There has been a longstanding debate regarding the origin of AMs. Previous studies have suggested that all tissue-resident macrophages, including AMs, originate from circulating monocytes (20–22). However, this theory has been revised during the past decade. Under pathological conditions like osteoarthritis, pancreatic cancer, and steatohepatitis, monocytes can infiltrate target tissues by expressing the C-C chemokine receptor type 2 (CCR2). Therefore, researchers hypothesized that mice lacking Ccr2 would have a lower count of AMs compared to control mice. However, the proportions and numbers of AMs were found to be similar between Ccr2 knockout and control mice, suggesting that circulating monocytes make only a minimal contribution to the AMs pool (23). Moreover, transcriptional and functional data revealed that circulating monocytes rarely serve as precursors of AMs, with different origins, functions, gene expression profiles, and strategies to regulate compartment size (24–26). In line with this, recent studies indicated that alveolar macrophages derive from embryonic monocyte progenitors, which originate from the yolk sac or fetal liver during embryonic development and then migrate into the lung, where they differentiate into alveolar macrophages (25, 27–29). However, it is also feasible that interstitial macrophages derived from circulating monocytes may become precursors of AMs during injury, depletion or explant culture (30–33). Taken together, these studies suggested that alveolar macrophages are capable of dividing, self-renewing, and sustaining themselves without relying on circulating monocytes (23–26).

Several cytokines are involved in AMs differentiation and maturation, including granulocyte-macrophage colony-stimulating factor (GM-CSF), transforming growth factor-beta (TGF-β), and interleukin-1α (IL-1α) ( Figure 1 ). GM-CSF serves as a key regulator of AMs terminal differentiation and maintenance, with studies showing that GM-CSF-deficient mice have a significant reduction in the number of mature AMs (15, 34, 35). Further discoveries in patients with rare CSF2RA mutations or anti-GM-CSF autoantibodies confirmed that the unique function of GM-CSF in the lung is fundamentally conserved in humans (4, 36, 37). Notably, GM-CSF triggered the development of AMs by upregulating the expression of PU.1 in AMs (35), and selectively inducing PPAR-γ in fetal monocytes in the lungs (35, 38, 39). In contrast to GM-CSF, although macrophage colony-stimulating factor (M-CSF) serves as a critical cytokine in regulating the development of monocyte and macrophage cells, it has minimal involvement in the differentiation of AMs (40). TGF-β is a multifunctional cytokine that is involved in many biological processes, including immune regulation (41). In contrast to other tissue-resident macrophages, AMs also require TGF-β signaling for development and homeostasis in an autocrine manner (42–44). Further studies indicated that TGF-β signaling leads to transcriptional changes in genes associated with AMs signature (Scgb1a1, Epcam, and Cyp4f18), lipid metabolism (Pparg, Apo-E, Olr1), and the phosphorylation of PPAR-γ (42, 45). IL-1α is another cytokine that has been implicated in AMs differentiation and maturation (46). It was investigated that the lack of IL-1α led to decreased proliferation and maturation of CD11b^low alveolar macrophages during granuloma formation, which ultimately resulted in compromised alveolar clearance and the onset of PAP (46).

In addition to cytokines, the transcription factor PU.1 has been shown to be critical for the terminal differentiation of AMs (47–49) ( Figure 1 ). Studies utilizing GM-CSF knockout mice have demonstrated that reduced expression of the PU.1 in AMs is associated with decreased expression of CD32, mannose receptor, and macrophage colony-stimulating factor receptor (M-CSFR), which results in the blockade of maturation and differentiation of AMs (47). Moreover, PU.1 is responsible for regulating the constitutive expression of FcγRs and opsonophagocytosis in AMs (49). Notably, GM-CSF signaling is required for the expression of PU.1 in AMs (47, 50). Kruppel-like factor 4 (KLF4) is another transcription factor that is involved in AM differentiation and maturation (51, 52). Further studies have demonstrated that KLF4 promotes AMs polarizing to M2 phenotype through the activation of the downstream STAT6 signaling (52).

The pulmonary surfactant (PS) is composed of lipids, including phosphatidylcholine, phosphatidylglycerol, and surfactant protein A-D (53). The vital function of PS is to decrease the surface tension of the alveoli during respiration, which is crucial in preventing lung collapse by reducing the pressure required to inflate the lungs during inhalation (54). Maintaining an appropriate size of the surfactant pool is imperative for proper lung function and is carefully regulated by the balanced processes of surfactant production, secretion, reuptake, recycling, and catabolism within the alveoli (55–57).

