The Intersection between Immune System and Idiopathic Pulmonary Fibrosis—A Concise Review

4.1. Monocyte/Macrophage Several studies involving IPF patients have implicated macrophages and monocytes in the pathogenesis and prognosis of the disease. Transcriptome data from peripheral blood mononuclear cell samples of IPF patients revealed that monocyte percentage above the mean was linked to shorter transplant-free survival [26]. This finding was further validated in a retrospective pooled analysis of four phase III randomized trials [27], involving 2067 patients from the ASCEND [28] and CAPACITY trial [29], which studied pirfenidone, and the INSPIRE trial [19], which evaluated interferon-gamma. Elevated monocyte percentages were significantly associated with one-year IPF progression (defined as a >10% absolute decline in FVC% predicted, a >50 m decline in six-minute walk distance, or death), as well as with increased risk of one-year all-cause hospitalization and mortality. Similar findings were observed in a systematic review and meta-analysis of patients with fibrotic ILD, as well as in a study involving patients with interstitial lung abnormalities detected through imaging [30,31]. In accordance, several studies have shown that mice with systemic monocytopenia are protected from the development of fibrosis. This protection has been observed in mice treated with intravenous liposomal clodronate, as well as in genetically modified CCR2−/− and CEBPd−/− knockout mice, which lack specific monocyte populations [32,33,34]. These findings suggest that circulating monocytes play a significant role in the fibrotic process and that their depletion or functional disruption can mitigate fibrosis development. 4.1.1. Tissue Resident Macrophages vs. Recruited Macrophages in Mouse Models of Pulmonary Fibrosis Alveolar macrophages (AMs) constitute the primary immune defense within the alveoli and airways, whereas lung interstitial macrophages (IMs) serve as pivotal regulators of the vasculature and lung interstitium. Both AMs and IMs are categorized as tissue-resident macrophages (TRMs), performing critical functions in maintaining homeostasis, facilitating metabolic processes, and mediating tissue repair in their respective organ environments. Additionally, these macrophages function as sentinel phagocytes within the immune system. During inflammatory conditions, monocytes are recruited to the lung, where they differentiate into recruited macrophages, exhibiting distinct transcriptional profiles and specialized functional roles [35]. Macrophage populations within tissues, derived from unique developmental lineages, express diverse surface markers that reflect their lineage and functional specificity. Classical monocytes are identified by the combination of MHC II^negative, CD64^low, CD11b^high, and Ly6C^high. Tissue resident interstitial macrophages are characterized by high expression of MHC II, CD64, CD11b, while being negative for Siglec F [35,36]. These macrophages can be further classified into subpopulations with distinct functional roles. Some are involved in maintaining tissue homeostasis and regulating vascular permeability, while others participate in immune surveillance and pro-inflammatory responses. The differentiation of these subpopulations is determined by surface markers such as LYVE-1 (lymphatic vessel endothelial hyaluronan receptor-1), Folr2 (folate receptor beta), CD206 (mannose receptor), Arg1 (arginase-1), and CX3CR1 [37,38]. Tissue-resident alveolar macrophages are identified by CD64^high, CD11c^high, F4/80 positive, MerTK positive, and Siglec F^high [36].Monocyte-derived macrophages are identified by CD64^high, F4/80 positive, MerTK positive, and Siglec F^low. During differentiation from monocyte-derived interstitial macrophages to monocyte-derived alveolar macrophages, they exhibit a decrease in CD11b and an increase in CD11c [35].Using various fate-mapping techniques, several groups have demonstrated that in naïve, unchallenged adult mice housed in clean facilities, tissue-resident alveolar macrophages maintain their population via proliferation in situ for months without contribution from circulating monocytes [39,40].Similarly, two clinical reports revealed that after lung transplantation, the alveolar macrophage population remains remarkably stable, with donor-derived cells making