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. 2012 Jan;122(1):337-47.
doi: 10.1172/JCI58393. Epub 2011 Dec 12.

Coordinate Regulation of Neutrophil Homeostasis by Liver X Receptors in Mice

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Free PMC article

Coordinate Regulation of Neutrophil Homeostasis by Liver X Receptors in Mice

Cynthia Hong et al. J Clin Invest. .
Free PMC article

Abstract

The most abundant immune cell type is the neutrophil, a key first responder after pathogen invasion. Neutrophil numbers in the periphery are tightly regulated to prevent opportunistic infections and aberrant inflammation. In healthy individuals, more than 1 × 10⁹ neutrophils per kilogram body weight are released from the bone marrow every 24 hours. To maintain homeostatic levels, an equivalent number of senescent cells must be cleared from circulation. Recent studies indicate that clearance of senescent neutrophils by resident tissue macrophages and DCs helps to set homeostatic levels of neutrophils via effects on the IL-23/IL-17/G-CSF cytokine axis, which stimulates neutrophil production in the bone marrow. However, the molecular events in phagocytes underlying this feedback loop have remained indeterminate. Liver X receptors (LXRs) are members of the nuclear receptor superfamily that regulate both lipid metabolic and inflammatory gene expression. Here, we demonstrate that LXRs contribute to the control of neutrophil homeostasis. Using gain- and loss-of-function models, we found that LXR signaling regulated the efficient clearance of senescent neutrophils by peripheral tissue APCs in a Mer-dependent manner. Furthermore, activation of LXR by engulfed neutrophils directly repressed the IL-23/IL-17/G-CSF granulopoietic cytokine cascade. These results provide mechanistic insight into the molecular events orchestrating neutrophil homeostasis and advance our understanding of LXRs as integrators of phagocyte function, lipid metabolism, and cytokine gene expression.

