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Current perspectives on the immunopathogenesis of sarcoidosis

  • Silvia Lee
    Correspondence
    Corresponding author. Harry Perkins Institute of Medical Research, Murdoch, 6150, Western Australia, Australia.
    Affiliations
    Department of Microbiology, Pathwest Laboratory Medicine, Perth, Australia

    Department of Advanced Clinical and Translational Cardiovascular Imaging, Harry Perkins Institute of Medical Research, Murdoch, Australia
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  • David Birnie
    Affiliations
    University of Ottawa Heart Institute, Ottawa, Canada
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  • Girish Dwivedi
    Affiliations
    Department of Advanced Clinical and Translational Cardiovascular Imaging, Harry Perkins Institute of Medical Research, Murdoch, Australia

    School of Medicine, University of Western Australia, Australia

    Department of Cardiology, Fiona Stanley Hospital, Murdoch, Western Australia, Australia
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Open ArchivePublished:September 22, 2020DOI:https://doi.org/10.1016/j.rmed.2020.106161

      Highlights

      • Innate and adaptive immune responses influence progression and determine clinical outcome in sarcoidosis.
      • Recent studies support an autoimmune component and also the role of microbial organisms as causative agents in sarcoidosis.
      • Establishment of recent animal and in vitro human models have provided insight in the immunopathogenesis of sarcoidosis.

      Abstract

      Sarcoidosis is an inflammatory systemic disease that commonly affects the lungs or lymph nodes but can manifest in other organs. Herein, we review the latest evidence establishing how innate and adaptive immune responses contribute to the pathogenesis and clinical course of sarcoidosis. We discuss the possible role of microbial organisms as etiologic agents in sarcoidosis and the evidence supporting sarcoidosis as an autoimmune disease. We also discuss how animal and in vitro human models have advanced our understanding of the immunopathogenesis of sarcoidosis. Finally, we discuss therapeutics for sarcoidosis and the effects on the immune system.

      Keywords

      1. Introduction

      Sarcoidosis is an inflammatory systemic disease that most commonly affects the lungs or lymph nodes (80–90%) but can manifest in other organs. For example, asymptomatic cardiac involvement is reported in up to 25% of sarcoidosis [
      • Birnie D.H.
      • Kandolin R.
      • Nery P.B.
      • Kupari M.
      Cardiac manifestations of sarcoidosis: diagnosis and management.
      ]. Diagnosis of sarcoidosis relies on both non-invasive imaging methods (e.g. positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI)) and invasive biopsies for histopathological examination [
      • Ungprasert P.
      • Ryu J.H.
      • Matteson E.L.
      Clinical manifestations, diagnosis, and treatment of sarcoidosis.
      ]. Advanced imaging modalities such as 18F-fluorodeoxyglucose (18F-FDG) PET/CT is often utilised to identify cardiac involvement [
      • Erthal F.
      • Juneau D.
      • Lim S.P.
      • et al.
      Imaging of cardiac sarcoidosis.
      ] or active disease in extra-cardiac sites, as FDG accumulates in activated inflammatory cells [
      • Akaike G.
      • Itani M.
      • Shah H.
      • et al.
      PET/CT in the diagnosis and workup of sarcoidosis: focus on atypical manifestations.
      ]. Using whole-body 18F-FDG PET/CT, we reported a 3.2% and 10% prevalence of sarcoidosis that involved only the heart in Canada [
      • Juneau D.
      • Nery P.
      • Russo J.
      • et al.
      How common is isolated cardiac sarcoidosis? Extra-cardiac and cardiac findings on clinical examination and whole-body (18)F-fluorodeoxyglucose positron emission tomography.
      ] and Western Australia, respectively [
      • Giudicatti L.
      • Marangou J.
      • Nolan D.
      • Dembo L.
      • Baumwol J.
      • Dwivedi G.
      The utility of whole body (18)F-FDG PET-CT.
      ]. The aetiology of sarcoidosis is unknown but involves an immune-mediated response against a putative antigen in genetically predisposed individuals leading to the formation of non-caseating granulomas in the affected organ. In most patients, the disease is acute and the granulomas spontaneously regress, but up to 40% of patients develop chronic disease (i.e. lasting >2 years) with persistent inflammation and tissue fibrosis. Currently, there is no cure for sarcoidosis and existing treatment options include corticosteroids, immunosuppressive agents or biologic agents to reduce inflammation [
      • Ungprasert P.
      • Ryu J.H.
      • Matteson E.L.
      Clinical manifestations, diagnosis, and treatment of sarcoidosis.
      ].
      Several reviews have discussed the critical role of the host immune response during sarcoidosis [
      • Inaoka P.T.
      • Shono M.
      • Kamada M.
      • Espinoza J.L.
      Host-microbe interactions in the pathogenesis and clinical course of sarcoidosis.
      ,
      • Patterson K.C.
      • Chen E.S.
      The pathogenesis of pulmonary sarcoidosis and implications for treatment.
      ,
      • Grunewald J.
      • Grutters J.C.
      • Arkema E.V.
      • Saketkoo L.A.
      • Moller D.R.
      • Muller-Quernheim J.
      Sarcoidosis.
      ,
      • Bennett D.
      • Bargagli E.
      • Refini R.M.
      • Rottoli P.
      New concepts in the pathogenesis of sarcoidosis.
      ]. Herein, we provide a comprehensive update mainly focussed on: (1) how innate and adaptive immune responses contribute to the pathogenesis and clinical course of sarcoidosis, (2) the possible role of micro-organisms as causative agents and the evidence supporting an autoimmune component in sarcoidosis, and (3) how animal and in vitro human models improve our understanding of the immunopathogenesis in sarcoidosis.

