Inefficient N2-Like Neutrophils Are Promoted by Androgens During Infection

Abstract

Neutrophils are major effectors of acute inflammation against infection and tissue damage, with ability to adapt their phenotype according to the microenvironment. Although sex hormones regulate adaptive immune cells, which explains sex differences in immunity and infection, little information is available about the effects of androgens on neutrophils. We therefore aimed to examine neutrophil recruitment and plasticity in androgen-dependent and -independent sites under androgen manipulation. By using a bacterial model of prostate inflammation, we showed that neutrophil recruitment was higher in testosterone-treated rats, with neutrophil accumulation being positively correlated to serum levels of testosterone and associated to stronger inflammatory signs and tissue damage. Testosterone also promoted LPS-induced neutrophil recruitment to the prostate, peritoneum, and liver sinusoids, as revealed by histopathology, flow cytometry, and intravital microscopy. Strikingly, neutrophils in presence of testosterone exhibited an impaired bactericidal ability and a reduced myeloperoxidase activity. This inefficient cellular profile was accompanied by high expression of the anti-inflammatory cytokines IL10 and TGFβ1, which is compatible with the "N2-like" neutrophil phenotype previously reported in the tumor microenvironment. These data reveal an intriguing role for testosterone promoting inefficient, anti-inflammatory neutrophils that prolong bacterial inflammation, generating a pathogenic environment for several conditions. However, these immunomodulatory properties might be beneficially exploited in autoimmune and other non-bacterial diseases.

Keywords: androgens; bacterial prostatitis; infection; neutrophils; testosterone.

Figure 1

Androgens increase neutrophil infiltration and tissue damage in bacterial infection of the prostate gland. Rats were orchidectomized (OX) and treated with testosterone 2 mg/kg/day (T) or 10 mg/kg/day (high testosterone-TT) before being inoculated with E. coli intraprostatically. (A) H&E staining at day 5 after infection shows not only interstitial inflammatory infiltrates but also a massive neutrophil invasion to prostatic acini in testosterone-treated animals. Bar = 100 μm. (B) Correlative analysis showing serum testosterone levels and prostatic neutrophil counts (n = 16, Pearson's correlation test). Neutrophils counts were calculated on H&E sections at day 5 post-infection. (C) The inflammatory parameters TNFα, SP-D, and HBD-1 are strongly increased in the prostate from testosterone-treated animals, with the TT group showing the highest levels (mean ± SEM; n = 4 per group; *p < 0.05; **p < 0.01; ***p < 0.001). (D) Representative images of immunocytochemistry for PBP and ACTA2, at day 5 post-infection, displaying a loss in epithelial secretory function when testosterone levels remain high (top images). ACTA2, as a marker for stromal organization, shows a weak expression and disruptions in the periacinar layer (arrows in bottom) of these animals. Bar = 100 μm. (E) At ultrastructural level, prostatic damage was related to the presence of numerous bacteria in the lumen (Lu) and invading epithelial cells (arrowheads, bottom). Orchidectomized rats show conserved morphology, with the conservation of secretory organelles such as Golgi complexes (g). EC, epithelial cell; N, neutrophil. Bar = 2 μm.

Figure 2

Testosterone signaling favors LPS-induced neutrophil recruitment in both androgen-dependent and –independent sites.(A)–(B) Rats were orchidectomized (OX) and treated with testosterone 2 mg/kg/day (T) before being inoculated with 1 mg of LPS intraprostatically for 24 h. (A) Representative H&E staining showing an intense neutrophil infiltration in testosterone-treated animals. Bar = 100 μm. (B) Quantification of neutrophil recruitment to the prostate in H&E-stained slides (left) and by flow cytometry using a FITC anti-Gr antibody (right). (C) Peritoneal neutrophil recruitment was achieved by injecting LPS 1 mg/kg for 12 h, with the quantification of neutrophils being performed by hemocytometer (left) and flow cytometry (right). (D) Neutrophil recruitment in the liver observed by intravital microscopy after an i.p. LPS 0.5 mg/kg injection. Mice were previously treated with testosterone (T; 10 mg/kg/day) or with the inhibitor of androgen signaling flutamide (FLUT; 7 mg/kg/day). The presence of testosterone increases neutrophil recruitment to the liver sinosoids. Left: representative images (see Supplementary Movie 1). Right: quantification of Ly6G (+) neutrophils per field of view. Data represent the mean ± SEM from at least three independent animals. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3

