Mouse macrophage RAW 264.7 cell lines were kindly provided by Cell Bank, Chinese Academy of Sciences. RAW 264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Cat. BC-M-005, Bio-Channel) containing 10% Fetal Bovine Serum (FBS) (Cat. BC-SE-FBS07, Bio-Channel), and 1% penicillin/streptomycin (Cat. BL505A, Biosharp) at 37°C with 5% CO₂.

Proteins were harvested from cells using a lysate solution and then centrifuged at 12,000 rpm for 10 min at a temperature of 4°C. The supernatant was collected, and the protein concentration of each sample was determined using the bicinchoninic acid (BCA) method and adjusted to achieve consistent protein concentrations across samples. Subsequently, the proteins were denatured by heating at 100°C for 10 min after mixing with 5x protein loading buffer. The protein samples were then separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes using a transfer buffer. These membranes were incubated overnight at 4°C with specific antibodies, which were diluted in a solution of 5% BSA in Tris-buffered saline containing 0.1% Tween-20 (TBST). Enhanced chemiluminescence reagents (NcmECL Ultra; Cat. E422-01; Vazyme Biotech Co., Ltd) were used to visualize the bound antibodies, and images were captured using the Vilber Fusion FX system.

The following primary antibodies were used: anti-IκB-α (1:1000, Cat. ET1603-6), anti-NF-κB p65 (1:1000, Cat. ET1603-12), and anti-p38 (1:1000, Cat. ET1702-65) antibodies, which were obtained from HUABIO. The anti-p-p38(Thr180/Tyr182) (1:1000, Cat. #AF4001) and anti-p-IRAK4(Thr345/Ser346) (1:1000, Cat. #DF7567) antibodies were purchased from Affinity Biosciences. The anti-p-NF-κB p65(Ser468) (1:2000, Cat. 82335-1-RR) and anti-IRAK1 (1:1000, Cat. 10478-2-AP) antibodies were purchased from Proteintech. The anti-p-ERK1/2(Thr202/Tyr204) (1:2000, Cat. #4370), anti-ERK1/2 (1:2000, Cat. #4695) and anti-IRAK4 (1:1000, Cat. #4363) antibodies were purchased from Cell Signaling Technology.

Conventional real-time qPCR was used to assess the mRNA levels in RAW 264.7 cells and mouse lung tissues. The following primer sequences were used.

Mouse Tnfα: Forward Primer (5′-3′) TGATCCGCGACGTGGAA; Reverse Primer (5′-3′) ACC​GCC​TGG​AGT​TCT​GGA​A.

Mouse Il6: Forward Primer (5′-3′) GAG​GAT​ACC​ACT​CCC​AAC​AGA​CC; Reverse Primer (5′-3′) AAG​TGC​ATC​ATC​GTT​GTT​CAT​ACA.

Mouse Gapdh: Forward Primer (5′-3′) GGA​GCG​AGA​TCC​CTC​CAA​AAT; Reverse Primer (5′-3′) GGC​TGT​TGT​CAT​ACT​TCT​CAT​GG.

The protocol is similar to previous study as described (Cai et al., 2024).

The harvested culture medium and bronchoalveolar lavage fluid (BALF) were subjected to analysis for the detection of TNF-α (Cat. #88-7324, ThermoFisher) and IL-6 (Cat. #88–7064, ThermoFisher). This was achieved using ELISA in strict accordance with the manufacturer’s guidelines.

Furthermore, a staining protocol was implemented to monitor the translocation of the NF-κB p65 subunit within cells to assess NF-κB activation. RAW 264.7 cells were fixed with a 2% formaldehyde solution in PBS for 20 min. This was followed by permeabilization using a 0.5% Triton X-100 solution in PBS for 10 min. Subsequently, the cells were blocked with a 5% BSA solution in PBS for an hour.

After fixation, the cells were incubated with anti-NF-κB p65 antibody (1:100) overnight. The cells were then washed three times with PBS and stained with Alexa Fluor 647-conjugated secondary antibodies (1:500, Cat. A0468; Beyotime Biotechnology) for an hour, and with DAPI (Cat. C1002, Beyotime Biotechnology) for 10 min.

Cell imaging was performed using an EVOS M7000 cell imaging system from ThermoFisher. The statistical analysis of colocalization was executed using the colocalization finder plugin in ImageJ.

