Cigarette Smoke Induces the Pro-inflammatory Response of Lung Macrophages via Activation of the IL-33/ST2-Dependent Pathway

Abstract

Cigarette smoke broadly affects the immune system, but the mechanisms by which it disrupts lung innate immunity and impairs macrophage-mediated homeostasis in healthy individuals remain unclear. This study investigates airway inflammation induced by chronic cigarette smoke extract (CSE) exposure by profiling pulmonary macrophages. CSE was generated by bubbling smoke from ten Marlboro cigarettes into 10 ml PBS to obtain 100% CSE, and C57BL/6 mice received 10% CSE intranasally 3 times per week for 4 wk. Chronic CSE exposure induced neutrophilic airway inflammation and augmented pulmonary Th17 and Th1 responses. Among macrophage subsets, CSE exposure promoted the expansion of CD11c⁺CD11b⁻ alveolar macrophages (AMs) and upregulated IL-17A across macrophage subsets in lung tissue. In the lung, epithelial-derived IL-33 was significantly increased and was associated with enhanced IL-33/ST2 axis activity in IL-17A⁺ macrophages. In AMs, sequential CSE and IL-33 stimulation significantly increased ST2 expression and an IL-33/ST2-mediated pro-inflammatory response marked by elevated IL-6. Moreover, CSE exposure reprogrammed AMs with enhanced MAPK and NF-κB signaling. Notably, IL-33-stimulated macrophages after CSE exposure skewed naive CD4⁺ T cells toward Th17 differentiation. These findings suggest that CSE-activated, IL-33/ST2-driven pro-inflammatory macrophages drive neutrophilic inflammation and Th17 skewing, linking innate and adaptive immune responses in airway inflammation.

Graphical Abstract

INTRODUCTION

Smoking is a major risk factor for impaired lung function, and numerous clinical studies have demonstrated a strong association between cigarette smoke (CS) exposure and the development of various lung diseases (1,2). CS induces not only structural damage to the lungs but also oxidative stress and inflammation, thereby contributing to the development and progression of chronic respiratory diseases (3,4,5,6). However, the mechanisms by which CS alters the innate immune system in healthy lungs and how these alterations promote lung disease remain insufficiently understood.

Macrophages are the most abundant immune cells in the lung and play a central role in maintaining pulmonary homeostasis by serving as the first line of defense against inhaled particles and Ags (7). These innate immune cells can adopt distinct functional phenotypes depending on environmental cues, thereby orchestrating appropriate immune responses. Recent studies have shown that in smokers, lung macrophages exhibit reduced expression of phagocytic receptors involved in the recognition of foreign particles and apoptotic cells, along with enhanced pro-inflammatory activity (8,9,10,11). Furthermore, macrophages from smokers are more prone to infection and aberrant immune responses, including autoimmunity (12,13,14). Thus, understanding how CS alters macrophage function is critical for elucidating the pathogenesis of smoke-induced lung diseases.

CS is known to exacerbate Th17 cell responses by upregulating key Th17-associated cytokines, including IL-17A, IL-6, and IL-23, in lung tissue and PBMCs (15,16). Although several studies have investigated the mechanisms underlying smoking-induced Th17 differentiation, the contribution of macrophages to this process remains largely unexplored (17,18).

In this study, we found that exposure to cigarette smoke extract (CSE) reprogrammed macrophages and enhanced pro-inflammatory signaling via the IL-33 and ST2 axis, which was associated with augmented IL-6-mediated type 3 inflammatory responses. These alterations in macrophage profiles suggest that innate immune activation by CSE may amplify adaptive immune responses. Collectively, our findings suggest that CSE-induced macrophage reprogramming may represent a key mechanism contributing to the development of chronic lung inflammation.