      1.1 Innate immune responses in sarcoidosis

      Whilst the focus of many previous studies has been the identification of antigen-specific adaptive immune responses in order to elucidate the inflammatory processes involved in sarcoidosis, it has becoming increasingly apparent that innate immunity also contributes to the initiation and also the progression of the disease [
      • Inaoka P.T.
      • Shono M.
      • Kamada M.
      • Espinoza J.L.
      Host-microbe interactions in the pathogenesis and clinical course of sarcoidosis.
      ,
      • Patterson K.C.
      • Chen E.S.
      The pathogenesis of pulmonary sarcoidosis and implications for treatment.
      ,
      • Grunewald J.
      • Grutters J.C.
      • Arkema E.V.
      • Saketkoo L.A.
      • Moller D.R.
      • Muller-Quernheim J.
      Sarcoidosis.
      ,
      • Bennett D.
      • Bargagli E.
      • Refini R.M.
      • Rottoli P.
      New concepts in the pathogenesis of sarcoidosis.
      ] (Table 1).
      Table 1Innate immune responses in sarcoidosis.
      SampleFindingsRef
      Heart, lung, skin tissuesInflammasomes (NLRP3, ASC, caspase-1) detected13,14
       BALFIncreased IL-1β production by macrophages14,15
      LN/non-lymphatic tissuesIncreased M2 macrophages20
       BloodDecreased % MAIT cells23
       BALFDecreased % NK and NKT cells24
       BloodPolymorphisms in TLR3 and NOD25,26
       BloodIncreased IL-8, TNFα production by macrophages26
       SerumIncreased NGAL levels27
       BALFmiR-21, miR-146a and miR-150 detected28
      Serum, skin tissuesTARC/CCL17 levels and expression36
       BloodIncreased % CD14++CD16+ monocytes37
      Abbreviations: BALF, bronchalveolar lavage fluid; LN, lymph nodes; NLRP3, NOD-like receptor family pyrin domain containing 3; ASC, apoptosis speck-like protein containing a caspase-1 recruiting domain; MAIT, mucosal-associated invariant T-cells; NK, natural killer; NKT, natural killer T-cell; NGAL, neutrophil gelatinase-associated lipocalin, miR, microRNA; TLR3, toll-like receptor 3; NOD, nucleotide-binding oligomerization domain.
      The innate immune system provides a first line of defence against invading microbes. Host cells express receptors on the cell surface, endosomal membrane or in the cytoplasm that are able to recognise pathogen-associated molecular patterns found in bacteria, viruses, fungi and protozoa [
      • Maeda K.
      • Caldez M.J.
      • Akira S.
      Innate immunity in allergy.
      ]. These include toll-like receptors (TLRs), nucleotide-binding oligomerization domain and leucine-rich repeat-containing receptors (NLRs), the AIM2-like receptors (ALRs) and RIG-I-like receptors [
      • Maeda K.
      • Caldez M.J.
      • Akira S.
      Innate immunity in allergy.
      ]. NLRs constitute one component of the NLRP inflammasome, which is a large multiprotein complex that also comprise of adaptor proteins (e.g. apoptosis speck-like protein containing a caspase-1 recruiting domain, ASC) [
      • Maeda K.
      • Caldez M.J.
      • Akira S.
      Innate immunity in allergy.
      ,
      • Evavold C.L.
      • Kagan J.C.
      Inflammasomes: threat-assessment organelles of the innate immune system.
      ]. Assembly of these proteins results in the activation of effector proteins (e.g. caspase-1) which promotes the cleavage of inactive forms of interleukin (IL)-1β and IL-18 into their active forms [
      • Maeda K.
      • Caldez M.J.
      • Akira S.
      Innate immunity in allergy.
      ,
      • Evavold C.L.
      • Kagan J.C.
      Inflammasomes: threat-assessment organelles of the innate immune system.
      ].
      Using immunostaining techniques for the adaptor protein ASC, Kron et al. identified the presence of inflammasome aggregates in heart tissue sections from three patients diagnosed with cardiac sarcoidosis [
      • Kron J.
      • Mauro A.G.
      • Bonaventura A.
      • et al.
      Inflammasome formation in granulomas in cardiac sarcoidosis.
      ]. Furthermore, they demonstrated that staining for ASC coincided with detection of NLRP3 and caspase-1 within the granulomas. Similarly, Huppertz et al. confirmed positive staining for caspase-1 and IL-1β together with CD68-positive macrophages within sarcoid granulomas from transbronchial and skin biopsies [
      • Huppertz C.
      • Jager B.
      • Wieczorek G.
      • et al.
      The NLRP3 inflammasome pathway is activated in sarcoidosis and involved in granuloma formation.
      ].
      Two recent studies reported higher levels of IL-1β released in the supernatant by unstimulated and lipopolysaccharide-stimulated alveolar macrophages and monocytes from sarcoidosis patients compared to healthy controls [
      • Huppertz C.
      • Jager B.
      • Wieczorek G.
      • et al.
      The NLRP3 inflammasome pathway is activated in sarcoidosis and involved in granuloma formation.
      ,
      • Talreja J.
      • Talwar H.
      • Bauerfeld C.
      • et al.
      HIF-1alpha regulates IL-1 beta and IL-17 in sarcoidosis.
      ]. Talreja et al. also showed that the transcription factor, hypoxia-inducible factor 1-alpha (HIF-1α) play an important role in increasing IL-1β and IL-17 production, as expression of HIF-1α was localised in the center of granulomatous tissue and multinucleated giant cells in lung biopsies from sarcoidosis patients and inhibition of HIF-1α reduced levels of these pro-inflammatory cytokines [
      • Talreja J.
      • Talwar H.
      • Bauerfeld C.
      • et al.
      HIF-1alpha regulates IL-1 beta and IL-17 in sarcoidosis.
      ]. This is consistent with another study, where inhibition of HIF-1α with small interfering RNA reduced levels of IL-1β and also inflammasome-associated proteins such as NLRP3, ASC and caspase-1 in a cell culture model of bleomycin-induced acute lung injury [
      • Huang J.J.
      • Xia J.
      • Huang L.L.
      • Li Y.C.
      HIF1alpha promotes NLRP3 inflammasome activation in bleomycininduced acute lung injury.
      ]. HIF-1α was originally thought to have an essential role in oxygen homeostasis and energy metabolism, but accumulating evidence suggests that HIF-1α may also be important in the regulation of immune responses during infection or inflammation [
      • Knight M.
      • Stanley S.
      HIF-1alpha as a central mediator of cellular resistance to intracellular pathogens.
      ,
      • Stothers C.L.
      • Luan L.
      • Fensterheim B.A.
      • Bohannon J.K.
      Hypoxia-inducible factor-1alpha regulation of myeloid cells.
      ]. Together, these studies confirm a role of inflammasomes and IL-1 in the pathogenesis of sarcoidosis and provide alternative pathways for future development of novel therapeutics.
      Depending on the cytokine microenvironment, macrophages can be classified as M1 or M2. M1 macrophages are activated by lipopolysaccharide and interferon (IFN)γ and produce IL-12 and tumor necrosis factor (TNF)α whereas M2 macrophages are generated in the presence of Th2 cytokines (IL-4 and IL-13) and produce immunosuppressive and immunoregulatory cytokines (e.g. IL-10) [
      • Ruytinx P.
      • Proost P.
      • Van Damme J.
      • Struyf S.
      Chemokine-induced macrophage polarization in inflammatory conditions.
      ]. Shamaei et al. demonstrated increased numbers of M2 macrophages in lymph nodes and non-lymphatic tissues from sarcoidosis patients compared to tuberculosis patients, whereas numbers of monocytes and M1 macrophages were similar in tissues from both patient groups [
      • Shamaei M.
      • Mortaz E.
      • Pourabdollah M.
      • et al.
      Evidence for M2 macrophages in granulomas from pulmonary sarcoidosis: a new aspect of macrophage heterogeneity.
      ].
      In a recent study, Lepzien et al. provided an in-depth analyses of monocytes and dendritic cell subsets isolated from bronchoalveolar lavage fluid (BALF), endobronchial biopsies, lung-draining lymph nodes and blood from within the same sarcoidosis patient presenting with either an acute onset (also known as Lofgren's syndrome and is characterised by arthritis, erythema nodosum and bilateral hilar lymphadenopathy on chest radiography) or a gradual onset (non-Lofgren's syndrome) [
      • Lepzien R.
      • Rankin G.
      • Pourazar J.
      • et al.
      Mapping mononuclear phagocytes in blood, lungs, and lymph nodes of sarcoidosis patients.
      ]. The investigators detected the presence of monocytes and dendritic cells in all anatomical compartments but the different cell subsets displayed differential distribution, maturation status and expression of chemokine receptors depending on their location. Furthermore, frequencies of plasmacytoid dendritic cells differed between patient groups, with lower percentages seen in the lung-draining lymph nodes from Lofgren's syndrome compared to non-Lofgren's syndrome patients [
      • Lepzien R.
      • Rankin G.
      • Pourazar J.
      • et al.
      Mapping mononuclear phagocytes in blood, lungs, and lymph nodes of sarcoidosis patients.
      ]. The study emphasises the importance of examining different compartments and further studies are necessary to determine the precise function of these cells in these locations.
      Mucosal-associated invariant T-cells (MAIT) are innate cells that constitute up to 10% of circulating T-cells and are involved in cancer, autoimmune disorders, and in various diseases caused by bacteria, viruses and parasites [
      • Godfrey D.I.
      • Koay H.F.
      • McCluskey J.
      • Gherardin N.A.
      The biology and functional importance of MAIT cells.
      ]. Upon activation, MAIT cells can produce pro-inflammatory cytokines including IFNγ and TNFα. Matsuyama et al. found reduced frequencies of MAIT cells in the peripheral blood from sarcoidosis patients compared to healthy controls, but expression of immune activation markers (e.g. CD69) and programmed cell death-1 (PD-1) on MAIT cells were higher in patients [
      • Matsuyama H.
      • Isshiki T.
      • Chiba A.
      • et al.
      Activation of mucosal-associated invariant T cells in the lungs of sarcoidosis patients.
      ]. The significance of increased PD-1 expression will be discussed later as this molecule has also been shown to be upregulated on adaptive immune cells from sarcoidosis patients. The investigators also reported increased numbers and frequencies of MAIT cells in the BALF from the lungs of patients with parenchymal infiltration compared to BALF from patients without parenchymal infiltration [
      • Matsuyama H.
      • Isshiki T.
      • Chiba A.
      • et al.
      Activation of mucosal-associated invariant T cells in the lungs of sarcoidosis patients.
      ].
      Frequencies of other innate/innate-like immune cells including NK and NKT cells have been found to be reduced in the BALF from patients with sarcoidosis compared to BALF from healthy controls and patients with other forms of interstitial lung diseases including chronic fibrotic hypersensitivity pneumonitis, non-specific interstitial pneumonia and idiopathic pulmonary fibrosis [
      • Bergantini L.
      • Cameli P.
      • d'Alessandro M.
      • et al.
      NK and NKT-like cells in granulomatous and fibrotic lung diseases.
      ].
      A polymorphism in TLR3 (L412F, rs3775291) have been shown to be associated with clinical disease in a population of Irish and American Caucasian patients with pulmonary sarcoidosis [
      • Cooke G.
      • Kamal I.
      • Strengert M.
      • et al.
      Toll-like receptor 3 L412F polymorphism promotes a persistent clinical phenotype in pulmonary sarcoidosis.
      ]. Carriage of the F allele was associated with disease persistence. Mechanistically, the authors demonstrated that activation of TLR3 in primary lung fibroblasts from patients homozygous for 412F resulted in decreased IFNβ expression, inhibition of apoptosis and dysregulated proliferative responses compared to cells from wild-type patients [
      • Cooke G.
      • Kamal I.
      • Strengert M.
      • et al.
      Toll-like receptor 3 L412F polymorphism promotes a persistent clinical phenotype in pulmonary sarcoidosis.
      ]. In a study of familial sarcoidosis, a polymorphism in NOD (G908R, rs2066845) as well as variants in the IL17A, KALRN and EPHA2 genes has been identified in patients who developed pulmonary disease [
      • Besnard V.
      • Calender A.
      • Bouvry D.
      • et al.
      G908R NOD2 variant in a family with sarcoidosis.
      ]. Additionally, macrophages from sarcoidosis patients displayed elevated IL-8 and TNFα mRNA levels despite reduced activation of the transcription factor NF-κB under basal condition [
      • Besnard V.
      • Calender A.
      • Bouvry D.
      • et al.
      G908R NOD2 variant in a family with sarcoidosis.
      ]. The results suggest an interplay between these genes in the development of sarcoidosis by promoting a heightened pro-inflammatory state in macrophages.
      Neutrophil gelatinase-associated lipocalin (NGAL) is a glycoprotein secreted from activated neutrophils and is important in the innate immune response. Sarcoidosis patients have been found to have elevated levels of NGAL compared to controls but levels did not correlate with markers of disease activity (e.g. angiotensin converting enzyme (ACE), CD4/CD8 ratio in BALF) [
      • Kato S.
      • Inui N.
      • Hozumi H.
      • et al.
      Neutrophil gelatinase-associated lipocalin in patients with sarcoidosis.
      ]. Additionally, Kishore et al. identified several microRNAs (miRNAs) present in extracellular vesicles (miR-146a and miR-150) and cells (miR-21) isolated from BALF from patients with pulmonary sarcoidosis [
      • Kishore A.
      • Navratilova Z.
      • Kolek V.
      • et al.
      Expression analysis of extracellular microRNA in bronchoalveolar lavage fluid from patients with pulmonary sarcoidosis.
      ]. Expression of miR-146a and miR-150 in extracellular vesicles correlated inversely with pulmonary function in patients with parenchymal involvement [
      • Kishore A.
      • Navratilova Z.
      • Kolek V.
      • et al.
      Expression analysis of extracellular microRNA in bronchoalveolar lavage fluid from patients with pulmonary sarcoidosis.
      ]. miRNAs are small single-stranded noncoding RNAs that regulate gene expression and play an essential role in various cellular functions including proliferation, apoptosis, differentiation, inflammation and innate immune responses (see Review [
      • Momen-Heravi F.
      • Bala S.
      miRNA regulation of innate immunity.
      ]). miR-21 and miR-146a have been demonstrated to negatively regulate inflammatory responses and thereby promote resolution of inflammation. Zhou et al. recently showed that miR-146a targeted IL-1 receptor-associated kinase l (IRAK1) following TLR4 stimulation and inhibited NF-κB activation resulting in reduced production of IL-6 and TNF-α in human monocytic cell line [
      • Zhou C.
      • Zhao L.
      • Wang K.
      • et al.
      MicroRNA-146a inhibits NF-kappaB activation and pro-inflammatory cytokine production by regulating IRAK1 expression in THP-1 cells.
      ]. Similarly, Li et al. reported inhibition of the NF-κB pathway and reduced levels of pro-inflammatory cytokines following over-expression of miR-150 in human pulmonary epithelial cell line [
      • Li P.
      • Yao Y.
      • Ma Y.
      • Chen Y.
      MiR-150 attenuates LPS-induced acute lung injury via targeting AKT3.
      ]. The study identified the serine/threonine protein kinase AKT3 as the potential target for miR-150 [
      • Li P.
      • Yao Y.
      • Ma Y.
      • Chen Y.
      MiR-150 attenuates LPS-induced acute lung injury via targeting AKT3.
      ]. These novel biomarkers warrant further investigation in larger cohorts and longitudinal studies to determine their clinical usefulness for sarcoidosis diagnosis or prognosis.

