Skip to main content
NCBI home page
Springer logoLink to Springer
. 2024 Dec 17;33(3):1473–1489. doi: 10.1007/s10787-024-01625-6

Anti-inflammatory effect of nestorone in a lipopolysaccharide-induced acute lung injury model through regulation of the TLR-4/Myd88/NF-κB signaling pathway

Aying Ma 1,#, Jieyun Zhou 2,#, Hui Zou 3, Li Yuan 1, Ruihua Zhong 2, Yan Zhu 2,✉, Chao Gao 1,✉
PMCID: PMC11913939  PMID: 39690363

Abstract

Progesterone plays a crucial and indispensable role in regulating immunity and attenuating inflammation. Nestorone® (NES, segesterone acetate) is a steroidal progestin and a 19-norprogesterone derivative with no -CH3 group radical at the 6-position. Here, we showed that NES enhanced the viability of lipopolysaccharide (LPS)-stimulated THP-1 cell-derived macrophages, potently inhibiting both arms of the Toll-like receptor 4 (TLR-4) signaling cascade triggered by LPS, especially the TLR-4/MyD88/NF-κB pathway. In addition, NES exerted an anti-inflammatory effect by significantly decreasing the secretion of inflammatory cytokines and chemokines in type II alveolar epithelial A549 cells and THP-1 cell-derived macrophages stimulated by LPS. Furthermore, we evaluated the potential of NES pre-treatment, administered 2 h prior to LPS exposure, to mitigate acute lung injury induced by LPS, using an LPS-induced acute lung injury (ALI) mouse model. In this study, NES alleviated lung inflammation and damage by reducing leukocyte infiltration and inflammatory cytokines in the bronchoalveolar lavage fluid (BALF) and lung tissues of mice. Interestingly, our findings indicate that NES at a dosage of 1 mg/kg (91.67%) was more effective than at dosages of 0.1 mg/kg (70.83%) or 10 mg/kg (87.50%), as well as more effective than dexamethasone (DEX, 5 mg/kg, 83.34%), in extending survival in mice subjected to lethal LPS-induced injury. Additionally, this dosage was more successful in reducing acute lung inflammation and alleviating diffuse alveolar damage in the lungs of C57 mice. Our study indicates that concentration is a critical determinant of the anti-inflammatory efficacy of NES. Consequently, NES emerges as a potentially promising therapeutic agent for the treatment of pulmonary inflammatory conditions through the modulation of TLR-4 signaling pathways.

Keywords: Nestorone, A549, Reporter cell, Acute lung injury, Anti-inflammatory, Toll-like receptor

Introduction

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new coronavirus responsible for the coronavirus disease 2019 (COVID-19) which caused a worldwide pandemic. The majority of people with COVID-19 have relatively mild respiratory symptoms (Marks and Gulick 2023); however, a small percentage of COVID-19 patients develop severe pneumonia with multiple organs affected (Swets et al. 2023; Bastos et al. 2023). The fluid accumulation in COVID-19-associated pneumonia is characterized by an exaggerated immune response with hypercytokinemia leading to inflammatory cell infiltration of the lung resulting in acute lung injury (ALI). Alveolar macrophages are activated via Toll-like receptor (TLR) and nucleotide oligomerization domain (NOD)-like receptor (NLR) signaling pathways that lead to further recruitment of macrophages and circulating neutrophils. Immune cells (including macrophages and neutrophils) accumulate in the lung and release pro-inflammatory cytokines, such as interferons (IFNs), tumor necrosis factors (TNFs), interleukins (ILs), and chemokines (Anwar et al. 2024; Gorman et al. 2022; Zhang et al. 2024a; Somasundaram 2023). The lung epithelium, specifically type II alveolar cells, is damaged by these cells and their products, with resultant disruption of the alveolar-capillary interface and increased pulmonary microvascular permeability (Zhang et al. 2024b). The resulting pulmonary edema impairs gas exchange, which in some cases may lead to respiratory failure (Scholten et al. 2017). This can eventually lead to acute respiratory distress syndrome (ARDS) which is the primary contributor to mortality in COVID-19-positive patients (Vora et al. 2021; Merad et al. 2022; Somasundaram 2023). Because ARDS is not an isolated pulmonary process, the release of oxidants and proteases from the activated neutrophils and macrophages can also cause distant tissue and organ damage and dysfunction (Chew et al. 2023; Bos and Ware 2022). Discovery of novel therapeutic drugs is imperative to halt the progression of this systemic damage.

It is well-known that LPS activates the TLR-4/NF-κB and TLR-4/IRF signaling pathways, thereby initiating the transcription of downstream pro-inflammatory cytokines, including IL-6, TNF-α, KC, IL-1β, IFN-γ, and MCP-1. These cytokines serve as critical indicators of a severe inflammatory response in ALI/ARDS (Fan et al. 2018; Huecksteadt et al. 2024; Priyandoko et al. 2024). Notably, IL-6 and TNF-α play pivotal roles in a cytokine storm phenomenon, contributing significantly to the exacerbation of disease aggravation (Zhang et al. 2024b). KC and MCP-1 are major chemotactic factors which contribute to the overall inflammatory response by inducing leukocyte infiltration (Schaaf et al. 2024; Taghavi et al. 2024; Holloman et al. 2024). IFN-γ is a pleiotropic cytokine that plays a critical role in mediating an array of immune responses, particularly in promoting antiviral activity (Ng et al. 2023; Merad et al. 2022; Fan et al. 2018). Upon the elevation of these cytokines, a diverse array of inflammatory cells, such as neutrophils and monocytes, are recruited. This recruitment ultimately leads to an increase in vascular permeability, thereby exacerbating the inflammatory response characteristic of such inflammatory diseases (Swets et al. 2023; Merad et al. 2022). Treatments for a cytokine storm can significantly enhance the body’s ability to fight infectious diseases. Interestingly, the risk of severe COVID-19 is consistently lower in women than men worldwide, suggesting that female biological sex is instrumental in protection (Vijayasingham et al. 2021; Mauvais-Jarvis et al. 2020). Previous studies have demonstrated the immunomodulatory and anti-inflammatory actions of high physiological concentrations of the steroids 17β-estradiol (E2) and progesterone (P4) (Blakely 2019). It is already known that E2 and P4 favor a state of decreased innate immune inflammatory response while enhancing immune tolerance and antibody production (Blakely 2019; Lee et al. 2022). This led to a novel consideration of the biological forces that are protective in women compared to men, and to therapeutic harnessing of these factors to mitigate ALI/ARDS morbidity and mortality.

Progesterone (P4) is an endogenous steroid hormone synthesized by the corpus luteum, playing a pivotal role in the reproductive system, mammary gland, and central nervous system through its interaction with specific receptors. Recent research has elucidated additional pleiotropic effects of P4 in various tissues, highlighting its capacity to inhibit inflammation and oxidative stress in conditions such as central nervous system disorders, traumatic brain injury, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis and autoimmune diseases (Lee et al. 2022). Thus new clinical uses of P4 and its synthetic analogues have emerged. Dexamethasone (DEX) is a glucocorticoid that acts as a synthetic version of the naturally-occurring hormone cortisol (Sinha et al. 2022). It has the same anti-inflammatory effects as cortisol, namely inhibition of the release of inflammatory chemokines by immune cells (Villar et al. 2020). This has the potential to reduce inflammation in the lungs, thus improving patient prognosis by decreasing the severity of ALI/ARDS (Horby et al. 2021). Studies have shown that glucocorticoids such as DEX have significant effects on ARDS caused by viruses (Sinha et al. 2022).

