The Pathogenesis of Pulmonary Sarcoidosis and Implications for Treatment

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1 Q1 Q31 Q2 Q3 [ Recent Advances in Chest Medicine ] The Pathogenesis of Pulmonary Sarcoidosis and Implications for Treatment Karen C. Patterson, MD; and Edward S. Chen Thoracic sarcoidosis is the most common form of sarcoidosis, encompassing a heterogeneous group of patients with a wide range of clinical features and associated outcomes. The distinction between isolated thoracic lymphadenopathy and pulmonary involvement matters. Morbidity is often higher, and long-term outcomes are worse for the latter. Although inflammatory infiltrates in pulmonary sarcoidosis may resolve, persistent disease activity is common and can result in lung fibrosis. Given the distinct clinical features and natural history of pulmonary sarcoidosis, its pathogenesis may differ in important ways from other sarcoidosis manifestations. This review highlights recent advances in the pathogenesis of pulmonary sarcoidosis, including the nature of the sarcoidosis antigen, the role of serum amyloid A and other host factors that contribute to alterations in innate immunity, factors that shape adaptive T-cell profiles in the lung, and how these mechanisms influence the maintenance of granulomatous inflammation in sarcoidosis. We discuss questions raised by recent findings, including the role of innate immunity in the pathogenesis, the meaning of immune cell exhaustion, and mechanisms that may contribute to lung fibrosis in sarcoidosis. We conclude with a reflection on when and how immunosuppressive therapies may be helpful for pulmonary sarcoidosis and a consideration of nonpharmacologic management strategies and a survey of potential novel therapeutic targets for this vexing disease. CHEST 2017; -(-):--- KEY WORDS: granulomatous inflammation; pulmonary fibrosis; sarcoidosis Sarcoidosis is a systemic disease marked by sterile granulomatous inflammation in affected organs. The cause remains uncertain, but a constellation of archetypical clinical and immunologic features characterize the disease. 1 Although sarcoidosis can affect nearly any organ in the body, thoracic disease, when it includes lymphadenopathy, is the most common site. Although a global disease, the prevalence of sarcoidosis varies by ancestral ABBREVIATIONS: IFN = interferon; NLR = nucleotide-binding oligomerization domain-like receptor; SAA = serum amyloid A; TLR = Q4 toll-like receptor; TNF = tumor necrosis factor; Treg = regulatory T AFFILIATIONS: From the Pulmonary, Allergy & Critical Care Division Q5 Q6 (Dr Patterson), University of Pennsylvania, Pennsylvania, PA; the Brighton & Sussex Medical School (Dr Patterson), Brighton, England; and the Johns Hopkins University School of Medicine (Dr Chen), Baltimore, MD. background. 2 In the United States, a nationwide survey of a medical database found a prevalence of 60 per 100,000 adults, with a threefold higher rate in blacks compared with whites, and a low prevalence among Hispanics and Asians. 3 The diagnosis of sarcoidosis is established by a combination of histopathologic and clinical findings. 4 Although many patients enter clinical remission within a few years of CORRESPONDENCE TO: Karen C. Patterson, MD, BSMS Teaching Building, University of Sussex, Falmer, BN1 9PX, UK. ; k.patterson@ Q7 bsms.ac.uk Copyright Ó 2017 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved. DOI: Q8 Q chestjournal.org 1

2 diagnosis, approximately a third experience chronic disease. Long-term outcomes relating to morbidity and mortality are closely linked to clinical phenotypes. The acute presentation of hilar lymphadenopathy, called Löfgren syndrome when occurring with fever and erythema nodosum, typically confers an excellent prognosis. Outcomes are generally less favorable for parenchymal pulmonary sarcoidosis compared with lymph node limited disease (Table 1). In a recent study, relapsing disease was most common among patients with pulmonary involvement. 5 In addition, pulmonary sarcoidosis can progress to pulmonary fibrosis, with its attendant respiratory impairment and secondary complications, including pulmonary hypertension and aspergillosis. 