Innate and Adaptive Immunity in Non-Infectious Granulomatous Lung Disease

Abstract

Sarcoidosis and chronic beryllium disease are non-infectious lung diseases that are characterized by the presence of non-caseating granulomatous inflammation. Chronic beryllium disease is caused by occupational exposure to beryllium containing particles, while the etiology of sarcoidosis is not known. Genetic susceptibility for both diseases is associated with particular MHC class II alleles, and CD4⁺ T cells are implicated in their pathogenesis. The innate immune system plays a critical role in the initiation of pathogenic CD4⁺ T cell responses as well as the transition to active lung disease and disease progression. In this review, we will highlight recent insights into antigen recognition in CBD and sarcoidosis. In addition, we will discuss the current understanding of the dynamic interactions between the innate and adaptive immune systems and their impact on disease pathogenesis.

Introduction

Granulomatous lung diseases include a spectrum of disorders initiated by infectious and non-infectious agents. Mycobacterial and fungal infections are the most common causes of infectious granulomatous lung disease whereas non-infectious etiologies include exposure to damaging particles, hypersensitivity pneumonitis, genetic disorders, and autoimmune diseases among others (1). Sarcoidosis and chronic beryllium disease (CBD)² are non-infectious granulomatous lung diseases with similar lung pathology (2). While CBD is a lung restricted disorder caused by occupational exposure to beryllium, sarcoidosis is a highly variable systemic disease of unknown etiology that leads to development of lung granulomas in ~90% of cases (3, 4). Genetic risk factors for both diseases include select sets of MHC class II molecules, particular HLA-DPB1 alleles in CBD (5, 6) and HLA-DRB1 alleles in sarcoidosis (7–10). Alterations in the immune system are evident in pulmonary sarcoidosis and CBD, including the accumulation of activated CD4⁺ T cells, macrophages, and B cells in the lung (11–15). Furthermore, lung pathology in both disorders is defined by the presence of compact, non-caseating granulomas consisting of central epithelioid macrophages and/or multinucleated-giant cells surrounded by a layer of lymphocytes (16). Current research suggests that coordinated interactions between the innate and adaptive immune response and their dysregulation are central to the pathogenesis of granulomatous lung disease (7, 17).

Central role of CD4⁺ T cells in CBD

Beryllium is a lightweight metal with unique chemical and physical properties. Beryllium confers strength, thermal conductivity and corrosion resistance in metal alloys and ceramics that are essential in a variety of aerospace, weapons, computer, and electronic applications. Occupational and indirect community exposure to beryllium in processing, manufacturing, and recycling industries can lead to the development of CBD (18–21). Beryllium was identified as a threat to respiratory health after its use was incorporated into manufacturing processes in the US in the 1940s and 1950s. Initially, its toxicity was linked to severe pneumonitis and granulomatous inflammation, referred to as “acute berylliosis”. This form of beryllium-induced lung disease has largely been eliminated due to the implementation of exposure limits in the workplace (18, 22–24). However, a less severe but progressive and chronic form of berylliosis (CBD) continues to impact a percentage of beryllium exposed workers (25). Genetic susceptibility to CBD is strongly linked to HLA-DPB1 alleles possessing a glutamic acid at the 69^th position of the β-chain (βGlu69), and the most prevalent βGlu69-containing molecule is HLA-DP2 (5, 6). The strong HLA association, the identification of delayed hypersensitivity reactions to beryllium in individuals with CBD (26), and the observation that a population of CD4⁺ T cells is expanded in the lungs of CBD patients that respond to beryllium as an “antigen” in an MHC class II dependent manner (11) established that beryllium specific CD4⁺ T cells are central players in the pathogenesis of CBD.

The natural history of CBD includes distinct phases that lead to progressive worsening of disease (Figure 1, left panel). Initiation of disease is asymptomatic and often includes a latent phase followed by the expansion of beryllium specific CD4⁺ Th1 cells in secondary lymphoid organs and their recirculation in the blood (beryllium sensitization) (27). The prevalence of beryllium sensitization is highly variable, occurring in 1–16% of beryllium exposed workers (28–36). A percentage of beryllium sensitized individuals (~6–8% per year) will transition to active lung disease, and in some cases, this occurs decades after beryllium exposure has ceased (37, 38). Early CBD is characterized by the infiltration of beryllium specific CD4⁺ Th1 effector memory cells and mononuclear phagocytes into the peribronchovascular regions of the lung (27, 39). The onset of symptoms occurs in established lung disease as macrophage/lymphocyte aggregates expand in a peribronchovascular distribution and form granulomas (40). In severe disease, worsening inflammation causes tissue damage and fibrosis in the lung. Most individuals with CBD will exhibit a decline in respiratory function over time, and historically 20–30% will develop pulmonary fibrosis and respiratory failure (38, 41).

