The emergence of inflammatory microglia during gut inflammation is not affected by FFAR2 expression in intestinal epithelial cells or peripheral myeloid cells.

Gut inflammation can trigger neuroinflammation and is linked to mood disorders. Microbiota-derived short-chain fatty acids (SCFAs) can modulate microglia, yet the mechanism remains elusive. Since microglia do not express free-fatty acid receptor (FFAR)2, but intestinal epithelial cells (IEC) and peripheral myeloid cells do, we hypothesized that SCFA-mediated FFAR2 activation within the gut or peripheral myeloid cells may impact microglia inflammation. To test this hypothesis, we developed a tamoxifen-inducible conditional knockout mouse model targeting FFAR2 exclusively on IEC and induced intestinal inflammation with dextran sodium sulfate (DSS), a well-established colitis model. Given FFAR2's high expression in myeloid cells, we also investigated its role by selectively deleting it in these populations of cells. In an initial study, male and female wild-type mice received 0 or 2% DSS for 5d and microglia were isolated 3d later to assess inflammatory status. DSS induced intestinal inflammation and upregulated inflammatory gene expression in microglia, indicating inflammatory signaling via the gut-brain axis. Despite the lack of significant effects of sex in the intestinal phenotype, male mice showed higher microglial inflammatory response than females. Subsequent studies using FFAR2 knockout models revealed that FFAR2 expression in IECs or immune myeloid cells did not affect DSS-induced colonic pathology (i.e. clinical and histological scores and colon length), or colonic expression of inflammatory genes. However, FFAR2 knockout led to an upregulation of several microglial inflammatory genes in control mice and downregulation in DSS-treated mice, suggesting that FFAR2 may constrain neuroinflammatory gene expression under healthy homeostatic conditions but may permit it during intestinal inflammation. No interactions with sex were observed, suggesting sex does not play a role on FFAR2 potential function in gut-brain communication in the context of colitis. To evaluate the role of FFAR2 activated by microbiota-derived SCFAs, we employed the same knockout and DSS models adding fermentable dietary fiber (0 or 2.5% inulin for 8 wks). Despite no genotype or fiber main effects, contrary to our hypothesis, inulin feeding augmented DSS-induced inflammation and signs of colitis, suggesting context-dependent effects of fiber. These findings highlight microglial involvement in colitis-associated neuroinflammation and advance our understanding of FFAR2's role in the gut-brain axis. Although not integral, we observed that the role of FFAR2 differs between homeostatic and inflammatory conditions, underscoring the need to consider different inflammatory conditions and disease contexts when investigating the role of FFAR2 and SCFAs in the gut-brain axis.

Functional characterization of the biogenic amine transporters on human macrophages.

Monocyte-derived macrophages (MDMs) are key players in tissue homeostasis and diseases regulated by a variety of signaling molecules. Recent literature has highlighted the ability for biogenic amines to regulate macrophage functions, but the mechanisms governing biogenic amine signaling in and around immune cells remain nebulous. In the CNS, biogenic amine transporters are regarded as the master regulators of neurotransmitter signaling. While we and others have shown that macrophages express these transporters, relatively little is known of their function in these cells. To address these knowledge gaps, we investigated the function of norepinephrine transporter (NET) and dopamine transporter (DAT) on human MDMs. We found that both NET and DAT are present and can uptake substrate from the extracellular space at baseline. Not only was DAT expressed in cultured MDMs, but it was also detected in a subset of intestinal macrophages in situ. Surprisingly, we discovered a NET-independent, DAT-mediated immunomodulatory mechanism in response to LPS. LPS induced reverse transport of dopamine through DAT, engaging an autocrine/paracrine signaling loop that regulated the macrophage response. Removing this signaling loop enhanced the proinflammatory response to LPS. Our data introduce a potential role for DAT in the regulation of innate immunity.

Targeting neurotransmitter-mediated inflammatory mechanisms of psychiatric drugs to mitigate the double burden of multimorbidity and polypharmacy.