6 Improving outcomes in sarcoidosis requires a better understanding of the pathogenesis. Fortunately, this is a dynamic period in sarcoidosis research. Several recent publications have started to challenge long-held assumptions about the pathophysiology. Further, a variety of recent publications clarify findings associated with the particularly common and morbid phenotype of pulmonary sarcoidosis. This review summarizes these findings. Specifically, we highlight the results of studies relating to antigen presentation, innate and adaptive immune responses, and granuloma biology in pulmonary sarcoidosis. The application of these findings to outcomes of chronic inflammation and pulmonary fibrosis is explored. We conclude with a discussion of clinical management and potential novel therapeutic domains. TABLE 1 ] Clinical Features of Pulmonary Sarcoidosis Inflammatory pulmonary sarcoidosis Fibrotic sarcoidosis Immunopathogenesis of Pulmonary Sarcoidosis Pulmonary sarcoidosis is a highly orchestrated immune response involving, sequentially, antigen-driven CD4þ T-cell activation, chemokine-driven recruitment of activated T cells to the lung, local macrophage accumulation, and granuloma formation (Fig 1). Although macrophages may assume antigen presenting capabilities in the lung, dendritic cells traffic to regional lymph nodes for the initial presentation of antigen, leading to activation of quiescent circulating CD4þ lymphocytes. 7 Adefining feature of active sarcoidosis is the dominant expression of interferon (IFN) gamma in affected organs; IL-2, IL-12, and tumor necrosis factor (TNF)-alpha are other important cytokines. The role of pulmonary-derived exosomes, extracellular vesicles of cellular material capable of promoting inflammation locally and distally, as biomarkers and as possible disease modifiers, is being explored. 8 Even as the antigen remains elusive, the clonal amplification of CD4þ T cells strongly supports the tenet that a pathogenic antigen contributes to disease. The resulting CD4þ T-cell alveolitis serves as a biomarker, closely tracking the rise and fall of disease activity. 9 Typically noncaseating and tightly formed, sarcoidosis granulomas are sterile and in the lungs primarily locate along lymphatic tracks (Fig 2). 10 Antigen Considerations The long-standing search to identify the sarcoidosis antigen continues. One hypothesis is that of Pulmonary Manifestations Imaging Findings Pulmonary Function Exertional dyspnea, dry cough, chest tightness Less common: chest pain, pleural rub Mild to severe exertional dyspnea, dry cough, chest tightness, hypoxemia Less common: inspiratory crackles, bronchiectasis-related sputum production, hemoptysis related to Aspergillosis species Bilateral hilar adenopathy (often with calcifications) Focal to extensive reticulonodular opacities along interlobular septa and bronchovascular bundles, upper zone predominant Less common: diffuse ground glass opacities, pneumothorax, pleural effusion Faint reticulations to dense linear bands Cystic lung disease, traction bronchiectasis, and airway distortion Less common: extensive parenchymal destruction, mycetoma Often normal Mild to moderate obstruction, restriction, or mixed pattern of disease Bronchial hyperreactivity Restriction and decreased diffusion capacity common Severity related to extent of structural lung disease and presence of coexisting pulmonary hypertension Nonreactive airflow obstruction common when airway distortion is present Q29 Q Recent Advances in Chest Medicine [ - # - CHEST ]

3 print & web 4C=FPO Ag Step 1: Antigen exposure and internalization, with presentation in regional lymph nodes. APCs TNF-α IL-12 IL-17 IFN-γ IL-2 CD4+ T cells Step 2: CD4+ cell activation, proliferation and Th1 polarization induced by antigen recognition + co-simulator signals. + Step 3: Newly activated circulating CD4+ cells hone, via chemokines, to tissue sites with antigen, and stimulate macrophages to organize into granulomas. Macrophage activation Granuloma formation Step 4: Cellular mediators of fibrosis are largely unknown but chronic inflammation is an important risk factor.? Pulmonary fibrosis Figure 1 Schematic depiction of key events in the pathogenesis of pulmonary sarcoidosis. Antigens of the (still unidentified) sarcoidosis particle are Q24 Q presented by antigen-presenting cells with a major histocompatibility complex capable of antigen binding. In a proinflammatory milieu (cytokines and Q display of costimulatory molecules), CD4þ cells with a T-cell receptor capable of antigen recognition are activated in regional lymph nodes. They traffic Q to the lung and engage with local antigen, releasing interferon gamma and directing the aggregation of macrophages into granulomas. Fibrosis may develop as a wound healing response in chronic disease. IFN ¼ interferon; TNF ¼ tumor necrosis factor. Q autoimmunity. Raising the possibility of an autoantigenic role for vimectin, significantly increased reactivity of circulating lymphocytes to this protein was observed in a subset of patients with pulmonary sarcoidosis; however, other studies have not yet have confirmed this finding. 11,12 However, disease features of sarcoidosis are generally distinct from those of autoimmune disease, even as there may be overlap in constitutional symptoms such as fatigue, arthralgia, and erythema nodosum. 13 High titers of autoantibodies are Figure 2 Axial image from a computed tomography scan of a patients with pulmonary sarcoidosis. The scattered macroscopic lesions represent an 329 amalgamation of microscopic granulomatous nodules. The peribronchial distribution is typical of pulmonary sarcoidosis, where disease occurs along 330 lymphatic tracks chestjournal.org 3

