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. 2009 May 11;206(5):1181-99.
doi: 10.1084/jem.20082521. Epub 2009 May 4.

Neutropenia with impaired host defense against microbial infection in mice lacking androgen receptor

Kuang-Hsiang Chuang  1 , Saleh Altuwaijri, Gonghui Li, Jiann-Jyh Lai, Chin-Yi Chu, Kuo-Pao Lai, Hung-Yun Lin, Jong-Wei Hsu, Peter Keng, Ming-Chi Wu, Chawnshang Chang
Affiliations

Neutropenia with impaired host defense against microbial infection in mice lacking androgen receptor

Kuang-Hsiang Chuang et al. J Exp Med. .

Abstract

Neutrophils, the major phagocytes that form the first line of cell-mediated defense against microbial infection, are produced in the bone marrow and released into the circulation in response to granulocyte-colony stimulating factor (G-CSF). Here, we report that androgen receptor knockout (ARKO) mice are neutropenic and susceptible to acute bacterial infection, whereas castration only results in moderate neutrophil reduction in mice and humans. Androgen supplement can restore neutrophil counts via stabilizing AR in castrated mice, but not in ARKO and testicular feminization mutant (Tfm) mice. Our results show that deletion of the AR gene does not influence myeloid lineage commitment, but significantly reduces the proliferative activity of neutrophil precursors and retards neutrophil maturation. CXCR2-dependent migration is also decreased in ARKO neutrophils as compared with wild-type controls. G-CSF is unable to delay apoptosis in ARKO neutrophils, and ARKO mice show a poor granulopoietic response to exogenous G-CSF injection. In addition, AR can restore G-CSF-dependent granulocytic differentiation upon transduction into ARKO progenitors. We further found that AR augments G-CSF signaling by activating extracellular signal-regulated kinase 1/2 and also by sustaining Stat3 activity via diminishing the inhibitory binding of PIAS3 to Stat3. Collectively, our findings demonstrate an essential role for AR in granulopoiesis and host defense against microbial infection.

