Immune Mechanisms in Fibrotic Interstitial Lung Disease

SUMMARY

Fibrotic interstitial lung diseases (fILDs) have poor survival rates and lack effective therapies. Despite evidence for immune mechanisms in lung fibrosis, immunotherapies have been unsuccessful for major types of fILD. Here, we review immunological mechanisms in lung fibrosis that have the potential to impact clinical practice. We first examine innate immunity, which is broadly involved across fILD subtypes. We illustrate how innate immunity in fILD involves a complex interplay of multiple cell subpopulations and molecular pathways. We then review the growing evidence for adaptive immunity in lung fibrosis, to provoke a re-examination of its role in clinical fILD. We close with future directions to address key knowledge gaps in fILD pathobiology: 1) longitudinal studies emphasizing early-stage clinical disease, 2) immune mechanisms of acute exacerbations, and 3) next-generation immunophenotyping integrating spatial, genetic, and single-cell approaches. Advances in these areas are essential for the future of precision medicine and immunotherapy in fILD.

eTOC blurb

Fibrotic Interstitial lung diseases have poor survival rates, with current immunotherapies proving ineffective. This review examines underlying innate and adaptive immune mechanisms and potential clinical impacts. It lays the groundwork for future studies on early disease stages, acute exacerbation mechanisms, and advanced immunophenotyping to enhance therapeutic outcomes.

INTRODUCTION

Fibrosing interstitial lung diseases (fILDs) encompass several distinct clinical entities that share pathologic hallmarks of lung injury, inflammation, and fibrosis (Figure 1). The global prevalence of ILD is estimated to be 4.7 million people in 2019¹, the majority of whom had fILD. Clinically, fILD carries a poor prognosis, with 5-year survival rates below 50% depending on the underlying diagnosis^2,3. These survival rates remain low, owing, in part, to a lack of effective, targeted therapies.

Figure 1. Fibrosing interstitial lung disease (fILD).

A. In fILD alveolar injury and abnormal repair leads to fibroblast accumulation, extracellular matrix deposition, and fibrosis of the interstitial spaces. The resulting end-stage “honeycombing” is seen in: B. lung histology; and C. high resolution chest CT imaging. B, C: images are obtained by authors.

Tremendous progress has been made toward understanding the pathobiology of fILD. Contemporary research efforts have focused on the contributions of non-immune cell populations, primarily epithelial and fibroblast cells. However, immune-mediated mechanisms have long been recognized. Here, we review recent advances in the immunobiology of fILD. After introducing fILD, we examine the innate immunology of fILD. Next, we discuss adaptive immune mechanisms in fILD. We review evidence that clinical subtypes of fILD defy simple classification regarding the involvement of adaptive immunity. We close with a discussion of how the future research agenda in fILD can address the gaps in knowledge preventing a more personalized approach to therapy.

CLASSIFICATION OF FIBROTIC ILD (fILD)

Over 200 distinct clinical disorders comprise the family of interstitial lung diseases. Of these, fILD are the most common and severe, with a median survival of 3–7 years³. Patients with fILD present with breathlessness, exercise intolerance, and cough. Clinically, these patients are subclassified based on associated medical comorbidities, radiographic findings, or tissue histology (Table 1)⁴. Regardless of classification, progressive disease leads to end-stage organ dysfunction due to excessive extracellular matrix (ECM) production and lung fibrosis. In the following subsections, we discuss specific morphological and clinical classifications of fILD that have particular relevance to our review.

Table 1.

Fibrosing interstitial lung diseases.

Disease (year of diagnostic guideline)	Prevalence	Most common age of onset	Epidemiology, potential risk factors	Prognosis	HRCT findings	Histopathology	Clinical Characteristics	Auto-antibodies	Associated immune-related genetic variants
IPF (idiopathic UIP pattern) (2022)4	5 per 100,000 people, 3 million (worldwide)	50–70 years old	Male dominant (male:female = 2:1), associated with cigarette smoking, environmental exposures, chronic viral infections	Estimated five-year survival: 45–70%, Median survival; 5 years	UIP pattern; subpleural and basal predominance, honeycombing, reticular abnormality, absence of features inconsistent with UIP pattern (e.g. cysts, marked mosaic attenuation, predominant GGO)	Fibrosis and honeycombing predominantly in subpleural and paraseptal area, absence of certain features (e.g. hyaline membranes, organizing pneumonia, granulomas, marked interstitial inflammatory cell infiltrates, airway predominant)	No environmental exposure known to cause HP No SARD	Anti-periplakin169, anti-HSPA2168 reported.	TOLLIP112, TLR3113, IL1RN191, IL8192, IL4193, TGFB1194,
NSIP202	1 to 9 per 100,000 people Further classified as cellular- (20%) and fibrosing- (80%) NSIP	50–60 years old	Female dominant (60–70%), 60–70% were never smokers	Five-year survival: 82.3%, Fibrosing has worse prognosis than cellular NSIP	NSIP pattern (basilar predominant GGO with or without subpleural sparing, reticular abnormality)	Temporally homogeneous inflammation and/or fibrosis, diffuse alveolar wall thickening by uniform fibrosis, Fibrotic foci and peripheral attenuation are typically absent	No history of exposure associated with HP No SARD	Not present.	Not reported.
SARD-ILD7,203 (distinct criteria for each subtype)	Varies by SARD (RA, 11%; SSc, 47%; MCTD, 56%; IIM, 41%; SLE, 6%)	Varies Smoking, male gender, presence of RF and anti-CCP antibody in RA-ILD	Varies	Varies	Varies (UIP predominant in RA-ILD, NSIP more common in other SARDs)	Varies	Concomitant with SARDs, such as rheumatoid arthritis, SSc, MCTD, SS, IIM, SLE	Auto-antibodies common*	RA-ILD; TOLLIP204, HLA-DRB1, SSc-ILD; STAT4205, IRF5205
HP11 (2020)	0.3–0.9 per 100,000 population	50–60 years old	No gender preference Less common in current and past smokers Antigen exposure (e.g., fungal, bacterial, animal, protozoal, and insect)	Varies among the subtypes. 58% of patients with fibrotic HP stayed alive 7 years after diagnosis	Acute HP (< 6 week) and subacute HP (6–23 week): upper lobe predominant GGO, mosaic attenuation, air trapping Chronic HP (>= 24 wks): upper lobe predominant reticular abnormality, peribronchovascular interstitial thickening, honeycombing, mosaic attenuation, air trapping, centrilobular nodules, relative sparing of the basis	Nonfibrotic HP: cellular interstitial pneumonia, cellular bronchiolitis, poorly formed non-necrotizing granulomas Fibrotic HP: In addition to the above findings, chronic fibrosing interstitial pneumonia and/or airway-centered fibrosis are foci of organizing pneumonia	History of environmental exposure known to cause HP No SARD	Not present	HLA-DRB1*04206, TOLLIP207, TNF206
uILD208,209 (2013)	10–25% in ILD cohorts	50–60 years old	Varies	Estimated five-year survival rate is 46–70%	Varies	Varies	No history of exposure associated with HP No SARDs	Not present	Not assessed

^*Common autoantibodies in SARD include e.g., RF, anti-CCP, anti-Scl70 (SSc), anti- centromere, anti-RNA polymerase III, anti-U1 RNP, anti-SS-A, anti-Jo-1 (myositis), anti-dsDNA. IPF, idiopathic pulmonary fibrosis; UIP, usual interstitial pneumonia; GGO, ground glass opacity; HP, hypersensitivity pneumonitis; SARD, systemic autoimmune rheumatic disease; iNSIP, idiopathic non-specific interstitial pneumonia; RA, rheumatoid arthritis; SSc, systemic sclerosis; MCTD, mixed connective tissue disorders; IIM, idiopathic inflammatory myopathy; SLE, systemic lupus erythematosus; RF, rheumatoid factor; CCP, cyclic citrullinated peptide; Scl-70, scleroderma-70; RA-ILD, rheumatoid arthritis associated interstitial lung disease; RNP, ribonucleoprotein; SS-A, Sjögren-syndrome-related antigen A; dsDNA, double stranded DNA; uILD, unclassifiable interstitial lung disease.

Morphological classifications

Usual interstitial pneumonia (UIP).

UIP is the archetypal histopathological lesion and chest imaging pattern found in fILD patients⁴. UIP is often idiopathic, that is, without a defined etiology. Histologically, UIP is defined by temporal and spatial heterogeneity of fibrotic lesions where fibroblastic infiltrates, mature fibrosis, “honeycomb” cysts, and scar tissue alternate with areas of normal lung parenchyma (Table 1)⁴. On chest high resolution computed tomography (HRCT) imaging, the UIP pattern consists of reticular opacities from thickened interstitium, honeycomb changes from cystic dilation and alveolar wall destruction, and enlarging airways caused by contraction of the fibrotic lung tissue (i.e., traction bronchiectasis). A key feature of UIP is that these findings arise predominantly in basilar and subpleural patterns. In the clinic, patients with UIP without known etiology carry the diagnosis idiopathic pulmonary fibrosis (IPF). IPF is the most common subtype of fILD and is the major focus of translational and experimental studies. UIP morphology can also manifest in a subpopulation of other fILD, such as systemic autoimmune rheumatic disease-related ILD (SARD-ILD).

Non-specific interstitial pneumonia (NSIP).

Radiographically, NSIP is defined by a pattern of inflammation and fibrosis without the architectural distortion found in UIP. Histologically, NSIP is more inflammatory than UIP, consisting of varying amounts of lymphocytes, plasma cells, and fibrosis characterized by collagen bundles with few fibroblasts. Unlike UIP, the lesions in NSIP exhibit temporal uniformity⁵. NSIP can be subcategorized into cellular (primarily inflammatory) and fibrotic (primarily fibrotic or mixed cellular and fibrotic) phenotypes. Cellular NSIP is more responsive to immunosuppressive treatment and has a better prognosis. Fibrotic NSIP is less reversible and has a poorer prognosis⁶. Like UIP, the morphology of NSIP can be idiopathic or can be found in a subpopulation of other clinical entities.