MATERIALS AND METHODS

Murine model and CSE treatment

Female 8-wk-old C57BL/6 mice (Orient Bio, Anyang, Korea) were used for this study. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Seoul National University (IACUC No. SNU-200327-1-3). Mice were assigned to one of 2 groups (n=5 per group): PBS control group and a CSE-treated group.

Marlboro Red cigarettes were used to prepare CSE. After removing the filter, cigarettes were combusted for 6–8 min each using Variable-Flox Peristaltic Pumps (Fisherbrand, Shanghai, China). Smoke from 10 cigarettes was bubbled into 10 ml of PBS and passed through a 0.75 µm filter, yielding 100% CSE (Supplementary Fig. 1). For chronic exposure, mice received intranasal instillation of 10% CSE 3 times per week for 4 wk (Fig. 1A).

Figure 1. Effects of CSE on airway inflammatory responses.

(A) Experimental protocol for long-term cigarette smoke exposure in mice. (B) Numbers of inflammatory cells in BALF, including macrophages, neutrophils, eosinophils, and lymphocytes. (C) H&E staining (×400) and quantitative scoring of lung histology. (D, E) Percentages of neutrophils, eosinophils, and their subtypes in lung tissue assessed by flow cytometry. Statistical analysis was performed by the Mann-Whitney U test.

ns, not significant.

^*p<0.05, ^**p<0.01.

Measurement of airway inflammation

Bronchoalveolar lavage fluid (BALF) was collected by washing the lungs with 1.8 ml of PBS (Biowest, Nuaillé, France). Recovered cells were centrifuged onto glass slides at 1,000 rpm for 5 min using a cytospin (Shandon CytoSpin III; Thermo Shandon, Runcorn, UK) and stained with Diff-Quik (Sysmex Co., Kobe, Japan). A minimum of 300 cells, including macrophages, neutrophils, eosinophils, and lymphocytes, were counted and classified.

Histological analysis

The left lobe was fixed in 4% paraformaldehyde at 4°C for 24 h, embedded in paraffin, and sectioned. H&E staining was performed by the Pathology Laboratory at the Seoul National University Hospital Biomedical Research Institute.

Flow cytometry

Lung tissue was minced and digested at 37°C for 90 min in RPMI 1640 medium (Biowest) supplemented with 1 mg/ml collagenase type IV (Worthington, Lakewood, NJ, USA), 10% FBS (Biowest), and 1% penicillin–streptomycin (Biowest). The digested tissue was passed through a 40 µm sterile cell strainer to obtain a single-cell suspension. RBCs were lysed using RBC lysis buffer (Sigma, St. Louis, MO, USA). The 1×10⁶ cells were stained with fluorochrome-conjugated Abs (BioLegend, San Diego, CA, USA) listed in Supplementary Table 1.

For intracellular cytokine staining, cells were stimulated in RPMI 1640 medium containing 1 µg/ml ionomycin (Sigma), 100 ng/ml phorbol 12-myristate 13-acetate (Sigma), and 1 µl/ml GolgiStop (BD Biosciences, San Jose, CA, USA) at 37°C for 3.5 h. For intracellular signaling pathway staining, cells were stimulated for 10 min and then stained. Surface staining was followed by fixation and permeabilization using the Cytofix/Cytoperm kit (BD Biosciences) for 30 min. All staining was performed after Fc receptor blocking with an Fc receptor binding inhibitor Ab (BD Biosciences) for 5 min at 4°C. Flow cytometry was performed using an LSR Fortessa X-20 (BD Biosciences), cell sorting was performed using a FACSAria III (BD Biosciences), and data were analyzed using FlowJo v10.

The flow cytometry gating strategy for macrophages, innate lymphoid cells (ILCs), and Th cells is presented in Supplementary Figs. 2 and 3. Neutrophils and eosinophils were analyzed in detail with a focus on the SiglecF marker (19).

