Female and male C57BL/6 mice (6–8 weeks of age) were purchased from SPF Biotechnology Co., Ltd. (Beijing, China) and bred under specific pathogen-free conditions according to the guidelines. All procedures were approved by the Ethics Review Committee of the National Institute for Communicable Disease Control and Prevention at the Chinese Center for Disease Control and Prevention.

N. farcinica IFM 10152 was purchased from the German Resource Centre for Biological Materials. Bacteria were grown in BHI broth (Oxoid Ltd, Hants, UK) at 37°C to exponential phase before experiments. Female and male C57BL/6 WT mice were injected intraperitoneally with a uniform bacterial suspension (100 µl) containing approximately 2×10⁸ colony-forming units (CFU), and mortality was assessed for 14 consecutive days. For inflammatory studies, 1×10⁷ CFU of N. farcinica IFM 10152 (50 µl) or 50 µl PBS was intranasally infected under anesthesia.

Mouse weight and body temperature were quantified immediately prior to N. farcinica IFM 10152 infection and 1 day post-infection. Mice were weighed to hundredth of a gram accuracy, and body temperature was monitored with an Animal Thermometer (KEW, Nanjing, China), which steadily assesses rectal temperature to the nearest 0.1°C in 3–5 seconds.

After the mice were sacrificed by cervical dislocation, pulmonary bronchoalveolar lavage fluid (BALF) was obtained through 3 successive lavages of the bronchi with 1 mL of ice-cold PBS under a sterile environment, and the protein content was assessed using Bradford reagent (TIANGEN, Beijing, China) following the manufacturer’s instructions. Whole lung and spleen tissue was collected and homogenized in 1 ml of PBS. For enumerating bacterial counts, serial dilutions of homogenate were plated on BHI agar plates, and the number of N. farcinica IFM 10152 CFUs was counted after 48 hours of incubation at 37°C.

Lungs were fixed in 4% paraformaldehyde overnight, embedded in paraffin and cut into 5-μm sections. Slides were stained using hematoxylin and eosin and then viewed using a biological microscope (Nikon, Eclipse Ci-L, Japan) according to the manufacturers’ instructions.

For time course experiments, animals were randomly assigned to be euthanized at 1, 7, or 14 days. Supernatants from lung homogenates were used to measure IL-4, IL-6, IL-10, IL-12, TNF-α and IFN-γ by quantitative ELISA (BD OptEIA™, San Diego, CA, USA). The assays were conducted as recommended by the manufacturer, and all cultures were processed in triplicate.

To examine the direct effect of E2 on N. farcinica IFM 10152 growth, E2 at different concentrations (10 nM, 50 nM, 250 nM) was added to the growth curve plate at 37°C. The OD₆₀₀ value was tested half an hour for 48 hours using an automatic growth curve analyzer (Bioscreen, Finland).

Ovaries of 6-week-old female mice were removed through bilateral incisions over the dorsum under anesthesia. For the sham operation, the ovaries were identified, and an equal volume of paraovarian adipose tissue was removed. Ten days after incisions were sutured, mice were injected subcutaneously with 100 μL of sesame seed oil with or without 100 ng of E2 (Sigma, St. Louis, MO) at 10:00 am for ten consecutive days before N. farcinica IFM 10152 infection. Then, E2 concentrations in serum were measured at 1 day postinfection using a Mouse E2 ELISA kit (MEIMIAN, Wuhan, China). Bacterial burden and protein content were determined as described above.

Primary alveolar macrophages were obtained by centrifuging BALF and were resuspended in phenol-free DMEM (BBI, Shanghai, China) supplemented with 10% fetal bovine serum (FBS; Gibco, USA). After 2 hours of incubation in the cell culture dishes at 37°C, the supernatant was discarded, and the adherent cells obtained were cultured with new medium. The mouse and human cell lines RAW264.7 and A549 (National Infrastructure of Cell Line Resource, Beijing, China) were cultured in phenol-free DMEM with 10% FBS at 37°C. In each experiment, wells were washed three times with PBS and seeded with or without 50 nM E2 at 37°C for 16–18 h in a CO₂ incubator before infection, and the cell suspension containing N. farcinica IFM 10152 was treated at an MOI of 10:1.

For the adhesion assay, A549 and RAW264.7 cells were seeded into 24-well microplates with or without round glass coverslips at a density of 3×10⁵ cells per well. After 1 h postinfection at 37°C, for electron microscopic observation, cells were washed three times with PBS and then fixed with methanol for 8 min at room temperature. After methanol was removed, cells were stained with Giemsa stain solution and left for 30 min at room temperature. Coverslips were then washed and removed from the petri plate and examined using an Echo Revolve Generation 2 (ECHO, USA). For bacterial adhesive determination, cells were washed twice with PBS to eliminate extracellular bacteria and lysed with 1 ml of H₂O to disrupt the cells and release the intracellular bacteria. Finally, cell lysates were serially diluted 10-fold for CFU determination and plated on BHI agar plates.