4 rare in sarcoidosis; more common is the detection of low titers secondary to a generalized hypergammaglobulinemia. 13 The presence of CD4þ T cells bearing host antigenic peptides has been observed, but it remains unclear whether this indicates an autoimmune process or reflects normal immune system surveillance activities. 14 There is greater evidence to implicate an infectious agent in sarcoidosis. The results of A Case Control Etiologic Study of Sarcoidosis 15 found an association of sarcoidosis and exposure to insecticides, mold and mildew, home central air conditioning, and birds, possibly representing contact with microbe-rich environments. Negative culture data and favorable responses to immunosuppression argue against an intact microbe in sarcoidosis. However, poorly degradable remnant microbial particles can be sequestered within phagocytes, avoid detection by standard microbial tests, and in the case of sarcoidosis, account for ongoing immune stimulation. A convergence of data support a role for remnant mycobacterial products in particular. Compared with control lung samples, the detection of residual mycobacterial genetic elements (DNA) was substantially greater in sarcoidosis samples. 16 Further, mycobacterial proteins mkatg and ESAT6 have been identified in sarcoidosis tissues, with sarcoidosis blood and lung T cells demonstrating increased reactivity against these proteins, particularly in active disease. 12,17,18 These findings helped form the premise for the recently completed Genomic Research in Alpha- 1 Antitrypsin Deficiency and Sarcoidosis study 19 to evaluate the lung microbiome and its relationship to host immune responses and clinical disease phenotype. Results of the Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis study are anticipated in the coming year. Innate Immunity: Antigen Presentation, Sequestration, and Clearance Expressed on antigen-presenting cells, pattern recognition receptors are a family of innate receptors that include toll-like receptors (TLRs) and nucleotidebinding oligomerization domain-like receptors (NLRs). These detect conserved chemical moieties and, when engaged, trigger a cascade of intracellular events. These events include the activation of mediators and kinases, such as p38, which control the activity of key transcription factors for inflammatory programs. 20 Although sarcoidosis immunopathology research has, historically, focused on the adaptive immune system, there is recent and growing interest in alterations in the innate immune system which may predispose or amplify sarcoidosis inflammation. Compared with control subjects, lung and blood cells of monocyte-macrophage lineage in sarcoidosis demonstrated increased levels of TLR2. 21,22 Lung epithelial cells also express TLR2 that is induced, in part, by local TNF-alpha and IFN gamma, representing an example of how adaptive immune responses may enhance innate immunity in pulmonary sarcoidosis. 23 For example, the increased expression of TNF-alpha by sarcoidosis monocytes and macrophages compared with control samples reveals a feed-forward mechanism through which adaptive and innate responses synergistically amplify granulomatous responses in sarcoidosis. Beyond TLR2, upregulation of the TLR9 pathway also has been observed in sarcoidosis BAL cells. 24 Similar to finding for TLRs, alterations in NLR signaling have been identified in sarcoidosis. BAL cells from patients predominantly affected by pulmonary disease had exaggerated responses to NLR (NOD1) ligands, with enhanced expression of the granuloma-supporting cytokines TNF-alpha and IL12/IL23p Exaggerated NLR responses were associated with higher basal activity of (intracellular) p38, which in turn were associated with enhanced expression of IRAK and Rip2, upstream kinases to p38, in sarcoidosis alveolar macrophages. 26 These results reflect alterations in many of the key signaling steps of innate immune activation in sarcoidosis. Although most available data support enhanced innate activity in lung macrophages, weak responses have also been identified in a subset of patients. In one study, cytokine production from circulating regulatory cells after TLR stimulation was diminished in patients with symptomatic compared with asymptomatic pulmonary sarcoidosis. 