Clinical classifications

Systemic autoimmune rheumatic disease-related ILD (SARD-ILD).

While most cases of fILD are idiopathic, pulmonary fibrosis is also a serious complication of many SARDs. A recent meta-analysis determined the prevalence of clinically-apparent ILD in patients with SARDs ranges from 6–60%, with highest prevalence in mixed connective tissue disease (MCTD) and systemic sclerosis (SSc)^7,8. SARD-ILD patients generally have better prognosis than patients with IPF. Lung pathology among SARD-ILD patients is heterogeneous, with fibrotic phenotypes contributing to excess morbidity and mortality⁹. Conversely, ~34% of patients with ILD can have features of SARDs, such as characteristic physical exam findings or the presence of autoantibodies¹⁰, without meeting full diagnostic criteria for a specific SARD. These patients with “interstitial pneumonia with autoimmune features” (IPAF) have a mildly better prognosis than IPF patients, but worse than SARD-ILD patients. The prevalence of histological subtypes, such as UIP or NSIP morphologies, differs among SARDs (Table 1)⁹.

Hypersensitivity pneumonitis (HP).

Like SARD-ILD, HP is an immune-mediated inflammatory and/or fibrotic lung disease. HP primarily affects the lung parenchyma and small airways arising from an immune reaction to inhaled antigens¹¹. Histologically, HP can share fibrotic subfeatures of UIP, but also demonstrates peribronchiolar metaplasia and poorly formed, non-necrotizing granulomas. HRCT classifies HP as either non-fibrotic or fibrotic, and the extent of fibrosis correlates with symptom severity, disease progression, and patient survival¹¹.

Despite differences in morphological and clinical classification schemes for fILD, progression of fibrosis remains the prime determinant of lung function and mortality. For this reason, clustering patients by shared lung morphology is gaining traction in the field. For example, some patients with rheumatoid arthritis-associated ILD (RA-ILD) resemble IPF patients with radiologic and histologic UIP patterns. These RA-UIP patients have increased mortality that more resembles the natural history of IPF patients than that of RA-ILD patients without UIP⁹. Hence, rather than classifying IPF as its own separate category, clinicians and researchers may benefit from considering IPF, RA-UIP, and other fILD with UIP features as one UIP group, distinct from RA patients without UIP. In the future, the current histopathological and radiological definitions of morphological subtypes should be refined by more granular immunophenotyping.

RESEARCH TOOLS

Working knowledge of clinical and experimental methods used to study immunological pathways in fILD is essential to understanding current efforts to refine our understanding of morphological subtypes. Over the past ten years, advances in biospecimen procurement, bioinformatic analysis, and experimental models have transformed our ability to study the immunology of fILD (Figure 2). Among these, human cohorts and animal models of fibrotic lung disease are indispensable. A growing number of established human cohorts and biorepositories helps define the natural history of disease and provides opportunities for molecular immunophenotyping. These translational findings can then be investigated in relevant animal models to test for novel mechanisms and potential treatment targets. The pairing of human cohort data with mechanistic studies in animal models has vastly improved our ability to translate laboratory findings to clinical practice.

Figure 2. Research methods in lung fibrosis.

Human samples for translational studies of patients with fibrotic lung interstitial lung disease include bronchoalveolar lavage fluid (BAL), lung tissue obtained during biopsy or explant, and blood. Biobanked specimens can be analyzed by “omic” approaches, while primary cells may be isolated from fresh tissue samples for conventional two-dimensional tissue culture studies or three-dimensional organoids or complex in vitro model systems like “lung on a chip”. Lung tissue can also be used to generate precision cut lung slices (PCLS). Animal models induce lung fibrosis via bleomycin or silica exposure.

Human cohorts

Human research cohorts provide a rich resource of clinical data and biospecimens for the study of fILD (Table 2). Retrospective studies are useful to define incidence, prevalence, risk factors, and natural history of ILD in population-based studies. In smaller-scale retrospective studies, a detailed review of clinical data, including imaging and pathology results, is feasible for careful phenotyping of ILD. Prospective studies can standardize clinical data collection, including additional research questionnaires, physical exams, as well as provide research biospecimens, such as peripheral blood, bronchoalveolar lavage fluid (BALF), and lung tissue. Molecular studies rely on a range of biobanks, from private institutions, like the Mass General Brigham Biobank, to national institutes, like the UK Biobank, and the Lung Tissue Research Consortium (LTRC) at the U.S. National Institutes of Health (available via the NIH BioLINCC, biolincc.nhlbi.nih.gov/studies/ltrc/).

Table 2.

Selected human cohorts investigating fILD prevalence, incidence, and progression.

Study name	Study design	Location and sample size estimate	ILD pheno typing	Bio-specimen
Rochester Epidemiology Project210	Population-based retrospective study	Olmsted County, MN, USA (n=500,000)	UIP	None
Taiwanese National Health Insurance Research Database211	Population-based retrospective study	Taiwan (n=23,000,000)	ILD	None
Viral RNA in UIP212	Retrospective study with retrieval of lung biopsy specimens	Birmingham, AL, USA (n=17)	UIP	Lung
Bronchoalveolar lavage in ILD213	Prospective study of bronchoalveolar lavage	Fukuoka, Japan (n=31)	ILD	BAL, blood
LTRC214	Prospective study of lung tissue	Multi-site, USA (n=4,200)	ILD, quantitative CT	Lung
UK Biobank215	Prospective biobank study	Multi-site, United Kingdom (n=500,000)	UIP	Blood
MGB Biobank216	Prospective biobank study	Boston, MA, USA (n=140,000)	ILD	Blood
Framingham Heart Study217	Prospective general population study	Framingham, MA, USA (n=5,209)	ILA, quantitative CT	Blood
MESA218	Prospective general population study	Multi-site, USA (n=6,814)	Quantitative CT	Blood
AGES176	Prospective general population study	Multi-site, Iceland (n=5,320)	ILA	Blood
COPDGene176	Prospective study of smokers	Multi-site, USA (10,371)	ILA, quantitative CT	Blood
Unaffected first-degree relatives of patients with pulmonary fibrosis175	Prospective study among relatives of fILD	Boston, MA, USA (n=105)	ILA	Blood
UKILD-Long COVID177	Prospective study of suspected ILD after COVID-19	Multi-site, United Kingdom (n=12,000)	ILA, quantitative chest imaging	BAL, blood
VARA219	Prospective registry of veterans with RA	Multi-site, USA (n=2,328)	ILD	Blood
Subclinical ILD in RA220	Prospective study of patients with RA	Denver, CO, USA (n=194)	ILA, quantitative CT	Blood
BRASS-Lung178	Prospective study of patients with RA	Boston, MA, USA (n=120)	ILA	Blood
SAIL-RA	Prospective study of patients with early RA	Multi-site, USA (n=160)	ILA	Blood
ANCHOR-RA	Prospective study of patients with RA	Multi-national (n=1,200)	ILA	Blood

BAL, bronchoalveolar lavage fluid; CT, computed tomography; ILA, interstitial lung abnormalities; ILD, interstitial lung disease; UIP, usual interstitial pneumonia; RA, rheumatoid arthritis.

Experimental models of fibrotic ILD

Nearly all experimental studies of fILD employ rodent models. These models include administration of silica, asbestos, fluorescein isothiocyanate, thoracic radiation, and, most commonly, bleomycin^12,13. In 2017, an American Thoracic Society workshop report described the bleomycin model as the best characterized model of lung fibrosis with highly relevant pathobiology¹². Bleomycin is a chemotherapeutic glycopeptide that cleaves DNA thereby precipitating cellular injury. The most common model is a single bleomycin dose delivered to the lung. Epithelial cell injury is followed by inflammatory cell recruitment during the first 7 days. Ultimately, acute inflammation subsides leaving a chronic inflammatory milieu with fibrosis evident by day 14. Most studies end on day 21 (peak fibrogenic response), and fibrosis partially resolves over the subsequent month¹⁴. Bleomycin can also be delivered systemically by injection or osmotic pump. In these systemic models, initial injury is to the vasculature. After a single-dose of intrapulmonary bleomycin, the mouse lung shows patchy alveolar damage, alveolar hyperplasia, deposition of provisional matrix, inflammation, fibroproliferation, induction of senescence, and even areas of traction bronchiectasis¹⁵. In support of bleomycin’s clinical relevance, transitional epithelial cells noted in human UIP pathology have also been identified following bleomycin exposure in mouse lung^16,17. However, a key feature of UIP pathology is fibroblastic foci, which are not reliably reproduced by this model. The partial resolution of fibrosis over time is also poorly representative of progressive fILD. Hence, a growing number of investigators use repeated dosing of bleomycin¹⁸ to model more persistent lung fibrosis.

While advances in gene sequencing and bench immunology have revolutionized our ability to investigate human disease, human cohorts and animal models have played an essential part in investigating the immunology of fILD. Continued advances in both their development and utilization is crucial for future discoveries of pathogenic immune axes and therapeutic targets.

NON-IMMUNE AXES IN LUNG FIBROSIS

Our review seeks to motivate further work in the immunology of fILD in part because the field has made the most progress on non-immune mechanisms of lung fibrosis. In normal lung, type 1 alveolar epithelial cells (AT1) are responsible for gas exchange and are renewed by type 2 alveolar epithelial cells (AT2), which possess stem cell-like properties. Recurrent injury or host-susceptibility factors, like age or early senescence, can exhaust AT2 renewal capacity and impair alveolar integrity¹⁹ (Figure 3). Consequently, dysfunctional lung epithelium produces pro-fibrotic cytokines and chemokines, such as transforming growth factor-β (TGF-β), platelet derived growth factor (PDGF), and matrix metalloproteinases (e.g., MMP-1 and MMP-7). Additionally, lung epithelial cells acquire mesenchymal phenotypes. Functionally, epithelial-to-mesenchymal transition is the direct transformation of epithelial cells into mesenchymal cells, contributing to the formation of myofibroblasts in pulmonary fibrosis¹⁹. Myofibroblasts express contractile proteins and secrete excessive ECM, thereby causing tissue fibrosis¹⁹. Compared to the substantial work investigating epithelial biology in fILD, understanding of the immune pathways in translational and experimental fILD represents a relative gap in knowledge in the pathobiology of fILD fibrosis.

