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    • To determine the functional consequences

    2022-06-21

    To determine the functional consequences of HIV gp120 binding to vaginal epithelial cells, an annotation clustering of the differentially expressed genes was performed. The top enriched biological processes included an inflammatory immune response, defense response and the pathways included the SODD/TNFR1 signaling pathway and the NOD-like receptor signaling pathway. Most of the genes in the immune response cluster were of the proinflammatory type such as cytokines TNFα, LIF, IL-1 family member 9 as well as chemokines such as CXCL-1, CXCL-2. Serum amyloid A is an acute phase protein expressed in response to inflammation and tissue injury. Further, the epithelial specific E74 like factor 3 transcription factor transactivates collagenases and mediates inflammation. The activation of BIRC-3 and a decrease in caspase 8, would swing the SODD/TNFR1 pathway in favor of NF-κB activation, and proinflammatory cytokine induction. These processes would act in concert, contributing to large scale local inflammation in the vaginal epithelium. In vivo, inflammation has been found to be a major determinant enhancing HIV sexual transmission and accelerating viral dissemination. Further, genital tract viral load has also been found to correlate with inflammation and increased levels of proinflammatory cytokines like TNFα [32], [33]. Increased local concentrations of proinflammatory cytokines such as TNFα, have been shown to disrupt and cause permeability breaches in retinal, renal, airway, intestinal and endometrial epithelia through loss of cell junctions [34], [35] and a decrease in epithelial resistance [36]. The observations made herein that HIV gp120 binding to vaginal epithelial cells, induces an inflammatory milieu, would facilitate permeability breaches along the epithelium leading to HIV entry. Proteases were another cluster of genes that are of functional significance in HIV infection. Interestingly the increased mRNA expression of several proteases was corroborated by an increase in intracellular and extracellular protease activity in gp120 treated Dexlansoprazole as compared to controls. The proteases which were highly enriched included cysteine dependent endopeptidases, as well as metalloproteases. Cysteine dependent proteases included cathepsin (CTSB), and the calpains which are cytosolic non-lysosomal proteases. The metalloproteases included the secreted matrix-metalloproteinases and adamalysin which were upregulated. The membrane anchored metalloproteases including MME and dipeptidases which have broad proteolytic activity were also dysregulated. Other proteases upregulated included SUMO proteins, SENP3. Sumoylation of proteins, by SUMO-specific proteases, such as SENP3 is known to occur as a stress response in cells [37]. Kallikrein-mediated proteolysis which plays a role in processing of antimicrobials such as the cathelicidins found in CVL were altered, which may dysregulate host defense responses [38]. The proteases which would impact the barrier properties of the epithelium are of pathological significance and included the calpains, MMPs and adamalysin. CAPN12 and CAPN14 were among the most highly enriched proteases in the protease cluster. The levels of calpastatin which is an endogenous inhibitor of calpains was unchanged. Calpains are known to loosen cell contacts through proteolysis of focal adhesion complexes leading to cell detachment, and also mediate degradation of tight junction proteins in cervix [39] and lung epithelial cells [40]. The increase in calpains may promote a weakening of cell–cell contacts of the vaginal epithelium. Besides calpains, MMP-7 was another protease upregulated in response to HIV gp120 treatment. MMP-9 was another metalloprotease that has also been previously reported by us to exhibit increased transcription and activity following gp120 binding to vaginal epithelial cells [14]. MMPs mediate degradation of tight junction components as well as adherens junction, leading to increased permeability of epithelia [41], [42], [43], [44]. The levels of Tissue Inhibitors of Matrix Metalloproteinases, TIMP-1, TIMP-2 and TIMP-3 were found to be unchanged (unpublished data). The MMP:TIMP ratio shifts the balance in favor of increased metalloprotease activity. The adamalysins which included the ADAMTS (a disintegrin like and metalloproteinase with thrombospondin type1 motifs) Zn dependent proteases are secreted in the extracellular proteinaceous matrix, and involved in matrix remodeling and proteolysis. The increased production of these proteases would lead to possible degradation of tight junctions and the extracellular matrix in the vaginal epithelium and basement membrane, leading to breaches in the epithelium. Supporting this hypothesis of epithelial barrier leakiness, studies have demonstrated that genital epithelial cells exhibit disruption of tight junctions, and increased permeability following binding of gp120 [36].