Endotoxin-free S100A8/A9 heterodimer (Calprotectin) was purchased from R&D Systems (Cat# 2220-S8-050). The following function-blocking monoclonal antibodies or western blotting antibodies were used: anti-TLR4 (InvivoGen Cat# mabg-htlr4-2) and anti-RAGE (R&D Systems, Cat # 1145-RG). Anti-total IκBα (Cat# 4814) and β-actin (Cat# 4970) were purchased from Cell Signaling Technology. HRP-conjugated secondary antibodies were from Cell Abcam.

Primary HBE cells were obtained from Lonza (Cat# CC-2540) and cultured in BEGM medium (Lonza, Cat# CC-3170) as described previously (Qin et al., 2022). For experiments, HBE cells were cultured onto collagen-coated plates (Corning) and grown to 80–90% confluence. Twenty-four hours prior to bacterial infection or stimulation, the culture medium was replaced with antibiotic-free BEGM basal medium supplemented with only the BEGM SingleQuots™ Triiodothyronine, Transferrin, and Insulin (T/T/I) supplements to minimize confounding effects from serum or other growth factors.

Klebsiella pneumoniae serotype K2 (ATCC 43816) was used as the wild-type (WT) strain. An isogenic acapsular mutant (Δcps) of a K2 strain, a gift of Dr. Lisong Chen (University of South China), was used as a control. Bacteria were grown from frozen glycerol stocks in LB broth at 37 °C for 12 h with shaking (220 rpm). For infection experiments, the overnight culture was subcultured at a 1:100 ratio in fresh LB broth and grown to mid-logarithmic phase. Cells were centrifugated and washed with PBS, and resuspended in antibiotic-free BEBM basal medium. Concentration was determined from OD₆₀₀ (1.0 ≈ 1 × 10⁹ CFU/mL) and confirmed by plating.

Prior to infection, HBE cell monolayers at 80–90% confluence were washed with PBS. Bacteria were inoculated at various Multiplicity of Infection (MOI) and infection was synchronized via low-speed centrifugation before cultured at 37 °C in 5% CO₂. For S100A8/A9 stimulating experiment, cells were incubated with endotoxin-free recombinant human S100A8/A9 protein (R&D Systems) at the indicated concentrations for specified durations. Culture supernatants were harvested for cytokine assays; or cells were lysed for PCR or western blotting.

Following RNA extraction from HBE cells using the RNeasy Mini Kit (QIAGEN) with on-column DNase I digestion, total RNA was quantified and 1 μg was reverse-transcribed into cDNA (Applied Biosystems, Cat#4368814). Gene expression was quantified by real-time PCR on a StepOnePlus system with SYBR Green Master Mix (Applied Biosystems, Cat#A25742). Data were analyzed using the 2^−ΔΔCt method (Luo et al., 2021). For genes showing a decrease in expression (where 2^−ΔΔCt<1), the results are reported as fold reduction by calculating the negative inverse of the fold change (−1/2^−ΔΔCt), according to the standard protocol by Schmittgen and Livak (2008). All primer sequences are provided in Table 1 (Sangon Biotech) (Vogl et al., 2018).

Cell culture supernatants were collected at specified time points. The concentrations of secreted S100A8/A9 heterodimer, IL-8, and IL-6 were quantified by commercial Quantikine ELISA kits (R&D Systems, Cat# DS8900 for S100A8/A9, D8000C for IL-8, and D6050B for IL-6). Following the kit instructions, absorbance was measured (F50, TECAN).

To overcome the transfection challenge in primary epithelial cells, HBE cells were subjected to siRNA-mediated S100A9 knockdown via the Amaxa™ 4D-Nucleofector™ System (Lonza). For each electroporation, 2 × 10⁶ cells in 100 μL P3 Primary Cell Solution were combined with 200 pmol of S100A9-targeting or non-targeting control siRNA (Horizon Discovery). After applying the manufacturer’s recommended pulse program for airway cells, transfected cells were replated in BEGM medium and allowed to recover for 48 ~ 72 h. Successful knockdown was validated at both the transcriptional (qRT-PCR) and secretory protein (ELISA) levels.

To block specific receptors, HBE cells were separately pre-treated for 1 h with either an anti-human TLR4 (10 μg/mL) or an anti-human RAGE (100 μg/mL) neutralizing antibody, or their respective isotype controls prior to stimulation with recombinant S100A8/A9. Following stimulation, supernatants were collected for cytokine measurement by ELISA.

Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. Following protein quantification (BCA assay), samples (20–30 μg) were subjected to SDS-PAGE and transferred to PVDF membranes. For immunodetection, membranes were blocked in 5% milk and then incubated with specific primary antibodies overnight at 4 °C. After washing, HRP-conjugated secondary antibodies were applied, and proteins were visualized by chemiluminescence (SuperSignal™ West Pico PLUS) on a ChemiDoc™ imager.

Following transfection and infection on coverslips, HBE cells were fixed, permeabilized, and blocked. NF-κB p65 was detected using a specific primary antibody and an Alexa Fluor 488-conjugated secondary antibody, with DAPI to identify nuclei. Imaging was performed on a Nikon Ts2R microscope.

Primary human neutrophils were isolated from healthy donors via Ficoll-Paque density gradient centrifugation. Chemotaxis assays were performed using Transwell inserts with a 3 μm pore size. Conditioned media (CM) were harvested from HBE cells following 24 h of infection or specific treatments. CM samples or fMLP (100 nM, as a positive control) were loaded into the lower chambers, and neutrophils (5 × 10⁵ cells) were introduced into the upper chambers and allowed to migrate for 90 min at 37 °C. Transmigrated cells were collected and quantified by flow cytometry (BD FACSCalibur) using CountBright™ Absolute Counting Beads (Thermo Fisher Scientific) as an internal standard to determine absolute cell counts relative to the beads within a defined gate.

The study was conducted in accordance with the Declaration of Helsinki. The study protocol involving human participants was reviewed and approved by the Ethics Committee of the University of South China (Approval No. 2023–007). All healthy donors provided informed written consent prior to blood collection for the isolation of primary neutrophils.

Data represent at least three biologically independent experiments. Values are shown as mean ± SEM. Statistical analyses were performed using GraphPad Prism 9 Software. Comparisons between two groups were made using an unpaired, two-tailed Student’s t-test. Comparisons among three or more groups were performed using one-way or two-way analysis of variance (ANOVA) followed by Tukey’s or Dunnett’s post-hoc test for multiple comparisons. p < 0.05 was considered statistically significant.

To investigate whether the airway epithelium is a direct source of the alarmin S100A8/A9 during bacterial infection, HBE cells were infected with wild-type K. pneumoniae. Quantitative RT-PCR showed that K. pneumoniae infection for 8 h induced an MOI-dependent increase in the mRNA of S100A8 and S100A9 (Figures 1A,B). Furthermore, the transcription of S100A8 and S100A9 exhibited a time-dependent increase following infection with K. pneumoniae at an MOI of 100 (Figures 1C,D). This transcriptional upregulation was followed by a marked increase in the secretion of the S100A8/A9 heterodimer into the culture supernatant, as quantified by ELISA (Figure 1E). A direct statistical comparison between the bacterial strains revealed that the isogenic acapsular mutant (Δcps) elicited significantly higher levels of S100A8/A9 protein secretion compared to the WT strain at equivalent MOI (Figure 1E). These data provide concrete evidence that the bacterial capsule functions as a physical shield that masks the bacterial ligands responsible for triggering the alarmin response in HBE cells.

To explore whether bacterial infection could alter the sensitivity of epithelial cells to endogenous alarmins, the expression of the S100A8/A9 receptor TLR4 was examined post-infection. Following infection of HBE cells with WT K. pneumoniae, qRT-PCR analysis demonstrated a time-dependent upregulation of TLR4 mRNA, which peaked at 12 h, a trend that was also observed for total TLR4 protein by Western blot (Figures 2A–C). We next sought to determine if HBE cells, in addition to producing S100A8/A9, could also respond to it. Uninfected HBE cells were treated with recombinant human S100A8/A9. This stimulation resulted in a robust secretion of the IL-8 and IL-6 (Figures 2D,E). These data suggest that K. pneumoniae infection not only induces the production of S100A8/A9 but also enhances the expression of its cognate receptor, potentially sensitizing the epithelium to subsequent S100A8/A9 signaling.

