Immune Response during Saccharopolyspora rectivirgula Induced Farmer’s Lung Disease

Abstract

Repeated exposures to Saccharopolyspora rectivirgula in some individuals can lead to a hypersensitivity reaction where a pro-inflammatory feedback loop can occur in the interstitial space in the alveoli of the lungs that can ultimately lead to granuloma formation and fibrosis, referred to as Hypersensitivity pneumonitis or Farmer’s Lung Disease. The pathogenesis of FLD is complex and incompletely understood. S. rectivirgula induces an immune response, triggering neutrophil influx into the lung followed by lymphocyte influx of CD8+ and CD4+ T cells. The cytokine IL17A has been shown to be critical for the development of S. rectivirgula induced Hypersensitivity pneumonitis. This chapter will review the immune response leading to the development of S. rectivirgula induced Hypersensitivity pneumonitis.

1. Introduction

Bacteria were one of the very first organisms to inhabit the Earth three billion years ago [1, 2]. Using today’s current technological approaches to determine the total mass of all the organisms on Earth, would reveal that bacteria are second most dominant organism on the planet [3]. Bacteria are important for human health, providing nutrients, protecting against developing diseases, aiding in food production, energy storage, helping to train the immune system and have the potential to capture carbon dioxide to reduce greenhouse gas emissions [4]. One of the most important discoveries of bacteria is that they can make antibiotics to treat bacterial diseases. The discovery of novel bacteria was prominent throughout the 1920’s until the 1970’s where the antibiotic resistant diseases were starting to take a foothold in the world [5, 6]. The discovery of new bacteria that have the potential to alleviate antimicrobial resistance is still an issue today. The phylum Actinobacteria are among the largest groups of bacteria and are one the major sources of antibiotics [7]. Actinobacteria are found in a wide variety of environments such as soils, deep oceans, plants, caves, and stones, among others [7, 8]. Rare actinobacteria such as Saccharopolyspora have the potential to be tapped for novel therapeutics [9]. However, some Saccharopolyspora species are also the cause of disease in humans [10].

1.1 History of Saccharopolyspora and its isolation

The genus Saccharopolyspora was first described by Lacey and Goodfellow in 1975 and later revised by Korn-Wendisch in 1989 [11, 12]. In 1971, Lacey was researching bacteria that may be associated with causing Bagassosis, a respiratory disease similar to farmer’s lung induced by the inhalation of dust from bagasse or sugar cane that had been crushed, liquid removed, dried, and baled for further processing [13, 14, 15]. The storage of bagasse at high humidity and high temperature encouraged the growth of mold and bacteria [15]. They suspected that thermophilic Actinomycetes may be the culprit due other thermophilic Actinomycetes involvement in farmer’s lung or mushroom worker’s lung [13, 16]. Furthermore, previous work had shown that serum from patients that were diagnosed with Bagassosis reacted against extracts of thermophilic Actinomycetes [15]. Lacey was able to isolate Thermoactinomyces vulgaris from moldy baggase, along with an unknown bacterium similar to other thermophilic Actinomycetes, but that was different enough from T. vulgaris to be considered a new species. Lacey named it Thermoactinomyces sacchari [13]. Three years later Lacey and Goodfellow reported on their observation of isolates of T. sacchari when grown at 40°C also had growth of an unidentified actinomycete, which was different from other taxa suggesting this was a new species. Lacey and Goodfellow called the unknown isolate Saccharopolyspora hirsute gen. et. sp. nov [11]. What followed Lacey and Goodfellow’s discovery was a reclassification of strains to other genera by the scientific community. In 1989 Korn-Wendisch completed a detailed taxonomic study comparing the genera Faenia and Saccharopolyspora finding that Faenia rectivirgula should be transferred to the genus Saccharopolyspora with its nomenclature now being S. rectivirgula [12].

