Systems biology analyses reveal enhanced chronic morphine distortion of gut-brain interrelationships in simian human immunodeficiency virus infected rhesus macaques

Abstract

BackgroundCommonly used opioids, such as morphine have been implicated in augmented SIV/HIV persistence within the central nervous system (CNS). However, the extent of myeloid cell polarization and viral persistence in different brain regions remains unclear. Additionally, the additive effects of morphine on SIV/HIV dysregulation of gut-brain crosstalk remain underexplored. Therefore, studies focused on understanding how drugs of abuse such as morphine affect immune dynamics, viral persistence and gut-brain interrelationships are warranted.Materials and methodsFor a total of 9 weeks, rhesus macaques were ramped-up, and twice daily injections of either morphine (n = 4) or saline (n = 4) administered. This was later followed with infection with SHIVAD8EO variants. At necropsy, mononuclear cells were isolated from diverse brain [frontal lobe, cerebellum, medulla, putamen, hippocampus (HIP) and subventricular zone (SVZ)] and gut [lamina propria (LP) and muscularis (MUSC) of ascending colon, duodenum, and ileum] regions. Multiparametric flow cytometry was used to were profile for myeloid cell polarity/activation and results corroborated with indirect immunofluorescence assays. Simian human immunodeficiency virus (SHIV) DNA levels were measured with aid of the digital droplet polymerase chain reaction (PCR) assay. Luminex assays were then used to evaluate soluble plasma/CSF biomarker levels. Finally, changes in the fecal microbiome were evaluated using 16S rRNA on the Illumina NovaSeq platform.ResultsFlow Cytometry-based semi-supervised analysis revealed that morphine exposure led to exacerbated M1 (CD14/CD16)/M2 (CD163/CD206) polarization in activated microglia that spanned across diverse brain regions. This was accompanied by elevated SHIV DNA within the sites of neurogenesis–HIP and SVZ. HIP/SVZ CD16+ activated microglia positively correlated with SHIV DNA levels in the brain (r = 0.548, p = 0.042). Simultaneously, morphine dependence depleted butyrate-producing bacteria, including Ruminococcus (p = 0.05), Lachnospira (p = 0.068) genera and Roseburia_sp_831b (p = 0.068). Finally, morphine also altered the regulation of CNS inflammation by reducing the levels of IL1 Receptor antagonist (IL1Ra).ConclusionThese findings are suggestive that morphine promotes CNS inflammation by altering receptor modulation, increasing myeloid brain activation, distorting gut-brain crosstalk, and causing selective enhancement of SHIV persistence in sites of neurogenesis.

Urinary cytokine measurements do not reflect surgery‐induced inflammation in rhesus macaques

“…A total of 33 different cytokines were measured in urine using the Luminex system (Table 1). All tested biomarkers have been successfully measured from plasma in prior studies of macaques (Bidokhti et al, 2018;Höglind et al, 2017;Joas et al, 2020;Olwenyi et al, 2022;Pantoja et al, 2017;Pérez-Guzmán et al, 2019;Singh et al, 2021;Woollard et al, 2018). The assay included evaluation of processed as described in Giavedoni (2005).…”

“…A total of 33 different cytokines were measured in urine using the Luminex system (Table 1). All tested biomarkers have been successfully measured from plasma in prior studies of macaques (Bidokhti et al, 2018; Höglind et al, 2017; Joas et al, 2020; Olwenyi et al, 2022; Pantoja et al, 2017; Pérez‐Guzmán et al, 2019; Singh et al, 2021; Woollard et al, 2018). The assay included evaluation of the following analytes: B‐cell activating factor (BAFF); B lymphocyte chemoattractant (BLC, CXCL13); C‐reactive protein (CRP); eotaxin‐1 (CCL11); granulocyte colony‐stimulating factor (G‐CSF); growth‐related oncogene‐a (GRO‐a, CXCL1); indoleamine‐pyrrole 2,3‐dioxygenase (IDO); interferon‐α (IFN‐α); IFN‐γ; interferon‐inducible T‐cell α chemoattractant (I‐TAC, CXCL11); interferon‐γ‐induced protein 10 kDa (IP‐10, CXCL10); interleukin‐1 beta (IL‐1β); IL‐1 receptor antagonist (IL‐1RA); IL‐4; IL‐8; IL‐10; IL‐12 p40 and p70; IL‐15; IL‐18; IL‐22; IL‐33 receptor (IL33R; ST2); lectin‐type oxidized low‐density lipoprotein receptor 1 (LOX‐1); macrophage migration inhibitory factor (MIF); monokine induced by γ interferon (MIG, CXCL9); macrophage inflammatory protein 1‐α (MIP‐1α, CCL3), MIP‐1β (CCL4); monocyte chemoattractant protein 1 (MCP‐1, CCL2); myeloperoxidase (MPO); perforin; regulated on activation, normal T cell expressed and secreted CCL5 (RANTES); tumor necrosis factor‐α (TNF‐α); soluble CD40 ligand (sCD40L); soluble intercellular adhesion molecule 1 (sICAM‐1); and vascular endothelial growth factor A (VEGF‐A).…”

“…Biomarkers were those typically measured in blood in clinical studies, and which are known to be effective biomarkers that rise in response to infection and inflammation. They have all been measured from plasma and used in prior studies of macaques (Bidokhti et al, 2018; Giavedoni, 2005; Höglind et al, 2017; Joas et al, 2020; Olwenyi et al, 2022; Pantoja et al, 2017; Pérez‐Guzmán et al, 2019; Singh et al, 2021; Woollard et al, 2018). We found that many (13/33) of these biomarkers were of measurable concentration in less than 50% of urine samples.…”