      1.2 Adaptive immune responses in sarcoidosis

      In addition to macrophages and dendritic cells, sarcoid granulomas comprise of infiltrating T- and B-cells. Analysis of lymphocyte profiles in lymph nodes prepared from endobronchial ultrasound-guided transbronchial needle aspiration revealed higher CD4/CD8 ratio compared to BALF from patients with sarcoidosis [
      • Akao K.
      • Minezawa T.
      • Yamamoto N.
      • et al.
      Flow cytometric analysis of lymphocyte profiles in mediastinal lymphadenopathy of sarcoidosis.
      ]. Several recent studies have demonstrated an important role for cells of the adaptive immune system in the development and progression of sarcoidosis (Table 2).
      Table 2Adaptive immune responses in sarcoidosis.
      SampleFindingsRef
      Blood, BALFIncreased Th1 cytokines; decreased proliferation; increased apoptosis and PD-1 expression on CD4+ T-cells33
       SerumIncreased CXCL9, CXCL10 and CXCL11 levels34
       BloodIncreased % CCR4+CD4+ T-cells36
       BloodIncreased TGFβ production by PD-1+ Th17 CD4+ T-cells39
       LNIncreased % Th17.1 T-cells43
       BloodIncreased % Treg cells44,45
      Blood, BALFIncreased % CD8+ T-cells expressing perforin/granzyme B54
      BALF, serumIncreased vimentin antibody levels56
       SerumIncreased BAFF levels46,57
       BloodIncreased % naïve B-cells, decreased % memory B-cells46,47
      Abbreviations: BALF, bronchalveolar lavage fluid; LN, lymph nodes; Th1, T helper 1; Th17, T helper 17; PD-1, programmed death-1; Treg, regulatory T-cells; BAFF, B-cell activating factor; TARC, thymus- and activation-regulated chemokine.

