Dopamine-driven increase in IL-1β in myeloid cells is mediated by differential dopamine receptor expression and exacerbated by HIV

Abstract

The catecholamine neurotransmitter dopamine is classically known for regulation of central nervous system (CNS) functions such as reward, movement, and cognition. Increasing evidence also indicates that dopamine regulates critical functions in peripheral organs and is an important immunoregulatory factor. We have previously shown that dopamine increases NF-κB activity, inflammasome activation, and the production of inflammatory cytokines such as IL-1β in human macrophages. As myeloid lineage cells are central to the initiation and resolution of acute inflammatory responses, dopamine-mediated dysregulation of these functions could both impair the innate immune response and exacerbate chronic inflammation. However, the exact pathways by which dopamine drives myeloid inflammation are not well defined, and studies in both rodent and human systems indicate that dopamine can impact the production of inflammatory mediators through both D1-like dopamine receptors (DRD1, DRD5) and D2-like dopamine receptors (DRD2, DRD3, and DRD4). Therefore, we hypothesized that dopamine-mediated production of IL-1β in myeloid cells is regulated by the ratio of different dopamine receptors that are activated. Our data in primary human monocyte-derived macrophages (hMDM) indicate that DRD1 expression is necessary for dopamine-mediated increases in IL-1β, and that changes in the expression of DRD2 and other dopamine receptors can alter the magnitude of the dopamine-mediated increase in IL-1β. Mature hMDM have a high D1-like to D2-like receptor ratio, which is different relative to monocytes and peripheral blood mononuclear cells (PBMCs). We further confirm in human microglia cell lines that a high ratio of D1-like to D2-like receptors promotes dopamine-induced increases in IL-1β gene and protein expression using pharmacological inhibition or overexpression of dopamine receptors. RNA-sequencing of dopamine-treated microglia shows that genes encoding functions in IL-1β signaling pathways, microglia activation, and neurotransmission increased with dopamine treatment. Finally, using HIV as an example of a chronic inflammatory disease that is substantively worsened by comorbid substance use disorders (SUDs) that impact dopaminergic signaling, we show increased effects of dopamine on inflammasome activation and IL-1β in the presence of HIV in both human macrophages and microglia. These data suggest that use of addictive substances and dopamine-modulating therapeutics could dysregulate the innate inflammatory response and exacerbate chronic neuroimmunological conditions like HIV. Thus, a detailed understanding of dopamine-mediated changes in inflammation, in particular pathways regulating IL-1β, will be critical to effectively tailor medication regimens.

Fig. 1

Dopamine-mediated increase in IL-1β production in primary macrophages varies with expression of dopamine receptor subtypes. Primary human monocyte-derived macrophages (hMDM) were treated for 24 h with 10^–6 M dopamine. Lysates were collected and analyzed for IL-1β using AlphaLISAs. Results were normalized to protein concentration. qPCR detected mRNA for all subtypes of dopamine receptors (DRD1, DRD2, DRD3, DRD4 and DRD5). In our 42 donors, which together significantly respond to dopamine and increase IL-1β production (A), most express DRD1 and DRD2, whereas a much smaller proportion express DRD3 and DRD4 (B). The connection between dopamine receptors and IL-1β was determined by analysis of IL-1β levels in hMDM that did or did not express (C) DRD1, (D) DRD2, (E) DRD3, and (F) DRD4. Although all express DRD5, we examined DRD5’s impact on dopamine-induced IL-1β production by splitting expression into quartiles: low, medium, high, and highest expression (G). Significance was determined using Wilcoxon and Mann–Whitney tests, *p < 0.05, ***p < 0.001, and ****p < 0.0001

Fig. 2

Dopamine receptor expression levels in primary human myeloid cells change with maturation. Primary human peripheral blood mononuclear cells (PBMCs), monocytes, and monocyte-derived macrophages (hMDM) were collected for subsequent mRNA analysis. 19 of these donors were paired across the three cell types. A qPCR detected mRNA for all subtypes of dopamine receptors (DRD1, DRD2, DRD3, DRD4 and DRD5) that were differentially expressed in these cell types (N = 33–42). B Using a D1-like to D2-like ratio (DRD1 + DRD5 / (DRD2 + DRD3 + DRD4), hMDM had a significantly higher ratio compared to monocytes or PBMCs. Significance was determined using Kruskal–Wallis tests and post-hocs with Dunn’s multiple comparisons,*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