S. rectivirgula is an aerobic, non-motile aerobic gram-positive bacteria part of the class Actinomycetes [9]. It grows up to 5 mm in diameter at temperature ranges of 50–55°C [17], in a variety of environments such as soil, plants and moldy hay [9, 18]. S. rectivirgula is one of the causative agents of a type of extrinsic allergic alveolitis, farmer’s lung disease. This review will focus on the immune response to S. rectivirgula induced hypersensitivity pneumonitis (HP) better or farmer’s lung disease [19, 20].

1.2 Hypersensitivity pneumonitis (HP) or farmer’s lung disease (FLD)

HP was first described by Bernardino Ramazzini da Capri in the early 1700s when he encountered workers that sifted grain developed a dry cough coupled with edema and weight loss [21]. He suggested the workers developed the cough due to the humid environments inside the wheat dust [21]. In the early 1930s farmer’s lung disease was first described by John Campbell after seeing five patients whom had pulmonary difficulties due to their working conditions [22]. All five patients were farmer laborers working with hay that was improperly stored to keep out moisture. Dr. Campbell obtained samples of “dust” from the hay and discovered fungi present. However, he was unable to discover the origin of disease, and found no correlation with the presence of fungi and his patients’ sputum samples. Today, we know HP as a collection of lung diseases that can occur in an occupational, recreational or home settings. HP is caused by the repeated exposure to organic or inorganic agents such as avian proteins, chemicals, molds, and bacteria, among others where the subset of HP is categorized by the offending agent [19, 20, 23]. Individuals are asymptomatic or symptomatic leading to a hypersensitive reaction. Historically, HP has been characterized into three stages, acute, sub-acute and chronic but the field seems to be moving towards categorizing HP in a sensitization phase and challenge phase [24]. The onset of symptoms are fever, cough, wheezing, chills and if left untreated patients can experience dyspnea, fatigue, weight loss and fibrosis [23]. Some individuals develop poorly formed granulomas that advance to pulmonary fibrosis [25, 26, 27, 28], which can lead to irreversible dysfunction with an increase in mortality [29], and some patients may even develop emphysema [30, 31]. Early diagnosis and treatment of HP and farmer’s lung disease may prevent long term effects. However, HP is difficult to diagnose due to patients’ symptoms that have similar pathologies to other respiratory diseases such as asthma or idiopathic pulmonary fibrosis coupled with a lack of a standardized diagnostic testing [23, 26, 32].

Diagnosis of patients with HP including farmer’s lung disease is a multi-factorial process. A clinical history is taken to determine the patients’ symptoms, descriptions of the environment they interact with such as work and home [33]. Samples from the patients’ environment may be undertaken to undergo microbiological identification. When an offending agent is unknown, in some cases diagnosis would include provoking a physiological response from the patient by conducting an inhalation challenge with various potential triggers to try to determine the nature of the offending agent [19, 33, 34, 35, 36]. Serum antibody tests are used to determine if the patient has been sensitized to the offending agent. IgG has been shown to be associated with farmer’s lung patients that develop symptoms or those that asymptomatic [19, 33]. A standardized enzyme-linked immunosorbent assay (ELISA) test has yet to be developed and adapted in the clinical setting or able to differentiate between patients who have been exposed from those that have actual manifestation of the disease [23, 37]. Sputum or serum samples may be collected to determine lymphocytosis or alveolitis in bronchial lavage fluid (BAL) [33]. High resolution CT of the chest or bronchoscopy may be used to differentiate FLD from other diseases that have similar clinical features [19, 33]. Lung biopsy is reserved for patients where there has been no identification of the specific antigen exposure [37].

Treatment options are limited for all subsets of HP. The best treatment for patients is to avoid the offending antigen or utilize personal protective equipment. Determining the offending agent can be challenging and avoiding it may not be feasible for resolution for all [23, 38]. Personal protective equipment is successful in reducing inhalation of antigens but might be financially challenging or have low compliance rates [38]. Even in the cases of FLD, improvement of hay packing techniques meant to reduce bacterial or fungi growth do not always work [39, 40]. Furthermore, in some cases when avoidance of the offending agent is successful some patients may still have a decline in lung function [23, 35]. Corticosteroids may be prescribed for certain patient demographics as they reduce the immune response but is not beneficial for long term usage [41].