      1.2.1 CD4+ T-cells

      CD4+ T-cells from pulmonary sarcoidosis patients have been shown to have increased spontaneous T helper 1 (Th1) cytokine production, decreased proliferative responses following T-cell receptor (TCR) stimulation and increased apoptosis and expression of PD-1 compared to healthy controls [
      • Hawkins C.
      • Shaginurova G.
      • Shelton D.A.
      • et al.
      Local and systemic CD4(+) T cell exhaustion reverses with clinical resolution of pulmonary sarcoidosis.
      ]. These immune dysfunction were more pronounced in matching BALF compared to peripheral blood from sarcoidosis patients and also in patients who had active disease compared to those who had resolved their disease [
      • Hawkins C.
      • Shaginurova G.
      • Shelton D.A.
      • et al.
      Local and systemic CD4(+) T cell exhaustion reverses with clinical resolution of pulmonary sarcoidosis.
      ]. The role for Th1 immune response in sarcoidosis was confirmed in another study that reported increased serum levels of IFNγ-inducible chemokines CXCL9, CXCL10 and CXCL11 in sarcoidosis patients compared to heathy controls [
      • Arger N.K.
      • Ho M.
      • Woodruff P.G.
      • Koth L.L.
      Serum CXCL11 correlates with pulmonary outcomes and disease burden in sarcoidosis.
      ]. Furthermore, CXCL11 levels were associated with measures of sarcoidosis disease burden including pulmonary function abnormalities and the number of organs involved [
      • Arger N.K.
      • Ho M.
      • Woodruff P.G.
      • Koth L.L.
      Serum CXCL11 correlates with pulmonary outcomes and disease burden in sarcoidosis.
      ]. In a subsequent study, the investigators found a correlation between serum levels of CXCL9 with systemic organ involvement whereas CXCL10 levels were associated with pulmonary function and respiratory symptoms [
      • Arger N.K.
      • Ho M.E.
      • Allen I.E.
      • Benn B.S.
      • Woodruff P.G.
      • Koth L.L.
      CXCL9 and CXCL10 are differentially associated with systemic organ involvement and pulmonary disease severity in sarcoidosis.
      ].
      T helper 2 (Th2) immune responses have also been implicated in sarcoidosis with high frequencies of CCR4+CD4+ T-cells observed in patients compared to healthy controls [
      • Nguyen C.T.H.
      • Kambe N.
      • Ueda-Hayakawa I.
      • et al.
      TARC expression in the circulation and cutaneous granulomas correlates with disease severity and indicates Th2-mediated progression in patients with sarcoidosis.
      ]. Both CCR4 and its ligand, thymus- and activation-regulated chemokine (TARC/CCL17), have been demonstrated in epithelioid cells of skin lesions from sarcoidosis patients [
      • Nguyen C.T.H.
      • Kambe N.
      • Ueda-Hayakawa I.
      • et al.
      TARC expression in the circulation and cutaneous granulomas correlates with disease severity and indicates Th2-mediated progression in patients with sarcoidosis.
      ]. Furthermore, elevated serum levels of TARC/CCL17 in sarcoidosis patients correlated with disease activity (e.g. ACE, lysozyme and sIL-2R) [
      • Nguyen C.T.H.
      • Kambe N.
      • Ueda-Hayakawa I.
      • et al.
      TARC expression in the circulation and cutaneous granulomas correlates with disease severity and indicates Th2-mediated progression in patients with sarcoidosis.
      ]. Circulating intermediate monocytes (defined as CD14++CD16+) have been shown to be a source of TARC/CCL17 and frequencies of these pro-inflammatory cells are increased in sarcoidosis patients compared to controls [
      • Kishimoto I.
      • Nguyen C.T.H.
      • Kambe N.
      • et al.
      Circulating intermediate monocytes produce TARC in sarcoidosis.
      ].
      Using mass cytometry, increased expression of immunoregulatory receptors including cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), inducible T-cell costimulatory (ICOS) and PD-1 were observed on CD4+ T-cells isolated from BALF from patients with Lofgren's syndrome whereas elevated expression of activation and effector molecules (e.g. HLA-DR, CD127, CD39 and CD44) were characteristic of CD4+ T-cells from patients with non-Lofgren's syndrome [
      • Kaiser Y.
      • Lakshmikanth T.
      • Chen Y.
      • et al.
      Mass cytometry identifies distinct lung CD4(+) T cell patterns in lofgren's syndrome and non-lofgren's syndrome sarcoidosis.
      ]. These data suggests that evaluating not only CD4+ T-cell percentages but profiling these cells may be valuable in predicting disease outcome.
      Celada et al. recently demonstrated that percentages of Th17 CD4+ T-cells from sarcoidosis patients with increased PD-1 expression produced higher amounts of TGFβ compared to healthy controls [
      • Celada L.J.
      • Kropski J.A.
      • Herazo-Maya J.D.
      • et al.
      PD-1 up-regulation on CD4(+) T cells promotes pulmonary fibrosis through STAT3-mediated IL-17A and TGF-beta 1 production.
      ]. Blockade of PD-1 reduced IL-17 and TGFβ expression on CD4+ T-cells and also secretion of TGFβ and collagen-1 in co-cultures of human lung fibroblasts and CD4+ T-cells from sarcoidosis patients [
      • Celada L.J.
      • Kropski J.A.
      • Herazo-Maya J.D.
      • et al.
      PD-1 up-regulation on CD4(+) T cells promotes pulmonary fibrosis through STAT3-mediated IL-17A and TGF-beta 1 production.
      ]. Reduced IL-17 and collagen-1 expression following PD-1 blockade was shown to be mediated via decreased STAT3 expression [
      • Celada L.J.
      • Kropski J.A.
      • Herazo-Maya J.D.
      • et al.
      PD-1 up-regulation on CD4(+) T cells promotes pulmonary fibrosis through STAT3-mediated IL-17A and TGF-beta 1 production.
      ].
      It is clearly evident from several of these recent studies that up-regulation of PD-1 expression on innate and adaptive immune cells is a prominent feature of sarcoidosis. PD-1 is a cell-surface protein expressed on activated immune cells with a role in regulating the immune response by down-regulating the immune system and promoting peripheral tolerance by suppressing T-cell inflammation [
      • Qin W.
      • Hu L.
      • Zhang X.
      • et al.
      The diverse function of PD-1/PD-L pathway beyond cancer.
      ]. The importance of PD-1 in the pathogenesis of sarcoidosis has also been established in several case studies of patients with malignancies receiving PD-1 inhibitors (see Review [
      • Rambhia P.H.
      • Reichert B.
      • Scott J.F.
      • et al.
      Immune checkpoint inhibitor-induced sarcoidosis-like granulomas.
      ]). Sarcoid-like granulomas that can affect one or more organs have been shown to develop in a minority of patients and this is likely due to activation of the Th1/Th17 pathway [
      • Rambhia P.H.
      • Reichert B.
      • Scott J.F.
      • et al.
      Immune checkpoint inhibitor-induced sarcoidosis-like granulomas.
      ].
      Under inflammatory conditions, Th17 T-cells can convert to highly pathogenic Th17.1 T-cells (CCR6+CXCR3+CCR4-) that can also produce IFNγ [
      • Paulissen S.M.
      • van Hamburg J.P.
      • Dankers W.
      • Lubberts E.
      The role and modulation of CCR6+ Th17 cell populations in rheumatoid arthritis.
      ]. Broos et al. reported increased frequencies of Th17.1 cells in the mediastinal lymph nodes from patients with sarcoidosis compared to healthy controls [
      • Broos C.E.
      • Koth L.L.
      • van Nimwegen M.
      • et al.
      Increased T-helper 17.1 cells in sarcoidosis mediastinal lymph nodes.
      ]. These cells accumulated at the site of inflammation as percentages were higher in BALF compared to mediastinal lymph nodes and peripheral blood [
      • Broos C.E.
      • Koth L.L.
      • van Nimwegen M.
      • et al.
      Increased T-helper 17.1 cells in sarcoidosis mediastinal lymph nodes.
      ]. Moreover, percentages of Th17.1 cells in BALF were higher in patients with chronic disease compared to patients who resolved the disease within the 2 years of clinical follow-up [
      • Broos C.E.
      • Koth L.L.
      • van Nimwegen M.
      • et al.
      Increased T-helper 17.1 cells in sarcoidosis mediastinal lymph nodes.
      ].
      Together, these studies suggest that sarcoidosis is a dynamic process that involves multiple different Th-cell subsets and that the regulation of the Th1/Th17/Th2 balance may be critical in determining disease burden and progression.
      Proportions of regulatory T-cells (Tregs, CD25+FOXP3+) have been found to be increased in pulmonary sarcoidosis patients compared to healthy controls [
      • Ding J.
      • Dai J.
      • Cai H.
      • Gao Q.
      • Wen Y.
      Extensively disturbance of regulatory T cells - Th17 cells balance in stage II pulmonary sarcoidosis.
      ,
      • Kachamakova-Trojanowska N.
      • Jazwa-Kusior A.
      • Szade K.
      • et al.
      Molecular profiling of regulatory T cells in pulmonary sarcoidosis.
      ] and displayed differential miRNA expression profiles between peripheral blood and BALF [
      • Kachamakova-Trojanowska N.
      • Jazwa-Kusior A.
      • Szade K.
      • et al.
      Molecular profiling of regulatory T cells in pulmonary sarcoidosis.
      ]. Furthermore, increased expression of genes involved in signalling through TLR pathway, proliferation and apoptosis were demonstrated in not only Tregs but in total peripheral blood cell population from sarcoidosis patients compared to controls [
      • Kachamakova-Trojanowska N.
      • Jazwa-Kusior A.
      • Szade K.
      • et al.
      Molecular profiling of regulatory T cells in pulmonary sarcoidosis.
      ]. Following glucocorticoid treatment, frequencies of Tregs and FOXP3 expression returned to levels seen in healthy controls [
      • Ding J.
      • Dai J.
      • Cai H.
      • Gao Q.
      • Wen Y.
      Extensively disturbance of regulatory T cells - Th17 cells balance in stage II pulmonary sarcoidosis.
      ].
      T follicular helper T (TFH) cells are a distinct subset of CD4+ T-cells characterised by high expression of CXCR5 with a specialised role in the formation and maintenance of germinal centers and the generation of high-affinity antibodies by B-cells. Frequencies of circulating TFH cells have been found to be reduced in sarcoidosis patients compared to healthy controls and these cells were present in sarcoid skin lesions suggestive of recruitment of these cells to affected tissues [
      • Ly N.T.M.
      • Ueda-Hayakawa I.
      • Nguyen C.T.H.
      • Okamoto H.
      Exploring the imbalance of circulating follicular helper CD4(+) T cells in sarcoidosis patients.
      ]. However another study reported increased proportions of circulating central memory (CD45RACD27+) TFH cells in patients with sarcoidosis compared to healthy controls [
      • Kudryavtsev I.
      • Serebriakova M.
      • Starshinova A.
      • et al.
      Imbalance in B cell and T Follicular helper cell subsets in pulmonary sarcoidosis.
      ].
      Several genetic variations in the human leukocyte antigen (HLA) region have been associated with the clinical course of sarcoidosis [
      • Kishore A.
      • Petrek M.
      Next-generation sequencing based HLA typing: deciphering immunogenetic aspects of sarcoidosis.
      ]. Spontaneous resolving disease was associated with HLA-DRB1*03 whereas extra-pulmonary involvement have been linked to carriage of HLA-DRB1*15 [
      • Kishore A.
      • Petrek M.
      Next-generation sequencing based HLA typing: deciphering immunogenetic aspects of sarcoidosis.
      ]. In a recent case control study of firefighters with World Trade Center (WTC)-related sarcoidosis, a genetic variant in HLA-B was associated with extra-thoracic involvement [
      • Cleven K.L.
      • Ye K.
      • Zeig-Owens R.
      • et al.
      Genetic variants associated with FDNY WTC-related sarcoidosis.
      ]. Using whole exome sequencing, Lahtela et al. reported variations in leukocyte immunoglobulin-like receptor subfamily B member 4 (LILRB4) was associated with good prognosis in Finnish sarcoidosis patients having the good prognosis HLA class II markers (HLA-DRB1*03:01 and/or HLA-DRB1*04:01-DPB1*04:01) [
      • Lahtela E.
      • Kankainen M.
      • Sinisalo J.
      • Selroos O.
      • Lokki M.L.
      Exome sequencing identifies susceptibility loci for sarcoidosis prognosis.
      ]. In a large Scandinavian cohort of >600 patients with sarcoidosis, the magnitude of expansion of CD4+ T-cells expressing the TCR Vα2.3 in the BALF have been shown to be not only associated with better prognosis, younger age and with the HLA-DRB1*03 allele [
      • Darlington P.
      • Kullberg S.
      • Eklund A.
      • Grunewald J.
      Subpopulations of cells from bronchoalveolar lavage can predict prognosis in sarcoidosis.
      ], but was also useful as a diagnostic tool [
      • Darlington P.
      • Kullberg S.
      • Eklund A.
      • Grunewald J.
      Lung CD4+ Valpha2.3+ T-cells in sarcoidosis cohorts with Lofgren's syndrome.
      ]. Karakaya et al. found that HLA-DRB1*03 was associated with high CD4+/CD8+ ratio, low percentages of lymphocytes (in Lofgren's syndrome patients), and low CD103+CD4+/CD4+ ratio (in non-Lofgren's syndrome patients) in the BALF [
      • Karakaya B.
      • Schimmelpennink M.C.
      • Kocourkova L.
      • et al.
      Bronchoalveolar lavage characteristics correlate with HLA tag SNPs in patients with Lofgren's syndrome and other sarcoidosis.
      ]. Cells expressing CD103 are terminally differentiated effector cells implicated in lung fibrosis and thus reduced CD103+CD4+/CD4+ ratio may predict a benign course of disease [
      • Karakaya B.
      • Schimmelpennink M.C.
      • Kocourkova L.
      • et al.
      Bronchoalveolar lavage characteristics correlate with HLA tag SNPs in patients with Lofgren's syndrome and other sarcoidosis.
      ].

