SARS-CoV-2 Spike Protein Destabilizes Microvascular Homeostasis.

SARS-CoV-2 infection can cause compromised respiratory function and thrombotic events. SARS-CoV-2 binds to and mediates downregulation of angiotensin converting enzyme 2 (ACE2) on cells that it infects. Theoretically, diminished enzymatic activity of ACE2 may result in increased concentrations of pro-inflammatory molecules, angiotensin II, and Bradykinin, contributing to SARS-CoV-2 pathology. Using immunofluorescence microscopy of lung tissues from uninfected, and SARS-CoV-2 infected individuals, we find evidence that ACE2 is highly expressed in human pulmonary alveolar epithelial cells and significantly reduced along the alveolar lining of SARS-CoV-2 infected lungs. Ex vivo analyses of primary human cells, indicated that ACE2 is readily detected in pulmonary alveolar epithelial and aortic endothelial cells. Exposure of these cells to spike protein of SARS-CoV-2 was sufficient to reduce ACE2 expression. Moreover, exposure of endothelial cells to spike protein-induced dysfunction, caspase activation, and apoptosis. Exposure of endothelial cells to bradykinin caused calcium signaling and endothelial dysfunction (increased expression of von Willibrand Factor and decreased expression of Krüppel-like Factor 2) but did not adversely affect viability in primary human aortic endothelial cells. Computer-assisted analyses of molecules with potential to bind bradykinin receptor B2 (BKRB2), suggested a potential role for aspirin as a BK antagonist. When tested in our in vitro model, we found evidence that aspirin can blunt cell signaling and endothelial dysfunction caused by bradykinin in these cells. Interference with interactions of spike protein or bradykinin with endothelial cells may serve as an important strategy to stabilize microvascular homeostasis in COVID-19 disease. IMPORTANCE SARS-CoV-2 causes complex effects on microvascular homeostasis that potentially contribute to organ dysfunction and coagulopathies. SARS-CoV-2 binds to, and causes downregulation of angiotensin converting enzyme 2 (ACE2) on cells that it infects. It is thought that reduced ACE2 enzymatic activity can contribute to inflammation and pathology in the lung. Our studies add to this understanding by providing evidence that spike protein alone can mediate adverse effects on vascular cells. Understanding these mechanisms of pathogenesis may provide rationale for interventions that could limit microvascular events associated with SARS-CoV-2 infection.

CD8+ CD73+ T cells in the tumor microenvironment of head and neck cancer patients are linked to diminished T cell infiltration and activation in tumor tissue.

Recent studies have implicated a role for adenosine-dependent immunosuppression in head and neck tumor microenvironments. We describe expression of CD73, an enzyme critical to the generation of adenosine from extracellular AMP, in T cells and other cell types within human head and neck tumors. Flow cytometric analyses of tumor-infiltrating cells indicate that CD3+ cells are the predominant source of CD73 among immune infiltrating cells and that CD73 expression, especially among CD8+ T cells, is inversely related to indices of T cell infiltration and T cell activation in the microenvironment of head and neck tumors. We provide evidence that CD73 expression on peripheral T cells and levels of soluble CD73 in circulation are correlated with CD73 expression on CD8+ T cells in tumors. Moreover, fluorescent microscopy studies reveal that CD8+ CD73+ cells are observed in close proximity to tumor cells as well as in surrounding tissue. In vitro studies with peripheral blood T cells indicate that anti-CD3-stimulation causes loss of CD73 expression, especially among cells that undergo proliferation and that exogenous AMP can impair T cell proliferation, while sustaining CD73 expression. These data suggest that CD8+ CD73+ T cells may be especially important mediators of immunosuppression in human head and neck cancer.

Highly oxidized low-density lipoprotein mediates activation of monocytes but does not confer interleukin-1ß secretion nor interleukin-15 transpresentation function.

