### MATERIALS AND METHODS

### Animals

Wistar strain male rats, 12 weeks old, and weighing 250– 350 g, were housed at the Animal Research Facility of the Universidad Nacional de Cordoba, in air-conditioned quarters, under a controlled photoperiod (14 h light/10 h darkness) with free access to rodent food and tap water. C57BL/6 mice were from Centro de Bioterismo in Universidade Federal de Minas Gerais (CEBIO, UFMG, Brazil). Animal care and experiments were conducted following the recommendations of the International Guiding Principles for Biomedical Research Involving Animals and approved by the local CICUAL (FCM-UNC, Argentina) Ethical Committee.

### Androgen Manipulation and Prostatitis Models

Rats were orchidectomized via the scrotal route under ketamine (80 mg/kg)/ xylazine (8 mg/kg) and divided into three groups, receiving immediately testosterone s.c. at physiological (2 mg/kg/day; T group; Sustanon, Organon, Argentina) or supraphysiological doses (10 mg/kg/day; TT group), or vehicle alone (sunflower oil; OX group). To confirm the androgen status, serum total testosterone levels of individual rats were determined by electrochemiluminescence immunoassay using a Roche Elecsys E170 immunoassay analyzer (Roche Diagnostics GmbH, Mannheim, Germany).

An acute bacterial prostatitis model was performed according to a protocol from our laboratory (21, 22). Briefly, two days after castration, OX, T, and TT rats were anesthetized and subjected to a laparotomy to expose the ventral prostate; infection was induced by an intraprostatic injection of 200 µL of Uropathogenic E. coli (108 CFU/ml, isolated from a patient with complicated urinary tract infection), with a 30-gauge needle. Animals were killed at 1, 3, and 5 days after infection, with the ventral prostate being processed for morphological, biochemical, and microbiological analyses. As controls, rats subjected to the same surgical procedures were used, replacing the bacterial suspension with sterile PBS.

On the other hand, a lipopolysaccharide (LPS)-induced model for prostatic inflammation was carried out by inoculating 50 µl of a solution of LPS from E. coli 055:B5 (20 mg/ml, Sigma– Aldrich, St. Louis, MO) in OX and T groups using the same surgical procedure described for bacterial prostatitis. Control animals received the vehicles. Ventral prostates were harvested and processed at 24 h after inoculation.

### Neutrophil Recruitment to the Peritoneum and Microbial Killing Assay

Neutrophil recruitment was induced by a single i.p. injection of thioglycollate (3 ml of a 3% solution; Sigma–Aldrich, St. Louis, MO) or LPS from E. coli 055:B5 (1 mg/kg) in rats of OX and T groups at day 3 post-castration. Sterile saline-injected animals were used as controls. Animals were anesthetized 4 h after thioglycollate or 12 h after LPS injection and the peritoneal lavage was harvested for analysis by injection of 10 mL of sterile PBS containing 0.835 UI/mL sodium heparin. The abdomen was gently massaged and the blood-free cell suspension was carefully aspirated with a syringe. The cell suspension was then spun for 6 min, 300×g, at 20◦C. After removal of the supernatant, residual red blood cells were removed by hypotonic lysis and the cells were spun again for 6 min, 300×g, at 20◦C. The cell pellet was washed and resuspended in 2 ml HBSS and total leukocyte counts were performed immediately in peritoneal lavage samples using a Neubauer chamber. To evaluate the neutrophil percentage, the differential leukocyte population was analyzed in cytospins after May-Grünwald-Giemsa staining (Biopur, Rosario, Argentina); a minimum of 500 leukocytes were counted, containing >85% pure populations of neutrophils. Additionally, peritoneal cells were stained with a FITC anti-rat granulocyte (Gr) antibody that recognizes rat neutrophils (BD Biosciences, San Jose, CA) and analyzed by flow cytometry using a FACSCanto II cytometer (Becton Dickinson, San Jose, CA).

For assessing ex vivo neutrophil bactericidal activity, thioglycollate-recruited neutrophils from OX and T groups were counted manually using a standard hemocytometer. Cytospins confirmed >90% neutrophils. Uropathogenic E. coli (the same used for prostatitis model) were pre-opsonized in 10% mouse serum on ice for 15 min. Neutrophils in RPMI 1640 (Sigma–Aldrich, St. Louis, MO) were plated onto 24 well plates at 1 × 106 neutrophils/well and infected with equal volume of E. coli in serum at a multiplicity of infection of 1 bacterium:1 neutrophil. Following 10 and 40 min incubation times, 50 µl of the suspension were taken for bacterial colony-forming units (CFU) quantification by serial agar plating.

### Intravital Liver Imaging

Mice were treated with testosterone (i.p. 10 mg/kg/day) or flutamide (s.c 7 mg/kg/day; Sigma–Aldrich, St. Louis, MO) for 3 days. Then, neutrophil recruitment was induced by a single i.p. injection of LPS from E. coli O111:B4 (0.5 mg/kg, Sigma–Aldrich, St. Louis, MO). Six hours after, mice received a single i.v. dose of a FITC anti-mouse Ly-6G antibody (4 µg/mouse; BioLegend, San Diego, CA), diluted in sterile saline (in a total volume of 100 µl), and confocal intravital imaging was performed as described (23, 24). In brief, mice were anesthetized (i.p.) with a mixture of ketamine (60 mg/kg) and xylazine (15 mg/kg) and a midline laparotomy was performed to expose the liver for imaging. Mice were imaged using Nikon Eclipse Ti (Nikon, Tokyo, Japan) with a C2 confocal head equipped with three different lasers (excitation at three wavelengths: 405, 488, and 543 nm) and emission bandpass filters at 450/50, 515/30, and 584/50 nm. The z-position is controlled by an automated device and 10X objective was used on the required resolution. Ten-minute movies were taken from each mouse and Ly-6G (+) neutrophil quantification was performed using Volocity 6.3 software (PerkinElmer, Waltham, MA).

## Histopathological Analysis and Immunocytochemistry

Tissue samples of ventral prostates were formalin-fixed and paraffin-embedded for routine hematoxylin–eosin (H&E) staining (Biopur, Rosario, Argentina). An Axiostar Plus microscope equipped with a digital camera (Zeiss, Oberkochen, Germany) was used to acquire 60X photographs, which were examined using Fiji software (NIH, Bethesda, MD). For prostate neutrophil quantification, 20 fields of 2 different sections from the same gland were analyzed, with at least three animals per experimental group.

Immunocytochemistry was performed on slides from paraffin-embedded prostates, which were cleared with xylene and rehydrated in a descending concentration series of ethanol. Samples were then incubated in EDTA pH 9.0 to perform antigen retrieval using microwave pre-treatment (except for detection of E. coli). To block the endogenous peroxidase activity, slides were treated with H2O2 in methanol for 15 min. Sections were treated with PBS-BSA 5% to block non-specific binding for 30 min, followed by an overnight incubation with the primary antibody (diluted in 1% PBS-BSA) at 4◦C in a humidified chamber. Afterwards, slides were incubated for 1 h with a specific biotinylated secondary antibody (at 1:180; Amersham Pharmacia, Buckinghamshire, UK) followed by 30 min in ABC complex (Vector, Burlingame, CA). Diaminobenzidine (Sigma–Aldrich, St. Louis, MO) was used as a chromogen substrate for 10 min and Harris hematoxylin as a counterstaining solution. Primary antibodies used for this study were: anti-PBP (rabbit polyclonal at 1:2000, developed by Dr. Maccioni (22), anti-ACTA2 (mouse monoclonal at 1:50; Novocastra, Newcastle, UK), and anti-E. coli antigen (rabbit polyclonal at 1:250, Affinity BioReagents, Golden, CO). For negative controls, antibodies were pre-absorbed with specific blocking peptides or replaced by rabbit or mouse normal serum.

### Transmission Electron Microscopy

Tissue blocks (1 mm3 ) from ventral prostate and pellets of peritoneal neutrophils were fixed in Karnovsky's mixture containing 2% (v/v) glutaraldehyde (EMS, Hatfield, PA) and 4% (w/v) formaldehyde in 0.1 M cacodylate buffer, pH 7.3, at 4◦C for 24 h. Samples were post-fixed in 1% osmium tetroxide (EMS, Hatfield, PA) for 2 h and washed in 0.1 M cacodylate buffer, before being dehydrated through a graded series of cold acetone and embedded in Araldite epoxy resin (EMS, Hatfield, PA) as previously published (21, 22, 25). Ultrathin sections were cut in a JUM-7 ultramicrotome (Jeol, Tokyo, Japan) and examined in a Zeiss LEO 906E electron microscope (Zeiss, Oberkochen, Germany) with digital acquisition of images.

### Sorting of Prostate Infiltrating Neutrophils by Flow Cytometry

Ventral prostates from rats with LPS-induced prostatitis were quickly excised, rinsed in fresh saline and weighted. Cell dissociation and neutrophil purification were performed using an adaptation of a published protocol (26). Briefly, tissues were minced into small fragments and treated with a digestion solution containing 200 U/ml collagenase type IA (Sigma Aldrich, St. Louis, MO) and 0.05% deoxyribonuclease type I (Sigma Aldrich, St. Louis, MO) in HBSS without calcium, magnesium or phenol red, pH 7.4 for 30 min, at 37◦C, and rocking at 60 rpm. The digested tissue was then passed through a 70-µm pore cell strainer with fresh sterile PBS. The cell suspension was then spun for 6 min, 300×g at 20◦C. After removal of the supernatant, residual red blood cells were removed by hypotonic lysis and cells were spun again for 6 min, 300×g, at 20◦C, washed, and resuspended in 1 ml of PBS. Cells were counted by collecting events for a fixed time (90 s) on a FACSCanto II cytometer. Neutrophil percentage was calculated by using a FITC anti-rat Gr monoclonal antibody (1/500 for 1 h at 4◦C).

To purify prostate-infiltrating neutrophils, Gr (+) cells were sorted in a FACSAria II cell sorter (BD Biosciences, San Jose, CA), with a purity of >97% being achieved and confirmed by electron microscopy.

