Viral transmission and pathogenesis in human tissues

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1. Upon release, HIV-1 incorporates cellular membrane proteins from infected cells. The functions of these proteins in virions remain largely unknown. We analyzed, in plasma of HIV-infected individuals, macrophage-derived HIV-1 virions bearing the host cell marker CD36. We found, using flow virometry, our technology that allows characterization of single viral particles, that some of these HIV-1 virions carry the immunosuppressive cytokine TGF- bound through TSP-1 to CD36. This cytokine remains bioactive, as shown by incubation of the virions with reporter cells. Bioactive cytokines carried on HIV-1 virions may change the immune status of HIV-1 target cells. In summary, our work demonstrated for the first time the existence of HIV-1 virions that are associated with an important cytokine, TGF-. Thus, not only cytokines produced by the human body cells, but also cytokines carried by virions may contribute to HIV infection. The importance of cytokines in HIV-1 infection was further emphasized in studying HIV-1 sexual transmission. We compared the cytokine profiles in blood and semen of infected individuals to investigate whether they are associated with HIV transmission to their sexual partner who became infected with a phylogenetically linked HIV strain (transmitters) or did not acquire HIV (non-transmitter). We found that the cytokine spectra in blood and semen of infected individuals are different, and focused on the latter. Semen provides an immunological milieu, largely determined by cytokines, in which HIV-1 variants are selected. Our analysis revealed that the cytokine profiles between transmitters and non-transmitters were statistically different in semen but not in blood. The unique cytokine profile in the semen of the transmitter group suggests these cytokines are likely to be an important determinant of HIV-1 transmission. Rather than a single cytokine being associated with transmission, we found a group of cytokines, namely IFN-gamma, IL-13, M-CSF, IL-17, and GM-CSF. This study provides the first correlations between seminal cytokines in the transmitting partner and HIV transmission, thus providing new targets for the development of HIV-1 preventive strategies. 2. Atherosclerosis is known to be an immunologic, inflammatory disease and is common in people living with HIV and in the general aging population where it is an important cause of morbidity. Atherosclerosis is associated with a dysregulation of the cytokine network leading to migration of T cells, in particular CD8+, to the area of plaque formation. We collected plasma samples from 48 volunteers without cardiovascular disease and 62 patients with ST-elevated myocardial infarction (STEMI). Samples were separated into soluble and EV fractions and analysed for 33 cytokine expressions using a multiplexed bead-based assay. Concentrations of evaluated cytokines were different in patients with STEMI and in control individuals. Next, we compared the frequencies of detection for the cytokines released in either soluble or EV-associated forms in patients with STEMI with those in controls. The frequencies of detection of soluble IL-1, IL-6, IL-8, IL-12p70, and TNF- were significantly higher in patients with STEMI than in controls. Furthermore, we analyzed the distribution of cytokines between soluble and EV-associated forms, and found that in control volunteers, multiple cytokines and some chemokines were found more often in the EV fraction than in the soluble fraction, while in patients with STEMI only a few were detected in EV-associated form more often than in soluble form. In control subjects without cardiovascular disease, the majority of cytokines were released independently either in soluble form or in EVs, while in patients with STEMI half of the measured cytokines were simultaneously released in the same individuals in both EV-associated and soluble forms. We performed a cluster analysis of cytokines and demonstrated that soluble cytokines in patients with STEMI are upregulated in a coordinated fashion, in contrast to the mainly unaffected system of EV-associated cytokines. Identification of cytokine clusters in STEMI permits investigation of their distinct contributions to this pathology, in particular to T-cell migration into the plaques. In pursuing this goal, we investigated mechanisms of T-cell migration in carotid arteries and plaques of SIV- or SHIV-infected non-human primates and in HIV-infected humans. We showed that expression of both CX3CL1 and IL-15 was greater in the aortas of SHIV/SIV-infected animals than controls. Furthermore, there was a trend toward more CX3CL1 expression, significantly greater IL-15 expression, and a significant increase in CD8 T cells in human atherosclerotic tissues. Finally, we showed that CX3CR1/CX3CL1 interactions promote chemoattraction of IL-15exposed CD8 T cells to endothelial cells. We concluded that IL-15 drives a pro-chemotactic phenotype on CD8 T cells that are then primed for endothelium localization via CX3CR1 ligation. Once CD8 T cells are in close proximity to endothelial cells, they induce activation and dysfunction of these cells. Plaque formation is also associated with migration of CD4 T cells. In HIV-1infected cells this migration was enhanced by co-infection with cytomegalovirus. In a cohort of HIV-infected individuals (under therapy) with CMV coinfection, we found that many CD4 memory T cells have a CD57+ phenotype and that CD57 expression was highest within the effector memory and terminally differentiated compartments. Thus, dysregulation of cytokines is translated into migration of both CD4 and CD8 cells into atherosclerotic plaques, possibly leading to their destabilization. Understanding these mechanisms may result in development of new therapeutic strategies. 3. SARS CoV-2 infects lung epithelia triggering severe pathology. Investigation of the mechanisms of SARS CoV-2 tissue pathogenesis and development of antiviral strategies, in particular, requires a deep understanding of SARS CoV-2 pathogenesis in the context of human tissue systems under laboratory-controlled conditions. We developed such a system. Human lung tissue is dissected into 2mm3 blocks and cultured at the air-liquid interface. Histology of these blocks revealed well preserved structural elements including alveoli with epithelial cells. Flow cytometry of cells from these blocks confirmed their viability and expression of ACE-2 receptor. Next, we obtained SARS CoV-2 pseudoviruses expressing S protein and encoding eGFP. These one-cycle viruses were tested for infectivity on cell lines expressing ACE-2 and lung tissues and proven to be infectious. Development of human lung tissue ex vivo pseudoviruses now allows us to characterize SARS CoV-2 main target cells with high-parameter flow cytometry.