27 Because there may be discordances between circulating and pulmonary immune responses in sarcoidosis, ongoing studies will help clarify the meaning of enhanced innate function in the lung. In addition, it is not clear if these alterations are primary or secondary events. Gene studies are therefore of particular interest. In studies of patients with sarcoidosis not stratified by clinical phenotype, polymorphisms in MyD88, a universal adaptor protein used by nearly all TLRs, were associated with sarcoidosis in a Greek population, and the NOD1 786A haplotype was overrepresented in a Japanese cohort. 28,29 Although these results suggest that aberrant innate activity may play a primary role in sarcoidosis, genomic studies have Q10 Q Recent Advances in Chest Medicine [ - # - CHEST ]

5 Q12 Q13 otherwise failed to firmly associate gene polymorphisms in pattern recognition receptors with sarcoidosis. Further research is needed to be able to answer the fundamental question of whether innate immune function is primarily altered in sarcoidosis. If so, then dysregulated responses to potentially a variety of elements, rather than to a single pathogen, may underlie the pathophysiology of sarcoidosis. Adaptive Immunity: T-Cell Responses Expanded populations of effector T cells are found in organs where sarcoidosis is active. In deference to a Th1/ Th2 paradigm, the dominant expression of IFN gamma and IL12p40 in the lung historically defined pulmonary sarcoidosis as a Th1-polarized disease. However, other T-cell lineages may also contribute to sarcoidosis inflammation. Several groups have identified the presence of Th17 cells in sarcoidosis lung tissue and BAL fluid. 30,31 Noted for their proinflammatory role in autoimmune diseases, cells bearing a Th17 phenotype also may enhance IFN gamma-driven immunopathology in sarcoidosis. Such nonclassical Th1-capable cells of Th17 origin can acquire the ability to produce IFN gamma, in part, through the effects of locally expressed TNF-alpha and IL-2, 32 via intrinsic functional plasticity. Designated as Th17.1 cells, these cells were predominant in pulmonary sarcoidosis samples, and may account for most disease-promoting IFN gamma production at sites of active disease. 33 However, in a study comparing Löfgren and non- Löfgren syndrome, and chronic and nonchronic phenotypes, the presence of Th17.1 cells was associated with the more favorable phenotypes. 34 Further investigation is needed to determine whether these cells have a pathogenic or protective role. The activity of effector lymphocytes has a central role in the pathogenesis of sarcoidosis. However, a recent study identified reduced proliferative responses of lungderived lymphocytes in sarcoidosis compared with control subjects. 35 In addition, the magnitude of augmented IL-2 and IFN gamma production after T-cell stimulation was lower in BAL and blood samples from sarcoidosis (mainly pulmonary) compared with control subjects. In another study, deficits in the IL-2 signaling pathway and increased PD-1 signaling were identified as mechanisms for these impairments. 36 Such findings of waning T-cell responses are consistent with the concept of lymphocyte exhaustion, used to describe the less exuberant lymphocyte function observed in conditions of long-standing inflammation. 37 In spite of such anergic findings in sarcoidosis, spontaneous cytokine release was still hyperactive in sarcoidosis compared with control subjects, and amplified CD4þ cell activity has been demonstrated in all phases of active disease. Exaggerated T-cell function may even be a risk factor of nonresolving inflammation in sarcoidosis. In a German cohort, the frequency of a polymorphism in BTNL2, a negative T-cell costimulator molecule, was associated with increased risk for chronic disease; however, this genotype-phenotype association was not replicated in a French cohort. 38,39 Ongoing work to define the evolution of effector T-cell function over the course of disease, and to identify whether primary alterations guide disease outcomes, is important. Finally, there is growing interest in the possible diseasemodifying role of regulatory T (Treg) cells in sarcoidosis. Treg cells are present in sarcoidosis tissues, and circulating Treg-cell counts are generally expanded. 