In healthy individuals, pulmonary surfactant is produced and recycled by type II alveolar epithelial cells (AECs), with around 30% of the surfactant being taken up and catabolized by AMs (13, 58). Low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), and oxidized lipoproteins (Ox-L) are taken up by AMs through various mechanisms, including macropinocytosis, phagocytosis, and scavenger receptor-mediated pathways (59) ( Figure 2 ). The lipids taken up by AMs are degraded into free cholesterol and free fatty acids in lysosomes. Subsequently, the free cholesterol is re-esterified into cholesterol esters in the endoplasmic reticulum (ER) and stored in the cytoplasm as lipid droplets (60). The accumulation of intracellular cholesterol activates transcription factors such as liver X receptor α and β (LXRα/LXRβ), Retinoid X receptor (RXR), and Peroxisome proliferator-activated receptor α and γ (PPARα and PPARγ), which in turn promote the transcription and expression of lipid transporters ATP binding cassette subfamily A member 1(ABCA1) and ATP-binding cassette transporter G1(ABCG1) (61–64). These transporters subsequently regulate the efflux of free cholesterol and the maturation of HDLs (65). In addition, the efflux of free cholesterol is facilitated by simple diffusion or SR-BI-mediated facilitated diffusion (66).

Pulmonary alveolar proteinosis (PAP) is characterized by a reduction in AMs-mediated surfactant clearance, resulting in the formation of dysfunctional foamy AMs and accumulation of alveolar surfactant (67, 68). GM-CSF plays a key role in surfactant clearance of AMs, as evidenced by the development of hereditary PAP in patients with deficiencies in either CSF2RA or CSF2RB resulting in a lack of AMs (4, 69), and the development of autoimmune PAP in patients with non-functional AMs due to neutralizing auto-antibodies against GM-CSF (70). The esterified and free cholesterol are mainly accumulated by AMs in PAP, while the surfactant component displays a higher proportion of cholesterol (71). Mechanistically, the primary defect in cholesterol clearance caused by disruption of GM-CSF signaling leads to a secondary reduction in both surfactant uptake and clearance (71) ( Figure 2 ). Moreover, disruption of GM-CSF signaling also caused a decrease in mRNA levels of several cholesterol homeostasis factors that are critical to AMs, including Abcg1, Lxrα, Acat1, Neutral cholesterol ester hydrolase 1(Nceh1), and Lipase A (Lipa) (71, 72). In line with this, replenishing PPAR-γ in AMs of GM-CSF knockout mice results in a decrease in intracellular lipid accumulation and an increase in cholesterol efflux activity by upregulating ABCG1 (73). Similar studies showed that PPAR-γ is implicated in the mitigation of foamy AMs formation by reducing the expression of ABCG1, CD36, Oxidized low-density lipoprotein receptor 1(Olr1), Fatty acid-binding proteins 1(Fabp1), Fabp4, and Cell Death Inducing DFFA Like Effector C(Cidec), which play crucial roles in the uptake, transport, storage, and processing of lipids (38, 74–76). Interestingly, lipid metabolism factors ABCA1 and LXRβ are inadequate in preventing surfactant accumulation in AMs (75). In addition to PPAR-γ, another crucial transcription factors regulated by GM-CSF is PU.1, which is known to modulate the expression of lipid transporters ABCA1 and ABCG1 (72, 76). Collectively, these studies indicate that GM-CSF signaling is necessary for cholesterol clearance in AMs and identify impaired cholesterol clearance as the main AMs abnormality underlying the development of PAP.

The alveolar immune microenvironment is a complex, multi-cellular system consisting of alveolar macrophages, dendritic cells, lymphocytes, and epithelial cells (77). These cells collaborate to maintain pulmonary homeostasis and defend against inhaled pathogens and environmental insults (78). Alveolar macrophages, as the predominant phagocytic cells in the lungs, play an essential role in the removal of inhaled particles and pathogens (79). Additionally, they release cytokines and chemokines that orchestrate the recruitment and activation of other immune cells at the site of infection (80).