For the invasion assay, A549 and RAW264.7 cells were incubated in a 24-well microplate at a density of 3×10⁵ cells per well, and primary alveolar macrophages were incubated at a density of 3×10⁴ cells per well. After 1 h of infection, cells were washed twice with PBS to eliminate extracellular bacteria and incubated in DMEM containing 50 μg/ml amikacin (20) and 2% FBS. For bacterial survival determination, cell lysates were plated on BHI agar plates after serially diluted. After 48 h of incubation, the colonies were counted.

Cytotoxicity assays of the E2-treated group and control group at 8 h postinfection were conducted using a CytoTox 96^® Non-Radioactive Cytotoxicity Assay (Promega, Madison, USA) as previously described (21). The absorbance data at 490 nm were collected using a microplate reader (BioTek, USA) according to the manufacturer’s instructions.

To block estrogen receptors, RAW264.7 cells were pretreated for 1 h at 37°C with the ER antagonist ICI 182780 (which blocks both nuclear and nonnuclear ERs, APEBixo, USA), the ERα-specific antagonist MPP (APEBixo, USA) or the ERβ-specific antagonist PHTPP (APEBixo, USA) prior to E2 exposure. CFU determination in RAW 264.7 cells was counted as described before.

For Western blot analysis, whole-cell extracts were harvested using RIPA lysis buffer (strong) (CWBIO, Beijing, China) containing protease inhibitor cocktail (CWBIO, Beijing, China) and phosphatase inhibitor cocktail (CWBIO, Beijing, China) at 30 min, 60 min, and 120 min postinfection, separated by SDS–PAGE and transferred onto PVDF membranes (Millipore, Darmstadt, Germany). Subsequently, the membranes were incubated with primary antibodies against monoclonal mouse anti-β-actin (1:4000, TransGen, China), rabbit anti-p-p44/42 MAPK (1:1000, CST, USA), rabbit anti-p-SAPK/JNK (1:1000, CST, USA) or rabbit anti-p-p38 (1:1000, CST, USA) overnight, followed by incubation with HRP-conjugated goat anti-rabbit IgG (1:1000, Beyotime, China) or HRP-conjugated goat anti-mouse IgG (1:4000, ZSGB-BIO, China). Finally, the bands were visualized using Amersham^® Hyperfilm^® ECL™ and MP Autoradiography Films (GE Healthcare).

To block MAPK signaling, RAW264.7 cells were pretreated for 1 h at 37°C with inhibitors of 20 μM p38 (SB 203580, Sigma, USA), 20 μM ERK (PD 98059, Sigma, USA) or 20 μM JNK (SP 600125, Sigma, USA) prior to E2 exposure. CFU determination was counted as described before.

Survival and growth curves were analyzed using GraphPad Prism 9.0.0. Group means and standard deviations (SDs) were analyzed by Student’s t test. Grayscale values of protein bands were analyzed by Image J. For all tests, difference was considered statistically significant if the p value is less than 0.05.

Following N. farcinica IFM10152 inoculation, female mice died significantly earlier than male mice, with survival differences noted as early as 24 h post-infection ( Figure 1A ). While 90% of male mice were able to survive, only 50% of female mice survived for 14 consecutive days.

To determine potential differences in lung infection and inflammation between sexes, we further established a nonlethal acute pneumonia model in age-matched C57BL/6 female and male mice with 1 ×10⁷ CFU N. farcinica IFM 10152 by intranasal infection. At 24 h postinfection, we observed that female mice had decreased body weight (4.04% decrease vs. 2.32%, Figure 1B ) and increased body temperature (0.7°C increase vs. 0.1°C, Figure 1C ) compared with male mice. Except for the dramatic physical changes, female mice had more abundant protein content than male mice in their airways (P < 0.05), as a sign of lung injury ( Figure 1D ). Consistent with the poor prognosis, female mice displayed a higher bacterial burden in lung tissue (P < 0.001) than male mice ( Figure 1E ), but no bacteria were found in the spleen tissue in either male or female mice (data not shown).

Examination of lung histopathology revealed exacerbated pathology in the N. farcinica IFM 10152-infected female mice compared with male mice ( Figure 1F ). The lungs of infected female mice showed marked thickening of the alveolar wall with large amounts of lymphocyte, neutrophil and macrophage infiltration, and some necrotic cell debris and hemorrhaging were also observed in the bronchial lumen. In addition, there was also evidence of inflammatory cells infiltrating into a ring around the vessel, forming a vascular sleeve ( Figure 1F ; black arrow). Male mouse-infected lungs showed evidence of lymphocyte and neutrophil infiltration, as well as slight thickening of the alveolar wall. And no obvious necrotic cell debris and vascular sleeves were observed in the bronchial lumen.