40 However, data on pulmonary Treg-cell function are mixed. The ratio of BAL Treg cell/effector T cells was significantly higher among patients with clinical remission vs chronic pulmonary disease on long-term follow-up. 41 In addition, after exposure to inhaled vasoactive intestinal peptide, an increase in BAL Treg-cell function was associated with improvements in clinical measures. These findings suggest that Treg cells may modulate sarcoid inflammation. However, in another study, subjects who developed chronic pulmonary disease vs spontaneous remission demonstrated higher circulating Treg-cell counts at diagnosis. 42 In the same study, circulating Treg cells in samples from sarcoidosis compared with control subjects demonstrated enhanced susceptibility to apoptosis. Ultimately, whether inflammation develops and persists may rest on the balance between the relative capacities of suppressive and proinflammatory immune responses. 43,44 In a recent study, the homeostatic ratio of Treg/Th17 cells trended with disease activity, decreasing in those who developed relapsing pulmonary sarcoidosis and increasing back to normal ranges with treatment. 5 Granuloma Biology in Sarcoidosis The cardinal histologic feature of sarcoidosis is the compact epithelioid granuloma, and disease morbidity is intimately associated with the mechanisms that govern granulomatous inflammation. 10 Distinct from foreign body granulomas, sarcoidosis granulomas develop in an adaptive immune milieu, with CD4þ T cells orchestrating macrophage activity. These epithelioid Q chestjournal.org 5

6 Q15 macrophages have a predominantly secretory rather than phagocytic role. Focal areas of necrosis may be observed, but the presence of significant caseation should prompt consideration of an alternative diagnosis. 45 Granuloma formation is a highly orchestrated event. TNF-alpha is a key granuloma-promoting cytokine; however, a concert of other signaling events is involved. 46 Infectious models provide insight regarding interactions between granulomas and their environment, including protein interrogation and sequestration, which may regulate the persistence of granulomas. 47 This functional characteristic supports knowledge gleaned from studies of sarcoidosis tissues, including the discovery of potential mediators of granulomatous inflammation through studies of Kveim- Siltzbach reagent, an extract of sarcoidosis tissues. Recent work has identified a possible role for serum amyloid A (SAA) protein, a component of the host inflammatory response in sarcoidosis. Compared with control subjects, BAL cells from sarcoidosis samples demonstrated increased reactivity to SAA, including increased TNF-alpha production, suggesting a pathogenic role for SAA in disease. 21 In addition, the histologic distribution of poorly soluble SAA proteins within sarcoidosis granulomas is distinguishable from other inflammatory lung diseases and may be a diseasedefining characteristic. 21,48 Levels of SAA appear correlated with the burden of pulmonary disease as indicated by Scadding chest radiograph stage. Finally, SAA enhances the persistence of experimental granulomatous inflammation in mice, offering insight into the role of SAA in chronic pulmonary sarcoidosis. Continued work is needed to identify what signals are essential for granuloma formation and maintenance in sarcoidosis. Antigen eradication appears to be an elusive goal in sarcoidosis. However, beyond the persistence of antigen, several factors may contribute to sustaining granulomatous inflammation. The persistence in sarcoidosis tissues of SAA may, via protein aggregation and antigen trapping, represent a risk factor for chronic disease. In a recent study, chronic activation of mtorc1, a metabolic checkpoint in macrophages, was identified in sarcoidosis and in experimental disease models as another possible mechanism of sustained granulomatous inflammation. 49 These findings shed insight into the complex regulation of granulomatous inflammation, and emphasize the central role of granuloma biology in sarcoidosis. Coherent Model of Chronic and Fibrotic Pulmonary Sarcoidosis Nonresolving inflammation in pulmonary sarcoidosis is the most critical risk factor for pulmonary fibrosis. Therefore, understanding the mechanisms of nonresolving inflammation is key to improving outcomes for patients with chronic inflammatory and fibrotic pulmonary sarcoidosis. The recent trend in research to phenotype patients according to disease chronicity and features reflects the growing interest to identify these mechanisms. Several findings relating innate and adaptive immune function with disease chronicity are highlighted earlier in the text. Beyond these, genetic associations have been identified between HLA haplotypes and long-term outcomes, including remitting (DRB1*0301) and persistent (DRB1*1501) disease. 