Figure 1. Natural histories of CBD and sarcoidosis.

Initiation of disease consists of an asymptomatic phase triggered by an exposure (beryllium in CBD; unknown agent (?) in sarcoidosis) that leads to activation of pathogenic adaptive immune responses in lymph nodes. Transition to subclinical lung disease occurs when monocytes and antigen experienced adaptive immune cells are recruited into the lung interstitium and the alveolus (alveolitis). This is followed by development of interstitial lymphoid aggregates and granuloma maturation, and the onset of clinical symptoms. In resolving sarcoidosis, granulomas involute, inflammation ceases, and symptoms decline as lung tissue returns to a healthy, homeostatic state. In progressive CBD and sarcoidosis, granulomas persist, and chronic inflammation is maintained. Failure to modulate granulomas inflammation reduces alveolar surface area and diminish lung function. Progression to end stage disease occurs tissue damage, deposition of collagen and pulmonary fibrosis, that can lead to respiratory failure.

An estimated 1,000,000 people are at risk for developing CBD worldwide (40), and efforts have been made to decrease risks of beryllium exposure in workers. However, there is no cure or preventive treatment that limits disease progression. Despite a thorough understanding of innate and adaptive immune processes in CBD, the factors that impact disease initiation, transition from sensitization to active disease and disease progression have not been defined. Several pre-clinical mouse models have been developed to identify targets for early interventions that could pave the way to limit progression to severe disease in patients (42–45). However, it is firmly established that beryllium sensitization is a prerequisite for developing CBD and has only been demonstrated in mice that express HLA-DP2 molecules on antigen presenting cells. Exposure of HLA-DP2 transgenic (Tg) mice (comprised of HLA-DPA1*01:03 and HLA-DPB1*02:01 alleles) to beryllium (42) results in the development of beryllium specific CD4⁺ T cell responses with the same functional phenotype and TCR specificity observed in HLA-DP2-expressing CBD patients. In this model, beryllium specific CD4⁺ T cells orchestrate and maintain alveolitis and peribronchovascular inflammation that is similar in cell composition to that seen in early stages of the human disease (42). Thus, the animal model confirms a central role for beryllium specific CD4⁺ T cells in CBD and has enhanced our understanding of the sequential interactions between cells of the innate and adaptive immune systems in the initiation and progression of beryllium induced granulomatous inflammation.

Neoantigen generation in CBD

After education in the thymus, the naïve T cell subset includes CD4⁺ T cells selected to bind with high affinity to foreign peptides presented on MHC class II molecules and with low affinity to the same MHC class II molecules presenting self-peptides. In vitro studies of human CD4⁺ T cells suggested that beryllium ions interact with certain MHC class II-self-peptide complexes to form a complete, high affinity ligand for a subset of TCRs. Analysis of TCR usage in CD4⁺ T cells from CBD patients indicated that beryllium specific T cells are oligoclonal and that related CDR3 amino acid sequences are present across HLA-DP2-expressing CBD patients (46). The structure of HLA-DP2 revealed a unique solvent-exposed acidic pocket composed of glutamic acid residues at positions 26, 68 and 69 of the β-chain (47). In vitro studies suggested that self-peptides are required for beryllium recognition, and using a non-biased peptide library approach, Falta et al. (48, 49) delineated multiple self-peptides that complete the αβTCR ligand in CBD. These include constitutively expressed plexin A structural proteins (PLXNA2, A3 and A4) that share nearly identical epitope sequences and peptides derived from the chemokines CCL3 and CCL4 that are expressed during inflammation. Fluorescently labeled HLA-DP2 tetramers loaded with either plexin A4, CCL3, or CCL4 peptides bound to CD4⁺ T cells from CBD patients only in the presence of beryllium ions and detected subsets of beryllium specific CD4⁺ T cells in CBD patients and beryllium exposed HLA-DP2 Tg mice (48–50). A common feature of these peptides is the presence of negatively charged amino acids at the p4 and p7 positions, which are adjacent to the acidic pocket. Structural analysis of the HLA-DP2-plexinA4 peptide/beryllium complex showed that beryllium was coordinated by negatively charged amino acids derived from the peptide and HLA-DP2 β-chain (51). Mutation of these beryllium coordinating amino acids abrogated T cell activation, confirming the role of these peptides in beryllium coordination (52). Crystallization of a multi-molecular complex of a beryllium specific TCR interacting with a beryllium loaded HLA-DP2/peptide complex (51) revealed that the TCR does not directly interact with the beryllium ion itself; rather, it contacts conformational changes in the topology of the HLA-DP2/plexin A4 complex induced only in the presence of beryllium. Thus, beryllium ions create neoantigens that are absent during thymic selection and are recognized by CD4⁺ T cells circulating in the naïve TCR repertoire. In this way, CBD has features of both a metal induced hypersensitivity and autoimmunity.