Farmer’s lung affects approximately up to 20 percent of individuals that typically work in agricultural settings (i.e. dairy farms) where environmental and genetic factors influence the susceptibility of workers, and even in some cases that of their families [23, 27, 35, 42, 43, 44]. However, not all exposed individuals develop FLD suggesting a genetic component that lead to developing symptoms [42]. The environmental factors and genetic factors that contribute to the development of FLD remains incompletely understood.

1.3 Environmental impact of FLD

Secondary infections, pesticides, air quality, among others have all been attributed to increased susceptibility to developing HP including FLD [45, 46, 47]. Interestingly, smokers have a decreased risk of developing HP but non-smokers have a lower risk of developing emphysema [30, 48]. The reduction of risk with smokers developing HP has been attributed to nicotine’s ability to suppress immune responses [48]. There are a variety of antigens that can lead to the development of HP. In some cases, patients are exposed to a mixture of antigens [49, 50, 51]. However, few studies evaluate the immune response when there is a mixture of antigens involved in disease pathogenesis, but there have been some studies looking at co-infection with viruses [46, 51, 52, 53]. For example, in mouse models, Cormier et al., found that single round of infection with Sendai virus following S. rectivirgula exposure enhances the immune response to S. rectivirgula, with increased BAL cell number, TNF-α and IL-1α compared control and S. rectivirgula only exposed mice [47]. Histological analysis of lungs also showed clear formation of granulomas in S. rectivirgula mice inoculated with Sendai virus. Thus, viral or secondary infections may contribute to the triggering of an immune response to S. rectivirgula.

Our environmental exposure during upbringing impacts our risk of developing allergic diseases whereby increased microbial exposure in early life can reduce the risk of developing allergies or allergic disease in adulthood [54, 55, 56]. The microbiome can modify host-immune responses and dysbiosis of the microbiome can lead to disease [2, 57]. Previous research has shown children that grow up in an agricultural setting have a reduction in developing allergic diseases. Interestingly children have been diagnosed with HP albeit there a very few studies that examine children with HP [58]. Thus, environmental upbringing may not fully explain risk factors to developing FLD. A genetic component may play a role in those with healthy microbiomes that leads to their susceptibility of disease. The microbiome has also been suggested to be able to affect the development of HP including farmer’s lung disease. Russell et al. investigated this effect on the development of FLD using antibiotic-mediated microbial shifts [59]. In experiments where mice were treated prenatally or perinatally with vancomycin or streptomycin and exposed to S. rectivirgula antigen for 3 days per week for 3 weeks, they found that streptomycin treated mice had increased IFN-γ and IL-17A production, increased lymphocytes in the BAL and more severe lung pathology compared to untreated or vancomycin treated mice. However, the vancomycin treated group had the biggest change microbial shift in the gut at the family level, with reduction of Bacteroidetes, but streptomycin treated mice had an increase in Bacteroidetes. Taken together antibiotic treatments that shifts the microbiome can result in increased severity of FLD. Increasing the diversity or specific group of bacteria may result in less severity of FLD but further testing needs to be conducted.

1.4 Genetic contribution to FLD

HP including FLD is such a rare disease the studies that genetic analysis of susceptibility to disease are few and far in between. In addition, the majority of studies that have evaluated genetic susceptibility have small sample size in their respective patient cohorts, and do not delineate the causative agent of HP. These reasons, among others, partly explains why there is no consensus on the genetic polymorphisms that may cause individuals to be sensitive to S. rectivirgula induced HP, yet they still may offer clues to a better understanding of specific intricacies FLD. Studies that were conducted hypothesize that polymorphisms in tumor necrosis factor alpha (TNF-α), major histocompatibility complex (MHC)/human leukocyte antigen (HLA) and transporter for antigen presentation (TAP), or pulmonary surfactant may play a role in sensitivities to S. rectivirgula [60, 61].

