Influence of plasma exosomes from women living with HIV Stratified by HAND on monocyte subpopulations from healthy women without HIV

HIV-associated neurocognitive disorders (HAND) continue to be a major cause of morbidity and mortality in PLWH (Heaton et al. 2010; Letendre et al. 2010; Vivithanaporn et al. 2010). Despite the success of antiretroviral therapy (ART) in improving the life expectancies of PLWH, the prevalence of HAND in milder forms is currently estimated at 20–50% (Eggers et al. 2017; Heaton et al. 2010; Letendre et al. 2010; Sacktor et al. 2016; Saylor et al. 2016; Simioni et al. 2010; Vivithanaporn et al. 2010). The pathophysiology of HAND involves increased blood–brain barrier (BBB) permeability, persistence of monocyte activation, systemic and central nervous system (CNS) inflammation, oxidative stress, neuronal dysfunction, and cell death (Cantres-Rosario et al. 2013; Gannon et al. 2011; Liner et al. 2010; McArthur et al. 2010). Currently, the associations of altered proportions of monocyte subpopulations based on the expression of CD14/CD16 surface markers with HIV neuropathogenesis remain to be investigated.

Studies have shown that despite ART, the number of inflammatory activated CD14 + CD16 + monocyte subsets increases in the peripheral blood, and these subsets cross the BBB and infect CNS macrophages and microglia, thus contributing to the development of HAND and the brain's viral reservoir (Buckner et al. 2011; Campbell et al. 2014; Fischer-Smith 2001; Ndhlovu et al. 2015; Valcour et al. 2011; Veenstra et al. 2017, 2019; Williams et al. 2014; Williams et al. 2012, 2013; Wong et al. 2019). However, the phenotype of the monocyte subpopulation that migrates into the brain and that is associated with HAND remains to be clarified.

Human monocytes are subdivided into three subsets based on the surface expression of CD14 and CD16: classical (CD14 + + CD16-), proinflammatory intermediate (CD14 + + CD16 +) and “patrolling” nonclassical (CD14 + CD16 +) subsets, with each subset representing approximately 90%, 5%, and 5% of the total circulating monocytes, respectively (Ożańska et al. 2020). Studies using grafted human classical monocytes in a humanized mouse model revealed that this subpopulation sequentially differentiates into intermediate and nonclassical monocyte subpopulations (Patel et al. 2017). Variations in the percentages of monocyte subpopulations can be observed in inflammatory diseases and infections. For example, a previous study in patients with sepsis or chronic inflammatory disease revealed that classical monocytes exhibit phagocytic functions without inflammatory attributes, intermediate monocytes display both phagocytic and inflammatory responses, and nonclassical monocytes exhibit inflammatory characteristics upon activation (Mukherjee et al. 2015). Furthermore, the levels of intermediate and nonclassical monocytes are increased in patients with sepsis, and only the nonclassical subpopulation is elevated in patients with chronic inflammatory diseases ((Mukherjee et al. 2015).

