AECs respond to environmental exposures by releasing alarmins, including thymic stromal lymphopoietin (TSLP), IL-25, and IL-33, which activate type 2 innate lymphoid cells (ILC2s), eosinophils, and mast cells (MCs), driving inflammation and immune cell recruitment (11). In asthma-chronic obstructive pulmonary disease overlap, AECs increase the secretion of other Damage-Associated Molecular Patterns (DAMPs), such as high mobility group box 1 protein (HMGB1), 70-kilodalton heat shock proteins (HSP70), cathelicidin LL-37 (LL-37), and calcium-binding protein A8 (A8), leading to the recruitment of effector cells and the initiation of inflammation (12). Additionally, epithelial cells and other cell types, such as basal, club, ciliated, goblet, ionocytes, endothelial, and fibroblast cells, are induced by IL-4, IL-13, and TGF-β to secrete periostin. The overexpression of periostin results in mucus hypersecretion, goblet cell hyperplasia, and increased matrix metalloproteinase (MMP)-9 secretion, contributing to eosinophil inflammation and permanent airway obstruction (13). IL-4 and IL-13 also trigger AECs to produce eotaxin, which attracts fibroblasts and eosinophils to the inflamed site (14). Moreover, activated AECs, neutrophils, and macrophages can secrete YKL-40, correlating with airway inflammation and remodeling (15).

Cilia on AECs are vital in respiratory disease. Motile cilia play a critical role in lung homeostasis by beating the mucus layer, which traps inhaled pathogens, toxins, and particles out of the airway. Abnormalities in ciliary function, quantity, and structure impair mucus clearance, increasing susceptibility to infections and promoting chronic inflammation (16, 17). Primary cilia act as cellular antennas, influencing the differentiation, migration, and cell fate control of AECs, airway smooth muscle (ASM) cells, and fibroblasts (18). Interestingly, although the quantity of primary cilia does not differ in normal epithelium, it significantly increases in COPD, specifically in the remodeling area (18). The increased antennas may contribute to dysregulated tissue repair, leading to abnormal differentiation and proliferation, ultimately resulting in airway thickening.

In the alveoli, alveolar type 1 cells (AT1) facilitate gas exchange, while alveolar type 2 cells (AT2) repair tissue damage through self-renewal, differentiation into AT1, secretion of TGF-β, and production of surfactant. Cigarette smoke and PM_2.5 have been shown to induce apoptosis in AECs and disrupt the differentiation of AT2 into AT1, leading to pulmonary emphysema in COPD murine models (19).

DCs identify ligands through pattern recognition receptors (PRRs), which trigger them to co-activate naïve helper T cells (Th0) via antigen presentation and cytokine secretion. The cytokines produced by DCs depend on the types of antigens and the subpopulations of DCs, creating the microenvironment around the site of inflammation (20). cDCs can be classified into conventional DCs type 1 (cDC1), conventional DCs type 2 (cDC2), plasmacytoid DCs (pDCs), and monocyte-derived DCs (moDCs) (21). cDC1s and moDCs are the primary sources of IL-12, IFN-α, and IFN-β, which activate T helper type 1 (Th1) polarization. Additionally, they cross-present antigens to CD8⁺ cytotoxic T cells (Tc), leading to tissue damage in the lungs (20, 21). In contrast, studies in asthmatic mice have shown that cDC2s and moDCs play significant roles in T helper type 2 (Th2) polarization (22). cDC2s also contribute to promoting T helper type 17 (Th17) polarization in lung asthmatic mouse models (23). pDCs produce type-I IFN, TNF-α, and IL-6, inducing Th1, Th17, and regulatory T cell (Treg) polarization (24).

Dysfunction of DCs contributes to the progression of asthma, COPD, and ACO. An imbalance in DC subpopulations correlates with COPD severity, as indicated by the ratio of OX40L to programmed death-ligand 1 (PD-L1) (25). Moreover, reduced DC activation leads to a decrease in Th2 and Th17 signaling, as demonstrated by a reduction in mucus production and cellular recruitment (26).