Under physiological conditions, AMs demonstrate heightened phagocytic activity, low inflammatory cytokine production, and overall suppression of inflammation and adaptive immunity (80, 81) ( Figure 3 ). AMs facilitate the resolution of inflammation by phagocytosing apoptotic and necrotic cells (a process known as efferocytosis), which in turn prevents the release of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α), Interleukin-1(IL-1), IL-8, and leukotriene C4 in the alveolar microenvironment (82–84). Consistent with this, efferocytosis prompts AMs to secrete anti-inflammatory cytokines such as TGF-β, prostaglandin E2 (PGE2), and platelet-activating factor (PAF), thereby preventing the onset of unnecessary inflammatory responses (85–87). Apart from its role in efferocytosis, an alternative mechanism through which AMs inhibit inflammation is by activating the regulatory T cells (Treg) immune response (88). It was demonstrated that the isolation of mouse-derived AMs, which were subsequently stimulated with ovalbumin and co-cultured with antigen-specific naive CD4⁺ T cells, led to generation of Foxp3⁺ Treg cells (89). Notably, this functional role of AMs was partially reversed by either inhibiting the binding of retinoic acid (RA) to its receptor (RAR), or by impeding the signaling of TGF-β (89). In addition to directly affecting Treg cell conversion by influencing the components of RA and TGF-β, AMs may also initiate the generation of Tregs through an indirect pathway (90–92). In the context of respiratory viral infections, AMs generate the immunoregulatory cytokine IL-27 via the induction of IL-6, which in turn recruits Ly6C⁺ monocytes and facilitates the local differentiation of Tregs (92). Communication with the alveolar microenvironment also modulates the immunological functions of AMs. The binding of CD200 and TGF-β, present on the cell membrane of alveolar epithelial cells, to their respective receptors, CD200R and TGF-βR, expressed on AMs, functions as a negative regulator of AM pro-inflammatory activity (93–95).

In patients with PAP, the lack of GM-CSF signaling impairs the immunological functions of AMs and neutrophils, making them more vulnerable to superimposed infections caused by fungal pathogens, nocardia, cytomegalovirus, pneumocystis carinii, anaerobes, and mycobacteria (96–102). Generally, up to 13% of patients with PAP may experience secondary pulmonary infections, with fungal infections being more commonly diagnosed and mycobacterial infections having the lowest mortality rate (99). About 20% of deaths in PAP patients are caused by secondary infections (103). Similarly, AMs from GM-CSF-deficient mice exhibit reduced phagocytosis, pathogen killing, expression and release of pro-inflammatory cytokines (35). Mechanistically, the impaired maturation and function of AMs resulting from GM-CSF signaling deficiency is the primary factor of secondary infections in PAP (50) ( Figure 3 ). It was found that GM-CSF promotes the uptake and clearance of adenovirus by AMs through a PU.1-dependent transcriptional program that redirects virion trafficking from the nucleus to lysosomes (104). Moreover, GM-CSF also regulates the production of genes that facilitate multiple immune and non-immune functions by stimulating the expression of PU.1 in AMs (50). It was demonstrated that the expression of PU.1 in GM-CSF-deficient AMs significantly increase the expression of mannose receptor (MR) and Toll-like receptor 2/4 (TLR-2/4), which are involved in the phagocytic internalization of microorganisms and pro-inflammatory signaling responses (35, 105). In line with this, the expression of PU.1 in GM-CSF-deficient AMs mediates a dose-dependent increase in the release of TNF-α and IL-6 (35). In addition, GM-CSF/PU.1 signaling orchestrates the molecular crosstalk between innate and adaptive immunity by modulating the production of IL-12, IL-18, and IFN-γ, consisting with the regulation of AMs phagocytosis via Fc-γ receptors (49). Together, these studies suggest that AMs have a critical function in preserving pulmonary immunological equilibrium in PAP patients, underscoring the essentiality of meticulous monitoring for infectious complications.

The administration of GM-CSF is a promising therapeutic strategy for autoimmune PAP, as it neutralizes the anti-GM-CSF antibodies and restores AM function. However, the effectiveness of GM-CSF therapy in secondary PAP remains to be established (112). A number of case series have reported that administering exogenous GM-CSF therapy has led to clinical improvement in patients with PAP ( Table 1 ). Recombinant GM-CSF is commonly administered through subcutaneous injection or inhalation, with various dosing regimens, including low-dose treatment (125 μg/day) and high-dose treatment (250 or 300 μg/day), etc (113–116, 118, 120–123). Therapeutic response of GM-CSF administration was evaluated via disease severity (alveolar arterial gradient (A-a) Do₂, oxygenation, etc.), pulmonary function tests, serum biomarkers of PAP, HRCT score, respiratory symptom scores, etc. The majority of the case series documented a response rate of 30-60%, with remission of respiratory symptoms and improvement in pulmonary function (113–116, 118, 120–123). Intriguingly, Campo et al. demonstrated that inhaled recombinant GM-CSF was well tolerated and effective in a PAP, and combination therapy proved to be more effective than WLL alone (119). Moreover, Tazawa et al. conducted a study in 3 successfully treated patients with idiopathic PAP, which found that the cell number, expressions of surface mannose receptor and PU.1, and phagocytic ability of AMs were all restored to control levels (124). Notably, the treatment resulted in a significant reduction in the neutralizing capacity and the levels of anti-GM-CSF autoantibodies in BALF of patients. Similarly, other studies also revealed that the administration of GM-CSF restored the morphology and adhesive function of AMs, upregulated the expression of GM-CSF receptor and PU.1, and improves lung clearance in PAP (47, 117, 125).