Nestorone® (NES, segesterone acetate), a synthetic progestin that selectively binds to the progesterone receptor (PR) and circumvents androgenic, estrogenic, and glucocorticoid activities associated with other synthetic progestins, stands as an ideal candidate for hormone replacement therapy and contraception (Lee et al. 2022). In vivo bioassays in rats and rabbits demonstrated significant binding of NES to the glucocorticoid receptor (Acevedo-Rodriguez et al. 2018). Equally important, NES also exerts neuroprotective, neuroregenerative and injury healing effects in animals against a myriad of neurological diseases characterized by demyelination, motor neuron cell death, and inflammatory pathological pathways, holds promise as a supplementary treatment for multiple sclerosis, stroke, spinal cord injury and amyotrophic lateral sclerosis (Acevedo-Rodriguez et al. 2018; Andrabi et al. 2017; Blakely 2019; Guennoun 2020; Guennoun et al. 2015; Rossetti et al. 2016). NES 3-week treatment stimulated the recruitment of oligodendrocyte progenitor cells, the replenishment of mature oligodendrocytes, and myelin regeneration in the female mouse brain (El-Etr et al. 2021). Previous studies (Fréchou et al. 2021; Lee et al. 2022; Sitruk-Ware et al. 2024) showed that intranasal delivery of NES offers a good bioavailability and neuroprotective efficacy after experimental stroke, and have also demonstrated that progesterone receptors (PR) are essential for cerebroprotection by endogenous progesterone and by progesterone treatment. NES has no oral activity; however, when given parenterally it requires only small amounts to achieve efficacy, and is thus well suited for use in inhaler delivery systems (Lee et al. 2022; Renke et al. 2023). Inhalation therapy allows drugs to be conveyed directly into the airways and enhances the bioavailability and efficacy of NES in the target cells of the lung while also reducing potential systematic side effects.

In this study, we conducted an initial assessment of the effects of NES on THP-1 cell-derived macrophages to investigate the activation of both branches of the TLR-4 signaling pathway cascade, specifically the nuclear factor kappa B (NF-κB)/AP-1 and IRF pathways. The evaluation aimed to determine its impact on the inhibitory activity triggered by lipopolysaccharide (LPS). We also utilized type II alveolar epithelial A549 cells and macrophages derived from THP-1 cells to identify the effects of NES on pro-inflammatory cytokines, employing enzyme-linked immunosorbent assay (ELISA) for quantification. We further investigated the in vivo efficacy of NES using a classical LPS-induced ALI murine model. In addition, we explored the effects of stand-alone pre-treatment with either NES or DEX in isolation on the survival of mice after LPS stimulation, aiming to assess whether NES improves survival outcomes in ALI/ARDS. Our results collectively indicated that NES exhibited strong anti-inflammatory activity against LPS-induced inflammation both in vitro and in vivo, which will aid in the development of an effective novel hormone therapy to ameliorate the devastating inflammation in ALI/ARDS, especially when caused by viral infections such as SARS-CoV-2.

Materials and methods

Materials

Nestorone (C23H30O4; molecular weight: 370.48; abs811713), dexamethasone acetate (abs44100471), and progesterone (abs42156041) were procured from Absin Bioscience Inc. (Shanghai, China). Dimethyl sulfoxide (DMSO) was obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). For in vitro experiments, NES, DEX, and progesterone were dissolved in DMSO, with the final DMSO concentration in the culture medium being ≤ 0.1%. For in vivo experiments, NES was dissolved in phosphate-buffered saline (PBS) at concentrations of 0.01, 0.1, and 1 mg/mL. Control mice were administered PBS at a volume of 0.01 mL/g, and followed the same injection schedule.

Cell culture and drug treatment

The A549 cell line, originating from human type II alveolar epithelial cells, was procured from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China. The cells were maintained in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS), at a temperature of 37 °C under a 5% CO2 atmosphere. Prior to the administration of LPS dissolved in PBS, the cells were pretreated for 2 h with NES dissolved in PBS. Sampling was conducted 24 h after LPS treatment.

MTS assay

Cells were initially seeded at a density of 8 × 103 cells per well into 96-well plates and allowed to adhere overnight. Subsequently, a range of reagent concentrations was added to the wells for durations of 6, 12, 24, or 48 h. Following this, 50 μL of MTS was introduced to each well and incubated for approximately 3 h at a temperature of 37 °C in an environment of 5% CO2. Absorbance values at a wavelength of 490 nm were then quantified using an automated multi-well spectrophotometer (SpectraMax iD3, Molecular Devices, Sunnyvale, CA, USA).

Reporter cell culture, NF-κB/AP-1 and IRF activation analysis

THP1-Blue™ and THP1-Dual™ cell lines were obtained from InvivoGen (San Diego, CA, USA) and maintained in culture following the supplier’s guidelines and established protocols. The THP1-Blue™ cells were propagated in complete RPMI medium supplemented with Zeocin (100 μg/mL, InvivoGen) to ensure the selection of cells expressing the SEAP and NF-κB/AP-1 reporter genes (Składanowski et al. 2016). Similarly, the THP1-Dual™ cells were cultured in complete RPMI medium with the addition of selective antibiotics, specifically Zeocin (100 μg/mL, InvivoGen) and blasticidin (10 μg/mL, InvivoGen) (Pirttiniemi et al. 2023). Following selection, 104 cells per well were seeded into 96-well plates and subjected to differentiation into macrophages using 50 ng/mL phorbol-12-myristate-13-acetate (PMA). These macrophages were then exposed to LPS at a concentration of 10 ng/mL, either in the presence or absence of NES, DEX, or progesterone at varying concentrations (0.01, 0.1, 1, 10, and 100 μmol/L) for a duration of 24 h. Subsequently, the culture medium from each well was collected, and the supernatants were stored for further analysis.

To activate NF-κB/AP-1, 20 μL of supernatant from THP1-Blue™ cells were incubated with 180 μL of QUANTI-Blue solution at 37 °C for approximately 2 h in a 96-well flat-bottomed plate (Składanowski et al. 2016). Similarly, for IRF activation, 20 μL of supernatant from THP1-Dual™ cells were subjected to the same incubation process (Pirttiniemi et al. 2023). The resultant colorimetric alteration of the solution was quantitatively assessed by measuring its absorbance at 655 nm utilizing a microplate reader (SpectraMax iD3, Molecular Devices, Sunnyvale, CA, USA).

Animals, LPS stimulation and drug administration

Male C57BL/6 mice, aged 7 weeks and weighing 18 ± 2 g, were procured from SLAC Laboratory Animal Co. Ltd. in Shanghai, China, and were subsequently maitained under specific-pathogen-free conditions. The experimental protocol was granted approval by the Shanghai Institute for Biomedical and Pharmaceutical Technologies Review Board (Permit Number: 2022–44. Shanghai, China). To ensure adaptation for use in the study, all mice were housed under standard conditions for 1 week. In the in vivo experiments, the mice were randomly allocated into six groups (n = 8 per group): the normal control group (Control), the LPS group (10 mg/kg), the DEX group (5 mg/kg), and three groups receiving LPS (10 mg/kg) in combination with NES at varying dosages (0.1 mg/kg, 1 mg/kg or 10 mg/kg). After a 2-h period, mice were treated with LPS at a dose of 5 mg/kg, which was diluted to a concentration of 0.5 mg/mL. A separate group of mice received an equivalent volume of PBS, serving as the normal control group. Twenty-four hours after LPS stimulation, the mice were anesthetized and sacrificed to collect bronchoalveolar lavage fluid (BALF), blood, and lung tissue for the purpose of analyzing lung inflammation and injury. Every possible measure was taken to ensure the optimal welfare conditions of the mice before, during, and after each experiment, and their general condition was monitored on a daily basis. Additionally, the weight of the mice was measured every other day. All experiments were conducted with an effort to minimize suffering and the number of animals used.

Bronchoalveolar lavage fluid (BALF) analysis

BALF was collected by the slow infusion and careful extraction of 0.8 mL pre-cooled PBS three times through the trachea. The volume of retrieved fluid was > 60% of the injected volume. The total and differential counts of cells, including neutrophils, macrophages, and lymphocytes, were conducted using light microscopy. The BALF total protein content was measured using a Bicinchoninic acid (BCA)-protein quantification assay kit (Beyotime, Shanghai, China). The BALF supernatants were stored in a − 80 °C freezer for further cytokine analysis.

