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Phospholipid Metabolism Is Associated with HIV Rebound Upon Treatment Interruption

Lipids are biologically active molecules involved in a variety of cellular processes and immunological functions, including inflammation. It was recently shown that phospholipids and their derivatives, lysophospholipids, can reactivate latent (dormant) tumor cells, causing cancer recurrence. However, the potential link between lipids and HIV latency, persistence, and viral rebound after cessation of antiretroviral therapy (ART) has never been investigated. We explored the links between plasma lipids and the burden of HIV during ART. We profiled the circulating lipidome from the plasma of 24 chronically HIV-infected individuals on suppressive ART who subsequently underwent an analytic treatment interruption (ATI) without concurrent immunotherapies. The pre-ATI viral burden was estimated as time-to-viral-rebound and viral load setpoints post-ATI. We found that higher pre-ATI levels of lysophospholipids, including the pro-inflammatory lysophosphatidylcholine, were associated with faster time-to-viral-rebound and higher viral setpoints upon ART cessation. Furthermore, higher pre-ATI levels of the pro-inflammatory byproduct of intestinal lysophosphatidylcholine metabolism, trimethylamine-N-oxide (TMAO), were also linked to faster viral rebound post-ART. Finally, pre-ATI levels of several phosphatidylcholine species (lysophosphatidylcholine precursors) correlated strongly with higher pre-ATI levels of HIV DNA in peripheral CD4+ T cells. Our proof-of-concept data point to phospholipids and lysophospholipids as plausible pro-inflammatory contributors to HIV persistence and rapid post-ART HIV rebound. The potential interplay between phospholipid metabolism and both the establishment and maintenance of HIV latent reservoirs during- and post-ART warrants further investigation. IMPORTANCE The likelihood of HIV rebound after stopping antiretroviral therapy (ART) is a function of the interplay between the size of HIV reservoirs that persist despite ART and the host immunological and inflammatory factors that modulate these reservoirs. There is a need to comprehensively understand these host factors to develop a strategy to cure HIV infection and prevent viral rebound post-ART. Lipids are important biologically active molecules that are known to mediate several cellular functions, including reactivating latent tumor cells; however, their role in HIV latency, persistence, and post-ART rebound has never been investigated. We observed significant links between higher levels of the pro-inflammatory lysophosphatidylcholine and its intestinal metabolic byproduct, trimethylamine-N-oxide, and both faster time-to-viral rebound and higher viral load setpoint post ART. These data highlight the need for further studies to understand the potential contribution of phosphatidylcholine and lysophosphatidylcholine metabolism in shaping host immunological and inflammatory milieu during- and post-ART.

ABSTRACT: Lipids are biologically active molecules involved in a variety of cellular processes and immunological functions, including inflammation. It was recently shown that phospholipids and their derivatives, lysophospholipids, can reactivate latent (dormant) tumor cells, causing cancer recurrence. However, the potential link between lipids and HIV latency, persistence, and viral rebound after cessation of antiretroviral therapy (ART) has never been investigated. We explored the links between plasma lipids and the burden of HIV during ART. We profiled the circulating lipidome from the plasma of 24 chronically HIV-infected individuals on suppressive ART who subsequently underwent an analytic treatment interruption (ATI) without concurrent immunotherapies. The pre-ATI viral burden was estimated as time-to-viral-rebound and viral load setpoints post-ATI. We found that higher pre-ATI levels of lysophospholipids, including the pro-inflammatory lysophosphatidylcholine, were associated with faster time-to-viral-rebound and higher viral setpoints upon ART cessation. Furthermore, higher pre-ATI levels of the proinflammatory byproduct of intestinal lysophosphatidylcholine metabolism, trimethylamine-Noxide (TMAO), were also linked to faster viral rebound post-ART. Finally, pre-ATI levels of several phosphatidylcholine species (lysophosphatidylcholine precursors) correlated strongly with higher pre-ATI levels of HIV DNA in peripheral CD4+ T cells. Our proof-of-concept data point to phospholipids and lysophospholipids as plausible pro-inflammatory contributors to HIV persistence and rapid post-ART HIV rebound. The potential interplay between phospholipid metabolism and both the establishment and maintenance of HIV latent reservoirs during-and post-ART warrants further investigation.

IMPORTANCE:
The likelihood of HIV rebound after stopping antiretroviral therapy (ART) is a function of the interplay between the size of HIV reservoirs that persist despite ART and the host immunological and inflammatory factors that modulate these reservoirs. There is a need to comprehensively understand these host factors to develop a strategy to cure HIV infection and prevent viral rebound post-ART. Lipids are important biologically active molecules that are known to mediate several cellular functions, including reactivating latent tumor cells; however, their role in HIV latency, persistence, and post-ART rebound has never been investigated. We observed significant links between higher levels of the pro-inflammatory lysophosphatidylcholine and its intestinal metabolic byproduct, trimethylamine-N-oxide, and both faster time-to-viral rebound and higher viral load setpoint post ART. These data highlight the need for further studies to understand the potential contribution of phosphatidylcholine and lysophosphatidylcholine metabolism in shaping host immunological and inflammatory milieu during-and post-ART.  (4). While the interplay between lipids and both HIV and ART has been studied in the context of the development of inflammation-associated comorbidities, particularly subclinical atherosclerosis (5-9), the potential impact of lipids on HIV latency, persistence, and post-ART rebound has never been investigated.