AMs capture and eliminate foreign antigens while also repairing damaged tissue. Upon activation, they polarize into two subtypes: classically activated macrophages (M1) and alternatively activated macrophages (M2) (27). M1 is triggered by lipopolysaccharide (LPS) and cytokines such as TNF-α, IFN-γ, and IL-12. This subclass can eliminate bacteria, damaged cells, and tumors, and it plays a role in Th1 activation through the secretion of IL-1β, IL-6, IL-12, IL-18, IL-23, TNF-α, inducible nitric oxide synthase (iNOS), reactive oxygen species (ROS), CXCL-1, CXCL-2, CXCL-3, CXCL-9, and CXCL-10 (27–30). Conversely, M2 is notable for its role in tissue repair and maintaining immune homeostasis through the secretion of IL-10, TGF-β, and vascular endothelial growth factor (VEGF) (29, 30). M2 can be divided into four main subgroups: M2a, M2b, M2c, and M2d. M2a polarization, influenced by IL-4 and IL-13, functions in tissue repair by secreting TGF-β, insulin-like growth factor, and fibronectin. M2b is induced by immune complexes, toll-like receptor (TLR) ligands, or IL-1R ligands and regulates the immune response through IL-10. M2c requires IL-10 and TGF-β to differentiate, producing the anti-inflammatory cytokine IL-10 and the tissue remodeling cytokine TGF-β. M2d, induced by IL-6, TLR ligands, and the A2 adenosine receptor (A2R), produces IL-10, TGF-β, monocyte colony-stimulating factor (M-CSF), and VEGF (31–33).

At a steady state of lung tissue, the major population of AMs exhibits a hybrid of M1 and M2 surface markers, allowing them to polarize swiftly (34). However, dysregulation in M1/M2 polarization is implicated in asthma, COPD, and ACO. Studies of small airway tissue and bronchoalveolar lavage (BAL) samples from smokers and COPD populations indicate that M1 predominates in the airway wall, while M2 phenotypes and their associated cytokines are elevated in the luminal space compared to normal controls. This suggests that the overexpression of M2 switching in the lumen produces excessive tissue repair following M1-mediated tissue damage, contributing to lung fibrosis and permanent obstruction (35).

Eosinophils respond to IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF), all of which are crucial for their proliferation, maturation, survival, and activation. They are also activated by TSLP and IL-33. This activation leads to inflammation, tissue injury, and airway remodeling through their adhesion, degranulation, cytokine secretion, and chemotaxis involving CCL-1, CCL-5, CCL-7, and CCL-8 (36–39). Eosinophil granules contain proteins, including major basic protein (MBP), eosinophil cation protein (ECP), eosinophil peroxidase (EPO), and eosinophil-derived neurotoxin (EDN), which affect the immunopathogenesis of ACO through their cytotoxic functions and induce inflammation (38, 40, 41). Moreover, recent reports indicate that cadherin L, derived from eosinophils, also promotes emphysema in COPD with eosinophilia (42).

Eosinophil-derived mediators, such as TGF-β1, cysteinyl leukotrienes, IL-4, and IL-13, drive the proliferation of ASM and fibroblasts, leading to long-term structural changes (38). Additionally, eosinophils interact with the neuroimmune system by forming eosinophil extracellular traps (EETs), which activate pulmonary neuroendocrine cells (PNCEs) to secrete calcitonin gene-related peptide (CGRP) and γ-aminobutyric acid (GABA), promoting cell infiltration and cytokine release, thereby increasing inflammation and mucus production (4). These non-selective mechanisms of eosinophil granule toxicity cause tissue damage and hyperresponsiveness, resulting in an increased exacerbation rate of ACO (40, 41, 43).