Lung wet/dry weight (W/D) ratio

The right lung was isolated and the fluid covering the excised lung was delicately removed, then the weight of the lung was measured as the wet lung weight. Subsequently, the lung tissue was heated in an 80 °C oven for 72 h, then weighed using an electronic scale, and the weight was recorded as the dry lung weight. Finally, the ratio of the dry lung weight to the wet lung weight was calculated to determine the lung wet/dry (W/D) weight ratio, which serves as an indicator of lung edema index, as previously described (Zhu et al. 2023).

Lung pathology

One portion of the upper left lung tissue was preserved in a fresh solution of 4% formaldehyde, followed by dehydration and embedding in paraffin. Subsequently, tissue sections with a thickness of 5 μm were prepared and subjected to staining using hematoxylin and eosin (H&E). For each mouse, three sections per lung sample were evaluated, with ten areas assessed per section. Lung histopathology was examined under a light microscope, following the methodology described previously (Fan et al. 2018).

Cytokine analysis

The levels of cytokines in BALF and serum were quantified using a Bio-plex pro mouse cytokine 23-plex panel kit (M60009RDPD; Bio-Rad, Hercules, CA, USA). The cytokines measured were IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-17, Eotaxin, granulocyte colony-stimulating factors (G-CSF), granulocyte–macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), keratinocyte-derived chemokine (KC), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), macrophage inflammatory protein-1β (MIP-1β), regulated upon activation normal T cell expressed and secreted (RANTES) and tumor necrosis factor-α (TNF-α). Multiplex assay was conducted utilizing the proprietary software of the Bio-Plex 200 system (Bio-Rad), following the manufacturer’s instructions.

Survival curve

A total of 24 mice (eight mice per group, repeated three times) were subjected to intranasal administration of a lethal dose of LPS at a concentration of 20 mg/kg to induce severe ALI. The survival rate and body weight of the mice in each group were meticulously recorded on a daily basis. Mice that survived the initial LPS challenge were subsequently subjected to an increased LPS dose of 25 mg/kg on Day 10. The survival of the mice was systematically monitored each day until the conclusion of the experiment on Day 21.

Statistical analysis

All data are presented as mean values of at least three independently-performed experiments ± standard error of the mean (SEM). Statistical significance was assessed by performing one-way analysis-of-variance (ANOVA) followed by the Bonferroni post-hoc test or Tukey’s post-hoc test, as applicable. Analyses were performed using GraphPad Prism 8.0 software (GraphPad Software Inc., San Diego, CA, USA). For all tests, #p < 0.05, ##p < 0.01, and ###p < 0.001 compared to control, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 compared to LPS were considered significant.

Results

NES protects cell viability against damage induced by LPS and increases anti-inflammatory activity in THP1-Blue™ cells.

To mimic LPS-induced lung injury, the effect of LPS at various concentrations (ranging from 0.001 ng/mL to 1 µg/mL) on the viability of THP1-Blue™ (vendor and location) positive cells was determined by MTS assay. After 48 h, the viability was significantly decreased by LPS stimulation (0.1–1000 ng/mL), whereas stimulation for 6, 12, or 24 h did not affect cell viability (p > 0.05) except at concentrations of 100 and 1000 ng/mL (Figs. 1A–D). These results suggested that LPS suppressed the growth of THP1-Blue™ cells in a dose- and time-dependent manner.

Fig. 1.

Fig. 1

Open in a new tab

LPS and progestogen as modulators of TLR-4/NF-κB/AP-1 signaling. A–D The viability of THP1-Blue™ cells stimulated by LPS at different concentrations (0.001–1000 ng/mL) for 6, 12, 24, or 48 h. E–H NF-κB/AP-1 activation in response to different doses of LPS. Effects of a concentration gradient of DEX, P4 and NES on I the viability of THP1-Blue™ cells, J the viability of THP1-Blue™ cells after LPS (10 ng/mL) stimulation, and K the NF-kB/AP-1 activation after 24 h. The results are presented as the mean ± SD from three separate experiments with triplet repeat represented by each data point. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. vehicle, and *p < 0.05, **p < 0.01, ***p < 0.001 vs. LPS

To confirm that LPS had an inhibitory effect on TLR-4/NF-κB signaling, we employed a reporter cell system to monitor NF-κB/AP-1 activation as described previously (Yang et al. 2015). As shown in Fig. 1B, LPS (10 and 100 ng/mL) exhibited the greatest effect on increasing NF-κB activation after 24 h compared to the other times and concentrations tested. Based on these results, we chose 10 ng/mL as the optimal LPS concentration for stimulation and 24 h as the optimal time for further experiments.

NES (0.001–10 µmol/L) had no significant effect on cell viability at 24 h when used independently (p < 0.05, Fig. 1I). In addition, treatment with DEX, serving as a positive control in this study, caused significant inhibition of the proliferation of THP-1 cell-derived macrophages in a dose-dependent manner (10, 100, and 1000 µmol/L). Simultaneously, the MTS cell viability assay demonstrated that the reduction in viability following a 24-h stimulation with LPS (10 ng/mL) was ameliorated by pre-treatment with NES or P4 at concentrations of 1, 10, and 100 µmol/L, with statistical significance (p < 0.05, Fig. 1J). These data suggested that NES had a protective effect on THP1-Blue™ cells against injury induced by LPS (10 ng/mL) at 24 h.

TLR-4 initiates signaling through both MyD88-dependent and MyD88-independent (TRIF-dependent) pathways. Initially, we assessed the impact of NES on the MyD88-dependent pathways of NF-κB activation induced by LPS at a concentration of 10 ng/mL (Fig. 1K). Two hours before LPS stimulation, the reporter cells were treated with DEX, P4, or NES (0.01–100 µmol/L). We found that NES (at concentrations of 1–100 µmol/L) significantly reduced the LPS-induced NF-κB/AP-1 activation. This inhibitory effect was not attributable to cytotoxicity (Figs. 1I and J).

NES protects the viability of THP1-Dual™ positive cells against damage induced by LPS and enhances anti-inflammatory activity in vitro

The MyD88-independent pathway signals through the TIR domain containing adapter inducing interferon-beta (TRIF) leading to activation of interferon regulatory factor 3 (IRF-3) together with late-phase activation of NF-κB driving secretion of IFN-β and other cytokines.

We evaluated the effect of NES on the MyD88-independent pathways of IRF triggered by LPS, by measuring cell viability and IRF activation in THP1-Dual™ cells at different time-points. MTS assay showed that when stimulated for 6, 12, or 24 h, cell death caused by LPS was similar at different concentrations (0.001 to 1000 ng/mL, viability (% of vehicle) > 95%, p > 0.05). However, after stimulating for 48 h, LPS (10, 100, and 1000 ng/mL) caused a significant increase in cell death compared to vehicle (Figs. 2A–D). LPS did not cause cellular toxicity at the experimental concentrations of 0.001–1000 ng/mL after 6, 12, or 24 h of treatment (Figs. 2A–D).

Fig. 2.

Fig. 2

Open in a new tab

NES as a modulator of TLR-4/IRF signaling. A–D The viability of THP-1 cell-derived macrophages after stimulation with 0.001–1000 ng/mL LPS for 6, 12, 24, or 48 h. E–H The effect of different doses of LPS on IRF activation at 6, 12, 24, and 48 h. I–K The effect of DEX, P4, and NES on THP1-Dual™ cells with or without LPS (10 ng/mL) stimulation for 24 h: I viability of THP-1 cell-derived macrophages was assessed at 24 h after treatment with different concentrations (0.0001–1000 µmol/L) of DEX, P4, or NES. J Viability of THP-1 cell-derived macrophages treated with the indicated concentrations (0.01–100 µmol/L) of DEX, P4, or NES for 24 h after 2 h of LPS (10 ng/mL) stimulation. K Effects of DEX, P4, and NES (0.01–100 µmol/L) on LPS (10 ng/mL)-induced IRF activation. Values shown are from triplicate wells per data point obtained in three separate cultures. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. vehicle, and *p < 0.05, **p < 0.01, ***p < 0.001 vs. LPS

Next, we employed a reporter cell system to monitor activation of IRF in the TLR-4 pathway at all analyzed time-points. As shown in Fig. 2G, LPS (10 ng/mL) stimulation for 24 h induced the greatest IRF activation compared to other concentrations or time-points. Taking these results together with the cell viability results, we chose LPS (10 ng/mL) stimulation for 24 h as the optimal treatment for further experiments.