Polymorphisms in the TNF-α gene have been linked to elevated inflammation, and single nucleotide polymorphisms (SNPs) at −308 position in the promoter region of the TNF gene has been associated with an increased risk to inflammatory diseases. Similarly to TNF-308A, TNF-238G has also been shown to be associated with increased production of plasma TNF-α in systemic lupus erythematous patients [62]. Furthermore, TNF-308A has been shown to be associated with high levels of TNF-α in vitro investigated in connection with susceptibility to developing FLD [61]. Patients with farmer’s lung disease that had been exposed to moldy hay and developed elevated responses had a higher frequency of both TNFA2 homozygous or heterozygous alleles compared to healthy controls. There were no significant differences in the allele frequencies of TNF-β intron 1 gene polymorphisms between the healthy controls compared to patients with HP. These studies suggest patients that develop HP including farmer’s lung may have a genetic predisposition to elevated TNF-α production.

MHC class I (HLA-DR) and II (HLA-DQ) play a critical role in the adaptive immune response by presenting epitopes from foreign or self-antigens to CD8⁺ or CD4⁺ T cells respectively [63]. MHC class I utilizes ATP-binding cassette proteins, transporters associated with antigen processing-1 and 2 (TAP1 and TAP2) to aide in the process of antigen presentation [57]. The genetic regions that encode for MHC are polygenic and incredibly polymorphic, allowing for the recognition of many different epitopes; polymorphism that occur in these gene regions also serve as risk factors in susceptibility to disease [36, 57]. Both TAP1 and TAP2 genes also map within the MHC class II region [64]. Polymorphisms in HLA-DR and DQ have been associated with susceptibility to developing HP, however it is unclear if this association extends to those who develop farmer’s lung [36]. Patients that have HP have been reported to have a significant increase in the frequency of the TAP1 genotypes Asp-637/Gly-637 and Pro-661/Pro-661 among patient cohorts that had bird fancier’s lung compared to healthy controls [64]. Familial studies of HP investigated polymorphisms in MHC class II genes HLA -DRB1/2/3/4/5, -DQA1, -DQB1, -DPA1, -DPB1, -DMA, and -DMB in healthy and HP patients with one patient having been diagnosed with FLD, have found that DRB1*04 alleles and haplotypes could be contributing the susceptibility to HP [65]. This suggests MHC and TAP might have a role in patient susceptibility to disease. However, we do not know if these polymorphisms are only prevalent for those whom may be afflicted with bird fancier’s lung compared to FLD.

Phospholipids and glycoproteins play a large role in maintaining healthy lung function. Dysfunction of these material can increase risk of developing pulmonary diseases. Surfactant is a complex material composed of phospholipids and proteins that reduce the surface tension in the alveoli and bronchiole of fully developed lungs secreted by type II alveolar cells. Pulmonary surfactants, SP-A, SP-B, SP-C and SP-D are critical for normal lung function by maintaining the integrity of alveoli [32, 66]. Surfactants can regulate phagocytosis in alveolar macrophages or bind to pathogens and allergens [67, 68]. The decrease or absence of surfactant that may occur during inflammatory conditions can lead to respiratory failure [32]. There is variability in the levels of surfactant reported in HP [69, 70, 71, 72]. One study found elevated levels of SP-A in patients with farmer’s lung [71]. Surfactants from HP patients have a reduce capacity to inhibit the proliferation of PBMCs [73]. The surfactant protein C gene has been associated with familial interstitial lung diseases.

2. Immune response to S. rectivirgula during development of FLD

The immune response to S. rectivirgula induced FLD is complex and remains incompletely understood. It is still unclear which innate immune cells trigger the inflammatory process in FLD. It is generally accepted that upon inhalation of an offending agent leads to its deposition in the interstitial spaces of the lung in the alveoli where alveolar macrophage most likely recognizes S. rectivirgula, leading to cytokine production to recruit neutrophils or induce the differentiation of T_H1, T_H2, or T_H17 cells (Figure 1) [20, 57]. It has been reported that the cytokines: IFN-γ, TNF-α, IL-1β, IL-6, IL-8, IL-10, IL-12, IL-13, IL-17A are all implicated in the disease [23, 28, 74, 75]. However, the cellular sources of these cytokines are still ambiguous [76], and there are conflicting reports as to which cells and cytokines are important for disease pathogenesis. Neutrophils, T_H2, T_H1, T_H17 and Tregs along with the cytokines IFN-γ, TNF-α, IL-10, or IL-17A have all been considered critical for disease progression [74, 77]. In addition, B cells develop into plasma cells that can secrete IgG to form antigen-antibody complexes with antigens from S. rectivirgula [77]. The varying experimental approaches for inducing FLD, such as mouse strains, the amount and route of S. rectivirgula exposure may account for discrepancies. Understanding the immune response to FLD will reveal mechanisms that enable better treatments for this disease as well as other pulmonary allergic diseases.