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Figures

Figure 1.
Figure 1.
AR-targeted disruption and analysis of peripheral neutrophils in ARKO mice. (A) Schematic representation of murine AR protein domains and corresponding exons in AR mRNA. The AR mRNA is composed of seven exons; exon 2 is removed in ARKO mice. DBD, DNA-binding domain. LBD, ligand-binding domain. (B) The purity of isolated bone marrow neutrophils from WT and ARKO mice is >90%. (C) Using the primers 5′-AATGGGACCTTGGATGGAGAAC-3′ and 5′-TCCCTGCTTCATAACATTTCCG-3′, an AR transcript of 305 bp will be obtained from WT AR, but only 153 bp when floxed exon 2 is deleted. Full-length AR mRNA is not expressed in neutrophils isolated from bone marrow of AR-deficient mice. (D) Intracellular AR protein is not detected in neutrophils isolated from bone marrow of AR-deficient mice. Nonspecific IgG was used as primary antibody for the control. (E) FACS analysis of peripheral blood from 8-wk-old WT and ARKO mice. FSC and SSC of peripheral leukocytes show significant decreases of high-SSC granulocytic population in ARKO mice (n = 4) compared with WT littermates (n = 4). (F) FACS analyses with neutrophil-specific antibodies (clone 7/4) and anti-CD11b antibodies. Neutrophils are drastically reduced in ARKO mice (n = 4) compared with WT littermates (n = 4). (G) Representative photomicrographs of Wright-Giemsa–stained cells on cytospins from blood after erythrocyte lysis. Neutrophils (arrow heads) are rarely found on leukocyte cytospins from ARKO mice compared with WT controls. Bar, 10 µm. (H) Manual differential counting of neutrophils was performed on blood smears from 9-wk-old sex-matched (WT), ARKO (KO), and Tfm male mice. Results are representative of four separate experiments (n = 4 per group). Data represent the mean ± SD. **, P < 0.01 compared with WT mice. ***, P < 0.001 compared with WT mice.
Figure 2.
Figure 2.
Analysis of bone marrow neutrophils in ARKO mice. (A) FSC/SSC analysis of bone marrow cells shows reduction of a cell population with granulocyte characteristics in 8-wk-old ARKO mice (n = 4) compared with WT littermates (n = 4). (B) Gated with CD45+, FACS analyses show that bone marrow neutrophils are significantly decreased in AR-deficient mice (n = 4) compared with WT littermates (n = 4). (C) FACS analysis with antibodies against neutrophils in peripheral blood from 10-wk-old sham-operated WT, castrated (WT-Cas), castrated mice implanted with DHT-releasing pellet (WT-Cas+DHT), sham-operated ARKO (ARKO), and sham-operated ARKO mice implanted with DHT-releasing pellet (ARKO+DHT). For each group, 105 cells from each of four mice were pooled and analyzed. Data were analyzed using CellQuest software; neutrophil populations are shown as histograms. DHT supplementation restores neutrophil numbers in peripheral blood of castrated mice, but not in ARKO mice. (D) DHT supplementation restores bone marrow neutrophils (neutrophil-specific antibody, clone 7/4, labeled) in castrated mice, but not in ARKO mice. (E) DHT supplementation also restores neutrophils (Gr-1 labeled) in bone marrow of castrated mice, but not in those of Tfm mice. (F) Neutrophil differential counts are only moderately reduced in patients after androgen ablation therapy. Blood analysis data were collected from 33 advanced prostate cancer patients before and after surgical castration. Neutrophil differential counts are shown and presented as the mean ± SD. Animal experiments were independently performed at least three times, and representative results from one experiment are shown.
Figure 3.
Figure 3.
AR knockdown in WT myeloid progenitors by RNA interference (RNAi) suppresses neutrophil differentiation, and restoring AR in ARKO cells induces neutrophil differentiation. (A) Bone marrow cells were plated in methylcellulose-containing media supplemented with the indicated cytokine and hematopoietic colonies containing >30 cells were scored after 7 d. We analyzed bone marrow cells from five 9-wk-old animals of each genotype. (B) Effect of adding various concentrations of DHT on colony formation of mouse bone marrow cells in methylcellulose-containing media supplemented with G-CSF. (C) The populations of GMPs and CMPs are comparable in bone marrow between WT and ARKO mice. GMPs were further isolated from WT and ARKO bone marrow using FACS sorting. (D) Representative photomicrographs of isolated neutrophils and precursors from bone marrow stained with Wright-Giemsa. A decrease of nuclei-segmented neutrophils (arrow heads) in ARKO bone marrow is observed when compared with the control. Bar, 10 µm. (E) GMPs were infected with retroviruses carrying pBabe vector, pBabe-AR, pSuperior vector, pSuperior-siAR, and pSuperior-scramble. Transduced GMPs were cultured in the presence of G-CSF (10 ng/ml) with or without 10 nM DHT for 7 d. Green fluorescent colony cells were then collected and counted. 200-count manual leukocyte differentials were examined after Wright-Giemsa staining. Numbers of neutrophils and precursor cells were calculated by multiplying neutrophil differential ratios by the total green fluorescent cell count. Triplicate experiments were performed and the error bars represent the SD.
Figure 4.
Figure 4.
Proliferative potential of neutrophil precursor and progenitor cells. (A) Cell cycle analysis of total bone marrow Gr-1+ cells from WT and ARKO mice. Comparison of cell cycle distributions shows that 9-wk-old ARKO mice have a lower percentage of cells in the S-phase and the G2/M phase of the cell cycle compared with WT littermates. Data are presented as the mean ± standard deviations (SD) of three independent experiments in the right panel. (B) Kinetics of BrdU labeled neutrophils in the blood of WT and ARKO mice after BrdU injection. (C) Comparison of BrdU incorporation in WT and ARKO blood neutrophils. At 96 h post injection, the ratio of BrdUdim in blood neutrophils is lower in 9-wk-old AR-deficient mice (n = 4) as compared with WT littermates (n = 4). For each group, 105 cells from each of four mice were pooled and used for analysis. The results are presented as percentage of blood neutrophils from four-replicate experiments and the error bars represent the SD in the right panel.