### Measurement of Myeloperoxidase (MPO) Activity

Whole ventral prostate glands were removed, frozen in liquid nitrogen, and stored at −80◦C. After thawing, tissue was weighed and homogenized on ice in 50 mM potassium phosphate buffer (1 g in 10 ml, pH 6.0). Homogenates were centrifuged (30.000×g, 15 min, 4◦C) and pellets were resuspended in hexadecyltrimethylammonium bromide (HTAB; Sigma Aldrich, St. Louis, MO) buffer (0.5% HTAB in 50 mM phosphate buffer, pH 6.0). Lysates were sonicated twice for 10 s each and freeze-thawed three times, after which sonication was repeated. Suspensions were then centrifuged (20.000×g, 15 min, 4 ◦C) and the resulting supernatants were assayed for MPO activity. Supernatants (30 µl) were added to 970 µl of 50 mM phosphate buffer (pH 6.0) containing 0.167 mg/ml o-dianisidine hydrochloride (Sigma Aldrich, St. Louis, MO) and 0.0005% H2O2 and the change in absorbance at 460 nm (A460) was measured. One unit (U) of MPO activity was defined as that degrading 1 µmol of H2O2 per minute at 25◦C and results were expressed as U MPO activity/g prostate and U MPO activity/mg protein, determined by the Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Hercules, CA).

To assess MPO activity in peritoneal cells, cellular MPO was extracted with 0.5% HTAB buffer from 4 × 106 cells obtained after hypotonic lysis of residual red blood cells. Lysates were then sonicated twice for 10 s each and spun for 15 min, 40.000×g, at 4◦C. Resulting supernatant was assayed for MPO activity as described, with results being expressed as U MPO activity/mg protein and µU MPO activity/cells.

### Microbiological Studies

To evaluate in vivo the growth of E. coli, pieces of ventral prostate from each rat were weighed, minced and gently homogenized in tryptic soy broth (1 g of tissue in 20 ml) in sterile conditions. Then, serial dilutions, i.e., 1/5 to 1/50, were made and 100 µl of these solutions were spread on Mueller Hinton agar, with the plates being incubated overnight at 37◦C. Finally, bacterial counting was expressed as CFU per mg of prostatic tissue.

### Ex vivo Determination of IL10 Production

Wistar rats were treated with flutamide (s.c 7.5 mg/kg/day) or its vehicle for 5 days. Peritoneal cells were harvested after 4 h of thyoglycollate i.p injection, washed, and resuspended in HBSS as described above. Total cells were then plated onto 24 well plates (1 × 106 neutrophils/well in RPMI 1640) and pulsed ex vivo with LPS O55:B5 1µg/ml for 24 h to elicit cytokine secretion. In vitro assays maintained the same conditions as in vivo (i.e., flutamide stimulation). Immunofluorescence for IL10 in neutrophils was carried out on methanol-fixed, permeabilized cells on coverslides using PE anti-rat IL10 (1/50; BD Biosciences, San Jose, CA) and FITC anti-rat Gr (1/50) antibodies.

For flow cytometry, cells were treated with cycloheximide 100µM (Sigma Aldrich, St. Louis, MO) 3 h before the analysis. The cell concentration was adjusted to 5 × 105 cells/ml and labeled for 30 min with the following antibodies: PE anti-rat IL10 (1/60), APC anti-rat CD11b (1/100; BD Biosciences, San Jose, CA), and FITC anti-rat Gr (1/150). Signals were acquired in a FACSCanto II cytometer and analyzed using FlowJo X software (Tree Star, Ashland, OR).

### Immunoblotting

Prostate tissues were minced and homogenized on ice with a teflon-glass tissue grinder in 2 ml cold PBS containing 1.25% Igepal CA-630, 1 mM EDTA, 2 mM PMSF, 10 ug/ml leupeptin, and 10 ug/ml aprotinin. The lysate was centrifuged at 14,000×g for 20 min at 4◦C and the supernatant was withdrawn and stored in aliquots frozen at −70◦C until required. Prostatic lysates from triplicate experimental conditions were pooled before loading into electrophoresis gels. Total protein concentration was measured with a Bio-Rad Protein Assay kit. For western blot, denatured protein samples (45 µg/lane) were separated on 12% SDS polyacrylamide gel and blotted to a Hybond-C membrane (Amersham Pharmacia, Freiburg, Germany). Incubation steps were performed in 5% defatted dry milk in PBS/0.1% Tween 20. Blots were incubated for the detection of SP-D (rabbit polyclonal antibody at 1:1000; Chemicon, Temecula, CA) during 3 h. After that, membranes were treated with peroxidase-conjugated goat anti-rabbit antibodies (Jackson, West Grove, PA) and visualized applying the chemiluminescence technique. The expression of β-actin (mouse monoclonal antibody at 1:5000, Sigma Aldrich, St. Louis, MO) was used as an internal control to confirm the equivalent total protein loading.

The peptide βdefensin-1 (HBD-1) was tested in homogenates of ventral prostates by dot blot. For that purpose, prostatic lysates were matched at a concentration of 100µg/ml, and 4 µl of each sample was spotted onto a Hybond C Super membrane (Amersham-Pharmacia, Freiburg, Germany). The membrane was then treated as explained above for western blot using an anti-HBD-1 (at 1:250, Santa Cruz Biotech, Santa Cruz, CA) as primary antibody.

Semiquantitative signals were derived by densitometric analysis from western and dot blots using Fiji software and data were displayed as area units.

## ELISA

In order to quantify TNFα in prostate homogenates, tissues were minced and homogenized on ice in cold PBS, as described for immunoblotting, centrifuged at 4◦C, 1.400 rpm for 15 min and stored at −20◦C until the day of the assay. TNFα amount was measured by a commercially available sandwich ELISA kit (eBioscience, San Diego, CA), following the manufacturer's instructions.

### Measurement of Gene Expression by Quantitative Real-Time Polymerase Chain Reaction (qPCR)

Total RNA samples were extracted from peritoneal neutrophils and prostate Gr (+)- and Gr (–)-sorted cells using TRIzol (Thermo Fisher, Carlsbad, CA). RNA was subsequently purified using Direct-zol RNA miniprep kit (Zymo Res., Irvine, CA) according to manufacturer's instructions. Then, 0.5 µg of RNA was used as a template for reverse transcription using EpiScriptTM Reverse Transcriptase (Epicentre, Madison, WI) with random hexamer primers (Fermentas, Thermo Fisher, Carlsbad, CA). qPCR was performed with power SYBR green PCR master mix (Applied Biosystems, Foster City, CA) in a ABI Prism 7500 detection system (Thermo Fisher, Carlsbad, CA). The expression of ACTB was chosen as housekeeping gene. Data analysis was based on the 2−11Ct method for normalization of raw data. All primers used are described in the **Supplementary Table 1.**

### Statistical Analysis

The characterization of data was accomplished by comparing their mean values ± standard error of the mean (SEM) from at least four independent protocols. Statistical differences between means were analyzed by unpaired Student's t-test. Data from more than two groups were analyzed using analysis of variance (ANOVA) with Tukey as the post-test to compare all pairs of columns. Significant differences were considered at p < 0.05. Statistical analyses and graphics were performed using SPSS, version 23.0 (SPSS Inc., Chicago, IL) and GraphPad Prism 6 (La Jolla, CA).

## RESULTS

### Androgen Withdrawal Modifies the Progression of the Inflammatory Response in the Prostate Gland

In order to study the effects of testosterone on prostatitis progression, castrated animals treated with different doses of testosterone were subjected to intraprostatic bacterial inoculation and analyzed at days 1, 3, and 5 after infection. As shown in **Figure 1A**, the prostate from rats supplemented with testosterone exhibited a massive neutrophil infiltration and invasion into the prostatic acini; this effect was more evident at day 5 post-infection. Moreover, serum androgen levels positively correlated to the number of neutrophils infiltrating the gland (**Figure 1B**). In contrast, rats with low testosterone levels displayed reduced inflammatory signs, with few focal infiltrates in the prostatic interstitium. Accordingly, the prostatic levels of TNF-α, SP-D, and HBD-1 were higher in rats treated with high doses of testosterone (**Figure 1C**), demonstrating a stronger inflammatory reaction in these animals. Considering the deleterious effects of neutrophils on tissue function, we assessed the histoarchitecture as well as the expression of prostatic binding protein (PBP) and α-smooth muscle actin (ACTA2, markers of epithelial secretory function and stromal integrity respectively) of the prostate gland. At day 5 post-infection, both the secretory and the stromal compartments of the ventral prostate were conserved in the castrated animals as judged by PBP and ACTA2 expressions (**Figure 1D**). This was consistent with the conservation of ultrastructural features, including a prominent Golgi complex, developed endoplasmic reticulum, and secretory vesicles (**Figure 1E**). Conversely, the epithelial layer of the testosterone-treated rats exhibited atrophy, desquamation, and invasion of neutrophils, suggesting that the exacerbated presence of neutrophils is associated to more inflammatory signs and tissue damage in the prostate gland of animals with high levels of testosterone. Taking together, these results evidence adverse effects of testosterone in a dose dependent manner during bacterial acute prostatitis.

### Testosterone Increases Neutrophil Recruitment Independently of the Stimulus Nature or the Site of Injury

The higher amount of neutrophils observed in testosteronetreated animals could be attributed to an androgen-related neutrophil malfunction which, unable to eradicate bacteria, maintains a constant recruitment of inflammatory cells. In order to further understand this, we used bacterial LPS instead of the live bacterium as stimulus. In line with the bacterial-induced prostatitis model, LPS promoted an intense inflammatory infiltration in testosterone-treated animals, resulting in a higher number of neutrophils within the prostate, assessed in histological slides as well as by flow cytometry using a specific antibody (anti-Gr) for rat neutrophils (**Figures 2A,B**).

Testosterone-treated rats also displayed a higher thioglycollate-induced neutrophil recruitment to the peritoneum (not shown), with this effect being even stronger when neutrophil recruitment was elicited by LPS for 12 h (**Figure 2C**), indicating that testosterone is associated to a higher recruitment of neutrophils not only in androgen-dependent but also in androgen-independent sites. Indeed, to corroborate this notion, a widely validated intravital mouse model (23, 24, 27) for visualization of LPS-induced neutrophil recruitment to the liver was performed. In mice treated with testosterone, LPS injection caused a massive neutrophil adhesion to hepatic sinusoids, which was reduced by the testosterone inhibitor flutamide (**Figure 2D** and **Supplementary Movie 1**).

Taking in mind that neutrophil recruitment from peripheral blood is a consequence of local chemokine production, mainly CXCL1 and CXCL2 (28), their mRNA expressions were analyzed in cells from prostates and peritoneal cavity after LPS challenge. As shown in **Figure 3A**, both chemokines increased after testosterone restoration in resident prostatic

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 = 100µm. (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 = 100µm. (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 = 2µm.

cells. Moreover, CXCL1 and CXCL2 were enhanced in Gr(+) sorted neutrophils infiltrating the gland in the LPS-induced prostatitis model (**Figure 3B**) as well as in LPS-elicited peritoneal cells from animals treated with testosterone (**Figure 3C**). Interestingly, the expression levels of MCP-1 did not change significantly between groups (**Figure 3B**). Altogether, these data indicate that androgens promote neutrophil recruitment in both androgen-dependent and –independent sites by increasing the

expression of specific chemokines in local cells as well as in neutrophils.

