Female C57BL/6 mice (aged 6–8 weeks, weighing 20  ± 2 g) were purchased from Guangdong Medical Laboratory Animal Center (Guangdong Province, China). All mice were maintained under specific pathogen-free conditions with a 12-hour light/dark cycle at 20–22°C. The animals were allowed free access to drinking water and food. The study complied with the guidelines of the Institutional Animal Care and Use Committees (IACUCs) of the Southern University of Science and Technology and was approved by the Animal Subjects Committee of Shenzhen People’s Hospital (IACUC Number: AUP-220704-LN-0417-01).

M. abscessus strain ATCC 410212027 was grown at 37°C on Middlebrook 7H10 agar supplemented with 10% OADC (oleic acid albumin dextrose catalase enriched) and 0.05% Tween-80 (Sigma-Aldrich). M. abscessus was collected by centrifugation and resuspended in Middlebrook 7H9 broth, supplemented with 0.2% glycerol, 0.05% Tween 80, and 10% ADC. Middlebrook 7H9 broth, Middlebrook 7H10 agar, OADC, and ADC were purchased from BD Pharmingen (San Diego, CA). The number of CFU bacteria was counted on Middlebrook 7H10 agar.

C57BL/6J mice were randomly assigned to the following groups (12 mice per group): 1) the control group (non-M. abscessus infected animals and treated with saline, intraperitoneal injection with saline); 2) M. abscessus infected group (animals infected with M. abscessus and intraperitoneally injected with saline); and 3) the ouabain-treated group (M. abscessus infected animals and intraperitoneal injection with ouabain). M. abscessus-infected and ouabain-treated groups were infected with 1.5 × 10⁷ CFU/50 μL M. abscessus via intratracheal intubation. An equal volume of saline was administered to the mice in the control group. The animals in the ouabain-treated group were intraperitoneally injected with 0.56 mg/Kg ouabain, based on previous in vivo work, for three consecutive days before M. abscessus infection (26). The control group and the M. abscessus-infected group were given equal volumes of saline intraperitoneally for three days. Ouabain octahydrate was purchased from Sigma-Aldrich as a >95% (HPLC) powder (Cat. no. 11018-89-6).

Blood plasma collected from the eyes of mice was used to determine IL-1β, TNFα, IL-6 and IL-10 concentrations using a mouse-specific ELISA kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions. Optical density was measured using a microplate spectrophotometer at 450 nm (VersaMax microplate reader, tunable, BN 2529, Molecular Devices).

The ligated upper lobe of the left lung was separated, immobilized with 4% paraformaldehyde and dehydrated with alcohol. The lung was then embedded in paraffin and cut into 4 μm thick sections. The tissues were treated with a hematoxylin and eosin kit (Beyotime Biotech, Shanghai, China) and stained and viewed with a light microscope.

The paraffin sections were transparent and were dewaxed with solvents (such as petrol and turpentine). The above samples were dehydrated with gradient ethanol and stained with an acid-fast Bacillus (AFB) kit according to the manufacturer’s instructions. Finally, the cells were observed under an oil microscope (Carl Zeiss Jena A1; Carl Zeiss Jena Company, Oberkochen, Germany).

Sections of mouse lung tissue were deparaffinized and washed with double distilled water. A pre-warmed citrate solution (pH = 6.0) was used for 15 minutes in the microwave for antigen retrieval. After cooling for 30 min, the sections were removed, washed twice with double distilled water (5 min/time) and rinsed three times with 1 × PBS (5 min/time). The endogenous peroxidase was blocked with the endogenous peroxidase blocker (3% H solution) and incubated for 30 min at RT. Then the sections were rinsed three times with 1 × PBS (5 min/time) and incubated for 1 hour with 5% normal donkey serum blocking buffer at RT. The serum was then discarded and the sections were incubated with the primary anti-NLRP3 antibody (Abcam, ab189494, 1:200) at 4°C overnight. Sections were then cleared three times with 1 × PBS (5 min/time) and incubated with HRP-labelled goat anti-rabbit IgG at 37°C in the dark for 2 hours. Sections were then cleared three times with 1 × PBS (5 min/time) and mounted with non-fluorescent buffered glycerol. Finally, the sections were viewed under a fluorescence microscope at high magnification.