The increased incidence of multimorbidities and polypharmacy is a major concern, particularly in the growing aging population. While polypharmacy can be beneficial, in many cases it can be more harmful than no treatment, especially in individuals suffering from psychiatric disorders, who have elevated risks of multimorbidity and polypharmacy. Age-related chronic inflammation and immunopathologies might contribute to these increased risks in this population, but the optimal clinical management of drug-drug interactions and the neuro-immune mechanisms that are involved warrants further investigation. Given that neurotransmitter systems, which psychiatric medications predominantly act on, can influence the development of inflammation and the regulation of immune function, it is important to better understand these interactions to develop more successful strategies to manage these comorbidities and complicated polypharmacy. I propose that expanding upon research in translationally relevant human in vitro models, in tandem with other preclinical models, is critical to defining the neurotransmitter-mediated mechanisms by which psychiatric drugs alter immune function. This will define more precisely the interactions of psychiatric drugs and other immunomodulatory drugs, used in combination, enabling identification of novel targets to be translated into more efficacious diagnostic, preventive, and therapeutic interventions. This interdisciplinary approach will aid in better precision polypharmacy for combating adverse events associated with multimorbidity and polypharmacy in the future.

Dopamine Levels Induced by Substance Abuse Alter Efficacy of Maraviroc and Expression of CCR5 Conformations on Myeloid Cells: Implications for NeuroHIV.

Despite widespread use of antiretroviral therapy (ART), HIV remains a major public health issue. Even with effective ART many infected individuals still suffer from the constellation of neurological symptoms now known as neuroHIV. These symptoms can be exacerbated by substance abuse, a common comorbidity among HIV-infected individuals. The mechanism(s) by which different types of drugs impact neuroHIV remains unclear, but all drugs of abuse increase central nervous system (CNS) dopamine and elevated dopamine increases HIV infection and inflammation in human myeloid cells including macrophages and microglia, the primary targets for HIV in the brain. Thus, drug-induced increases in CNS dopamine may be a common mechanism by which distinct addictive substances alter neuroHIV. Myeloid cells are generally infected by HIV strains that use the chemokine receptor CCR5 as a co-receptor, and our data indicate that in a subset of individuals, drug-induced levels of dopamine could interfere with the effectiveness of the CCR5 inhibitor Maraviroc. CCR5 can adopt distinct conformations that differentially regulate the efficiency of HIV entry and subsequent replication and using qPCR, flow cytometry, Western blotting and high content fluorescent imaging, we show that dopamine alters the expression of specific CCR5 conformations of CCR5 on the surface of human macrophages. These changes are not affected by association with lipid rafts, but do correlate with dopamine receptor gene expression levels, specifically higher levels of D1-like dopamine receptors. These data also demonstrate that dopamine increases HIV replication and alters CCR5 conformations in human microglia similarly to macrophages. These data support the importance of dopamine in the development of neuroHIV and indicate that dopamine signaling pathways should be examined as a target in antiretroviral therapies specifically tailored to HIV-infected drug abusers. Further, these studies show the potential immunomodulatory role of dopamine, suggesting changes in this neurotransmitter may also affect the progression of other diseases.

Methamphetamine Increases the Proportion of SIV-Infected Microglia/Macrophages, Alters Metabolic Pathways, and Elevates Cell Death Pathways: A Single-Cell Analysis.

Both substance use disorder and HIV infection continue to affect many individuals. Both have untoward effects on the brain, and the two conditions often co-exist. In the brain, macrophages and microglia are infectable by HIV, and these cells are also targets for the effects of drugs of abuse, such as the psychostimulant methamphetamine. To determine the interaction of HIV and methamphetamine, we isolated microglia and brain macrophages from SIV-infected rhesus monkeys that were treated with or without methamphetamine. Cells were subjected to single-cell RNA sequencing and results were analyzed by statistical and bioinformatic analysis. In the animals treated with methamphetamine, a significantly increased proportion of the microglia and/or macrophages were infected by SIV. In addition, gene encoding functions in cell death pathways were increased, and the brain-derived neurotropic factor pathway was inhibited. The gene expression patterns in infected cells did not cluster separately from uninfected cells, but clusters comprised of microglia and/or macrophages from methamphetamine-treated animals differed in neuroinflammatory and metabolic pathways from those comprised of cells from untreated animals. Methamphetamine increases CNS infection by SIV and has adverse effects on both infected and uninfected microglia and brain macrophages, highlighting the dual and interacting harms of HIV infection and drug abuse on the brain.