Figure 1.

Overview of FLD pathogenesis. Saccharopolyspora rectivirgula is inhaled in large quantities and deposited in the interstitial spaces of the lung. Alveolar macrophages produce inflammatory mediators upon recognition of S. rectivirgula by TLRs and potentially other pattern recognition receptors, triggering an immune response by producing pro-inflammatory mediators. In response to these inflammatory signals, neutrophils are recruited and lymphocytes, such as T_H17, Tregs, or T_H1 cells differentiate and produce their respective cytokines. As a susceptible individual continually comes into contact with S. rectivirgula, a pro-inflammatory feedback loop commences which can lead to fibrosis or granuloma formation, resulting in reduced lung function. (adaptive from “SARS-CoV-2: What We Know About Its Effects on Respiration”, by BioRender.com (2021) retrieved from https://app.biorender.com/biorender-templates).

Mouse models used have been utilized to study the immune response and pathology of FLD caused by S. rectivirgula. These models exposed mice to varying doses of S. rectivirgula and for varying lengths of time [78]. There are variations in the way S. rectivirgula is prepared for subsequent exposure to mice. In some cases, S. rectivirgula is collected and lyophilized or sonicated. This is often done so as to break down the bacterial cell wall and release internal contents for exposure to the immune system in PBS [47, 53, 76, 79, 80, 81, 82, 83, 84, 85]. Intranasal, oropharyngeal aspiration or intratracheal inoculations are common techniques used to exposing mice to S. rectivirgula [59, 74, 79]. The timeline of exposure is not uniform. Exposure times vary anywhere from 3 consecutive days per week for 3 weeks up to 12 weeks [59, 74, 76, 85, 86]. Regardless of the vagaries of the model, studies have shown that inhalation of S. rectivirgula results in the recognition of pathogen associated molecular patterns found on S. rectivirgula proteins or lipoproteins toll like receptors (TLR). TLRs are transmembrane glycoproteins that can recognize pathogen associated molecular patterns important for microbial functions, and can trigger host immune responses [87]. There are 12 different TLRs in mice and 10 in humans [88]. In vitro studies suggest S. rectivirgula can interact with TLR2 [84]. An unknown TLR ligand of S. rectivirgula’s can activate TLR2 or TLR9; TLR2 is can heterodimerize with TLR1 or TLR6 and detect lipoproteins from bacteria, whereas TLR9 recognizes unmethylated CpG motifs [87, 89, 90]. Through the immune signaling adaptor, MyD88, TLR2 or TLR9 activate different downstream signaling pathways that lead to the production of a variety of cytokines and chemokines such as TNF-α, IL-6, IL-1β, or CXCL2 by macrophages or dendritic cells; the cytokine and chemokine production leads to the recruitment of neutrophils, CD4⁺ or CD8⁺ T cells that further produce inflammatory mediators that may result in inflammation in the lung [27, 57, 91, 92].