Fig. 3

Dopamine receptor-dependent effects on IL-1β expression in human microglia. Dopamine receptor transcripts were measured by qPCR. C06 and C20 microglia express DRD1, DRD2, and DRD5 mRNA. A In C06 microglia, there was significantly higher DRD2 expression compared to the D1-like receptors, and in B C20 microglia, there was significantly higher expression of the D1-like receptors compared to DRD2. C C06 and D C20 microglia were treated with dopamine (10^–6 M) or LPS (10 ng/mL) as a positive control for 3 h and examined for IL-1β mRNA. There was no effect in C06 microglia, but in C20 microglia, dopamine significantly increased IL-1β mRNA. E C06 and F C20 microglia were treated with dopamine (10^–6 M) or poly I:C (10 ug/mL) as a positive control for 4 h, then lysates were collected and examined for IL-1β production by AlphaLISA. There was no effect in C06 microglia. However, in C20 microglia dopamine significantly increased IL-1β production. G C06 and H C20 microglia were pretreated with flupentixol (Flux, 10–6 M) or vehicle for 30 min in the presence of absence of 3 h dopamine (DA, 10^–6 M) treatment and examined for IL-1β mRNA. There was no effect of treatment on C06 microglia, but in C20 microglia Flux significantly blocked the DA-mediated increase in IL-1β. Significance was determined by paired t-tests, Wilcoxon tests, Kruskal–Wallis tests, post-hoc with Dunn’s multiple comparisons, and a two-way mixed effects model and post-hoc with Tukey’s multiple comparisons, *p < 0.05, **p < 0.01, ***p < 0.001, and *****p < 0.0001. ND = not detected

Fig. 4

Differential gene expression of dopamine-treated human microglia. A Dopamine induced both up-regulated (776) and down-regulated (828) genes (fold change >|1.1| and FDR < 0.05) in C20 microglia. Violin plots showing the relative expression of genes in the two different conditions (vehicle or dopamine), including (B) genes previously found to be upregulated by dopamine in myeloid cells, (C) genes associated with modulation inf inflammation, (D) genes associated with regulation of dopaminergic signaling, (E) genes associated with Wnt signaling, and (F) genes associated with microglia identity and activation

Fig. 5

Dopamine increases IL-1β and inflammasome expression in HIV-infected primary macrophages. A Representative immunofluorescent images at 20X objective (DAPI, blue; Phalloidin, red; p24, green) show human monocyte-derived macrophages (hMDM) infected with 0.5 ng/mL HIV_ADA and treated with dopamine (10^–6 M) for 7 days. Higher levels of cell fusion and giant cell formation as well as p24 expression (white arrows) are seen in infected dopamine-treated cultures relative to cultures only infected with HIV. hMDM were also infected with 0.5 ng/mL HIV_ADA for 7 days and then treated with dopamine (10^–6 M) for 3 h and examined for (B) IL-1β, (C) NLRP3, (D) NLRC4, and (E) AIM2 expression by qPCR. There was a significant increase in IL-1β, NLRP3, and NLRC4 and a trending increase in AIM2 expression with HIV + Dopamine relative to HIV alone. With these same samples we simultaneously extracted protein and examined (F) NLRP3, (G) NLRC4, and (H) AIM2 expression by Western Blot. There was only a significant increase in NLRP3 expression with HIV + Dopamine relative to HIV alone. Significance was determined by paired t-tests and Wilcoxon tests, *p < 0.05 and **p < 0.01

Fig. 6

Dopamine increases IL-1β and NLRP3 in HIV-infected microglia. C20 microglia were infected with 2.5 ng/mL HIV_ADA for 48 h, then treated with dopamine (10^–6 M) for 3 h and examined for (A) IL-1β and (B) NLRP3 mRNA expression by qPCR. For both IL-1β and NLRP3 there was a significant increase with HIV + Dopamine relative to HIV alone. C iMicroglia were infected with 1 ng/mL of HIV_ADA for 7 days with or without dopamine (10^–6 M) and representative immunofluorescent images at 60X objective (DAPI, blue; CellMask, red; p24, green) show higher levels of cell fusion and giant cell formation as well as p24 expression (white arrows) in infected dopamine-treated cultures relative to cultures only infected with HIV. iMicroglia were also infected with 1 ng/mL of HIV_ADA for 7 days and then treated with dopamine (10⁻⁶ M) for either 3 h to examine IL-1β mRNA expression with qPCR or 4 h to examine IL-1β production by AlphaLISA. D Analysis of IL-1β mRNA levels show dopamine increases IL-1β in HIV-infected cells. E Analysis of lysate IL-1β levels by AlphaLISA show there is a trend that dopamine increases IL-1β in HIV-infected cells. Significance was determined by paired t-tests and Wilcoxon tests, *p < 0.05 and **p < 0.01