Oxidized low-density lipoprotein (LDL) contributes to cardiovascular disease in part by mediating activation and maturation of monocytes and macrophages. Furthermore, co-localization studies using histochemical approaches have implicated a potential role for oxidized LDL as a mediator of interleukin-15 (IL-15) expression in myeloid cells of atherosclerotic plaque. The latter activity could be an important pro-inflammatory mechanism that mediates myeloid cell/T-cell crosstalk. Here, we examined the responses of primary human monocytes to highly oxidized LDL molecules. Oxidized LDL readily induced secretion of chemokines MCP-1 (CCL2) and GRO-α (CXCL1) but unlike lipopolysaccharide (LPS), has limited capacity to induce a variety of other cytokines including tumor necrosis factor-α, IL-6, IL-1ß and interferon-γ-induced protein-10 and also displayed a poor capacity to induce p-Akt or P-S6 signaling. Failure of oxidized LDL to induce IL-1ß secretion was associated with limited induction of caspase-1 activation. Furthermore, despite finding evidence that oxidized LDL could enhance the expression of IL-15 and IL-15 receptor expression in monocytes, we found no evidence that it could confer IL-15 transpresentation capability to these cells. This observation contrasted with induction of IL-15 transpresentation in lipopolysaccharide-stimulated monocytes. Overall, our data suggest that highly oxidized LDL is a selective inducer of monocyte activation. Sterile inflammatory mediators, particularly those implicated in Toll-like receptor 4 signaling, may play a role in vascular pathology but the activities of these agents are not uniform.

Exosomes derived from HIV-1-infected cells promote growth and progression of cancer via HIV TAR RNA.

People living with HIV/AIDS on antiretroviral therapy have increased risk of non-AIDS-defining cancers (NADCs). However, the underlying mechanism for development and progression of certain NADCs remains obscure. Here we show that exosomes released from HIV-infected T cells and those purified from blood of HIV-positive patients stimulate proliferation, migration and invasion of oral/oropharyngeal and lung cancer cells. The HIV transactivation response (TAR) element RNA in HIV-infected T-cell exosomes is responsible for promoting cancer cell proliferation and inducing expression of proto-oncogenes and Toll-like receptor 3 (TLR3)-inducible genes. These effects depend on the loop/bulge region of the molecule. HIV-infected T-cell exosomes rapidly enter recipient cells through epidermal growth factor receptor (EGFR) and stimulate ERK1/2 phosphorylation via the EGFR/TLR3 axis. Thus, our findings indicate that TAR RNA-containing exosomes from HIV-infected T cells promote growth and progression of particular NADCs through activation of the ERK cascade in an EGFR/TLR3-dependent manner.

Proteome and Protein Network Analyses of Memory T Cells Find Altered Translation and Cell Stress Signaling in Treated Human Immunodeficiency Virus Patients Exhibiting Poor CD4 Recovery.

Background. Human immunodeficiency virus (HIV) patients who experience poor CD4 T-cell recovery despite viral suppression during antiretroviral therapy (ART) are known as immunological nonresponders. The molecular mechanism(s) underlying incomplete immune restoration during ART is not fully understood. Methods. Label-free quantitative proteomics on single-cell type central memory T cells were used to reveal relative protein abundance changes between nonresponder, responder (good CD4 recovery during ART), and healthy individuals. Proteome changes were analyzed by protein pathway and network analyses and verified by selected reaction monitoring mass spectrometry. Results. Proteomic analysis across groups detected 155 significant proteins from 1500 nonredundant proteins. Pathway and network analyses revealed dysregulation in mammalian target of rapamycin and protein translation-related proteins and decreases in stress response-related proteins for nonresponder subjects compared with responders and controls. Actin cytoskeleton signaling was increased for HIV responders and nonresponders alike. Conclusions. Memory T cells from immunologic nonresponders have increases in proteins related to motility and protein translation and decreases in proteins capable of responding to cellular stresses compared with responders and controls. The potential for T cells to manage stress and modulate metabolism may contribute to their capacity to reconstitute a lymphopenic host.

Recycled IL-7 Can Be Delivered to Neighboring T Cells.

IL-7 is a key homeostatic cytokine that provides signals for T cell survival and proliferation in vivo. In this article, we provide evidence that IL-7 utilization is enhanced by a novel mechanism of cytokine "recycling" during which T cells treated with rIL-7 are rapidly induced to express p-STAT5 and are subsequently able to recycle biologically active cytokine for release to neighboring cells in soluble form. Our observations indicate that the ability of cells to recycle IL-7 is dependent on IL-7R α-chain (CD127) and endocytosis, consistent with a model whereby IL-7 is internalized via receptor interactions before recycling. These observations provide evidence of a novel mechanism that enables cells to optimally use IL-7.

Decreased IL-7 responsiveness is related to oxidative stress in HIV disease.

