Unique Ontogeny of Macrophages in the Adult Mouse Cornea

Abstract

Purpose : Macrophages (MF) in adult physiological conditions are arguably the most functionally diverse and abundant cell population of the mononuclear phagocyte system. The developmental lineage of MFs is largely binary. One set of origins derives from “primitive” hematopoiesis. Primitive MFs are established prenatally in tissues and are unique in their longevity and maintenance throughout adulthood via self-renewal. The second origin comes from “definitive” hematopoiesis. Definitive MFs are transient and maintained via turnover from circulating monocytes. MF ontogeny is important because developmental origins help shape functional specialization. As such, the current study was aimed to characterize the ontogeny of corneal (k) MFs in healthy adult mice.

Methods : We relied on lineage tracing mouse lines that can discriminate ontogeny at the genetic level. We applied Runx1^MerCreMer; eYFP^flox pregnant dams that were tamoxifen (tam) pulsed at E7.5, to identify primitive MFs. Conversely, to identify definitive MFs, we used Flt3^Cre; TdTomato^flox mice. To determine MF longevity, thought to be a unique feature of primitive MFs, we used tam pulse/wash-out Cxr3cr1^eYFP-CreER; TdTomato^flox mice. Corneal whole mounts were prepared from these adult mice and immunolabeled for F4/80, CD11b, Iba-1, or CD206. Colocalization of Cre-reporters were evaluated by confocal microscopy.

Results : From pulsed-labeled Runx1^MerCreMer; eYFP^flox dams, the adult progeny had rare eYFP-positive kMFs, suggesting against the presence of primitive MFs in the cornea. Congruently, Cx3cr1^eYFP-CreER; TdTomato^flox adult mice that were tam pulsed and assayed 1 year later, had rare tomato-positive kMFs, again suggesting against the presence of primitive MFs. By contrast, in Flt3^Cre; TdTomato^flox mice, the vast majority of kMFs were TdTomato-positive, indicative of a definitive lineage. Strikingly, however, in Cx3cr1^eYFP-CreER; TdTomato^flox adult mice that were tam pulsed and assayed 7 weeks later, the majority of kMFs were tomato-positive, potentially suggesting the presence of a definitive MF with intermediate longevity.

Conclusions : Our findings reveal that kMFs are definitively derived in adult mice. However, these MFs persist longer than the usual definitive MF lifespan, a characteristic more akin with primitive MFs. This challenges the definition that all definitive MFs are short-lived in adults and raises new questions about MF physiology, which may be unique to the cornea.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.