Fresh mouse lung tissue was selected for RNA sequencing, with three replicates. Total RNA was extracted using TRIzol reagent (15596-026, Life Technologies) according to the manufacturer’s instructions and RNA integrity was assessed using an Agilent Bioanalyzer 2100 (Agilent Technologies). The qualified total RNA was then purified using the RNA Clean XP Kit and the RNase-Free DNase Set. Nine libraries were prepared using the VAHTS Total RNA-seq (H/M/R) Library Prep Kit for Illumina (NR603-02; Vazyme). The library was summarized and sequenced as 150 bp paired-end sequencing reads using an Illumina HiSeq machine. After removing low-quality tags and various contaminants from the original sequencing reads, the clean dataset was processed with mirDeep29 (v2.0.0.5), annotated in the miR-Base database 30 (version 20) as a miRNA reference and aligned with the mouse reference genome (mm10) to predict novel miRNAs. Differentially expressed miRNAs between the four analysis time points were identified by log2Fold-Change| (|log2FC|) ≥1 and a false discovery rate (FDR) <0.05. All predicted target genes were annotated in the database. The web tool DAVID (Visualisation and Integrated Discovery) v6.8 was used for the enrichment analysis of the Gene Ontology (GO) and the Kyoto Encyclopaedia of Genes and Genomes (KEGG) with a significance of FDR <0.05.

According to the instructions for use, single cell suspensions from the lung were prepared and stained as follows: 0.1 μL zombie dye was added to each tube and incubated at room temperature for 10–15 minutes protected from light. The Fc receptor was blocked with CD16/CD32 (Mouse Fc Block, Clone 2.4 G2) for 10 minutes. Mice containing PE anti-CD45 (clone GL-1, Biolegend, San Diego, CA), BV421 anti-mouse F4/80 (clone T45-2342, BD Biosciences, Franklin Lakes, San Diego, CA) and PE-Cy7 anti-mouse CD11b (clone M1/70, BD Biosciences)-specific antibodies that stain extracellular surface markers were combined and stained in the dark for 30 minutes. Unstained cells were used to set the flow cytometer settings. Fluorescence minus one control (FMO) was used to determine the gating limits. Flow cytometric data were acquired using a CYTEK Northern Lights-CLC (Becton and Dickinson, Franklin Lakes, NJ) and analyzed using FlowJo 7.6 software and GraphPad Prism 9.0.

Lung tissue was obtained from the terminal sacrifice of mice and cut into 1–2 mm³ cubes. The lung tissue samples and infected cells were fixed with a solution of 2% paraformaldehyde (v/v) and 2.5% glutaraldehyde (v/v) in 0.05 M cacodylate-HCl buffer (pH 7.2) for 2–4 hours. After fixation, the samples were processed using conventional methods. The sections were analyzed using a transmission electron microscope (JEOL, Tokyo, Japan) at an accelerating voltage of 80 kV.

Mouse bone marrow-derived macrophages (BMDMs) were isolated and differentiated as previously described (26). C57BL/6 mice were sacrificed by dislocation of the spine, bone marrow from the femurs was rinsed and cultured in DMEM (10% FBS + 15% L929 cell-conditioned medium). After 7 days of culture, BMDMs were obtained as a homogeneous population of adherent cells. The number of cells was analyzed with a cell counter and the cells were divided into three groups: Negative control group (same dose of PBS), M. abscessus infection group (Multiplicity of Infection (MOI) = 5), and the group treated with Ouabain (the cells were pretreated with ouabain 1 hour before M. abscessus infection). 100 nmol/mL was chosen because this concentration was effective in a previous in vitro study (27).