C57BL/6 mice, each with an approximate weight of 20 g, were procured from the Experimental Animal Center of Hangzhou Medical College. The mice were housed in a controlled environment with a consistent temperature and a structured light-dark cycle. They were provided with a nutritionally balanced diet and access to fresh water. The welfare of these animals was meticulously upheld, adhering to the rigorous ethical guidelines set forth by the Animal Ethics Committee of Hangzhou Medical College.

In the described experimental setup, mice were allocated into five distinct groups, with each group comprising eight mice:

I. Control mice (Con);

For the treatment, KT-474 and JH-I-25 were administered intragastrically at a dosage of 200 μL/20 g body weight. The control and LPS groups received an equivalent volume of vehicle solution, which consisted of 5% DMSO, 30% PEG-400, and 65% saline. Following a 12-h treatment period with the vehicle, KT-474, or JH-I-25, the mice were then challenged with 5 mg/kg of LPS through intratracheal instillation except for the control group. Six hours after LPS administration, the mice were euthanized, and BALF, serum, and major organ tissue samples were collected for subsequent testing. The collected BALF samples were subjected to centrifugation at 1000 rpm for 10 min at a temperature of 4°C. The supernatant was collected and the concentrations of cytokines TNF-α and IL-6 were detected. These cytokines are key markers of inflammation, and their levels in the BALF can provide insight into the efficacy of the treatments in reducing lung inflammation in the context of LPS-induced ALI.

The lung tissues were first fixed in 4% paraformaldehyde for a period of 48 h. After fixation, the tissues were dehydrated through a graded series of alcohol concentrations and then embedded in paraffin. Subsequently, 5 µm-thick sections of the paraffin-embedded tissues were prepared for immunohistochemical staining. H&E staining was performed to visualize the tissue morphology and assess histopathological changes. The stained tissue sections were then examined under a light microscope. For a more objective and quantitative assessment, the images were analyzed using ImageJ software. Lung injury was quantified using the Smith scoring method, consistent with previously published studies (Xiao et al., 2025).

For F4/80 and Myeloperoxidase (MPO) staining, lung tissue that was embedded in paraffin was subjected to deparaffinization and rehydration, followed by antigen retrieval using a citrate buffer (pH 6.0) heated to boiling for 10 min. Thereafter, endogenous peroxidase activity was neutralized with a 3% hydrogen peroxide solution. The sections were then exposed to 5% bovine serum albumin (BSA) for 1 h to block non-specific binding, and then incubated with the F4/80 or MPO primary antibodies for 12 h on a shaker maintained at 4°C. The sections were treated with an HRP-conjugated secondary antibody for 15 min, developed with DAB chromogen, and counterstained with hematoxylin. The samples were observed under an inverted fluorescent microscope. The intensity of the histochemical staining was quantitatively analyzed using ImageJ software (NIH).

The data presented in this study was collected from a minimum of three independent trials and was expressed as the mean ± standard error of the mean (SEM). Statistical analysis was conducted using GraphPad Prism (GraphPad).

For comparisons between two sets of data, a two-tailed unpaired Student’s t-test was employed. When more than two data groups were compared, one-way ANOVA followed by Dunnett’s post hoc test was used. The colocalization level of the NF-κB p65 subunit was calculated for each individually transfected cell using the ImageJ colocalization finder plugin, with the Pearson value indicating the degree of colocalization.

The threshold for statistical significance was set at P < 0.05. Post-tests were only conducted if the F statistic achieved a P-value less than 0.05, assuming the absence of significant discrepancies in variance homogeneity.

Considering the critical function of IRAK4, the phosphorylation of IRAK4 was evaluated in an LPS-induced acute lung injury (ALI) model. According to the data in Figures 1A–D, the gene expression of pro-inflammatory markers Il6, Tnfα, Vcam, and Icam was found to be upregulated in the ALI model. Additionally, LPS-induced ALI mice exhibited elevated levels of TNF-α and IL-6 in both serum and BALF (Figures 1E–H). Histopathological analysis of hematoxylin and eosin (HE)-stained lung tissues (upper Figures 1I,J) revealed erythrocyte accumulation in the pulmonary capillaries, thickening of the pulmonary septum, fibrin exudation in the alveolar spaces, and disrupted alveolar architecture, confirming the successful induction of an inflammatory response by LPS. Moreover, immunohistochemistry analysis revealed an accumulation of macrophages in the lungs, as indicated by an increase in F4/80, a specific macrophage marker, suggesting the occurrence of an inflammatory cascade (lower Figures 1I,K). Significantly, IRAK4 phosphorylation was found to be significantly elevated in tissue samples induced by ALI (Figure 1L), consistent with its critical role in the LPS-stimulated TLR4 signaling pathway.