The MTS cell viability assay showed that NES (0.0001–100 µmol/L) had no significant effect on cell viability at 24 h when used alone (Fig. 2C), while at a concentration of 1000 µmol/L, cell death was significantly enhanced (p < 0.001 compared to vehicle). These data suggested that a high concentration of NES (1000 µmol/L) was severely toxic to cells. Next, we evaluated whether NES had a protective effect against LPS-induced death of THP1-Dual™ positive cells. As expected pretreatment with NES (0.1–100 µmol/L) increased the viability of THP1-Dual™ cells after stimulation with LPS (10 ng/mL) at 24 h, while 0.01 µmol/L NES had no effect (p > 0.05, Fig. 2G).

To evaluate the inhibitory effect of NES on LPS-induced THP1-Dual™ positive cells, we determined IRF activation, which is activated by the MyD88-independent pathway. Figures 2E–H show the LPS dose responsiveness of IRF activation. Two hours before LPS stimulation, the THP1-Dual™ reporter cells were treated with NES (0.01–100 µmol/L). We found that NES at these concentrations significantly reduced LPS-induced IRF activation. The inhibitory effect was not due to cellular toxicity (Fig. 1A). These observations substantiated that NES exhibited significant inhibitory effects on both signaling pathways downstream of TLR-4. Among them, NES strongly inhibited NF-κB activation, while it had a weaker inhibitory effect on IRF activation.

Effect of NES on the inhibition of TLR-4 signaling and associated inflammatory response

The anti-inflammatory activity of NES was next verified in A549 type II alveolar epithelial cells, and THP1-Dual™ cell-derived macrophages. The levels of pro-inflammatory cytokines were analyzed after treatment with LPS alone or together with DEX, P4, or NES for 24 h (Fig. 3). Through assessment of the pro-inflammatory cytokine levels, we found that all three drugs were able to reduce LPS-induced KC (keratinocyte-derived chemokine, or chemokine (C-X-C motif) ligand 1, CXCL-1), IL-6, tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein-1 (MCP-1) production, where NES exhibited a much stronger inhibitory effect on IL-6, KC, TNF-α, and MCP-1 secretion than DEX or P4 (Fig. 3). There was no statistically significant difference observed in the inhibitory activity between DEX and P4. Although these results suggested that concentration was important for the inhibitory activity of the drugs, increasing the dose (100 µmol/L) did not help further enhance this inhibitory effect. As a result, the in vitro evaluation revealed that the inhibitory activity of NES on LPS-induced NF-κB/AP-1 and IRF activation, as well as IL-6, KC, TNF-α, and MCP-1 secretion, was significantly higher than that of DEX or P4 at the same concentration (1, 10, or 100 µmol/L).

Fig. 3.

Fig. 3

Open in a new tab

The anti-inflammatory effect of NES in type II alveolar epithelial A549 cells and THP1-Dual™ cell-derived macrophages. The levels of pro-inflammatory cytokines IL-6, KC, TNF-α, and MCP-1 in A549 cells (A–D) or THP-1 cell-derived macrophages (E–H) stimulated with LPS (10 ng/mL) for 24 h in the presence and absence of DEX, P4 or NES treatment. These results were typical of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. LPS

NES shows potent therapeutic activity in an LPS-induced ALI mouse model

Since P4 exhibited strong ability to ameliorate inflammation and eliminate excessive oxidative stress(Kolatorova et al. 2022; Taraborrelli 2015), we anticipated that NES as one of the progestogens, which has a high affinity for progesterone receptors, might effectively reduce LPS-triggered lung injury in vivo. Herein, we further validated this hypothesis in an LPS-induced ALI mouse model. To exclude the influence of progesterone, we chose male mice as the animal model for this study. We adopted a classical mouse model of ALI caused by a 10 mg/kg dose of LPS administered intranasally (i.n.) to induce acute injury within lung tissue. NES at doses of 0.1, 1, and 10 mg/kg, DEX at a dose of 5 mg/kg, or PBS as the control were administered via intratracheal (i.t.) instillation 2 h prior to LPS stimulation. Subsequent analyses were performed 24 h after the LPS challenge (Fig. 4A).

Fig. 4.

Fig. 4

Open in a new tab

The inhibitory activity of NES in an ALI mouse model induced by LPS. A Schematic diagram of the animal experimental protocol. Mice were pretreated with DEX (5 mg/kg) or NES (0.1, 1, and 10 mg/kg) 2 h before LPS challenge, then sacrificed 24 h after stimulation with LPS (10 mg/kg), and biological measurements including differential BALF cell counting and epithelial permeability were performed for further analysis. C–F BALF in each group was collected for evaluation of C the total number of cells, as well as numbers of D neutrophils, E macrophages, F lymphocytes, G total protein content, and H lung W/D ratio. n = 8/group, *p < 0.05, **p < 0.01, ***p < 0.001 relative to the LPS group

First, we quantified inflammatory cell infiltration and secretion of certain cytokines within BALF to evaluate the airway inflammatory response. After 24 h of LPS infusion, we found that NES (0.1, 1, and 10 mg/kg) and DEX (5 mg/kg) significantly reduced the total numbers of inflammatory cells, neutrophils and macrophages, but slightly reduced the lymphocytes in BALF of ALI mice compared to the control group (Figs. 4B–D). These results suggested that NES pretreatment promoted macrophage infiltration while suppressing neutrophil recruitment.

As a key element in the pathogenesis of ALI, we then assessed the permeability of the alveolocapillary membrane by measuring total proteins in BALF and the wet-to-dry (W/D) weight ratio of the right lung. Treatment with progestogen effectively inhibited the LPS-induced increase in the content of total proteins in BALF (Fig. 4F), especially at a high concentration of NES (10 mg/kg). As shown in Figs. 4H and I, NES pretreatment significantly decreased the W/D ratio within lungs of ALI mice. These results demonstrated that progestogen was potent in ameliorating the injury caused to the alveolocapillary membrane in the early stage of ALI. NES was more potent than DEX in the management of lung inflammation. Taken together, these data suggest that progestogen may regulate inflammatory responses and mitigate diffuse alveolar damage in the context of ALI.

Anti-inflammatory effects of NES in lung tissue of LPS-treated ALI mice

We further evaluated the histopathological characteristics of lung tissue following an LPS challenge, both with and without pretreatment using NES and DEX. Progestogen was able to reduce total cell counts and neutrophil counts in the BALF, and markedly ameliorated pathological changes of lung tissues. The severity of lung injury and inflammation was manifested by five independent histological indexes: neutrophils in the alveolar space, neutrophils in the interstitial space, formation of hyaline membranes, proteinaceous debris filling the airspaces, and alveolar septal thickening (Figs. 5A–E), and the lung injury score was obtained based on these features. It was found that the average injury score of the NES-treated group (1 mg/kg) was significantly lower than that of the groups treated with DEX (0.1 mg/kg) or NES (10 mg/kg) (Fig. 5F). Specifically, NES at 1 mg/kg resulted in fewer interstitial neutrophils and less alveolar septal thickening than NES at 10 mg/kg (Figs. 5G and H). These data suggested that NES (1 mg/kg) was superior to either DEX (5 mg/kg) or NES (0.1 and 10 mg/kg) in inhibiting inflammatory responses and reducing diffuse alveolar damage in lungs with ALI.