50 These observations may be influenced by signals from non-hla genes within or adjacent to the major histocompatibility complex region, such as RAGE. 51 In addition, polymorphisms in the TLR10- TLR1-TLR6 gene cluster and apoptotic gene pathways have been implicated as risk factors for chronic active sarcoidosis. 52,53 There may be an inherited susceptibility to development of fibrosis in sarcoidosis as well; however, this requires further study. 54 Although important, all of these findings, individually and collectively, do not complete the model of end-stage disease. Herein we posit additional processes, intimately involved in the underlying pathogenesis of sarcoidosis, which may be relevant to outcomes of nonresolving inflammation and fibrosis. For example, a major conceptual challenge is to reconcile how fibrosis occurs in the presence of an antifibrotic milieu of sarcoidosis characterized by upregulation of IFN gamma, which generally inhibits collagen expression. 55 A recent study associated an IFN gamma-induced gene expression signature with correlates of disease severity and chronicity in sarcoidosis. 56 This finding is in line with the key role of IFN gamma in the pathogenesis, but leaves open the question of how fibrosis develops in this setting. The plasticity of pulmonary macrophages, well established in other models of chronic disease models, also may be relevant to outcomes in sarcoidosis. In acute sarcoidosis, pulmonary macrophages typically demonstrate findings consistent with classical activation. These include involvement of IFN gamma and expression of CD80, reflecting efforts associated with pathogen containment and clearance. 57 In contrast, macrophages activated through nonclassical stimuli Q16 Q Recent Advances in Chest Medicine [ - # - CHEST ]

7 Q18 display diverse functions and can contribute to fibrosis in the context of wound healing. 58 Although IL-4/IL-13 (M2a) or IL-10 (M2c) activated macrophages, so-called alternatively activated macrophages, have yet to be detected in pulmonary sarcoidosis, a mix of macrophages with M2a and M2c features were histologically localized to areas of myofibrosis in neuromuscular sarcoidosis. 59 The relevance of the immunoregulatory M2a phenotype, induced by IL-4/IL- 13, in the context of a Th1-dominant process such as active sarcoidosis is unclear. However, the M2c phenotype, induced by IL-10, can participate in tissue remodeling and fibrosis. Such macrophages express high levels of CCL18, 60 inducing collagen expression in lung fibroblasts. 61 CCL18 is highly expressed in patients with fibrotic lung diseases, including fibrotic sarcoidosis. 62 Further research to better define macrophage function across the trajectory of pulmonary sarcoidosis, particularly in relation to the development of fibrotic sarcoidosis, is urgently needed. Considered collectively, data from sarcoidosis research support a model of persistent inflammation related to several possible abnormalities. These include impairments in antigen clearance or sequestration, hyperresponsive effector T cells, and abnormally persistent macrophage activation. Whether deficiencies in regulatory immune responses have a primary or merely accessory role is an important question for future research. Likely, combinations of these factors account for the wide range of clinical features and outcomes of pulmonary sarcoidosis. An important unmet need in the field, the transition from chronic inflammation to fibrosis remains poorly understood; however, macrophage function is likely to have a key role. Implications for Clinical Management Chronic empirical treatment in asymptomatic patients with pulmonary sarcoidosis is not a feasible strategy in most cases. However, for symptomatic patients with active disease, immunosuppressive therapy is often indicated. When the status of disease activity is unclear, CT imaging is often a valuable test. 63 Although acknowledging the difficulty in reconciling clinical practice with a lack of rigorous studies, a variety of reviews address general treatment options. 64 Although corticosteroids are generally effective for suppressing active sarcoidosis, maintaining a dose high enough for clinical efficacy may be impractical because of intolerable side effects; however, in this context, low-dose corticosteroids may be ineffective in a subset of patients. 65 In addition, the response to a given treatment may be phenotype specific. Currently available and potential treatments for pulmonary sarcoidosis are subsequently considered. Nonpharmacologic interventions are also reviewed. Systemic corticosteroids remain the first-line treatment for sarcoidosis. Their success is likely related to broadspectrum immunosuppressive activity, affecting an array of immune cell lines. 