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theses

Classically and alternatively activated macrophages and inflammatory mediators they release play a key role in the pathogenesis of ozone-induced lung injury. In these studies, we investigated the origin of these cells and mechanisms regulating their accumulation in the lung following ozone exposure. We hypothesized that macrophages originate in the bone marrow and the spleen, and that chemokine receptors CCR2 and CX3CR1 mediate their migration to the lung; moreover, macrophage activation is controlled, in part, by the nuclear receptor FXR. To test this hypothesis, we analyzed the effects of ozone on splenectomized mice, CCR2 knockout mice and FXR knockout mice. Following ozone exposure, increased numbers of pro-inflammatory CD11b+Ly6CHi and anti-inflammatory CD11b+Ly6CLo macrophages were observed in lungs of control (CTL) mice. Splenectomy resulted in decreases in pro-inflammatory macrophages in the lung and down regulation of CCR2, CCL2, and CCL4, but increases in CD11b+Ly6CLo anti-inflammatory macrophages. After ozone exposure, we also observed increases in lung macrophages staining positively for CCR2, a chemokine receptor known to mediate trafficking of pro-inflammatory macrophages from the bone marrow to sites of injury. Loss of CCR2 was associated with reduced numbers of CD11b+Ly6CHi and iNOS+ pro-inflammatory macrophages in the lung and decreased expression of the pro-inflammatory cytokines, IL-1β and TNFα. Decreases in proinflammatory/cytotoxic lung macrophages in SPX and CCR2-/- mice were correlated with reduced ozone toxicity and oxidative stress, demonstrating that these cells originate in both the spleen and bone marrow. To further investigate macrophage trafficking from the bone marrow, we generated GFP+ chimeric mice by adoptive transfer of 2x106 bone marrow (BM) cells from GFP+ mice into irradiated CTL mice. After 4 weeks, approximately 98% of BM cells were GFP+, while only 5% of lung macrophages were GFP+. Ozone exposure resulted in an increase in pro-inflammatory GFP+CD11b+Ly6CHi and anti-inflammatory GFP+CD11b+Ly6CLo macrophages in the lung at 24 h. Whereas GFP+Ly6CHi macrophages remained elevated for 72 h, increases in GFP+Ly6CLo macrophages were transient. These studies suggest that bone marrow contributes both pro- and anti-inflammatory macrophages to lung macrophage pools responding to ozone. This was confirmed using CX3CR1+/GFP reporter mice and by staining lung macrophages for CCR2. These data suggest that multiple macrophage subpopulations play distinct roles in ozone-induced lung injury. To investigate potential mechanisms regulating macrophage activation, we used transgenic mice lacking FXR, a nuclear receptor with anti-inflammatory activity. Treatment of WT mice with ozone resulted in increased FXR expression in the lung, most notably in macrophages. Loss of FXR resulted in increased numbers of pro-inflammatory Ly6CHi macrophages in the lung and prolonged up-regulation of iNOS, indicating chronic inflammation and macrophage activation. Conversely, numbers of MR+, YM1+, and Arg I+ anti-inflammatory macrophages were decreased. These data indicate that FXR plays a role in limiting lung inflammatory responses to ozone. Taken together, these studies demonstrate that multiple mechanisms contribute to pro-inflammatory and anti-inflammatory lung macrophage accumulation and activation in the lung following ozone exposure. Identification of the origin of inflammatory macrophages and of mechanism mediating their activation may be important in the development of novel therapeutics aimed at selectively targeting these cells and reducing inflammatory lung injury.

ETD_7967

Ph.D.

Includes bibliographical references

by Mary Francis

doi:10.7282/T3RR223F

ETD doctoral

The author owns the copyright to this work.