Endothelial cell infection and dysfunction, immune activation in severe COVID-19.

Rationale: Pulmonary vascular endotheliitis, perivascular inflammation, and immune activation are observed in COVID-19 patients. While the initial SARS-CoV-2 infection mainly infects lung epithelial cells, whether it also infects endothelial cells (ECs) and to what extent SARS-CoV-2-mediated pulmonary vascular endotheliitis is associated with immune activation remain to be determined. Methods: To address these questions, we studied SARS-CoV-2-infected K18-hACE2 (K18) mice, a severe COVID-19 mouse model, as well as lung samples from SARS-CoV-2-infected nonhuman primates (NHP) and patient deceased from COVID-19. We used immunostaining, RNAscope, and electron microscopy to analyze the organs collected from animals and patient. We conducted bulk and single cell (sc) RNA-seq analyses, and cytokine profiling of lungs or serum of the severe COVID-19 mice. Results: We show that SARS-CoV-2-infected K18 mice develop severe COVID-19, including progressive body weight loss and fatality at 7 days, severe lung interstitial inflammation, edema, hemorrhage, perivascular inflammation, systemic lymphocytopenia, and eosinopenia. Body weight loss in K18 mice correlated with the severity of pneumonia, but not with brain infection. We also observed endothelial activation and dysfunction in pulmonary vessels evidenced by the up-regulation of VCAM1 and ICAM1 and the downregulation of VE-cadherin. We detected SARS-CoV-2 in capillary ECs, activation and adhesion of platelets and immune cells to the vascular wall of the alveolar septa, and increased complement deposition in the lungs, in both COVID-19-murine and NHP models. We also revealed that pathways of coagulation, complement, K-ras signaling, and genes of ICAM1 and VCAM1 related to EC dysfunction and injury were upregulated, and were associated with massive immune activation in the lung and circulation. Conclusion: Together, our results indicate that SARS-CoV-2 causes endotheliitis via both infection and infection-mediated immune activation, which may contribute to the pathogenesis of severe COVID-19 disease.

SARS-CoV-2 Infects Endothelial Cells In Vivo and In Vitro.

SARS-CoV-2 infection can cause fatal inflammatory lung pathology, including thrombosis and increased pulmonary vascular permeability leading to edema and hemorrhage. In addition to the lung, cytokine storm-induced inflammatory cascade also affects other organs. SARS-CoV-2 infection-related vascular inflammation is characterized by endotheliopathy in the lung and other organs. Whether SARS-CoV-2 causes endotheliopathy by directly infecting endothelial cells is not known and is the focus of the present study. We observed 1) the co-localization of SARS-CoV-2 with the endothelial cell marker CD31 in the lungs of SARS-CoV-2-infected mice expressing hACE2 in the lung by intranasal delivery of adenovirus 5-hACE2 (Ad5-hACE2 mice) and non-human primates at both the protein and RNA levels, and 2) SARS-CoV-2 proteins in endothelial cells by immunogold labeling and electron microscopic analysis. We also detected the co-localization of SARS-CoV-2 with CD31 in autopsied lung tissue obtained from patients who died from severe COVID-19. Comparative analysis of RNA sequencing data of the lungs of infected Ad5-hACE2 and Ad5-empty (control) mice revealed upregulated KRAS signaling pathway, a well-known pathway for cellular activation and dysfunction. Further, we showed that SARS-CoV-2 directly infects mature mouse aortic endothelial cells (AoECs) that were activated by performing an aortic sprouting assay prior to exposure to SARS-CoV-2. This was demonstrated by co-localization of SARS-CoV-2 and CD34 by immunostaining and detection of viral particles in electron microscopic studies. Moreover, the activated AoECs became positive for ACE-2 but not quiescent AoECs. Together, our results indicate that in addition to pneumocytes, SARS-CoV-2 also directly infects mature vascular endothelial cells in vivo and ex vivo, which may contribute to cardiovascular complications in SARS-CoV-2 infection, including multipleorgan failure.

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. Vß 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.

Interferon-α inhibits CD4 T cell responses to interleukin-7 and interleukin-2 and selectively interferes with Akt signaling.

Persistent type I IFN production occurs during chronic viral infections, such as HIV disease. As type I IFNs have antiproliferative activity, it is possible that chronic exposure to these cytokines could adversely affect T cell homeostasis. We investigated the capacity of IFN-α to impair T cell proliferation induced by the homeostatic cytokine, IL-7, or another common γ-chain cytokine, IL-2, in cells from healthy human donors. We found that IL-7- or IL-2-induced proliferation of CD4(+) T cells was partially inhibited in the presence of IFN-α. The CD4(+) T cells that were exposed to IFN-α also displayed attenuated induction of IL-2 and CD40L following TCR stimulation. Analyses of signaling pathways indicated that IL-7 and IL-2 induced a delayed and sustained P-Akt signal that lasted for several days and was partially inhibited by IFN-α. In contrast, IL-7-induced P-STAT5 was not affected by IFN-α. Furthermore, IFN-α had no detectable effect on P-Akt that was induced by the chemokine SDF-1. Both inhibitors of P-Akt and P-STAT5 blocked IL-7-induced T cell proliferation, confirming that both signaling pathways are important for IL-7-induced T cell proliferation. These results demonstrate that IFN-α can selectively inhibit cytokine-induced P-Akt as a potential mechanism to disrupt homeostasis of T lymphocytes.