Figure 5.
Figure 5.
Insensitivity to G-CSF signaling in ARKO neutrophils. (A) Only few apoptotic neutrophils can be found in freshly isolated WT and ARKO bone marrow cells. (B) G-CSF protection against apoptosis in ARKO bone marrow neutrophils was less than in WT cells in vitro. Isolated mature bone marrow neutrophils were cultured in RPMI medium with 20% FCS without or with G-CSF (100 ng/ml) for 48 h. Neutrophils were then stained with Annexin V and 7-AAD, and analyzed by flow cytometry. (C) A lower increasing rate of blood neutrophils was found in ARKO mice in response to G-CSF injection. (D) G-CSF target genes are down-regulated in ARKO neutrophils. Data represent mean ± SD for three separate experiments. *, P < 0.05; **, P < 0.01. (E) SOCS3 protein expression is decreased in ARKO neutrophils. (F) mRNA expression of G-CSFR is comparable between WT and ARKO neutrophils. (G) G-CSF binding to WT and ARKO neutrophils is similar.
Figure 6.
Figure 6.
Stat3 transcriptional activity is reduced in ARKO neutrophils. (A) PIAS3, Stat3, and pY-Stat3 proteins are comparable in WT and ARKO bone marrow neutrophils. Cells were isolated from mice and then incubated with G-CSF (40 ng/ml) at 37°C for the indicated period. (B) AR protein expression is lower in bone marrow neutrophils of castrated mice than those of WT mice, while Stat3 and pY-Stat3 levels are comparable. Bone marrow Gr-1+ cells were isolated from mice and then incubated with G-CSF (40 ng/ml) at 37°C for 30 min. (C) AR interacts with the GST-PIAS3 in the GST pull-down assay. (D) Binding of Stat3 by PIAS3 is more in ARKO bone marrow neutrophils. The result of band intensities of Stat3, pY-Stat3, and AR in the coimmunoprecipitation experiment was normalized with band intensities of PIAS3 and shown in the right panel. (E) AR can relieve the PIAS3 inhibition of activated Stat3 action. The results are presented as fold induction of luciferase activity from triplicate experiments using CV-1 cells and the error bars represent the SD. ***, P < 0.001. (F) CA-Stat3 induces but DN-Stat3 inhibits neutrophil maturation. (G) PIAS3 inhibits neutrophil maturation, while increased AR rescues the inhibition. (H) PIAS3-siRNA increases neutrophil maturation. Results are presented as cell numbers of neutrophils and precursors from triplicate experiments and the error bars represent the SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 7.
Figure 7.
AR is essential for ERK1/2 activation in G-CSF induced neutrophil proliferation. (A) With G-CSF (40 ng/ml) treatment for the indicated times, ERK1/2 phosphorylation status was examined in bone marrow neutrophils of WT, castrated WT, and ARKO mice. (B) AR is required for G-CSF-induced ERK1/2 phosphorylation. By immunoblotting, the expression status of phosphorylated ERK1/2 and total ERK1/2 were compared among each group of G-CSF–induced granulocytes derived from WT or ARKO bone marrow cells infected with retroviruses carrying AR, AR-siRNA, or control vectors. v, vector alone; Sc, pSuperior-scrambled siRNA; Si-AR, pSuperior-AR siRNA. (C) U0126 almost abolishes G-CSF–induced proliferation of bone marrow cells. In the presence or absence of G-CSF (10 ng/ml), bone marrow cells (106/ml) were incubated with or without 1 µM U0126 for 3 d. Proliferative activity was then examined by 3H-thymidine incorporation. (D) Effects of AR deficiency on the human myeloblastic cell line, KG-1. Stably transfected cells were plated at 5 × 105 cells/ml in RPMI medium and the number of total viable cells was calculated each day. Results are the mean of three independent experiments and the error bars represent the SD. (E) Schematic representation of the role of AR in G-CSF signaling.
Figure 8.
Figure 8.
Neutrophil function and host defense against infection in the absence of AR. (A) Uptake of fluorescein-labeled E. coli by WT and ARKO neutrophils at 37°C. (B) Flow cytometric analysis of oxidant production and subsequent 2′,7′-dichlorofluorescein generation in WT and ARKO granulocytes after PMA stimulation. (C) Relative mRNA expression of chemokines and cytokines in isolated WT and ARKO bone marrow granulocytes. *, P < 0.05. (D) Mature bone marrow neutrophils from WT or ARKO mice were tested for migration in Transwell chambers toward the indicated chemoattractant (200 ng/ml MIP-2, 1 µg/ml KC, or 100 µM fMLP). The number of cells migrated through the Transwell was determined by cell counting. Data from six independent experiments are presented and the error bars represent the SD. *, P < 0.05; **, P < 0.01. (E) Neutrophil degranulation of WT and ARKO neutrophils was examined without or with stimulation by fMLP, C5a, or PMA. (top) The release of the primary granules was examined using the β-glucuronidase activity in the supernatant of stimulated neutrophils. Data are presented as the percentage of total cellular β-glucuronidase content. (bottom) Release of the specific granule marker lactoferrin in the supernatant of stimulated neutrophils was quantified by ELISA. Both degranulation assays were performed in six independent experiments. (F) Representative photograph of WT and ARKO mice after 12-h intraperitoneal injection with E. coli. ARKO mice showed signs of severe infection, such as hunchback, ruffled fur, and eye infection; however, WT mice remained normal. (G) Kaplan-Meier survival curve of WT and ARKO mice in response to intraperitoneal challenge with E. coli. We examined the time course of survival following E. coli infection. Survival over time is shown as percentage mice remaining.
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