Cytokine release syndrome, also referred to as hypercytokinemia or a ‘cytokine storm’, is a consequence of a dysregulated immune response and has been linked to the pathology seen in individuals presenting with severe COVID-19 (1). High levels of pro-inflammatory cytokines cause symptoms such as fever, hemodynamic instability, coagulopathy, splenomegaly, hepatitis, and multi-organ failure, which can be fatal (1). One of the common causes of mortality in COVID-19 is hypercoagulability (2), with some patients presenting with strokes, myocardial infarcts, pulmonary emboli, mesenteric ischemia, and limb thrombosis (3). Microemboli have also been associated with myocarditis and cardiac failure (2).

While it is still unclear whether the virus itself has intrinsic procoagulant effects, coagulopathy likely stems from the inflammatory response observed in these individuals and the ensuing endothelial activation or damage (4). Some authors have suggested that COVID-19 should be viewed and treated as a ‘true vascular disease’ and the importance of endothelium-platelet interactions is increasingly recognised (5, 6).

Although the main function of platelets is to maintain vascular integrity through coagulation and angiogenesis, their role in both the innate and adaptive immune response is also well recognised (7). Platelets are involved in the first-line response to invading pathogens, activating an innate immune response through Toll-like receptors (TLRs) (7). Toll-like receptor-7 plays an important role in protecting against viral infections in the innate immune system (8). SARS-CoV-2, a single-stranded ribonucleic acid (ssRNA) virus, binds to TLR-7 within endosomal compartments of cells, including platelets (9). Following binding of ssRNA viruses to TLR-7, the MyD88-dependent pathway is initiated, resulting in upregulated expression of inflammatory cytokines, most notably interleukin (IL)-1, IL-6, IL-10, IL-12, and tumor necrosis factor (TNF)-α, by various immune cells including T-cells, B-cells, macrophages, monocytes, fibroblasts, plasmacytoid dendritic cells, and endothelial cells (10). The upregulated expression of these cytokines is consistent with a skewing of the immune response towards a T helper (Th)2 or Th17 phenotype (10).

It has been shown that platelets interact directly with SARS-CoV-2 and enhance uptake of the virus into various cell types (9). Platelets are hyperactivated during SARS-CoV-2 infection through mechanisms as diverse as epithelial damage, hypoxia, neutrophil extracellular trap formation, interactions between SARS-CoV-2 spike protein and platelets, autoimmune responses, and autocrine activation (6). Activated platelets undergo degranulation and release a number of inflammatory mediators that are stored in their granules (11). In addition, activated platelets also exhibit upregulated expression of adhesion molecules, including platelet-derived growth factor (PDGF) and regulated upon activation normal T-cell expressed and secreted (RANTES), which are both stored in and secreted from platelet α-granules (12).

South Africa has one of highest prevalences of HIV infection in the world, estimated at 13% in the general and 18.7% in the adult population (13). From the start of the pandemic, it was feared that HIV-associated immunodeficiency might predispose people living with HIV (PLWH) to infection with SARS-CoV-2, and that the persistent, systemic immune activation caused by HIV might exacerbate hyperinflammation during COVID-19. Both scenarios could lead to poor outcomes. Notably, an increase in thrombotic events (up to ten-fold higher) has been reported in PLWH receiving antiretroviral therapy (ART) compared to those of the healthy, uninfected population (14). This vulnerable population is, therefore, potentially at an even higher risk of developing thrombosis if co-infected with SARS-CoV-2.

Nevertheless, despite the existence of an extensive body of literature, predominantly encompassing clinical studies and systematic reviews focused on comparisons of outcomes of patients hospitalized with COVID-19 without and with HIV co-infection, few of these studies have included detailed comparisons of the levels of biomarkers of systemic innate immune activation and their associations with organ dysfunction and outcome. To address this issue, the present study compared associations between the systemic levels of biomarkers of platelet and endothelial activation with clinical parameters and treatment outcomes in people living with and without HIV infection, admitted to hospital with COVID-19 in Tshwane, South Africa.

As expected, COVID-19 was characterized by high levels of systemic inflammation, as reflected by the acute phase reactant, CRP, which is produced by the liver in response to increased expression of IL-6. Increased levels of CRP lead to elevated concentrations of IL-1β and TNF-α that, in turn, lead to the increased expression of adhesion molecules such as ICAM-1 (15). All the platelet and endothelial markers, with the exception of PDGF-BB, were significantly higher in people with COVID-19 than in the group of healthy controls, indicating the extent of platelet and endothelial activation elicited by SARS-CoV-2 (6).

Overall, levels of inflammation were equivalent between COVID-19 patients with and without HIV infection. While the levels of pro-inflammatory markers, such as CRP, IL-6, IL-1Ra, and TNF-α, were slightly higher, and anti-inflammatory cytokines, such as TGF-β1, were lower in COVID-19/PLWH than in those without HIV, none of these differences was significant. The platelet chemokine, RANTES, was significantly lower in PLWH but, after adjusting for other factors significantly associated with RANTES, such as age and sex, this difference lost significance. The initial difference observed was therefore likely due to the dissimilar demographic profiles of the patients with and without HIV infection.