Inflammatory cytokines drive CD4+ T-cell cycling and impaired responsiveness to interleukin 7: implications for immune failure in HIV disease.

BACKGROUND: Systemic inflammation has been linked to a failure to normalize CD4(+) T-cell numbers in treated human immunodeficiency virus (HIV) infection. Although inflammatory cytokines such as interleukin 6 (IL-6) are predictors of disease progression in treated HIV infection, it is not clear how or whether inflammatory mediators contribute to immune restoration failure. METHODS: We examined the in vitro effects of IL-6 and interleukin 1ß (IL-1ß) on peripheral blood T-cell cycling and CD127 surface expression. RESULTS: The proinflammatory cytokine IL-1ß induces cell cycling and turnover of memory CD4(+) T cells, and IL-6 can induce low-level cycling of naive T cells. Both IL-1ß and IL-6 can decrease T-cell surface expression and RNA levels of CD127, the interleukin 7 receptor α chain (IL-7Rα). Preexposure of healthy peripheral blood mononuclear cells (PBMCs) to IL-6 or IL-1ß attenuates IL-7-induced Stat5 phosphorylation and induction of the prosurvival factor Bcl-2 and the gut homing integrin α4ß7. We found elevated expression of IL-1ß in the lymphoid tissues of patients with HIV infection that did not normalize with antiretroviral therapy. CONCLUSIONS: Induction of CD4(+) T-cell turnover and diminished T-cell responsiveness to IL-7 by IL-1ß and IL-6 exposure may contribute to the lack of CD4(+) T-cell reconstitution in treated HIV-infected subjects.

Impaired T-cell responses to sphingosine-1-phosphate in HIV-1 infected lymph nodes.

The determinants of HIV-1-associated lymphadenopathy are poorly understood. We hypothesized that lymphocytes could be sequestered in the HIV-1+ lymph node (LN) through impairments in sphingosine-1-phosphate (S1P) responsiveness. To test this hypothesis, we developed novel assays for S1P-induced Akt phosphorylation and actin polymerization. In the HIV-1+ LN, naïve CD4 T cells and central memory CD4 and CD8 T cells had impaired Akt phosphorylation in response to S1P, whereas actin polymerization responses to S1P were impaired dramatically in all LN maturation subsets. These defects were improved with antiretroviral therapy. LN T cells expressing CD69 were unable to respond to S1P in either assay, yet impaired S1P responses were also seen in HIV-1+ LN T cells lacking CD69 expression. Microbial elements, HIV-1, and interferon α - putative drivers of HIV-1 associated immune activation all tended to increase CD69 expression and reduce T-cell responses to S1P in vitro. Impairment in T-cell egress from lymph nodes through decreased S1P responsiveness may contribute to HIV-1-associated LN enlargement and to immune dysregulation in a key organ of immune homeostasis.

Shared monocyte subset phenotypes in HIV-1 infection and in uninfected subjects with acute coronary syndrome.

The mechanisms responsible for increased cardiovascular risk associated with HIV-1 infection are incompletely defined. Using flow cytometry, in the present study, we examined activation phenotypes of monocyte subpopulations in patients with HIV-1 infection or acute coronary syndrome to find common cellular profiles. Nonclassic (CD14(+)CD16(++)) and intermediate (CD14(++)CD16(+)) monocytes are proportionally increased and express high levels of tissue factor and CD62P in HIV-1 infection. These proportions are related to viremia, T-cell activation, and plasma levels of IL-6. In vitro exposure of whole blood samples from uninfected control donors to lipopolysaccharide increased surface tissue factor expression on all monocyte subsets, but exposure to HIV-1 resulted in activation only of nonclassic monocytes. Remarkably, the profile of monocyte activation in uncontrolled HIV-1 disease mirrors that of acute coronary syndrome in uninfected persons. Therefore, drivers of immune activation and inflammation in HIV-1 disease may alter monocyte subpopulations and activation phenotype, contributing to a pro-atherothrombotic state that may drive cardiovascular risk in HIV-1 infection.

Down-regulation of serum gonadotropins is as effective as estrogen replacement at improving menopause-associated cognitive deficits.

Declining levels of estrogen in women result in increases in gonadotropins such as luteinizing hormone (LH) through loss of feedback inhibition. LH, like estrogen, is modulated by hormone replacement therapy. However, the role of post-menopausal gonadotropin increases on cognition has not been evaluated. Here, we demonstrate that the down-regulation of ovariectomy-driven LH elevations using the gonadotropin releasing hormone super-analogue, leuprolide acetate, improves cognitive function in the Morris water maze and Y-maze tests in the absence of E2. Furthermore, our data suggest that these effects are independent of the modulation of estrogen receptors alpha and beta, or activation of CYP19 and StAR, associated with the production of endogenous E2. Importantly, pathways associated with improved cognition such as CaMKII and GluR1-Ser831 are up-regulated by leuprolide treatment but not by chronic long-term E2 replacement suggesting independent cognition-modulating properties. Our findings suggest that down-regulation of gonadotropins is as effective as E2 in modulating cognition but likely acts through different molecular mechanisms. These findings provide a potential novel protective strategy to treat menopause/age-related cognitive decline and/or prevent the development of AD.