Androgens augment LPS-induced, neutrophil-specific chemokine mRNA expression. (A–B) Rats were orchidectomized (OX) and treated with testosterone 2 mg/kg/day (T) before being inoculated with 1 mg of LPS intraprostatically for 24 h. Cell sorting was carried out to isolate Gr (+) neutrophils from prostatic cells and mRNA expression was evaluated by qPCR. (A) Testosterone treatment promotes an increase in CXCL1 and CXCL2 in prostatic cells. (B) Neutrophils exhibit a similar behavior, with no changes in Monocyte Chemoattractant Protein-1 (MCP1), the key chemokine regulating migration of monocytes/macrophages. (C) LPS-induced peritoneal neutrophils from testosterone-treated animals (T) showing an increase in CXCL1 and CXCL2. In all cases, the mRNA levels are relative to those of ACTB. Mean ± SEM, each dot represents one animal. *p < 0.05; **p < 0.01.

Figure 4

Testosterone treatment impairs neutrophil ability to kill bacteria. (A-B) Rats were orchidectomized and treated with testosterone at 2 mg/kg/day or 10 mg/kg/day (high testosterone) before being inoculated with E. coli intraprostatically. (A) Representative electron microscopy images showing apoptotic neutrophils (AN) free of bacteria (left), while prostate infiltrating neutrophils (N) in testosterone-treated rats display intact phagocytosed bacteria (right, arrowheads) after 5 days of infection. EC: epithelial cell. Bar = 5 μm. (B) This is consistent with the intense E. coli immunostaining, localized in intracinar neutrophils in testosterone- and high testosterone-treated rats. Bar = 100 μm. (C–D) Bacterial growth after being co-incubated ex vivo with peritoneal neutrophils from orchidectomized (OX) and testosterone-treated (T) rats. The reduced bactericidal ability of neutrophils from T rats depicting in (C) is also seen at ultrastructural level (D) where abundant intact free bacteria are observed in presence of testosterone. Data are representative from at least 3 independent experiments. **p < 0.01.

Figure 5

Androgens modulate neutrophil phenotype. Rats were orchidectomized (OX) and treated with testosterone 2 mg/kg/day (T) before being inoculated with 1 mg of LPS intraprostatically for 24 h (A, C, D, F). To elicit peritoneal neutrophils, a single i.p. injection of LPS 1 mg/kg was applied (B, E). (A) Myeloperoxidase (MPO) activity in prostatic tissue is impaired in testosterone-treated animals, referred per mg of proteins (left) as well as per g of tissue (right). (B) Peritoneal neutrophils also show a decrease in MPO activity in animals treated with T. Data are mean ± SEM, from n = 4 per group. *p < 0.05; **p < 0.01. (C) The mRNA expression for MPO show no changes between groups. (D) Cytokine profiling of Gr (+)-sorted prostatic neutrophils by qPCR, depicting that cells from testosterone-treated animals express high levels of anti-inflammatory TGFβ and IL10, while pro-inflammatory cytokines are reduced. (E) Peritoneal neutrophils from testosterone-treated rats also have an anti-inflammatory/ immunomodulatory/“N2-like” phenotype, compatible to that reported by Fridlender et al. (9) and characterized by high expression of TGFβ and IL10 along with low levels of IL1b, IL12p40, and TNFα. ACTB was used as reference mRNA. Mean ± SEM, each dot represents one animal. *p < 0.05; **p < 0.01. (F) Representative images of prostatic neutrophils showing cellular edema and vacuolization in testosterone-treated animals. Bar = 5 μm.

Figure 6

Antiandrogen therapy inhibits IL10 production. Rats were treated with flutamide (7.5 mg/kg/day) for 5 days and thioglycollate-elicited peritoneal cells were pulsed ex vivo with LPS 1 μg/ml for 24 h. (A) Immunofluorescence for IL10 in absence of antiandrogens, analyzed by confocal microscopy. Most of Gr (+) peritoneal neutrophils express IL10. Bar = 50 μm. (B) Flow cytometry showing a decrease in the granulocytic population (R1) along with an increase in the frequency of mononuclear cells (R2) after flutamide treatment (representative dot plots; n = 3; top panels). The Gr (+) CD11b (+) R1 neutrophil population shows a decrease in IL10 expression after flutamide treatment (center panels, left) while the monocytic R2 population also displays low expression of IL10 in the same group (bottom panels). Data are representative of n = 3.