Cell Cross-Talk in Alveolar Microenvironment: From Lung Injury to Fibrosis

Abstract

Alveoli are complex microenvironments composed of various cell types, including epithelial, fibroblast, endothelial, and immune cells, which work together to maintain a delicate balance in the lung environment, ensuring proper growth, development, and an effective response to lung injuries. However, prolonged inflammation or aging can disrupt normal interactions among these cells, leading to impaired repair processes and a substantial decline in lung function. Therefore, it is essential to understand the key mechanisms underlying the interactions among the major cell types within the alveolar microenvironment. We explored the key mechanisms underlying the interactions among the major cell types within the alveolar microenvironment. These interactions occur through the secretion of signaling factors and play crucial roles in the response to injury, repair mechanisms, and the development of fibrosis in the lungs. Specifically, we focused on the regulation of alveolar type 2 cells by fibroblasts, endothelial cells, and macrophages. In addition, we explored the diverse phenotypes of fibroblasts at different stages of life and in response to lung injury, highlighting their impact on matrix production and immune functions. Furthermore, we summarize the various phenotypes of macrophages in lung injury and fibrosis as well as their intricate interplay with other cell types. This interplay can either contribute to the restoration of immune homeostasis in the alveoli or impede the repair process. Through a comprehensive exploration of these cell interactions, we aim to reveal new insights into the molecular mechanisms that drive lung injury toward fibrosis and identify potential targets for therapeutic intervention.

The alveolar microenvironment is composed of various cell types that communicate closely, including epithelial cells, endothelial cells, fibroblasts (Fbs), and immune cells. These cells, together with the extracellular matrix (ECM), support alveolar architecture by expressing factors that aid in epithelial injury repair, maintain the stemness potential of alveolar type 2 (AT2) stem cells, and facilitate appropriate cell fate and migration (1, 2). Recent studies have revealed the mechanisms underlying the diverse responses of these cells to lung injury, providing insights into why the alveolar microenvironment can either regenerate or deteriorate within weeks after severe lung injury (3). These findings hold promise for improving clinical treatments by providing a deeper understanding of the intricate interactions between different cells.

In this review we specifically examine the features of the alveolar microenvironment during different stages of injury. Single-cell transcriptome analysis has enabled the detailed investigation and visualization of different cell subtypes and their interactions within the alveoli. These advances have substantially contributed to the understanding of lung repair processes after lung injuries, as well as the development of lung diseases.

Major Cells and Their Regulations in Alveolar Development and Homeostasis

Central Role of AT2 Cells

Lung development involves coordinated regulation of cell development by lineage-specific transcriptional networks. Multipotent endodermal progenitor cells differentiate into Sox2⁺ proximal or Id2⁺/Sox9⁺ distal endodermal progenitors before the pseudoglandular stage. The proximal and distal endodermal progenitors undergo distinct lineage differentiation into conducting airways and lung parenchyma, respectively, following the axial patterning program for branching morphogenesis and growth (4). Sox9⁺ distal lung progenitor cells acquire early alveolar type 1 (AT1) and AT2 expression characteristics during the pseudoglandular stage. FGF10 (Fb growth factor 10) expressed by Fbs acts on the epithelial surface of Fgfr2b to promote branching (5). At this stage, the primary source of VEGF (vascular endothelial growth factor) begins to transition from mesenchyme to epithelium (6). During the canalicular stage, the primitive pulmonary terminal sac forms, and differentiating Fbs release Wnt, which can promote the differentiation of the terminal sac epithelium into AT1 and AT2 cells. The terminal sac epithelium continues to separate during the saccular stage and becomes enveloped by capillaries (Figure 1).

Figure 1.

Cell communications during lung development. Alveolar EP cells arise from the endodermal lineage, while the lung mesoderm gives rise to cell types including ECs and fibroblasts (Fbs). Fb progenitors expressing Tbx4 influence the behavior and morphogenesis of EP cells. Fb progenitors mainly differentiate into ACTA2⁺ and WNT2⁺ subsets. PDGFα (platelet-derived growth factor α) in AT2 cells leads to paracrine activation of PDGFRα (PDGF receptor α) signaling in alveolar myofibroblast progenitors. WNT signaling from WNT2⁺ Fbs maintains progenitor self-renewal and regulates branching morphogenesis and proximal-distal patterning. In the pseudoglandular stage, CD206⁺ MACs interact with SOX9⁺ epithelial progenitors and influence their differentiation through IL-1β secretion. The fetal liver provides another source of MAC in the pseudoglandular stage, which together with monocyte-derived MACs after birth make up interstitial MAC populations. c-Kit⁺ EC progenitors are regulated by Fbs and EP cells. VEGF from Fbs and epithelium specify EC subsets, while Ang-1 from epithelial progenitors drives angiogenesis, expanding and stabilizing capillary networks. EC progenitors eventually differentiate into gCap and aerocyte subsets, forming capillaries surrounding the alveoli. AM = alveolar macrophage; Ang1 = angiopoietin-1; AT1 = alveolar type 1; AT2 = alveolar type 2; gCap = general capillary; IM = interstitial macrophage; MAC = macrophage; Tbx4 = T-box transcription factor 4; VEGF = vascular endothelial growth factor.