Primary cell isolation

Naive CD4⁺ T cells were isolated from mouse splenocytes using the MojoSort Mouse CD4 Naive T Cell Isolation Kit (BioLegend). Isolated cells were incubated at 37°C for 24 h and then co-cultured with CSE- and IL-33-stimulated macrophages at a 1:1 ratio for 24 h.

Cell culture

CRL-2019 (American Type Culture Collection, Manassas, VA, USA) were seeded at 5×10⁴ cells per well. After 2 h, 0.2% CSE was added. After 48 h of CSE treatment, the culture medium was replaced with fresh medium containing 20 ng/ml recombinant IL-33 (R&D Systems, Minneapolis, MN, USA), and cells were incubated for an additional 24 h.

Quantitative real-time PCR

Total RNA was extracted using TRIzol (Thermo Fisher Scientific, Waltham, MA, USA), and cDNA synthesis was performed using the SensiMix II probe kit (Bioline, London, UK). Quantitative PCR was performed using SYBR Green (Bioline). Relative expression levels were normalized to Hprt1. Primer sequences are listed in Supplementary Table 2.

Statistical analysis

Data are presented as mean ± SEM. Statistical significance was determined using the Mann–Whitney U test or one-way ANOVA followed by Tukey’s post hoc test. Analyses were performed using GraphPad Prism 10 (GraphPad Software, San Diego, CA, USA). A p-value <0.05 was considered statistically significant.

RESULTS

CSE exposure increases pulmonary inflammation and skews the macrophage landscape

Chronic CSE exposure significantly increased neutrophil counts in BALF (Fig. 1A and B). Histological analysis revealed marked neutrophilic infiltration and exacerbated airway inflammation (Fig. 1C). Similar to the BALF analysis, flow cytometry showed that neutrophil and eosinophil populations were increased, with a notable expansion of the SiglecF⁺ neutrophil subpopulation (Fig. 1D and E). These results demonstrate that chronic CSE exposure induces neutrophilic inflammation in the lung.

CSE-exposed inflammation is skewed toward Th17 and Th1 immune responses

Next, we investigated lymphocyte effector functions to further characterize the immune landscape within the lung. No significant changes in total ILCs or their subsets were observed following CSE exposure (Fig. 2A). CSE exposure led to a significant increase in total CD4⁺ T cells in the lung, particularly Th17 and Th1 subsets, whereas Th2 and Treg populations remained unchanged (Fig. 2B-E). Taken together, these results indicate that chronic CSE exposure induces Th17 and Th1 immune responses in the lung.

Figure 2. Changes in ILCs and CD4+ T cells in the lung after CSE exposure.

(A) Percentage of total ILCs and their subtypes in the CSE 4-wk exposure model. (B) Percentages of total T cells, CD4⁺ T cells, and CD8⁺ T cells. (C) Percentages of Th17 and Th1 cells in the lungs and representative dot plots. (D) Percentages of Th2 cells in the lungs and representative dot plots. (E) Percentages of Foxp3⁺CD25⁺ regulatory T cells in the lungs and representative dot plots. Statistical analysis was performed by the Mann-Whitney U test, and one-way ANOVA followed by Tukey’s test as a post-hoc test.

^*p<0.05, ^**p<0.01.

Chronic CSE exposure reshapes the macrophage landscape characterized by alveolar macrophage (AM) activation

We next examined alterations in lung macrophage populations following chronic CSE exposure (Fig. 3A). Although total macrophage numbers increased, this expansion was driven primarily by CD11c⁺CD11b⁻ AM, irrespective of MHCII expression, rather than interstitial macrophages (Fig. 3B-D). We next explored the influx of monocyte-derived macrophages (MoMs) following sustained CSE exposure. In contrast to AMs, CSE exposure did not significantly increase MoMs or alter subsets defined by MHC II expression (Fig. 3E). However, based on CD206 and CD86 expression, both CD206⁻CD86⁺ and CD206⁺CD86⁻ macrophage populations within MoMs were significantly increased in CSE-exposed lungs (Fig. 3F). Specifically, the M2c-like subset (CD206⁺CD86⁻MHCII⁻), associated with tissue remodeling, was markedly elevated (Fig. 3G). Notably, CSE exposure significantly increased IL-17A and IL-13 expression in AMs, with a more modest increase in IL-17A expression observed in CD11c⁻CD11b⁺ MoMs (Fig. 3H and I). These findings indicate that chronic CSE exposure reshapes the lung macrophage landscape, enhancing the pro-inflammatory and tissue-remodeling potential of AMs through increased expression of Th2- and Th17-associated cytokines.