HIV disease results in decreased IL-7 receptor expression and IL-7 responsiveness in T cells. To explore mechanisms of these deficiencies, we compared CD127 expression and IL-7 induction of P-STAT5 in T cells from HIV-infected persons with serum concentrations of cytokines (IL-7, IL-6 and IL-15), markers of microbial translocation (sCD14 and LPS), and with an indicator of oxidative stress (malondialdehyde (MDA) adducts). CD127 expression was directly related to IL-7 responsiveness in most CD8+ T cell subsets but not in CD4+ T cells from HIV-infected persons. MDA adducts were increased in serum of HIV-infected patients and were inversely related to IL-7 responsiveness in CD8+ T cells and in central memory CD4+ T cells. Incubation of T cells from healthy controls with hydrogen peroxide resulted in impairments in IL-7 induction of P-STAT5. These findings suggest that oxidative stress that is characteristic of HIV disease could contribute to impairments in IL-7 responsiveness and disrupt T cell homeostasis.

Microparticle delivery of Interleukin-7 to boost T-cell proliferation and survival.

In HIV infections, homoeostasis of T cells is dysregulated such that there is a depletion of CD4(+) T cells and a progressive loss of naïve CD4(+) and CD8(+) T cells. Methodologies that can improve the function of some or all of these cells will likely enhance immune responsiveness in HIV infection. Interleukin-7 (IL-7) is a cytokine that has been shown to be critical in homeostatic expansion of naïve CD8(+) and CD4(+) cells in lymphopenic hosts, as well as regulating effector T cell to memory T-cell transition and memory T-cell homeostasis. In animal studies and clinical trials, repeated injections of IL-7 are used to boost both CD4(+) and CD8(+) cell counts. Daily injections, however, are painful, inconvenient, and provide a frequent route for pathogen entry. We developed a poly (D,L-lactide-co-glycolide; PLGA) microparticle controlled release system to administer IL-7 in which a single injection of microparticles can provide therapeutic delivery of IL-7. IL-7 encapsulated PLGA microparticles were first synthesized using a water/organic/water double emulsion method, release from the particles was then optimized using in vitro release studies and therapeutic effectiveness was finally studied in animal studies. These PLGA microparticles showed effective delivery of IL-7 for 1 week in vitro. These results were translated to in vivo delivery as well, which was followed for 9 days. Controlled release of IL-7 in mice demonstrated biological activity in both CD4(+) and CD8(+) T cells in mice, which was consistent with previously reported results using daily injections.

Frequencies of FoxP3+ naive T cells are related to both viral load and naive T cell proliferation responses in HIV disease.

HIV infection results in depletion and dysfunction of naïve CD4(+) T cells. The mechanisms underlying these deficiencies are not understood. We investigated the frequencies of CD4(+) naïve subsets in HIV disease as defined by expression of CD25 and/or FoxP3 and the relationship of these frequencies to naïve T cell proliferation function. We observed increased proportions of CD25(+)FoxP3(+) and CD25(+)FoxP3(-) cells and decreased proportions of CD25(-)FoxP3(-) cells within the naïve CD4(+) cell compartment from HIV-infected persons compared with findings in healthy donors. These perturbations were related to higher plasma HIV RNA levels but not with higher immune activation, as measured by the proportions of CD38(+) memory CD4(+) T cells. Naïve T cell proliferation responses to mitogen stimulation were inversely related to the frequencies and absolute numbers of FoxP3(+) naïve T cells. MDA, a marker of oxidative stress, and sCD14, a marker of monocyte activation and a surrogate for microbial translocation, were increased in serum samples from HIV(+) donors; however, neither marker was related to naïve T cell function in HIV(+) donors. These observations suggest that alterations in naïve T cell subset frequencies could contribute to naïve T cell dysfunction in HIV disease, but these alterations are not necessarily the result of chronic immune activation.

Interleukin-7 receptor signaling is deficient in CD4+ T cells from HIV-infected persons and is inversely associated with aging.