Substantial structural differences exist between the distal airways of humans and mice. Anatomically, mouse airways are directly connected to the alveoli, where distal airway stem cells expressing Trp63 and keratin 5 (DASC [Trp63/Krt5]) exist. Upon transplantation into the infected lungs, these cells differentiate into AT1, AT2, and bronchiolar secretory cells (7). This process effectively mitigates the structural effects of the impact of endogenous stem cell loss. In contrast, humans and other large mammals possess transitional structures, such as respiratory bronchioles (transitional bronchioles) and alveolar ducts, which harbor a distinct population of secretory cells separate from those found in the larger proximal airways. Organoid models have demonstrated that these respiratory secretory cells function as progenitors for AT2 cells, which play a pivotal role in the maintenance and regeneration of the alveolar niche (8); however, these specific cell types are absent in mouse lungs.

In adult alveoli, squamous AT1 cells cover the surface area responsible for gas exchange and maintain close contact with the capillary endothelial cells. In contrast, AT2 cells are the primary producers of surfactants, which are stored in lamellar bodies. AT2 cells play an active role in the innate immunity by secreting factors that inhibit bacterial growth and activate alveolar macrophages (AMs) to combat infections (9, 10). Furthermore, a subset of AT2 cells with high regenerative capacity can respond uniquely to regenerative signals triggered by viral infections, exposure to substances such as bleomycin, pneumonectomy, and other stimuli, enabling rapid expansion and regeneration of the alveolar epithelium (11–14).

AT2 cell behavior, including quiescence, proliferation, and differentiation, is governed by a complex network of signaling pathways from lung development to homeostasis maintenance in adults. These pathways consist of Wnt, Notch, Hippo/Yap, bone morphogenetic protein, cytokines (IL-1β, IL-4, IL-13, and IL-6), and growth factors (such as TGF-β [transforming growth factor-β], FGFs, PDGF [platelet-derived growth factor], EGF [epidermal growth factor], and VEGF) (2, 13, 15–18). These factors are produced by neighboring cells, including epithelial, Fb, neuronal, neuroendocrine, endothelial, and immune cells, and the ECM, and control the differentiation and proliferation of AT2 cells through various mechanisms, such as cell–cell contact, paracrine signaling, and autocrine signaling. Notably, bidirectional communication between epithelial cells and Fbs is crucial for these mechanisms (19).

Phenotypes of Fbs and Their Cross-Talk with AT2 Cells

Most of the factors mentioned above are secreted by crucial components that surround AT2 and Fb cross-talk. Fbs are a group of cells that exhibit high heterogeneity and can quickly change their phenotypes and transform into one another. Cells of the same type may present varying marker expression in different diseases, making it challenging to define Fbs using a single marker (20). In addition to genetic markers, functional properties are the ultimate criteria for distinguishing common features from unique ones in Fb biology. These cells can be classified into three functionally distinct Fb populations: myofibroblasts (myoFbs), lipofibroblasts, and matrix Fbs (Figure 2) (10). Dynamic changes in these three cell types indicate different requirements for development and repair (Figure 3). They provide the necessary scaffolding and paracrine signaling for epithelial cell proliferation, differentiation, and ECM component production, thereby promoting tissue repair and regeneration (21). Two cell types, mesenchymal alveolar niche cells and LGR5, which are very similar to lipofibroblasts, are located in alveolar niches and are capable of modulating AT2 function through the WNT and FGF pathways (22, 23). Zepp and Morrisey (24) summarized the interstitial Fbs in alveolar niche as Axin2-positive myogenic precursors, Wnt2-expressing PDGFR-positive cells, and mesenchymal alveolar niche cells. Axin2-positive myogenic precursors transit into myoFbs during injury and promote a fibrotic response. To a large extent, these cell types likely overlap; however, as mentioned above, the plasticity of Fbs makes their nomenclature and distinction in different environments and states extremely challenging.

Figure 2.

Cell cross-talk is a crucial process for maintaining a balanced internal environment in the alveoli after birth. In a healthy state, epithelial cells, Fbs, and macrophages interact coordinately to remove aging cells and promote the renewal of structural cells. Lipofibroblasts and matrix Fbs, as well as endothelial cells, regulate the behavior of epithelial progenitors by secreting various growth factors that can either stimulate proliferation or promote differentiation, often with opposing effects. Epithelial cells, on the other hand, can influence the transformation of Fbs by secreting PDGFRα. Fbs and epithelial cells affect the maturation and activation of macrophages through the GM-CSF pathway. Once matured, alveolar macrophages regulate surfactant production by AT2 cells. AT2 cells communicate with AMs through TGF-β, IL-10, CD200, and SIRP1α to keep AMs in a quiescent state and allow adaptive response to external stimuli. Activating the NOTCH pathway induces PDGFA secretion from AT2 cells, initiating Fb differentiation, while blocking the Notch pathway promotes alveolar epithelial cell generation. FGF7 = fibroblast growth factor 7; GM-CSF = granulocyte–macrophage colony–stimulating factor; TGF-β = transforming growth factor-β.

Figure 3.

Subtypes and functional changes of various types of cells in alveolar niches from lung injury to fibrosis. AT0 = alveolar type 0; ECM = extracellular matrix; Lipo.Fb = lipofibroblast; Matrix.Fb = matrix fibroblast; MMP = matrix metalloprotease; Myo.Fb = myofibroblast.