up the majority of the alveolar macrophage pool even five years post-transplant [41,42]. Although in the healthy, unperturbed lung at steady state, the origin of alveolar macrophages—whether from fetal or adult sources or from differentiated alveolar macrophages—does not influence their ability to occupy the lung niche [43,44], tissue-resident macrophages and monocyte-derived macrophages frequently display distinct, sometimes opposite, roles in various injury models, including cardiac injury [45], schistosomiasis (a parasite infection) [46]. Evidence suggested that tissue-resident alveolar macrophages (TR-AMs) are not actively involved in the fibrotic response, whereas monocyte-derived alveolar macrophages (Mo-AM) play a more significant role. In a study conducted by Gibbons et al., the depletion of circulating monocytes during the fibrotic phase of bleomycin-induced lung fibrosis resulted in reduced fibrosis [32]. Specifically, the depletion of the “inflammatory” Ly6C^high monocyte subset significantly decreased the number of Ym1-positive alternatively activated macrophages in the lungs. The adoptive transfer of Ly6C^high monocytes into bleomycin-treated mice during the fibrotic phase exacerbated lung fibrosis, as evidenced by increased collagen deposition and a higher number of Ym1-positive macrophages. Using bone marrow chimera mice, Misharin et al. demonstrated that in bleomycin-induced lung fibrosis, circulating monocytes are recruited to the lung and differentiate into Mo-AMs [47]. By utilizing CD11cCre Casp8flox/flox and LysMCre Casp8flox/flox mice to selectively deplete caspase-8, a suppressor of necroptosis, to markedly reduce the Mo-AM population, fibrosis was attenuated. In contrast, the depletion of TR-AMs using intratracheal liposomal clodronate did not affect fibrosis severity. Using similar CD11cCreCasp8flox/flox mice in an asbestos induced lung fibrosis model, Joshi et al. further demonstrated monocyte-derived alveolar macrophages were specifically located in these fibrotic areas, around bronchoalveolar duct junctions where asbestos fibers lodged, where they co-localized with fibroblasts, forming a fibrotic niche [48]. These macrophages expressed high levels of markers associated with fibrosis, such as Csf1, Pdgfa, and Mrc1, supporting their role in driving fibrosis through their interactions with fibroblasts. A different strategy to study the contribution of Mo-AMs in pulmonary fibrosis was used by McCubbrey et al. [49]. Their approach used hCD68rtTAcre (reverse tetracycline-controlled transactivator) floxed c-FILP (cellular FADD-like IL-1β–converting enzyme–inhibitory protein) which allowed for the inducible deletion of c-FLIP specifically in CD11b^high macrophages, making them susceptible to apoptosis upon administration of doxycycline. The study found that conditional deletion of c-FLIP in CD11b^high macrophages resulted in their significant depletion from the lung protecting the mice from developing lung fibrosis as reflected in the histology and lung compliance. In summary, these studies collectively demonstrate the pivotal role that monocyte derived macrophage populations play in the development and progression of lung fibrosis. A summary of monocyte and macrophage dynamics in steady-state and fibrosis in the mouse pulmonary fibrosis model is illustrated in Figure 1. 4.1.2. Macrophage Heterogeneity in IPF

The Classic M1/M2 Polarization Concept

Given the significant overlap in surface marker expression between different macrophage subsets, an effective approach to their characterization has been the analysis of specific gene expression profiles following cytokine or microbial stimulation. Classically activated macrophages (M1) are primarily involved in host defense mechanisms against bacteria, protozoa, and viruses, and they also play a key role in antitumor immunity. In contrast, alternatively activated macrophages (M2) are known for their anti-inflammatory properties and their contributions to tissue repair and wound healing [50]. Notably, using surgical lung biopsies, studies have demonstrated that usual interstitial pneumonia is associated with significantly higher levels of IL-13 and its receptor subunits, IL-13Ra2 and IL-13Ra1, particularly in fibroblastic foci [51,52]. Given their role in tissue remodeling, fibrosis has traditionally been considered an M2-dominated