Title : Assessing the role of osteopontin in regulating cytokine expression among macrophages and neuroinflammatory pathways in HIV-Associated Neurocognitive Disorder (HAND)
Previous work in humanized mouse models of human immunodeficiency virus type-1 (HIV-1) has demonstrated that knocking down the matricellular protein Osteopontin (OPN) in vivo leads to a robust increase in the expression of proinflammatory markers TSPO and AIF-1, suggesting that OPN might have a physiological role as a molecular brake in suppressing the inflammatory response to viral infection (Mahmud et al., 2020). To further characterize how OPN specifically regulates the immune response during HIV infection, we knocked down OPN in human macrophages in the presence and absence of HIV infection, examining the corresponding changes in bulk RNA expression across protein-encoding genes. After finding a significant increase in expression of pro-inflammatory cytokines like IL6, TNFα, AIF1, IL1β, and a significant decrease in expression of anti-inflammatory cytokines like TGFβ in response to knocking down OPN, we propose that OPN may specifically regulate the immune response in HIV-infected macrophages by suppressing the expression of certain cytokines. Next, we conducted rt-PCR using macrophage RNA and found robust increases in amplification for IL6 and TNFα specifically in HIV-infected macrophages with OPN knockdown, supporting our hypothesis that OPN may physiologically suppress inflammation. Finally, we evaluated many canonical pathways that were disrupted by knocking down OPN and found increased T-helper cell activation by macrophages, increased macrophage trafficking and diapedesis, but also increased neuroinflammation. Together, this offers strong evidence for the role of OPN in regulating the immune response to HIV infection in macrophages and may more broadly substantiate the role of OPN signaling on modulating host cell factors critical for immune activation and maintaining the inflammatory burden of HIV.
The goal of this study was to investigate whether OPN functions as a molecular brake regulating the inflammatory response in human macrophages infected with HIV, given previous studies which validated this hypothesis in humanized mouse models for HIV. We produced additional evidence that knocking down OPN results in the overexpression of pro-inflammatory cytokines IL6, TNFα, AIF1, IL1β, and a significant decrease in the expression of anti-inflammatory cytokines like TGFβ. Additionally, we found more activation in inflammatory signaling pathways like activation of T-helper cells, increased immune trafficking and transcellular migration across endothelial layers, and even increased neuroinflammation via astrogliosis (data not shown). Given that IL6 and TNFα are known to cause astrocytosis in the brain and weaken the blood- brain barrier (Nair et al, 2008), increased VCAM-1 signaling may suggest that knocking down OPN may promote the mobilization of peripheral immune elements like macrophages across the blood-brain barrier via the upregulation of certain pro-inflammatory cytokines. This is important because it may suggest a similar pathophysiological basis for HIV-associated neurocognitive disorder and MS. Lastly, we confirmed in our rt-PCR studies that IL6 and TNFα primer sequences amplified significantly more when OPN was knocked down in macrophages infected with HIV, while controlling for basal and nonspecific amplification, further validating the hypothesis that OPN plays a strong role in the homeostatic regulation of cytokine activity. These results are crucial to understanding the molecular basis for the maintenance of neuroinflammation and neuropathology in the presence of viral infection, and may more broadly suggest that the downregulation of OPN may at least partly explain the overwhelming neuroinflammatory burden seen in HAND.