During the pseudoglandular stage, mesenchymal progenitors expressing Tbx4 (T-box transcription factor 4) migrate to distinct mesenchymal niches to initiate differentiation (25). Airway smooth muscle progenitors are localized exclusively in the mesenchyme, preceding the formation of airway buds. Lineage tracing of Tcf21 (Fb-expressing transcription factor 21) showed that the progenitor lineage was similar to that of FGF10⁺ cells. Tcf21⁺ cells have an airway smooth muscle differentiation program during early development and a LIF program at later stages (26). During the canalicular stage, ACTA2⁺ Fbs loosely encircle these structures, with the stalk epithelium exposed to WNT2⁺ Fbs, thereby promoting AT2 differentiation. MyoFbs secrete the Wnt inhibitor NOTUM, which provides a spatial pattern for airway development. Notch2 signaling in AT2 induces PDGF, which is necessary for elastic fiber secretion by myoFbs (Figure 1) and is deposited at the tips of septal crests to provide a scaffold for secondary septation (5, 27). Zepp and colleagues (28) showed that Acta2⁺ progenitors at Embryonic Day 15.5 lie in close proximity to early AT1 progenitors. They generate committed transient secondary crest myoFbs, which exert substantially more traction force and are a functionally specialized Fb lineage that remodels the alveolus. Transcription factors, such as Foxa and Tead, regulate AT1 to derive Shh and Wnt, which are essential for the specification and fate maintenance of the force-exerting secondary crest myoFbs (Figure 1). The data suggest that the loss of the AT1 signaling node in pediatric diseases may result in abnormal intercellular communication within the alveolus, thereby contributing to respiratory insufficiency. The early activation of Fbs can facilitate a rapid reparative response to injury. FGFR2 is critical for the maintenance and recovery of AT2 cells after acute lung injury (ALI), impairment of which can result in airspace enlargement, collagen metabolism disorder, and higher postinjury mortality rates (29, 30). Epithelial cells produce the WNT ligand WNT7b, which induces the expression of transmembrane receptors PDGFRα and PDGFRβ in adjacent Fb progenitor cells (31). PDGFR expression varies among different subsets of Fbs (32) and during different stages of development (33). Consequently, PDGFRα⁺ cells comprise a heterogeneous cell population with varying contributions to lung maturation and injury responses (32, 34).

Interactions between Endothelial Cells and AT2 Cells

Epithelial and endothelial cells closely interact during lung development. Epithelial cells play crucial roles in promoting blood vessel growth and branching (35). VEGF is a key signaling molecule that facilitates communication between epithelial and endothelial cells (35–37). Epithelial-secreted angiopoietin-1 binds to Tie-2 receptors on endothelial cells and regulates their growth (Figure 1) (38). Endothelial cells consist of two mixed cell types: “aerocytes” for gas exchange and leukocytes trafficking in the lung (39), and “general capillaries,” to regulate vascular tone and function as stem/progenitor cells in capillary homeostasis, immune regulation, and repair (24, 39, 40) (Figure 3). These cell types develop from bipotent progenitors but mature differentially during disease and aging, responding to specific signals from other alveolar cell types. c-KIT⁺ endothelial progenitor cells are abundant in embryonic and neonatal lungs, playing a crucial role in increasing pulmonary angiogenesis and preventing alveolar simplification (41). Wnt produced by AT1 cells regulates endothelial differentiation in early postnatal life, reflecting the close relationship between AT1 and endothelial cells in the alveolus (42). Ding and colleagues (43) observed that Vegfr2 and Fgfr1 on endothelial cells mediate signals to produce MMP14 (matrix metalloprotease 14), which is important for AT2 proliferation and alveolar regeneration. We (44) and Lee and colleagues (45) found that thrombospondin-1 promotes AT2 cell proliferation (Figure 2). Further research is needed to explore how different subtypes of endothelial cells communicate with epithelial cells to promote the stability of the blood–gas barrier and efficiency of gas exchange.

Macrophage Source and Fate Affect Alveolar Homeostasis

Alveolar-resident macrophages originate from the yolk sac during early fetal development (Figure 1). Before birth, macrophages exhibit high proliferative activity and localize specifically around small blood vessels, indicating a potential role of macrophages in modulating the development of pulmonary vasculature (46). CD206⁺ macrophages exist in fetal lung tissue throughout development and are in direct communication with SOX9⁺ epithelial tip progenitors. These macrophages secret IL-1β, which may lead to SOX9⁺ epithelial tip progenitors to differentiation (47) (Figure 1). Macrophages are divided into two subtypes: AMs and interstitial macrophages (IMs) (48, 49). IMs can arise from the bone marrow, fetal liver, and embryonic progenitor cells and are generally known for their strong immune-regulating and antigen-presenting abilities. AM maturation is closely tied to the secretion of GM-CSF (granulocyte–macrophage colony–stimulating factor) by AT2 and is regulated by TGF-β (50) (Figure 2). Under normal conditions, AMs maintain environmental stability and adopt immunosuppressive states. They also regulate pulmonary surfactant homeostasis through the GM-CSFR-PPAR-c pathway, which is crucial for lung function and immunity (51). In accordance with the ligands expressed by AT2 cells, AMs express various receptors, including TGF-βR, IL-10R, CD200R, and SIRP1α, which keep them in a quiescent state and allow adaptive response to external stimuli (Figure 2). However, the lung microenvironment is complex, and multiple factors influence and modify the gene transcriptional program of pulmonary macrophages. Macrophages undergo profound transcriptional reprogramming, and their response to inflammation is just one aspect of their function. The traditional classification of M1 and M2 macrophages based solely on inflammation is insufficient for current research needs; therefore, this phenotypic dichotomy cannot easily define their diverse characteristics (52).