Figure 3. Immune and stromal cell interactions drive fibrosis in fILD.

Epithelial cells exhibit aberrant repair responses after recurrent injury in the setting of risk factors like age, smoke exposure, or increased cell senescence. The dysfunctional epithelial cells profibrotic mediators, leading to a cascade of cell-cell interactions among the epithelium, endothelium, mesenchyme, and immune system drives lung fibrosis (i.e., deposition of extracellular matrix [ECM] in the lung interstitium by myofibroblasts). In addition to the proliferation of fibroblasts, the transition of type 2 alveolar epithelial cells (AT2) and endothelial cells (EMT and Endo-MT, respectively) to mesenchymal cells contribute to the accumulation of fibroblasts.

INNATE IMMUNE AXES IN LUNG FIBROSIS

Macrophage populations in lung fibrosis

Several lines of evidence implicate macrophages in a range of clinical fILD. Increased alveolar macrophages are observed in fibrotic lung tissue from both IPF and SARD-ILD patients¹⁵. Elevated monocyte counts in BALF are associated with poor prognosis in IPF patients²⁰. In SSc-ILD patients, circulating classical CD14⁺ monocytes with dysregulated pro-fibrotic programs accumulate in fibrotic foci in their lungs²¹. Lung macrophages can be categorized by both cell lineage history and their anatomical location (i.e., airway, alveolar, and interstitial). For example, transcriptional and lineage analyses show that resident alveolar macrophages are self-renewing and distinct from monocyte-derived macrophages recruited from extra-pulmonary locations. These different myeloid cell populations likely have distinct contributions to the pathophysiology of lung fibrosis. For example, experimental models indicate monocyte-derived macrophages are more important drivers of disease than resident macrophages. Monocyte-derived alveolar macrophages reside near areas of fibrosis and express pro-fibrotic and extracellular matrix remodeling genes^22–24. In line with these findings, deletion of monocyte-derived lung macrophages attenuates lung injury^22–24, while deletion of resident alveolar macrophages did not affect fibrosis in an asbestosis-induced model of pulmonary fibrosis²⁴. Monocyte-derived macrophages undergo apoptosis and decline in number during the later, fibrotic stages of murine lung fibrosis²⁵.

M1 and M2 macrophages

Macrophages have been dichotomized into M1 and M2 phenotypes (Figure 4). M1 macrophages were originally defined as nitric oxide producing cells and are considered pro-inflammatory, a phenotype resembling “classically activated” by IFNγ, TNFα, GM-CSF, or lipopolysaccharide stimulation. M2 macrophages were originally defined as arginase-1 expressing cells and are anti-inflammatory, and include macrophages “alternatively activated” by IL-4 or IL-13²⁶. Th1 cells produce IFNγ to stimulate M1 macrophages to produce pro-inflammatory cytokines, like IL-1β and TNFα. These stimuli induce characteristic M1 functions, such as upregulation of inducible nitric oxide synthase 2 (iNOS) and production of reactive oxygen species (ROS), inflammatory cytokines (e.g., IL-1β, IL-6, IL-12, TNFα, and CXCL10), and surface expression of MHC II, CD80, and CD86. M1 macrophages with their pro-inflammatory and ROS generating capabilities are critical to clearing infection, particularly of intracellular organisms and viruses. M1 macrophages also are implicated in numerous non-infectious, inflammatory contexts, ranging from atherosclerosis to autoimmune demyelinating disease. Conversely, M2 macrophages help resolve inflammation and augment tissue repair through production of TGF-β and various growth factors, such as fibroblast derived growth factor, PDGF, and vascular endothelial growth factor.

Figure 4. Th1/2, M1/M2, and Th17 axes in fILD.

A. Naive T cells, also known as T-helper type 0 (Th0) cells, can differentiate into Th1 (via stimulation by IL-12) or Th2 cells (via IL-4). Th1 cells are on balance protective against lung fibrosis. Th1 cells promote M1 and inhibit M2 phenotypes in macrophages. M1 macrophages contribute to pathogenesis pathogenic by prolonged production of IL-1β that inhibits healthy epithelial cell renewal and inflammatory stimulation of fibroblast migration and proliferation. Th2 cells are pathogenic and drive M2 phenotypes. M2 macrophages secrete TGF-β that promotes the differentiation of fibroblasts into myofibroblasts producing ECM. B. Th17 cells are pathogenic and produce IL-17 that activates neutrophils and augments EMT. These mesenchymal cells migrate, proliferate, and differentiate into myofibroblasts producing ECM.

The M1/M2 classification has been an important framework for macrophage studies in fibrosis for over 20 years. M2 macrophages promote extracellular matrix deposition, angiogenesis, and tissue remodeling as part of wound healing. However, these homeostatic functions by M2 macrophages can go awry, leading to pathogenic fibrosis in a range of organs, including skin, kidney, liver and lung. Consistent with the disease paradigm of aberrant lung repair leading to fibrosis, M2 macrophages and monocytes are increased in fILD patients. Markers of M2 macrophages, like CD163, are elevated on lung macrophages in IPF patients²⁷, and SSc-ILD patients have increased CD163⁺ M2 monocytes in circulation²⁸. M2 macrophages promote myofibroblast differentiation via Wnt/β-catenin signaling²⁹ or secretion of profibrotic mediators like TGF-β, galectin-3³⁰, and S100A4³¹. Investigation of M2 macrophages continues to reveal important pathologic mechanisms, including the recent discovery of the AP-1 transcription factor Fra-2 driving type VI collagen expression and lung fibrosis³².

Considering fILD as a strictly “M2” disease is too simplistic, however. The failure of IPF clinical trials targeting the canonical M2 cytokine IL-13^33,34 underscores this perspective. Beyond the general redundancy of pathological mechanisms in lung fibrosis, at least two factors likely contribute to the failure of targeting M2-related pathways. First, M2 macrophages can have different effects depending on the time course of injury. These cells can reduce injury and promote healing without scarring, as in experimental myocardial infarction³⁵ and skeletal muscle injury³⁶. In experimental liver injury, macrophage depletion during the early healing phase worsened fibrosis, while macrophage depletion during the later fibrotic phase reduced myofibroblast accumulation and scarring³⁷. Second, the stimuli driving M2 macrophages are more complex than simply cytokines like IL-13. Additional upstream factors, including M1 macrophages, modulate the M2 response. For example, in the murine bleomycin model, adoptive transfer of Ly6c^hi (“inflammatory”) monocytes increases the abundance of M2 macrophages in the lung and promotes fibrosis²⁷.

Pro-inflammatory cytokines produced by M1 macrophages, such as IL-1β^38,39 and TNFα⁴⁰, are also implicated in the pathogenesis of fILD. M1 macrophages are likely major sources of the increased IL-1β found in BALF from IPF patients³⁸. In the bleomycin model, knocking out the IL-1β receptor, IL1R1, attenuates both lung inflammation and fibrosis³⁹. While some amount of interstitial macrophage-derived IL-1β is necessary for physiologic type 1 alveolar epithelial cells differentiation and tissue repair, sustained IL-1β production inhibits regeneration and promotes the dysfunctional repair that is a hallmark of lung fibrosis⁴¹. Production of mature IL-1β requires the inflammasome, and both NLRP3 and AIM2 inflammasomes are activated in alveolar macrophages in human and murine lung fibrosis^42,43. In line with a pathogenic role for M1 macrophages generally and the inflammasome specifically, mice deficient in NLRP3 were resistant to lung inflammation and fibrosis⁴⁴

As an alternative to the M1/M2 scheme, macrophages can be classified as CCR2⁺ (“inflammatory”) or CX3CR1⁺ (“reparative”) phenotypes. This scheme may provide a more robust understanding of macrophage contributions to fILD. CCR2 is a cell-surface receptor expressed by macrophages and T cells that binds CCL2, CCL7, CCL8, CCL13, and other ligands. CCR2⁺ macrophages drive inflammation in a range of contexts, including the early phase of wound healing, myocardial infarction, and infection²⁶. CX3CR1 is a cell-surface receptor that binds CX3CL1 (fractalkine). CX3CR1⁺ macrophages promote tissue repair in both normal and pathological fibrosis across several organs. Both CCR2⁺ and CX3CR1⁺ subpopulations are pathogenic in lung fibrosis. Expression of CCR2 and CX3CR1 ligands (CCL2 and CX3CL1, respectively) is increased in the lungs of IPF patients^45,46. In experimental models, Ccr2-deficient mice had less macrophage infiltration into the lung and were protected from bleomycin-induced fibrosis⁴⁷, with the important caveats that Ccr2-deficient mice have broad deficits in the recruitment of myeloid cells and cytokine production from T cells⁴⁸. In the bleomycin model, mice with global deletion of CX3CR1 had attenuated pulmonary fibrosis with reduced infiltration of bone marrow-derived fibrocytes and CD206⁺ macrophages in the lung⁴⁹. Mice with targeted deletion of CX3CR1⁺SiglecF⁺ macrophages were protected from lung fibrosis, with a reduction in Pdgfra⁺ and Pdgfrb⁺ fibroblasts⁵⁰. Hence, pathogenic macrophages in fILD come from both traditional “inflammatory” and “reparative” subsets, whether classified by the M1/M2 or CCR2/CX3CR1 schema.