Neutrophils are stimulated by extracellular matrix (ECM) proteins, cytokines, and microorganisms at inflammatory sites. Optimal activation requires a two-step process involving priming and activating stimuli. Several factors, such as TNF-α, GM-CSF, IFN-γ, IL-6, IL-17, CXCL-1, and CXCL-8, have been identified as priming agents that facilitate full activation alongside DAMPs and pathogen-associated molecular patterns (PAMPs) (44, 45). Activated neutrophils release serine proteases, including neutrophil elastase (NE), cathepsin G (CG), proteinase 3 (PR3), reactive oxygen species (ROS), myeloperoxidase (MPO), and neutrophil extracellular traps (NETs). These proteases, along with ROS formation and NETosis, degrade elastin, collagen, fibronectin, and proteoglycans, contributing to epithelial cell destruction and structural changes (46). Moreover, neutrophils can initiate tissue repair through heat shock signaling, controlling the movement of the matrix from healthy areas into injured tissue (47).

Recent research shows that ECM proteins influence neutrophil functions, migration, ROS production, MPO secretion, and NET formation. Type III collagen in lung tissue reduces neutrophil migration while enhancing ROS production, leading to tissue damage and prolonged neutrophil presence in the lung tissue, contributing to long-term inflammation and airway remodeling. This may relate to the increased production of type I and type III collagen in the airway during the early stages of COPD (48). ECM also determines the velocity and direction of neutrophil migration, guiding them to the injury site and causing them to remain in the tissue (49). These neutrophil functions may explain the severity of ACO patients due to increased neutrophil attraction and activation at the inflamed site.

MC activation occurs due to various stimuli, including IL-3, IL-9, IL-33, IgE, and DAMPs (50). Once activated, they produce histamine, tryptase, chymase, leukotrienes C4, and prostaglandins D2, IL-4, IL-5, IL-13, VEGF-A, VEGF-C, and CXCL-1. These molecules are associated with inflammation and airway remodeling (50–52). Interestingly, MC-released mediators can crosstalk with neurons, inducing them to release the neuropeptide substance P, thereby triggering MC degranulation (51).

MCs have two main subpopulations defined by their content granules of tryptase and chymase: MC_T (tryptase-positive) and MC_TC (both tryptase and chymase-positive). In asthma and COPD, MC_TC populations are increased, affecting bronchial epithelial cells by altering their migration, velocity, proliferation, and morphology, thereby disrupting the epithelial barrier through the production of tryptase and chymase (53). The granules of MCs play a significant role in airway hyperresponsiveness, inflammation, and airway thickening. Histamine induces ASM contraction, while leukotrienes and prostaglandins mediate bronchoconstriction and remodeling (54, 55).

ILCs are innate immune cells that differentiate into five subsets: ILC1, ILC2, ILC3, NK cells, and lymphoid tissue inducer (LTi) cells. They express cytokines similar to Th1, Th2, and Th17 but are distinguished from T cells by their lack of specific antigen receptors, especially T cell receptors (TCRs) (56). Although the mechanisms of ILCs in ACO are still being explored, their cytokines can induce several cells to function, such as neutrophils, eosinophils, MCs, AMs, T cells, and B cells, leading to airway inflammation and remodeling in asthma and COPD. ILC1 and NK cells share the expression of IFN-γ and TNF-α via T-bet driven by IL-12 (52, 57). The cytokines released by ILC1s, such as IFN-γ and TNF-α, increase neutrophilic inflammation (58, 59). NK cells contribute to tissue damage by releasing perforin and granzyme A and B, which correlates with the risk of COPD exacerbation (60). ILC2s release IL-4, IL-5, IL-9, and IL-13 through GATA-3 activation when exposed to TSLP, IL-25, and IL-33 (56, 61). ILC2 and type 2 cytokines are increased in the peripheral blood of COPD patients (62). The increased eosinophil count correlates with ILC2 levels in asthmatic patients (63). Moreover, ILC2 also plays a role in epithelial barrier destruction, inducing inflammation in murine asthma lung tissue (64). ILC3 and LTi secrete IL-17 and IL-22 through ROR-γt activation when stimulated with IL-2 and IL-23, contributing to neutrophilic inflammation and recruitment (52, 65, 66). A positive correlation has been observed between the ILC3 population, neutrophils, and AM1 in severe asthma (67).