Total RNA was extracted from lung tissue and cell pellets using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. One microgram of total RNA was reverse transcribed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA) to generate cDNA. cDNA was then amplified using the SYBR Green Master Mix (TaKaRa, Mountain View, CA) according to the manufacturer’s protocol. Quantitative real-time PCR was performed using the CFX384 Fluorescent Quantitative Real-Time PCR System (Bio-Rad, Hercules, CA). Relative mRNA concentrations were calculated using the 2-ΔΔCt method, and GAPDH was used as an internal control. The primers used for cDNA synthesis and qPCR are listed in Supplementary Table S1 .

All statistical analyses were performed using GraphPad Prism software (Prism 9.0). Flow cytometry data were analyzed using FlowJo 10 software. Data are expressed as mean ± SEM, and statistical significance was determined using a two-way analysis of variance (ANOVA) followed by Student’s t-test. Statistical significance was set at P < 0.05.

To investigate the anti-inflammatory effect of ouabain against M. abscessus infection, we first analyzed the cytokine responses. M. abscessus infection elicited a robust systemic inflammatory response, as evidenced by significantly increased plasma levels of pro-inflammatory cytokines including IL-1β (p < 0.0001, Figure 1A ), IL-6 (p < 0.0001, Figure 1B ) and TNF-α (p < 0.0001, Figure 1C ) compared to saline-treated controls. Interestingly, the anti-inflammatory cytokine IL-10 also showed increased levels post-infection (p < 0.0008, Figure 1D ). Treatment with ouabain effectively attenuated the cytokine storm triggered by the infection and significantly suppressed the upregulation of pro-inflammatory mediators (p < 0.05, Figures 1A–C ). Of note, while ouabain reduced overall cytokine production, it maintained elevated IL-10 levels (p < 0.0087, Figure 1D ), suggesting a possible mechanism for its anti-inflammatory effects. Ouabain treatment significantly reduced the intensity of AFB staining in lung sections ( Figure 1E ) compared to infected controls (no staining was observed in uninfected animals). This correlated with a 2.3-fold decrease in the number of viable bacteria (p < 0.0001, Figure 1F ), indicating a significant reduction in bacterial burden in the lungs. H&E-stained lung sections showed that M. abscessus infection induced severe granulomatous inflammation with extensive inflammatory infiltration. However, treatment with ouabain reduced the infiltration of inflammatory cells by 58%, maintained alveolar integrity and significantly suppressed the development of granulomas ( Figures 1G, H ). Compared to the saline-treated controls, a separate group of mice received ouabain alone (0.56 mg/Kg), which did not induce systemic toxicity or pulmonary inflammation, consistent with previous reports (28). For the sake of conciseness and to focus on infection-related outcomes, these control data are not presented. Therefore, our results indicate that a successful animal model of M. abscessus infection was established, and ouabain exerts potent anti-inflammatory effects in M. abscessus pulmonary infection.

As key mediators of innate immunity, neutrophils and macrophages play crucial roles in host defense against microbial pathogens. To characterize ouabain’s immunomodulatory effects, we performed flow cytometric analysis of leukocyte populations in the M. abscessus infection model. We immunostained for double-positive Ly6G/CD11b neutrophils, F4/80/CD11b macrophages, CD11b/CD19 B cells, and CD3/CD8 T cells ( Supplementary Figure S1A ). Gating of BALF CD45⁺ leukocytes, as shown in Supplementary Figures S1B, C , and myeloid cells were further analyzed for specific macrophage and neutrophil markers. The percentages of F4/80⁺CD11b⁺ and Ly6G⁺CD11b⁺ subsets showed a significant increase in BALF induced by M. abscessus infection compared to the control (p < 0.05). However, ouabain treatment decreased the upregulation of the F4/80⁺CD11b⁺ and Ly6G⁺CD11b⁺ subsets in M. abscessus-infected lung tissues (p < 0.05, Figures 2A–D ). We also measured the F4/80⁺CD11b⁺, Ly6G⁺CD11b⁺, CD11b⁺CD19⁺, and CD3⁺CD8⁺ subsets in the murine lung tissues. The percentages of neutrophils and macrophages in the ouabain-treated group (5.29% and 7.45%, respectively) were significantly lower than those in the M. abscessus infection group (10.72% and 12.5%) (p < 0.05, Figures 2E–H ). We also found that the percentages of CD11b⁺CD19⁺ B cell subsets and CD3⁺CD8⁺ T cell subsets were not significantly different between the M. abscessus-infected and ouabain-treated groups ( Supplementary Figures S1D – S3G ), which were all lower than those in the control group. These data demonstrate that ouabain specifically modulates innate immune cell recruitment without affecting adaptive immune populations, suggesting targeted regulation of myeloid cell trafficking during M. abscessus infection.