Alterations in monocyte subsets have also been observed in PLWH with HAND. The CD14 + CD16 + monocyte subpopulation is critical in the neuropathogenesis of HIV infection, and the proportion of this subpopulation can be increased to ∼40% in PLWH associated with HAND, despite ART (Campbell et al. 2014; Valcour et al. 2011; Williams et al. 2014; Williams et al. 2013). The factors that are responsible for the persistent changes in the monocyte subset phenotypic profiles of virologically suppressed PLWH are not clearly understood. Exosomes have emerged as being possible mediators of immune system activation (Chettimada et al. 2018; Li et al. 2019). These structures are membranous nanovesicles (with a size range of ~ 40–160 nm) of endosomal origin that are released from most cells and that are recognized as being mediators of cellular communication in normal physiological processes, such as the balance of the immune response, regulation of central and peripheral immunity, and cellular differentiation (Kalluri and LeBleu 2020; McKelvey et al. 2015; Saeedi et al. 2019; Ståhl et al. 2019). Exosomes can play a key role in different pathological conditions, including those linked to long-term inflammation associated with chronic immune activation, such as HIV infection and neurological diseases (Pérez et al. 2019; Ståhl et al. 2019). Exosomes may bind to receptors on target cells, thereby transducing a signal or transferring functional receptors, proteins, lipids, DNA, mRNAs, or miRNAs from parent cells to recipient cells, thus affecting cell function and behavior (Ståhl et al. 2019). For example, Freeman et al. revealed that monocyte subpopulations from female donors without allergies were able to internalize exosomes collected from the plasma of individuals with type 2 diabetes mellitus, thus inducing alterations in monocytic function, including increased expression of genes associated with immune function and inflammation, apoptosis and oxidative stress (Freeman et al. 2018). Moreover, studies have reported that exosomes are more abundant in the plasma of PLWH than in healthy controls (Pérez et al. 2019). Chettimada et al. showed that plasma exosomes from ART-treated PLWH containing immune activation markers such as CD14, C-reactive protein (CRP), human leukocyte antigen-A (HLA-A), and HLA-B were internalized by the human acute monocytic leukemia cell line THP-1 and induced the expression of genes associated with interferon responses, innate immune responses, and inflammation (Chettimada et al. 2018).

Previously, we reported that exosomes isolated from the plasma of WLWH with HAND in our Hispanic/Latino Longitudinal Cohort (HLLC) exhibited significantly increased levels of soluble insulin receptor (sIR), HIV-1 Tat, and ROS compared with those in exosomes isolated from the plasma of healthy controls (Cantres-Rosario et al. 2022). However, the influence of these exosomes on the phenotypic changes of HIV-uninfected monocytes has never been explored. In this study, we investigated whether the phenotypic profile of monocytes from healthy women without HIV was influenced by the uptake of plasma exosomes collected from WLWH stratified by HAND.

Different monocyte subpopulations are associated with HIV neuropathogenesis. Numerous studies suggest that an inflammatory monocyte subpopulation (CD14 + CD16 +) is persistent in the blood circulation of ART-treated PLWH and is associated with HAND by transmigrating across the BBB into the CNS (Buckner et al. 2011; Campbell et al. 2014; Fischer-Smith 2001; Ndhlovu et al. 2015; Valcour et al. 2011; Veenstra et al. 2017, 2019; Williams et al. 2014; Williams et al. 2012, 2013; Wong et al. 2019). Although many studies have reported an expansion of circulating intermediate and nonclassical monocytes in PLWH despite ART, further data are needed to understand the etiology of the CD14 + CD16 + subpopulation that is associated with HAND. Specifically, the factors that are responsible for the variations in the levels of CD14/CD16 monocyte subpopulations in ART-treated WLWH are not clearly understood.

Exosomes released from cells can exert regulatory influences on other cells, such as the modulation of host immune responses and microbial pathogenesis (Bellingham et al. 2012). Their secretion from HIV-infected cells may involve different cargo molecules and responses than those of uninfected cells. Previously, we reported in our HLLC cohort that HIV infection significantly increased the number of plasma exosomes containing reactive oxygen species, HIV-1 Tat, and soluble insulin receptor (Cantres-Rosario et al. 2022). However, their possible role in influencing the phenotypic changes of HIV-uninfected monocyte subpopulations has never been explored. In this study, we investigated whether plasma exosomes from WLWH stratified according to HAND can be internalized by monocytes from healthy female donors and induce changes in their CD14/CD16 phenotypic profiles (including classical, nonclassical, and intermediate subsets).