Advanced macrophage phenotyping

Enabled by high-throughput methods in RNA sequencing at single-cell resolution (scRNA-seq), there is a growing appreciation for macrophage populations distinguished by markers other than traditional subsets. Single-cell and other analyses have highlighted the expression of two monocyte-derived macrophage subpopulations – heparin-binding EGF-like growth factor (HBEGF⁺) macrophages⁵¹ and platelet-derived growth factor A (PDGFA⁺)⁵⁰ macrophages – in both IPF lung and experimental lung fibrosis. HBEGF^51,52 and PDGFA⁵⁰ are expressed in myeloid cells from multiple organs and are known to promote fibrogenesis, stromal chemotaxis, and wound healing.

Single-cell transcriptomic studies performed on IPF lung also identified an expanded population of SPP1⁺ alveolar macrophages adjacent to areas of fibrosis^53–55. Secreted phosphoprotein 1 (SPP1, osteopontin), which is found both in the ECM and intracellularly, is expressed in a wide range of cell types, including bone, smooth muscle, endothelium and myeloid cells. SPP1⁺ macrophages are implicated in the pathogenesis of fibrosis in multiple organs other than lung, such as heart, kidney, liver, and skin⁵⁶. Inhibition of SPP1 with intranasal administration of small interfering RNA (siRNA) decreases pulmonary fibrosis in vivo. The profibrotic functions of SPP1 include the promotion of TGF-β-mediated epithelial-to-mesenchymal differentiation⁵⁷, and migration of lung fibroblasts in vitro⁵⁸. Beyond SPP1 itself, SPP1⁺ macrophages express other pro-fibrotic genes, such as IL1RN (IL-1 receptor antagonist), MMP9, and CHI3L1^53,55. The biology of SPP1⁺ lung macrophages may be informed by studies in other organs. For example, in a murine model of myocardial infarction, platelet-derived CXCL4 was critical for SPP1⁺ macrophage differentiation, and Cxcl4-deficient mice were protected against fibrosis after myocardial infarction and kidney ischemiareperfusion injury⁵⁹. Platelet-bound antigen-antibody immune complexes induced SPP1 expression in monocytes in vitro through CSF1 (macrophage colony-stimulating factor)⁵⁹. In lung macrophages, IL-6 cooperated with CSF1 to induce SPP1 expression⁵⁸. When SSc patients with and without ILD were treated with tocilizumab (anti-IL-6 receptor blocking monoclonal antibody [mAb]), the circulating levels of SPP1 were reduced⁵⁸. A role for CSF1 may not be limited to SARD-ILD, as CSF1 expression by macrophages increased at the terminus of the “cell trajectory” analysis of scRNA-seq of IPF lung, which raises the possibility of an autocrine loop amplifying macrophage recruitment, activation, and expression of SPP1⁵⁵.

Macrophage axes in clinical fILD

The subtypes of fILD vary in their relative amounts of inflammation and fibrosis (Table 1), and SARD-ILD and IPF clearly have distinct underlying immune drivers. However, these fILD share several innate immune axes centered on myeloid cells. First, SARD-ILD and IPF patients can share the same M2-like axes. Serum expression of CHI3L1 (a marker of M2 macrophages) predicts worsening pulmonary function in IPF patients⁶⁰ and the development of ILD in patients with RA⁶¹. Experimentally, CHI3L1 promotes alternative activation of macrophages⁶², fibroblast proliferation, and expression of profibrotic transcriptional programs⁶⁰. As another example, in patients with SSc-ILD, peripheral monocytes are primed for increased expression of MMP9 (a prominent biomarker in IPF patients⁶³) when stimulated by profibrotic cytokines TGF-β, IL-4, IL-10, and IL-13²¹.

Second, both IPF patients⁶⁴ and SSc-ILD⁶⁵ patients have increased numbers of M1-like monocytes in circulation. IPF patients have circulating monocytes with an inflammatory phenotype characterized by increased expression of type I interferon (IFN)-responses and of CD64 (FcγR1), an IFNγ-response gene⁶⁴. In addition, a subpopulation of CD64⁺ monocytes with increased expression of CCL2 and type I IFN genes were expanded in IPF lungs. The abundance of peripheral blood monocytes, or their expression of CD64, have also been associated with worse clinical outcomes, including decreased forced vital capacity (lung function), 6-minute-walk distance²⁰, and radiographic fibrosis⁶⁴. This macrophage/CCL2 axis identified in IPF has also been described in SSc patients, where increased levels of CCL2 in serum⁶⁶ and BALF⁶⁷ were associated with clinical ILD and reduced pulmonary function.

Third, increases in SPP1⁺ macrophages have been identified in lungs from SSc-ILD and HP patients, as well as IPF patients^{53–55,68,69}. Elevated serum levels of SPP1 correlate with lung function decline in SSc-ILD^58,60. The continued expansion of single-cell studies to include SARD-ILD and HP patients, along with the application of new technologies like spatial transcriptomics and proteomics, will likely reveal shared macrophage populations and functional pathways across fILD in increasingly finer detail. For example, HBEGF⁺ macrophages are a population in IPF lung, but HBEGF⁺ macrophages also mediate fibroblast invasiveness in the synovia of rheumatoid arthritis patients⁵², therefore meriting examination in future studies of RA-ILD.

In sum, fILD is driven by a broad spectrum of traditional inflammatory and reparative macrophage populations, along with new subpopulations, such as SPP1⁺ or HBEGF⁺ macrophages that have been uncovered using single-cell analysis. While IPF and SARD-ILD are distinct clinical entities with different treatment approaches, these diseases share macrophage axes and candidate therapeutic targets. A key challenge is translating insights obtained from studying a brief and self-resolving injury in mice to understanding the progression of clinical fILD over years. We hypothesize that many patients with earlier-stages of fILD, including both IPF and SARD-ILD, have both the inflammatory and reparative myeloid populations identified in experimental lung fibrosis. The spatial distribution of these functionally distinct myeloid populations may influence the differentiation of these disease subtypes. A critical question is how the mixture of myeloid populations evolve in patients with rapid progression of lung fibrosis. Some patients may have local exacerbations of their inflammatory myeloid populations, while other patients may be dominated by a ‘runaway’ expansion of their “reparative” monocytes and macrophages. We hypothesize that patients with both types of progression are found across disease subtypes, such as both IPF and SARD-ILD, but these speculations require further translational study across larger patient cohorts.

Neutrophils

Along with macrophages, neutrophils are key effector cells in acute lung inflammation and the early response to lung infection. Neutrophils respond to infection with anti-microbial granules, extracellular traps (NETs) and phagocytosis of microbes. Although IPF patients have increased infiltration of neutrophils in the lung⁷⁰, neutrophils are far less studied in fILD than macrophages. Neutrophils may drive lung fibrosis by release of neutrophil elastase^71,72, MMPs⁶³, leukotrienes, and NETs^73–75. These mechanisms are also implicated in lung fibrosis. For example, small molecule inhibition or genetic deletion of neutrophil elastase reduced lung fibrosis in murine models of lung fibrosis^71,72. Neutrophil elastase acts on multiple cell types in the lung, promoting proliferation of fibroblasts and their differentiation into myofibroblasts⁷². In acute lung injury and cystic fibrosis, the protease activity of neutrophil elastase acts on lung epithelial cells to potentiate the expression of inflammatory cytokines and chemokines, epithelial cell senescence, and cell death. These mechanisms may also apply to lung fibrosis. Neutrophils are also a major source of MMPs, which regulate ECM degradation and tissue remodeling in fibrosis. The levels of multiple MMPs, such as MMP3, MMP8, MMP9, are elevated in both the BALF and lung tissue of IPF patients, and the MMP levels correlate with disease activity⁶³. In vivo experiments support a profibrotic role for MMPs. For example, MMP3- and MMP8-deficient mice were protected from bleomycin-induced pulmonary fibrosis^76,77. MMP8-deficient mice had greater lung inflammation, with increased expression of Cxcl10 and Ccl3 (MIP-1α). Deletion of either chemokine in MMP8-deficient mice reversed their phenotype, with reduced inflammation and increased fibrosis in the bleomycin model⁷⁷.

To fight infection, neutrophils release NETs, an extracellular network of chromatin fibers studded with antimicrobial peptides and enzymes. In the murine bleomycin model, neutrophil activation and NETs correlated with disease severity⁷⁸. NETs directly augment lung fibroblast proliferation and myofibroblast transdifferentiation in vitro. NETs contain several components, such myeloperoxidase, histone 3, and IL-17, that have profibrotic effects on fibroblasts^73,79. In vitro, NETs promoted the secretion of inflammatory cytokines, such as IL-1β, TNFα, and IFNγ from macrophages, contributing to both tissue injury and downstream recruitment of neutrophils and macrophages^80,81.

We lack a definitive demonstration of the role of neutrophils in experimental pulmonary fibrosis, as it is unclear whether prior studies⁸² achieved complete neutrophil depletion⁷⁸. Further functional studies in vivo are critical to interpret the growing descriptive findings for neutrophils in clinical fILD. IPF patients have an increased number of neutrophils in their lungs⁸³, driven by elevated chemoattractants (e.g., CXCL1, CXCL8, and G-CSF)^84,85. Elevation of these neutrophil-attracting chemokines is associated with increased mortality in IPF⁸⁶. In IPF lung, neutrophils co-localize with SPP1⁺ profibrotic macrophages. These SPP1⁺ macrophages had increased expression of TREM2 and CD9, which are markers for profibrotic, “scar-associated macrophages” in other organs⁶⁸. Neutrophils adjacent to SPP1⁺ macrophages secreted MMP9, IL-17A, and GM-CSF. IL-17A can have profibrotic interactions with epithelial cells and fibroblasts in vitro⁸⁷ and in vivo^38,88. GM-CSF inhibited collagen deposition in the bleomycin model⁸⁹ but may be profibrotic in other contexts. The sum effect of neutrophil-derived cytokines is yet to be determined. The limited translational studies to date have focused on neutrophils in IPF, but neutrophils likely influence other types of fILD. For example, in lungs with the NSIP pattern of lung fibrosis, neutrophil elastase and NETs are located near myofibroblasts and fibrotic lesions⁷³. As short-lived cells, neutrophil populations can rapidly rise and fall and change phenotype. Hence, there is a particular need to understand the role of neutrophils in acute exacerbations of fILD.

