Michael L. Freeman

Partners in Crime: The Role of CMV in Immune Dysregulation and Clinical Outcome During HIV Infection.

In the current era of combination antiretroviral therapy (ART), human immunodeficiency virus (HIV)-infected individuals are living longer and healthier lives. Nevertheless, HIV-infected persons are at greater risk for age-related disorders, which have been linked to residual immune dysfunction and inflammation. HIV-infected individuals are almost universally co-infected with cytomegalovirus (CMV) and both viruses are associated with inflammation-related morbidities. Therefore, a detailed investigation of the relationship between CMV and aging-related morbidities emerging during chronic HIV infection is warranted. Here, we review the literature on how CMV co-infection affects HIV infection and host immunity and we discuss the gaps in our knowledge that need elucidation.

Inflammation perturbs the IL-7 axis, promoting senescence and exhaustion that broadly characterize immune failure in treated HIV infection

Background:HIV-infected patients who fail to normalize CD4 T cells despite suppressive antiretroviral therapy have impaired immune homeostasis: diminished naive T-cell numbers, elevated T-cell turnover, senescence, and inflammation. Methods:Blood samples from immune failures (n = 60), immune successes (n = 20), and healthy controls (n = 20) were examined for plasma interleukin (IL)-7 levels, for cellular expression of the IL-7R&agr; chain (CD127), for the exhaustion and senescence markers programed death 1 (PD-1) and CD57, and for the survival factor Bcl2. Because both inflammatory and homeostatic cytokines can induce T-cell cycling, we also examined the effects of these mediators on exhaustion and senescence markers. Results:Plasma levels of IL-7 were elevated and both CD4 and CD8 T-cell CD127 expression was decreased in immune failure. Plasma levels of IL-7 correlated directly with naive CD4 T-cell counts in immune success and inversely with T-cell cycling (Ki67) in healthy controls and immune success, but not in immune failure. CD4 T-cell density of PD-1 was increased and Bcl2+ CD4 T cells were decreased in immune failure but not in immune success, whereas the proportion of T cells expressing CD57 was increased in immune failure. PD-1 and CD57 were induced on CD4 but not CD8 T cells by stimulation in vitro with inflammatory IL-1&bgr; or homeostatic (IL-7) cytokines. Conclusions:Perturbation of the IL-7/IL-7 receptor axis, increased T-cell turnover, and increased senescence may reflect dysregulated responses to both homeostatic and inflammatory cytokines in immune failure patients.

IL-15 promotes activation and expansion of CD8 + T cells in HIV-1 infection

In HIV-1-infected patients, increased numbers of circulating CD8+ T cells are linked to increased risk of morbidity and mortality. Here, we identified a bystander mechanism that promotes CD8 T cell activation and expansion in untreated HIV-1-infected patients. Compared with healthy controls, untreated HIV-1-infected patients have an increased population of proliferating, granzyme B+, CD8+ T cells in circulation. Vβ expression and deep sequencing of CDR3 revealed that in untreated HIV-1 infection, cycling memory CD8 T cells possess a broad T cell repertoire that reflects the repertoire of the resting population. This suggests that cycling is driven by bystander activation, rather than specific antigen exposure. Treatment of peripheral blood mononuclear cells with IL-15 induced a cycling, granzyme B+ phenotype in CD8+ T cells. Moreover, elevated IL-15 expression in the lymph nodes of untreated HIV-1-infected patients correlated with circulating CD8+ T cell counts and was normalized in these patients following antiretroviral therapy. Together, these results suggest that IL-15 drives bystander activation of CD8+ T cells, which predicts disease progression in untreated HIV-1-infected patients and suggests that elevated IL-15 may also drive CD8+ T cell expansion that is linked to increased morbidity and mortality in treated patients.