      1.2.2 CD8+ T-cells

      Examination of CD8+ cytotoxic T-cells isolated from peripheral blood and BALF revealed increased frequencies of circulating CD8+ cytotoxic T-cells expressing perforin and granzyme B and displayed higher target cell lysis in sarcoidosis patients compared to healthy controls [
      • Parasa V.R.
      • Forsslund H.
      • Enger T.
      • et al.
      Enhanced CD8(+) cytolytic T cell responses in the peripheral circulation of patients with sarcoidosis and non-Lofgren's disease.
      ]. Increased proportions and functions of CD8+ cytotoxic T-cells were more pronounced in sarcoidosis patients without Lofgren's syndrome compared to patients with the acute form of the disease suggesting a role for these cells in the development of chronic disease [
      • Parasa V.R.
      • Forsslund H.
      • Enger T.
      • et al.
      Enhanced CD8(+) cytolytic T cell responses in the peripheral circulation of patients with sarcoidosis and non-Lofgren's disease.
      ]. This is in line with a previous study that found higher expression of Ki-67, a marker associated with cell proliferation, in CD8+ T-cells compared to CD4+ T-cells [
      • Kaiser Y.
      • Lakshmikanth T.
      • Chen Y.
      • et al.
      Mass cytometry identifies distinct lung CD4(+) T cell patterns in lofgren's syndrome and non-lofgren's syndrome sarcoidosis.
      ].