We performed mRNA transcriptome sequencing to elucidate and identify the possible anti-inflammatory mechanism of ouabain in an M. abscessus-infected mouse model. Transcriptome data revealed 3731 DEGs between the control and M. abscessus-infected groups, including 1,450 downregulated and 2,281 upregulated genes. 479 downregulated and 293 upregulated genes were identified in the ouabain-treated group ( Supplementary Figure S2A ). Moreover, we observed the intersections of these four gene sets, which generated a Venn plot ( Figure 3A ), indicating that ouabain has a strong and specific regulatory effect on M. abscessus-induced inflammation. Remarkably, 89.8% (430/479) of the downregulated genes in the ouabain-treated group were upregulated following M. abscessus infection. Conversely, 75.8% (222/293) of the up-regulated genes in the ouabain-treated group were among the M. abscessus-infected down-regulated gene sets. The heat map showed that most differentially expressed genes related to the inflammatory response were downregulated in the ouabain-treated group compared with those in the M. abscessus-infected group ( Figure 3B ). These genes were highlighted in volcano plots: inflammasome pathway correlation genes were upregulated in the lungs of M. abscessus-infected mice compared to the control group ( Figure 3C ). All increases were downregulated after the Ouabain administration ( Figure 3D ). Protein-protein interaction (PPI) analysis was performed using the STRING database (I suppose this is a subheading), which also revealed a strong correlation between the NLRP3 inflammasome and pro-inflammatory factors such as iNOS, IL-1β, IL-6, and TNFα ( Figure 3E ). These results provide compelling evidence that ouabain’s therapeutic effects involve transcriptional regulation of the NLRP3 inflammasome pathway and associated inflammatory mediators during M. abscessus infection.

To determine whether ouabain alleviated the NOD-like signaling pathway induced by M. abscessus infection, GO analysis and KEGG pathway enrichment of 222 candidate targets were performed. GO analysis highlighted GO terms with high enrichment of DEGs (>100 genes), which showed that upregulated genes induced by M. abscessus were mainly associated with the following functions: response to adaptive immune response (ontology: biological process), external side of the plasma membrane (ontology: cellular component), and receptor regulatory activity (ontology: molecular function) ( Figure 4A ). Depending on the outcome of the M. abscessus-infected group, the enriched biological process ontologies in the ouabain-treated group were negative regulation of lymphocyte activation, T cell activation, and activation of the immune response. These enriched molecular functional entities are mainly receptor regulation and receptor-ligand activity. The cell component analysis revealed that the external side of the plasma membrane accounted for the largest proportion ( Figure 4B ). KEGG pathway analysis identified 20 remarkable pathways in the M. abscessus-infected group, presenting the pathways of upregulated genes: viral, tuberculosis, phagosome, NOD-like receptor signaling pathway (cellular processes), and immune system (organismal systems) ( Figure 4C ). The enriched KEGG pathways of the ouabain-downregulated genes included the NOD-like receptor signaling pathway, Tuberculosis, Toll-like receptor signaling pathway, NF-κB signaling, and cell adhesion molecules ( Figure 4D ). Given the above results, we submitted the complete gene expression datasets to Gene Set Enrichment Analysis (GSEA) and extracted biological knowledge. Judging from the gene rank distribution and enrichment scores of the rank association matrix in the gene list, both the activated and repressed gene sets showed relatively reliable enrichment. These gene sets were significantly enriched and mainly related to the inflammatory response ( Figures 4E, F ) and cellular response to molecules of bacterial origin ( Supplementary Figures S2B, C ). These findings implied that ouabain has potent, targeted regulatory effects in the M. abscessus-infected mouse model, principally influencing infection- and inflammation-associated pathways, such as the NOD-like receptor signaling pathway.