disease. In experimental models of lung fibrosis, various therapeutic interventions have focused on reducing the presence or activity of M2 macrophages. These interventions include targeting TNF-alpha [53], inhibiting Gab1 and Gab2 adaptor proteins, which are key players in the IL-4 signaling pathway [7], and neutralizing IL-33 [54]. Additionally, the use of microcystin-leucine arginine (microcystin-LR), an environmental cyanobacterial toxin, in bleomycin-induced pulmonary fibrosis models, and serum amyloid P (SAP) in TGF-beta overexpression models have also shown efficacy in reducing lung fibrosis [55]. Despite the encouraging results from these preclinical studies that aimed to interfere with M2 macrophages, clinical phase III trials in humans have largely yielded disappointing outcomes. One possible explanation for these results is that macrophage polarization and immune cell differentiation in the disease state are not mutually exclusive or as well-defined as in healthy conditions. Indeed, research has shown that monocytes isolated from the peripheral blood of IPF patients exhibit a significantly upregulated Type I interferon (IFN) response, marked by the overexpression of interferon-stimulated genes such as MX1, ISG15, and OASL [56]. Additionally, resident memory T cells from explanted lungs of IPF patients have been found to exhibit IFN-γ–mediated responses, further complicating the immune landscape in IPF [57]. It is important to note that the decreased production of certain cytokines or the presence of skewed immune cell populations in fibrotic lungs may represent an altered lung microenvironment rather than an inherent defect in cytokine production. Consequently, the administration of exogenous cytokines alone has proven insufficient to fully re-differentiate immune cells in the fibrotic milieu [58]. This suggests that fibrosis involves a complex interplay of immune cells and signaling pathways that cannot be corrected by targeting a single cytokine or cell type, highlighting the need for more comprehensive therapeutic strategies.

The Pathological Macrophage Subsets in IPF Revealed by High-Dimensional Transcriptomics

Single-cell RNA sequencing has revealed multiple distinct macrophage populations in the lung tissue of IPF patients, including a recruited macrophage subpopulation characterized by high expression of SPP1 (osteopontin) and CHIT1 (chitinase 1) [59]. In healthy lungs, FABP4^high macrophages constitute the predominant alveolar macrophage subpopulation, while SPP1^high macrophages are present at low levels. However, in fibrotic regions of IPF lungs, the expansion of SPP1^high macrophages, accompanied by a reduction in FABP4^high macrophages, particularly in the lower lung lobes, correlates with poor lung function, as evidenced by lower forced vital capacity percentage predicted (FVC% pred) [60]. Trajectory analysis indicates that these SPP1^high macrophages originate from circulating CD14+ monocytes, not tissue-resident macrophages, underscoring their role as recruited monocyte-derived macrophages central to the fibrotic process in IPF [59]. In fact, studies across disease models and species identify pathological macrophage subsets localized near ECM-producing fibroblasts, commonly referred to as scar-associated macrophages (SAMs), fibrosis-associated macrophages, or matrisome-associated macrophages. These subsets, which commonly express SPP1, TREM2, CD9, FABP5 (Fatty Acid Binding Protein 5), and GPNMB (Glycoprotein Non-Metastatic B), are implicated in various fibrotic diseases other than pulmonary fibrosis, including mouse models of skeletal muscle fibrosis [61], metabolic-associated fatty liver disease/nonalcoholic steatohepatitis [62,63,64], myocardial infarction [65], ureteric obstruction models [66], and human conditions such as acne keloidalis [67], liver cirrhosis [68,69], nonalcoholic fatty liver disease/steatohepatitis [70,71], post-COVID fibrosis [72], dilated cardiomyopathy [73], and myocardial infarction [74]. These findings underscore the conserved role of these macrophages in fibrotic diseases across tissues and species, highlighting them as potential therapeutic targets in fibrosis modulation. 4.1.3. Macrophage Recruitment Macrophage recruitment to sites of tissue injury and fibrosis is a complex process influenced by various chemical and mechanical signals.