      1.2.3 B-cells

      Perturbations in the B-cell compartment are also evident in patients with sarcoidosis. Frequencies of circulating naïve and transitional B-cells have been found to be increased whereas proportions of memory B-cells were reduced and plasmablast percentages were similar in sarcoidosis patients compared to healthy controls [
      • Ly N.T.M.
      • Ueda-Hayakawa I.
      • Nguyen C.T.H.
      • Okamoto H.
      Exploring the imbalance of circulating follicular helper CD4(+) T cells in sarcoidosis patients.
      ,
      • Kudryavtsev I.
      • Serebriakova M.
      • Starshinova A.
      • et al.
      Imbalance in B cell and T Follicular helper cell subsets in pulmonary sarcoidosis.
      ].
      Immunoglobulin levels have been shown to be elevated in patients with extra-pulmonary involvement and in patients without Lofgren's syndrome but had limited usefulness in identifying patients who required corticosteroid treatment or the occurrence of relapse in treated patients [
      • Belhomme N.
      • Jouneau S.
      • Bouzille G.
      • et al.
      Role of serum immunoglobulins for predicting sarcoidosis outcome: a cohort study.
      ]. Levels of antibodies to vimentin, a protein of the intermediate filament family found in mesenchymal cells, were found to correlate with clonal expansion of T-cells expressing TCR Vα2.3/Vβ in the BALF and were higher in patients with the HLA-DRB1*03 allele compared to patients not carrying HLA-DRB1*03 [
      • Kinloch A.J.
      • Kaiser Y.
      • Wolfgeher D.
      • et al.
      In situ humoral immunity to vimentin in HLA-drb1*03(+) patients with pulmonary sarcoidosis.
      ].
      Serum levels of B-cell activating factor (BAFF) have been found to be higher in sarcoidosis patients compared to healthy controls [
      • Ly N.T.M.
      • Ueda-Hayakawa I.
      • Nguyen C.T.H.
      • Okamoto H.
      Exploring the imbalance of circulating follicular helper CD4(+) T cells in sarcoidosis patients.
      ,
      • Ando M.
      • Goto A.
      • Takeno Y.
      • et al.
      Significant elevation of the levels of B-cell activating factor (BAFF) in patients with sarcoidosis.
      ], and BAFF levels in serum and BALF correlated with disease activity (e.g. ACE, lysozyme, sIL-2R) and severity (e.g. number of organs affected, pulmonary function tests) [
      • Ando M.
      • Goto A.
      • Takeno Y.
      • et al.
      Significant elevation of the levels of B-cell activating factor (BAFF) in patients with sarcoidosis.
      ].

      1.3 Causative agents of sarcoidosis

      The precise aetiology of sarcoidosis remains unknown but involves an immune-mediated response against a putative antigen in genetically predisposed individuals leading to the formation of non-caseating granulomas in the affected organ. Previous studies have suggested an association between the presence of specific pathogens and sarcoidosis. As both tuberculosis and sarcoidosis are granulomatous diseases that share similar clinical and radiological features, T-cell immune responses to mycobacterial antigens and genomic or protein material from mycobacteria have been identified in sarcoid granulomas [
      • Terziroli Beretta-Piccoli B.
      • Mainetti C.
      • Peeters M.A.
      • Laffitte E.
      Cutaneous granulomatosis: a comprehensive review.
      ]. The relationship between tuberculosis and sarcoidosis was recently demonstrated in a large nationwide cohort study of 31,221 tuberculosis patients and 62,442 non-tuberculosis patients [
      • Wang S.H.
      • Chung C.H.
      • Huang T.W.
      • et al.
      Bidirectional association between tuberculosis and sarcoidosis.
      ]. The investigators reported an 8.08-fold higher risk of developing sarcoidosis in patients with tuberculosis compared to the general population [
      • Wang S.H.
      • Chung C.H.
      • Huang T.W.
      • et al.
      Bidirectional association between tuberculosis and sarcoidosis.
      ]. Furthermore, Mycobacterium tuberculosis and non-tuberculous mycobacterial DNA have been identified in 67% of cutaneous lesions from sarcoidosis patients [
      • Lee H.
      • Eom M.
      • Kim S.H.
      • Wang H.Y.
      • Lee H.
      • Choi E.H.
      Identification of Mycobacterium tuberculosis and non-tuberculous mycobacteria from cutaneous sarcoidosis lesions by reverse blot hybridization assay.
      ].
      Another common pathogen detected in sarcoidosis patients is Propionibacterium acnes. P. acnes-derived insoluble immune complexes were frequently detected in sinus macrophages in sarcoid lymph nodes compared to non-sarcoid lymph nodes [
      • Suzuki Y.
      • Uchida K.
      • Takemura T.
      • et al.
      Propionibacterium acnes-derived insoluble immune complexes in sinus macrophages of lymph nodes affected by sarcoidosis.
      ]. In a recent study using a novel P. acnes-specific antibody (PAC), Yamamoto et al. reported higher Propionibacterium catalase expression in cultured isolates from sarcoid compared to isolates from non-sarcoid lymph nodes and this was primarily detected in sinus macrophages [
      • Yamamoto K.
      • Uchida K.
      • Furukawa A.
      • et al.
      Catalase expression of Propionibacterium acnes may contribute to intracellular persistence of the bacterium in sinus macrophages of lymph nodes affected by sarcoidosis.
      ]. Bacterial catalase is essential for intracellular survival and persistence of the bacteria in host cells during latent infection. It has also been implicated in the formation of granulomas and promoting Th1 immune responses in sarcoidosis patients [
      • Yorozu P.
      • Furukawa A.
      • Uchida K.
      • et al.
      Propionibacterium acnes catalase induces increased Th1 immune response in sarcoidosis patients.
      ].
      These recent findings are consistent with a large body of evidence showing microbial involvement in triggering inflammation and granuloma formation [
      • Clarke E.L.
      • Lauder A.P.
      • Hofstaedter C.E.
      • et al.
      Microbial lineages in sarcoidosis. A metagenomic analysis tailored for low-microbial content samples.
      ]. However, not all granulomas and not every sarcoidosis patients showed the presence of microbial DNA or protein indicating that other factors may contribute to granuloma development [
      • Inaoka P.T.
      • Shono M.
      • Kamada M.
      • Espinoza J.L.
      Host-microbe interactions in the pathogenesis and clinical course of sarcoidosis.
      ]. Furthermore, failure to establish an association between infectious agents and sarcoidosis relied on methods such as histological stains and microbial cultures which have not been as successful compared to the more sensitive polymerase chain reaction and sequencing in identifying and detecting the presence of microbes in sarcoid granulomas [
      • Grunewald J.
      • Grutters J.C.
      • Arkema E.V.
      • Saketkoo L.A.
      • Moller D.R.
      • Muller-Quernheim J.
      Sarcoidosis.
      ]. Additionally, biopsy specimens collected from different organs, stages of sarcoidosis and differences in the ethnic populations studied could also explain varying results in previous studies. For example, P. acnes was more commonly detected in the Japanese population whereas Mycobacterium species were more frequently found in patients from the United States and Europe [
      • Esteves T.
      • Aparicio G.
      • Garcia-Patos V.
      Is there any association between Sarcoidosis and infectious agents?: a systematic review and meta-analysis.
      ]. Advancements in microbial genomics and proteomics-based methods will help confirm the role of M. tuberculosis and P. acnes as etiologic agents in sarcoidosis and also elucidate other potential infectious agents.