Herring roe oil supplementation alters microglial cell gene expression and reduces peripheral inflammation after immune activation in a neonatal piglet model.

Neonatal brain development can be disrupted by infection that results in microglial cell activation and neuroinflammation. Studies indicate that polyunsaturated fatty acids (PUFAs) and their metabolites can resolve inflammation. It is not known if dietary PUFA increases lipid metabolites in brain or reduces neuroinflammation in neonates. We hypothesized that dietary PUFAs might suppress neuroinflammation by inhibiting pro-inflammatory cytokine over-production and promoting inflammatory resolution in the periphery and brain. Piglets were obtained on postnatal day (PD) 2 and randomly assigned to herring roe oil (HRO) or control (CON) diet. HRO was included at 2 g/kg powdered diet. HRO increased DHA levels in occipital lobe and the DHA to arachidonic acid (ARA) ratio in hippocampal tissue. HRO decreased ARA metabolites in occipital lobe. HRO failed to attenuate microglial pro-inflammatory cytokine production ex vivo. HRO did not affect fever or circulating resolvin D1 levels. HRO decreased circulating neutrophils and liver inflammatory gene expression, but increased resolution marker gene expression in liver post LPS. HRO upregulated CXCL16, TGFBR1, and C1QA in microglial cells. HRO supplementation exerted beneficial effects on inflammation in the periphery, but further studies are needed to evaluate the specific effects of omega-3 supplementation on microglial cell physiology in the neonate.

Dietary Iron Deficiency Impaired Peripheral Immunity but Did Not Alter Brain Microglia in PRRSV-Infected Neonatal Piglets.

During the postnatal period the developing brain is vulnerable to insults including nutrient insufficiency and infection that may lead to disrupted development and cognitive dysfunction. Since iron deficiency (ID) often presents with immunodeficiency, the objective of this study was to investigate peripheral viremia and inflammation as well as brain microglial phenotype and function when ID and respiratory infection occur simultaneously in a neonatal piglet model. On postnatal day 2 (PD 2) male and female piglets were assigned to one of four treatments and fed either control or ID milk replacer. On PD 8 half the pigs on each diet were inoculated with either vehicle or porcine reproductive and respiratory syndrome virus (PRRSV; P-129). Blood samples were collected prior to inoculation (PD 7) and repeated once weekly. Rectal temperature, feeding score, and sickness behavior were measured daily until PD 28. Hematocrit, hemoglobin, and serum iron were reduced by ID but not PRRSV infection. PRRSV-infected piglets displayed viremia by PD 14; however, those fed control diet had lower viral titer on PD 28, while circulating virus remained elevated in those fed an ID diet, suggesting that ID either impaired immune function necessary for viral clearance or increased viral replication. ID piglets infected with PRRSV displayed reduced sickness behavior compared to those fed control diet on PD 13-15 and 18-20. While ID piglet sickness behavior progressively worsened, piglets fed control diet displayed improved sickness score after PD 21. Microglia isolated from PRRSV piglets had increased MHCII expression and phagocytic activity ex vivo compared to uninfected piglets. ID did not alter microglial activation or phagocytic activity. Similarly, microglial cytokine expression was increased by PRRSV but unaffected by ID, in stark contrast to peripheral blood mononuclear cell (PBMC) cytokine expression, which was increased by infection and generally decreased by ID. Taken together, these data suggest that ID decreases peripheral immune function leading to increased viremia, but immune activity in the brain is protected from acute ID.