Chemical Signals

One important chemoattractant identified in macrophage recruitment is FIZZ1 (Found in Inflammatory Zone 1). According to migration assays, FIZZ1 has been shown to possess chemoattractant activity for bone marrow-derived cells, including macrophages. Studies have demonstrated that bone marrow cells from both PBS- and bleomycin-treated mice migrated toward FIZZ1, although the response was stronger in control mice. In vivo experiments further reinforced the importance of FIZZ1 in fibrosis. FIZZ1 knockout mice exhibited significantly reduced recruitment of bone marrow cells to the lungs following bleomycin treatment. This reduction in bone marrow cell recruitment also correlated with lower numbers of inflammatory cells, including macrophages, in the bronchoalveolar lavage fluid, compared to wild-type mice [75]. Another important signaling pathway involved in macrophage recruitment is the CCL2/CCR2 axis. CCL2 is a potent chemoattractant responsible for the recruitment of fibrocytes and profibrotic macrophages to sites of tissue injury. Elevated levels of CCL2 have been identified in patients with idiopathic pulmonary fibrosis, highlighting its role in fibrosis progression [56]. In mouse models, the disruption of this signaling pathway has provided protection against lung fibrosis. For instance, CCR2 knockout mice were shown to be protected from lung fibrosis induced by bleomycin treatment [33,76] and intrathecal instillation of FITC (Fluorescein Isothiocyanate) [77,78]. These findings suggest that inhibiting the CCL2/CCR2 axis could be a potential therapeutic target in preventing fibrosis. However, the translation of these findings into human therapies has proven to be more complex. For instance, a phase 2 randomized, double-blind placebo-controlled trial using carlumab, a human immunoglobulin G1κ monoclonal antibody designed to specifically neutralize the profibrotic activities of CCL2, demonstrated an unexpected outcome. Patients treated with carlumab showed a decline in forced vital capacity (FVC), a key indicator of lung function. Moreover, contrary to expectations, both total and free CCL2 levels were found to be elevated in the treated patients [24]. This paradoxical increase suggests that compensatory mechanisms may come into play upon blockade of the CCL2/CCR2 pathway. In line with this observation, a CCL12 knockout mouse model—where CCL12 is a murine homolog of CCL2—showed a compensatory increase in the expression of other CCR2 ligands, including CCL2 and CCL7, and did not exhibit protection from fibrosis following bleomycin treatment [79]. These findings underscore the complexity of chemokine signaling in fibrosis and highlight the potential limitations of targeting individual pathways without addressing compensatory mechanisms.

Mechanical Signals

In addition to chemical signals, mechanical cues in the extracellular matrix (ECM) have emerged as critical regulators of macrophage morphology, migration, activation, and function. Surface topographic features and stiffness significantly influence macrophage behavior and have been extensively studied in the context of foreign body reactions [80,81,82]. The mechanical properties of the fibrotic lung undergo significant changes during fibrosis progression. These include increased tissue stiffness (with healthy lung tissue having a Young’s modulus of approximately 1.96 kPa compared to up to 16.5 kPa in fibrotic lungs), altered viscoelastic properties, elevated surface tension, and regional heterogeneity [83]. In addition, the fibrotic ECM is characterized by excessive fibrillar collagen aligned in dense bundles, a result of active ECM remodeling by contractile myofibroblasts. These structural changes provide macrophages with additional topographic and mechanical cues that modulate their behavior [84]. Xu et al. explored how macrophages prepolarized with IL-4/13 respond to such cues in pulmonary fibrosis. Using a collagen hydrogel system with varying stiffness and fiber alignment, the authors demonstrated that macrophages in regions of high ECM stiffness or strong collagen alignment exhibited elongated morphologies and aligned along collagen fibers, displaying enhanced migratory behavior. These macrophages polarized into a pro-fibrotic phenotype, characterized by increased secretion of transforming growth factor-beta (TGF-β) and interleukin-6 (IL-6). Mechanistically, this response was mediated through integrin αMβ2 and cytoskeletal remodeling