SIV/SHIV Infection Triggers Vascular Inflammation, Diminished Expression of Krüppel-like Factor 2 and Endothelial Dysfunction

BACKGROUND Human immunodeficiency virus (HIV) infection is associated with increased risk of thromboembolic and cardiovascular comorbid conditions. Although systemic inflammation is linked to cardiovascular risk, direct evidence of vascular inflammation and endothelial dysfunction is lacking. METHODS We examined by immunofluorescence microscopy thoracic aortas from 16 simian immunodeficiency virus (SIV)- or simian-human immunodeficiency virus (SHIV)-infected and 16 uninfected rhesus macaques. RESULTS Focal endothelial proliferation and subendothelial inflammatory cells were found in sections of all infected animals, compared with minimal changes in sections from the 16 uninfected controls. In the infected animals, we detected increased endothelial levels of bacterial 16s ribosomal DNA as well as increased subendothelial accumulation of CD68(+) monocytes/macrophages (P< .001) and CD8(+) T lymphocytes (P< .001). Endothelial dysfunction was manifested by decreased levels of endothelial nitric oxide synthase (P< .005) and Krüppel-like factor 2 (KLF2) (P< .005). KLF2 expression was decreased in primary human aortic endothelial cells exposed to bacterial lipopolysaccharide or to oxidized low-density lipoprotein in vitro, and this could be prevented by simvastatin. CONCLUSIONS SIV and SHIV infection lead to endothelial inflammation, dysfunction, and decreased KLF2 expression reflecting early atherosclerotic changes. Translocated bacterial components and lipid oxidation products may induce endothelial dysfunction in HIV infection that could be prevented by statin treatment.

Cytokines and T-Cell Homeostasis in HIV Infection

Untreated human immunodeficiency virus (HIV) infection is characterized by progressive CD4(+) T-cell depletion and CD8(+) T-cell expansion, and CD4(+) T-cell depletion is linked directly to the risk for opportunistic infections and infection-associated mortality. With suppression of HIV replication by antiretroviral therapy, circulating CD4(+) Tcell numbers typically improve while CD8(+) T-cell expansion persists, and both CD4(+) T-cell cytopenia and CD8(+) T-cell expansion are associated with morbidity and mortality. In this brief review, we report on the role that selected homeostatic and inflammatory cytokines may play both in the failure of CD4(+) T-cell restoration and the CD8(+) T-cell expansion that characterize HIV infection.

Elevated Red Cell Distribution Width (RDW) Identifies Elevated Cardiovascular Disease Risk in Patients with HIV infection.

Abstract: Red cell distribution width (RDW) is linked to cardiovascular risk in the general population, an association that might be driven by inflammation. Whether this relationship holds for patients with HIV infection has not been previously studied. Using a large clinical registry, we show that elevated RDW (>14.5%) is independently associated with increased risk of coronary artery disease {odds ratio [OR] 1.39 [95% confidence interval (CI): 1.25 to 1.55]}, peripheral vascular disease [OR 1.41 (95% CI: 1.29 to 1.53)], myocardial infarction [1.43 (95% CI: 1.25 to 1.63)], heart failure [OR 2.23 (95% CI: 1.99 to 2.49)], and atrial fibrillation [OR 1.96 (95% CI: 1.64 to 2.33)]. In conclusion, in the context of the inflammatory milieu that accompanies HIV infection, RDW remains a powerful marker of cardiovascular disease.

Responsiveness to IL-7 but not to IFN-α is diminished in CD4+ T cells from treated HIV infected patients who experience poor CD4+ T-cell recovery.