### Androgens Decrease the Bactericidal Ability of Neutrophils

Ultrastructural analysis of the prostate revealed frequent undigested bacteria within neutrophils and invading the epithelial layer in animals treated with testosterone after 5 days of bacterial infection (**Figure 4A**). This was consistent with an intense immunostaining for E. coli in prostates from testosterone-treated rats compared to those from castrated animals (**Figure 4B**); this was also confirmed by bacterial cultures (data not shown), indicating an abnormal clearance of bacteria in presence of androgens. In contrast, most of neutrophils from castrated animals exhibited apoptotic features and absence of phagocytosed bacteria (**Figure 4A**), along with a weak E. coli immunostaining, all signs compatible with an accurate resolution phase of inflammation. In addition, thioglycollateelicited peritoneal neutrophils from castrated or testosteronetreated rats were challenged ex vivo to E. coli and bacterial counting and electron microscopy was performed at 10 and 40 min after coincubation. As shown in **Figure 4C**, neutrophils from testosterone-treated rats decreased their bactericidal ability. Moreover, the ultrastructural analysis of these cells revealed

frequent undigested live bacteria in both the extracellular and intracellular compartments (**Figure 4D**), confirming that androgens regulate negatively neutrophil activity even in androgen-independent sites.

MPO is packaged in the azurophilic granules of neutrophils and released into phagosomes when they uptake and kill bacteria (1). We wondered whether the decreased bactericidal effect of neutrophils associated to testosterone could be related to an alteration in the activity of MPO. Prostatic tissues from rats with LPS-induced prostatitis and low level of testosterone showed a higher MPO activity compared to those with normal androgen status (**Figure 5A**). Consistently, a lower MPO activity per neutrophil was observed in LPS-induced peritoneal neutrophils of animals treated with testosterone (**Figure 5B**). Interestingly, when the MPO mRNA expression was assessed in Gr (+) sorted prostate infiltrating neutrophils, no differences were found between groups (**Figure 5C**), suggesting that androgens regulate MPO at post-transcriptional level.

### Testosterone Favors a "N2-Like" Neutrophil Phenotype, With High Expression of Anti-inflammatory Cytokines

Considering the existence of different neutrophil phenotypes, particularly in the tumor microenvironment (9), we wondered if testosterone manipulation could result in a shift of cytokine expression by neutrophils in the context of acute inflammation. By using the LPS-induced prostatitis model, performed in castrated or testosterone-treated rats, the mRNA expression for "N1-" and "N2-like" neutrophils was assessed by qPCR in Gr (+)-sorted cells. Prostatic neutrophils from animals supplemented with testosterone displayed a higher expression of IL10 and TGFβ1 along with a lower IL12 expression compared to those with low testosterone (**Figure 5D**). When analyzing LPS-recruited peritoneal neutrophils, similar findings were observed, with high testosterone levels being related to high IL10 and TGFβ expressions and to a decrease in the pro-inflammatory cytokines IL12, IL1β, and TNFα (**Figure 5E**). On the other hand, the morphological evaluation at ultrastructural level showed the existence of clear alterations in LPS-recruited prostatic neutrophils from testosterone-treated animals, including cellular swelling and the occurrence of vacuoles (**Figure 5F**). These findings suggest that in acute inflammatory scenarios, androgens promote "N2-like" antiinflammatory and dysfunctional neutrophils, which extend the inflammatory process.

Together, these data reveal a stimulatory effect of testosterone on neutrophil-produced anti-inflammatory cytokines. To further corroborate these results, rats were treated with flutamide (an antiandrogen widely used in therapy) for 5 days; peritoneal cells were harvested after 4 h of thioglycollate and pulsed ex vivo with LPS for 24 h to elicit cytokine secretion. As expected, flow cytometry analysis demonstrated that flutamide decreased the expression of IL10, not only in the granulocytic population but also in mononuclear cells (whose frequency was increased by the anti-androgen, as shown in **Figure 6**).

### DISCUSSION

Sex differences in mortality and immunocompetence are well documented in humans and other vertebrates (29). Males are at higher risk of developing acute respiratory distress, sepsis, and multiorgan failure after traumatic hemorrhagic shock and thermal injury, in part due to abnormal activation of neutrophils (5). Although morphological differences in granulocytes from men and women have been known since 1954 (30), the need to expose factors explaining functional differences has resurfaced recently in order to adapt new treatments depending on the characteristics of the pathophysiological process in each sex. In this sense, various studies have uncovered important immune regulatory functions for androgens, including effects on neutrophil accumulation (31–33), maturation, activation (34), and survival (35). In line with this evidence, we here report that testosterone increases local chemokine expression, leading to a higher recruitment of neutrophils to the site of infection, but at the same time, these cells exhibit a "N2-like" phenotype with a reduced efficiency in killing bacteria and high expression of immunomodulatory molecules such as IL10 and TGFβ1.

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 = 5µm. (B) This is consistent with the intense E. coli immunostaining, localized in intracinar neutrophils in testosterone- and high testosterone-treated rats. Bar = 100µm. (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.

Neutrophil recruitment is an important early step in inflammatory response against pathogenic invasion or sterile tissue damage. The recruitment of neutrophils into the tissue is initiated by neutrophil-active chemoattractants, mainly CXCR2 ligands CXCL1 and CXCL2, released from danger signal-activated professional tissue-resident sentinel cells or stromal cells (28). Recruited neutrophils into injury site can also deliver active chemokines directly contributing to their own recruitment (36). In the present study, we observed that the presence of testosterone leads to an increased bacterial-induced mRNA expression of CXCL1 and CXCL2, which was associated to a higher neutrophil recruitment. In agreement, androgen supplementation has been shown to augment neutrophil infiltration in penile urethroplasty (33) and after myocardial infarction in both male and females (31). Human studies demonstrated that decreasing testosterone levels results in an attenuation of exercise-induced neutrophil accumulation in muscles (37), indicating a strong positive correlation between androgen levels and neutrophil infiltration. Furthermore, hyperandrogenemia is associated to a higher neutrophil count in steady state conditions in women with polycystic ovary syndrome (38). The excessive or aberrant

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 = 5µm.

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.

neutrophil infiltration in the presence of high androgen levels seems to favor tissue damage and organ dysfunction not only after infection but also in non-infectious conditions (31, 33). However, novel evidence suggests that neutrophil accumulation could also play a positive role in organ homeostasis by resolving inflammation (39–42), with a phenotypic characterization (i.e., pro-inflammatory vs. pro-resolving neutrophils) being necessary in order to predict the final effect of these cells on damage progression and tissue function.

Unlike other immune cells, existence of clearly defined neutrophils subtypes remains unclear. Accumulating evidence suggests that neutrophils may exhibit certain plasticity according to the microenvironment (8, 9, 43, 44). For instance, different subtypes of neutrophils were identified during infection with methicillin-resistant S. aureus (MRSA), associated either to resistance or to susceptibility to infection in mice (8). Neutrophils from MRSA-resistant hosts show a pro-inflammatory phenotype, with IL12 and CCL3 production, whereas those from MRSA- susceptible mice are anti-inflammatory in nature (IL10+/CCL2+), inducing M2 macrophages (8). Neutrophil polarization to an IL10-producing anti-inflammatory phenotype has also been reported by different pathogens (43, 45) as well as by serum amyloid A1 (46). Nevertheless, neutrophil subsets have mainly been characterized in tumoral conditions, where constitutively produced cytokines and growth factors can promote polarization of cells recruited into the tumor (9, 47). Of note, IFNβ and TGFβ1 induce neutrophils to acquire anti-tumoral (N1) or pro-tumoral (N2) phenotypes respectively (9, 47). N2 neutrophils are postulated to have a main role in promoting tumoral growth by increasing extracellular matrix deposition and by dampening a proper immune response (9). In this context, TGFβ1 appears as a central player in the tumor microenvironment orchestrating diverse pro-tumoral, antiinflammatory, and immunomodulatory actions, including the induction and maintenance of an N2 phenotype. The induction of "N2-like" neutrophils in the presence of testosterone in our study can also be explained by overexpression of TGFβ1 since its promoter activity is directly regulated by androgens through the androgen receptor (AR) (48, 49). However, whether TGFβ1 acts directly or by other mechanisms to induce N2 neutrophil maturation by androgens deserves further research.

The ability of androgens to promote antiinflammatory/immunomodulatory phenotypes has been previously recognized for professional immune cells (16, 20). In general, testosterone tends to inhibit pro-inflammatory molecules such as TNFα, iNOS, and NO whereas induces IL10 and TGFβ1 anti-inflammatory signaling (19, 50–52). Accordingly, we found that androgens favor a higher IL10 and TGFβ1 expression along with a lower IL12 expression on recruited neutrophils. Strikingly, this phenotype was accompanied by a reduction in both MPO activity and bactericidal ability in vivo as well as ex vivo, resulting in an inadequate bacterial clearance. In accordance, it has been reported that, in vitro, testosterone decreases the microbicidal activity of human neutrophils by dampening the production of reactive oxygen species (53, 54). In contrast, after trauma or burn injury, androgens have been reported to enhance neutrophil activation in blood, as judged by CD11b expression and respiratory burst activity (32), which might explain why males are more susceptible to acute shock aggressiveness than females. These ambivalent results could be attributed to the type of stimuli used and the pathological conditions, with scarce data available on the bactericidal ability of neutrophils after androgen manipulation. In any case, the promotion of "N2-like" neutrophils by androgens may be deleterious as well as favorable, depending on the particular context; albeit being inefficient in killing bacteria, their ability to generate TGFβ1, as described before (39, 40), could be beneficial in repairing tissues and resolving inflammation (39, 40).

Our results indicate that testosterone modulates neutrophil activity within the prostate and in androgen-independent sites as well. In a bacterial milieu, testosterone promotes, in a dose dependent manner, a recruitment of malfunctioning neutrophil that amplifies and prolongs the inflammatory response, with the persistence of their toxic products destroying cellular components and generating a favorable environment for the development of pathologies (14, 55). On the other hand, the testosterone-induced anti-inflammatory profile displayed by these neutrophils could be beneficial in some non-bacterial types of inflammation.