Our initial results revealed that exosomes from the plasma of WLWH patients or healthy controls were internalized by monocytes from healthy female donors in a time-dependent manner. Differences were observed in the monocytic uptake of exosomes from WLWH patients compared with that from healthy controls. Although monocytes exposed to both groups of exosomes exhibited significantly increased uptake at 24 h of incubation, the exosomes from WLWH increased monocytic uptake at an early time point of 6 h. When we evaluated exosome uptake in all three monocyte subpopulations, we observed an increase in all of the subsets in a time-dependent manner, with the increase being greater at 24 h. However, when we compared monocytes exposed to HIV-negative exosomes with those incubated with HIV-positive exosomes, the classical and intermediate monocyte subsets exhibited increased intracellular exosome levels (MFIs) when exposed to exosomes collected from WLWH (Fig. 2).

These findings suggest the presence of different modified cargos between HIV-positive and HIV-negative exosomes or the presence of different membrane receptors in monocyte subsets that can influence internalization. For example, studies have revealed that exosomes derived from HIV-infected cells rapidly enter recipient cells through the epidermal growth factor receptor (Chen et al. 2018). These receptors are present in peripheral blood monocytes and monocyte-derived macrophages, thus making them susceptible to exosome internalization (Chan et al. 2009; Lamb et al. 2004). Further studies are needed to characterize and identify the differences in monocyte receptors that are responsible for the entrance of HIV-positive exosomes compared with those of HIV-negative controls.

Our findings also support the unique individual functions of classical, intermediate, and nonclassical monocyte subpopulations when exposed to exosomes from WLWH stratified into NC and SNI groups. Our data revealed differences in exosome uptake among all of the monocyte subsets exposed to both NC and SNI exosomes (Figs. 4 and 5). This finding is interesting due to the fact that variations in the functions and levels of monocyte subsets have been identified in PLWH and HAND patients. Veenhuis et al. reported that higher proportions of intermediate monocytes and lower proportions of classical monocytes are associated with poorer cognitive function in U.S. cohorts of virologically suppressed WLWH (Veenhuis et al. 2021). Other studies suggest that the intermediate monocyte subpopulation is more permissive to HIV infection because of relatively higher expression levels of the HIV coreceptor CCR5, and these cells preferentially transmigrate across the BBB via higher surface cell expression of CCR2. Moreover, the cells can transport HIV into the CNS and release viral proteins, cytokines, and chemokines that trigger neuronal damage (Buckner et al. 2011; Rao et al. 2014; Veenhuis et al. 2021; Williams et al. 2012).

We also observed a decrease in the percentage of classical monocytes from 1 to 24 h after exposure to HIV-positive exosomes, whereas the percentage of the intermediate subpopulation significantly increased during the same time period (Fig. 6). This shift in phenotype to a more intermediate state as the number of classical monocytes decreases may suggest a role in the cell differentiation induced by HIV-positive exosomes. Further studies are warranted to elucidate the role of plasma HIV-positive exosomes in the increased proportions of classical and intermediate monocytes and decreased proportions of nonclassical monocytes.

Interestingly, higher percentages of all of the monocyte subpopulations were observed in cells exposed to NC exosomes at 1 h, 6 h and 24 h than in those exposed to SNI exosomes (Table 4). This finding is very important and requires more attention, due to the fact that it may provide insights into an early transitional stage of monocytic subpopulations associated with HAND and possible novel biomarkers in PLWH. These novel results may also help to identify novel targets and mechanisms at early time points in the progression of this condition to prevent or delay the progression to HAND.

Our study has certain limitations, such as the lack of information related to the date when the WLWH who donated exosomes became infected, the small sample size, and the availability of exosomes that solely originated from NC or SNI groups. Additionally, the lack of inclusion of plasma exosomes from men living with HIV (MLWH) represents a weakness of the study, due to the fact that our HLLC is solely comprised of WLWH. However, the results obtained from this study may provide information for future research investigating gender differences between men and women. HAND is a spectrum of neurocognitive dysfunction that also affects PLWH with asymptomatic neurocognitive impairment (ANI). This group is equally important because it is identified as being a transition point between unimpaired and impaired cognition. However, our data support the importance of evaluating the role of plasma exosomes from WLWH stratified according to HAND in the etiology of monocyte subsets and the mechanisms associated with the cognitive impairment that is observed in these patients.