The production of cytokines by innate immune cells can also differ between the sexes in response to different stimuli, including bacterial infections. Analysis of the cytokine levels in the lung supernatant showed that at 1, 7 and 14 days postinfection, female mice had significantly elevated cytokine production levels (IL-4, IL-6, IL-10, IL-12, TNF-α and IFN-γ) in response to N. farcinica IFM 10152 compared with male mice, although all cytokines showed a decreasing trend during the days( Figure 2 ). These massive cytokine levels, which persisted for more than two weeks, are likely a consequence of the poor prognosis in the female mice.

Sex steroid hormones have been described to directly influence bacterial growth and metabolism. To test whether E2 could exert a direct effect on N. farcinica IFM 10152 growth, bacteria were grown in brain-heart infusion agar (BHI), with and without E2. Under the conditions tested, we found no quantitative change in the growth of N. farcinica IFM 10152, regardless of E2 concentration ( Figure 3A ).

Having identified no direct effect of E2 on N. farcinica IFM 10152 growth, we sought to determine whether E2 impacted the host inflammatory response. Female mice were sham ovariectomized (supplemented with sesame seed oil) or ovariectomized (supplemented with sesame seed oil or E2 at physiological doses) prior to challenge with N. farcinica IFM 10152. The results showed that serum E2 levels in female mice decreased significantly after ovariectomization but increased after exogenous E2 supplementation ( Figure 3B ). The E2 effect was confirmed by measuring the protein content in BALF and counting the bacterial burden in lung tissues. Concordant with the changes in E2 levels, E2-treated ovariectomized mice had a significant increase in protein content compared with oil-treated ovariectomized mice, which was essentially the same as that of oil-treated sham ovariectomized mice ( Figure 3C ). Furthermore, the lung bacterial burden of ovariectomized mice was significantly higher after supplementation with E2, although it was still significantly lower than that of sham ovariectomized mice ( Figure 3D ). These data demonstrated that ovariectomized mice supplemented with E2 exhibited impaired bacterial clearance compared to oil-treated ovariectomized mice.

Previous studies established that Nocardia grew as a facultative intracellular parasite in cultured alveolar macrophages (21, 22). We next determined the direct effects of E2 on N. farcinica IFM 10152 growth in alveolar macrophages by seeding N. farcinica IFM 10152 into phenol-free DMEM containing FBS with 50 nM E2 for 6 hours. The results showed that E2-treated cells had more N. farcinica CFUs in plates than controls ( Figure 3E ).

To further evaluate the effect of E2 on N. farcinica IFM 10152 growth in cells, A549 and RAW 264.7 cells were imaged by electron microscopy after 1 h of infection. The results showed that N. farcinica IFM 10152 adhered and proliferated better in the E2-treated group than in the control group ( Figures 4A, B ). As such, the bacterial burden of N. farcinica IFM 10152 in the E2-treated groups was higher than that in the control group at 6, 12 and 24 h postinfection ( Figure 4C ). In addition, we observed that the cytotoxicity of N. farcinica IFM 10152 was significantly higher in the E2-treated group than in the control group in both A549 and RAW264.7 cells ( Figure 4D ). To determine whether the difference in cells treated with E2 versus vehicle was ER-specific, RAW 264.7 cells were seeded and supplemented with three ER antagonists. The increased bacterial burden in E2-supplemented RAW 264.7 cells was attenuated in the presence of ICI 182,780, MPP and PHTPP ( Figure 4E ). Taken together, these findings support a specific role for nuclear ERs in the impact of E2 on promoting N. farcinica IFM 10152 infection.

To elucidate the mechanisms by which E2 promotes Nocardia survival in host cells, we examined mitogen-activated protein kinase (MAPK) activation in response to N. farcinica IFM 10152 infection. The results showed that the E2-treated group downregulated the phosphorylation levels of ERK (p-ERK), JNK (p-JNK), and p38 (p-p38) compared to the control group in both A549 and RAW 264.7 cells ( Figures 5A–D ). Then, we detected the relationship between bacterial survival in cells and the MAPK signaling pathway with MAPK inhibitors. The results showed increased bacterial survival in the SB 203580- and SP 600125-treated groups, although there was no detectable difference between the PD 98059-treated and control groups ( Figure 5E ). These results indicate that E2 promotes bacterial survival by inhibiting activation of the MAPK-mediated inflammatory response.