      1.4 Evidence of an autoimmune response in sarcoidosis

      Formation of immune complexes in response to antigens derived from healthy lung tissue extract in sarcoidosis patients but not in tuberculosis patients suggests an autoimmune component to the disease [
      • Starshinova A.
      • Zinchenko Y.
      • Filatov M.
      • et al.
      Specific features of immune complexes in patients with sarcoidosis and pulmonary tuberculosis.
      ]. Moreover, vimentin, which have been shown to induce T- and B-cell responses in sarcoidosis patients, have been shown to be involved in the pathogenesis of several autoimmune diseases [
      • Musaelyan A.
      • Lapin S.
      • Nazarov V.
      • et al.
      Vimentin as antigenic target in autoimmunity: a comprehensive review.
      ].

      1.5 Understanding immunopathogenesis of sarcoidosis using animal and in vitro human models

      A major limitation in identifying the precise pathogenesis of sarcoidosis is the current lack of an appropriate pre-clinical animal model for this disease. Recent advances have been made towards establishing models to fill in the knowledge gaps (Table 3).
      Table 3Recent animal and in vitro human models established to understand the immunopathogenesis of sarcoidosis.
      CauseFindingsRef
      Propionibacterium acnesMiceIncreased Th17 cells and decreased Treg cells in blood; Higher IL-17/IL-23 and lower IL-10/TGFβ production in BALF; inhibition of IL-17 reduced inflammation and granuloma development68
      Mycobacterial superoxide dismutase AMicePolarised Th1 profile; reduced frequencies and impaired function of Treg cells; activation of the PI3K/Akt signalling pathways in the lungs70
      Multiwall carbon nanotubeMiceActivation of T-cell signalling, IL-12/IL-17/IFNγ signalling, apoptosis and oxidative phosphorylation in BALF71
      Mycobacterium tuberculosis trehalose 6,6′-dimycolateMiceIncreased expression of IL-1β within granulomas; inhibition of NLRP3 or IL-1β antibodies reduced the formation of pulmonary granulomas14
      Purified protein derivative-coated polystyrene beadsPBMC from sarcoidosis patientsActivation of IL-13 gene pathways72
      Abbreviations: BALF, bronchalveolar lavage fluid; LN, lymph nodes; PBMC, peripheral blood mononuclear cells; NLRP3, NOD-like receptor family pyrin domain containing 3; Th1, T helper 1; Th17, T helper 17; Treg, regulatory T-cells; TGFβ, transforming growth factor β
      Using multiple low dose inoculations of Propionibacterium acnes, Song et al. established a mouse model of sarcoidosis-like granulomatosis that exhibited similar histopathological and immunological characteristics of human sarcoidosis [
      • Song J.
      • Zhao M.
      • Li Q.
      • et al.
      IL-17A can promote Propionibacterium acnes-induced sarcoidosis-like granulomatosis in mice.
      ]. The authors showed that percentages of Th17 cells were higher whereas Treg cells were lower in the blood from mice inoculated with Propionibacterium acnes compared to saline-treated control animals [
      • Song J.
      • Zhao M.
      • Li Q.
      • et al.
      IL-17A can promote Propionibacterium acnes-induced sarcoidosis-like granulomatosis in mice.
      ]. Increased levels of cytokines produced by Th17 cells (IL-17 and IL-23) and reduced levels of cytokines produced by Treg cells (IL-10 and TGFβ) were also evident in the BALF from mice inoculated with Propionibacterium acnes [
      • Song J.
      • Zhao M.
      • Li Q.
      • et al.
      IL-17A can promote Propionibacterium acnes-induced sarcoidosis-like granulomatosis in mice.
      ]. Furthermore, IL-17 knockout mice or inhibition of IL-17 using neutralising antibodies reduced inflammation and granuloma development [
      • Song J.
      • Zhao M.
      • Li Q.
      • et al.
      IL-17A can promote Propionibacterium acnes-induced sarcoidosis-like granulomatosis in mice.
      ].
      In another murine model using mycobacterial superoxide dismutase A to induce pulmonary sarcoidosis, polarised Th1 profile, reduced frequencies and impaired function of Treg cells and activation of the PI3K/Akt signalling pathways have been demonstrated in the lungs [
      • Zhang B.
      • Dai Q.
      • Jin X.
      • et al.
      Phosphoinositide 3-kinase/protein kinase B inhibition restores regulatory T cell's function in pulmonary sarcoidosis.
      ]. PI3K signalling is known to be crucial for maintaining Treg cell homeostasis and function. Using PI3K inhibitors, the investigators demonstrated reduced PI3K/Akt signalling, restoration of Treg cell percentages and function and reduced production of inflammatory cytokines [
      • Zhang B.
      • Dai Q.
      • Jin X.
      • et al.
      Phosphoinositide 3-kinase/protein kinase B inhibition restores regulatory T cell's function in pulmonary sarcoidosis.
      ]. Similarly, using the same experimental model, administration of IL-33 suppressed PI3K/Akt signalling and restored Treg cell homoeostasis and suppressive function [
      • Zhang B.
      • Zhao F.
      • Mao H.
      • et al.
      Interleukin 33 ameliorates disturbance of regulatory T cells in pulmonary sarcoidosis.
      ]. These pertinent studies provide alternative treatment strategies for sarcoidosis and warrants further investigation.
      Mohan et al. established a multiwall carbon nanotube-based murine model of granulomatous inflammation that displays similar histological and inflammatory responses to human sarcoidosis [
      • Mohan A.
      • Malur A.
      • McPeek M.
      • et al.
      Transcriptional survey of alveolar macrophages in a murine model of chronic granulomatous inflammation reveals common themes with human sarcoidosis.
      ]. The authors identified several common pathways that were activated including T-cell signalling, IL-12/IL-17/IFNγ signalling, apoptosis and oxidative phosphorylation in BALF samples [
      • Mohan A.
      • Malur A.
      • McPeek M.
      • et al.
      Transcriptional survey of alveolar macrophages in a murine model of chronic granulomatous inflammation reveals common themes with human sarcoidosis.
      ].
      In a murine pulmonary granuloma model induced by trehalose 6,6′-dimycolate (TDM) (a glycoplipid found in the cell wall of Mycobacterium tuberculosis), increased expression of IL-1β was observed within granulomas and in areas that also stained positive for macrophages [
      • Huppertz C.
      • Jager B.
      • Wieczorek G.
      • et al.
      The NLRP3 inflammasome pathway is activated in sarcoidosis and involved in granuloma formation.
      ]. Administration of the NLRP3 inflammasome pathway inhibitor MCC950 or anti-IL-1β antibodies to the TDM-treated mice reduced the formation of pulmonary granulomas [
      • Huppertz C.
      • Jager B.
      • Wieczorek G.
      • et al.
      The NLRP3 inflammasome pathway is activated in sarcoidosis and involved in granuloma formation.
      ]. Furthermore, the authors demonstrated the important role of miR-223 in regulating NLRP3 inflammasome activation, as formation of granulomas were increased in miR-223-deficient mice treated with TDM [
      • Huppertz C.
      • Jager B.
      • Wieczorek G.
      • et al.
      The NLRP3 inflammasome pathway is activated in sarcoidosis and involved in granuloma formation.
      ].
      Locke at al developed an in vitro model of sarcoidosis that involved the formation of granuloma-like multicellular aggregates following culture of peripheral blood mononuclear cells from sarcoidosis patients with purified protein derivative-coated polystyrene beads [
      • Locke L.W.
      • Crouser E.D.
      • White P.
      • et al.
      IL-13-regulated macrophage polarization during granuloma formation in an in vitro human sarcoidosis model.
      ]. Using an unbiased systems biology approach, the authors implicated a role for IL-13-regulated gene pathways and showed that these pathways were present in human sarcoidosis lung and mediastinal lymph node tissues [
      • Locke L.W.
      • Crouser E.D.
      • White P.
      • et al.
      IL-13-regulated macrophage polarization during granuloma formation in an in vitro human sarcoidosis model.
      ]. As previously mentioned, IL-13 is known to be involved in the induction of M2 macrophages. Indeed, M2 polarisation has been demonstrated within the sarcoid granuloma-like structures [
      • Locke L.W.
      • Crouser E.D.
      • White P.
      • et al.
      IL-13-regulated macrophage polarization during granuloma formation in an in vitro human sarcoidosis model.
      ].
      One of the significant drawbacks for many of the current animal models mentioned above is that they recapitulate key events in the immunopathogenesis of acute sarcoid granulomas. Future studies are essential to establish models that not only mimic multi-organ involvement but also certain attributes of chronic sarcoid granulomas including the development and accumulation of fibrotic tissues. Furthermore, the animal models described place a heavy emphasis on histological features of granuloma, but it is unknown whether sarcoidosis triggered by microbes or microbe-related agents can induce the same clinical manifestations evident in humans.