These ILC subtypes are not permanent and can switch to other subtypes (68). Research has found that ILCs increasingly transition into the ILC1 subclass, with the elevated ILC1 population associated with smoking status and disease severity in COPD (59). Interestingly, ILC2s can polarize into the ILC1 subtype in response to infections or exposure to toxic agents in COPD (68). This explains why individuals with the same phenotype exhibit different characteristics.

In asthma, the elevated number of ILC2s and the overexpression of type 2 cytokines correlate with greater disease severity and progression (63, 68–71). In contrast, the levels of ILC1s and ILC3s cytokines are elevated in ACO and COPD patients (72). Moreover, an increased ILC3 population and neutrophil inflammation are observed in the neutrophil asthma and COPD groups (66, 73). These changes are affected by the overexpression of specific cytokines, which can excessively drive ILC plasticity into subgroups under different conditions.

ELISA is a technique used to monitor inflammation in respiratory diseases by quantifying substances such as proteins and cytokines. For example, sputum periostin indicates fixed airflow obstruction and correlates with elevated levels of sputum IL-13 and eosinophils (13, 99). ELISA can also detect soluble cytokine receptors, such as sIL-2R levels, which are linked to disease severity in the COPD group (100). This method also measures the levels of EETs and NETs (4). Additionally, the ELISA method can detect specific IgE (sIgE), which is essential for allergy evaluation (101).

Flow cytometry is a technology used to analyze and classify cells based on their expressions, such as DNA/RNA content, CD markers, transcription factors, cytokines, and cell receptors. This provides more information about cell state, cell subtype, cell function, and cytokine production (4, 9).

Flow cytometry effectively quantifies the total number of specific cell types by highlighting their distinguishing features (34) (Figures 2A, B). It also reveals the percentages of surface receptors present on immune cells (Figures 2C, D). Markers such as IL-4R, IL-5R, CXCR1, and IL-17R are essential in severe obstructive lung disease, serving as targets for biological therapy that can potentially influence patients' dose response (102).

Furthermore, flow cytometry detects biomarkers using multiplex bead-based indirect immunofluorescence assays, determining biomarker levels based on fluorescent intensity on cytokine-specific beads (Figure 2E) (103). It also tracks cell proliferation, such as in the lymphocyte transformation test (LTT), which uses carboxyfluorescein diacetate succinimidyl ester (CFSE) dye to monitor lymphocyte generation. Flow cytometry also provides cell function analysis, such as the MCs activation test (MAT), which can improve the diagnosis of IgE-mediated allergic phenotypes and analyze EETs and NETs formation in specific cell populations (4, 104).

Molecular technologies, such as nucleic acid sequencing and real-time polymerase chain reaction (RT-PCR), shed light on how single nucleotide polymorphisms (SNPs) play a role in immune system dysregulation in various diseases. SNPs are variations in a single nucleotide within human DNA that can lead to differences in individual phenotypes. Numerous studies have demonstrated the significance of SNPs in assessing disease risk and severity, as well as in informing treatments for asthma, COPD, and ACO, which are critical for future clinical practice (108). For example, gene variations associated with the IL-4/IL-13 pathway are linked to asthma susceptibility. Chinese patients with four SNPs, including IL4 (rs2243250C>T), IL13 (rs1800925C>T), IL4R (rs1805010G>A), and STAT6 (rs3224011T>C), show an association with a high-risk genotype for asthma vulnerability by elevating the expression levels of IL4, IL13, and STAT6 (108). Variations in TNF (rs1800629G>A) are associated with an increased risk of COPD in Asian populations (109). The haplotype of VEGFA (rs833068G>A, rs833070T>C, rs3024994C>T, rs3024997G>A, and rs3025000C>T), designated as GCCAT, significantly increases COPD risk in the Mongolian population, with an odds ratio (OR) of 3.409 (110). Furthermore, research on type-2 phenotype asthma and COPD among the Japanese population indicates that IL4R (rs8832A > G), associated with IL-4Rα levels and frequent exacerbations, may predict the effectiveness of IL-4Rα antagonist drugs (111).