TLR2 can modulate neutrophil responses [93]. The absence of TLR2 in mice did not affect neutrophil influx in response to a single exposure to S. rectivirgula [84]. However, the absence of TLR2 led to a significant reduction in MIP-2 production suggesting another pathway may be more important for the recruitment of neutrophils. Signaling through TLR2 is dependent on MyD88, and MyD88 may be important for neutrophil recruitment into the lung during S. rectivirgula exposure. MyD88^−/− mice exhibited a reduction in neutrophils in the lungs, along with a decrease in MIP-2/CXCL2 and TNFα in the bronchoalveolar lavage fluid, that may partly explain the reduction of neutrophils. Further studies found that a single exposure of S. rectivirgula results in a significant decrease in CXCL2 mice lacking TLR2, TLR9, or both found, and upon repeated exposure to S. rectivirgula, TLR2^−/− and TLR2/9^−/− mutant mice exhibited reduced levels of lung CXCL2 and neutrophils. However, TLR2/9^−/− mice still developed granuloma as a result of exposure to S. rectivirgula. These data suggest TLR2 or TLR9 are not critical for the pathogenesis of FLD [91, 94]. The serine/threonine protein kinase (PK)D, can be activated downstream of MyD88, and has been shown to be activated by S. rectivirgula [94]. PKD1 activation leads to activation of MAPKs and NF-κB and other downstream signals that leading to the production of pro-inflammatory mediators in macrophages or dendritic cells that can recruit leukocytes [94]. When Young-In Kim et al. used a PKD inhibitor in a mouse model of S. rectivirgula induced famer’s lung disease, they found a significant reduction of neutrophils, MPO activity and that PKD inhibition also suppressed the development of alveolitis. This suggest PKD1 may be a potential target to reduce inflammation in FLD. Taken together, these data suggest that while TLR2 or TLR9 may not be critical, MyD88 dependent TLRs may play a role in the pathogenesis of FLD. Understanding which TLRs and the pathways they use that lead to disease progression could potentially lead to a therapeutic target.

TLRs are not the only way pathogenesis of FLD may occur. Antibodies such as IgG are able to tag S. rectivirgula as foreign also initiating an immune response. Patients with FLD have IgG antibodies and have been reported to have circulating immune complexes, suggesting antigen-antibody aggregates forms upon inhalation of SR [95]. In allergic diseases that provoke an IgG response, immune complexes may form when there is a large dose of inhalation of an allergen. When re-exposure of another large dose of allergen occurs, immune complexes, composed of antigen-antibody formation, may form in the walls of the alveoli, resulting in a reduction of O2 exchange due to possible fluid, protein or cells accumulation [57]. Upon exposure to S. rectivirgula, glycoproteins on S. rectivirgula’s surface are recognized by IgG antibodies, forming antigen-antibody complexes [96]. Antigen-antibody complexes mark S. rectivirgula as foreign to the host and can trigger alveolar macrophage or dendritic cells to produce pro-inflammatory mediators that can recruit neutrophils, CD8⁺, CD4⁺ cells. S. rectivirgula activation of dendritic cells or macrophages through TLR and the formation of antigen-antibody complexes induces a pro-inflammatory environment via the production of cytokines from CD4⁺ T helper cells.

CD4⁺ T helper cells can fine tune an immune response with their unique abilities to produce a wide variety of cytokines. Naïve CD4⁺ T cells encounter antigen and can differentiate into T helper (T_H) subsets such as T_H1, T_H2, T_H9, T_H17, T_H22, T follicular cells (T_FH), or T regulatory cells (Tregs) and Type 1 regulatory cells (Tr1), depending on signals from the microenvironment. Previous studies were unable to reach a consensus on which T_H cell(s) type are important for disease pathogenesis in FLD [28, 74, 76, 85, 86, 97, 98], however, subsequent studies have found a prominent role for T_H17 cells. The presence of lung lymphocytes and granuloma formation are classic hallmarks of FLD, and CD4⁺ and CD8⁺ T cells are found in the BAL of patients with FLD, suggesting they play a role in pathogenesis [79]. Granuloma formation has been historically associated with macrophages, T_H1 and T_H2 cells [99, 100]. T_H1 cells produce IFN-γ and promote macrophage responses against extracellular or intracellular bacteria [57]. IFN-γ may play an important role since IFN-γ^−/− mice (on a Balb/c background) that were exposed to S. rectivirgula did not develop granulomas, and when given exogenous IFN-γ, developed granulomas in the airways [81]. However, the absence of IFN-γ did not affect the S. rectivirgula induced increase in the number of cells that infiltrated the airways. The cytokine IL-12 can regulate IFN-γ [101], and mice (C57Bl/6) exposed to S. rectivirgula have elevated IL-12 and IFN-γ when compared to DBA/2 mice that are resistant against developing FLD. However, when DBA/2 mice are given recombinant IL-12 intranasally, they become sensitive to developing FLD [83]. While BAL and blood samples taken from patients with FLD or summer-type HP had elevated IFN-γ levels, it is unclear whether these were patients who were sensitive to S. rectivirgula [98]. The cellular sources of IFN-γ were likened to neutrophils and a small proportion of NK cells, and in the absence of neutrophils there is a reduction of the severity of alveolitis, although the recruitment of immune cells into the lung following S. rectivirgula is not completely eliminated [28], although more recent work suggest that neutrophils may not be important for the development of inflammation in the lung [102]. Experiments with Rag-1^−/− mice (lacking B and T cells) reconstituted with spleen cells from IFNγ^−/− or WT mice and exposed to S. rectivirgula, suggest that IFN-γ plays an important role in disease pathogenesis.