Changes in Cell Phenotypes and Cell Cross-Talk in ALI

The lung epithelium serves as an initial barrier against respiratory pathogens and toxins. Maintaining the balance of the alveolar microenvironment in adults and facilitating repair after injury requires changes in the behavior of epithelial populations at different anatomical locations and active interactions among various components, including Fbs and macrophages. However, because of the complexity and variability of these cell phenotypes, their roles in the acute phase of lung injury still unclear.

Injury and Regeneration of Alveolar Epithelial Cells

Epithelial progenitor cells within the alveoli play an essential role in the repair of lung injury. The mechanism underlying human alveolar regeneration after lung injury is far less understood than that in mice. Differences in distal airway anatomy between humans and mice lead to different disease processes. In human chronic obstructive pulmonary disease, respiratory secretory cells undergo changes corresponding to an abnormal state of AT2 cells, which is associated with lung damage caused by smoke exposure in ferrets (8). This can explain why mice have a considerably lower pathological response to cigarette smoke exposure, whereas ferrets and humans experience substantial respiratory bronchiole pathological damage due to smoke inhalation (8, 53). AT2 cells are common progenitor cells involved in alveolar repair in both humans and mice. In mice, AT2 cell–mediated repair requires glucose metabolism during bleomycin-induced lung injury (54). Lineage-negative epithelial stem/progenitor cells are primarily found in the small airways of mouse. In cases of severe injury caused by influenza or bleomycin, resting lineage-negative epithelial stem/progenitor cells are stimulated and transdifferentiated into basal-like pod cells expressing ΔNp63/Krt5 via a Notch-dependent pathway. These transdifferentiated cells then migrate to the wound area and assist in repairing the damaged epithelium (55). However, it is important to note that although this epithelization process can help maintain the integrity of the tissue barrier, it cannot generate a fully functional alveolar epithelium (24, 55). This limitation forms the basis for subsequent abnormal lung repair.

AT1 cells persist in areas where AT2 cells are lost because of viral infection and upregulate the IFN response gene (56). During the regeneration process after adult lung resection, there is a low rate of transition from AT1 to AT2 cells (57). However, in neonatal ALI, AT1 cells undergo redifferentiation into AT2 cells and promote alveolar regeneration. This marked plasticity is controlled primarily by Hippo signaling in the AT1 cell lineage (Figure 2). However, the regenerative capacity of AT2 cells for AT1 cell is restricted to mature lungs, which may contribute to the different responses to ALI observed in pediatric and adult patients (58). This study also revealed that AT1 cells exhibit plasticity in both neonatal and adult ALI, indicating a broader role for AT1 cells in acute injury and chronic degenerative diseases. The plasticity of AT1 cells increases the number of AT2 cells and enhances the flexibility of the alveolar niche in response to injury (58).

During the differentiation process of AT2 cells into AT1 cells, a pre-AT1 transition cell state (PATS) was characterized. PATS cells undergo extensive stretching and exhibit increased expression of TP53, TGF-β, DNA damage response signals, and cellular senescence signals. Lung tissues from adult patients with coronavirus disease (COVID-19)–related acute respiratory distress syndrome (ARDS) display diffuse alveolar edema, destruction of the epithelial barrier, and a substantially increased number of PATS or damage-associated transient progenitors (59, 60). This suggests that decreased AT2 proliferation or impaired AT2–AT1 cell differentiation after injury may contribute to the failed repair and regeneration of the alveolar epithelium (59). Aging of the alveolar epithelium may affect repair and inflammatory resolution, potentially leading to fibrosis. Pediatric patients generally recover fully from alveolar damage, whereas adult ARDS survivors experience severe lung function impairment characterized by permanent alveolar simplification and fibrosis (61). AT2 cells from elderly mice demonstrate a reduction in the expression of cell proliferation and apoptosis markers, as well as an increase in the expression of senescence and proinflammatory genes after lung injury, compared with young mice (62–64), which has also been observed in humans (64). However, the precise role of AT2 cell senescence in repair and adaptive remodeling remains unclear, as does the contribution of other cells to the alveolar microenvironment. Further research is needed to gain a better understanding of the impact of aging on alveolar repair and regeneration capacity, particularly in human alveoli. Moreover, exploring the therapeutic potential of targeting the aging pathway is necessary (12).

Activation and Differentiation of Fbs

The repair pathway is activated early during ARDS onset to promote the resolution of epithelial and endothelial damage, alveolar leakage, and appropriate proliferation and differentiation of epithelial cells (Figure 4). However, unresolved damage accompanied by a disordered immune response can lead to fibrotic remodeling (65). Epithelial cells injury releases cytokines and damage-associated molecular patterns, which trigger immediate TGF-β release and subsequent Fb activation (66). Activated Fbs sustain the recruitment of circulating immune cells to the site of injury by expressing chemokines (67). They also secrete ECM remodeling enzymes, especially the ECM protease ADAMTS4 (68). Continuous mechanical or biological stimulation leads to excessive release of the above factors; activated Fbs gradually show an aging phenotype, and their function is simpler, leading to scar repair (Figure 4).