Other myeloid cells

Beyond monocytes, macrophages, and neutrophils, less numerous granulocyte populations have been shown to make a demonstrable contribution to the pathobiology of pulmonary fibrosis. Plasmacytoid dendritic cells (pDC) distinguish SARD-ILD. pDCs are highly inflammatory cells in SARD, a finding not established in IPF. Increased number of pDC infiltrate the lung and skin tissue of SSc patients^90,91; and pDC are found in other inflamed tissue from SARD patients, such as the synovium in RA and skin lesions in systemic lupus erythematosus. pDC are the major source of CXCL4, a cytokine/chemokine that directly induces myofibroblast differentiation and collagen synthesis in stromal cells and endothelial cells in vitro⁹². CXCL4 can also promote differentiation of monocytes into pro-inflammatory and pro-fibrotic dendritic cells⁹³. Patients with SSc-ILD were found to have elevated serum levels of CXCL4 that correlated with the extent of both skin and lung fibrosis⁹⁴. This finding may account for the observation that macrophage populations in the SARD-ILD, like SSc-ILD, had increased type I IFN responses compared to IPF even though both clinical phenotypes share SPP1⁺ (more abundant in more fibrotic areas of lung) and FABP4⁺ (more abundant in the areas of lung with normal histology) macrophages⁶⁹. In experimental lung fibrosis, gene knockout or antibody blockade of CXCL4 reduced lung fibrosis⁹², suggesting a therapeutic avenue to break a pro-fibrotic positive feedback loop from pDCs to macrophages and fibroblasts.

Myeloid-derived suppressor cells (MDSCs) are a heterogenous population of myeloid cells characterized by their immunosuppressive activity, which plays a crucial role in the pathophysiology of various conditions such as infection, cancer, and autoimmune diseases. The two main subtypes of MDSCs are monocytic MDSCs and granulocytic MDSCs. Compared with control subjects and chronic obstructive pulmonary disease (COPD) patients, MDSCs are more abundant among peripheral blood mononuclear cells (PBMCs) of ILD patients, including IPF, HP, NSIP, and SARD-ILD. In particular, monocytic MDSCs, rather than granulocytic MDSCs, were more abundant in PBMC of IPF patients⁹⁵. MDSCs are protective in other organ fibrosis models, such as kidney and liver^96,97. As in these other models, MDSCs derived from IPF patients suppress T cell proliferation in vitro, a finding that suggests MDSCs modulate the expression of co-stimulatory molecules on peripheral T cells from IPF patients⁹⁵. Despite these observations, increased circulating MDSCs negatively correlate with lung function in IPF patients⁹⁵. This finding of more MDSCs in patients with worse fibrotic disease might seem to refute the in vitro data and findings in other organs; however, one can hypothesize that the MDSC mobilization is an insufficient homeostatic attempt to attenuate pulmonary fibrosis. Alternatively, T cell subsets can be either pathogenic or protective in lung fibrosis, as we describe further below, and MDSCs may have different effects on these T cell subpopulations.

Complement

Cell-specific chemokines and cytokines are not the only molecules that mediate cross-talk among innate immunity, epithelium and fibroblasts. Other innate immune mediators, such as complement and ligands of toll-like receptors (TLRs), are expressed across multiple cell types and contribute to the pathophysiology of lung fibrosis. IPF patients have elevated levels of the complement component 3a (C3a) and 5a (C5a) in the lung, and increased C1q deposition in the lung is associated with poor prognosis in IPF patients⁹⁸. Further, IPF lung epithelial cells express less of complement inhibitory proteins (e.g., CD46, CD55)⁹⁹. Like other innate immune pathways, complement is also implicated in other clinical fILD beyond IPF. In HP, immune complexes deposited in the lung can activate complement¹⁰⁰, which may then amplify the activation of innate immunity. Autoantibody production in SARD-ILD and IPAF patients also raises the possibility of increased complement activation in these diseases when compared to IPF; however, this hypothesis needs to be tested experimentally.

AT2, bronchiolar epithelial cells, alveolar macrophages, and polymorphonuclear leukocytes can synthesize complement proteins following inflammatory cytokine stimulation by IL-6, IL-1, TNFα, and IFNγ. Serine proteases from alveolar macrophages cleave and activate C5, which initiates a range of inflammatory signaling cascades. In the murine bleomycin model, C5-deficient mice¹⁰¹ and C1q deficient mice¹⁰² had reduced lung fibrosis, compared to control mice. Complement factors can drive disease via several cellular targets, such as the induction of neutrophil proteolytic enzymes and reprogramming of macrophages towards a M2 phenotype¹⁰³. Complement also acts directly on lung epithelium and stromal cells. C3a induced expression of TGFB by lung epithelial cells, and C3a and C5a amplified that effect by downregulating SMAD7, a negative regulator of TGF-β⁹⁹. C5a induces lung fibroblasts to express MMPs¹⁰¹, and C1q stimulates fibroblast proliferation¹⁰⁴. In other end-organs¹⁰⁵ complement can promote epithelial-to-mesenchymal transition, but this function is yet to be determined in lung fibrosis.

The effect of complement in different stages of lung fibrosis needs delineation. For example, despite an eventual reduction in lung fibrosis, C5-deficient mice had exacerbated acute lung inflammation, which suggests the role of complement varies between the early inflammatory and later fibrotic phrases. In a recurrent theme, understanding how the shorter time course of murine models correlates to human disease is a challenge. Like neutrophils, complement may be most relevant to acute exacerbations or acute injury responses to injury.

Toll-like receptors

TLRs are evolutionarily conserved pattern recognition receptors in innate immunity. Like complement, TLRs can mediate the earliest responses to infection, inflammation, and tissue injury. Among TLRs, work in fILD has focused on TLR4, which both activates inflammatory responses from immune cells and upregulates profibrotic molecules in lung fibroblasts. Inhibiting MD2, an accessory receptor for TLR4, reduced myofibroblast differentiation and ECM remodeling. Consistent with these results, TLR4 deletion in fibroblasts in vivo resulted in less lung and skin fibrosis following the systemic administration of bleomycin, a model of SSc¹⁰⁶. HMGB1 is a ligand for TLR4 that is released in broad inflammatory responses. HMGB1 promoted fibroblast proliferation in vitro, and anti-HMGB1 mAb reduced lung inflammation and fibrosis in the murine model¹⁰⁷. ECM proteins tenascin-c and fibronectin extra type III domain A (FN-EDA, a fibronectin splice variant) bind TLR4. Both tenascin-c¹⁰⁸ and FN-EDA¹⁰⁹ stimulate myofibroblast differentiation and promote lung fibrosis in vivo. Tenascin-c, in particular, was upregulated in the skin and serum samples obtained from SSc patients compared with those in healthy controls. Tenascin-c deficient mice showed attenuated collagen accumulation and fibrosis severity in both lung and skin, as well as spontaneous resolution of fibrosis in these organs in the repetitive subcutaneous bleomycin-induced systemic sclerosis model, suggesting the role of Tenascin-c in maintaining the persistent fibrosis¹⁰⁸. These data link TLR activation, inflammation and fibrogenesis. However, TLR signaling may also play a reparative, anti-fibrotic role following lung injury. For example, fibroblast-specific deletion of Tlr4 reduced bleomycin-induced lung fibrosis¹⁰⁶. Interestingly, mice with a global deficiency of Tlr4 had more severe lung fibrosis, perhaps due to impaired epithelial repair and capacity for renewal¹¹⁰. Thus, while dysregulated TLR signaling is evident in the pathophysiology of fILD, cellular heterogeneity in expression and response profiles makes therapeutic targeting complex.

There is evidence implicating TLR and their ligands in clinical fILD. BALF levels of HMGB1 are increased in patients with IPF and HP, compared with control¹⁰⁷. Tenascin-c and FN-EDA are increased in the lungs of patients with IPF^109,111, HP¹¹¹, and SSc-ILD¹⁰⁸. A genome-wide association study on European-American patients identified common variants in the Toll-interacting protein (TOLLIP) gene encoding a regulatory molecule of TLR-NF-kB signaling pathways, and their association with IPF susceptibility¹¹². Another study detected a loss-of-function variant in TLR3 associated with poor prognosis in IPF¹¹³. These associations are more striking in some SARD, where most genetic polymorphisms associated with increased risk of SSc are related to TLR or other aspects of innate immunity¹¹⁴. Polymorphism in TLR2 is associated with SSc and the development of pulmonary arterial hypertension, but an association between TLR polymorphisms and SSc-ILD has not been defined¹¹⁵. The extensive literature on the microbiome in pulmonary fibrosis suggests that the microbiome can induce inflammation via TLR signaling (as previously reviewed¹¹⁶). Multiple questions remain, such as the relative contribution of microbial and endogenous TLR ligands, the contribution of TLR over the course of clinical disease, and the mechanistic contribution, if any, of genetic associations. In a range of contexts, from infection to autoimmunity, innate immunity has a major role in activating and modulating the adaptive immune response, and vice versa. A direction for future studies is the cross-talk between innate immunity and adaptive immunity, which remains relatively undefined in clinical fILD.