### AUTHOR CONTRIBUTIONS

MS and AQ performed most protocols. MS, NP, MC, CL, and AQ analyzed data. JN carried out and analyzed qPCR experiments. GM performed, analyzed, and supervised intravital imaging. MS, CM, and AQ wrote the manuscript. CM and AQ designed the study and supervised the research work.

### ACKNOWLEDGMENTS

This work was funded by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET PIP 2014-2016 No. 112<PHONE_NUMBER>8 to CM), Secretaría de Ciencia y Tecnología Universidad Nacional de Córdoba (SECyT-UNC, 2014-2015, 2015-2017 to CM and AQ), and Agencia Nacional de Promoción Científica y Tecnológica–Ministerio de Ciencia y Tecnología (Foncyt PICT to AQ and CM).

The authors wish to acknowledge the excellent technical assistance of Elena Pereyra and Lucia Artino in TEM processing and the expert advice in live imaging by members of the Menezes' Lab.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2018.01980/full#supplementary-material

Supplementary Movie 1 | Testosterone increases LPS-induced neutrophil accumulation in the liver. Intravital microscopy after an i.p. LPS 0.5 mg/kg injection for the visualization of neutrophil recruitment to liver sinusoids. Mice were previously treated with the antiandrogen flutamide (7 mg/kg/day) or with testosterone (10 mg/kg/day) for 3 days. Quantification of Ly6G (+) neutrophils (green) is shown in Figure 2D.

Supplementary Table 1 | List of primers (Rattus norvegicus) used for different target mRNAs.

### REFERENCES

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer PM and handling Editor declared their shared affiliation.

Copyright © 2018 Scalerandi, Peinetti, Leimgruber, Cuello Rubio, Nicola, Menezes, Maldonado and Quintar. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Differential redox state contributes to sex Disparities in the response to influenza Virus infection in Male and Female Mice

*Ignacio Celestino1†, Paola Checconi 2†, Donatella Amatore1 , Marta De Angelis1 , Paolo Coluccio1 , Rosanna Dattilo3 , Danilo Alunni Fegatelli1 , Ann Maria Clemente4 , Paola Matarrese5 , Maria Gabriella Torcia4 , Romina Mancinelli6 , Caterina Loredana Mammola6 , Enrico Garaci 2 , Anna Rita Vestri1 , Walter Malorni5 , Anna Teresa Palamara1,2† and Lucia Nencioni1 \*†*

#### *Edited by:*

*Virginia Rider, Pittsburg State University, United States*

#### *Reviewed by:*

*Ross Vlahos, RMIT University, Australia Malav Suchin Trivedi, Northeastern University, United States*

#### *\*Correspondence:*

*Lucia Nencioni<EMAIL_ADDRESS>
*† These authors have contributed equally to this work.*

#### *Specialty section:*

*This article was submitted to Cytokines and Soluble Mediators in Immunity, a section of the journal Frontiers in Immunology*

*Received: 31 March 2018 Accepted: 16 July 2018 Published: 30 July 2018*

#### *Citation:*

*Celestino I, Checconi P, Amatore D, De Angelis M, Coluccio P, Dattilo R, Alunni Fegatelli D, Clemente AM, Matarrese P, Torcia MG, Mancinelli R, Mammola CL, Garaci E, Vestri AR, Malorni W, Palamara AT and Nencioni L (2018) Differential Redox State Contributes to Sex Disparities in the Response to Influenza Virus Infection in Male and Female Mice. Front. Immunol. 9:1747. doi: 10.3389/fimmu.2018.01747*

*1Department of Public Health and Infectious Diseases, Pasteur Institute Cenci Bolognetti Foundation, Sapienza University of Rome, Rome, Italy, 2San Raffaele Pisana, IRCCS, Telematic University, Rome, Italy, 3Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy, 4Department of Clinical and Experimental Medicine, University of Florence, Florence, Italy, 5Center for Gender-Specific Medicine, Istituto Superiore di Sanità, Rome, Italy, 6Department of Anatomical, Histological, Forensic Medicine and Orthopedic Sciences, Sapienza University of Rome, Rome, Italy*

Influenza virus replicates intracellularly exploiting several pathways involved in the regulation of host responses. The outcome and the severity of the infection are thus strongly conditioned by multiple host factors, including age, sex, metabolic, and redox conditions of the target cells. Hormones are also important determinants of host immune responses to influenza and are recently proposed in the prophylaxis and treatment. This study shows that female mice are less susceptible than males to mouse-adapted influenza virus (A/PR8/H1N1). Compared with males, PR8-infected females display higher survival rate (+36%), milder clinical disease, and less weight loss. They also have milder histopathological signs, especially free alveolar area is higher than that in males, even if pro-inflammatory cytokine production shows slight differences between sexes; hormone levels, moreover, do not vary significantly with infection in our model. Importantly, viral loads (both in terms of viral M1 RNA copies and tissue culture infectious dose 50%) are lower in PR8-infected females. An analysis of the mechanisms contributing to sex disparities observed during infection reveals that the female animals have higher total antioxidant power in serum and their lungs are characterized by increase in (i) the content and biosynthesis of glutathione, (ii) the expression and activity of antioxidant enzymes (peroxiredoxin 1, catalase, and glutathione peroxidase), and (iii) the expression of the anti-apoptotic protein Bcl-2. By contrast, infected males are characterized by high expression of NADPH oxidase 4 oxidase and phosphorylation of p38 MAPK, both enzymes promoting viral replication. All these factors are critical for cell homeostasis and susceptibility to infection. Reappraisal of the importance of the host cell redox state and sex-related effects may be useful in the attempt to develop more tailored therapeutic interventions in the fight against influenza.

Keywords: sex differences, gender, hormones, influenza virus, redox state, glutathione

## INTRODUCTION

Viruses replicate in the living cells of their hosts and use many intracellular pathways for their own advantage. Consequently, host factors like age, general health, metabolic, and redox conditions of the cells can have important repercussions on different steps of the virus life cycle (1–4). Furthermore, general redox state may also affect host immune response to viral replication (5–8). Cells containing high levels of thiols, e.g., glutathione (GSH) and cysteine content, or characterized by higher antioxidant defenses, as well as abundant expression of Bcl-2 proteins family, are less permissive to viral replication, including influenza (2). Moreover, our and other groups previously demonstrated that cells infected with influenza virus were characterized by low levels of GSH (2, 9–12) and by an increase of reactive oxygen species (ROS) production (10, 11, 13). During influenza virus infection, there is also a depletion of key antioxidant enzymes, due to their secretion or because of virus-induced loss of lung cells (14, 15). The oxidative stress is useful for the virus since many pathways involved in the regulation of viral replication and host responses to viral infection are highly responsive to even transient changes in the redox state of the cytoplasmic environment (16). In fact, some enzymes like protein disulfide isomerase (PDI) or NADPH oxidase 4 (NOX4) regulate specific steps of virus life cycle, including the folding and maturation of viral glycoprotein hemagglutinin (10) and the nuclear-cytoplasmic export of viral nucleoprotein (NP) (11, 17, 18).

Sex and gender, that refer to biology and behavior, respectively (19), also impact viral infections. Analysis of several epidemiological studies has highlighted that disease severity and fatality following exposure to influenza A viruses are generally higher in women than men (19, 20). The mechanisms underlying this sex/gender difference are several and tightly interconnected; behavioral, immunological, hormonal, and genetic factors are all included (20). Focusing on biological factors, it is known that females mount a higher immune response than males, which can accelerate virus clearance and reduce virus load, but can also make females more prone to immunopathology and to development of autoimmune disease (21). Klein et al. (20, 22, 23) reported that the exaggerated immunity and consequent immunopathology lead females to greater morbidity and mortality with respect to males. Such a response can be modulated by hormone concentrations, and so age may also affect the sex-related variability (24, 25). Epidemiological studies in which results were stratified by age in fact, report that hospitalization and morbidity rates due to influenza A viruses are higher in males than in females from birth to 15–19 years (26–31). Non-endocrine factors, as genetic ones, could prevail in the latter case. It has been shown that genetic variation in chromosome Y regulates susceptibility to influenza A virus, making specific variants in males mice more susceptible to infection (32).

Interesting parameters that also differently characterize cells isolated from male and female animals were the redox ones (33, 34). Malorni et al. (35) reported differences between vascular smooth muscle cells (VSMC) from male and female rats in terms of "basal" redox balance. Either H2O2 or O2 − levels were significantly lower in VSMC from females than those from male rats. Moreover, the intracellular GSH content was higher in female than in male rats. The same authors found that antioxidant enzyme activity was significantly higher in VSMC from female than in male, independently from the stimuli that induced stress (35, 36). Many redox-sensitive cell-signaling pathways are differently activated in both sexes (37).

On the basis of this evidence, in this study, we verified the hypothesis that host redox state plays a role in sex disparities in the outcome of influenza virus infection. To evaluate viral replication in male and female mice, we chose the Balb/c strain, which is considered a Th2-type strain (38), to better highlight the effect of the virus (as opposed to the immune response). Female and male mice were infected with a mouse-adapted strain of influenza A (H1N1) and the progression of disease was monitored by measuring some redox parameters usually altered during infection. We found that in terms of both survival and clinicopathological parameters of disease, the female mice displayed higher resistance to the infection, due to significant differences in the systemic and pulmonary "redox profiles" between female and male mice.

### MATERIALS AND METHODS

In accordance with national law, the experiments described in this manuscript were approved by the Italian Ministry of Health, which verified the ethical and scientific appropriateness of the research. All animals received humane treatment, and every effort was made to minimize their suffering. Unless otherwise stated, all commercial products cited were used in accordance with the manufacturers' instructions.

### Mice and Virus Infection

Balb/c 6-week-old mice [400 females, body weight (bw) range = 15–19 g; 400 males, bw = 19–23 g] were purchased from Harlan Laboratories (Milan, Italy). Animals were housed under specific pathogen-free conditions (5/cage, SmartFlow IVC Rack, Tecniplast, Varese, Italy) at 12:12 h light:dark cycle, and *ad libitum* access to food and water. After 1 week, each mouse was individually weighed and randomly assigned to an experimental group.