Figure 4.

Cellular interactions that contribute to the progression from ALI to fibrosis. In ALI, damaged AT2 cells release chemokines, cytokines, or damage-associated molecular patterns (DAMPs), which activate nearby Fbs and macrophages. The activated Fbs secrete various growth factors and collagen to contribute to the emergency repair of the injured site. Fb-secreting and epithelial cell–secreting cytokines drive the shift of monocyte-derived macrophages (Mon-MACs) to a proinflammatory phenotype to eliminate damaged cells and tissue debris rapidly. Macrophages secrete diverse repair factors to cooperate with Fbs in promoting fibrous repair. As endothelial cell injury is a significant feature of ALI, the interaction among endothelial cells, epithelial cells, and Fbs is critical. Endothelial cells actively interact with the ECM through MMP14 to repair extravascular matrix damage. Continuous inflammation triggers the ongoing transformation of various types of Fbs into myoFbs, leading to dysfunctional ECM metabolism because of the sustained release of growth factors and collagen. Mon-MACs induced by epithelial cells, Fbs, and endothelial cells serve as not only transmitters of inflammatory signals but also continue to release profibrosis-related factors such as PDGF and AREG that induce Fb proliferation and activation. Senescence and persistent inflammatory responses result in AT2 cells’ converting to a SASP, characterized by the persistent accumulation of intermediate states and impaired differentiation of AT1 cells. Aging AT2 cells continue to slowly release various inflammatory and profibrosis-related factors, perpetuating the activation of endothelial cells, Fbs, and macrophages and driving the vicious cycle of fibrosis. ALI = acute lung injury; Lipo-FB = lipofibroblast; Myo-FB = myofibroblast; SASP = senescence-associated secretory phenotype; TRB-sc = terminal and respiratory bronchioles secretory cell.

Paradoxical Roles of Macrophages on AT2 Cells

Cytokines and ILs secreted by injured epithelial cells primarily drive AM activation (69), which mediates inflammatory responses involved in lung injury (70) (Figure 2). In addition to decreasing alveolar fluid clearance in mice infected with influenza virus (71), AMs induce the apoptosis of epithelial cells after influenza infection, serving as a preemptive host defense mechanism to reduce susceptibility to influenza A virus infection (72). The coordination between macrophages and epithelial cells is critical for injury repair (73). A deficiency in CD200–CD200R cross-talk between AM and epithelial cells leads to inflammatory macrophage accumulation and enhanced sensitivity to influenza infection, resulting in delayed resolution of inflammation (74). During the resolution phase of ALI, AMs digest extrinsic apoptotic neutrophils. VEGF-C/VEGFR-3 signaling increases efferocytosis via the upregulation of integrin α_v in the macrophages (75). AMs establish direct communication with the alveolar epithelium via gap junctions, facilitating the transmission of antiinflammatory Ca²⁺ signals and thereby mitigating lung injury induced by LPS (69, 76). The removal of resident AMs leads to enhanced pulmonary inflammation and disruption of barrier function because of the loss of inhibition of epithelial cytokine secretion (77). AM depletion enhances the susceptibility of AT1 cells to influenza A virus infection through the arachidonic acid signaling pathway in the alveolar epithelium, thereby exacerbating lung injury (78), suggesting a protective effect of AMs on AT1 cells in an inflammatory environment. AMs rely on the trefoil factor 2/Wnt axis to drive epithelial cell proliferation after lung injury (79). IL-1β derived from IMs activates a subset of IL-1R1–expressing AT2 cells through the HIF1α-mediated glycolytic pathway, transforming them into intermediate cells necessary for the differentiation of mature AT1 cells. However, during chronic inflammation, the persistent presence of IL-1β prevents AT1 differentiation, leading to abnormal accumulation of intermediate states and impaired alveolar regeneration.

Therefore, the regulation of inflammatory cells such as macrophages in the process of lung regeneration is both intricate and delicate (13). The production of immunomodulatory cytokines such as IL-10, TGF-β, WNTs, VEGF, IGF-1, and SOD2 suggests that macrophages may have important immunomodulatory and reparative effects (50) (Figure 4). The paradoxical influence of macrophages suggests that the interaction between macrophages and AT2 cells in ALI is phenotype dependent. Optimal repair processes necessitate the suppression of macrophage proinflammatory response while simultaneously enhancing their prorepair and prophagocytic phenotypes.