To establish the functional importance of epithelial-derived S100A8/A9, siRNA was used to specifically silence S100A9 expression in HBE cells, which prevents the formation of the stable heterodimer. Successful knockdown was validated by qRT-PCR, showing a 3.18-fold reduction in S100A9 mRNA levels compared to control siRNA-transfected cells (Figure 3A), which was consistent with the decreased S100A8/A9 protein secretion (Figure 3B). To ensure that the attenuated inflammation was not due to altered bacterial load, we confirmed that S100A9 silencing did not affect the initial adherence or internalization of K. pneumoniae (Supplementary Figure S1A). Following infection with WT K. pneumoniae, S100A9-deficient cells exhibited a significantly attenuated inflammatory response, with a 65% reduction in IL-8 and a 50% reduction in IL-6 secretion compared to control cells (Figures 3C,D). Critically, this inhibitory effect was specific to bacterial challenge, as S100A9 deficiency did not impair the cytokine response to a non-bacterial stimulus, TNF-α (Supplementary Figures S1B,C). These findings collectively demonstrate that the epithelial S100A8/A9-TLR4 axis is a mandatory component of the early inflammatory cascade during infection.

Previous studies have established that the S100A8/A9 heterodimer can signal through either TLR4 or RAGE (Kang et al., 2015; Ma et al., 2017). To identify the specific receptor facilitating the autocrine loop in HBE cells, we performed targeted blocking assays. Initially, we tested the effect of exogenous rS100A8/A9 stimulation. Pre-incubation with a neutralizing anti-TLR4 antibody significantly abrogated the secretion of IL-8 and IL-6 induced by rS100A8/A9 (Figures 4A,B). In contrast, an anti-RAGE antibody had no significant inhibitory effect (Figures 4C,D). Critically, to validate this axis during an active infection, we performed receptor blockade during K. pneumoniae challenge. As shown in Figures 4C,D, the robust cytokine burst triggered by K. pneumoniae was significantly attenuated by the anti-TLR4 antibody, whereas the anti-RAGE antibody provided no protection. These results, confirm that TLR4 is the principal and mandatory signaling conduit for both the alarmin protein itself and the endogenous alarmin loop initiated by bacterial infection.

To delineate the molecular mechanism of the alarmin-mediated amplification loop, we utilized two complementary approaches. First, to determine if S100A8/A9 is a direct activator of the signaling cascade, uninfected HBE cells were stimulated with rS100A8/A9. This exogenous challenge triggered the degradation of IκB and the nuclear translocation of p65 (Figures 5A–C), confirming the potent signaling potential of alarmin. Second, to assess whether the endogenous alarmin loop is required for pathway activation during the host-pathogen interaction, we monitored NF-κB dynamics during K. pneumoniae infection in S100A9-deficient cells. As expected, the robust signaling response induced by K. pneumoniae was significantly attenuated when the endogenous alarmin system was silenced (Figures 5D,E). Consistent with these biochemical findings, immunofluorescence analysis revealed that the nuclear translocation of p65 induced by K. pneumoniae was markedly reduced in S100A9-deficient cells compared to control cells (Figure 5F). These results collectively demonstrate that the S100A8/A9 autocrine loop is the principal driver of strong NF-κB activation in epithelial cells following K. pneumoniae infection. Finally, to establish a direct functional link between this signaling axis and cytokine release, we utilized the pharmacological NF-κB inhibitor BAY 11–7,082. As shown in Figure 5G, pre-treatment with BAY 11–7,082 significantly abrogated the secretion of IL-8 and IL-6 induced by rS100A8/A9 stimulation. These data provide robust functional confirmation that the canonical NF-κB pathway is the mandatory signaling conduit for this amplification loop.

To determine the functional consequence of the epithelium-intrinsic S100A8/A9 amplification loop, we investigated whether supernatants from infected HBE cells could recruit neutrophils. A Transwell migration assay was performed using freshly isolated primary human neutrophils, with migrated cells quantified by flow cytometry. As shown in Figure 6A, conditioned supernatants from HBE cells infected with WT K. pneumoniae induced robust and significant neutrophil migration, which was substantially greater than that induced by supernatants from uninfected cells. Crucially, to establish the specific role of the epithelial S100A8/A9 loop in this process, we tested supernatants from S100A9-deficient HBE cells. The chemotactic potential of supernatants from infected, S100A9-deficient cells was significantly diminished, with the number of migrated neutrophils reduced by approximately 60% versus controls (Figure 6B). Collectively, these findings position the S100A8/A9-TLR4 autocrine signaling axis within the epithelium as a key mechanism for orchestrating neutrophil influx to infection sites.