Tudor domain-containing protein 9-targeting siRNA nanoparticles alleviate Pseudomonas aeruginosa lung injury in preclinical models by promoting neutrophil cuproptosis

Abstract

Pseudomonas aeruginosa pneumonia poses a significant therapeutic challenge. Nanoparticles serve as an effective tool for nucleic acid delivery to efficiently alleviate pneumonia. This study develops a hyaluronic acid (HA)-coated peptide nanoparticle system for targeted delivery of small interfering RNA (siRNA) against Tudor domain-containing protein 9 (TDRD9), identified via RNA sequencing of bronchoalveolar lavage fluid-derived neutrophils from 21 recruited patients (11 males/10 females). Adoptive transfer of TDRD9-silenced polymorphonuclear neutrophils into neutrophil-depleted male mice attenuates lung inflammation and edema. Mechanistically, TDRD9 suppresses neutrophil cuproptosis by upregulating programmed death ligand 1 (PD-L1) through interaction with CD80 to activate p38 mitogen-activated protein kinase (MAPK) signaling. HA-si-TDRD9 nanoparticles enhance neutrophil cuproptosis, reduce pulmonary neutrophil accumulation, and ameliorate lung injury via PD-L1/CD80/MAPK. Importantly, HA-si-TDRD9 nanoparticles reduce bacterial growth, apoptosis, and inflammation in human lung organoids. This work demonstrates that targeting TDRD9 with siRNA nanoparticle platform presents a promising therapeutic strategy for treating bacterial lung injury.

Fig. 1. Transcriptomic profiling of differentially expressed genes in pneumonia patients.

A Volcano plot and hierarchical clustering heatmap of differentially expressed genes (DEGs) between neutrophils from Control and Pseudomonas aeruginosa (PA)-pneumonia neutrophils. B Volcano plot and hierarchical clustering heatmap of DEGs between non-PA-pneumonia and PA-pneumonia neutrophils. C Male C57BL/6 mice received 50 μL of bacterial suspension of PA strain PAO1 (OD₆₀₀ = 0.6; 7 × 10⁶ CFU/mL in 0.9% sterile saline) via oropharyngeal intratracheal instillation. Control mice received an equal volume of sterile saline. Quantitative real-time PCR validation of upregulated genes (phosphodiesterase 4D [PDE4D], Tudor domain-containing protein 9 [TDRD9], sialic acid-binding Ig-like lectin 14 [SIGLEC14], integrin subunit alpha 7 [ITGA7]) in PA-pneumonia neutrophils versus Control. n = 10 mice per group. Data represent median ± interquartile range (IQR), and analyzed by two-sided t-test (C). Source data are provided as a Source Data file.

Fig. 2. Pseudomonas aeruginosa (PA) induces lung injury in mice.

A An experimental flowchart illustrating male C57BL/6 mice were intranasally inoculated with 50 μL of bacterial suspension of PA strain PAO1 (OD₆₀₀ = 0.6; 7 × 10⁶ CFU/mL in 0.9% sterile saline). Bronchoalveolar lavage fluid (BALF) and lung tissues were then harvested at 1-, 3-, and 7-day post-infection. B Body weight trajectories of mice over 7 days post-PAO1 infection. n = 12 mice per group. C Quantitative analysis of colony-forming unit (CFU) in lung tissues. n = 6 mice per group. D Quantification of pulmonary edema via lung dry-to-wet weight ratios. n = 6 mice per group. E Histopathological assessment of PAO1-induced lung injury by hematoxylin and eosin (HE) staining. Scale bar: 500 µm (top), 100 µm (middle), 25 µm (bottom). n = 6 mice per group. F Proinflammatory cytokine levels (tumor necrosis factor-alpha [TNF-α], interleukin-1 beta [IL-1β], and IL-6) in BALF measured by enzyme-linked immunosorbent assay (ELISA). n = 6 mice per group. G, H Flow cytometric quantification of neutrophils, macrophages, and B cells in lung tissues post-PAO1 infection. n = 6 mice per group. Data represent median ± interquartile range (IQR), and analyzed by two-sided two-way ANOVA with Tukey’s multiple comparisons test (B), or two-sided one-way ANOVA with Tukey’s multiple comparisons test (C–G). Source data are provided as a Source Data file.

Fig. 3. Pseudomonas aeruginosa (PA) suppresses neutrophil cuproptosis in mouse lungs.