66 Methotrexate is a popular second-line agent. Although the dosage used for chemotherapy inhibits T-cell proliferation, other mechanisms are invoked for the lower doses used in sarcoidosis. A review of methotrexate use in rheumatoid arthritis is likely applicable to therapeutic considerations in pulmonary sarcoidosis. 67 In a randomized clinical trial, the TNF-alpha inhibitor infliximab was associated with a significant, albeit modest, clinical improvement in pulmonary sarcoidosis. 68 It is not clear if the benefit of TNF-alpha blockade is caused by granuloma disruption 69 or other effects, including effects on dendritic cell function. 70 TNF-alpha genotypes may influence response to treatment; however, this requires further study. 71 Additionally, the development of antibodies to infliximab may lead to treatment failure, and necessitate switching to adalimumab. 72 Despite the central role of T cells in the pathogenesis, efficacy of lymphocyte-specific therapies is surprisingly poor. For example, calcineurin inhibitors are generally ineffective, and inhibitors of lymphocyte proliferation, such as azathioprine, should not be used as a single agent. 64 Several recent lines of investigation have been instructive, even if disappointing. IL-12 is a key cytokine in sarcoidosis, driving Th1 polarization and subsequent IFN gamma release. 73 A randomized controlled study of anti-il-12/il-23 ustekinumab in chronic sarcoidosis did not demonstrate a benefit 74 ; however, these results may reflect an inability to discern the clinical value of adding this biological agent to a maintenance regimen of corticosteroids. 75 Therapies to enhance clearance of sarcoidosis antigen(s) are highly desirable yet elusive. To date, there is no evidence that impaired T-cell responses precede chronic inflammation in sarcoidosis, or that enhancing inflammation will promote antigen clearance. To the contrary, therapies which augment immune function have been associated with increased or de novo sarcoidosis activity. 76 In a recent study, antimycobacterial treatment for cutaneous sarcoidosis was associated with clinical improvement. 77 Rather than pathogen eradication, anti-inflammatory effects were invoked as the possible therapeutic mechanism. Finally, chestjournal.org 7

8 in a small, nonrandomized clinical trial, rituximab was not associated with clinical improvement. 78 Although rituximab may have immunomodulatory effects beyond B-cell inhibition, there are no data to support its use in sarcoidosis at this time. 79 Looking ahead, potential novel therapeutic targets arise from recent findings (Fig 3). One study found that Q19 inhibitors to IRAK and RIP2 significantly reduced p38- dependent, Th1-related responses in sarcoidosis alveolar macrophages and peripheral blood leukocytes. 26 Inhibitors to SAA aggregation have recently been identified and may represent a novel approach to address persistent granulomatous inflammation in sarcoidosis. 80 Similarly, the recent identification of mtor1 as an agonist for experimental granulomatous inflammation supports the evaluation of newer granuloma-focused inhibitors in sarcoidosis. 49 In efforts to optimize the ratio of regulatory to inflammatory immune cell activity, inhaled vasoactive intestinal peptide and transcutaneously delivered nicotine exerted favorable immunomodulatory effects in sarcoidosis, including enhanced Treg-cell capacity. 27,41 Q28 Although immunosuppressive medications may slow or avert pulmonary fibrosis in sarcoidosis, it remains difficult to identify which patients are at greatest risk, and long-term empirical therapy to prevent this outcome is fraught with complications. 81 Although the mechanisms of fibrosis in sarcoidosis and idiopathic pulmonary fibrosis differ in important ways, a proper evaluation of disease-slowing, antifibrotic treatments (pirfenidone and nintedanib) used for idiopathic Figure 3 A multitude of cytokines and intracellular signaling molecules are highlighted as key mediators of T-cell, macrophage, and potentially fibroblast activation. Activation and regulation of these cells are, in turn, key determinants in the outcomes of active disease, remission, and fibrosis. Genetic associations of these factors and potential therapeutic targets are highlighted. SAA ¼ serum amyloid A; Treg ¼ regulatory T. See Figure 1 legend for expansion of other abbreviations. Organ dysfunction (fibrosis) pulmonary fibrosis may be important for sarcoidosis. 65 The development of clinical approaches to reliably identify patients most at risk of developing fibrosis is also critical. Exacerbations or recrudescence of disease is a common clinical challenge in pulmonary sarcoidosis. 82 Recent cessation of treatment is an important risk factor for recurrence. 