      1.6 Treatment of sarcoidosis

      Glucocorticoids remain as the first line of therapy for acute and chronic pulmonary sarcoidosis [
      • Culver D.A.
      • Judson M.A.
      New advances in the management of pulmonary sarcoidosis.
      ]. Glucocorticoids can act on various immune cells in the peripheral circulation. These include promoting an anti-inflammatory state on monocytes and macrophages and inhibiting neutrophil and T-cell activation by suppressing the production of pro-inflammmatory cytokines [
      • Cain D.W.
      • Cidlowski J.A.
      Immune regulation by glucocorticoids.
      ]. Using RNA-sequencing, Talreja et al. demonstrated genes involved in glycolysis, oxidative phosphorylation and lipid metabolisms were affected in alveolar macrophages whereas genes involved in cytokine-cytokine receptor interaction were affected in CD14+ monocytes isolated from sarcoidosis patients in response to ex vivo treatment with dexamethasome [
      • Talreja J.
      • Bauerfeld C.
      • Sendler E.
      • Pique-Regi R.
      • Luca F.
      • Samavati L.
      Derangement of metabolic and lysosomal gene profiles in response to dexamethasone treatment in sarcoidosis.
      ].
      Biologic agents are considered as the third line of treatment, particularly in patients who are refractory to conventional treatment. These include the TNF-α inhibitors, infliximab and adalimumab, and the chimeric monoclonal antibody against CD20, rituximab [
      • Culver D.A.
      • Judson M.A.
      New advances in the management of pulmonary sarcoidosis.
      ]. Kullberg et al. recently reported decreased CD4/CD8 ratio, reduced frequency of CD4+ T-cells expressing the activation marker CD69 and lower percentages of Tregs in BALF from patients following 6 months of infliximab therapy [
      • Kullberg S.
      • Rivera N.V.
      • Abo Al Hayja M.
      • Grunewald J.
      • Eklund A.
      Changes in lung immune cells related to clinical outcome during treatment with infliximab for sarcoidosis.
      ].
      A Phase II clinical trial is currently underway to test the safety and efficacy of abatacept, a CTLA-4–Ig fusion protein, in patients with chronic, steroid-refractory sarcoidosis [
      • Frye B.C.
      • Rump I.C.
      • Uhlmann A.
      • et al.
      Safety and efficacy of abatacept in patients with treatment-resistant SARCoidosis (ABASARC) - protocol for a multi-center, single-arm phase IIa trial.
      ]. In addition to clinical data, the trial will also examine change in immunological profiles in blood and BAL samples following abatacept therapy [
      • Frye B.C.
      • Rump I.C.
      • Uhlmann A.
      • et al.
      Safety and efficacy of abatacept in patients with treatment-resistant SARCoidosis (ABASARC) - protocol for a multi-center, single-arm phase IIa trial.
      ]. Abatacept has previously been used for treatment of rheumatoid and psoriatic arthritis, both diseases characterised by excessive Th1 immune responses [
      • Simon T.A.
      • Soule B.P.
      • Hochberg M.
      • et al.
      Safety of abatacept versus placebo in rheumatoid arthritis: integrated data analysis of nine clinical trials.
      ,
      • Lubrano E.
      • Scriffignano S.
      • Perrotta F.M.
      Abatacept for the treatment of psoriatic arthritis.
      ].
      Given the importance of Th17 cells in disease progression, targeting key transcription factors (e.g. RORγt) and cytokines (e.g. IL-23) involved in Th17 differentiation represent novel therapeutic targets for sarcoidosis. The RORγt inhibitor, VTP-43742, is currently in Phase II clinical trial for the treatment of psoriasis [
      • Sun N.
      • Guo H.
      • Wang Y.
      Retinoic acid receptor-related orphan receptor gamma-t (RORgammat) inhibitors in clinical development for the treatment of autoimmune diseases: a patent review (2016-present).
      ] and the IL-23 inhibitor, ustekinumab, has shown reduced disease activity in patients with psoriatic arthritis [
      • Rahman P.
      • Arendse R.
      • Khraishi M.
      • et al.
      Long-term effectiveness and safety of infliximab, golimumab and ustekinumab in patients with psoriatic arthritis from a Canadian prospective observational registry.
      ]. However, two recent case studies reported the development of sarcoidosis in patients with psoriasis following treatment with ustekinumab [
      • Kobak S.
      • Semiz H.
      Ustekinumab-induced sarcoidosis in a patient with psoriatic arthritis.
      ,
      • Gad M.M.
      • Bazarbashi N.
      • Kaur M.
      • Gupta A.
      Sarcoid- like Phenomenon - ustekinumab induced granulomatous reaction mimicking diffuse metastatic disease: a case report and review of the literature.
      ]. Therefore, further clinical research on the safety and effects of these biologic agents in modulating local and systemic immune functions are warranted.

      2. Conclusions

      The pathogenesis of sarcoidosis is poorly understood but involves cells from both innate and adaptive immune responses. Evidence from recent studies has revealed changes in frequencies and phenotypes of immune cell populations and also levels of soluble immune mediators in the peripheral blood and tissues from sarcoidosis patients that correlated with disease burden, stage and progression (Fig. 1). Longitudinal studies are warranted to determine whether evaluation of circulating immune cell subsets is a clinically useful tools for monitoring disease progression. Additionally, it is pertinent that future studies focus on identifying the antigen(s) responsible for triggering the inflammatory immune response so that early treatment can be initiated to prevent early stages of tissue fibrosis.
      Fig. 1
      Fig. 1Immunopathogenesis of granuloma formation in sarcoidosis. Recent studies support an autoimmune component and the possible role of microbial organisms as causative agents in sarcoidosis. Development and progression of sarcoid granulomas involves both innate and adaptive immune responses. Antigens are presented by antigen-presenting cells such as macrophages and dendritic cells (DC) to T-cells with a T-cell receptor (TCR) capable of antigen recognition. Cytokines, chemokines and other soluble mediators (e.g. NGAL and antibodies) are produced by innate and adaptive immune cells. The type of immune response generated is a key determinant in disease outcome: resolution or persistence. An effective immune response mounted against the antigen results in remission or resolution of sarcoidosis. In the absence of an efficient immune response, antigen persists and fibrosis can develop in chronic disease. Refer to Table 1, Table 2 for abbreviations.

      Declaration of competing interest

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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