ADAPTIVE IMMUNE AXES

The medical management of fILD depends on either immunosuppressive or antifibrotic therapies depending on the disease and the individual. These treatment decisions rely in part on clinical judgment, as the randomized controlled trial (RCT) data for immunosuppressive medications targeting adaptive immunity are limited and of lower certainty in fILD¹¹⁷. In certain scenarios, glucocorticoids and immunosuppressive medications are used for SARD-ILD patients¹¹⁸; hence, clinical practice suggests a possible role for adaptive immunity in some SARD-ILD patients. Supported by RCT studies, clinicians avoid glucocorticoid steroids and immunosuppressants in IPF patients¹¹⁹, regardless of disease stage or acute exacerbation. Thus, current clinical practice argues against a role for adaptive immunity in IPF. Furthermore, clinical trials targeting T cell-related pathways, such as the administration of IFNγ or anti-IL-13 blocking mAb, have failed in IPF^34,120. Rather than immunosuppressive medications, two anti-fibrotic drugs, nintedanib and pirfenidone, are indicated for patients with IPF⁴. Nintedanib is a small molecule tyrosine kinase inhibitor that attenuates PDGF, fibroblast derived growth factor, and vascular endothelial growth factor receptor signaling. Pirfenidone is a small molecule with a myriad of anti-fibrotic, anti-inflammatory, and anti-oxidant properties; its precise mechanism of action is unclear. Both drugs modestly slow the rate of lung function decline in IPF¹²¹, and nintedanib has a similar effect in SSc¹²².

Nonetheless, accumulating clinical and translational evidence suggests an adaptive immune component of IPF pathogenesis may have been underappreciated. Here, we seek to provoke reconsideration of adaptive immunity across the range of fILD, including IPF. We first review the contribution of T and B lymphocytes to lung fibrosis and place these findings in clinical context.

T lymphocytes

T helper (Th) cells

T cells are broadly classified into CD8⁺ cytotoxic T-cells, which interact with MHC class I, and CD4⁺ Th cells, which bind MHC class II. CD8⁺ T cells are primarily responsible for injuring infected or cancerous cells, while CD4⁺ T cells play a crucial role in coordinating immune responses by secreted cytokines. Traditional CD4⁺ T cell subsets, including Th1 (T-bet⁺ IFNγ⁺), Th2 (GATA3⁺), and Th17 (RORγT⁺), have been implicated in ILD (Figure 4 and reviewed in¹²³). Th1 cells, which are derived from Th0 cells upon stimulation with IL-12, play a central role in cell-mediated immunity. Th1 cells produce IFNγ and activate macrophages and neutrophils, contributing to anti-tumor immunity and host defense against intracellular pathogens. In contrast to Th1 cells, Th2 cells, which are induced upon stimulation with IL-4, potentiate humoral immunity. Through the production of cytokines such as IL-4 and IL-13, Th2 cells stimulate antibody production and activate eosinophils, as in allergic diseases¹²³. Th17 cells contribute to host defense against bacterial and fungal infections and have been implicated in autoimmune diseases and tissue injury in lung disease¹²³.

While inflammation is often viewed as a contributor to fibrosis progression, not all proinflammatory factors contribute to fibrosis. In contrast to pathogenic pro-inflammatory M1 or CCR2⁺ macrophages, pro-inflammatory Th1 pathways are protective in experimental lung fibrosis (Figure 3A). In the bleomycin model, administration of IL-12 attenuated lung fibrosis, while deficiency of the Th1 transcription factor Tbx21 (T-bet) in CD4⁺ cells exacerbated fibrosis¹²⁴. After treatment with bleomycin, T-bet-deficient mice had increased levels of Th2 cytokines and TGF-β. Unsurprisingly, given the profibrotic role of M2-like macrophages, Th2 cells potentiate experimental lung fibrosis. Th2-biased mouse strains, such as DBA/2, have more severe and prolonged pulmonary fibrosis compared to Th1-biased mice, like C57BL/6J, in the chronic bleomycin model¹²⁵ and the antigen-driven model of chronic HP¹²⁶. Furthermore, mice lacking IL-13 or IL-4Rα have less severe lung fibrosis¹²⁷. CRTH2 is a prominent receptor on Th2 cells and innate lymphoid type 2 cells. CRTH2 stimulation by chitinase 3 like 1 drives alternative activation of macrophages and experimental pulmonary fibrosis. This pathway likely occurs in human IPF⁶².

IL-17 producing T cells can promote lung fibrosis. IL-17A drove experimental lung and skin fibrosis following intratracheal and intradermal administration of bleomycin, respectively^38,88,128. IL-17A stimulates lung epithelial-to-mesenchymal transition, fibroblast recruitment, proliferation, and ECM production⁸⁷. IL-17A is also secreted by innate immune, epithelial, and endothelial cells. The relative contributions of IL-17A secreted by T cells, as opposed to these other cells, have not been well-defined. IL-23 stimulates IL-17A production by Th17 and γδ T cells in the early inflammatory phase of the murine bleomycin model, thereby augmenting lung fibrosis¹²⁹. Although IL-23 also activates fibroblasts, its adverse effects in the bleomycin model are likely mediated primarily by lymphoid cells, as IL-23 did not induce myofibroblast proliferation, differentiation, or ECM production in human lung fibroblasts in vitro¹³⁰.

As with macrophages, the pathogenic role of T cells and associated cytokines in fILD is more nuanced than classical phenotypic functions. T cell function changes over time in experimental lung fibrosis. For example IL-4 has an early anti-inflammatory role that changes to a profibrotic role later in the chronic stage¹³¹. Similarly, although IL-4 deficient mice have worse acute lung injury, they have less lung fibrosis over time¹³¹. This observation speaks to the importance of coordinated immunologic responses to lung injury where IL-4 modulates the early inflammatory response but drives an M2 macrophage-mediated pro-fibrotic response later in the disease course.

Th cells in clinical fILD

T cell populations clearly modulate the development and severity of lung fibrosis in experimental lung fibrosis. Correlating these insights with clinical disease has proven more difficult. In one example, IL-4 has a protective effect in the earlier, inflammatory phase of the murine model, the clinical correlation of this earlier inflammatory phase is uncertain¹³¹. Further, scRNA-seq analyses of end-stage IPF lung tissue showed comparatively little difference in the proportions of T cells and B cells compared to control, in contrast to the differences seen in macrophage populations^53–55. T cells have a more obvious role in autoimmune disease (i.e., SARD-ILD) or in disease driven by environmental antigens (i.e., HP), than in IPF. In support of a role for T cells in SARD-ILD, the Th2 cytokines IL-4 and IL-13 are elevated in lungs from SSc-ILD patients¹³² and IL-17RA is increased in RA-ILD⁸⁷. For clinical HP, the ratio of CD4⁺/CD8⁺ T cells in BALF is occasionally used to help distinguish this disease from other fILDs, like IPF or sarcoidosis. While Th1 profiles predominate in non-fibrotic types of HP, a transition to a Th2 phenotype is associated with fibrotic HP¹³³.

Although the role of T cells in IPF patients is less conspicuous than in SARD-ILD patients, a growing body of translational data brings forward the question of Th cells in IPF patients. First, CD4⁺ and CD8⁺ T cells are increased in IPF, and increased CD8⁺ T cells in surgical lung biopsies were associated with worse lung function¹³⁴. Experimental models have established the anti-fibrotic properties of Th1 cells and IFNγ and the pathogenic effect of Th2 cells. In concordance with this, IFNγ levels are decreased in the BALF or serum of IPF patients^135,136. These translational and experimental findings may suggest a beneficial impact of Th1 and other IFNγ-producing cells. IPF patients have elevated levels of the Th2 cytokines IL-4 and IL-13¹³⁶. Regarding Th17 cells, IPF patients have elevated expression of IL-17RA in their lungs⁸⁷ and Th17 pathway genes (e.g., STAT3) in their PBMCs. Increased expression of this Th17 axis was associated with poor prognosis¹³⁷. Hence, experimental and translational evidence has suggested a functional role for T cells in IPF. However, these findings must be reconciled with the lack of efficacy for glucocorticoids and other T cell-modulating medications in IPF. There are several possible explanations. Certainly, T cells may have little, if any, role in IPF. Alternatively, therapeutic intervention may require specific targeting of pathogenic T cells subsets rather than the nonspecific effects of glucocorticoid. Further, therapeutic intervention may need to be personalized to the phase of disease. For example, we can speculate that T cells have a role in the earliest stages of disease, prior to when most IPF patients first present to the clinic; but then, after non-immune axes are established, T cells then take a minor role in clinically apparent disease. In our discussion of “Future Directions,” we recommend longitudinal study of progressive IPF and its acute exacerbations, which may identify areas for more individualized intervention in adaptive immunity.

Regulatory T cells

Regulatory T cells (Treg) are CD4⁺ cells characterized by the expression of FOXP3 that play a crucial role in maintaining immune tolerance and immune homeostasis. Tregs manage other cell types by direct cytotoxicity, immune checkpoint activation, and secretion of cytokines like IL-10 and TGF-β1. The net role of Tregs in fibrosis is unclear. Adoptive transfer of Tregs attenuated pulmonary fibrosis after repeated doses of bleomycin in a chronic fibrosis model¹³⁸; however, adoptive transfer of Tregs exacerbated lung fibrosis after a single dose of bleomycin¹³⁹. Radiation-induced pulmonary fibrosis in mice was ameliorated by early depletion of Tregs with anti-CD25 mAb administered 2h after irradiation¹⁴⁰. Depletion of Tregs increased Th1 and Th17 responses¹⁴⁰. A protective role for Th17 cells in radiation-induced lung fibrosis that is distinct from their role in bleomycin models is just one of many possible explanations to reconcile the different results in the radiation, single dose bleomycin, and repeated dose bleomycin models.