CD4⁺ T cells in sarcoidosis

Sarcoidosis is a systemic, granulomatous disorder that affects the lung in approximately 90% of cases (4). The clinical presentation of sarcoidosis ranges from an incidental chest radiographic finding in asymptomatic subjects to lung fibrosis. In many cases, granulomatous inflammation resolves while in progressive disease, granulomas persist and can lead to pulmonary fibrosis (Figure 1, right panel). Sarcoidosis occurs worldwide, affecting all races, both sexes, and individuals of all ages, although it typically affects individuals between 20 and 50 years of age (4, 53, 54). Variable prevalence and disease severity occur in individuals of different races and ethnic backgrounds (55).

CD4⁺ T cells are increased in the bronchoalveolar lavage (BAL) and granulomas in sarcoidosis (13, 56, 57). Th1, Th17 cells and multifunctional cells that express both Th1 and Th17 cytokines, transcription factors and chemokine receptors (i.e., Th17.1) are increased in sarcoidosis lungs compared to healthy control subjects (reviewed in (58)). Furthermore, local accumulation of Th17.1 cells in the lungs and lung draining lymph nodes precedes development of granulomatous inflammation in early stages of disease (59). Thus, an asymptomatic phase, in which activation of adaptive immune cells against undefined antigens, likely occurs in the lymph nodes similar to the sensitization phase of CBD. Transcriptome analysis of sarcoid lung and BAL samples consistently show that MHC II dependent antigen presentation, T cell signaling and Th1/IFN-γ signaling pathways are associated with active pulmonary sarcoidosis and CBD (60–62).

Familial clustering of sarcoidosis supports a genetic contribution to disease (63), and several MHC class II alleles (e.g., HLA-DRB1*03:01, 04:01, 11:01, and 15:01) have been associated with disease susceptibility. A Case Control Etiologic Study of Sarcoidosis (ACCESS) showed that HLA-DRB1*11:01 (DR11) was significantly associated with sarcoidosis in African-Americans and Caucasians in the US (9), and a recent study noted its association with disease persistence (64). In fact, the incidence of DR11 expression is doubled in US sarcoidosis patients compared to case-matched controls. In Scandinavian subjects with an acute form of sarcoidosis known as Löfgren’s syndrome (LS), an association between the presence of HLA-DRB1*03:01 (DR3) and expansions of oligoclonal TRAV12–1-expressing CD4⁺ T cell populations in the lung has been described (65–69). Together, these data suggest that CD4⁺ T cells play an important role in the pathogenesis of sarcoidosis; however, the antigen specificity is not known. Defining antigen recognition in sarcoidosis could clarify the relationship between environmental exposures, genetic susceptibility, and natural history of the disease.