Fig. 5.

Fig. 5

Open in a new tab

The protective effect of NES on lung damage in LPS-induced ALI mice. A–F Representative histological images of lung sections from mice, stained with H&E for histological evaluation, are presented at magnifications of ×10 and ×40: A sham control group (administered PBS only), B LPS + PBS group, C LPS + DEX group (5 mg/kg), D LPS + NES group (0.1 mg/kg), E LPS + NES group (1 mg/kg), and F LPS + NES group (10 mg/kg). Arrows (red) denote representative regions, highlighting features such as alveolar septal thickening, the presence of neutrophils within the alveolar and interstitial spaces, hyaline membranes, and proteinaceous debris. The scale bar in the left panel = 200 μm while that in the right panel = 50 μm. Lung damage was assessed by examination of five pathophysiological features to obtain the total injury score (G–K). G NES markedly decreased the neutrophil count within the alveolar space. H Higher doses of NES (1 and 10 mg/kg) markedly decreased the neutrophil count within the interstitial spaces, I diminished the formation of hyaline membranes and J proteinaceous debris, and K attenuated alveolar septal thickening. n = 8 per group, ns not significant

Anti-inflammatory effects of NES in blood and BALF of mice with LPS-induced ALI

Next, an analysis to evaluate the global impacts of NES on cytokine production was performed in both BALF and serum of mice with LPS-induced ALI using a multiplex Luminex assay (vendor and address) (Figs. 6 and 7), to investigate the underlying mechanisms of the protective effects of NES. As shown in Figs. 6 and 7, we observed significant alterations in the expression of various pro-inflammatory cytokines and chemokines across the control group, the LPS group, the DEX-treated group (5 mg/kg), and the NES-treated group (0.1, 1, and 10 mg/kg). In comparison to the LPS challenge group, pre-treatment with NES, particularly at a dose of 1 mg/kg, or DEX, resulted in a significant reduction in the levels of pro-inflammatory cytokines and chemokines, including TNF-α, IL-6, IFN-γ, MCP-1, IL-1α, IL-1β, and KC. Additionally, there was a tendency for increased levels of IL-10, IL-17α, and granulocyte-colony stimulating factor (G-CSF) in both BALF and serum. Of note, the levels of G-CSF, IL-1β, and IFN-γ have been documented to have associations with neutrophils. Collectively, these findings indicate that, beyond its TLR-4 inhibitory properties, the anti-inflammatory effects of NES pretreatment may also play a role in neutrophil-mediated inflammatory processes in ALI/ARDS.

Fig. 6.

Fig. 6

Open in a new tab

The effect of NES on cytokine production in the BALF of mice with LPS-induced ALI. A Heat map showing cytokine profiles of the BALF under LPS challenge (10 mg/kg, 24 h) with/without NES pre-treatment (0.1, 1, and 10 mg/kg, 2 h before LPS stimulation) using a multiplexed cytokine assay. Different color codes represent the mean fold changes normalized to the PBS group, n = 6 (six were randomly selected from the BALF of eight mice in each group). B–G The levels of selected cytokines in the BALF: B IL-6, C G-CSF, D IFN-γ, E KC, F MCP-1, and G TNF-α. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. LPS group. ns not significant

Fig. 7.

Fig. 7

Open in a new tab

The effect of NES on cytokine production in the serum of mice with LPS-induced ALI. Multiplexed cytokine array profile of serum (A) from mice stimulated with LPS with or without NES (0.1, 1, and 10 mg/kg) pre-treatment, different color codes represent the mean fold changes normalized to the PBS group. B–G The levels of selected cytokines in the serum: B IL-6, C G-CSF, D IFN-γ, E KC, F MCP-1, and G TNF-α; n = 6. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. LPS group. ns not significant

Survival rate

DEX is one of the corticosteroid drugs that have shown a reduction in mortality of COVID-19 (Horby et al. 2021). In hospitalized patients with COVID-19 pneumonia without additional oxygen requirements and at risk of progressing to severe disease, DEX treatments might lead to a decrease in the development of ARDS and thereby reduce death. Considering that NES shows significant binding to glucocorticoid receptors (GR) in vivo (Lee et al. 2022), we investigated whether NES administration could affect the short-term and long-term survival rates in mice subjected to lethal doses of LPS (20 mg/kg) (Fig. 6A). The administration of the drugs, DEX and NES, occurred 2 h prior to the initial and subsequent LPS challenges, followed by administration on alternate days. Daily observations and recordings were made regarding changes in body weight and the survival status of the mice. As shown in Fig. 8C, the survival rates for the groups pretreated with NES at doses of 0.1, 1, and 10 mg/kg were 70.83% (17/24), 91.67% (22/24), and 87.50% (21/24), respectively, while in the DEX (5 mg/kg) group it was 83.34% (20/24), all of which were significantly higher survival rates than that in the LPS group at 66.67% (16/24, p < 0.001). Interestingly, it was observed that NES-treated mice that survived the initial LPS shock subsequently developed a sustained tolerance to a second lethal LPS challenge (25 mg/kg). This phenomenon occurred with the higher dose of NES (1 and 10 mg/kg) group, but not with the lower dose of NES (0.1 mg/kg, 50.00%, 12/24) or DEX (5 mg/kg, 62.50%, 15/24); the survival rate in the LPS group was only 37.50% (9/24, p < 0.001 vs. the other groups). The body weight and mental state of the PBS control mice were always good.

Fig. 8.

Fig. 8

Open in a new tab

Effect of NES or DEX on the short-term and long-term survival of LPS-treated ALI mice (A–D): A A scheme of the prophylactic treatments of NES (0.1, 1, and 10 mg/kg) or DEX (5 mg/kg) in an LPS-induced ALI model. The mice received an intratracheal pretreatment with NES, DEX or PBS 2 h prior to the initial LPS challenge (20 mg/kg) on Day 0. Subsequently, the surviving mice were administered a second LPS dose (25 mg/kg) on Day 10. The remainder of the time (days 2–8 and days 12–20) treatments were administered at a fixed time (9:00 am) every other day; B the mouse body weights before and during the experiments; C the effects of NES and DEX on the short-term survival of mice with severe ALI; D the long-term survival of mice with severe ALI after secondary LPS (25 mg/kg) challenge with prophylactic treatments with NES or DEX. n = 8 per group, repeated three times. E and F The augmented tolerance to LPS, elicited by DEX and NES in reporter THP-1 cell-derived macrophages, is characterized by the suppression of E NF-κB activation and F IRF activation following a second LPS exposure 24 h subsequent to the initial LPS stimulation. n = 3, repeated three times. Values shown were obtained in three separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. double LPS stimulus

In addition, the weight loss observed in mice treated with LPS (20 mg/kg) appeared to be exacerbated (Fig. 6D). These findings suggest that NES (1 mg/kg) not only enhances short-term survival rates but also induces a long-term protective effect against LPS challenge, while mitigating the side effect of weight loss. This indicates the potential clinical applicability of NES in the treatment of ALI/ARDS.

Such unexpected finding is consistent with previous studies (Dorneles et al. 2023) on LPS tolerance, suggesting that NES also has the ability to promote LPS tolerance. Subsequently, we assessed the effect of LPS tolerance by utilizing THP-1-derived macrophages equipped with reporting systems for NF-κB/AP-1 or IRF activation. In both the presence and absence of DEX or NES, the cells were initially stimulated with LPS at a concentration of 10 ng/mL. After a 24-h incubation period, the cells were subjected to a subsequent exposure to LPS at the same concentration. Twenty-four hours later, the reporter enzyme reaction was measured by colorimetry to quantify the activation of NF-κB/AP-1 or IRF (Figs. 8E and F). A previous study (Mauvais-Jarvis et al. 2020) found that cells showed tolerance to a second LPS stimulation after LPS-pretreatment, as evidenced by a significantly reduced activation of NF-κB/AP-1 compared to a one-time LPS exposure (Fig. 8E). Without intervention, the tolerance effect of LPS 24 h after the second stimulation was more pronounced with regard to NF than IRF. For IRF activation, DEX and NES had a slight effect on LPS tolerance, but the results were not significantly different. These results indicated that the LPS tolerance effect on IRF activation basically disappeared after 24 h of rest. Interestingly, only larger dose of NES (10 μmol/L and 100 μmol/L) showed an enhanced LPS tolerance effect in both arms of the signaling pathways, while DEX could not no matter which arm of the two signaling pathways was evaluated. This observation suggested that the NES concentration was important to induce stronger LPS tolerance in vitro, supporting our in vivo findings (Figs. 8C and D).