Figure 3. Effects of CSE on lung macrophages.

(A, B) Representative flow cytometry plots and percentages of total lung macrophages. (C) Proportion of CD64⁺CX3CR1⁺ IMs in the lung. (D, E) Proportions of CD11c⁺CD11b⁻ alveolar macrophages and CD11c⁻CD11b⁺ monocyte-derived macrophages in the lung and their MHCII expression. (F) Percentages of CD206⁻CD86⁺CD11c⁺ and CD206⁺CD86⁻CD11c⁻ macrophage subsets among CD11b⁺ macrophages. (G) Proportion of M2 macrophage subtypes within CD11b⁺ macrophages. (H, I) Representative flow cytometry plots showing IL-17A⁺ or IL-13⁺ populations in AMs and MoMs. Statistical analysis was performed by the Mann-Whitney U test.

IM, interstitial macrophage.

^*p<0.05, ^**p<0.01.

The epithelial IL-33–macrophage ST2 axis plays a critical role in CSE-induced lung inflammation

To investigate the epithelial damage-macrophage activation axis associated with CSE-induced type 3 immune responses, we assessed IL-33 and ST2 signaling in lungs exposed to CSE. IL-33 was localized primarily to EpCAM⁺ lung epithelial cells rather than macrophages, and chronic CSE exposure markedly increased IL-33 levels in the lung. Consistent with quantitative fluorescence intensity analysis, both IL-33 protein levels in BALF and Il33 transcript levels in lung tissue were significantly increased (Fig. 4A-C). In summary, chronic CSE exposure significantly induced IL-33 production in the lung, predominantly from epithelial cells.

Figure 4. IL-33/ST2 signaling in lung macrophages induced by CSE.

(A) Protocol for IL-33 analysis in in vivo models. (B) IF staining of PBS- or CSE-exposed lungs. Scale bar=20 μm. White arrowhead-F4/80⁺IL-33⁺, Yellow arrowhead- EpCAM⁺IL-33⁺, Red arrowhead-EpCAM⁺ F4/80⁺IL-33⁺. (C) Gene expression and protein levels of IL-33 in the lung. (D, E) Percentage of ST2⁺ macrophages and their scatter plots in AMs and MoMs. (F, G) Percentage of ST2⁺ macrophages in IL-17A⁺ or IL-13⁺ AMs and MoMs. Statistical analysis was performed by the Mann-Whitney U test.

^*p<0.05, ^**p<0.01, ^****p<0.0001.

Next, we investigated the association between ST2 expression, the receptor for IL-33, and IL-17A⁺ or IL-13⁺ macrophages. ST2 expression was elevated across AMs and MoMs and was markedly upregulated in IL-17A⁺ macrophages, but not in IL-13⁺ macrophages, following CSE exposure (Fig. 4D-G). Notably, IL-17A⁺ macrophages were rarely detected in the ST2⁻ fraction (Supplementary Fig. 4). These results suggest that CSE exposure enhances the pulmonary IL-33 and ST2 axis and that this pathway is more closely associated with type 3 inflammation than with type 2 polarization.