Loss of interleukin-7 (IL-2) receptor expression has been described in T lymphocytes from persons with human immunodeficiency virus (HIV) infection, potentially contributing to perturbations in T cell homeostasis. We investigated IL-7 receptor signaling by measuring signal transducer and activator of transcription 5 (STAT5) phosphorylation in CD4+ T cell subsets from HIV-infected persons. We determined that CD45RA- memory cell subsets (both CD27+ and CD27-) displayed the most robust immediate responses to IL-7, whereas naive CD4+ T cells sustained the signal most efficiently. Memory CD4+ T cells with a terminal phenotype (CD45RA+CD27-) responded poorly to IL-7 stimulation. Defects in signaling were observed in cells from viremic HIV-infected persons and were especially pronounced in CD45RA-CD27- memory subset. Although CD127 expression was diminished for T cells from HIV-infected persons, it was not directly related to IL-7 receptor signaling function. Instead, age was inversely related to IL-7 signaling in cells from both HIV-infected viremic subjects and healthy control subjects. Thus, HIV infection results in impaired IL-7 responsiveness, especially in memory CD4+ T cells, and this defect is likely compounded by aging.

S-phase entry leads to cell death in circulating T cells from HIV-infected persons.

Central memory T cells are thought to play a critical role in memory T cell homoestasis by undergoing self-renewal and by maturating into effector T cells that mediate immunity at tissue sites. Circulating T cells in S phase of the cell cycle are found at increased frequencies during HIV infection and are predominantly composed of cells with a central memory phenotype. Here, we tested the hypothesis that CD4 and CD8 S-phase T cells have different capacities to complete cell cycle and survive. S-phase T cells in peripheral blood from HIV-infected donors were identified by incubating whole blood with BrdU ex vivo. Upon in vitro cultivation, S-phase T cells were more likely to die than to complete mitotic division. Intrinsic differences were observed between CD4 and CD8 S-phase T cells during incubation. Higher frequencies of CD4+ S-phase T cell underwent apoptosis after incubation in medium alone or after TCR stimulation, and CD4+ S-phase T cells were less readily induced to proliferate after incubation with IL-2 than were CD8+ S-phase T cells. CD4+ and CD8+ S-phase T cells expressed low levels of Bcl-2, which could contribute to their heightened susceptibility to cell death. Intrinsic differences in the proliferation and survival of CD4+ and CD8+ S-phase T cells could influence the homeostatic maintenance of these T cell subsets in HIV disease.

Interleukin-7 enhances proliferation responses to T-cell receptor stimulation in naïve CD4+ T cells from human immunodeficiency virus-infected persons.

Proliferation responses of naïve CD4(+) T cells to T-cell receptor and interleukin-7 (IL-7) stimulation were evaluated by using cells from human immunodeficiency virus-positive (HIV(+)) donors. IL-7 enhanced responses to T-cell receptor stimulation, and the magnitude of this enhancement was similar in cells from healthy controls and from HIV(+) subjects. The overall response to T-cell receptor stimulation alone or in combination with IL-7, however, was diminished among viremic HIV(+) donors and occurred independent of antigen-presenting cells. Frequencies of CD127(+) cells were related to the magnitudes of proliferation enhancement that were mediated by IL-7. Thus, IL-7 enhances but does not fully restore the function of naïve CD4(+) T cells from HIV-infected persons.

Impaired induction of CD27 and CD28 predicts naive CD4 T cell proliferation defects in HIV disease.

Many immunological defects have been described in HIV disease, including a diminished capacity of naive CD4+ T cells to expand after TCR stimulation. The mechanisms underlying impaired naive CD4+ T cell expansion in HIV disease are not well described. Using a rigorous phenotypic definition of naive T cells, we found that cell cycle entry after TCR engagement was restricted to cells that increased surface expression of costimulatory molecules CD27 and CD28. Induction of these receptors, however, was not sufficient to result in cell cycle entry among the CD4+CD31- naive T cell subset. Analyses of cells from HIV-infected persons indicated that naive CD4+CD31+ T cells from these subjects were impaired in their ability to enter the cell cycle after stimulation and this impairment was predicted by the relatively poor induction of costimulatory molecules on these cells. Thus, failure to increase surface expression of costimulatory molecules may contribute to the naive T cell expansion failure that characterizes HIV infection.

Interferon-alpha differentially rescues CD4 and CD8 T cells from apoptosis in HIV infection.