Objective:To assess CD4+ T-cell responsiveness to IL-7 and IFN-&agr; in HIV-infected patients who experience poor recovery of CD4+ T-cell counts during therapy (immune failure patients). Design:Responses to IL-7 and IFN-&agr; were compared between HIV-infected immune failure (CD4+ cell counts <379 cells/&mgr;l) patients and immune success (CD4+ cell counts >500 cells/&mgr;l) as well as healthy control patients. Methods:Flow cytometry was used to assess peripheral blood mononuclear cells for IL-7-induced proliferation, CD25 expression, and signaling (signal transducer and activator of transcription 5 phosphorylation and Akt phosphorylation) in CD4+ T cells. Freshly isolated cells were characterized by expression of IL-7R&agr; (CD127) among CD4+ T-cell maturation subsets by flow cytometry and sorted CD3+ T cells were assessed for expression of IFN-&agr; and interferon stimulated genes (2′–5′-oligoadenylate synthetase-1 and myxovirus resistance A protein) by quantitative real-time PCR. Responses to IFN-&agr; were assessed by induction of signal transducer and activator of transcription 1 phosphorylation and inhibition of IL-7-induced CD4+ T-cell proliferation. Results:IL-7-induced proliferation and CD25 expression were decreased in CD4+ T cells from immune failure patients. CD127 expressing CD4+ T cells were decreased, whereas expression of 2′–5′-oligoadenylate synthetase-1, myxovirus resistance A protein, and IFN-&agr; mRNA were increased in total CD3+ T cells from immune failure patients. CD127 expression correlated with CD25 induction but not proliferation, whereas T-cell IFN-&agr; mRNA was associated with reduced proliferation in CD4+ T cells from immune failure patients. IFN-&agr;-mediated induction of signal transducer and activator of transcription 1 phosphorylation and inhibition of proliferation were not diminished in CD4+ T cells from immune failure patients. Conclusion:IL-7 responsiveness is impaired in immune failure patients and may be related to expression of CD127 and IFN-&agr;.

CD161 Expression on Mucosa-Associated Invariant T Cells is Reduced in HIV-Infected Subjects Undergoing Antiretroviral Therapy Who Do Not Recover CD4+ T Cells

Background: Mucosa-associated invariant T (MAIT) cells are a recently identified class of innate-like T cells that are involved in the mucosal immune response. MAIT cells are characterized by expression of TCR Vα7.2 and CD161. In HIV infection, there is a profound early loss of MAIT cells from the circulation that never fully recovers, even after prolonged viral control with antiretroviral therapy (ART). Methods: We analyzed PBMCs from fresh whole blood from HIV-negative or ART-treated HIV-positive donors with full (Immune Success) or impaired (Immune Failure) CD4+ T- cell recovery by flow cytometry for T-cell markers, TCR Vα7.2, and CD161. The PBMCs were cultured with or without TCR-mediated stimulation, and CD161 expression was assessed on Vα7.2+ T cells. Interferon-γ (IFNγ) production was assessed by intracellular cytokine staining. Results: We found a decrease in the percentage of CD3+ T cells that expressed CD161 and the percentage of Vα7.2+ T cells that expressed CD161, in HIV-infected individuals. We also found a significant increase in the percentage of T cells that were Vα7.2+CD161- in immune failure compared to controls, accompanied by an increase in the percentage of Vα7.2+CD161- T cells that express CD8+ in donors with immune failure, but not immune success. After TCR stimulation in vitro, Vα7.2+ T cells reduced expression of CD161, yet Vα7.2+ CD161- cells from immune failure donors retained the ability to express IFNγ on stimulation. Conclusions: Our findings suggest that in immune failure patients, the reduction in peripheral MAIT cells is due, at least in part, to a loss in CD161 expression, and is not merely the result of trafficking into mucosal tissues or cell death. These CD161- cells retain their function.

Reply to Barrett, et al

Author(s): Freeman, Michael L; Mudd, Joseph C; Shive, Carey L; Younes, Souheil-Antoine; Panigrahi, Soumya; Sieg, Scott F; Lee, Sulggi A; Hunt, Peter W; Calabrese, Leonard H; Gianella, Sara; Rodriguez, Benigno; Lederman, Michael M

A System of Cytokines Encapsulated in ExtraCellular Vesicles

Cytokines are soluble factors that mediate cell–cell communications in multicellular organisms. Recently, another system of cell–cell communication was discovered, which is mediated by extracellular vesicles (EVs). Here, we demonstrate that these two systems are not strictly separated, as many cytokines in vitro, ex vivo, and in vivo are released in EV-encapsulated forms and are capable of eliciting biological effects upon contact with sensitive cells. Association with EVs is not necessarily a property of a particular cytokine but rather of a biological system and can be changed upon system activation. EV-encapsulated cytokines were not detected by standard cytokine assays. Deciphering the regulatory mechanisms of EV-encapsulation will lead to a better understanding of cell–cell communications in health and disease.