Male C57BL/6 mice were intranasally inoculated with 50 μL of bacterial suspension of PA strain PAO1 (OD₆₀₀ = 0.6; 7 × 10⁶ CFU/mL in 0.9% sterile saline) for 1, 3, and 7 days. A Quantitative real-time PCR quantification of phosphodiesterase 4D (PDE4D), Tudor domain-containing protein 9 (TDRD9), and integrin subunit alpha 7 (ITGA7) in lung neutrophils. n = 6. mice per group. B Western blot analysis of PDE4D, TDRD9, and ITGA7 expression in lung neutrophils. n = 6 mice per group. C Western blot profiling of cuproptosis-related proteins (dihydrolipoamide S-acetyltransferase [DLAT], ferredoxin 1 [FDX1], lipoic acid synthetase [LIAS], copper-transporting ATPase A/B [ATP7A/B], solute carrier family 31 member 1 [SLC31A1], and tumor protein p53 [p53]) in neutrophils. n = 6 mice per group. D Immunofluorescence (IF) staining demonstrating co-localization of neutrophil marker LY6G (green) with Fe-S cluster proteins DLAT and FDX1 (red) in lung tissues. Nuclei were counterstained with DAPI (blue). Scale bar: 25 µm. n = 6 mice per group. Data represent median ± interquartile range (IQR), and analyzed by two-sided one-way ANOVA with Tukey’s multiple comparisons test (A–D). Source data are provided as a Source Data file.

Fig. 4. Neutrophil-specific Tudor domain-containing protein 9 (TDRD9) depletion attenuates Pseudomonas aeruginosa (PA)-induced pneumonia.

A An experimental flowchart illustrating that male C57BL/6 mice were intravenously administered 50 μg anti-Ly6G antibody and intravenously injected with neutrophils (1 × 10⁶ cells/mouse) 48 h prior to PA strain PAO1 (OD₆₀₀ = 0.6; 7 × 10⁶ CFU/mL in 0.9% sterile saline) infection. Neutrophils were pre-transducted with lentiviral-based short hairpin RNA (shRNA) knocking down TDRD9 (sh-TDRD9) or corresponding negative control vectors (sh-NC). B Body weight changes in mice over 3 days post-PAO1 infection. n = 12 mice per group. C Quantitative analysis of colony-forming unit (CFU) in lung tissues. n = 6 mice per group. D Lung dry-to-wet weight ratios evaluating edema severity. n = 6 mice per group. E Bronchoalveolar lavage fluid (BALF) cytokine levels (tumor necrosis factor-alpha [TNF-α], interleukin-1 beta [IL-1β], and IL-6) were quantified by enzyme-linked immunosorbent assay (ELISA). n = 6 mice per group. F Hematoxylin and eosin (HE) staining reveals inflammatory damage in lung tissues. Scale bar: 500 µm (top), 100 µm (middle), 25 µm (bottom). n = 6 mice per group. Data represent median ± interquartile range (IQR), and analyzed by two-sided two-way ANOVA with Tukey’s multiple comparisons test (B), or two-sided one-way ANOVA with Tukey’s multiple comparisons test (C–F). Source data are provided as a Source Data file.

Fig. 5. Tudor domain-containing protein 9 (TDRD9) suppresses neutrophil cuproptosis in vitro.

A Neutrophils were pretreated with escalating doses of the copper ionophore elesclomol (ES) or dimethyl sulfoxide (DMSO; vehicle control) for 24 h. Dose-dependent cytotoxicity of ES in human neutrophils. n = 3 independent experiments. B Neutrophils were pretreated with 10 μM ES for 24 h and challenged with Pseudomonas aeruginosa (PA) strain PAO1 at a multiplicity of infection (MOI) of 10 for 1 h. Viability of neutrophils treated with 10 μM ES ± PAO1 infection. n = 3 independent experiments. C Western blot analysis of cuproptosis-related proteins under PAO1/ES co-treatment. n = 3 independent experiments. D PAO1-induced TDRD9 upregulation. n = 3 independent experiments. E Correlation heatmap between TDRD9 and cuproptosis regulators. F Neutrophils were pre-transducted with lentiviral-based short hairpin RNA (shRNA) knocking down Tudor domain-containing protein 9 (TDRD9) (sh-TDRD9) and exposed to 20 μM tetrathiomolybdate (cuproptosis inhibitor; 2 h), 50 μM deferoxamine (DFO; iron chelator; 1 h), 10 μM ferrostatin-1 (ferroptosis inhibitor; 1 h), 5 μM Z-VAD-FMK (pan-caspase inhibitor; 1 h), 1 μM necrosulfonamide (necroptosis inhibitor; 1 h), and 500 μM 3-methyladenine (3-MA; autophagy inhibitor; 1 h). Neutrophil viability under pharmacological inhibition of alternative cell death pathways. n = 3 independent experiments. G Neutrophils were pre-transducted with sh-TDRD9, followed by treatment with 10 μM ES and PAO1. Quantitative analysis of colony-forming unit (CFU) in neutrophils. n = 3 independent experiments. H Neutrophils were pre-transducted with sh-TDRD9 or lentiviral TDRD9 overexpression construct (oe-TDRD9), followed by treatment with 10 μM ES and PAO1. Viability of PAO1/ES-treated neutrophils with TDRD9 knockdown/overexpression. n = 3 independent experiments. I Enzyme-linked immunosorbent assay (ELISA) analysis of tumor protein p53 (p53) levels in cell culture medium. n = 3 independent experiments. J Protein expression dynamics of cuproptosis mediators under genetic TDRD9 modulation. n = 3 independent experiments. Data represent median ± interquartile range (IQR), and analyzed by two-sided two-way ANOVA with Tukey’s multiple comparisons test (A), or two-sided one-way ANOVA with Tukey’s multiple comparisons test (B–D, F–J). Source data are provided as a Source Data file.