Active Endothelial Cell–AT2 Cell Communication

The injury to the alveolar capillary barrier is a defining characteristic of ARDS and directly contributes to the distinctive physiological abnormalities observed. Endothelial damage leads to the release of anticoagulant molecules from the endothelial surface and an increase in procoagulant molecules, thereby promoting the formation of microvascular thrombi (80). Capillary endothelial cell injury leads to decreased oxygen diffusion capacity, increased vascular permeability, and plasma exudation into the alveolar interstitium and alveolar space, eventually forming a hyaline membrane that covers the alveolar epithelial surface (65). However, the responses of different subsets of endothelial cells to ALI remain unclear. Immune endothelial cells are enriched in genes involved in immune responses such as major histocompatibility complex genes, while developmental endothelial cells are enriched in genes involved in vascular development, such as Sox17. This suggests that some endothelial cells are predisposed to immune signaling, whereas others are predisposed to endothelial regeneration (81). In ALI, epithelial cell–endothelial cell paracrine signaling can induce endothelial cell barrier failure (82). A specific population of cells in mice express high concentrations of Car4 can receive signals from AT1 cells during ALI and promote remodeling of the alveolar–vascular interface after injury (83). The endothelial cell chemokine receptor CXCR7 protects against epithelial cell injury and reduces fibrosis during the ALI stage (84). After injury, S1P (angiocrine sphingosine-1-phosphate) released by endothelial cells acts through the S1PR2–YAP signaling axis on AT2 cells to facilitate their differentiation into AT1 cells for alveolar repair (85). During the differentiation of AT2 to AT1 cells, Krt8⁺ PATS cells communicate with endothelial cells via Edn1 and PAI-1. Meanwhile, AT1 cells are capable of communicating with endothelial cells through VEGFα and Sema3e. These findings suggest that communication between endothelial and epithelial cells is dependent on the cell phenotype (86) (Figure 3).

Factors Driving ALI to Fibrosis

Impaired Alveolar Epithelial Repair

The late stages of severe ARDS are characterized by ineffective alveolar regeneration, prolonged immune disorders, and fibrosis (12, 59, 87). Although traditional concepts suggest that Fbs are responsible for pulmonary fibrosis because of the proliferation and deposition of ECM proteins in the parenchyma, emerging changes in the alveolar epithelium play a dominant role in fibrosis (2, 21, 88). Increased concentrations of epithelial-associated biomarkers, such as surfactant protein D, CA19-9, and CA-125, in the serum of patients with fibrosis correlate with disease severity and predict mortality (89). Recent advances in organoid and single-cell sequencing technologies have led to the discovery of new subsets of AT2 cells in structural lung diseases, such as developmental diseases and fibrosis. Dysfunctional AT2 cells resulting from repeated microinsults not only fail to sustain physiological lung regeneration but also promote aberrant epithelial-mesenchymal interactions that contribute to fibrosis rather than regeneration. Accumulation of the intermediate state alveolar type 0 (AT0) cells in ALI, chronic obstructive pulmonary disease, and fibrosis suggests that AT1 differentiation is incomplete and cannot effectively achieve alveolar regeneration. The loss of EGF plays an important role in the abnormal differentiation of AT2 cells, which eventually leads to the differentiation of AT0 cells into terminal and respiratory bronchioles secretory cells, forming the typical pathological structure of alveolar bronchiolization observed in fibrosis. Specific subtypes of Fbs such as LGR5⁺ play key roles in this process (3, 23). Because of the presence of respiratory bronchioles, the differentiation program of AT0 is significantly distinct from those observed in the alveolar region of the mouse lung, which involve a unique transient state known as PATS or damage-associated transient progenitors (13, 90). The shared driver AT2 cytopathic features of ARDS and fibrosis reflect the central issue of functional cell regeneration during lung injury repair (91) (Figure 4).

Fb fate Determines AT2 Differentiation in Fibrosis

Although the characteristics of Fbs in fibrotic progression have been extensively studied, it is crucial to recognize that complex interactions between upstream cells and the cellular microenvironment may play a vital role in this process. Different subsets of Fbs exhibit variations in their localization, activity, and gene expression, resulting in unique functions and responses to injury. However, all these subsets contribute to the progression of fibrosis to varying degrees and can undergo reversible phenotypic changes. After injury, Axin2⁺ myofibrogenic progenitors predominantly generate pathologically deleterious myoFbs that proliferate and produce excessive amounts of ECM (92). In the context of acute or chronic injury, lipofibroblasts transdifferentiate into myoFbs, leading to a substantial expansion of the myoFb population (93). Nevertheless, during fibrosis regression, some myoFbs revert to lipofibroblasts (94), indicating a certain degree of reversibility in Fbs during the repair process (21, 94) (Figures 3 and 4). Increasing PDGFRα expression in Fbs can inhibit the induction of ACTA2 and alter the fibrosis process (21). The differentiation of AT2 cells into AT1 cells is not supported by Fbs derived from aged mice or adult human donors. However, the addition of PDGF-A restored AT2–AT1 differentiation in cocultures of human lung organoids with aged or fibrosis Fbs, suggesting that restoring PDGFR⁺ Fbs may be beneficial for promoting epithelial differentiation into AT1 cells, a function that is impaired in fibrosis (95). MyoFbs are the primary cells driving fibrosis, in which normal repair matrix functionality is lost; therefore, reversing the transition from profibrotic to prorepair Fbs represents a key aspect in improving fibrosis prognosis (21).