CSE exposure induces increased IL-33 responsiveness, elevated IL-6, and activation of the MAPK and NF-κB signaling pathways in macrophages

We evaluated IL-6 as a potential mediator linking chronic CSE exposure-induced type 3 immune responses with macrophage reprogramming. In our model, chronic CSE exposure increased IL-6 levels in the lung (Fig. 5A). Next, we isolated CD11c⁺CD11b⁻ AMs from an in vivo model to assess whether CSE reprograms AMs to enhance IL-33 responsiveness and inflammatory activity and to determine whether these macrophages represent a major source of IL-6. CSE-conditioned AMs exhibited increased IL-6 secretion ex vivo (Fig. 5B), supporting macrophages as one cellular source contributing to the elevated IL-6 milieu.

Figure 5. Type 3 inflammation-induced effects of CSE exposure via AMs.

(A, B) Experimental protocols and changes in IL-6 levels in BALF and in the percentage of IL-6⁺ AMs isolated from lung tissue. (C) Experimental scheme and cellular signaling pathways in AMs. (D) Co-culture protocol of AMs and naive CD4⁺ T cells and changes in Th cell differentiation. Statistical analysis was performed by the Mann-Whitney U test, and one-way ANOVA followed by Tukey’s test as a post-hoc test.

^*p<0.05.

In addition, co-treatment of CD11c⁺ AMs isolated from control mice and a macrophage cell line with CSE and IL-33 significantly increased the expression of pro-inflammatory markers, including Il6 and Tnfa, as well as St2, Il1b, and Mcp1, compared with IL-33 alone (Supplementary Fig. 5A-C). These findings suggest that CSE exposure exacerbates macrophage responses, with enhanced IL-6 secretion.

CSE exposure significantly activated the MAPK and NF-κB signaling pathways in macrophages (Fig. 5C). These findings suggest that chronic CSE exposure reprograms AMs toward an IL-6-mediated pro-inflammatory state, potentially through engagement of these pathways.

Next, we assessed whether these reprogrammed macrophages could promote type 3 immune features. AMs from chronically CSE-exposed mice significantly skewed naive CD4⁺ T cells toward Th17 differentiation ex vivo (Fig. 5D). Furthermore, in a macrophage cell line under acute CSE exposure conditions, stimulation along the CSE/IL-33–ST2 pathway was associated with a Th17-biased inflammatory program (Supplementary Fig. 6). Taken together, these results suggest that the CSE-driven IL-33/ST2 axis may condition AMs to promote Th17 skewing through induction of inflammatory mediators such as IL-6.

DISCUSSION

This study investigates how chronic exposure to CSE is associated with alterations in the pulmonary macrophage landscape, including AM activation and neutrophilic inflammation. Our results suggest that chronic CSE exposure increases epithelial IL-33 expression and activates the IL-33/ST2 axis, which is accompanied by increased IL-17A, IL-13, and IL-6 levels in AMs and activation of MAPK and NF-κB signaling, collectively creating an environment favorable for Th17 inflammatory responses.

Th17 cells secrete pro-inflammatory cytokines, including IL-17, IL-22, and CXCL3, with IL-17A notably inducing neutrophil recruitment and thereby contributing to chronic inflammation and emphysema (15,20). Recent studies have determined a potential role for macrophage-derived cytokines in the activation of Th17 immune responses (21,22,23). Furthermore, Ly6c⁺ monocyte-derived macrophages in a bleomycin-induced lung injury model have been shown to induce Th17 differentiation (24). Given the established role of IL-17A, primarily secreted by Th17 cells, in activating airway epithelial cells to release neutrophil chemoattractants such as CXCL8 (15,25), IL-17A positivity in macrophages may reflect a smoke-driven type 3 inflammatory milieu rather than definitive evidence of active IL-17A secretion by macrophages. In line with these observations, we identified a skew toward Th17 and Th1 immune responses in CSE-exposed lungs.

