To investigate the role of the NKG2D-MICA/B axis in pulmonary fibrosis, we employed a well-established bleomycin (BLM)-induced mouse model. Male C57BL/6 mice (8–10 weeks old, 20–25 g, n=20) were anesthetized with pentobarbital sodium and subsequently administered BLM (1.5 mg/kg in 40 μL of PBS, n=10) or PBS via intratracheal instillation (n=10). Lung tissues were harvested 21 days post-induction for NKG2D-MICA/B detection via Western blot, RT–qPCR, and flow cytometry. To confirm the successful construction of the pulmonary fibrosis model, we examined the expression of fibronectin protein and hydroxyproline in mouse lung tissue. For flow cytometry analysis, NKG2D⁺ cells were gated as CD3^-NK1.1⁺NKG2D⁺ and subjected to semi-quantitative counting.

To study the cellular crosstalk critical to PF pathogenesis, we utilized an in vitro coculture system. Human NK-92MI cells were activated with IL-2 (100 IU/mL) and co-cultured with mitomycin C-treated K562 cells, a standard protocol to enhance NK cell effector functions, prior to coculture with fibroblasts. To explore the underlying mechanism, we first performed in vitro functional validation using the human lung fibroblast cell line MRC-5. MRC-5 were transfected with a MICA-overexpressing plasmid (PLVX-IRES-ZsGreen1-MICA-Stbl3, 2 μg/mL) via Lipofectamine 3000 (Invitrogen). Transfected MRC-5 cells were cocultured with NK-92MI cells (1:5 ratio) in RPMI-1640 medium supplemented with 10% FBS for 48 hours. NKG2D expression on NK-92MI cells was analyzed by flow cytometry.

To validate our findings in a model with greater physiological relevance to human lung fibrosis, we extended our studies to human fetal lung fibroblasts (HFL-1 cells). HFL-1 cells were pretreated with recombinant human TGF-β1 (5 ng/mL) for 24 hours to induce a profibrotic phenotype, simulating a key aspect of the fibrotic microenvironment. These activated fibroblasts were subsequently cocultured with pre-activated NK-92MI cells at a 1:5 ratio in serum-free DMEM for 72 hours. Following coculture, fibroblast expression of the NKG2D ligand MICB and classic fibrosis markers (including fibronectin and TGF-β1) was analyzed by Western blot and/or RT–qPCR. Concurrently, NK cell effector functions were evaluated by measuring the secretion of interferon-gamma (IFN-γ) by ELISA and cytolytic activity via a lactate dehydrogenase (LDH) release assay.

Adeno-associated virus 5 (AAV5) exhibits optimal pulmonary tropism among serotypes (AAV2/5/6/9) because of enhanced lung infection efficiency. AAV5-mediated NKG2D overexpression was achieved via a first-generation adenovirus system. E1/E3-deleted Ad5 plasmids were linearized with PacI, transfected into HEK293 cells (E1-expressing), and purified via CsCl gradient centrifugation. We obtained the following two adeno-associated viruses: pAAV[Exp] (20)-CAG>mKlrk1[NM_033078.4](ns):T2A:EGFP: WPRE, pAAV[Exp]-CAG>EGFP: WPRE. Male C57BL/6 mice (6-8 weeks old) were randomly assigned to six groups: the control group, BLM group, AAV5-EGFP group, AAV5-EGFP+BLM group, NKG2D-AAV5 group and NKG2D-AAV5+BLM group, with 10 mice in each group. On day 0, the mice were intratracheally administered 50 μL of empty vector or AAV5-NKG2D (2*10¹¹vg/ml). On day 14, BLM (0.75 mg/kg, Supplementary Figure 1.) or saline was delivered via intratracheal injection. Daily body weight monitoring was conducted. On day 35 (3 weeks post-BLM), the mice underwent pre-sampling CT imaging.

To evaluate the safety profile of AAV5, liver, spleen, and kidney tissues were harvested and weighed. For detailed characterization of the transduction efficiency and targeting specificity of AAV5, 3-μm-thick lung tissue sections were subjected to immunofluorescence (IF) staining to detect NKG2D expression, thereby directly validating the efficacy of AAV5-mediated gene delivery. Subsequently, histopathological assessments were performed on lung sections using hematoxylin and eosin (HE) staining and Masson’s trichrome staining to evaluate the severity of pulmonary fibrosis and inflammatory infiltration. Furthermore, total protein was extracted from the right lung tissue for the detection of fibrosis-related markers. Western blot (WB) analysis was conducted to examine the expression levels of the NKG2D receptor protein, as well as fibrosis indicators including collagen type I and fibronectin. These results provide additional protein-level confirmation of AAV5 transduction and its regulatory effects on the fibrotic process.