Based on the function of IRAK4 in the LPS-induced acute lung injury model, the efficacy of KT-474 (structure shown in Figure 2A) in suppressing inflammatory reactions was further explored in vitro. RAW 264.7 cells were pre-incubated with 500 nM KT-474 or a representative IRAK4 inhibitor JH-I-25 and stimulated with 0.5 μg/mL LPS for 1 h. At this concentration, neither compound exhibited significant cytotoxicity, ensuring that the observed effects were not confounded by cell viability issues (Supplementary Figure S1). Western blot analysis revealed that both KT-474 and JH-I-25 effectively inhibited the phosphorylation of IRAK4 (Figures 2B,C). However, only KT-474 was able to degrade IRAK4, thereby disrupting its scaffold function (Figures 2B,D). LPS stimulation activates IRAK4’s kinase activity, leading to downstream degradation of IRAK1 and phosphorylation of the MAPK signaling pathway. Both KT-474 and JH-I-25 were able to suppress these LPS-induced effects, indicating their capacity to interfere with IRAK4 kinase function-mediated signaling (Figures 2E–H). To determine the effect of KT-474 on the NFκB pathway, RAW 264.7 cells were pre-treated with KT-474, JH-I-25, and DMSO for 24 h and stimulated with LPS for 0, 15, 30, 60, 120 and 180 min (Figure 2I). KT-474 effectively blocked the phosphorylation of NF-κB p65 and showed inhibition of IκB-α degradation, reflecting the results of a previous study (Pereira et al., 2022). In contrast, IκB-α degradation occurred as early as 15 min in JH-I-25-treated macrophages (Figure 2I). In addition, KT-474 treatment significantly reduced LPS-induced NFκB p65 translocation into the nucleus compared to JH-I-25 treatment (Figure 2J). These results indicated that the IRAK4 degrader KT-474 alone effectively inhibited the NF-κB pathway, leading to better inhibition of TLR4 signaling pathway compared JH-I-25.

Considering that KT-474 suppresses both the MAPK and NF-κB pathways in LPS-stimulated RAW 264.7 cells, the potential inhibitory effects of KT-474 on the transcription and expression of key inflammatory cytokines IL-6 and TNF-α were analyzed. For this purpose, RAW 264.7 cells were pre-treated with various concentrations of KT-474 (20, 100, and 500 nM) or 500 nM JH-I-25 for 1 h, followed by stimulation with 0.5 μg/mL LPS for 6 h. Our results revealed that KT-474 treatment led to a dose-dependent decrease in the transcription and production of both IL-6 and TNF-α (Figures 2K–N). Notably, this inhibitory effect was more pronounced with KT-474 than with JH-I-25, even at the highest concentration of KT-474 used (500 nM). These findings further highlight the potential therapeutic advantages of KT-474 over conventional IRAK4 inhibitors in modulating inflammatory responses.

Considering dual role of IRAK4 in TLR signaling, straightforward inhibition might not fully harness its therapeutic potential. KT-474 emerges as a promising candidate, showing the capacity to effectively degrade IRAK4 in both in vitro and in vivo settings. Therefore, the IRAK4 degradation ability of KT-474 was further explored in RAW 264.7 cells. Our findings demonstrated that KT-474 degrades IRAK4 in a dose-dependent manner, with a DC₅₀ value of 4.034 ± 0.243 nM, indicating the concentration at which 50% of IRAK4 degradation occurs (Figure 3A).

Further experiments were conducted to investigate the mechanism behind KT-474-induced IRAK4 degradation. RAW 264.7 cells were pre-treated with JH-I-25 (an IRAK4 specific inhibitor), thalidomide (a cereblon (CRBN) E3 ligand), and MG132 (a proteasome inhibitor) for 2 h before 24 h of KT-474 treatment (Figures 3B–D). The prevention of IRAK4 degradation by these inhibitors suggests that the degradation is mediated by the ubiquitin-proteasome pathway. Specifically, the involvement of the CRBN ligand indicates that KT-474 may engage the CRBN E3 ligase complex to ubiquitinate IRAK4, marking it for subsequent degradation by the proteasome.

