• CD68-GFP reporter mice show GFP transgene expression in both monocytes and tissue resident macrophage populations.• Adoptively transferred CD68-GFP monocytes maintain GFP expression after recruitment in an ongoing inflammatory response.The recruitment of monocytes and their differentiation into macrophages at sites of inflammation are key events in determining the outcome of the inflammatory response and initiating the return to tissue homeostasis. To study monocyte trafficking and macrophage differentiation in vivo, we have generated a novel transgenic reporter mouse expressing a green fluorescent protein (GFP) under the control of the human CD68 promoter. CD68-GFP mice express high levels of GFP in both monocyte and embryoderived tissue resident macrophages in adult animals. The human CD68 promoter drives GFP expression in all CD115 1 monocytes of adult blood, spleen, and bone marrow; we took advantage of this to directly compare the trafficking of bone marrow-derived CD68-GFP monocytes to that of CX 3 CR1 GFP monocytes in vivo using a sterile zymosan peritonitis model. Unlike CX 3 CR1 GFP monocytes, which downregulate GFP expression on differentiation into macrophages in this model, CD68-GFP monocytes retain high-level GFP expression for 72 hours after differentiation into macrophages, allowing continued cell tracking during resolution of inflammation. In summary, this novel CD68-GFP transgenic reporter mouse line represents a powerful resource for analyzing monocyte mobilization and monocyte trafficking as well as studying the fate of recruited monocytes in models of acute and chronic inflammation. (Blood. 2014;124(15):e33-e44)

CYP4A11 transgenic mice (CYP4A11 Tg) were generated to examine in vivo regulation of the human CYP4A11 gene. Expression of CYP4A11 in mice yields liver and kidney P450 4A11 levels similar to those found in the corresponding human tissues and leads to an increased microsomal capacity for -hydroxylation of lauric acid. Fasted CYP4A11 Tg mice exhibit 2-3-fold increases in hepatic CYP4A11 mRNA and protein, and this response is absent in peroxisome proliferator-activated receptor ␣ (PPAR␣) null mice. Dietary administration of either of the PPAR␣ agonists, fenofibrate or clofibric acid, increases hepatic and renal CYP4A11 levels by 2-3-fold, and these responses were also abrogated in PPAR␣ null mice. Basal liver CYP4A11 levels are reduced differentially in PPAR␣ ؊/؊ females (>95%) and males (<50%) compared with PPAR␣ ؊/؉ mice. Quantitative and temporal differences in growth hormone secretion are known to alter hepatic lipid metabolism and to underlie sexually dimorphic gene expression, respectively. Continuous infusion of low levels of growth hormone reduced CYP4A11 expression by 50% in PPAR␣-proficient male and female transgenic mice. A larger decrease was observed for the expression of CYP4A11 in PPAR␣ ؊/؊ CYP4A11 Tg male mice to levels similar to that of female PPAR␣-deficient mice. These results suggest that PPAR␣ contributes to the maintenance of basal CYP4A11 expression and mediates CYP4A11 induction in response to fibrates or fasting. In contrast, increased exposure to growth hormone down-regulates CYP4A11 expression in liver.

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Cytochrome P450 1A1 (CYP1A1) is induced by halogenated and polycyclic aromatic hydrocarbons following activation of the aryl hydrocarbon receptor (AhR). Protein kinase C (PKC) has been implicated in the regulation of this response. In tissue culture, induction of PKC activity with phorbol esters synergizes the actions of TCDD-induced CYP1A1, while PKC inhibitors block induction of CYP1A1 by TCDD. Here, the actions of specific PKC inhibitors on CYP1A1 induction were examined using a HepG2 human cell line (TV101L) that carries a stably integrated firefly luciferase gene under control of the human CYP1A1 promoter (-1612/+293). TV101 cells were treated with TCDD and either the kinase inhibitor staurosporine or one of the PKC inhibitors GF109203X, Gö6983, or Gö6976. Aryl hydrocarbon receptor-dependent activation of CYP1A1-luciferase and cellular PKC activity were measured. TCDD treatment induced CYP1A1-luciferase activity in an AhR-dependent manner, as determined by binding of nuclear AhR to xenobiotic response elements (XREs). Dose-dependent inhibition of PKC activity by staurosporine was concordant with inhibition of TCDD-induced CYP1A1-luciferase activity. However, the PKC inhibitors GF109203X, Gö6983, and Gö6976 blocked PKC activity at concentrations independent of those necessary to block TCDD induction of CYP1A1-luciferase activity. For all inhibitors, reduction in CYP1A1-luciferase activity was independent of AhR activation, as determined by electrophoretic mobility shift analysis of TCDD-activated nuclear AhR. The specific PKC inhibitors did not significantly alter cytosolic or nuclear levels of AhR protein, whether alone or in combination with TCDD. These results suggested that PKC was not the sole factor responsible for regulation of CYP1A1.

Transgenic mice (CYP4A11‐Tg) were generated to examine regulation of the human CYP4A11 gene by PPARa agonists and fasting in vivo. Expression of the CYP4A11 transgene in mice gave liver and kidney CYP4A11 protein levels similar to those found in the corresponding human tissues. Fasted CYP4A11‐Tg exhibited a 2‐ to 3‐fold increase in hepatic CYP4A11 mRNA and protein as measured by qPCR and Western blots, respectively. Treatment with PPARa agonists, fenofibrate or clofibric acid, produced 2‐ to 4‐fold increases in hepatic CYP4A11 mRNA and protein. Collectively, these data show that fasting and PPARa agonists enhance CYP4A11 gene expression in CYP4A11‐Tg. The role of PPARa in this process was then tested in CYP4A11‐Tg that carried PPARa−/+ or PPARa−/− genotypes. Hepatic P450 4A11 protein and mRNA were induced 2‐fold by fenofibrate in PPARa−/+ CYP4A11‐Tg. Constitutive CYP4A11 levels were dramatically reduced (> 95%) in PPARa−/− females compared to PPARa−/+ females, whereas male PPARa−/− CYP4A11‐Tg retained >60% of the levels found in PPARa−/+ males. As neither CYP4A11 mRNA nor protein was inducible by fenofibrate in male or female PPARa−/− CYP4A11‐Tg, our data suggest that PPARa mediates induction of the CYP4A11 transgene by fenofibrate. Supported by NIH grants HD04445 (EFJ), GM49135 (RHT), and AA07842 (JML).