A mouse-adapted strain of influenza A/Puerto Rico/8/34 (H1N1; PR8) was used. In our experiments, 1 plaque-forming unit (PFU) of PR8 stock was equivalent to 2.9 × 103 genome copies, approximately 2.0 × 103 genome copies/tissue culture infectious dose 50% (TCID50) according to the relationship between TCID50 and PFU provided by the American Type Culture Collection. The 50% mouse lethal dose (MLD50) was determined in female and male mice that had been lightly anesthetized by isofluorane

**Abbreviations:** BALF, broncho-alveolar lavage fluid; CAT, catalase; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; Gusb, glucuronidase beta; GCL, glutamate cysteine ligase; GR, glutathione reductase; GSS, glutathione synthase; GSHPx, glutathione peroxidase; H&E, hematoxylin & eosin-stained; MLD50, 50% mouse lethal dose; NOX4, NADPH oxidase 4; NP, nucleoprotein; GSSG, oxidized glutathione; PRDX, peroxiredoxin; PFU, plaque-forming unit; PDI, protein disulfide isomerase; ROS, reactive oxygen species; GSH, reduced glutathione; Rpl13a, ribosomal protein L13A; SOD, superoxide dismutase; TCID50, tissue culture infectious dose 50%; TAC, total antioxidant capacity; VSMC, vascular smooth muscle cells.

(Esteve, Milan, Italy) inhalation and intranasally inoculated with PR8 at different doses (0.01–10 PFU/animal).

For assessment of morbidity and survival related to seasonallike influenza infections, the *inoculum* consisted of 50 µl of sterile phosphate-buffered saline (PBS), alone (mock-infected controls) or containing 0.5 PFU/mouse of PR8 (infected animals).

Infected and control animals were daily monitored up to 21 days post-infection (p.i.). Each animal was weighed, its rectal temperature was measured (Temp Thermocouple Meter, Oakton, USA), and the clinical severity of disease was scored using the following scale (39, 40): 0 = no visible signs of disease; 1 = slight ruffling of fur; 2 = ruffled fur, reduced mobility; 3 = ruffled fur, reduced mobility, rapid breathing; 4 = ruffled fur, minimal mobility, huddled appearance, rapid and/or labored breathing indicative of pneumonia.

At the end of the experiments, the mice were euthanized with an overdose of tiletamine/zolazepam (Virbac, Milan, Italy) (800 mg/kg bw) and xylazine (Bayer, Milan, Italy) (100 mg/kg bw). Specimens for analysis [blood, broncho-alveolar lavage fluid (BALF), and lungs] were then collected as described below.

### Blood

### Serum Total Antioxidant Capacity (TAC) Assay

On p.i. days 3, 6, 9, and 21, blood was collected from the retroorbital venous sinuses of control and PR8-infected mice. The recovery was made with a Pasteur pipette after ocular instillation of oxybuprocaine (1 drop/eye) (Novartis, Siena, Italy). The sample was allowed to clot for 45 min (to facilitate removal of all platelets and precipitates) and then centrifuged at 10,000 × *g* for 15 min at +4°C. The serum was stored at −80°C prior to assay with the TAC Kit (JaICA, Florence, Italy), which measures the sample's capacity to convert Cu+2 to Cu+1 .

### Sex Hormone Quantification

Testosterone and estradiol quantification was performed using a colorimetric competitive enzyme immunoassay kit purchased from Enzo Life Sciences (3V Chimica, Rome, Italy), according to the manufacturers' instructions.

### Broncho-Alveolar Lavage Fluid (BALF)

Mice were euthanized, and a sterile 23-G catheter was inserted into the exposed tracheal lumen. Two instillations of sterile PBS (0.8 ml) containing protease inhibitors (Sigma-Aldrich, Milan, Italy) were injected through the catheter and aspirated as previously described (41). The BALF samples were centrifuged at 1,000 × *g* for 15 min at +4°C and the supernatant stored at −80°C prior to analysis.

### Total Protein Content

For assessment of lung damage, the total protein content of each BALF specimen was measured with a standard Micro BCA Kit (Pierce, Monza, Italy). BALF samples (150 µl) were pipetted into a microplate well, working reagent (150 µl) was added, and the plate was incubated at 37°C for 2 h and cooled to room temperature. The optical density of each solution was measured at 570 nm with a Multiskan Ex Reader (Thermo Fisher Scientific, Monza, Italy).

### Cytokine Quantification

A multiplex assay was used to measure cytokine (IL-1, IL-6, TNF-α, IL-10, IFN-γ, CCL2-MCP1, and CCL3-MIP1) levels in each BALF sample. Plates were read on a Bio-Plex MAGPIX instrument, and data were analyzed with Bio-Prosoftware (Bio-Rad, Milan, Italy).

### Lungs

### Assay of Viral Titers

Whole lungs isolated from infected female and male mice were removed, weighed, frozen, and stored at −80°C. For the quantification of viral M1 RNA copies, total RNA was extracted from thawed lungs that had been homogenized in TRI Reagent (Sigma-Aldrich, Milan, Italy) (1 ml/75 mg of tissue) with a Polytron homogenizer. The RNA pellet was washed with 1 ml of 75% ethanol (7,500 × *g* for 5 min at +4°C) and air-dried for 30 min. Diethylpyrocarbonate water (100 µl) was added, and tube was heated to 55°C for 15 min to facilitate dissolution. The isolated RNA was treated with DNase I (Invitrogen, Life Technologies, Monza, Italy), and its quality and quantity were verified spectrophometrically (Pearl Nanophotometer, IMPLEN, Munich, Germany). The number of viral M1 RNA copies was determined by quantitative real time RT-PCR using the One Step Influenza A/B r-gene and Quanti FluA kits (BioMérieux, Florence, Italy). For the evaluation of TCID50, lungs were homogenized in RPMI 1640 medium, and homogenates were subjected to TCID50 assay on MDCK cells. The number of wells showing positive cytopathic effects was scored, and the titer was calculated as previously described (42).

### Histologic Examination

Lung histology was evaluated in female and male infected mice (*n* = 25/group). Mock-infected mice were used as controls. Mice were sacrificed at 3, 6, 9, and 21 days p.i. Each sacrifice was followed by complete necroscopy with macroscopic and microscopic examinations of the lungs.

For the histopathological and morphological examination, each lung was fixed in buffered formalin at room temperature for 48 h and embedded in paraffin with a melting point of 55–57°C. Sections (3-μm thick) were stained with hematoxylin and eosin and Masson's trichrome.

The samples were evaluated independently and blindly by three investigators (Caterina Loredana Mammola, Antonio Franchitto, and Romina Mancinelli), and necroinflammatory changes were scored as follows (43, 44): 0 = no lesions; 1 = mild focal inflammation; 2 = moderate–severe inflammation or necrosis affecting less than 25% of lung tissue examined; 3 = severe inflammation with necrosis or severe inflammation affecting 25–50% of lung tissue examined; 4 = severe inflammation with necrosis affecting more than 50% of the lung tissue examined. For each lung, at least five slides were analyzed. Briefly, serial paraffin sections were obtained per animal. For each sample, 10 fields were analyzed per section. Alveoli were identified and bordered to calculate the corresponding areas. All ambiguous structures, airways, and vascular structures were excluded. The tissue and airspace areas were tabulated using the IAS Delta Sistemi software (Rome, Italy) (10, 45–47).

### Assays of Thiols Levels and Antioxidant Enzyme Activities

A sterile 23-G butterfly needle was inserted into the euthanized mouse's right ventricle and connected to a peristaltic pump (Generalcontrol, Milan, Italy). The lungs were then perfused with PBS containing 50 U/ml heparin (Sigma-Aldrich, Milan, Italy) to remove erythrocytes and clots. Cuts were made in the liver to facilitate perfusate outflow. The lungs were then removed, weighed, frozen in liquid nitrogen, and stored at −80°C until assayed.

Intracellular glutathione (GSH) and oxidized forms [oxidized glutathione (GSSG)] were measured in lung homogenates with the Glutathione Assay Kit (Cayman Chemical, Florence, Italy) following the manufacturer's instructions, after deproteinization with metaphosphoric acid of the samples. For GSSG quantification, an aliquot of deproteinized samples was first incubated with 2-vinylpyridine to derivatize GSH. Reduced GSH levels were obtained by differences between total GSH and GSSG.

The total amount of free thiols in deproteinized samples from lung homogenates and in serum were measured by a standard colorimetric assay using Ellman's reagent (48).

Catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSHPx) activities were also measured with specific kits (Cayman Chemical, Florence, Italy). Calculation of enzymatic activity was determined following the manufacturer's instructions.

### RT-PCR Analysis of Pulmonary mRNA Levels

Total RNA was isolated from the lungs as described above and used as a template for generating cDNA (iScript cDNA Synthesis Kit, Bio-Rad, Milan, Italy). An aliquot of the cDNA was subjected to 40 cycles of RT-PCR amplification (95°C, 10 s; 60°C, 30 s) using iQ SYBR Green Supermix and a LightCycler iQ 5 (Bio-Rad, Milan, Italy). To ensure that the primers produced a single and specific PCR amplification product, a melting curve analysis was carried out at the end of the PCR cycle. The housekeeping genes glucuronidase beta (Gusb), ribosomal protein L13A (Rpl13a), and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) were used for normalization. Relative quantitative evaluation was performed by the comparative ΔΔCt method.

The following forward and reverse primers were used: glutathione reductase (GR) (TTCAGTTGGCATGTCATC forward; CCGTGGATAATTTCTATGTGA reverse), glutathione synthase (GSS) (GTGCTACTGATTGCTCAA forward; ACATG GATCTTCCTGTCT reverse), glutamate cysteine ligase (GCL) (AA GTCCCTCTTCTTTCCA forward; CCTTGAATATTGGCAC ATTG reverse), Bcl-2 (CCTACGGATTGACATTCTC forward; AT ACATAAGGCAACCACAC reverse), Rpl13a (ATGGGATGAAT CAGTTGAG forward; ATAGGGTACTTGGTCAGG reverse), Gapdh (TGCGACTTCAACAGCAACTC forward; ATGTAG GCCATGAGGTCCAC reverse), Gusb (GTACTCCTTGGAG GTGAA forward; TGAATCCTCGTGCTTATTG reverse). The results are presented as fold increases relative to levels observed in mock-infected control mice.