Antigen recognition in sarcoidosis

Speculation regarding causative agents in sarcoidosis have included self-antigens, such as vimentin (70, 71), bacterial-derived antigens from Mycobacterium sp. (72–76) or Cutibacterium sp. (77), as well as environmental antigens (38, 78). Using an unbiased pathway of antigen discovery, Greaves et al. (79) recently discovered an epitope derived from the NAD-dependent protein deacetylase hst4 (NDPD) of Aspergillus nidulans that was recognized by TRAV12–1-expressing CD4⁺ T cells from the lungs of HLA-DR3-expressing LS subjects, a distinct subset of acute sarcoidosis. Serum IgG antibodies specific to A. nidulans NDPD were also identified in these patients. Lung CD4⁺ T cells from HLA-DR3-expressing non-LS sarcoidosis patients also responded to the NDPD peptide (79), suggesting that both sarcoidosis patient groups expressing HLA-DR3 may represent a spectrum of the same disease, rather than two distinct clinical entities. Previous studies have linked exposure to fungal byproducts to more severe cases of sarcoidosis with higher fungal biomass levels found in residences of sarcoidosis patients compared to controls (80, 81). These studies suggest a potential role of A. nidulans in the pathogenesis of LS (82). However, the study raises important questions, such as the role of Aspergillus sp. in non-HLA-DR3 sarcoidosis and its role in countries where LS is uncommon. Thus, validation of these findings in other sarcoidosis cohorts is needed.

Innate and adaptive interactions in the initiation of CBD and sarcoidosis

There is no consistent dose response relationship that accurately predicts risk of beryllium sensitization (83, 84). Studies of occupational exposure and sensitization in mice suggest that this may, in part, be explained by the divergent effects of different physiochemical forms of beryllium on the release of danger signals that act as endogenous adjuvants in vivo (85–87). We and others have shown that low dose exposures of mice to crystalline and ionic forms of beryllium rapidly induce death of airway macrophages (AMs) that correlates with sensitizing doses in the HLA-DP2 Tg mouse model (Figure 2A) (85–87). In healthy lungs, AMs clear innocuous particles, release surfactants and secrete immunoregulatory cytokines, thereby maintaining an environment for efficient gas exchange (88). These immunoregulatory functions are disrupted when AMs phagocytose irregular particles or membrane damaging substances, leading to the secretion of pro-inflammatory cytokines and/or initiation of regulated cell death (87, 89, 90). Beryllium crystals disrupted lysosomal membranes within minutes of phagocytosis (89) and released TNF-α that enhanced intracellular bioavailability of damage-associated molecular pattern (DAMPs) in AM (e.g., full length IL-1α and fragmented nucleosomal DNA) (89–91). Subsequent lysosome dependent cell death led to DAMP release and activation of pulmonary DCs via IL-1R1 and TLR9 (42, 89). Using HLA-DP2 Tg MyD88^−/− bone marrow to reconstitute the immune compartment of lethally irradiated HLA-DP2 Tg recipient mice, Collins et al. (89) showed that MyD88-dependent pathways in immune cells were required for beryllium induced DC activation, beryllium sensitization and granuloma initiation. Furthermore, depletion of DCs and treatment of mice with TNF-α blocking antibodies prevented beryllium sensitization, confirming a critical early role of these pathway in breaking peripheral tolerance (89, 90).

Figure 2. Model of pathways implicated in the pathogenesis of CBD and sarcoidosis.

A. Innate pathways and disease initiation. Environmental exposure to microbes, a possibility in sarcoidosis, or particulate antigens in both CBD and sarcoidosis induce TNF-α, which amplifies intracellular DAMPs in AMs (e.g., IL-1α, fragmented chromatin, HMGB1) released upon cell death. DAMPs engage TLRs and IL-1R1 in DCs (dotted black arrow). In CBD, this pathway may enhance the release of beryllium ions and CCL3 and CCL4 that are engulfed by DCs and presented as neoantigens. B. Activation of pathogenic CD4⁺ Th1 cell responses. Activated DCs enter lymph nodes and migrate to T cell rich regions and promote expansion and survival of effector memory CD4⁺ Th1 cells (CXCR3⁺) that in sarcoidosis may co-express Th17 related cytokines that enter the circulation. C. Development of granulomatous lung disease. Disruption of barrier integrity in the airways allows entry of persistent antigen and danger signals into the lung interstitium. TNF-α induces upregulation of adhesion molecules on endothelial cells and local release of a spectrum of chemokines that recruit monocytes, T_EM, Treg, and B cells. Recruited monocytes develop into moDCs and recruited macrophages that process and present antigen. Local release of cytokines by moDCs may enhance polyfunctional cytokines secreted by Th1/Th17.1 cells. IFN-γ promotes macrophage activation and TNF-α release, creating a cycle of persistent inflammation that drives early granuloma formation. Pathways associated with resolution in sarcoidosis versus progression of granulomatous disease in CBD and progressive sarcoidosis and associated signaling pathways are shown. Pathways uniquely associated with sarcoidosis or CBD are highlighted in red or blue, respectively.

