The impact of age on vitamin D receptor expression, vitamin D metabolism and cytokine production in ex vivo Rhodococcus equi infection of equine alveolar macrophages.

Abstract

Rhodococcus equi (R. equi), a pneumonia-causing intracellular bacterium, results in significant morbidity and mortality in young foals, while healthy adult horses rarely develop disease. Survival and replication within alveolar macrophages (AM&#x3c6;) are the hallmarks of R. equi's pathogenicity. The vitamin D receptor (VDR) and its ligand, the active vitamin D metabolite 1,25(OH)D, are important in immune responses to intracellular bacteria. The vitamin D/VDR pathway regulates the downstream production of cytokines in infected human AM&#x3c6;. The immunomodulatory role of the vitamin D/VDR pathway in equine leukocytes is unknown. The objective of the current study was to determine the impact of R. equi infection and age on synthesis of 1,25(OH)D, VDR expression, and cytokine production in an ex vivo model of R. equi infection in equine AM&#x3c6;. AM&#x3c6; were collected from ten healthy foals at 2-, 4- and 8-weeks old and from nine healthy adult horses once via bronchoalveolar lavage. AM&#x3c6; were mock infected (CONTROL) or infected with a virulent laboratory strain of R. equi for 7 days (INFECTED). VDR expression was determined via RT-qPCR from cell lysates. 1,25(OH)D and cytokines were measured in cell supernatant by immunoassays. VDR expression was impacted by age (P&#xa0;=&#xa0;0.001) with higher expression in AM&#x3c6; from 8-week-old foals than from 2-week-old foals and adults. There was no significant effect of infection in foal AM&#x3c6;, but in adults, relative VDR expression was significantly lower in INFECTED AM&#x3c6; compared to CONTROL AM&#x3c6; (P&#xa0;=&#xa0;0.002). There was no effect of age or infection on 1,25(OH)D concentration (P&#xa0;>&#xa0;0.37). Mean TNF&#x3b1; production was significantly higher from INFECTED compared to CONTROL AM&#x3c6; from 4- and 8-week-old foals and adults (P&#xa0;<&#xa0;0.005). Mean IFN&#x3b3; production was significantly higher from AM&#x3c6; from foals at 8-weeks-old compared to 2-weeks-old (P&#xa0;=&#xa0;0.013) and higher from INFECTED AM&#x3c6; than from CONTROL AM&#x3c6; in foals at 4-weeks-old and in adults (P&#xa0;<&#xa0;0.027). The proportion of samples producing IL-1&#x3b2; and IL-10 was also significantly higher from INFECTED compared to CONTROL AM&#x3c6; isolated from 4-week-old foals (P < 0.008). Similarly, in adult samples, IL-17 was produced from a greater proportion of INFECTED compared to CONTROL samples (P&#xa0;=&#xa0;0.031). These data document age-associated changes in VDR expression and cytokine production in equine AM&#x3c6; in response to R. equi infection. This preliminary investigation supports the need for further research to fully elucidate if the vitamin D pathway has an immunomodulatory role in the horse.