### Western Blot Analysis

Whole lungs of female and male infected mice (*n* = 9/group) were homogenized in RIPA lysis buffer [20 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGDA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1% Triton X-100, and 0.1% sodium dodecyl sulfate (SDS)] supplemented with phenylmethylsulfonyl fluoride, protease inhibitor mixture, and phosphatase inhibitor (Sigma-Aldrich, Milan, Italy). Lung lysates were incubated for 30 min on ice and then centrifuged at 13,000 × *g* for 30 min. The protein concentration of the supernatants was determined with the Micro BCA Protein Assay Kit (Pierce, Monza, Italy). Samples were separated by SDS-PAGE, blotted onto nitrocellulose membranes, blocked with 10% non-fat dry milk, and stained with primary (see below) and secondary antibodies peroxidase-conjugated (Jackson ImmunoResearch, Milan, Italy). Blots were developed with the ECL-Plus Detection System (GE Healthcare, Milan, Italy) and subjected to densitometry with the Quantity One Program (Bio-Rad, Milan, Italy).

Primary antibodies included rabbit polyclonal anti-NOX4, anti-phospho-p38, anti-Bcl-2 (Santa Cruz Biotechnology, Dallas, TX, USA); rabbit polyclonal anti-PRDX1 (Abcam); and mouse monoclonal anti-actin (Sigma-Aldrich).

### Statistical Analyses

The long-rank test was used to assess the difference in the overall Kaplan–Meier survival curves. Variations on bw and temperature were assigned by using a linear mixed model for repeated measures adjusted by baseline value followed by *post hoc* analysis (Bonferroni's correction). The Wilcoxon test was performed to compare the values of Glutathione, viral M1 RNA copies, and protein concentrations in BALF in the two groups (all statistical analyses were performed using R version 3.3).

Unpaired two-tailed Student's *t*-test or one-way ANOVA test were used for antioxidant enzyme activity; gene and protein expression; cytokine levels; alveolar area in both sexes (statistical analysis was performed using GraphPad Prism™ software version 6.0).

*p*-Values of less than 0.05 (*p* ≤ 0.05) were considered statistically significant.

### RESULTS

### Female Mice Are More Resistant to Influenza Virus Infection Than Males

Female and male Balb/c mice were infected intranasally with 0.5 PFU/mouse and clinical signs of infection, bw, body temperature, and survival were monitored daily until 21 days after infection. The clinical responses and survival rates observed in female and male mice up to 21 days p.i are shown in **Figure 1**. The first symptoms of disease (piloerection, reduced food intake, and lethargy) appeared in both sexes 4 days p.i. and increased rapidly in intensity. The males exhibited more pronounced horripilation (as the first sign of pain) than the females and higher clinical scores at peak disease intensity (on p.i. days 6–12) (**Figure 1A**). In addition, the percentage of bw decreased rapidly in each male, whereas for some females the bw did not decrease considerably. The overall trend of bw loss at day 9 p.i. was higher in males than in females (26.6 and 23.2%, respectively), even if no statistically

with 10 male and 10 female animals. Scores ranged from 0 (no disease) to 4 (signs and symptoms that are indicative of pneumonia). See Section "Materials and Methods" for details. (B) Spaghetti plot of the daily body weight (expressed as percentage respect to day 0); the bold lines represent the overall trend. (C) Kaplan–Meier overall survival curves. Results represent data pooled from four independent experiments, each performed with 10 males and 10 females (*n* = 40/sex), \*\*\**p*-value = 0.001.

substantial differences were detected (**Figure 1B**; Figure S1A in Supplementary Material). In terms of body temperature, no differences were observed in the two groups as well (Figure S1B in Supplementary Material).

Nevertheless, the percentage of survival following infection was significantly lower among males in comparison to females (53.8 and 90%, respectively, log rank \*\*\**p*-value = 0.001) (**Figure 1C**). Furthermore, the average day of death occurred earlier in male than in female group (on p.i. day 7 vs. on p.i. day 10).

### Influenza Virus Causes More Severe Lung Damage in Male Mice

To look at the damage caused by PR8 infection in the lungs of mice, the animals were euthanized and lungs fixed in 10% buffered formalin prior to sectioning at 3 µm and staining with hematoxylin & eosin-stained (H&E) and Masson's trichrome as described in Section "Materials and Methods" (**Figure 2**).

The observation of lung tissues from uninfected control mice (*n* = 5/sex) did not highlight lesions in any of the sacrificed animals. No changes were found in the normal architecture of pulmonary parenchyma, as well as in the normal morphology of airways, alveolus, and alveolar septa. The free alveolar area for these animals (Ctr) compared with that measured in infected mice (I) is reported in **Figures 2** and **3A**. Results are shown for 3, 6, and 9 days p.i., since on day 21 p.i. both female and male mice that survived did not show significant differences. As reported on the table under the graph (**Figure 3A**), the percentage of reduction of free alveolar area in males was higher than in females.

In details, in PR8-infected male mice (*n* = 5) sacrificed after 3 days p.i. (**Figures 2B** and **3A**), we described initial alterations of lungs parenchyma; alveolar area resulted slightly reduced compared with control lungs with thickened alveolar septa. In addition, all samples showed peripheral edema and alteration of epithelium with inflammatory cells adhering to the surface of bronchioles. The mean ± SD of free alveolar area was 975 ± 235 with a % reduction respect to Ctr of 32 ± 16. Female mice lungs (*n* = 5) at 3 days p.i. (**Figures 2A** and **3A**) displayed a similar histopathological damage and the mean ± SD of alveolar area was 1,204 ± 238 with a % reduction of 18 ± 16.

display the same aspects, maintaining a greater alveolar area (original magnification 10×).

In infected male mice (*n* = 5) sacrificed after 6 days p.i. (**Figures 2B** and **3A**), we found widespread impairment of pulmonary parenchyma; the pictures of interstitial pneumonia were characterized by the presence of higher inflammatory exudate (interstitial and alveolar) with inflammatory cells, fibrin, cellular debris, and obvious vascular congestion and areas of necrosis. The alveolar area is greatly decreased if compared with the control lungs and strikingly, the lungs of the male mice displayed signs of more severe damage than those of the females consisting of bronchiolitis, peri-bronchiolitis, interstitial edema, alveolar wall thickening, dense interstitial granulocyte, and lymphocyte infiltrates, and the alveolar area was 708 ± 212 with a % reduction of 51 ± 14.8. By p.i. day 6, these lesions already involved over 25% of the considered parenchyma, and similar involvement was observed in survivors sacrificed 9 days p.i. Female mice lungs (*n* = 5) at 6 days p.i. (**Figures 2A** and **3A**) showed similar histopathological alterations from a qualitative point of view, but larger preserved parenchymal areas; therefore, the alveolar area was significantly higher than male mice and this difference persisted for the duration of the experiment, indicating that the virally induced inflammation had a lower impact on lung's female (alveolar area: mean ± SD 996 ± 286 with a % reduction of 32 ± 19.5).

mice were 10 (5/sex), infected mice on p.i. day 3 were 19 (9 females and 10 males), on p.i. day 6 were 19 (10 females and 9 males), on p.i. day 9 were 18 (9/sex), \*\**p*-value = 0.006.

In infected male mice lungs (*n* = 5) at 9 days p.i. (**Figures 2B** and **3A**), diffuse impairment of pulmonary parenchyma was observed. The "alveolar area" was greatly reduced compared with the lung of the controls but lightly reduced compared with the animals after 6 days p.i. (free alveolar area 625 ± 213 with a % reduction of 56 ± 14.9). Mice female lungs (*n* = 5) at 9 days p.i. (**Figures 2A** and **3A**) presented chronic flogistic infiltrate with prevalent interstitial localization activated by epithelial/endothelial lesions: the picture is similar to that of male animals, but there are a lower incidence of collagen and exudative deposition and necrosis; moreover, reconstitution areas of the alveolar epithelium is observed in female lungs (free alveolar area 837 ± 230 with a % reduction of 43 ± 15.7).

Finally, as an indirect measure of the diffuse alveolar damage, protein concentrations in BALF samples from PR8-infected mice and mock-infected controls (Ctr) were assessed. As shown in **Figure 3B**, increases were observed on p.i. day 6 in infected animals of both sexes. By p.i. day 9 (when maximal lung damage was noted in lung sections), BALF protein levels were significantly higher in the male group (\*\**p*-value = 0.006).

From a molecular point of view, a panel of different inflammatory cytokines and chemokines (IL-1, IL-6, TNF-α, IL-10, IFN-γ, CCL2-MCP1, and CCL3-MIP1) was evaluated in BALF from males and females mice. As shown in **Figure 4**, both sexes produced all the cytokines and their levels were higher than those measured in mock-infected mice. In fact, the pro-inflammatory cytokines IL-6, TNF-α, and IL-1 were increased in both sexes, the latter particularly in males. Instead, IFN-γ cytokine levels resulted more pronounced in females. Regarding chemokines CCL2-MCP1 and CCL3-MIP1, an increase was observed in both sexes, with MIP-1 higher in males. The immunosuppressive cytokine (IL-10) was increased in males on day 6 p.i.

These results apparently contradicted most of the literature that report that adult female mice experienced a greater morbidity and mortality after influenza virus infection than males, and this was correlated to immunopathology (24, 49); because hormones affect the immune response to viral infection, we wondered what are the hormonal levels in our model. We found plasma estradiol levels of 39.75 ± 18.6 pg/ml in control female

mice and 30.3 ± 9.5 pg/ml in infected females (p.i. day 6); regarding testosterone, we measured 18.74 ± 6.2 ng/ml in control males and 15.12 ± 1.6 ng/ml in infected males (p.i. day 6); therefore, no significant differences in hormone level between uninfected and infected mice were detectable.

As the lung damage appeared less severe in females and on the basis of the results from hormone quantification, which seemed not to change during infection, we finally looked at the viral replication. As displayed in **Figure 5A**, viral M1 RNA copies in lung homogenates were consistently higher in the male group, and this difference was statistically significant on p.i. day 6 (\*\**p*-value = 0.004). Similarly, viral M1 RNA copies measured in BALF samples were also considerably higher in males than in females, during peak illness (**Figure 5B**). Accordingly, on p.i. day 6, the TCID50 measured on lung homogenates obtained from infected male mice was higher than in female mice (1,582 ± 457 and 654 ± 32 U/ml, respectively).

Therefore, collectively these data suggest that the higher morbidity and, consequently, the lower survival, as well as the more severe and extended lung damage exhibited by PR8-infected male mice, may be the result of a higher replication of influenza virus in the lungs of the male mice.