83 Less is known about mediators of exacerbations while on treatment. Conditions which augment Th1 immunity can trigger de novo or worsening disease. In HIV-induced lymphopenia, immune reconstitution is associated with increased sarcoidosis activity. 84 Conditions which increase IFN signaling are also known to augment sarcoidosis inflammation. Close clinical follow-up is prudent for patients undergoing IFN-based therapies for diseases such as chronic hepatitis. 85 This recommendation may apply to other immune-enhancing therapies, including PD-1 inhibitors, which have also been associated with new and recurrent sarcoidosis. 76 We conclude this section with an acknowledgement that medication therapy is just one aspect of the clinical management of pulmonary sarcoidosis. Screening for pulmonary hypertension and heart disease, including cardiac sarcoidosis, is indicated for patients with persistent dyspnea. 86,87 Pulmonary rehabilitation is a rational recommendation for patients with impaired functionality. Sleep hygiene optimization and sleep apnea screening are indicated for patients with chronic fatigue. Side effects and complications of treatment, including occult infection, should be considered for any Antigen nicotine Treg Antigen VIP activation presentation IL2 IL10 MHC I / II RIP2 (-) BTNL2 NLRs Th1 / Th17 Macrophage APCs activation activation IL17 IL12/IL23p40 TNF CCR6 IFN γ MyD88 TLRs Antigen CXCR3 Granuloma sequestration formation mkatg, ESAT6, others? SAA Organ injury (inflammation)? pirfenidone TGFβ TORC1 nintedanib ANXA11 CCL18 CCL18 M1 vs M2 differentiation Dysregulated cytokines and mediators Genetic associations Fibrocyte recruitment Remission Potential therapeutic targets and agents Antigen clearance print & web 4C=FPO Recent Advances in Chest Medicine [ - # - CHEST ]

9 unexplained change in condition. Smoking cessation should be recommended for patients with nicotine dependence. Although smoking is associated with a decreased risk of sarcoidosis, among patients with sarcoidosis, lung function is worse for those who smoke, and smoking has not been shown to attenuate disease progression. 27,88,89 Finally, there is growing interest in the role of genetic testing to aid in prognosis. 90 However, there is currently no genetic test for sarcoidosis, and assessing for sarcoidosis in family members should be prompted by signs or symptoms suggestive of the disease. Conclusions Thoracic sarcoidosis refers nonspecifically to pulmonary or nodal disease, or both. The clinical features of pulmonary sarcoidosis are distinct from other phenotypes, suggesting important distinctions in its pathophysiology, and a need to refine the disease model based on evolving research. Recent work has identified the contribution of Th17-like cells to IFN gamma release in the adaptive immune response. An emerging body of literature suggests that innate immune responses may be important as well; however, whether alterations are primary or secondary events remains unclear. Relatedly, elucidating how antigen is processed and cleared is essential, particularly as it relates to chronic disease. Circumstantial evidence implicates a possible role for remnant mycobacterial proteins in the initiation of sarcoidosis, and SAA proteins may contribute to ongoing immune stimulation. Other immune events which impede dissolution of granulomas may mediate chronic disease, and need to be better understood. Finally, further studies to characterize T-cell function during the transition from acute to chronic disease, and to critically evaluate the implications of Treg-cell functional capacity, are important. In addition to these specific lines of investigation, the integration of high throughput techniques and advanced computational methods will help define disease endotypes and relevant biomarkers. 19 In parallel, phenotyping patients in research according to pulmonary involvement is essential, as are continuing efforts to better define the concepts of refractory, recurrent, and severe disease. Such multifaceted research is our great opportunity to discover novel therapeutic targets, which are desperately needed for this incurable disease, and to optimize patient care and outcomes. Finally, although immunosuppression has a central role in clinical management, nonpharmacologic interventions can also often improve functionality, and should be considered in all patients with pulmonary sarcoidosis. Acknowledgments Financial/nonfinancial disclosure: None declared. References Q21 Q22 Q23 1. Costabel U, Hunninghake GW. ATS/ERS/WASOG statement on sarcoidosis. Sarcoidosis Statement Committee. 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