driven by Rho-associated kinase 2 (ROCK2). The process was shown to be inhibited by pirfenidone, an FDA-approved anti-fibrotic drug, which disrupts integrin signaling and cytoskeletal reorganization [84]. Beyond static mechanical cues, the dynamic interplay between macrophages and fibroblasts plays an important role in regulating macrophage motility and phenotype. In dense collagen networks (DCNs), macrophages relied on tunnel-like conduits created by fibroblasts to migrate through the ECM. In loosely connected networks (LCNs), macrophages utilized fibroblast-aligned collagen fibers as directional tracks for movement [85].The mechanical remodeling of the ECM by fibroblasts not only facilitated macrophage migration but also guided their positioning in fibrotic regions. Dynamic mechanical cues, such as deformation fields generated by fibroblast contractions, further amplify macrophage recruitment. A study by Pakshir et al. demonstrated that macrophages detect the velocity of local substrate displacements and migrate toward regions of force generation, even in the absence of chemotactic gradients. This mechanosensing process, facilitated by integrin α2β1 and stretch-activated ion channels, highlights the sophisticated mechanical communication between contractile fibroblasts and macrophages. Importantly, dynamic forces were essential for macrophage migration, as static ECM cues alone, such as collagen alignment, were insufficient to drive their movement [86]. However, not all studies reach the same conclusions regarding macrophage mechanosensing in fibrotic environments. Using 3D collagen and polyacrylamide (PA) hydrogels to decouple ECM stiffness from collagen concentration, another study revealed that macrophages exhibit differential gene expression based on ECM stiffness. For example, the expression of Fizz1, a gene associated with tissue repair, was suppressed in stiffer ECM environments, while Arg1 expression remained unaffected. Unlike the integrin-dependent mechanisms described in previous studies, this process relied on cytoskeletal remodeling, which regulated chromatin accessibility to control mechanosensitive gene expression. Similar results are replicated in vivo using a bleomycin-induced pulmonary fibrosis model. The authors conclude that these findings suggest that macrophage sensing of ECM mechanics could provide a negative feedback mechanism to prevent excessive ECM deposition and maintain homeostasis [87]. Although these conclusions may initially appear contradictory, the differences likely stem from variations in experimental conditions, such as the biomechanical properties of substrates (e.g., differing thresholds of stiffness), architectural features (e.g., two-dimensional versus three-dimensional structures, or specific topographic characteristics), and the involvement of fibroblasts. Furthermore, these discrepancies underscore the complex and multifaceted role of macrophages in fibrosis and highlight the inherently resolving nature of the mouse bleomycin-induced pulmonary fibrosis model. Together, these findings emphasize the nuanced interplay of macrophages with mechanical and dynamic cues in the fibrotic microenvironment, offering insights into potential therapeutic strategies for modulating macrophage activity in fibrosis. 4.1.4. Macrophage Effects Once recruited, macrophages are able to influence fibrosis by multiple mechanisms: secretion of pro-fibrotic mediators, including TGF-beta [58], directly modulating extracellular matrix through matrix metalloproteinases (MMPs) [88,89] and indirectly orchestrating inflammatory responses [90]. 4.1.5. Fate of Macrophages Survival of macrophages is contingent on specific microenvironmental cues and cellular interactions. Tissue-resident alveolar macrophages depend on granulocyte-macrophage colony-stimulating factor (GM-CSF), which is primarily produced by alveolar type II (ATII) cells, while monocyte derived macrophages rely on macrophage colony-stimulating factor (M-CSF) for their maintenance [91,92,93]. The fate of monocyte-derived macrophages in the context of pulmonary fibrosis has been the subject of considerable research, with varying results across different models. Studies have reported divergent lifespans for these macrophages, ranging from less than 24 h [32] to up to a year [47]. The maintenance of macrophage populations may occur through either autocrine or paracrine signaling mechanisms, wherein cells either self-sustain by