### Enhanced Systemic Antioxidant Power Protects Female Mice During Viral Infection

Influenza virus infection is known to be strongly conditioned by host redox environment, including the intracellular GSH content, antioxidant defense, and expression of redox-regulated

Celestino et al. Sex Disparities in IAV Infection

cell pathways (2, 9–13, 17, 18). To determine whether these factors contributed to the sexual disparities in susceptibility to PR8 infection observed in our model, we first compared the TAC of female and male mice. The TAC reflects the abundance of antioxidant molecules and enzymes available in the blood to counteract the effects of ROS/reactive nitrogen species, such as those produced during viral infection. As shown in **Figure 6A**, mock-infected female mice displayed appreciably higher TAC than their male counterparts. More striking sex-related differences were seen in PR8-infected mice. The reduction potential of serum from female mice remained high (near baseline levels) throughout the viral infection, whereas that of the males dropped significantly. On p.i. days 3 and 6, the TAC recorded for the males was significantly lower than those of the females (unpaired *t*-test \*\**p*-value <0.01). By p.i. day 21, TAC of surviving animals had returned to their respective baseline levels, which were once again lower in males. Accordingly, the analysis

Figure 6 | Antioxidant defenses are higher in infected female mice. (A) Serum from mock-infected (Ctr) and PR8-infected mice was collected on p.i. days 3, 6, 9, and 21 and assayed with the potential antioxidant test, which measures the total antioxidant power in terms of the sample's ability to reduce copper. (B) Lung homogenates from Ctr and PR8-infected mice were assayed for CAT activity at the same time points than in panel (A). Each value reported represents the mean ± SD of results from two separate experiments, each performed in duplicate (*n* = 4), \**p*-value <0.05 and \*\**p*-value <0.01. (C) Peroxiredoxin (PRDX)1 protein expression was analyzed by western blotting in the lungs of infected female and male mice. Actin was used as loading control. Blot shows three animals for each sex (\**p*-value ≤0.05) and is representative of three independent experiments performed.

of free thiols in serum and lung homogenates from infected and mock-infected mice showed a slight reduction in infected males compared with mock infected, while no differences were detectable between infected and non-infected females (data not shown).

Next, we assessed antioxidant enzyme activities in lung homogenates. As shown in **Figure 6B** in mock-infected controls, CAT activity did not significantly differ among males and females. After PR8 infection, however, CAT activity in the lungs of male mice dropped substantially, reaching levels on p.i. days 3 and 6 that were significantly lower than those of the female group, which remained stable throughout the viral infection (unpaired *t*-test \**p*-value <0.05).

Activity of SOD increased appreciably in both sexes on p.i. day 3, but this change was not statistically significant. Essentially, PR8 infection was not associated with any significant change in pulmonary SOD activity in either the female or male mice, and no significant sex-related differences were observed at any of the time points (Figure S2 in Supplementary Material). Furthermore, we measured GSHPx activity, finding that it decreased significantly in both sexes but in greater extent in infected male mice than female. To note that female mock-infected mice showed significantly higher basal activity of this enzyme (Figure S3 in Supplementary Material). Finally, the expression of another antioxidant enzyme, peroxiredoxin (PRDX)1, was analyzed by western blot in lung of infected female and male mice at p.i. days 3 and 6 (time when the maximal difference in redox conditions was observed). As shown in **Figure 6C**, the expression of this enzyme was higher in females than in males. The densitometric analysis of ratio PRDX1/actin of three animals for each sex at 3 and 6 days p.i. was 1.5- and 3-fold higher, respectively, unpaired *t*-test \**p*-value ≤0.05, suggesting that females are more protected by influenza for the presence of reducing conditions.

### The Intracellular Content and Biosynthesis of GSH Are Preserved in Infected Female Mice

Influenza virus infection is associated with reductions in the GSH content of infected cells, which facilitate viral replication (2, 9–12). As shown in **Figure 7A**, basal levels of GSH in lung homogenates from the mock-infected control group were slightly higher and less variable in females than in males. As expected, levels decreased in both sexes after infection, but on p.i. day 6, there was a sharp drop in the GSH content of male lungs, which resulted in significantly lower levels than those found in females (\**p*-value = 0.034). Interestingly, this drop coincided with the time of viral loads peak in the lungs of the male mice.

Reduced glutathione depletion may be due to its buffering role against ROS that, during viral infection, essentially derive from NOX4 (11). Therefore, we evaluated the expression of this enzyme in the lung homogenate of females and males. Densitometric analysis of three animals for both sexes demonstrated that NOX4 was less expressed in females than in males (3 and 6 days p.i., 1.5-fold lower, unpaired *t*-test \**p*-value ≤0.05; **Figure 7B**).

\**p*-value = 0.034. (B) NADPH oxidase 4 (NOX4) expression was analyzed by western blotting in the lungs of infected female and male mice. Actin was used as loading control. Blots shown are one representative experiment of three performed (three animals for each sex, \**p*-value ≤0.05). (C) RT-PCR quantification of enzymes responsible for recycling and biosynthesis of GSH [glutathione reductase (GR), glutamate cysteine ligase (GCL), and glutathione synthase (GSS)]. Gene expression was measured in lung homogenates of animals sacrificed on p.i. days 3, 6, and 9. Box-plots represent the fold increases relative to levels observed in mock-infected controls (*n* = 8/sex). Unpaired *t*-test \**p*-value ≤0.05.

Intracellular GSH is regenerated from the oxidized form (GSSG) by GR or synthesized *ex novo* by the consecutive actions of GCL and GSS (50). Our next step was thus aimed at determining whether the sex-related differences in pulmonary GSH levels were also associated with differences in transcriptional expression of these three enzymes. As shown in **Figure 7C**, compared with their male counterparts, female PR8-infected mice showed a greater upregulation of GCL and GSS expression, suggesting more efficiency in counteracting PR8-induced GSH depletion (unpaired *t*-test \**p*-value <0.05). Collectively, these results indicate that female mice have an intrinsically higher antioxidant capacity, and during PR8 infection they are also capable of more efficient restoration of the physiological redox milieu in terms of GSH content that could be due to the upregulation of its synthesis.

### Lung Homogenates From Females Contain Higher Levels of the Anti-Apoptotic Bcl-2 Protein

Several intracellular redox-regulated pathways are involved in regulation of influenza virus replication, particularly the kinase p38MAP that is activated by NOX4-derived ROS (11). In cells that are highly permissive to viral infection, activated p38MAPK is entirely addressed to the nucleus, in which it participates efficiently in vRNP phosphorylation. In cells that are characterized by high levels of GSH and abundant expression of the anti-apoptotic protein Bcl-2, influenza virus replication is reduced (2). The inhibition is due to co-localization of activated p38MAPK with its cytosolic substrate (Bcl-2) and block of its translocation to the nucleus. As a consequence, NP is retained in the nucleus and viral replication is inhibited (18). Thus, we decided to evaluate whether the differences in viral load observed between the two sexes were also related to differences in p38MAPK activation and in Bcl-2 expression in the lungs. We found that p38MAPK was early activated in both groups on p.i. day 3 (**Figure 8A**). However, densitometric analysis of three different animals revealed that the kinase was more activated (almost twofold) in males compared to three homogenates of females (unpaired *t*-test \**p*-value = 0.02), thereby indicating more efficiency of p38MAPK in males. Afterward, we evaluated the expression of Bcl-2 (both mRNA and protein) in the lungs of females and males on p.i. days 3 and 6. We found that during viral infection, female mice exhibited more substantial *bcl-2* gene upregulation compared with males (**Figure 8B** unpaired *t*-test: \*\**p*-value = 0.0018; \*\*\**p*-value = 0.0002). Specifically, *bcl*-2 transcript levels in females were approximately two times as high as those found from mock-infected controls. On the contrary, there was no significant upregulation in the male mice. At the same time, densitometric analysis three different animals revealed that Bcl-2 protein levels found in the lung homogenates were also clearly higher in the female group (**Figure 8C**, 3 and 6 days p.i., unpaired *t*-test \**p*-value ≤0.05).

All these results indicate that during PR8 viral infection, females activate transcriptional processes to maintain high levels of Bcl-2 protein. This event might contribute to keep p38MAPK in the cytosol and to inhibit NP traffic and viral replication.

### DISCUSSION

In this article, we focused on one of the *in vivo* mechanisms contributing to sex-related disparities in influenza virus infection. In particular, we pointed at systemic and organ redox state as critical determinant for influenza virus replication.  These sex-based disparities correlate largely on differences between the redox conditions in the female and male animals. Mock-infected female mice have an intrinsically higher antioxidant capacity, measured as total serum antioxidant power and GSH content in lung homogenates. These

better physiological conditions persist during viral infection when we observed: upregulation of enzymes responsible for GSH biosynthesis, higher level of PRDX1, maintenance of CAT activity, and a less decrease of GSHPx activity. Infected females display also higher expression (at the mRNA and protein levels) of the anti-apoptotic protein Bcl-2, which is involved in the regulation of specific steps of influenza virus replication (2, 18). On the other hand, infected male mice displayed high expression of NOX4 enzyme, and increased levels of phosphorylated p38MAPK.

The impact of sex on susceptibility to viral infections has been hypothesized several years ago (51). Generally, females and males of various species respond differently to many DNA and RNA viruses. The mechanisms underpinning sex differences in response to viral infections are controversial, and roles for immunological, hormonal, behavioral, epigenetic, and genetic factors have all been proposed (20, 52).

It has been shown that females generate stronger innate and adaptive immune responses than males, with immune cells higher in number and activity, as well as with higher antibodies levels than males (21–23). This immunological advantage contributes to virus clearance, but on the other hand it makes females more prone to autoimmune diseases and to infectious disease-derived immunopathology (21–23). In fact, infectious diseases pathogenesis derives both from the pathogen and from the host immune response (22, 23). Influenza viruses can cause severe disease as interstitial pneumonia and bronchiolitis, characterized by typical inflammatory anatomical–pathological lesions and sometimes, massive hemorrhage, with interstitial, bronchiolar, and alveolar localization (53).

In addition to a massive cell infiltrates in the infected lungs, there is an overproduction of several pro-inflammatory cytokines and chemokines (54, 55). In fact, in this study, we observed histopathological alterations relative to interstitial pneumonia and in line with this observation, we found high levels of IL-1, IL-6, TNF-α, IL-10, IFN-γ, CCL2-MCP1, and CCL3-MIP1 in infected mice of both sexes.