Figures

Figure 1
Figure 1. LXR signaling regulates neutrophil homeostasis.
(AC) Absolute neutrophil (A), leukocyte (B), and rbc (C) counts in peripheral blood of 6- to 8-week-old WT and LXRαβ–/– mice. Each point represents an individual mouse. P values are shown. (D) Representative FACS plots of Ly6GhiBrdU+ cells gated through CD11b+ in peripheral blood of 6- to 8-week-old WT and LXRαβ–/– mice on day 3 after BrdU injection (2 mg i.p.). Percent Ly6GhiBrdU+ cells is indicated within plots. (E) Frequency of Ly6Ghi BrdU+ cells gated through CD11b+ in peripheral blood of 6- to 8-week-old WT or LXRαβ–/– mice on the indicated days after BrdU pulse. n = 4 per group. (F) Absolute number of neutrophils in peripheral blood of 6- to 8-week-old WT mice fed chow compounded with 0.012% GW3965 or control chow for 3.5 days. n = 3 per group. Data are representative of 2 experiments. **P < 0.01.
Figure 2
Figure 2. LXR does not intrinsically influence granulocyte development and survival.
(A) Quantification of monocytic (CFU-M), granulocytic (CFU-G), mixed granulocytic/monocytic (CFU-GM), and mixed granulocytic/erythroid/monocytic/megakaryocytic (CFU-GEMM) bone marrow colonies formed per 3 × 104 WT and LXRαβ–/– nucleated bone marrow cells on day 8 using standard differentiation conditions. Colonies were counted and analyzed by color and morphology to assign category. Data are representative of 2 experiments performed in triplicate. (B) FACS analysis of CD11bhiGR-1hi cells in bone marrow of 6- to 8-week-old WT and LXRαβ–/– mice. FACS data are representative of greater than 10 mice. (C) H&E stains of purified CD11bhiGR-1hi cells in bone marrow of 6- to 8-week-old WT and LXRαβ–/– mice. Original magnification, ×200. (D) Frequency of viable (PI-negative, annexin V–negative) purified bone marrow neutrophils cultured in media with 10% or 1% FBS for 18, 40, and 64 hours. Data are representative of 2 experiments performed in triplicate.
Figure 3
Figure 3. Accumulation of neutrophils in spleen and liver of LXRαβ–/– mice.
(A and B) FACS analysis and absolute numbers of CD11bhiGR-1hi cells in spleen of 6- to 8-week-old WT and LXRαβ–/– mice. (A) Percent cells in the circled region is indicated in each plot. (B) Each point represents an individual mouse. (C) H&E stains of FACS-sorted CD11bhiGR-1hi cells in spleen of 6-week-old WT and LXRαβ–/– mice. Original magnification, ×400, ×640 (insets). (D) FACS plot of CD11bhiGR-1hi cells in liver parenchyma from WT and LXRαβ–/– mice. Liver was perfused with saline and then collagenase for 5 minutes each prior to harvesting tissue. Percent cells in the circled region is indicated in each plot. (E and F) Expression and frequency of intracellular cleaved caspase-3 in splenic GR-1hiCD11bhi cells gated through MHC class II cells. Numbers in E denote intracellular cleaved caspase-3 frequency in the bracketed regions. FACS plots are representative of 8 mice. ***P < 0.001.
Figure 4
Figure 4. LXR signaling regulates the IL-23/IL-17 granulopoietic signaling axis.
(A) Il23a, Il12a, and Il12b expression in WT BMDCs treated with LPS and GW3965. On day 7, BMDCs were treated with 1 μM GW3965 or vehicle for 18 hours and then activated with 1 ng/ml LPS for 5 hours. (B) Basal Il23a, Il12a, and Il12b expression in WT and LXRαβ–/– BMDCs. (C) Il23a gene expression in WT and LXRαβ–/– thioglycolate-elicited macrophages activated with 1 ng/ml LPS for 5 hours and cocultured with purified aged neutrophils (AN). Experiments are representative of 2–4 experiments performed in triplicate. (D and E) Frequency of IL-17A+ WT and LXRαβ–/– T cells in spleen from 6- to 8-week-old mice. Cells were stimulated with PMA/ionomycin in the presence of brefeldin A for 5 hours ex vivo. Cells were stained with anti-Thy1, fixed, permeabilized, stained with anti–IL-17A, and analyzed by flow cytometry. Percent Thy1+IL-17+ cells in spleen is indicated. FACS plots are representative of 4 mice per group repeated twice. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 5
Figure 5. LXR regulates phagocytosis of aged neutrophils in vitro and clearance in vivo.
(A) Decreased phagocytosis of apoptotic neutrophils by LXRαβ–/– macrophages. Thioglycolate-elicited, aged peritoneal WT neutrophils were labeled with CMFDA (cell tracker green) and cocultured with thioglycolate-elicited macrophages (labeled red with CMPTX) for 90 minutes. Cultures were extensively washed with cold PBS and enzyme free cell dissociation buffer to remove non- and semiadherent neutrophils. Quantification of engulfed neutrophils was determined by confocal microscopy. In vitro engulfment assays are a composite of 5 individual experiments performed in triplicate. (B) LXR signaling promoted phagocytosis of apoptotic neutrophils. WT and LXRαβ–/– macrophages were pretreated with GW3965 (1 μM) for 18 hours and then cultured with apoptotic neutrophils as above. (C and D) In vivo clearance of purified lysM-EGFP+ bone marrow neutrophils from spleen of WT and LXRαβ–/– mice 40 hours after adoptive transfer. Percent GFP+Ly6G+ cells in total spleen is indicated. GFP neutrophil counts were computed by determining total nucleated splenocyte counts and multiplying by the frequency of GFP+Ly6G+ cells. In vivo neutrophil clearance FACS plots are representative of 4 mice. **P < 0.01, ***P < 0.001.
Figure 6
Figure 6. The LXR/MER axis regulates neutrophil clearance and homeostasis.
(A) Mertk, Gas6, and Axl gene expression from WT and LXRαβ–/– thioglycolate-elicited peritoneal macrophages treated with GW3965 (1 μM) or cocultured with aged neutrophils for 18 hours. Data are representative of 3 experiments performed in triplicate. (B and C) Confocal microscopy images (B) and quantification of neutrophil phagocytosis (C). WT thioglycolate-elicited peritoneal macrophages were treated with control IgG or anti-Mer Ab and then cocultured with aged neutrophils for 90 minutes. Subsequently, cultures were extensively washed with cold PBS and enzyme free cell dissociation buffer to remove non- and semiadherent neutrophils. Cells were stained for CD68 expression and Ly6G expression to distinguish macrophages from neutrophils (original magnification, ×400), and the phagocytic index was determined. Data are representative of 3 experiments performed in triplicate. (D and E) Frequency of blood GR-1hiCD11bhi cells from 5- to 6-week-old WT, Gas6–/–, or Mertk–/– mice. Percent cells in the circled region is indicated in each plot. FACS plots are representative of 4 mice per group.

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