Disturbed ECM Metabolism and Cell Communication Constitute a Vicious Cycle

Fibrosis is driven by disturbances in ECM metabolism. The ECM plays a critical role in regulating the normal functions of alveoli, as it acts as a reservoir for various growth factors and cytokines that influence the proliferation, migration, structural remodeling, and apoptosis of epithelial cells (96). In healthy lungs, stromal fibers are arranged neatly, with a uniform basal layer of alveolar epithelium. However, in fibrosis lungs, stromal fibers become disorganized, leading to the complete destruction of the basal layer of the alveolar epithelium. The balance between matrix protein synthesis and degradation, mediated primarily by MMPs, determines ECM accumulation in tissues. Increased ECM stiffness is strongly associated with Fb differentiation (96). TGF-β is a crucial mediator of pathological scar formation. Under mechanical stress, TGF-β is activated in inflammatory cells and Fbs, leading to phenotypic changes in lung Fbs and increased ECM secretion (97). In healthy lungs, MMP activity is tightly regulated but increases during tissue repair and pathological remodeling, such as in fibrosis. Lung tissues of patients with fibrosis showed upregulated expression of MMP1, MMP3, MMP7, MMP8, and MMP9, whereas downregulation was observed for MMP19 and MMP13, which have antifibrotic effects (98). The role of MMP14 remains controversial, as it is upregulated in patients with fibrosis; however, intervention with bleomycin in MMP14-null mice inhibits epithelial cell regeneration and exacerbates fibrotic lesion progression. MMP14 deletion also leads to an increased expression of age-related markers (99, 100) (Figure 4). All types of Fbs, not just myoFbs, contribute to ECM production (20), indicating that all mesenchymal subpopulations contribute to matrix gene expression during fibrosis (101). Our understanding of myoFbs as the primary pathological cell type in fibrosis is evolving (101).

Communication between Macrophages and Fbs in Fibrotic Progression

Macrophages have a considerable impact on fibrosis by interacting with epithelial cells and Fbs. Injured epithelial cells release signals that attract and activate macrophages at the injury site, leading to a proinflammatory environment (102). Engulfing dead cells or damage-associated molecular patterns triggers the release of growth factors, such as amphiregulin, PDGF, and TGF-β, by macrophages, which stimulate epithelial and vascular repair (103, 104). Nevertheless, these macrophages produce higher concentrations of proinflammatory factors, such as TNFα and IL-1β (105). If these processes become pathological, injured epithelial cells continue to communicate with monocyte-derived macrophages through MCP-1–CCR2 signaling, which transforms them into a profibrotic phenotype. This irregular interaction results in disrupted wound responses and excessive collagen deposition (66, 106) (Figure 4).

Recently, profibrotic macrophages have been identified in the BAL fluid of patients with severe ALI caused by COVID-19. These macrophages demonstrated high expression of the immune markers CD163, Lyve-1, and C1q, together with profibrotic genes. Reducing the population of monocyte-derived macrophages can alleviate bleomycin-induced pulmonary fibrosis (102, 107, 108). Hence, it can be inferred that profibrotic macrophages differentiate from monocytes and show a phenotype similar to IMs. In addition, IM infiltration into the air space promotes fibrosis (109, 110). Macrophages involved in pulmonary fibrosis express specific proteins, such as SPP1 (osteopontin), LGMN (legumain), and TGF-β1 (50, 111, 112). Notably, LGMN is an asparaginyl endopeptidase associated with MMP activation, TGF-β signaling, and ECM deposition (113) (Figure 3). SPP1 is a secreted ECM phosphoglycoprotein that stimulates Fbs to produce type I collagen (114). TGF-β1 is a major regulator of wound healing and organ fibrosis (112). Macrophage-derived TGF-β1 promotes collagen production in Fbs and inhibits collagen turnover by suppressing MMP14 (112, 115).

The function of profibrotic macrophages is regulated by the macrophage colony-stimulating factor produced by Fbs (116). These cells produce molecules promoting Fb proliferation (116). The interaction between these two cell types is essential for coordinating lung tissue repair after injury. If a homeostatic imbalance persists, particularly when AT2-to-AT1 differentiation fails, the interaction between macrophages and Fbs may lead to the formation of an epithelial–endothelial– Fb–macrophage fibrotic niche around the injured area (50). The fibrotic niche promotes Fb proliferation and activation by creating self-sustaining cellular circuits. This is sustained by growth factors released by macrophages and Fbs, resulting in excessive ECM production and fibrogenesis (107, 116, 117) (Figure 4). Considering the diverse origins, intricate functional alterations, overlapping characteristics of macrophages in acute and chronic injuries, and the failure of conventional M1/M2 classification of macrophages to meet the requirements of current multidimensional research, it is imperative to summarize the functional and spatial attributes of macrophages using extensive data analysis techniques, such as spatial transcriptomics and single-cell sequencing, to accurately define and explore their functions (Figure 3).

Emerging Role of Endothelial Cells in Fibrosis

Further studies are needed to examine the heterogeneity of endothelial cells and their specific role in fibrosis, as endothelial cells are the primary cellular components of the alveolar niche (24). After injury, endothelial cells upregulate MMP14, which breaks down the ECM to release EGF-like ligands and stimulate epithelial progenitor growth (118) (Figure 4). In a hypoxic metabolic model, endothelial cells promote collagen deposition in an HIF-1α–dependent manner (119). Under high-stress conditions, endothelial cells release TGF-β, which causes an increase in the concentration of miR-143-3p. This increase induces COL5A2 (120). During chronic injury, endothelial cells can recruit VEGFR1-expressing perivascular macrophages. These macrophages stimulate sustained upregulation of the Wnt/β-catenin–dependent Notch ligand Jagged1 (encoded by Jag1) in endothelial cells, further activating Notch signaling in perivascular Fbs and promoting fibrosis (84) (Figure 4). The presence of ACKR1⁺ endothelial cells in fibrosis is closely correlated with regions characterized by collagen deposition and inflammation. These are dominant in fibrotic areas and accumulate in regions where myoFbs accumulate, thereby affecting the progression (121) (Figure 3). Pericytes are closely related to Fbs and make extensive physical contact with the endothelium. Pericytes may substantially contribute to the myoFb pool in pulmonary fibrosis (122), which may be caused by signaling disruption in the Wnt pathway with the endothelium (123), leading to a profound profibrotic effect on pericytes and their transfer to myoFbs. Interaction network analysis suggested potential cross-talk between endothelial cells, macrophages, and stromal cells during fibrosis. This analysis focused on the VEGF pathway and highlighted the role of endothelial cells in recruiting circulating monocytes, inducing Fb proliferation, and promoting ECM production (124). Targeting the alveolar niche, where macrophages, endothelial cells, and perivascular Fbs interact, may facilitate the development of therapies that stimulate lung regeneration and alleviate fibrosis (Figure 4).