To elucidate the mechanism of action of NKG2D-AAV5 and assess the expression of downstream signaling molecules in the BLM-induced pulmonary fibrosis model, the following experiments were performed: DAP12 protein expression in lung tissue was determined. The interaction between NKG2D and DAP12 was analyzed via immunofluorescence colocalization and coimmunoprecipitation (co-IP). In addition, downstream signaling involves SYK detection (IHC) and aging-related proteins (p-p53, p21) via WB.

To validate the role of NKG2D in pulmonary fibrosis, we treated PF mice with NKG2D antibodies. Male C57BL/6 mice (8–10 weeks old) were subjected to a BLM-induced PF model via single intratracheal instillation of BLM (1.5 mg/kg). On day 0, the mice were randomized into four groups: the control group (n=10), model group (n=12), PFD group (300 mg/kg, qd, n=12), and NKG2D-Ab group (100 μg (21, 22), qod, n=12). Drug interventions were administered accordingly starting from day 1 and continued consecutively for 21 days. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected on day 21. Fibrosis severity was assessed via Masson’s trichrome staining, high-resolution micro-CT imaging, and collagen deposition analysis via western blotting (Fibronectin). Concurrently, inflammation was evaluated through BALF cell counts and histopathological examination via H&E staining. Furthermore, the protein expression of DAP12, SYK, and p21 in lung tissues was determined by IF, IHC, and WB. To clarify the regulatory role of SYK on p53, this study treated HEK293T cells overexpressing p53 with the SYK inhibitor R406 (10 μM), and then detected changes in p53 protein levels by WB.

Statistical analyses were performed using GraphPad Prism (version 9.0; San Diego, CA, USA) and R (version 4.3.1). Data are presented as the mean ± standard error of the mean (SEM). For comparisons between two groups, an unpaired two-tailed Student’s t−test was used after confirming normality with the Shapiro–Wilk test and homogeneity of variances with F−test; otherwise, the non−parametric Mann–Whitney U test was applied. For comparisons among multiple groups, one−way analysis of variance (ANOVA) was used when data met assumptions of normality and equal variance. If the ANOVA showed a significant overall effect (P < 0.05), post−hoc comparisons were conducted using Tukey’s honestly significant difference (HSD) test for all pairwise comparisons, or Dunnett’s test when comparing each treatment group against a single control group. A P−value < 0.05 was considered statistically significant.

Compared with the control group, the expression of fibronectin in the lung tissue of the model group was upregulated, and the content of hydroxyproline, a collagen-characteristic amino acid, was significantly increased, indicating that the animal model of pulmonary fibrosis was successfully constructed (Figures 1A, B). Both mRNA and protein levels of NKG2D and its ligands were significantly elevated in lung tissues compared to controls (Figures 1A, C, D). In mice, the NKG2D ligands H60c and RAET1L are functional homologs of the human MICA/B proteins studied here. Flow cytometric analysis confirmed a pronounced accumulation of NKG2D⁺ NK cells in fibrotic lungs (Figure 1E). Together, these data demonstrate a dysregulated NKG2D-MICA/B axis during PF pathogenesis.

On the basis of these observations, we investigated NK cell–fibroblast interactions via in vitro coculture systems. NK-92-MI cells were activated with IL-2 or cocultured with K562 chronic myeloid leukemia cells at a 1:5 effector-to-target ratio for 72 hours. Functional analysis revealed significant NK cell activation, marked by increased IFN-γ secretion, increased LDH release (Figure 2A), and a substantial increase in NKG2D⁺ cell populations, as detected via flow cytometry (Figure 2B). Collectively, these data confirm that K562 stimulation potently activates NK cells while increasing surface NKG2D expression.

Coculture with MICA-transfected MRC-5 fibroblasts further amplified NKG2D expression on NK cells (Figure 2C), demonstrating ligand-induced receptor modulation. Similarly, when activated NK-92MI cells (pre-stimulated with K562) were cocultured with HLF-1 fibroblasts, MICA/B expression increased (p<0.001, Figure 2D; Supplementary Figure 2). Concurrently, the levels of the fibrotic markers fibronectin and TGF-β were elevated in these coculture systems (Figures 2E, F), suggesting that bidirectional NK-fibroblast communication through the NKG2D-MICA/B axis may drive fibrotic progression.

