not normalize it in any of the treated groups (HIR and NIR patients). Treg normalization was observed in the blood of HIR patients following HAART, but did not occur in NIR patients. Treg from HIV-infected patients (treated or not) suppressed HIV and hCMV-specific T-cells from gut and blood. Plasma LPS levels and percentage of HLA-DR+CD38+ T-cells were significantly elevated in all infected groups compared to controls. Conclusions These findings suggest that control of viral replication is not sufficient to normalize gut Treg frequency in patients, independent of their response to HAART. Furthermore, persistence of functional Treg in the gut appears to be associated with the failure of HAART to repair mucosal damage.

HIV-1-neutrophil interactions trigger neutrophil activation and Toll-like receptor expression

Abstract Although neutrophils are the first-line of host defense against infection and express a wide number of pattern recognition receptors (PRRs), the function of these PRRs, including Toll-like receptors (TLRs), in HIV-1 infection remains unclear. TLRs play an important role in innate immunity, and while their involvement in viral immune pathogenesis was recently proposed, little is known about their expression and function during the neutrophil response to HIV-1 exposure. Here, we have shown that freshly isolated human neutrophils from healthy donors exhibited altered TLR expression, which may affect their function, after being challenged with HIV-1, alone or in the presence of TLR agonists. TLRs may promote neutrophil activation, pro-inflammatory cytokine secretion, and the production of reactive oxygen species. To our knowledge, this study is the first demonstration of functional TLR expression on neutrophils in response to HIV-1 treatment, suggesting a possible neutrophil/HIV-1 interaction through TLRs. Although additional studies are required to confirm the function of TLRs in neutrophils, our data clearly suggest that they play a role in the regulation of innate immunity by neutrophils, which could be engaged in HIV-1 pathogenesis or host defense.

Chronically HIV-1 Infected Patients Exhibit Low Frequencies of CD25+ Regulatory T Cells

The characterization of regulatory T cells (Treg) during HIV infection has become of particular interest considering their potential role in the pathogenesis of the acquired immunodeficiency syndrome. Different reports on Tregs in HIV-infected patients vary greatly, depending on the state of disease progression, anatomical compartment, and the phenotypic markers used to define this cell subpopulation. To determine the frequency of Tregs we included paired samples from peripheral blood and rectal biopsies from controls and chronic HIV patients with or without detectable viral load. Tregs were determined by flow cytometry using three different protocols: CD4+Foxp3+; CD4+Foxp3+CD127Low/-, and CD4+CD25+CD127Low/-. In addition, and with the purpose to compare the different protocols we also characterized Tregs in peripheral blood of HIV negative individuals with influenza like symptoms. Here, we report that Treg characterization in HIV-infected patients as CD4+Foxp3+ and CD4+Foxp3+CD127Low/- cells was similar, indicating that both protocols represent a suitable method to determine the frequency of Tregs in peripheral blood mononuclear cells (PBMC) and gut associated lymphoid tissue (GALT). In contrast, in HIV but not in flu-like patients, detection of Tregs as CD4+CD25+CD127Low/- cells resulted in a significantly lower percentage of these cells. In both, HIV patients and controls the frequency of Treg was significantly higher in GALT compared to PBMC. The frequency of Tregs in PBMC and GALT using CD4+Foxp3+ and CD4+Foxp3+CD127Low/- was higher in HIV patients than in controls. Similarly, the frequency of Treg using any protocol was higher in flu-like patients compared to controls. The results suggest that relying on the expression of CD25 could be unsuitable to characterize Tregs in PBMC and GALT samples from a chronic infection such as HIV.

Increase of Frequency and Modulation of Phenotype of Regulatory T Cells by Atorvastatin Is Associated with Decreased Lung Inflammatory Cell Infiltration in a Murine Model of Acute Allergic Asthma

Regulatory T cells (Tregs) play an important role by controlling allergic inflammation of airways. Recently, it has been shown that statins have immunomodulatory properties, probably mediated by their effects on Tregs. Therefore, we evaluated the in vivo effect of atorvastatin (ATV) on Tregs and its association with the inflammatory process in a model of allergic asthma. BALB/c mice were sensitized with ovalbumin (OVA) and then challenged with intranasal OVA. ATV (40 mg/kg) was delivered by daily intraperitoneal injection for 7 or 15 days before each OVA challenge. ATV treatment for 7 days increased the frequency of Tregs in mediastinal lymph nodes (MLN) and the interleukin (IL)-10 in lungs. After 15 days of treatment, ATV increased the percentage of glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR+) and programmed cell death protein 1 (PD-1+) Tregs in the lung, without enhancing their suppressive activity, but also increased the percentage of conventional T cells expressing GITR+, PD1+, and OX-40 (tumor necrosis factor receptor superfamily member 4). Although no significant changes were observed in the number of inflammatory cells in the bronchoalveolar lavage (BAL), OVA-specific immunoglobulin E in the serum, and type 2 helper (Th2) cytokines in the lungs, there was a significant decrease of peribronchial inflammation that negatively correlated with the Tregs in MLN and the concentration of IL-10 in the lung. These results suggest that ATV has an immunomodulatory role possibly mediated by their effects on Tregs, which could contribute to the control of inflammation during allergic asthma. Further studies are necessary to elucidate the contribution of Treg to immunomodulatory action of statins in the context of allergic asthma.