secreting their own survival factors or rely on signals from neighboring cells within the microenvironment. Recent studies have provided further insights. These analyses utilizing single-cell RNA sequencing and in situ RNA hybridization revealed that M-CSF receptor (M-CSFR) and its ligand, M-CSF, are upregulated in fibrotic macrophages. The detection of CSF1/CSF1R expression in alveolar macrophages suggests that these cells may be capable of maintaining their population in the fibrotic niche via autocrine production of M-CSF, thus becoming independent of external signals from other lung cells [48]. This autocrine loop likely contributes to the persistence of macrophages in the fibrotic lung, which in turn sustains fibrosis by continually promoting ECM production and remodeling. In a computational model exploring the interaction between macrophages and fibroblasts, researchers further elucidated the importance of M-CSF in sustaining the fibrotic process. The model demonstrated that reducing CSF1 levels led to a decrease in macrophage numbers, which, in turn, resulted in a reduction in fibroblast activity. This allowed the tissue to transition toward a more normal, less fibrotic state [94]. Interruption of this M-CSF/M-CSFR signaling pathway has shown therapeutic promise in preclinical models. Blockade of this signaling using an anti-M-CSF antibody or the small molecule PLX3397 led to a significant reduction in monocyte-derived macrophages and subsequently ameliorated fibrosis [48]. This finding highlights the potential of targeting macrophage survival, by targeting key signaling pathways, such as the M-CSF/M-CSFR axis, as a therapeutic strategy for fibrosis. 4.1.6. Therapeutics Targeting Macrophages Due to the pivotal role macrophages play in the progression of pulmonary fibrosis, targeted therapies focusing on these cells are highly desired. Of these approaches, nanoparticles and liposomes are among the most widely explored platforms for delivering therapeutic agents directly to macrophages. Their versatility lies in their ability to encapsulate diverse payloads, including small molecules, siRNA, mRNA, and proteins, which can modulate macrophage behavior effectively. To increase specificity, these carriers can be further engineered by modifying their surfaces with ligands or antibodies targeting macrophage-specific markers, such as CD206, a receptor highly expressed on M2 macrophages in fibrotic tissues. In a notable study, Singh et al. engineered mannosylated albumin nanoparticles (MANPs) to exploit the high expression of the mannose receptor (CD206) on profibrotic monocyte-derived macrophages. Using a bleomycin-induced pulmonary fibrosis mouse model, the researchers demonstrated that approximately 75% of the injected MANPs were taken up by monocyte-derived alveolar macrophages. This was confirmed using Cx3cr1CreERT2+, Tdtomato fl/fl genetic lineage tracing mice. To further explore therapeutic potential, MANPs were loaded with small-interfering RNA (siRNA) targeting TGFβ1. Treatment with MANPs significantly reduced collagen deposition and other fibrotic markers, including TGFβ1 and IL-1β, while improving lung function. These findings highlight the efficacy of macrophage-targeting nanoparticles in mitigating fibrosis and restoring lung architecture [95]. Similar results were reported by Hou et al., who utilized M2pep-modified Mn-curcumin metal-organic framework nanoparticles (M2NP-BLZ@Mn-Cur). These nanoparticles feature a Mn-curcumin metal-organic framework coupled with M2pep ligands to enhance targeting specificity toward M2 macrophages, and BLZ945, a CSF-1R inhibitor, to facilitate the selective depletion of profibrotic M2 macrophages [96]. Although studies specifically focusing on nanoparticle-mediated macrophage targeting for pulmonary fibrosis are limited, lessons can be drawn from their application in other diseases, such as atherosclerosis. Nanoparticles have been extensively explored in the treatment and diagnosis of atherosclerosis, with multiple clinical trials underway. As summarized in a review, macrophage-targeting nanoparticles in atherosclerosis have been employed to inhibit monocyte recruitment, suppress macrophage proliferation, restore efferocytosis, inhibit inflammation, and induce macrophage apoptosis [97]. These therapeutic mechanisms are highly relevant to pulmonary fibrosis. Folate receptor-beta (FR-β), a glycosylphosphatidylinositol-anchored glycoprotein, represents another promising