Fig. 6. Transcriptomic landscape of Tudor domain-containing protein 9 (TDRD9)-regulated pathways in human neutrophils.

A Human peripheral blood neutrophils were pre-transducted with lentiviral-based short hairpin RNA (shRNA) knocking down TDRD9 (sh-TDRD9) or corresponding negative control vectors (sh-NC), followed by treatment with Pseudomonas aeruginosa (PA) strain PAO1 at a multiplicity of infection (MOI) of 10 for 1 h. RNA-sequencing on PAO1-exposed neutrophils following TDRD9 knockdown. Principal component analysis (PCA) of transcriptional profiles from sh-TDRD9 versus sh-NC neutrophils. B Volcano plot visualizing differentially expressed genes (DEGs) (red: upregulated; blue: downregulated). C Hierarchical clustering of DEGs. D Gene ontology (GO) enrichment across biological processes, cellular components, and molecular functions. E STRING database-derived protein-protein interaction (PPI) network. Source data are provided as a Source Data file.

Fig. 7. The programmed death ligand-1(PD-L1)/cluster of differentiation 80 (CD80)/mitogen-activated protein kinase (MAPK) axis mediates Tudor domain-containing protein 9 (TDRD9)-dependent cuproptosis regulation.

A Neutrophils were pre-transducted with lentiviral-based short hairpin RNA (shRNA) knocking down TDRD9 (sh-TDRD9) and lentiviral PD-L1 overexpression construct (oe-PD-L1), followed by treatment with 10 μM elesclomol (ES) for 24 h and Pseudomonas aeruginosa (PA) strain PAO1 at a multiplicity of infection (MOI) of 10 for 1 h. Quantitative analysis of colony-forming unit (CFU) in neutrophils. n = 3 independent experiments. B PD-L1 overexpression rescued TDRD9 knockdown-induced pathway suppression. n = 3 independent experiments. C Enzyme-linked immunosorbent assay (ELISA) analysis of PD-L1 and sCD80 levels in cell culture medium. n = 3 independent experiments. D Viability restoration by PD-L1 overexpression in TDRD9-depleted neutrophils. n = 3 independent experiments. E Cuproptosis-related protein dynamics under TDRD9 knockdown ± PD-L1 overexpression. n = 3 independent experiments. F ELISA analysis of tumor protein p53 (p53) levels in cell culture medium. n = 3 independent experiments. G Neutrophils were pre-transducted with oe-PD-L1 and lentiviral-based shRNA knocking down CD80 (sh-CD80), followed by treatment with 10 μM elesclomol (ES) for 24 h and infection with PAO1 at an MOI of 10 for 1 h. Signaling pathway-related protein expressions under PD-L1 overexpression ± CD80 knockdown. n = 3 independent experiments. H ELISA analysis of PD-L1 and sCD80 levels in cell culture medium. n = 3 independent experiments. I Viability reduction by CD80 knockdown in PD-L1-overexpressed neutrophils. n = 3 independent experiments. J, K Cuproptosis-related protein dynamics under PD-L1 overexpression ± CD80 knockdown. n = 3 independent experiments. L PD-L1-CD80 interaction using co-immunoprecipitation assay. n = 3 independent experiments. Data represent median ± interquartile range (IQR), and were analyzed by two-sided one-way ANOVA with Tukey’s multiple comparisons test (A–K). Source data are provided as a Source Data file.

Fig. 8. HA-si-TDRD9 NPs ameliorate Pseudomonas aeruginosa (PA)-induced lung injury and modulate neutrophil cuproptosis.