Under homeostatic conditions, AMs maintain immune quiescence by suppressing excessive responses to inhaled Ags through intrinsic inhibitory mechanisms (26). CS disrupts this regulation by upregulating pattern recognition receptors, thereby activating innate immune responses and inducing a pro-inflammatory microenvironment in the airway (10,11,12,13,14,27). A previous study suggested that continuous CS exposure in asthmatic lungs increases AM recruitment and polarizes MoMs toward M1 or M2 phenotypes, potentially contributing to the development and exacerbation of asthma (28,29,30). In this study, AMs demonstrated the most significant increase upon exposure to CSE, and we explored the distinct characteristics of AMs and MoMs following CSE treatment.

The secretion of IL-13, which is implicated in airway inflammation and fibrosis, was elevated in CS-exposed AMs (31,32). Moreover, MoMs exhibited an expansion of a CD206⁺CD86⁻MHCII⁻ M2c-like population, which has been linked to tissue-remodeling contexts in prior models (33,34). These findings suggest that CS-induced polarization of macrophages toward M2c-like or IL-13⁺ phenotypes may contribute to tissue remodeling and chronic airway inflammation.

Recent studies highlight that IL-6 is a central instructive cytokine for Th17 lineage commitment and expansion (35,36). Furthermore, macrophage-derived IL-6 is increasingly recognized as a key upstream signal that shapes the inflammatory environment in the lung and promotes Th17 cell expansion (37,38). Specifically, CS exposure has been shown to exacerbate the IL-33/ST2 axis, leading to increased IL-6 production by circulating immune cells (39). This smoke-induced increase in IL-6 has been further associated with the induction of Th17 immune responses, and the marked elevation of IL-6 in smoke-exposed AMs suggests a potential mechanism underlying CS-driven type 3 inflammation (40). Similarly, exacerbated IL-6 reprogramming in monocyte-derived AMs following tobacco exposure highlights its potential role in the pathogenesis of type 3 inflammation and tissue remodeling (30). Our results demonstrate that chronically CSE-exposed AMs are associated with IL-6-mediated distortion of Th17 polarization. These findings support a potential role for reprogrammed AMs in CSE-induced type 3 inflammation.

IL-33 is generally considered a key player in the pathogenesis of asthma and allergic inflammatory diseases, with studies suggesting its influence on M2 macrophage, ILC2, and Th2 cell differentiation in allergic airway inflammation (41,42,43). In addition, IL-33 has been associated with chronic inflammatory diseases such as rheumatoid arthritis and has been suggested to promote Th17 immune responses (44). IL-33-matured dendritic cells produce cytokines such as IL-1β and IL-6, thereby inducing Th17 differentiation and promoting Treg-to-Th17 conversion, while IL-33-activated mast cells also contribute to Th17 differentiation and neutrophilic inflammation (45,46,47,48,49). Notably, CS exposure is known to induce the release of IL-33 from damaged airway epithelial cells (50,51,52,53,54,55). Recent studies have shown that CS skews type 2 inflammatory responses toward a more pro-inflammatory profile, as IL-33-induced ILC2-mediated type 2 immunity transitions toward type 1 immunity upon CS exposure, correlating with reduced ST2 and GATA3 expression (56). Our results show that CSE exposure increases lung IL-33 levels and upregulates ST2 expression in lung macrophages, consistent with enhanced IL-33 responsiveness in CSE-exposed macrophages. Of note, ST2 expression within the CSE-exposed lung was selectively increased in IL-17A⁺ macrophages, in contrast to IL-13⁺ macrophages, indicating that pro-inflammatory macrophages augmented by CSE via the IL-33/ST2 signaling axis are preferentially associated with an IL-17A⁺ phenotype. In addition, we propose that increased expression of pro-inflammatory cytokines such as Tnfa, Il6, and Il23a in smoke-conditioned macrophages may contribute to a Th17-polarizing cytokine milieu. Together with enhanced IL-33 and ST2 signaling in macrophages, these changes may be associated with amplified pro-inflammatory responses in the lung (57).

