Training of circulating monocytes with particulate matter pollutant induces a robust inflammatory response
We hypothesized that alteration of monocytes associated with exposure to PM2.5 is mediated via induction of trained immunity. To test our hypothesis, we enriched circulating monocytes from N=10 donors and then stimulated them for 24h with a well-characterized particulate pollutant, standard reference material (SRM1648a). Next, we rested the monocytes for 6 days and then stimulated them with lipopolysaccharide (LPS) or house dust mite (HDM) aeroallergen for 24h (20 ). Finally, the levels of pro-inflammatory mediators were measured in supernatants (Fig. 3A). Training of monocytes with SRM1648a induced a significant upregulation of TNF, IL-6, and IL-8 after LPS or HDM stimulation (Fig. 3B).
We next asked whether prior exposure to ambient PM2.5contributes to trained immunity in monocytes. To address this question, we analyzed the responses, based on exposure to extremely low (N=5) versus extremely high levels of PM2.5 (N=5). Monocytes from individuals highly exposed to ambient PM2.5displayed increased TNF, IL-6, and IL-8 levels compared to monocytes from individuals exposed to low PM2.5 levels after stimulation with LPS or HDM (Fig. 3C). Additionally, monocytes were trained with β-glucan as a positive control and demonstrated a robust induction of trained immunity (N=5 per group, data not shown). Next, we demonstrated that the trained immunity phenotype induced by SRM1648a is retained if the same pollutant is used as the secondary stimulant. Notably, the level of IL-6 did not significantly change upon pollutant re-exposure, suggestive of a specific pattern of trained immunity elicited by a second encounter with pollutants (Fig. 3D).
We further validated pollution-mediated trained immunity by measuring secreted mediators in supernatants from monocyte cultures 7 days after induction of trained immunity using a Luminex assay (N=5 subjects with lowest versus 5 subjects with highest exposure to PM2.5). Training of circulating monocytes by the SRM1648a promoted a hyper-inflammatory response upon stimulation with LPS or HDM compared to the control untrained group; specifically, we observed significant upregulation of pro-inflammatory cytokines TNF, IL-1β, IL-6, GM-CSF, and G-CSF (Fig. 4A). In addition, SRM1648a provoked a significant increase in the levels of chemokines including, IL-8, MIP-1α, MIP-1β, GRO, and IP-10 after LPS or HDM stimulation (Fig. 4B). Chemokine responses were relatively specific, as MCP-1 and MDC levels did not significantly change after training of monocytes with SRM1648a compared to the control group (Fig. 4C). Secretion of MCP-3 was significantly elevated upon pollutant training regardless of the type of stimulation, LPS or HDM (Fig. 4C). Finally, the levels of EGF and FGF-2, were significantly elevated by either LPS or HDM stimulation after pollutant training (Fig. 4C), whereas VEGF level did not change compared to the control group (data not shown). Multivariate profiling of mediators secreted from monocytes upon pollution-induced training further demonstrated the relationships between analytes during re-stimulation (Supplementary Fig. 5). The differences between the analyte levels for LPS and HDM suggest distinct hyper-inflammatory profiles dependent on the type of stimulant. Altogether, we found that a pollution model analogous to PM2.5 exposure robustly induces trained immunity in monocytes.