KEYWORDS
There is currently no standard method to measure the total body burden of the replicationcompetent HIV reservoir (1, 10). However, a possible way to estimate both the overall size of the HIV reservoir and the degree of viral control is by assessing time-to-viral-rebound and/or viral load setpoints upon cessation of ART. In this study, we profiled the circulating lipidome from the plasma of 24 chronically HIV-infected individuals on suppressive ART who subsequently underwent an analytic treatment interruption (ATI) (11,12). All 24 individuals underwent ATI without concurrent immunomodulatory agents that might confound our analysis. Lipidomic analysis was performed using liquid chromatography-mass spectrometry (LC-MS), as described previously (13), on plasma samples collected immediately before ATI. Both time-to-viralrebound and viral load setpoints were measured during ATI. This cohort had a wide distribution of viral rebound times (14 to 119 days; median=28) and viral load setpoints (median=13,675 copies/ml; Supplementary Table 1). Using these data, we investigated whether there is a link between pre-ATI lipid profiles and the body burden of HIV during ART (estimated as post-ATI time-to-viral-rebound and viral load setpoints).

Levels of plasma lysophospholipids measured pre-ATI associate with time-to-viral-rebound
post-ATI. We identified a total of 967 lipids, belong to 21 lipids classes (described in found that pre-ATI levels of several of these lipids significantly associated with a faster time-to-viral-rebound ( Figure 1A; lipids with a hazard ratio (HR)>5 and P<0.01 are labeled). We next examined whether these lipids belong to particular lipidomic pathways or classes. Pathway analysis of all lipids whose pre-ATI levels associated with time-to-viral-rebound with P<0.05

Supplementary
showed that the pathway most associated with viral rebound was glycerophospholipid metabolism ( Figure 1B). The pre-ART levels of three lipid classes were significantly (FDR<0.05) associated with faster time-to-viral-rebound ( Figure 1C and Supplementary Table   3): lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), and lysophospholipid acid (LPA). All three classes belong to the lysophospholipid group, which is a subgroup of the glycerophospholipid family shown in Figure 1B. Lysophospholipids are small bioactive lipid molecules known to play important roles in regulating several biological functions, including promoting inflammation (8,(14)(15)(16)(17)(18)(19). The significant associations between these lysophospholipid classes and faster time-to-viral-rebound were confirmed using two additional, independent analyses: Mantel-Cox survival test, after separating participants into low or high groups based on the median level of each of these lipid classes ( Figure 1D); and Spearman's rank correlation between the levels of these lipid classes and time-to-viral-rebound (Supplementary Table 3).
These data point, for the first time, to plausible links between phospholipid and lysophospholipid metabolism and HIV rebound post ART. Intriguingly, similar phospholipids and lysophospholipids were recently shown to reactivate latent (dormant) cancer cells (4). Our exploratory findings, that are consistent with the reported functions of these lysophospholipids, raise the question of whether these lysophospholipids condition the host environment with higher levels of inflammation that might impact viral reactivation, cellular processes, and immunological functions during-and/or post-ATI.

Levels of plasma trimethylamine-N-oxide (TMAO) measured pre-ATI associate with post-ATI
time-to-viral-rebound. The pro-inflammatory lipid class LPC can be hydrolyzed in the intestine to LPA and choline; choline can be metabolized into trimethylamine, which is converted to trimethylamine-N-oxide (TMAO) in the liver (20). TMAO induces several pro-inflammatory mediators and has been implicated in several inflammation-associated diseases (20)(21)(22)(23). Given that LPC and LPA lipids were among the lipids whose pre-ATI levels associated with faster viral rebound upon ART cessation (Figure 1A-D), we sought to examine if levels of TMAO associated with post-ATI time-to-viral rebound. We performed metabolomics analysis, using LC-MS, as described previously (24), on the same pre-ATI plasma samples. Indeed, pre-ATI levels of TMAO were higher in individuals with lower than the median days to viral rebound (fast rebounders) compared to individuals with higher than the median days to rebound (delayed rebounders) (Figure 1E)  (Figure 2A). Furthermore, pre-ATI LPC and LPE classes levels correlated with post-ATI viral load setpoint (Figure 2B-C, respectively). Finally, the pre-ATI levels of the LPC (24:0) lipid species, which was one of the individual lipids whose pre-ATI level correlated with time-to-viral-rebound (Figure 1A), also associated with post-ATI viral load setpoints ( Figure 2D). Notably, levels of LPC (20:4) during HIV infection have been shown to associate with the progression of carotid artery atherosclerosis, even after ART suppression (6).
These data indicate that pre-ATI phospholipid metabolism is linked to viral load setpoint upon ART cessation.

Pre-ATI phosphatidylcholines associate with pre-ATI HIV DNA in the periphery. Finally, we
examined the links between pre-ATI plasma lipidome and pre-ATI total HIV DNA measured in periphery CD4 + T by droplet digital PCR (ddPCR), as described (25). Levels of several phosphatidylcholine species (precursors of lysophosphatidylcholine) significantly correlated (FDR <10%) with CD4 + T cell-associated HIV DNA (Figure 2E-H). These data further highlight the potential links between phospholipid metabolism and HIV persistence.
Our exploratory study has limitations, including small sample size and sampling of blood. The   Supplementary Table 3. A list of lipid classes whose pre-ATI levels associate with faster time- to-viral-rebound upon ART cessation.