Phagocytosis, induction of CCL3 and CCL4 expression, and cell death in macrophages may contribute to neoantigen generation in CBD. The crystal structure of HLA-DP2 indicates that interactions with beryllium ions promote formation of neoantigen complexes as discussed above; however, beryllium oxide and metal dusts encountered in the workplace are highly insoluble. Macrophages incrementally increase the bioavailability of beryllium ions that dissociate from insoluble particles in lysosomes, where the low pH increases solubility. A mixture of beryllium ions and particles are subsequently released from the cells upon cell death and are ingested by neighboring phagocytic cells (48, 92–94). It is possible that repeated cycles of phagocytosis and cell death may enhance local availability of beryllium ions and chemokines for DCs (Figure 2A, B) (51). Thus, interactions between highly insoluble beryllium particles and macrophages may enhance neoantigen generation to meet a certain threshold over time. This proposed mechanism represents a possible explanation for the delayed onset of beryllium sensitization and CBD, occurring years to decades after occupational exposure has ended.

Human studies of T cell responses in pulmonary sarcoidosis are largely restricted to later stages of disease since patients are only identified after pulmonary inflammation is evident. Thus, there are gaps in our understanding of the role of innate immunity in disease initiation and activation of CD4⁺ T cells in sarcoidosis. Murine models have been developed that exhibit similar features to pulmonary sarcoidosis; unfortunately, these models do not replicate the systemic features of the disease (95). Since multiple organs can be impacted by sarcoidosis, it is possible that presentation of neoantigens, autoantigens, or antigens from colonizing or infectious microbes could be presented to the immune system by DCs located in tissues outside of the lung. However, since innate pathways are needed to break peripheral tolerance, it is likely that DAMPs and/or microbial pathogen-associated molecular patterns play a role in the initiation of CD4⁺ T cell responses. Innate signaling pathways through TLRs are enriched in the lung of sarcoidosis subjects (96), and those engaged by putative infectious or environmental etiological agents may play a primary role in addition to being enhanced during granulomatous inflammation. Gene association studies suggest that polymorphisms in MyD88, TLR2, and TLR9 may play a minor role in conferring susceptibility to sarcoidosis (97–99). TLR2 recognizes components of gram-positive bacteria, fungal cell walls, and serum amyloid A, all of which have been detected in sarcoid granulomas, while TLR9 is engaged by bacterial or nucleosomal DNA in endosomal compartments (72, 77, 81, 100). TLR2 expression is elevated on peripheral blood monocytes and expressed in innate and adaptive cells in the mediastinal lymph nodes of sarcoidosis subjects, and BAL cells from sarcoidosis lungs released increased cytokine in response to TLR2/1 ligation (101, 102). AMs from sarcoidosis subjects express increased TLR9 expression and secrete CXCL10 in response to CpG enriched DNA compared to AMs from healthy controls (103). In addition, mycobacterial heat shock proteins, endogenous heat shock proteins, and high mobility group box protein 1 (HMGB1) and associated signaling pathways are enhanced in airway macrophages in both CBD and sarcoidosis and are discussed in more detail below (104, 105).

Innate and adaptive pathways and the initiation of granulomatous lung disease

Granulomas serve as a barrier between toxic substances and healthy tissues, and studies of granulomas induced during mycobacterial infection have shown that interactions between innate and adaptive immune cells drive the formation and regulation of granulomatous inflammation to limit bystander damage and fibrosis (106). While beryllium sensitization may occur with any exposure in genetically susceptible individuals, progression to CBD is associated with higher cumulative exposures to aerosolized insoluble beryllium dusts (40, 107). Single cell transcriptome analysis of recruited BAL macrophages showed that pathways related to MHC class II mediated antigen presentation, complement, cytokine signaling, HSP signaling, and TLR signaling pathways were enriched in CBD compared to beryllium sensitized controls and in sarcoidosis compared to healthy controls (108). These associations have previously been reported in numerous genomic and epigenomic studies of lung biopsies and BAL and blood studies in sarcoidosis versus healthy control subjects (60–62, 96, 109–112).