Discussion

NES as a synthetic corticosteroid progestogen, a potent TLR-4 inhibitor for treating ALI/ARDS

Innate immune cells, particularly alveolar macrophages and rapidly recruited circulating neutrophils (Fan et al. 2018), play a pivotal role in the pathogenesis of ALI and ARDS. During the initial phase of ALI/ARDS, lung tissue-resident macrophages, referred to as alveolar macrophages, are promptly activated upon detection of microbial products and danger-associated molecular patterns released as a result of lung infections or tissue damage (Priyandoko et al. 2024; Bos and Ware 2022). These activated macrophages further drive the acute inflammatory responses by releasing cytotoxic substances, recruiting circulating neutrophils and monocytes to the lungs, and interacting with other cells (lymphocytes, epithelial and endothelial cells) to boost the inflammatory responses (Schaaf et al. 2024; Chernov et al. 2024). These inflammatory processes are critical for combating invading pathogens; however, unrestrained overwhelming inflammation eventually leads to lung damage and pulmonary edema (Ding et al. 2023). Consequently, modulation of the TLR-4 signaling pathway, a principal mediator of the inflammatory cascade during the initial phase of lung injury, may provide a promising avenue for the treatment of ALI/ARDS.

Current immunomodulatory interventions to control TLR-mediated pathological inflammation predominantly depend on the use of systemic immunosuppressive agents, notably corticosteroids such as DEX, which is recognized as one of the most potent anti-inflammatory pharmaceuticals currently available. However, this approach may compromise the host’s immune defense against infections (Sinha et al. 2022). Furthermore, the prolonged administration of DEX is associated with substantial adverse effects across multiple organ systems. In addition long-term use of DEX is accompanied by significant side-effects in almost every organ (Theoharides and Conti 2020). Therefore, DEX should only be used in infection cases if the benefits outweigh the risks. Moreover, DEX would block macrophages from clearing secondary, nosocomial, infections (Horby et al. 2021). Hence, DEX may be useful for short-term treatment in severe, intubated, COVID-19 patients, but could be outright dangerous during recovery because the virus will not only persist, but the body will be prevented from generating protective antibodies (Theoharides and Conti 2020; Villar et al. 2020; Merad et al. 2022). NES is a synthetic progestogen, a corticosteroid sex hormone, which has been used for long-term contraception and has shown favorable tolerability and minimal side effects. Subdermal NES implants effectively suppress ovulation for a duration of two years. Additionally, when combined with estrogen in a vaginal contraceptive system, NES has received approval from the the U.S. Food and Drug Administration (FDA). The pharmacological profile of NES aligns well with the safety and efficacy outcomes observed in clinical trials conducted to date.

In this study, through screening progestin has identified a new anti-inflammation reagents of NES. We have shown that NES effectively inhibits TLR-4 signaling-mediated immune/inflammatory responses in a highly-tunable manner in macrophages (Figs. 1 and 2). NES also showed protective and anti-inflammatory effects in in vitro cell culture systems, including THP-1 cell-derived macrophages and type II alveolar epithelial A549 cells (Fig. 3). NES pretreatment via intratracheal instillation alleviated LPS-induced lung inflammation and injury in an in vivo animal model of LPS-induced ALI (Fig. 4). NES obviously reduced the alveolar wall thickening, hemorrhage, and inflammatory cell infiltration within the interstitial lung tissue and reversed the increase of white blood cells (WBCs) in BALF caused by LPS (Fig. 5). In particular, NES strongly inhibited neutrophil recruitment and chemokine production (IL-6, IFN-γ, KC, MCP-1, and TNF-α) in the lung (Figs. 6 and 7). These chemokines are critical inflammatory mediators involved in the initiation and progression of ALI. By elevating the concentration to 1 mg/kg or 10 mg/kg, NES markedly enhanced both short-term survival rates (1 mg/kg: 91.67% [22/24]; 10 mg/kg: 87.50% [21/24]) and long-term survival rates (1 mg/kg: 87.50% [21/24]; 10 mg/kg: 79.17% [19/24]) in mice subjected to lethal doses of LPS (Fig. 8). NES also facilitated the maintenance of a normal diet and stable mental state in ALI mice, all of which may intained consistent body weights. This may have contributed to their enhanced long-term survival (Fig. 8). Therefore, combined with clinical trials (Renke et al. 2023) supporting the utility of NES as a contraceptive, they demonstrate the safety profile of NES under different delivery platforms in humans, guiding a lab-to-clinic pathway for advancing its use as a novel and promising treatment for inflammatory diseases with complex pathophysiology, such as ALI/ARDS.

Possible mechanism of modulation of TLR-4 signaling in vitro and in vivo by NES

In the present study, the inhibitory effects of DEX and NES were most prominent on the MyD88-dependent NF-κB signaling pathway within the TLR-4 signaling cascade. A more pronounced inhibition of NF-κB activation was observed in reporter cell assays (Figs. 1 and 2). This trend was also reflected in cytokine measurements (Fig. 3), whereas the MyD88-independent IRF signaling pathway exhibited a reduced response to DEX or NES treatment. NES significantly downregulated the contents of IL-6, TNF-α, MCP-1, IL-1β, KC, and IFN-γ in both BALF and serum of mice with ALI induced by LPS, as depicted in Figs. 6 and 7. These findings indicate that NES plays an important role in attenuating the expression of inflammation-related cytokines. Among the three different dosages tested, NES at 1 mg/kg demonstrated the most pronounced efficacy in reversing the LPS-induced upregulation of IL-6, TNF-α, MCP-1, IL-1β, KC, and IFN-γ. The most pronounced anti-inflammatory effects were observed in the NES middle dose group (1 mg/kg), potentially attributable to competitive binding of progesterone receptors. Notably, despite the high dose being ten times greater than the middle dose, it did not exhibit any significant adverse effects. This study demonstrated that NES potently inhibited TLR-4 signaling by mediating NF-κB and IRF-3 activation, thereby regulating inflammatory responses during ALI. Considering the complicated feedback regulation of hormones and the complexity of pathogenesis of ALI/ARDS, further investigation is needed to elucidate the mechanisms underlying the therapeutic effects of NES. Particular attention should be paid to its interaction with the progesterone receptor and the precise mechanisms of its action.

The accumulation and activation of neutrophils within tissues can exert a detrimental effect, as evidenced in various inflammatory diseases, including rheumatoid arthritis, ARDS, and COPD (Guennoun 2020). Recent advancements in elucidating the role of chemokines have significantly enhanced our understanding of neutrophil trafficking during inflammatory processes. Neutrophil activity is predominantly regulated by G-CSF (Chen et al. 2024). Of note, this study demonstrated that NES significantly elevated the levels of G-CSF, which has been previously reported to possess anti-inflammatory properties, in both the BALF and serum of mice with LPS-induced ALI. Therefore, it is crucial to regulate neutrophil levels to effectively manage infections and mitigate their potential deleterious effects on tissues (Bos and Ware 2022). NES may play an important role in vivo in modulating neutrophil survival at sites of inflammation.