Rituximab is a chimeric monoclonal antibody that specifically recognizes the B-lymphocyte antigen CD20 (126). It has been previously used to treat CD20⁺ B-cell lymphoma, as well as autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis (126, 127). Expanding on this, Rituximab has also been utilized as a B-cell depleting agent in order to eradicate anti-GM-CSF autoantibodies and subsequently serve as a therapeutic intervention for PAP. Multiple studies have reported positive outcomes in terms of improvement in lung function and imaging variables in cases of autoimmune PAP following treatment with Rituximab, as observed in both case series and isolated cases (128–131) ( Table 2 ). In addition, the administration of Rituximab therapy led to a consistent reduction in both CD19⁺ B-cells in peripheral blood and anti-GM-CSF autoantibodies in BALF (131). Mechanistically, Rituximab therapy ameliorates the lipid metabolism of AM in patients with PAP, as demonstrated by the upregulation of key lipid metabolism molecules, including PPAR-γ, ABCG1, and lysosomal phospholipase A2 (LPLA2), a critical enzyme for surfactant degradation (132). Furthermore, Oil Red O staining indicates a reduction in intracellular lipid droplets in AMs, leading to an increase in free cholesterol in BALF and maintaining lipid homeostasis in the alveoli (132).

Currently, the pharmacotherapies utilized to restore lipid homeostasis in AMs comprise Pioglitazone and Statins. In the development of PAP, the absence of key molecules PPAR-γ and ABCG1 in AM is a crucial factor contributing to the dysregulation of cholesterol transport (75). In line with this, restoration of PPAR-γ signaling leads to a subsequent upregulation of ABCG1 expression, which facilitates the efflux of intracellular cholesterol in AMs (73). Pioglitazone is an agonist of PPAR-γ, which has been historically employed for the treatment of type 2 diabetes and Alzheimer’s disease (133, 134). Recent research utilizing Csf2rb^-/- PAP mice has demonstrated that pioglitazone can increase the expression of ABCG1, enhance cholesterol efflux in AMs, and alleviate pulmonary symptoms in PAP mice (135). A comparable outcome was documented in an autoimmune PAP patient that was unresponsive to conventional first-line therapies but demonstrated improvement of lung function with pioglitazone treatment (30 mg taken once daily) (136). The clinical study investigating the efficacy of pioglitazone in treating PAP is underway (NCT03231033) (136).

Statin is primarily used to reduce the risk of cardiovascular diseases by lowering cholesterol levels in the blood (137). Notably, a study involving two autoimmune PAP patients who were unresponsive to WLL treatment found that statins could significantly improve the patients’ clinical symptoms, lung function, and radiological features, as well as reduce the intracellular cholesterol levels in AMs (138). In Csf2rb^−/− mice, statin treatment also promoted cholesterol efflux in AMs and alleviated disease symptoms (138). In addition, a prospective real-world observational study evaluated the therapeutic efficacy of statins on 40 non-hypercholesterolemic PAP patients (139). 65% of the patients showed a treatment response, characterized by an increase in PaO₂, DLCO%, and a decrease in disease severity score (DSS) and radiographic abnormalities (139). The cutoff dose was 67.5 mg daily, with a corresponding specificity of 64.3% and sensitivity of 96.2% (139).

In addition to pharmacological treatments aimed at restoring AM function, direct transplantation of gene-corrected AMs or iPSC-derived AMs has also shown promise in the treatment of hereditary PAP. A study was conducted using lentiviral-mediated overexpression of the Csf2ra gene to prepare gene-corrected AMs, which were then transplanted into Csf2ra^-/- mice. The results showed that the transplantation improved AM function, restore lipid homeostasis and surfactant metabolism, and ameliorated the cytological, histological, and biomarker abnormalities associated with PAP (140). To enable the translation of gene-corrected AM therapy to PAP patients, a self-inactivating (SIN) lentiviral vector expressing a codon-optimized human CSF2RA-cDNA driven by an EF1α short promoter (Lv.EFS.CSF2RAcoop) was created. Results indicated that Lv.EFS.CSF2Racoop restored GM-CSF signaling in AMs by reconstituting GM-CSF receptor expression, and did not elicit negative effects in the target cells (141). Similarly, by using TALEN-mediated genetic integration, a codon-optimized CSF2RA transgene was inserted into the AAVS1 locus of iPSCs derived from hereditary PAP patients. This restored GM-CSF receptor functionality and corrected the disease phenotype of monocytes/macrophages derived from hereditary PAP iPSCs in vitro ( 111). Additionally, it has been demonstrated that human iPSC-derived macrophages can be transplanted into the lungs and differentiate into AMs, leading to a decrease in alveolar proteinosis in a humanized PAP model (142, 143).