Beryllium sensitized CD4⁺ Th1 cells with an effector memory phenotype are present in the BAL in early stages of CBD (113–117). Disruption of the epithelial barrier in the inflamed airways may promote penetration of causative antigens(s) into the lung parenchyma along with danger signals and cytokines where they may be taken up by interstitial macrophages (Figure 2C). TLR and TNFR1 signaling induces upregulation of adhesion molecules on endothelial cells and release of proinflammatory chemokines that promote recruitment of monocytes, effector memory T cells and B cells to lung granulomas (118). Monocytes recruited to the lung can develop into monocyte derived macrophages and monocyte derived DCs (moDCs) that are evident in sarcoidosis granulomas and in mice chronically exposed to beryllium (43, 119). MoDCs interact with CD4⁺ T cells in early lymphoid aggregates in beryllium-exposed mice (43, 120) and are observed in lymphocyte rich regions of sarcoid granulomas (121). Release of IL-12 by moDCs within granulomas amplifies T cell release of TNF-α and IFN-γ. These cytokines divert phagocytosed antigens away from lysosomal compartments and into MHC class II compartments for presentation (122, 123), upregulate adhesion molecules, and amplify the release of TNF-α from macrophages, creating a positive feedback loop that sustains inflammatory cytokine production (Figure 2C) (123).

CD8⁺ T_EM cells are present in sarcoid granulomas, and B cells are present in the BAL and are detected in CBD and sarcoidosis lung biopsies (15, 124); however, their role in disease pathogenesis is not known. B cells in the lungs of individuals with CBD and sarcoidosis exhibit a unique phenotype (CD11c^hi CD21^loTbet^lo) that is shared by autoreactive B cells in systemic lupus erythematosus and other autoimmune diseases (15). Antibodies and autoantibodies, complement, and immune complexes are elevated in individuals with sarcoidosis compared to healthy control subjects (43, 70, 125). B cells accumulate in BAL and early granulomas in beryllium exposed mice in response to innate signaling pathways (14). Treatment of mice with B cell depleting antibody did not impact beryllium sensitization or antibody levels in the HLA-DP2 Tg CBD mouse model; however, infiltrating CD4⁺ T cells and macrophages failed to form aggregates resulting in a pattern of diffuse inflammation and increased airway injury (14). These data suggest that B cells may play a role in the formation of lymphoid aggregates and highlight the protective role of granulomas in sequestering damaging particles. In addition, disease resolution has been reported in refractory sarcoidosis patients treated with rituximab (126, 127). Paradoxically, sarcoidosis-like granulomas were reported in an autoimmune patient treated with rituximab (128). Thus, further study is needed to understand the role of B cells and antibodies in pathogenesis of CBD and sarcoidosis (129).

In LS and in many non-LS sarcoidosis patients, granulomas resolve (Figure 2C). Effective antigen clearance, potent effector T cell responses followed by downmodulation of macrophage activation are associated with resolution. There are conflicting data regarding the role of Th17 cytokines in resolving sarcoidosis (reviewed in (130)). In LS, a resolving form of the sarcoidosis, Th17.1 cells that express high levels of IFN-γ, IL-17A, IL-10, and IL-22 are elevated in patients with a better prognosis (131). Conversely, in non-LS sarcoidosis cohorts, Th17.1 cells have been associated with disease progression and are less polyfunctional and produce high levels of IFN-γ and low levels of IL-17A (59). Based upon previous associations between potent effector T cell responses in self-limited sarcoidosis (111) and the association of fungal antigen recognition by CD4⁺ T cells in HLA-DR3 expressing LS patients (7, 79), it is tempting to speculate that Th17 cytokines are more effective at clearing the causative agents in LS versus those driving disease progression in non-LS sarcoidosis. While neutrophils are recruited by IL-17A cells, longitudinal studies or development of a relevant mouse model may be needed to define their role due to their short half-life and clearance by resident AMs in vivo.