OBJECTIVE: To examine the effects of interferon-alpha (IFN-alpha) on T cell survival and activation in HIV infection. DESIGN: The effects of IFN-alpha on spontaneous apoptosis and CD38 expression among T cell subsets were determined in vitro and studied in relation to CD4 cell counts, plasma HIV RNA levels and the age of the subjects. METHODS: Peripheral blood mononuclear cells from 48 HIV-infected persons and 17 healthy donors were incubated in vitro overnight with or without the addition of IFN-alpha. Percentages of apoptotic cells (positive for annexin V) and CD38 cells were determined among T cell subsets. RESULTS: IFN-alpha inhibited spontaneous apoptosis of CD4 and CD8 T lymphocytes. This protective activity was impaired in CD4 T cells from HIV-infected persons. The reduced protection of IFN-alpha among CD4 cells from HIV-infected persons was not related to the percentages of activated (CD38 or CD45RO+CD38+) cells. Surprisingly, IFN-alpha induced CD38 expression among CD8 T cells from HIV-infected persons, and the magnitude of this effect was directly related to circulating CD4 T cell count. The CD8 T cell subset that expressed CD38 in response to IFN-alpha was defined as CD28 negative, CD62 ligand (CD62L) intermediate/negative. CONCLUSIONS: Heightened expression of IFN-alpha in HIV infection may contribute to the phenotypic activation state that characterizes chronic infection while a diminished responsiveness of CD4 T cells to the protective effect of this cytokine may contribute to differential survival of CD4 and CD8 T cells in HIV disease.

Peripheral S-phase T cells in HIV disease have a central memory phenotype and rarely have evidence of recent T cell receptor engagement.

Heightened proliferation and death of T lymphocytes may play a key role in human immunodeficiency virus (HIV) pathogenesis; however, the mechanism that mediates this effect and the phenotype of the proliferating T cells have not been clearly determined. We assessed S-phase cell frequencies and phenotype by ex vivo bromodeoxyuridine incorporation and flow-cytometric analysis in a group of 35 HIV-infected individuals. Frequencies of S-phase T cells were increased in HIV disease and were related to plasma HIV RNA levels but not to CD4 cell, total T cell, or total lymphocyte counts. S-phase cells were phenotypically defined as "central memory" cells (CD45RO+CD62L+CCR7+). Although activated (CD38+), S-phase cells lacked CD69 expression, rarely expressed CD25, and were not overrepresented among HIV-specific cells, as might have been expected if these cells had recently been activated by HIV antigens. Thus, in HIV infection, central memory T cells may be highly susceptible to bystander mechanisms of immune activation, leading to S-phase entry.

Impaired TCR-mediated induction of Ki67 by naive CD4+ T cells is only occasionally corrected by exogenous IL-2 in HIV-1 infection.

Perturbations in naive T cell homeostasis and function may play a major role in the immunodeficiency that accompanies HIV infection. By examining naive CD4(+) T cell function on a single cell basis, we provide evidence that these cells have significant qualitative defects in HIV disease. Ki67, a molecule expressed during cell cycle progression, is induced less efficiently among naive CD4(+) T cells from HIV-infected individuals following activation with anti-TCR Ab. The impairment in Ki67 expression is evident even when a separate function, CD62L down-modulation, is within normal ranges. Moreover, the defects in Ki67 induction are only sometimes corrected by the addition of rIL-2 to cell cultures. An initial assessment of IL-2 unresponsiveness in cells from selected HIV-infected individuals suggests that the defect is not a consequence of impaired IL-2R expression or IL-2R signaling capability. Qualitative defects in naive T cells that cannot be routinely corrected by IL-2 have significant implications for disease pathogenesis and for strategies using IL-2 as a vaccine adjuvant in HIV disease.

Close link between CD4+ and CD8+ T cell proliferation defects in patients with human immunodeficiency virus disease and relationship to extended periods of CD4+ lymphopenia.

T cell proliferation failure is commonly associated with human immunodeficiency virus (HIV) infection. By examining T cell function on a single-cell basis, we found that CD4(+) T cell proliferation failure was often accompanied by CD8(+) T cell proliferation defects in patients with HIV disease. The defects are characterized by a proportional failure and reduced efficiency of precursor T cell proliferation after stimulation. In this study, patients who historically had low levels of circulating CD4(+) T cells were most likely to demonstrate cellular proliferation failure, regardless of current CD4(+) T cell counts. In contrast, neither historical nor current plasma HIV RNA levels were predictive of proliferation failure. These results suggest that mechanisms of T cell proliferation failure are more complex than can be explained by the direct effects of HIV replication and that therapeutic intervention to avoid prolonged periods of CD4(+) lymphopenia may be desirable for the preservation of immune function in patients with HIV disease.