Consistent with a protective role, the number of CD4⁺CD25⁺FOXP3⁺ Tregs in PBMC and BALF was decreased in ILD patients, including those with IPF and SARD-ILD. Tregs isolated from ILD patients had reduced proliferation and decreased ability to suppress Th1 and Th2 cells. More severely impaired Tregs were isolated from patients with more severe disease¹⁴¹. However, a different study found an increased proportion of activated Tregs in PBMC and BALF from IPF patients, compared with that of SSc patients and healthy controls¹⁴². These two studies reflect both a need for more fine immunophenotyping of Treg subpopulations in clinical fILD, but also a simple need for larger patient cohorts, as the latter study examined BALF from only 7 IPF patients. The latter study in particular examined patients with milder disease severity, so studies of Tregs in both earlier and later-stage clinical disease are needed. Finally, studies of the lung tissue itself are important. In contrast to the BALF findings, scRNA-seq demonstrated increased abundance of Treg in SSc-ILD lungs compared to that of healthy controls¹⁴³.

Tissue-resident T cells

Just as resident lung macrophages are distinct from monocyte-derived infiltrating macrophages, tissue resident memory T cells (T_RM) also have a distinct role in fILD. T_RM cells persist long-term in peripheral tissues without ongoing antigen presentation. T_RM cells populate mucosal organs such as the lung, skin, gut, and reproductive tract in both humans and mice. By being “on-site,” T_RM can mount rapid defenses against respiratory infections. T_RM cells promote proinflammatory responses in a variety of diseases, including psoriasis, asthma, and inflammatory bowel diseases. Similarly, increased CD8⁺ T_RM cells have been found in the lungs of SSc-ILD patients¹⁴³. Multiomic single-cell analysis with mass cytometry by time-of-flight (CyTOF) and single-cell RNA-seq identified increased relative abundance of CD103⁺CD4⁺ T_RM, CD8⁺ T_RM, and T effector memory cells expressing CD45RA (TEMRA) among T cells in IPF lung compared to control¹⁴⁴. In IPF lung, these T_RM cells demonstrated an activated IFNγ response phenotype and were associated with increased expression of TNF and perforin 1 (PRF1)¹⁴⁴. Experimental models suggest a protective role for more inflammatory T cell axes. Consistent with this model, Th1 cell expansion with vaccinia vaccination after bleomycin improved fibrosis resolution, which was associated with increased lung T_RM cells¹⁴⁵. In another murine model, expansion of CD103^lo CD4⁺ T_RM by intranasal exposure to A. fumigatus drove fibrosis by enhanced production of IL-5 and IL-13¹⁴⁶. These two studies demonstrate the need for further investigation of the heterogeneity of T_RM contributions to the pathobiology of fILD.

Coactivating and immune checkpoint receptors

Coactivating receptors and immune checkpoints regulate the activation of immune responses and return to homeostasis. Recent studies raise the question whether these receptors have a role in fILD. Coactivating receptors may potentiate pathogenic T cells, as a study of 59 IPF patients found that increased expression of CD28 and ICOS in PBMCs was associated with worse lung transplant-free survival¹⁴⁷. In lungs from IPF patients, another coactivator, CD40L, was expressed on activated T cells and B cells in tertiary lymphoid tissue¹⁴⁸. Animal studies with anti-CD40L neutralizing mAb supported a pathogenic role for CD40L in autoimmune¹⁴⁹ and radiation-induced pulmonary fibrosis¹⁵⁰. CD40L⁺ T cells most likely interact with CD40 expressed on myeloid cells; however, CD40L⁺ T cells could also interact with fibroblasts, whose expression of CD40⁺ can be induced by IL-13^148,151. Experimentally, engagement of CD40 stimulated fibroblast proliferation, adhesion, and IL-6 production^152,153. The role of these and other coactivators on T cells merit further study in both IPF and SARD-ILD. For example, a polymorphism in the coactivator TNFSF4 (OX40L) is associated with susceptibility to SSc¹⁵⁴, and OX40L was upregulated in the serum and fibrotic skin of SSc patients and correlated with worse prognosis in SSc-ILD¹⁵⁵.

Recent studies have explored how immune checkpoints contribute to lung fibrosis. The immune checkpoint PD-1 is upregulated on T cells in the peripheral blood of IPF patients^137,156. Several other subsets of PD-1^hi T cells are increased in clinical fILD besides PD-1^hi CD4⁺ T cells. PD-1^hi Th17 subpopulations are increased in the lungs of sarcoidosis and IPF patients. Similarly, PD-1^hiCD28null T cells are associated with poor prognosis in IPF patients¹⁴⁷, and anti-PD-1 mAb accelerated fibrosis in humanized NSG mice given CD28null T cells¹⁵⁷. Therapies targeting immune checkpoints may be beneficial in lung fibrosis. For example, PD-1^hi CD4⁺ T cells from patients with sarcoidosis or IPF expressed TGF-β and stimulated collagen production in cocultured fibroblasts. This pathogenic effect was attenuated by PD-1 blockade¹³⁷. In the bleomycin model, PD-1 knockout mice or mice treated with anti-PD-L1 mAb have reduced lung fibrosis¹³⁷. Some studies of immune checkpoint blockade should be interpreted carefully, as these therapies may not act on T cell axes. A mass cytometric analysis of UIP lung tissue identified coexpression of CD47, FSP1, and PD-L1 in ACTA2⁺ fibroblasts in areas of lung fibrosis¹⁵⁸, and fibroblasts with an invasive phenotype have especially high levels of PD-L1¹⁵⁹. Treatment with anti-PD-L1 mAb had direct effects on fibroblast function¹⁵⁹.

Like the role of T cells themselves, the role of their coactivators and immune checkpoints remains controversial in clinical fILD, particularly IPF. T cell coactivators and immune checkpoints merit further investigation via careful immunophenotyping with high-dimensional approaches, such as single-cell or spatial omics, that are coming to the fore. Certainly, any treatments modulating T cells would need to be approached cautiously, given the experience in oncology of treatment with immune checkpoint inhibitors causing ILD. Pre-existing interstitial lung abnormalities are known risk factors for ILD induced by immune checkpoint inhibitors¹⁶⁰, but the mechanism of this association is not yet understood.

B lymphocytes

While it is not surprising that B cells contribute to SARD-ILD, they have also been implicated in UIP. IgA⁺ memory B cells are more abundant in the peripheral blood and lungs of IPF patients. In IPF lung, IgA⁺ memory B cells are colocalized with activated PD-1⁺ follicular T helper (Tfh)¹⁶¹. Before activated B cells terminally differentiate into plasma cells, plasmablasts are an intermediate cell type that express both B cell (CD19⁺) and plasma cell (CD38⁺CD27⁺) surface markers. Compared to healthy controls, IPF patients have an increased relative abundance of plasmablasts in circulation, which negatively correlates with pulmonary function¹⁶². Lung biopsies from IPF patients frequently contain CD20⁺ B cell aggregates and plasma cell infiltration^163,164. In IPF lung, B cells can also organize into bronchus-associated lymphoid tissue (BALT). BALT is tertiary lymphoid organ with follicles containing germinal centers and follicular dendritic cells, and BALT is also prominent in many SSc and RA patients¹⁶⁴. The presence of BALT correlates with increased expression of cytokines known to orchestrate interactions between T and B cells, such as CCL21, CXCL13, BAFF, and ICOSL¹⁶⁴. Serum CXCL13 is associated with more severe disease and, together with MMP7, SPP1, CHI3L1, and CA-125, predicts transplant-free survival in IPF patients^165,166. In addition to BALT, enlargement of lung-draining lymph nodes is common and predicts worse survival in fILD, including IPF¹⁶⁷. In the future, mechanistic hypotheses for B cells can be tested in the murine bleomycin model, which develops germinal centers containing IgA⁺ and CD19⁺CD95⁺IgD^low B cells and plasma cells¹⁶¹.

The expansion of BALT and lymph nodes in clinical fILD raises the question of lung injury driven by antibodies. Serum and BALF IgG, including autoreactive antibodies, are increased in IPF patients¹⁶⁸; and increased serum autoreactive IgA levels are associated with worse pulmonary function¹⁶¹. Furthermore, perivascular deposition of immune complexes is seen in UIP lung¹⁶⁸. Several potential self-antigens for these antibodies have emerged. In one study, 40% of IPF patients had IgG auto-antibodies against periplakin, a component of desmosomes on lung epithelium; the level of this IgG auto-antibody correlated with decreased pulmonary function¹⁶⁹. By contrast, IgG auto-antibodies against HSPA2 were associated with better survival in IPF patients, although the mechanism is unclear¹⁶⁸. The potential pathobiological mechanisms for auto-antibodies require further study. In one hypothesis, anti-periplakin antibodies suppress alveolar epithelial cell migration in vitro and so may contribute to an aberrant lung injury repair¹⁶⁹. Beyond their role in antibody production, peripheral B cells from IPF patients released more IL-6, IL-8, and MMP7 compared to healthy control after stimulation with microbial danger signals like CpG or β-glucan. These activated B cells also promoted the migration, proliferation, and activation of fibroblasts in vitro¹⁷⁰. In support of a pathogenic role for B cells, CD19, a positive regulator of B cell activation, exacerbated lung injury in the murine bleomycin model¹⁷¹. In sum, while IPF patients are not considered to have an autoimmune disease, evidence suggests that B cells may be pathogenic in at least a subset of fILD without systemic autoimmune disease.

While T cells are relatively understudied in fILD, B cells are even less investigated and will benefit from inquiry with newer “omics” technologies. Bulk RNA-seq analysis of lung tissue from IPF patients identified increased expression of POU2AF1, a transcription factor mainly expressed in B cells. Mice lacking POU2AF1 were protected against bleomycin-induced pulmonary fibrosis¹⁷². More powerful discovery methods, like scRNA-seq, promise to finely define the role of B cells in IPF and other fILD. Single-cell approaches to B cell receptor and T cell receptor repertoire can detail the diverse range of lymphocyte receptors that enable recognition of antigens. Computational methods have been employed to elucidate shared specificity within B cell or T cell receptors, facilitating insights to immune responses. In addition, phage display techniques allow for the screening of target antigens¹⁷³. Insights from other fibrotic organs may also drive future developments, particularly in how B cells interact with other cell types. For example, in a model of myocardial infarction, activated B cells secrete CCL7 and drive cardiac fibrosis by recruiting pro-fibrotic macrophages¹⁷⁴. Such insights should inspire important investigations to examine similar B cell-macrophage interactions in fILD.