Furthermore, the pathological assessment conducted in this study revealed significant infiltration and accumulation of white blood cells, particularly neutrophils, within both the interstitial and alveolar spaces (Fig. 5). These cells serve as critical inflammatory mediators in the initiation and progression of LPS-induced ALI in a mouse model. Notably, this infiltration was attenuated by the administration of NES. Additionally, NES demonstrated efficacy in decreasing the permeability of the alveolocapillary membrane, which is typically compromised by the inflammatory mediators associated with LPS exposure (Fig. 4). Collectively, these data provide new evidence that NES play important but specific roles in regulation of the neutrophils response following LPS stimulation.

NES-enhanced LPS tolerance and beneficial effects for COVID-19

Diffuse alveolar damage with hyaline membranes, hallmarks of ARDS, have been found on pulmonary histological examination of patients with COVID-19 (Sinha et al. 2022). Furthermore, an uncontrolled inflammatory state is frequent with COVID-19 and may contribute to multi-organ failure in these patients (Swets et al. 2023). Many studies have found that corticosteroids play an important role in controlling this exacerbated response (McEvoy et al. 2024; Horby et al. 2021). In this study, NES exerts more significant inhibition than DEX in lung injury. NES may, to some extent, improve and/or replace the glucocorticoid (DEX) sensitivity in the treatment of pulmonary inflammatory diseases. Further research is necessary to elucidate the mechanisms through which the reduction of inflammatory cells by NES in pulmonary histology influences the regulation of inflammatory factor production.

LPS tolerance is a state of repressed responsiveness to repeated LPS stimulation, which may serve as a protective mechanism to avoid overwhelming immune responses due to continuous and repeated endotoxin exposure (Dorneles et al. 2023). The results of the present study demonstrated the protective effects of NES on ALI. Administration of NES at a concentration of 1 mg/kg, significantly improved both short-term (91.67%, 22/24) and long-term (87.5%, 21/24) survival rates in mice subjected to lethal doses of LPS. Additionally, NES changed LPS tolerance in reporter cells. These characteristics and therapeutic interventions establish NES as a promising candidate for further development as a novel therapeutic agent for the treatment of various inflammatory conditions, including ALI/ARDS, particularly those associated with viral infections such as SARS-CoV-2.

Many studies using the single-cell atlas have defined COVID-19-enriched neutrophil states and molecular mechanisms of DEX action, enabling development of targeted immunotherapies for severe COVID-19 ALI/ARDS (Sinha et al. 2022; Villar et al. 2020; Weeratunga et al. 2023). One study found that male patients had higher proportions of IFN (active) neutrophils and preferential steroid-induced immature neutrophil expansion, potentially affecting severe COVID-19-related ALI/ARDS outcomes (Dorneles et al. 2023). The opposite is true for females, which may be related to women’s innate progesterone, which supports our observations in the current study. This suggests that NES has the potential to function as a anti-inflammatory therapeutic agent for the treatment of acute and chronic inflammatory disorders. And the anti-inflammatory potential of NES should be investigated in other ALI models and in all ages animals (Fig. 9).

Fig. 9.

Fig. 9

Open in a new tab

Schematic diagram showing that LPS, as a ligand of TLR-4, can activate the TLR-4/MyD88/NF-κB and TLR-4/IRF-3 pathways and then upregulate the level of IL-6, TNF-α, MCP-1, IFN-γ, IL-1β, and KC, promoting lung damage. NES reverses this effect of LPS and protects against LPS-induced acute lung injury (ALI) due to its anti-inflammatory and immunomodulatory actions, which are possibly mediated by modulation of the TLR-4/NF-κB signaling pathways

Conclusion

In summary, NES is a pharmacological agent with multiple targets that operates through various pathways, notably exerting significant anti-inflammatory effects. Consequently, it is expected to be a preventive and/or therapeutic agent for ALI/ARDS. Our study identified the TLR-4/NF-κB signaling pathway as a critical mechanism through which NES mediates its anti-inflammatory effects in ALI/ARDS. Future research will focus on elucidating the mechanisms by which NES activates the TLR-4 pathway and the associated LPS tolerance in the context of ALI/ARDS.

Acknowledgements

We thank professor Aiwu Ke for the constructive discussions for the revisions of this manuscript. This study was funded in part through the National Key Research and Development Program of China (No. 2023YFC3402700), the Shanghai Municipal Natural Science Foundation (No. 22ZR1411200) and the Youth Foundation of Zhongshan Hospital Fudan University (No. 2023ZSQN26).

Author contributions

A.M., J.Z., Y.Z., and C.G. designed the experiments. A.M., J.Z., and H.Z. acquired and analyzed the data. A.M., J.Z., H.Z., L.Y., and R.Z. conducted the experiments. A.M., J.Z., and C.G. wrote the original manuscript draft. A.M., Y.Z., and C.G. reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

Data availability

Not applicable.

Declarations

Conflict of interest

The authors declare no conflict of interest.

Institutional review board statement

The animal study protocol was approved by the Ethics Committee for the Use of Animals of Shanghai Institute for Biomedical and Pharmaceutical Technologies (protocol code 2022–44, Shanghai, China).

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Aying Ma and Jieyun Zhou contributed to this work equally.

Contributor Information

Yan Zhu, Email: zhuyan@sippr.org.

Chao Gao, Email: gao.chao@zs-hospital.sh.cn.