Dysregulation of innate and adaptive responses in progressive granulomatous lung disease

Persistent release of Th1 cytokines, STAT1 signaling pathways, chronic inflammation, and alterations in TCR signaling are consistently associated with CBD and progressive sarcoidosis (60–62, 109–112). Antibody-mediated depletion of Tregs in the HLA-DP2 Tg mouse model of CBD results in persistent and progressive granulomatous inflammation with evidence of centralized multinuclear giant cells and enhanced deposition of collagen over time (42). In the lung, Tregs in progressive sarcoidosis and CBD are present but have reduced suppressive activity (132–134). Tregs from the lungs of subjects with non-resolving sarcoidosis exhibit reduced capacity to suppress inflammatory cytokine production compared to Tregs from the lungs of subjects with resolving sarcoidosis (132, 135–138). Single cell transcriptome analysis of peripheral blood Tregs from sarcoidosis subjects suggests that dysfunctional p53, cell death, and TNFR2 signaling pathways in Tregs may limit their survival and ability to suppress macrophage activation (139). TNFR2 promotes Treg survival and suppressive function in inflammatory environments (140–142), and restored Treg function have been associated with resolution of granulomatous inflammation in sarcoidosis (143, 144).

Dysregulation of key macrophage pathways that regulate macrophage immunometabolism are associated with disease progression in sarcoidosis. PPARγ, a negative regulator of inflammatory gene expression and antigen presentation in macrophages, is reduced in progressive sarcoidosis (145). Furthermore, PPARγ deficient mice exposed to mycobacterial peptide/particle conjugates exhibit exacerbated granulomatous inflammation and fibrosis in the lung. Conversely, dysfunctional activation of the mTOR pathway was associated with disease progression in sarcoidosis (146). Mechanistic analysis of this pathway in mice showed that hyperactivation of mTORC1 in macrophages enhanced their proliferation and survival, inhibited autophagic repair, and disrupted lysosome function. These mice spontaneously developed sarcoidosis-like granulomas in the lungs and other tissues, an effect that was prevented by an mTORC1 inhibitor (146). Single-cell RNA-sequencing analysis of differentially expressed pathways in classical monocytes from sarcoidosis subjects showed increased expression of inflammatory genes enhanced by pattern recognition receptors (TLR2/HMGB1) and cytokine receptor (TNFR1, IL-6, GMCSF) that could be regulated with immunosuppressive agents (139). In contrast, predictive modeling suggested that enhanced mTOR signaling and TGFβ are key drivers of monocyte hyperactivation in sarcoidosis and were not altered in the presence of immunosuppressive agents (139). Together, these data suggest that progression to persistent granulomatous inflammation is associated with a failure to regulate hyperactivated macrophages (147, 148).

Persistent TCR stimulation in CBD and sarcoidosis promotes T cell upregulation of PD-1 and PD-L1, reduced T cell proliferation and anergy (117, 149). PD-1/PD-L1 interactions between exhausted T cells and macrophages may in part promote disease progression by impairing the ability of macrophages to degrade particulate antigens (150). PD-1 and TGFβ enhance STAT-3 dependent pathways that promote production of collagen by human lung fibroblasts (151). In addition, expression of genes, including thioredoxin, CXCL2, and CXCL3, were significantly reduced in macrophages in CBD and progressive sarcoidosis (108, 152). Recent results from the multicenter Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS) study suggest that the combination of signatures in the BAL may reflect distinct clinical involvements (153). For example, T cell activation pathways were enriched in subjects with lymphadenopathy, enhanced TGFβ1 and mTOR signaling pathways were enriched in individuals with interstitial granulomatous disease, and innate and adaptive immune pathways were enriched in individuals with multiorgan involvement.

Conclusions

The discovery of CD4⁺ T cells that recognize CCL3 and CCL4 peptide-containing neoantigens in the context of HLA-DP2/Be²⁺ provides a direct link between innate and adaptive immunity in CBD. A similar approach used to identify CD4⁺ T cell responses specific to an HLA-DR3 presented peptide from A. nidulans in LS provides a pathway to facilitate discovery of the inciting antigens that drive CD4⁺ T cell alveolitis and granulomatous inflammation in non-LS sarcoidosis. Persistent antigen presentation, chronic macrophage activation and Treg dysfunction are associated with disease progression in both CBD and sarcoidosis. The functional analysis of novel pathways in larger cohorts is needed to better understand the pathogenesis of CBD and sarcoidosis and to define useful biomarkers, diagnostic tools and novel therapies.