This mechanism underscores the nuanced approach of KT-474, which does not merely inhibit IRAK4 activity but actively directs its degradation, potentially overcoming the limitations of simple inhibition strategies. The findings indicate the therapeutic potential of KT-474, leveraging the ubiquitin-proteasome system to modulate IRAK4 levels and, by extension, TLR signaling in disease contexts.

KT-474 has shown significant anti-inflammatory activity, exceptional kinome selectivity, and oral bioavailability (Ackerman et al., 2023; Zheng et al., 2024), and has emerged as a potential candidate for the in vivo LPS-induced ALI model. C57BL/6 mice were pre-treated with KT-474 at doses of 10 mg/kg and 20 mg/kg before the LPS challenge, with JH-I-25, a parent IRAK4 kinase inhibitor, serving as a positive control. Following intratracheal LPS administration (5 mg/kg), serum, BALF, lung and other major organ tissue samples were collected, as displayed in Figure 4A. Histological analysis (H&E staining) of major organs, along with serum biochemical parameters, indicated that neither KT-474 nor JH-I-25 caused significant toxicity or adverse effects in mice, supporting their safety profiles under the experimental conditions (Supplementary Figures S2A–C). LPS induced significant pulmonary injury and edema, leading to an increase in total cells in BALF (Figure 4B) and an elevated lung wet/dry weight ratio (Figure 4C). Notably, KT-474 exhibited a more potent inhibitory effect compared to JH-I-25, demonstrating its superior efficacy in mitigating lung injury. The impact of KT-474 on downstream MAPK and NF-κB activities in vivo was also evaluated. Immunoblotting of mouse lung tissue samples revealed that LPS challenge triggered phosphorylation of NFκB p65 and ERK while decreasing IκB-α levels (Figure 4D). JH-I-25 demonstrated significant in vivo MAPK inhibition, as evidenced by markedly reduced p-p38 levels; however, it did not inhibit IκB-α degradation and NFκB p65 phosphorylation. Consistent with in vitro findings, KT-474 showed superior inhibitory activity against NF-κB compared to the kinase inhibitor JH-I-25.

Furthermore, KT-474 can more effectively suppress the transcription of inflammatory markers (Figures 4E–H). JH-I-25 effectively reduced the expression levels of TNF-α and IL-6 in lung tissues, while KT-474 significantly diminished the LPS-induced rise in pro-inflammatory cytokines (TNFα and IL-6) in serum and BALF samples (Figures 4I–L). Histopathological examination of lung sections revealed that KT-474 attenuated the structural damage of lung tissues caused by LPS exposure (Figure 4M). Immunohistochemical analysis targeting F4/80 (macrophage marker) and MPO (neutrophil marker), were conducted to assess the impact of KT-474 on macrophage presence in the LPS-induced ALI model. The results indicated an upsurge in F4/80 and MPO immunoreactivity in the lungs of mice subjected to LPS, whereas KT-474 treatment notably reduced F4/80-positive macrophage and MPO-positive neutrophil accumulation compared to both the LPS-stimuli and the JH-I-25-treated groups (Figure 4N; Supplementary Figure S2D).

ALI is a grave condition leading to approximately 30%–45% mortality in patients (Meyer et al., 2021), and the impact of KT-474 on the survival of mice with acute inflammation was evaluated. A sepsis mouse model was established by an intravenous injection of 32 mg/kg LPS. Daily intraperitoneal injections of KT-474 (at 10 mg/kg and 20 mg/kg), 20 mg/kg JH-I-25, and vehicle control were administered (Figure 5A). The 7-day observation period revealed a significantly lower mortality rate in the KT-474- and JH-I-25-treated groups compared to the sepsis model group. Notably, the effectiveness of KT-474 surpassed that of JH-I-25. According to our results, KT-474 significantly enhanced the survival of mice with acute bacterial inflammation, aligning with the observation that KT-474 counters the severe inflammatory response triggered by LPS in ALI (Figure 5B). These findings underscore the protective effects of KT-474 on LPS-induced ALI.