Transcriptional, Epigenetic, and Functional Reprogramming of Monocytes From Non-Human Primates Following Chronic Alcohol Drinking

Abstract

Chronic heavy drinking (CHD) of alcohol is a known risk factor for increased susceptibility to bacterial and viral infection as well as impaired wound healing. Evidence suggests that these defects are mediated by a dysregulated inflammatory response originating from myeloid cells, notably monocytes and macrophages, but the mechanisms remain poorly understood. Our ability to study CHD is impacted by the complexities of human drinking patterns and behavior as well as comorbidities and confounding risk factors for patients with alcohol use disorders. To overcome these challenges, we utilized a translational rhesus macaque model of voluntary ethanol self-administration that closely recapitulates human drinking patterns and chronicity. In this study, we examined the effects of CHD on blood monocytes in control and CHD female macaques after 12 months of daily ethanol consumption. While monocytes from CHD female macaques generated a hyper-inflammatory response to ex vivo LPS stimulation, their response to E. coli was dampened. In depth scRNA-Seq analysis of purified monocytes revealed significant shifts in classical monocyte subsets with accumulation of cells expressing markers of hypoxia (HIF1A) and inflammation (NFkB signaling pathway) in CHD macaques. The increased presence of monocyte subsets skewed towards inflammatory phenotypes was complemented by epigenetic analysis, which revealed higher accessibility of promoter regions that regulate genes involved in cytokine signaling pathways. Collectively, data presented in this manuscript demonstrate that CHD shifts classical monocyte subset composition and primes the monocytes towards a more hyper-inflammatory response to LPS, but compromised pathogen response.

Transcriptional and Epigenetic Regulation of Monocyte and Macrophage Dysfunction by Chronic Alcohol Consumption

“…These three classical clusters differentially expressed markers including CD14, MHC class II, TLR8, and CD86 ( 124 ). Interestingly, this high level of heterogeneity in classical monocytes was also observed in alcohol-fed NHPs with the identification of seven classical monocyte clusters by single cell RNA sequencing analysis ( 68 ). Two of these clusters were exclusively composed of cells from chronic heavy drinking macaques: HIF1A hi and SOD2 hi .…”

“…Upon LPS stimulation, monocytes of alcohol-fed NHPs upregulate genes associated with myeloid inflammatory pathways ( TNFSF21 , TLR2 ). These transcriptional pro-inflammatory changes were confirmed at the protein level with the increased production of pro-inflammatory mediators TNF-α, IL-6, IL-15, and CCL4 in LPS-stimulated monocytes ( 68 ). In splenic macrophages isolated from male macaques classified as chronic heavy drinkers, LPS stimulation induced greater production of TNF-α, MIP-1α, MIP-1β, IL-1β, IL-6, IL-8, and GM-CSF ( 22 ).…”

“…While this experimental model does not induce any overt liver damage over a 12-month period, signs of fatty liver disease were observed in cynomolgus macaques exposed to an open access period of 6 months followed by 12 months of abstinence that was followed by another open access period of up to 19 months (67). This rhesus macaque model was used to define the impact of chronic drinking on circulating and tissue-resident immune cells showing that the circulating innate immune cells bear the largest burden of chronic heavy drinking (21,22,(68)(69)(70)(71)(72)(73). However due to the inherent variation in alcohol consumed in the voluntary drinking models, a model of gastric infusion of alcohol was developed in order to deliver a consistent alcohol dose in treated rhesus macaques, where animals are infused with 30% ethanol in water (w/v) via an intragastric catheter either 4 days per week or 7 days per week during 3 months resulting in blood alcohol levels of 2.3 g/kg (74)(75)(76).…”

“…Defects in anti-microbial functions of monocytes and macrophages (Figure 1) have been reported to play a critical role in the observed increased vulnerability to infectious diseases due to chronic drinking resulting in reduced phagocytic activity and pathogenfighting function in Kupffer cells, microglia, alveolar and splenic macrophages (22,107,(125)(126)(127)(128). Under homeostatic conditions, chronic alcohol leads to a transcriptional downregulation of genes associated with immune and anti-viral responses (MHC class II, antigen processing and presentation, and IFNg signaling pathways) in non-classical monocytes of alcohol-fed NHPs (68). In contrast to the hyper-inflammatory response to LPS generated by monocytes/ macrophages from heavy drinking humans or animals, chronic alcohol exposure leads to a dampened production of immune mediators by macaque monocytes stimulated with E.coli bacteria, including reduced production of IL-1b, IL-5, IL-6, IL-15, CCL4 and CXCL11 (68).…”

“…Marker genes of these clusters enriched to gene ontology terms related to myeloid cell differentiation, cytokine signaling, and response to alcohol. Trajectory analysis showed that chronic heavy drinking was associated with a specific lineage that culminated in the HIF1A hi cluster, indicating that heavy drinking dysregulates monocyte differentiation states thereby affecting monocyte response to inflammation ( 68 ). In addition, chronic heavy drinking was associated with downregulated expression of MHC class II genes but upregulated expression of IFN-inducible genes and activation of NF-κB signaling pathway in classical monocytes ( 68 ).…”