References

  1. Acevedo-Rodriguez A, Kauffman AS, Cherrington BD et al (2018) Emerging insights into hypothalamic-pituitary-gonadal axis regulation and interaction with stress signalling. J Neuroendocrinol 30:e12590 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andrabi SS, Parvez S, Tabassum H (2017) Neurosteroids and ischemic stroke: progesterone a promising agent in reducing the brain injury in ischemic stroke. J Environ Pathol Toxicol Oncol 36:191–205 [DOI] [PubMed] [Google Scholar]
  3. Anwar HM, Salem GEM, Abd El-Latief HM et al (2024) Therapeutic potential of proteases in acute lung injury and respiratory distress syndrome via TLR4/Nrf2/NF-kb signaling modulation. Int J Biol Macromol 267:131153 [DOI] [PubMed] [Google Scholar]
  4. Bastos LSL, Hamacher S, Kurtz P et al (2023) The association between prepandemic icu performance and mortality variation in COVID-19: a multicenter cohort study of 35,619 critically ill patients. Chest 165:870–880 [DOI] [PubMed] [Google Scholar]
  5. Blakely KK (2019) A new year-long combination hormonal contraceptive. Nurs Womens Health 23:172–176 [DOI] [PubMed] [Google Scholar]
  6. Bos LDJ, Ware LB (2022) Acute respiratory distress syndrome: causes, pathophysiology, and phenotypes. Lancet 400:1145–1156 [DOI] [PubMed] [Google Scholar]
  7. Chen L, Gong P, Su Y et al (2024) Angiotensin II type 2 receptor agonist attenuates LPS-induced acute lung injury through modulating THP-1-derived macrophage reprogramming. Naunyn Schmiedebergs Arch Pharmacol 397:99–108 [DOI] [PubMed] [Google Scholar]
  8. Chernov AS, Rodionov MV, Kazakov VA et al (2024) CCR5/CXCR3 antagonist TAK-779 prevents diffuse alveolar damage of the lung in the murine model of the acute respiratory distress syndrome. Front Pharmacol 15:1351655 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chew KW, Malani PN, Gandhi RT (2023) COVID-19 therapeutics for nonhospitalized patients—updates and future directions. JAMA 330:1519–1520 [DOI] [PubMed] [Google Scholar]
  10. Ding Q, Wang Y, Yang C et al (2023) Clinical utility of the sivelestat for the treatment of ALI/ARDS: moving on in the controversy? Intensive Care Res 3:12–17 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dorneles GP, Teixeira PC, Peres A et al (2023) Endotoxin tolerance and low activation of TLR-4/NF-κb axis in monocytes of COVID-19 patients. J Mol Med (Berl) 101:183–195 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. El-Etr M, Akwa Y, Rame M et al (2021) Nestorone(®), a 19nor-progesterone derivative boosts remyelination in an animal model of demyelination. CNS Neurosci Ther 27:464–469 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fan E, Brodie D, Slutsky AS (2018) Acute respiratory distress syndrome: advances in diagnosis and treatment. JAMA 319:698–710 [DOI] [PubMed] [Google Scholar]
  14. Fréchou M, Zhu X, Kumar N et al (2021) Sex differences in the cerebroprotection by nestorone intranasal delivery following stroke in mice. Neuropharmacology 198:108760 [DOI] [PubMed] [Google Scholar]
  15. Gorman EA, O’Kane CM, McAuley DF (2022) Acute respiratory distress syndrome in adults: diagnosis, outcomes, long-term sequelae, and management. Lancet 400:1157–1170 [DOI] [PubMed] [Google Scholar]
  16. Guennoun R (2020) Progesterone in the brain: hormone, neurosteroid and neuroprotectant. Int J Mol Sci 21:5271 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Guennoun R, Labombarda F, Gonzalez Deniselle MC et al (2015) Progesterone and allopregnanolone in the central nervous system: response to injury and implication for neuroprotection. J Steroid Biochem Mol Biol 146:48–61 [DOI] [PubMed] [Google Scholar]
  18. Holloman BL, Wilson K, Cannon A et al (2024) Indole-3-carbinol attenuates lipopolysaccharide-induced acute respiratory distress syndrome through activation of ahr: role oF CCR2+ monocyte activation and recruitment in the regulation of CXCR2+ neutrophils in the lungs. Front Immunol 15:1330373 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Horby P, Lim WS, Emberson JR et al (2021) Dexamethasone in hospitalized patients with COVID-19. N Engl J Med 384:693–704 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Huecksteadt TP, Myers EJ, Aamodt SE et al (2024) An evaluation of type 1 interferon related genes in male and female-matched, SARS-CoV-2 infected individuals early in the COVID-19 pandemic. Viruses 16:472 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kolatorova L, Vitku J, Suchopar J et al (2022) Progesterone: a steroid with wide range of effects in physiology as well as human medicine. Int J Mol Sci 23:7989 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lee JY, Castelli V, Kumar N et al (2022) Contraceptive drug, nestorone, enhances stem cell-mediated remodeling of the stroke brain by dampening inflammation and rescuing mitochondria. Free Radic Biol Med 183:138–145 [DOI] [PubMed] [Google Scholar]
  23. Marks KM, Gulick RM (2023) Covid-19. Ann Intern Med 176:Itc145–Itc160 [DOI] [PubMed] [Google Scholar]
  24. Mauvais-Jarvis F, Klein SL, Levin ER (2020) Estradiol, progesterone, immunomodulation, and COVID-19 outcomes. Endocrinology. 10.1210/endocr/bqaa127 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. McEvoy N, Lowry J, O’Dowd A et al (2024) High-dose corticosteroid therapy in COVID-19: the recovery trial. Lancet 403:1337 [DOI] [PubMed] [Google Scholar]
  26. Merad M, Blish CA, Sallusto F et al (2022) The immunology and immunopathology of COVID-19. Science 375:1122–1127 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ng CT, Fong LY, Abdullah MNH (2023) Interferon-gamma (IFN-γ): reviewing its mechanisms and signaling pathways on the regulation of endothelial barrier function. Cytokine 166:156208 [DOI] [PubMed] [Google Scholar]
  28. Pirttiniemi A, Adeshara K, Happonen N et al (2023) Long-chain polyphosphates inhibit type i interferon signaling and augment lps-induced cytokine secretion in human leukocytes. J Leukoc Biol 114:250–265 [DOI] [PubMed] [Google Scholar]
  29. Priyandoko D, Widowati W, Lenny L et al (2024) Green tea extract reduced lipopolysaccharide-induced inflammation in l2 cells as acute respiratory distress syndrome model through genes and cytokine pro-inflammatory. Avicenna J Med Biotechnol 16:57–65 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Renke G, Callizo C, Paes R et al (2023) Clinical approaches to nestorone subdermal implant therapy in women’s health. Biomedicines 11:2586 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rossetti MF, Cambiasso MJ, Holschbach MA et al (2016) Oestrogens and progestagens: synthesis and action in the brain. J Neuroendocrinol. 10.1111/jne.12402 [DOI] [PubMed] [Google Scholar]
  32. Schaaf KR, Landstreet SR, Pugazenthi S et al (2024) Cell-free hemoglobin triggers macrophage cytokine production via TLR4 and MYD88. Am J Physiol Lung Cell Mol Physiol 326:L29-l38 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Scholten EL, Beitler JR, Prisk GK et al (2017) Treatment of ards with prone positioning. Chest 151:215–224 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sinha S, Rosin NL, Arora R et al (2022) Dexamethasone modulates immature neutrophils and interferon programming in severe COVID-19. Nat Med 28:201–211 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sitruk-Ware R, Sussman H, Brinton R et al (2024) Nestorone (segesterone acetate) effects on neuroregeneration. Front Neuroendocrinol 73:101136 [DOI] [PubMed] [Google Scholar]
  36. Składanowsk M, Golinska P, Rudnicka K et al (2016) Evaluation of cytotoxicity, immune compatibility and antibacterial activity of biogenic silver nanoparticles. Med Microbiol Immunol 205:603–613 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Somasundaram NP (2023) COVID-19 and endocrinology. Best Pract Res Clin Endocrinol Metab 37:101809 [DOI] [PubMed] [Google Scholar]
  38. Swets MC, Kerr S, Scott-Brown J et al (2023) Evaluation of pragmatic oxygenation measurement as a proxy for COVID-19 severity. Nat Commun 14:7374 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Taghavi S, Campbell A, Engelhardt D et al (2024) Dimethyl malonate protects the lung in a murine model of acute respiratory distress syndrome. J Trauma Acute Care Surg 96:386–393 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Taraborrelli S (2015) Physiology, production and action of progesterone. Acta Obstet Gynecol Scand 94(Suppl 161):8–16 [DOI] [PubMed] [Google Scholar]
  41. Theoharides TC, Conti P (2020) Dexamethasone for COVID-19? Not so fast. J Biol Regul Homeost Agents 34:1241–1243 [DOI] [PubMed] [Google Scholar]
  42. Vijayasingham L, Bischof E, Wolfe J (2021) Sex-disaggregated data in covid-19 vaccine trials. Lancet 397:966–967 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Villar J, Añón JM, Ferrando C et al (2020) Efficacy of dexamethasone treatment for patients with the acute respiratory distress syndrome caused by covid-19: study protocol for a randomized controlled superiority trial. Trials 21:717 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Vora SM, Lieberman J, Wu H (2021) Inflammasome activation at the crux of severe COVID-19. Nat Rev Immunol 21:694–703 [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Weeratunga P, Denney L, Bull JA et al (2023) Single cell spatial analysis reveals inflammatory foci of immature neutrophil and CD8 T cells in COVID-19 lungs. Nat Commun 14:7216 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Yang H, Fung SY, Xu S, Sutherland DP, Kollmann TR, Liu M, Turvey SE (2015) Amino acid-dependent attenuation of toll-like receptor signaling by peptide-gold nanoparticle hybrids. ACS Nano 9:6774–6784 [DOI] [PubMed] [Google Scholar]
  47. Zhang PH, Zhang WW, Wang SS et al (2024a) Efficient pulmonary lymphatic drainage is necessary for inflammation resolution in ards. JCI Insight. 10.1172/jci.insight.173440 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zhang S, Liu Y, Zhang XL et al (2024b) ANKRD22 aggravates sepsis-induced ards and promotes pulmonary m1 macrophage polarization. J Transl Autoimmun 8:100228 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Zhu Y, Luo L, Zhang M et al (2023) Xuanfei baidu formula attenuates LPS-induced acute lung injury by inhibiting the nf-κb signaling pathway. J Ethnopharmacol 301:115833 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Not applicable.

Articles from Inflammopharmacology are provided here courtesy of Springer

RESOURCES