To characterize the immunomodulatory potential of ouabain in M. abscessus infection, we first evaluated macrophage polarization patterns in infected murine lungs. Our qPCR results revealed a distinct M1 macrophage polarization profile in response to M. abscessus infection. Compared to uninfected controls, infected lung tissues exhibited significant upregulation of key pro-inflammatory markers: inducible nitric oxide synthase (iNOS, 3.8-fold), interleukin-6 (IL-6, 4.2-fold), interleukin-1β (IL-1β, 4.3-fold), and tumor necrosis factor-α (TNF-α, 3.5-fold) ( Figures 5A–D ). Remarkably, ouabain treatment substantially mitigated this inflammatory response, decreasing expression of these markers by more than 2-fold compared to untreated infected mice. These transcriptional changes were corroborated at the protein level. Lung tissues were homogenized in PBS and centrifuged at 12,000 × g for 15 min at 4°C. The resulting supernatants (lung tissue homogenate supernatants) were collected for cytokine analysis. ELISA measurements demonstrated that M. abscessus infection significantly elevated lung tissue supernatant concentrations of IL-1β (385 ± 42 pg/mL), IL-6 (320 ± 38 pg/mL), and TNF-α (245 ± 30 pg/mL) (all p < 0.001 versus uninfected controls). Ouabain treatment effectively normalized these cytokine levels to 185 ± 22 pg/mL (IL-1β), 160 ± 18 pg/mL (IL-6), and 112 ± 15 pg/mL (TNF-α) (p < 0.01 versus infected untreated group; Figures 5E–G ). Notably, neither infection nor ouabain treatment significantly altered IL-10 levels ( Figure 5H ). Transmission electron microscopy (TEM) analysis demonstrated that M. abscessus infection triggered significant activation of pulmonary macrophages, manifested by distinct ultrastructural alterations including: (1) markedly increased cytoplasmic electron density, (2) prominent proliferation of intracellular organelles (particularly mitochondria [M], indicated by red asterisks), and (3) substantial accumulation of both autolysosomes (ASS, red arrows) and azurophilic granules (AG, blue arrows) ( Figure 5I ). Notably, ouabain treatment effectively preserved macrophage ultrastructural integrity, significantly attenuating both organelle stress and the formation of inflammatory particles.

Our transcriptomic analysis identified NLRP3 inflammasome pathway modulation as the principal mechanism underlying ouabain’s anti-inflammatory activity during M. abscessus infection. This finding was substantiated by immunohistochemical analysis of lung tissues, which revealed significant downregulation of NLRP3, IL-1β, ASC, and Caspase-1 expression in ouabain-treated animals relative to infected controls ( Figures 6A–D ). These data collectively confirmed ouabain’s suppression of functional inflammasome activation. While ouabain monotherapy significantly reduced inflammation, the incomplete normalization of NLRP3/IL-1β levels suggests two complementary strategies: Low-dose corticosteroids could augment ouabain’s immunomodulation while minimizing toxicity, as shown in tuberculosis models. Following established protocols (27), bone marrow-derived macrophages (BMDMs) were differentiated into M0 macrophages (PMA-stimulated) and challenged with M. abscessus. The infection induced pronounced M1 polarization, as demonstrated by 3-4-fold upregulation of characteristic M1 markers (iNOS, IL-6, and TNF-α; p < 0.01 versus unstimulated M0 controls, Figures 6E–I ). Notably, co-treatment with 100 nM ouabain attenuated these pro-inflammatory responses by ≥50% (p < 0.05). These findings collectively demonstrate that ouabain mitigates M. abscessus-triggered inflammatory responses by specifically inhibiting NLRP3 inflammasome activation and subsequent IL-1β production in macrophages.