macrophage-specific target. FR-β mediates the unidirectional transport of folate into cells and is overexpressed on activated macrophages, while its expression is undetectable on resting macrophages [98]. Researchers have utilized this receptor to deliver therapeutic agents selectively to macrophages in fibrotic tissues. In one example, a folate-linked TLR7 agonist (FA-TLR7-54) was developed to reprogram profibrotic macrophages. When administered intravenously to bleomycin-treated mice, FA-TLR7-54 predominantly accumulated in the lungs. Flow cytometry confirmed that the nanoparticles were selectively taken up by FR-β-positive macrophages, primarily monocyte-derived alveolar macrophages. FA-TLR7-54 effectively shifted macrophages from an M2-like profibrotic phenotype to an M1-like antifibrotic state. This polarization shift resulted in the suppression of profibrotic cytokines such as CCL18, IL-1β, and Arg1, while promoting antifibrotic markers like CXCL10, IL-6, and IFNγ. Notably, the treatment reduced fibrosis without inducing significant systemic toxicity, as evidenced by stable body weight, normal plasma cytokine levels, and unaffected histology in major organs [99]. Elevated miR-33 levels in bronchoalveolar lavage (BAL) cells and lung macrophages of idiopathic pulmonary fibrosis (IPF) patients suggest its involvement in disease progression. Ahangari et al. developed a peptide nucleic acid (PNA)-based miR-33 inhibitor (PNA-33) that can be delivered both intranasally and intravenously. While intranasal delivery was more efficient, systemic administration also showed therapeutic benefits. Pharmacological inhibition of miR-33 with PNA-33 improved mitochondrial homeostasis and enhanced autophagy in macrophages. These effects translated into significant attenuation of fibrosis in bleomycin-induced pulmonary fibrosis mouse models. The study underscores the importance of targeting macrophage metabolic pathways and mitochondrial function to mitigate fibrotic progression [100]. While each of these strategies focuses on distinct molecular pathways, they collectively highlight the promise of macrophage-targeted therapies in addressing pulmonary fibrosis by offering high precision and efficacy while minimizing systemic toxicity. The ability to reprogram macrophages from a profibrotic to an antifibrotic state provides a multifaceted approach to tackling this complex disease. Future studies should build on these findings to optimize delivery systems, explore combination therapies, and translate these promising preclinical results into clinical applications. 4.2. Innate Lymphoid Cells Innate lymphoid cells (ILCs) are a recently identified group of immune cells that, unlike T and B lymphocytes, do not express recombination-activating genes, which are responsible for the somatic recombination of antigen receptors. ILCs are classified into three major subsets: ILC1, ILC2, and ILC3, based on their cytokine production profiles and transcription factor dependencies, paralleling the functional characteristics of CD4+ T helper cell subsets [101]. While the role of ILCs in immune responses is relatively underexplored compared to other lymphocytes, growing evidence points to the significant involvement of group 2 innate lymphoid cells (ILC2s) in the pathogenesis of pulmonary fibrosis. ILC2s are primarily activated by a group of epithelial-derived cytokines known as “alarmins”, which include IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) [101]. Once activated, ILC2s secrete large amounts of type 2 cytokines, including IL-5, IL-9, and IL-13 [102]. These cytokines are crucial mediators of fibrotic responses, as mentioned above in the M1/M2 polarization and Th1/Th2 cell section. Experimental models using bleomycin-induced pulmonary fibrosis have provided evidence for the involvement of ILC2s in fibrotic processes. Mice genetically deficient in ILC2s, such as staggerer mice (Rorasg/sg) that lack the transcription factor RORα essential for ILC2 development [103], as well as mice deficient in the IL-33 receptor ST2 [104], exhibit significantly reduced fibrosis compared to wild-type controls. However, despite these promising findings, the precise mechanisms by which ILC2s contribute to fibrosis remain incompletely understood, necessitating further studies to elucidate the complex signaling pathways involved and to explore potential therapeutic strategies targeting ILC2s or their upstream activators in fibrotic lung diseases.