However, the picture in female mice appeared different because of larger preserved parenchymal areas and a consequently total alveolar area significantly higher than in male mice. This difference persisted during the infection, suggesting that the virally induced inflammation had a lower impact on lung's female. Hormones exert a complex role in inflammation, in particular, estradiol enhances inflammation at low doses, but reduces it at higher concentrations (56), while low concentration of testosterone has reported to have a negative impact on the outcome of influenza disease (57). In our model, we found basal levels of estradiol, which did not significantly change with the infection, similar to that reported by Robinson et al. (24). Although a slight decrease, testosterone as well did not significantly vary with the infection. Therefore, in the attempt to explain the less severe lung damage that we observed in females, especially between 6 and 9 days p.i., we looked at the pathogen: we found lower viral titer in lung homogenates and BALF from female mice, with a lower infectivity, as shown by TCID50. So, these results lead us to argue that the lower impact observed in females is related to a less extent of virus replication and spread, more than a host immune effect.

 Female and male mice differed remarkably in terms of their basal redox state and their ability to counteract virus-associated oxidative imbalance. Prior to inoculation, more reducing conditions were found in the female animals in terms of GSH levels in the lungs and the TAC. These differences are in line with those reported in VSMC isolated from the aortas of male and female rats (35). *In vitro* data suggest that this sexual dimorphism can be maintained after the induction of oxidative stress, which results in females displaying greater resistance to oxidative injury and an increased capacity to counteract it (35). For example, some authors report different sex-dependent susceptibility to cytotoxic agents and treatments that is related to the incapacity of XY neurons to maintain GSH intracellular levels (58). Our *in vivo* findings support this view: during the course of influenza virus infection, the intrinsic redox balance, i.e., reducing conditions, was more effectively maintained in the female mice. In these animals, inoculation was promptly followed by the activation of enzymes involved in biosynthesis of GSH aimed at counteracting the GSH depletion induced by the virus. The period of upregulated GSH synthesis and higher levels of GSH in the females coincided with the period characterized by peak viral loads in males.

Thiols are key players in conditions of oxidative stress. Most non-protein antioxidants as well as antioxidant enzymes are thiol based (59). GSH acts as radical scavenger by directly neutralizing a variety of reactive molecules, like superoxide anion and hydroxylradicals (60), and indirectly through enzymatic reactions being a cofactor of GSHPx (61). Here, we found in male mice, higher levels of NOX4, one of the major enzymes producing ROS, thus suggesting that GSH depletion in males might be due to its consumption for its ROS buffering function. In fact, we have previously demonstrated that inhibition of NOX4 activity through chemical inhibitors or RNA silencing blocks the influenza virusinduced ROS increase, restores the content of GSH, and inhibits viral replication (11). Interestingly, several studies demonstrate that estrogens inhibit ROS production (56) by modulating antioxidant enzyme activities (62); estrogen levels have been shown also to be positively correlated to GSHPx activity in women, while no significant correlation was observed with SOD (63, 64) that in our model did not change between sexes. Moreover, estradiol has been shown to increase expression of GCL (65), that is the rate-limiting enzyme for the synthesis of GSH (60) and therefore, together with GSS and GSHPx, closely linked to the GSH levels. On the contrary, testosterone has been shown to have pro-oxidant effect (66, 67) and so we cannot exclude that it could contribute to viral replication in males by activating redox-sensitive pathways. We also found a drop in CAT activity in infected males, especially when the viral replication peaked at 6 days p.i. Accordingly, a time-dependent decrease in CAT activity has been observed in parallel to increase in influenza NS1-protein expression (13).

Several authors report that by restoring reducing conditions, viral replication and virus-induced host damage are inhibited, suggesting antioxidant therapy as a potential antiviral strategy (8, 9, 68–70). Indeed, various synthesized or natural compounds characterized by antioxidant activity have been proposed as anti-influenza agents (17, 71–76). For example, our group has shown that GSH treatment strongly inhibits viral replication by impairing glycoprotein folding (10); on the other hand, we have recently shown that GSH depletion increased influenza virus replication by preventing activation of innate antiviral response (7). Indeed, the role of GSH in modulating immune response is well known (8, 77–79). For example, in antigen-presenting cells, GSH depletion correlates with defective antigen processing and reduced secretion of T helper 1 (Th1) cytokines, thus favoring polarization from the typical Th1 profile toward a Th2 response (8). Furthermore, in T lymphocytes, intracellular GSH content is critical for their proliferation as well as extracellular thiols for their activation and function. Angelini et al. (80) demonstrated that exogenous thiols, i.e., free cysteine and thioredoxin, were released by monocyte-derived human dendritic cells (DCs) in the extracellular space to provide a reducing microenvironment required for T lymphocyte activation and an efficient immune response. In our study, we found a slight decrease of free thiols in the lung homogenates and serum of infected males, while no changes were observed in infected females compared with control. It would be interesting to investigate whether the observed decrement in males is due to a dysfunction of DCs and to impairment in T lymphocyte activation.

The imbalance in the redox state is fundamental for the activation of many cell factors, involved in the regulation of host response and in the control of influenza virus life cycle (16). Among them, MAPKs and Bcl-2 protein regulate the intracellular trafficking of the viral NP (18). In this study, we found that phosphorylation of p38MAPK is highly expressed in lung homogenate of males, suggesting that this phenomenon could explain in part the high viral load measured in males. Conversely, we found Bcl-2 to be highly expressed in the lungs of infected female mice, at both transcriptional and translational level. Furthermore, overexpression of Bcl-2 protein has been hypothesized to be associated with increased GSH levels (81, 82), and these characteristics have been found in lung homogenates of female infected mice. Based on this evidence, we can hypothesize that in females the more resistance to oxidative damage during PR8 infection may impair virus replication probably by blocking viral protein maturation and vRNP complex formation.

A final point to be considered in this scenario concerns the hypothesized role of autophagy, a cytoprotective host process that is subverted by the influenza virus to ensure its own replication (83). Metabolic stress appears to bolster a stronger, more sustained autophagic response in cells from females than in those collected from males (84). Therefore, we cannot exclude the possibility that more effective autophagic cytoprotection in lung cells from female mice led to a "weaker" cytopathological cascade.

In conclusion, our data suggest that the mechanisms underlying the sexual disparities observed in the host response to influenza can be ascribed in part to differences in their capacity to maintain redox homeostasis. In fact, in our model, we have found that females are more resistant to the influenza virus due to their ability to maintain reduced conditions during infection, thereby hindering completion of the virus life cycle and inhibiting viral replication. Therefore, although further studies are needed to fine characterize redox mechanisms underlying sex disparities in infections, i.e., the use of different antioxidants like *N*-acetylcysteine, GSH, or natural polyphenols, as well as the silencing of antioxidant enzymes that regulate viral replication, our findings may contribute to the identification of new targets for sex-based antiviral therapies. Indeed, generally, sex-related differences are not considered in current strategies for the prevention, management, and treatment of many diseases (85). Instead, a more detailed knowledge of the metabolic conditions that characterize the two sexes could ultimately improve our ability to

### REFERENCES

provide patients with individualized therapies and cost-effective solutions.

### ETHICS STATEMENT

In accordance with national law, the experiments described in this manuscript were approved by the Italian Ministry of Health, which verified the ethical and scientific appropriateness of the research. All animals received humane treatment, and every effort was made to minimize their suffering.

### AUTHOR CONTRIBUTIONS

IC, PChecconi, DA, MA, PColuccio, RD, and PM performed experiments. DF, AC, MT, RM, CM, AV, WM, and LN analyzed data. IC, PChecconi, EG, ATP, and LN designed and supervised experiments. IC, PChecconi, ATP, and LN wrote the paper.

### ACKNOWLEDGMENTS

The authors thank Andrea Martinelli and Flavio Torriani for their helpful technical assistance and Professor Antonio Franchitto for the supervision of the manuscript.

### FUNDING

This work was supported in part by the Italian Ministry of Instruction, Universities and Research – MIUR PRIN 2015 W729WH (ATP) and Ateneo (LN and ATP) grants.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at https://www.frontiersin.org/articles/10.3389/fimmu.2018.01747/ full#supplementary-material.

Figure S1 | Body weight (bw) and temperature in female and male infected mice. (A) Spaghetti plot of the daily bw (expressed in grams); the bold lines represent the overall trend. (B) Spaghetti plot of the daily body temperature, the bold lines represent the overall trend. Results represent data pooled from 4 independent experiments, each performed with 10 males and 10 females (*n* = 40/sex).

Figure S2 | No differences in superoxide dismutase (SOD) activity were observed in infected female and male mice. Lung homogenates from Ctr and PR8-infected mice were assayed for SOD activity on p.i. days 3, 6, 9, and 21. Each value reported represents the mean ± SD of results from two separate experiments, each performed in duplicate (*n* = 4).

Figure S3 | Glutathione peroxidase (GSHPx) activity is less reduced in infected female mice. Lung homogenates from Ctr and PR8-infected mice were assayed for GSHPx activity on p.i. day 6. Each value reported represents the mean ± SD of results from 3 mice/sex, each performed in triplicate (*n* = 9). One-way ANOVA test \*\*p-value <0.01; \*\*\*p-value <0.001.

lung inflammation. *PLoS Pathog* (2011) 7:e1001271. doi:10.1371/journal.ppat. 1001271

Ontario by age and gender. *Epidemiol Infect* (2007) 135:253–61. doi:10.1017/ S095026880600690X

**Conflict of Interest Statement:** The authors declare that the research was carried out in the absence of any personal, commercial, or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Celestino, Checconi, Amatore, De Angelis, Coluccio, Dattilo, Alunni Fegatelli, Clemente, Matarrese, Torcia, Mancinelli, Mammola, Garaci, Vestri,*  *Malorni, Palamara and Nencioni. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*


**5. Social and cultural factors**

[1, 2] ecological framework of the different systems that impact on a child's development, could also help researchers and practitioners to better understand the situation of parents who are taking care of a child with ASD. He posits five different systems that can influence childhood development: the Microsystem, Mesosystem, Exosystem, Macrosystem and Chronosystem. A change in any one of these components can cause a change in the other components. Micro‐ systems are those closest to the child-such as family and school-as they directly impact on the child. These Microsystems are interrelated and together form a higher level system: the Mesosystem. However these take place within an Exosystem of community services and supports that in turn is influenced by the wider cultural Macrosystem, including ideologies, laws, economic and social policies and religious attitudes. All of these systems and the interactions between them can change over the course of a child's life which is captured in the concept of the Chronosystem.

As [33] argued, in an ecological model the main emphasis has to be on "fixing" the multiple ecological environments, rather than "fixing" the child with disability and having him/her fit into different social layers of family, community and society, which is the aim of the medical model. This suggests a shift of attitude to change the focus away from the child with disability as an independent entity towards a more global view which covers both children and their families. According to an ecological model, the focus is on a transformed ecology in which children with different types of disabilities can develop by using their skills in interaction with a responsive environment [33].