Our innovative animal model combining NKG2D-AAV5 with BLM induction provides compelling evidence for the pathogenic role of NKG2D. Notably, no significant differences in body weight or organ weight were observed among the experimental groups of mice, suggesting a favorable safety profile for NKG2D-AAV5. IF analysis of whole lung sections revealed colocalization of the NKG2D protein (red fluorescence) and NK1.1⁺ cells (green fluorescence), as evidenced by merged yellow signals. This result specifically confirms that NKG2D expression is restricted to NK cells within murine lung tissue (Figure 3D). Compared with that in the BLM group, the fluorescence intensity of NKG2D at positive sites in both the NKG2D-AAV5 group and the NKG2D-AAV5+BLM combination group was significantly greater (Figure 3D). Collectively, these findings validate the efficient and targeted expression of NKG2D via AAV5 transduction, as well as its precise functional localization to NK cells in this experimental setting.

Advanced imaging and histological analyses provided robust evidence of disease exacerbation. CT scans and Masson’s trichrome staining revealed significantly higher fibrosis scores in the NKG2D-AAV5+BLM group than in the BLM-alone group (Figures 4A, B, D, E). Furthermore, compared with the control group, the combined use of AAV5-NKG2D and BLM significantly increased the protein expression of hydroxyproline in mouse lung tissue (Supplementary Figure 3). Consistently, H&E staining confirmed elevated inflammatory infiltration in the NKG2D-AAV5+BLM group (Figures 4C, F). Molecular analyses further supported these findings, with BALF cell counts significantly increased in the NKG2D-AAV5+BLM group (Figure 4G). WB analysis revealed markedly increased protein expression of collagen-I and fibronectin in NKG2D-AAV5+BLM-treated lungs (Figure 4H). These results collectively establish that NKG2D activation, delivered efficiently and specifically by AAV5, synergizes with BLM-induced injury to accelerate PF through multiple pathological cascades.

Mechanistically, co-immunoprecipitation assays in K562 cells confirmed a robust interaction between NKG2D and its adaptor protein DAP12 (Figure 5A). In vivo, protein expression of DAP12 was significantly increased in the NKG2D-AAV5+BLM group (Figures 5B, C), validating the relevance of this axis. Analysis of downstream signaling revealed pronounced upregulation in the combination group: immunohistochemistry showed elevated SYK expression (Figure 5D), while Western blotting detected increased levels of the senescence-associated markers phospho-p53 (Ser15) and p21 (Figure 5E). These findings indicate that NKG2D overexpression accelerates PF by activating the DAP12-SYK-p53-p21 cellular senescence axis.

Therapeutic intervention with an anti-NKG2D monoclonal antibody in BLM-induced PF mice significantly ameliorated disease progression. Micro-CT quantification showed an approximately 40% reduction in fibrotic lesion volume compared to the BLM model group (Figures 6A, B); histopathological evaluation indicated attenuated lung inflammation and diminished collagen deposition following antibody treatment (Figures 6C–F); and WB further confirmed significant downregulation of the fibrosis marker fibronectin in treated versus untreated PF mice (Figures 6G, H). Collectively, these integrated findings demonstrate that anti-NKG2D antibody therapy effectively reduces PF progression through multifaceted modulation of inflammatory and fibrotic pathways.

We further assessed downstream signaling molecules of NKG2D in lung tissues from PF mice before and after therapeutic intervention. IF analysis revealed significant colocalization of NKG2D and DAP12 proteins in fibrotic lungs, with both proteins markedly downregulated (Figures 7A, B) following anti-NKG2D antibody treatment. Consistently, IHC and WB demonstrated concomitant suppression of SYK and p21 expression (Figures 7C–F), indicating effective blockade of the NKG2D-DAP12-SYK-p53 signaling axis. To investigate the role of SYK in regulating p53, we performed an experiment in HEK293T cells overexpressing p53, treating them with the SYK inhibitor R406. Western blot analysis revealed that R406 treatment significantly reduced p53 protein levels (Figure 7G). This result suggests that SYK activity may positively regulate p53 expression.