A An experimental flowchart illustrating intravenous injections of low-dose (0.1 μmol) or high-dose (0.2 μmol) hyaluronic acid (HA)-coated peptide nanoparticle (NP) system for targeted small interfering RNA (siRNA) delivery against Tudor domain-containing protein 9 (TDRD9) (HA-si-TDRD9 NPs) three days after PA strain PAO1 (OD₆₀₀ = 0.6; 7 × 10⁶ CFU/mL in 0.9% sterile saline) infection. B Body weight dynamics in PAO1-infected mice treated with HA-si-NC NPs or HA-si-TDRD9 NPs (0.1/0.2 μmol). n = 12 mice per group. C Quantitative analysis of colony-forming unit (CFU) in lung tissues. n = 6 mice per group. D Lung edema quantification via wet-to-dry weight ratios. n = 6 mice per group. E Bronchoalveolar lavage fluid (BALF) cytokine levels. n = 6 mice per group. E TDRD9 mRNA expression in lung tissue. n = 6 mice per group. F Representative hematoxylin and eosin (HE)-stained lung images. Scale bar: 500 µm (top), 100 µm (middle), 25 µm (bottom). n = 6 mice per group. G, H Programmed death ligand-1(PD-L1)/cluster of differentiation 80 (CD80)/mitogen-activated protein kinase (MAPK) signaling pathway-related indicators in lung neutrophils. n = 6. I Cuproptosis-related protein expression in lung neutrophils. n = 6 mice per group. Data represent median ± interquartile range (IQR), and analyzed by two-sided two-way ANOVA with Tukey’s multiple comparisons test (B), or two-sided one-way ANOVA with Tukey’s multiple comparisons test (B–D, C–I). Source data are provided as a Source Data file.

Fig. 9. HA-si-TDRD9 NPs reduce HLO susceptibility to PA infection.

A A spherical 3D human lung organoid (HLO) structure was successfully established in Matrigel, and its growth was observed under an optical microscope for 7 days. Representative bright-field images of HLOs cultured for 7 days. Scale bars: 200 μm (top), 100 μm (bottom). n= 3 independent experiments. B Hematoxylin and eosin (HE) staining comparing histological features of native human lung tissue and HLOs. Scale bar: 60 μm. n = 3 independent experiments. C Immunohistochemistry analysis of Ki67, NK2 homeobox 1 (NKX2.1), and SRY-box transcription factor 9 (SOX9) expression in native lung tissue and HLOs. Scale bar: 60 μm. n = 3 independent experiments. D HLOs were incubated with 0.1 μM hyaluronic acid (HA)-coated peptide nanoparticle (NP) system for targeted small interfering RNA (siRNA) delivery against Tudor domain-containing protein 9 (TDRD9) (HA-si-TDRD9 NPs) or HA-si-NC NPs, followed by infection with Pseudomonas aeruginosa (PA) strain PAO1. HE staining showing HA-si-TDRD9-mediated preservation of HLO morphology following PA challenge. n= 3 independent experiments. E Bacterial growth curves in HLO cultures. n = 3. F Quantitative real-time PCR analysis of TDRD9 mRNA levels in HLOs. n = 3 independent experiments. G Western blot analysis of TDRD9, programmed death ligand-1(PD-L1), cluster of differentiation 80 (CD80), and mitogen-activated protein kinase (MAPK) expression. n = 3 independent experiments. H TUNEL assay quantifying apoptotic cells in HLOs. Scale bar: 25 μm. n = 3 independent experiments. I Western blot of cleaved caspase-3, -8, and -9 levels. n = 3 independent experiments. J Enzyme-linked immunosorbent assay (ELISA) quantification of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and IL-6 levels in HLO supernatants. n = 3 independent experiments. Data represent median ± interquartile range (IQR), and analyzed by two-sided two-way ANOVA with Tukey’s multiple comparisons test (E), or two-sided one-way ANOVA with Tukey’s multiple comparisons test (F–J). Source data are provided as a Source Data file.

Fig. 10. A graphic abstract summarizes that HA-si-TDRD9 NPs attenuate Pseudomonas aeruginosa (PA)-driven lung injury.

Pathogenic gene Tudor domain-containing protein 9 (TDRD9) was identified through RNA sequencing of neutrophils collected from bronchoalveolar lavage fluid (BALF) of 21 recruited patients. TDRD9-targeting siRNA was encapsulated into hyaluronic acid (HA)-coated amphiphilic peptide p5RHH nanoparticles (NPs). Administration of HA-si-TDRD9 nanoparticles to PA-infected mice or human lung organoids demonstrated that these NPs disrupt the programmed death ligand-1(PD-L1)/cluster of differentiation 80 (CD80)/mitogen-activated protein kinase (MAPK) signaling pathway, promote neutrophil cuproptosis, and ultimately ameliorate inflammation and lung injury.