Senescence and Inflammation Promote Fibrosis

Genes such as TGFB, VEGF, PDGFRA, and FGF are considered “culprit” genes that promote the occurrence and development of fibrosis. Therefore, a series of antifibrotic drugs have been developed to target them (125). Notably, these genes are essential for lung development, regeneration, and maintenance of normal function. Their dysregulated expression in pulmonary fibrosis was initially intended to facilitate the rapid repair of damaged areas. Several factors contribute to the disruption of this process, with age being the foremost factor (126). The regenerative function of epithelial cells, the immune regulatory function of macrophages, and the tissue repair capacity of Fbs decline considerably with age. Fbs derived from aging lungs exhibit sustained activation and promote ECM remodeling and collagen deposition compared with those derived from young lungs (107, 127, 128). This challenges the intricate interplay among multiple cell types, and the fragile microenvironment of the alveoli struggles to regain balance after various insults (26), which explains why pulmonary fibrosis typically occurs after the age of 65 years (129, 130). In addition to a substantial decrease in proliferation and differentiation (131), senescent epithelial cells exhibit senescence-associated secretory phenotype, in which various inflammatory factors are secreted, and the secretion and metabolism of chemokines, MMP, and various growth factors are disrupted (132). Senescent AT2 plays vital roles in lung Fb activation and proliferation (2, 133). Under a given condition, the WNT/IL-1b/NF-κB signaling axis is a powerful inducer of AT2 proliferation in ALI (2, 134, 135), and direct exposure of pulmonary Fbs to IL-1β may inhibit TGF-β–induced myoFb formation, collagen synthesis, MMP production, and lysyl oxidase (136). However, the continuous release of inflammatory factors leads to persistent activation of Fbs and macrophages. This further impairs the proliferation and differentiation potential of epithelial cells, making it difficult to effectively treat severe lung injury. The number and function of AMs decline with age, and there is substantial accumulation of proinflammatory and profibrotic monomacrophages, which may contribute to impaired lung injury repair in elderly individuals (12, 105, 137). The impaired regenerative capacity of endothelial cells is considered a significant contributing factor to the persistent development of pulmonary fibrosis in elderly patients with fibrosis (138). The continuous stimulation of epithelial and endothelial cells by inflammation leads to the inability of the alveolar–capillary barrier to effectively recover, resulting in constant permeation of plasma into the alveoli (65). This is accompanied by the migration of many inflammatory cells into the alveoli, which further worsens the imbalance in the microenvironment within the alveoli. These findings suggest that prolonged pulmonary fibrosis is associated with age-related immune and collagen metabolism disorders, leading to impaired lung injury repair in elderly individuals (Figure 4).

Conclusion and Future Directions

We explored the communication between AT2-centered endogenous stem cells and various subtypes of surrounding Fbs, macrophages, and endothelial cells under normal conditions, ALI, and fibrosis to gain insights into the maintenance of microenvironment homeostasis and recovery after injury. Although recent advances in single-cell sequencing and bioinformatics technology have enabled networked cell interaction analysis and effective exploration of potential receptor–ligand pairs, most of the transcriptome-level inferences still require evidence at the molecular level to determine their significance in cellular fate, except for certain well-established receptor–ligand pairs. The maintenance of microenvironmental homeostasis and recovery after injury is a complex and precise process that involves coordination and cooperation among multiple components. Abnormal cellular communication during the onset of ALI can lead to fibrosis. Therefore, it is crucial to consider ensuring normal alveolar repair after the removal of injury-causing factors in early-stage treatment to improve lung function. The numerous and different subtypes of receptor–ligand pairs in the alveolar microenvironment require precise selection of target pairs for promoting accurate epithelial repair and avoiding interference with other normal cellular processes (139). Fzd5–Wnt signals in the epithelium, other than in Fbs or endothelium, are promising for promoting alveolar epithelial stem cell regeneration without exacerbating fibrosis during lung injury. Controlling the inflammatory response is fundamental to facilitating normal interactions between different cell types and maintaining homeostasis in the microenvironment. Restoring the proportion and function of alveolar-resident macrophages is key to maintaining alveolar immune balance. Promoting the transformation of aging Fbs into stromal cells with diverse functions and balanced distribution represents an ideal state for sustaining normal ECM metabolism as well as proper proliferation and differentiation of epithelial cells. These insights provide a theoretical basis for exploring clinical antifibrosis therapies aimed at restoring microenvironmental homeostasis and promoting alveolar repair.