Initially, IL-33 was thought to predominantly induce type 2 immune responses; however, accumulating evidence has demonstrated its involvement in both innate and adaptive immunity, with the IL-33/ST2 signaling axis engaging downstream pathways such as MAPK and NF-κB (58,59,60). In particular, the MAPK signaling pathway, which has been identified as a major driver of IL-6-mediated inflammation in response to CS exposure, is evident from the perspective of macrophage reprogramming (39). Interestingly, the signaling pathways that amplify the IL-33 and IL-6 axis differ by cell type, involving p42/p44 ERK in human bronchial epithelial cells but p38 MAPK in human PBMCs (61). These findings support the notion that IL-33 and ST2-mediated changes in lung AMs are associated with activation of the p38 MAPK pathway.

More recently, NF-κB signaling has also been recognized as a key downstream component of IL-33 and ST2 signaling in macrophages, particularly in the context of inflammatory priming. It has been established that co-stimulation of macrophages with IL-33 and lipopolysaccharide leads to pro-inflammatory responses through amplification of TLR4/Myd88 signaling pathways (62). In the CSE-only exposure group, Myd88 and Nfκb gene expression was upregulated; however, overt pro-inflammatory responses were observed only upon subsequent IL-33 stimulation, suggesting that CS exposure primes macrophages for enhanced IL-33 responsiveness via Myd88/NF-κB signaling.

It remains unclear how CSE-conditioned macrophages mechanistically regulate type 3 inflammation in vivo. The identity and functional relevance of IL-17A⁺ macrophages require further characterization. In particular, additional studies are needed to determine whether macrophage-associated IL-17A reflects active secretion of a bioactive cytokine and whether it functionally contributes to macrophage reprogramming or downstream type 3 immune responses under chronic CSE exposure. Moreover, additional work is needed to clarify the relationship between the expanded M2c-like population and type 3 inflammation, and to determine whether this subset contributes to tissue remodeling. While our model was designed to study smoke-induced inflammatory reprogramming in otherwise healthy lungs and does not recapitulate fibrotic remodeling, future studies using refined, remodeling-focused models will be required to interrogate M2c-like macrophages and to clarify whether and how IL-33/ST2 signaling contributes to a Th17-skewing inflammatory milieu in this context.

Our findings suggest that chronic CSE exposure induces macrophage reprogramming in the lung via the IL-33/ST2 signaling pathway, skewing macrophages toward an inflammatory AM phenotype characterized by elevated MHCII expression and increased IL-6 production. This reprogramming may contribute to CSE-induced type 3 inflammation. In conclusion, our results provide insight into the fundamental mechanisms underlying airway inflammation triggered by CSE exposure in the lung under homeostatic conditions.

Abbreviations

AM

alveolar macrophage

BALF

bronchoalveolar lavage fluid

CS

cigarette smoke

CSE

cigarette smoke extract

FSC-A

forward scatter area

FSC-H

forward scatter height

FSC-W

forward scatter width

IACUC

Institutional Animal Care and Use Committee

ILC

innate lymphoid cell

MoM

monocyte-derived macrophage

SSC-A

side scatter area

SSC-H

side scatter height

SSC-W

side scatter width

SUPPLEMENTARY MATERIALS

Supplementary Table 1

Flow cytometry and immunofluorescence staining antibody list

Supplementary Table 2

Primer sequences used in quantitative PCR amplification

Supplementary Figure 1

A schematic diagram of CSE preparation.

Supplementary Figure 2

FACS gating strategy for macrophage populations.

Supplementary Figure 3

FACS gating strategy for T cells and ILCs.

Supplementary Figure 4

Cytokine profile in ST2^- lung macrophages.

Supplementary Figure 5

Changes in IL-33/ST2 axis, signaling pathways, and cytokines in AMs following CSE pre-exposure in vitro.

Supplementary Figure 6

The CSE/IL-33 axis promotes the in vitro induction of type 3 inflammation in macrophages.