The aforementioned systemic, pro-inflammatory biomarker data reflected, and probably explain, the essentially comparable clinical data, which characterized the groups of SARS-CoV-2-HIV-infected and –uninfected patients. While patients with COVID-19 without HIV infection appeared to have a more coordinated immune response, as reflected by the larger number of significant correlations between the immunological markers, this did not translate into differences in levels of inflammation or clinical outcomes. There were, however, a few exceptions. PLWH had lower levels of the acute phase reactant, ferritin, possibly secondary to less hypoxia (supported by the fact that PLWH needed less oxygen supplementation). In contrast, PLWH had higher levels of the coagulation marker, INR, together with non-significantly elevated levels of D-dimer. D-Dimer levels increase on recurrent cycles of coagulation and fibrinolysis, are associated with thrombosis and thromboembolic conditions and are, therefore, considered an important predictor of these events in COVID-19 patients (16). A correlation between D-Dimer and various physiological processes recognized in PLWH have been reported. These include endothelial dysfunction, microbial translocation, and active viral replication, as measured by VL (17–19). Interestingly, an equivalent number of clinical thrombotic events was reported in the two groups, although an important caveat is the absence of specialized scans that could have enabled diagnosis of occult thrombosis.

After multivariable logistic regression that accounted for age, gender, and differences in the prevalence of diabetes between the groups, VEGF emerged as a marker of interest. VEGF is a potent angiogenic factor and inducer of vascular permeability (20). It is released during hypoxia or in inflammatory conditions in response to endothelial injury and has been shown to be significantly increased in patients with COVID-19, correlating with disease severity (20, 21).

The early stage of endothelial activation is characterized by increased expression of ICAM-1 on the surface of endothelial cells to allow trans-endothelial migration of leukocytes to sites of inflammation (20). This is followed by elevation of plasminogen activator inhibitor-1 (PA-1) and soluble thrombomodulin that initiate alterations in the coagulation process, as well as VEGF, which drives angiogenesis (20). The interaction of these biomarkers with leukocytes, smooth muscle cells, and other proinflammatory cytokines, leads to remodeling of the vessel wall and subsequent vasculopathy (22). In this regard, it is interesting to note that VEGF was strongly and positively correlated with the pro-inflammatory cytokines, IL-1β, TNF-α and the chemokine, MIP-1α, in both groups, indicating the recruitment of inflammatory cells to the newly formed vessels.

HIV has long been known to be associated with higher levels of VEGF than uninfected controls (23). This is generally assumed to be detrimental and associated with the presence of Kaposi’s sarcoma, as well as HIV-associated encephalopathy (24). It has, however, also been postulated that VEGF may play a role in maintaining vasculature and protecting against the development of age-related cognitive decline, and may hence represent an appropriate response under conditions of hypoxia (25). In this regard, it is interesting to note that, in our study, VEGF was higher in PLWH but that their oxygen saturation was higher and FiO2 lower, indicating less hypoxia. This suggests that the association between VEGF and HIV is unrelated to hypoxia, a contention also supported by others (26). It is intriguing to consider whether the higher levels of VEGF observed in PLWH could be related to the lower oxygen requirement in this population.

It would seem that the elevated levels of VEGF were caused by the interaction between HIV and SARS-CoV-2, since levels were significantly higher in the presence of co-infection than with HIV infection alone. This interaction was, however, evident only in patients with good HIV control, since levels in the co-infected group with active viral replication had median VEGF levels at the lower limit of detection of the assay. This is further supported by the fact that the difference in VEGF between COVID-19/PLWH and control PLWH was only evident in the presence of an undetectable VL. It is therefore interesting to note that some authors have proposed that VEGF might be a biomarker of a more preserved immune system (27). In this regard, it is noteworthy that PLWH with a low CD4 count and unsuppressed VL, common features of those who had demised, had significantly lower levels of VEGF.

COVID-19/PLWH with a CD4 count ≤200 cells/μL could be distinguished from those with higher CD4 counts by lower levels of VEGF and higher levels of IL-6. While their inflammatory markers were similar, it is notable that all four of the COVID-19/PLWH who died were in this category. This finding should be considered in light of the significant lymphopenia observed in all COVID-19 patients in this study.

In conclusion, this study of patients hospitalized with COVID-19 demonstrated a relatively low mortality and good outcomes overall, both in people with and without HIV infection. Although significantly elevated, no significant differences were observed in the levels of cytokines, chemokines, and growth factors for platelet- and endothelial-associated markers between patients with and without HIV infection, which is in keeping with the clinical findings. The exception was VEGF, which was higher in PLWH. VEGF is a potent angiogenic factor, which could be advantageous under hypoxic conditions. Interestingly, VEGF concentrations were lower in PLWH with a low CD4 count and unsuppressed VL, which could be an indication that these patients are less able to mount an appropriate immune response.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

Ethics approval was obtained from the Research Ethics Committee of the Faculty of Health Sciences of the University of Pretoria (247/2020). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained for participation from the participants or the participants’ legal guardians/next of kin.

TR, FA and VU conceptualized the study. MM, ZB and HS prepared samples and performed the biomarker assays. FA, VU and ZB recruited participants and collected clinical data. MM, ZB, HS, RA and TR wrote the final manuscript. All authors contributed to the article and approved the submitted version.