KNOWLEDGE GAPS AND FUTURE DIRECTIONS

A clear conclusion is the complexity of the immune response in fILD and the heterogeneity among patients and diseases. We recommend the fILD research agenda prioritize the following gaps in knowledge: First, immune mechanisms in earlier-stage fILD are poorly understood. Longitudinal studies of at-risk patients provide a valuable opportunity to study the initiation of disease and identify diagnostic and prognostic biomarkers. By screening patients before clinically-apparent ILD, one can detect subclinical or “preclinical” ILD. To investigate “preclinical” disease and its earliest-stages, prospective studies can enroll patients without a clinical diagnosis of ILD that are at elevated risk. Studies taking this approach target family members of patients with fILD¹⁷⁵, smokers (COPDGene cohort¹⁷⁶), post-COVID-19 patients¹⁷⁷, and patients with SARDs without known ILD (BRASS-Lung, SAIL-RA, and ANCHOR-RA cohorts¹⁷⁸). Collectively, observational studies inform the natural history of fibrotic lung disease, while associated biospecimens facilitate mechanistic investigations into its biologic underpinnings. Longitudinal studies also identify fILD patients that worsen at different rates. Identification of the regulators of disease progression, and their biomarkers, is important not only for the development of therapeutics but also for refinement of clinical trials. One study compared IPF patients with more stable disease to IPF patients with rapid progression of disease¹⁷⁹. The lungs of IPF patients with rapid progression had increased expression of genes already implicated in disease, such as surfactant proteins and CCN1. Surprisingly, IPF patients with rapidly progressive disease showed the greatest differential transcriptional activity around the lung vasculature, rather than in fibroblastic foci¹⁷⁹. This example highlights that longitudinal study of patients can illuminate new mechanisms not appreciated in end-stage patients that comprise the majority of single-cell analyses to date. However, opportunities for translational studies are highly limited, as fILD patients rarely undergo surgical lung biopsy and even bronchoscopy is becoming less common. One solution is multicenter efforts to pool biospecimens from patients undergoing lung surgery for ILD diagnosis or for removal of a lung cancer in the context of underlying fILD. Lung tissue from these clinically indicated surgeries should be complemented by research protocols that enroll patients for BAL and cryobiopsy or other endoscopic sampling of the lung tissue. While relatively few IPF patients have been immunophenotyped by high-throughput methods, there is an even more urgent need for immunophenotyping of non-IPF fILD.

Second, the most abrupt changes in lung function occur during acute exacerbations of pulmonary fibrosis, and the underlying mechanisms of acute exacerbation are poorly understood. Acute exacerbation is a rapid clinical deterioration that can be either idiopathic or associated with possible triggers, like infection or lung surgery. The pathology of acute exacerbation is typically diffuse alveolar damage superimposed on the pre-existing lung fibrosis. Although acute exacerbation is a major cause of morbidity and mortality in fILD, few studies have investigated the underlying molecular mechanisms. In contrast, acute exacerbations of obstructive lung diseases, like asthma or COPD, have been better defined at the cellular and molecular level. The innate immune system has been implicated in the pathogenesis of acute exacerbations. Acute exacerbation of IPF involves an increase in BALF levels of IL-23¹⁸⁰ and neutrophils¹⁸¹, along with serum levels of neutrophil elastase¹⁸². Further, the number of BALF neutrophils correlated with patient mortality⁸⁶. In an experimental model of acute ILD exacerbation induced by lipopolysaccharide, treatment with anti-IL-23 mAb was given to interrupt the activation of neutrophils. This treatment led to decreases in IL-17A levels, airway inflammation, and fibrosis¹⁸⁰. Macrophages are also implicated in acute exacerbations. In scRNA-seq analysis of IPF lung, CCL18⁺ and SPP1⁺ macrophages partially overlap⁵⁸, and CCL18⁺ macrophages are also found in SSc-ILD lung¹⁸³. BALF levels of CCL18 predicted acute exacerbation of IPF patients¹⁸¹, and elevated levels of serum CCL18 was associated with mortality¹⁸⁴. As one pathogenic mechanism, CCL18 can directly induce the production of collagen from lung fibroblasts¹⁸⁵. To date, only bulk RNA-seq analysis, rather than more cutting-edge methods such as single-cell analysis, have been used to study acute exacerbations. Bulk RNA-seq analysis of BAL from IPF patients demonstrated increased expression of innate immune chemokines and their signaling during acute exacerbation¹⁸⁶. Owing to smaller numbers of affected patients and the acuity of exacerbations, a fuller understanding of acute exacerbations will require both the assembly of large, multi-center biorepositories for sample collection, and the application of newer “omic” approaches.

Third, as highlighted above, currently available immunophenotyping data in clinical fILD is often based on older technologies. Newer, high-dimensional methods, such as single-cell analysis or high-resolution spatial ‘omics’ methods will provide a more granular profile of immune cells. Spatial approaches are important given the lung’s complex architecture, the spatial heterogeneity of fibrosis, and cell-cell crosstalk within fibrotic niches¹⁸⁷. For example, spatial transcriptomic analyses of UIP lung found that the expression of regulatory genes, such as NFKBIZ, were associated with histological features of fibrosis¹⁸⁸, but even histologically uninvolved areas had dysregulated gene expression patterns¹⁸⁹. The early focus of these spatial studies have been epithelial and stromal cells; however, spatial “omic” methods are also ideal for examination of immune cells. Spatial transcriptomic analysis of human fILD lung will help contrast the cell-to-cell interactions for B cells within BALT, lymph nodes and elsewhere in the lung. Multimodal approaches will be of particular importance for immune cells. Combining single-cell transcriptomics with proteomics is highly effective for examining lymphoid subpopulations. To complement RNA-seq, single-cell assay for transposase-accessible chromatin (ATAC)-seq can identify the regulatory elements for immune cells, which are less defined than their effector programs. A critical advance will be combining genetic and single-cell approaches to define expression quantitative trait loci (eQTLs) in immune cell subpopulations. An example in healthy individuals combining genomic variants with single-cell analysis found that the behavior of eQTL variants depended on the T cell subpopulation¹⁹⁰. Genome-wide association studies in IPF identified the most common genetic risk factor, MUC5B, as well as many rare but highly pathogenic variants (e.g., TERT, TERC) that do not have obvious immune-related mechanisms¹⁹. However, there are also mutational variants within several immune-related genes that have clear associations with IPF, such as TOLLIP¹¹², TLR3¹¹³, IL1RN¹⁹¹, IL8¹⁹², IL4¹⁹³, TGFB1¹⁹⁴. Single-cell and other approaches can identify the cell types expressing these and other genomic associations. Subsequently, murine models with these genetic variants can test their function, as has been done for non-immune axes. For example, mice expressing human polymorphisms associated with epithelial dysfunction develop spontaneous lung fibrosis and ILD^195–197. These findings could have therapeutic implications, as industry considers genomic associations to significantly de-risk therapeutic development for a candidate target gene.

Research initiatives to address the preceding gaps in knowledge should be complemented by two underutilized research approaches. First, the overlap of pro-fibrotic immunological programs across organ systems should be explored. Advances in high-throughput immunophenotyping may reveal fibrotic programs shared by lung and other organs. For example, our cross-tissue human fibroblast atlas at single-cell resolution of fILD lung, RA synovium, inflammatory bowel disease bowel, and Sjogren’s lip biopsies identified two fibroblast cell states shared across these four diseases – CXCL10⁺CCL19⁺ inflammatory fibroblasts near T cells and SPARC⁺COL3A1⁺ perivascular fibroblasts¹⁹⁸. These add to known cross-organ findings, such as the expansion of SPP1⁺ macrophages in rheumatoid arthritis synovium, chronic kidney disease and heart failure⁵⁹, CD9⁺TREM2⁺ macrophages in hepatic fibrosis⁶⁸, or HBEGF⁺ macrophages in both lung fibrosis and rheumatoid arthritis synovium^52,199. Finding pathogenic genes affecting multiple organs is important for drug discovery, as a therapeutic target with multiple disease indications is a more attractive investment for drug development. Second, how we can simplify yet replicate immune interactions in experimentally tractable model systems is essential for therapeutic development. Next-generation immunophenotyping will lead to an increasing number of candidate genes whose mechanisms need validation at the bench. Despite extensive efforts, most promising drug targets for fILD have failed in human clinical trial phases II and III. These trial failures stem from the complex pathobiology of ILDs, personalized drug responses, and over-reliance on simple in vitro and animal models. Consequently, research attention has turned towards newer in vitro models, like three-dimensional (3D) approaches ranging from precision cut lung slices (PCLS) and organoids to “lung-on-a-chip”²⁰⁰ (Figure 2). PCLS treated with a profibrotic cocktail recapitulated some of the transcriptional profiles of IPF lungs at single-cell resolution, including an increase in aberrant basaloid cells, collagen expressing ectopic endothelial cells and the gene signature of SPP1⁺ macrophages²⁰¹. To better understand the interaction of immune cells with the epithelium and stroma, we encourage the co-culture of PCLS with immune cells. Since PCLS has its own weaknesses, combining newer in vitro models with murine studies will help complement their respective limitations and may improve their clinical translation.

Our review of the immune response in clinical and experimental fILD underscores its mechanistic complexity. Although tremendous progress has been made in defining immune cell types driving pulmonary fibrosis, important knowledge gaps have limited the clinical translation of immunotherapies for these diseases. Future immunotherapies will need to be tailored to individual patients and their stage of disease. Such “personalized medicine” will require earlier disease recognition, advanced immunophenotyping, and more representative experimental model systems. Through these ongoing investigative efforts, we are optimistic the field will develop approaches to reprogram profibrotic immune responses for patients with fILD, for whom treatment options are limited.