2.1 Th17 immune response to S. rectivirgula induced FLD

In 2005, a new T_H cell type was discovered and determined to be an independent subset from T_H1 and T_H2 cells [103]. This cell type, the T_H17 cell, develops in the presence of TGF-β, IL-1, IL-6, IL-21, or IL-23, and has been shown to protect against extracellular bacteria by producing the pro-inflammatory cytokines IL-17A, IL-17F, and IL-22 [57, 104]. Analysis of gene expression profiles of lung biopsies of patients with HP found an upregulation of the IL-17 receptor IL-17RC, suggesting that T_H17 cells may play a role in HP pathogenesis [105]. In a mouse model of S. rectivirgula induced FLD, Joshi et al. found an increase in IFN-γ and IL-12p35 mRNA in S. rectivirgula exposed mice, along with significant increase in IFN-γ, IL-4, IL-13, IL-6 and IL-17A in lung homogenates. However, when they exposed IL-17^−/− mice they found a decrease in alveolitis and lower production of cytokines and chemokines compared to WT mice [85]. Simonian et al. investigated the role of T_H17 cells in S. rectivirgula induced FLD over the course of 4 weeks [74], comparing WT, TCRβ^−/− (that lack CD4⁺ or CD8⁺ T cells), or IL-17ra^−/− mice for collagen deposition and cytokine expression in lung homogenates. They found that T cells are critical for disease since TCRβ^−/− mice do not develop pulmonary fibrosis, but still had collagen deposits albeit significantly less than WT mice. Reconstitution of mice lacking both αβ and γδ T cells (TCRβ^−/−δ^−/− mice) with CD4⁺ or CD8⁺ T cells followed by exposure to S. rectivirgula indicated that αβ CD4⁺ T cells contribute significantly to collagen deposit, influx of cells into the lung, increase in IL-17 production, along with pulmonary fibrosis compared to CD8⁺ T cells. Analysis of T_H1, T_H2, or T_H17 cytokines and TGF-β revealed that IL-17A was predominant in the airways of S. rectivirgula exposed mice. Furthermore, IL17ra^−/− mice exposed to S. rectivirgula exhibited a significant decrease in lymphocyte influx and lung fibrosis, and little production of IFN-γ by T cells after 2 weeks of S. rectivirgula exposure. Interestingly, S. rectivirgula exposure of T-bet^−/− mice led to more severe disease, with an enhanced T_H17 response and increases in collagen production compared to WT mice [97]. However, it should be noted that IFN-γ production is not entirely dependent on T-bet [97]. Taken together these data suggest T_H17 cells and their production of IL-17A are important for the pathogenesis of FLD.

T_H17 cells are not the only cell type that can produce IL-17A. Neutrophils, γδ T cells, invariant natural killer cells (iNKT), and group 3 innate lymphoid cells (ILC3s) have all been shown to produce IL-17A [106, 107, 108, 109]. Studies with γδ T cells have shown that they produce IL-17A in S. rectivirgula induced FLD but are not important for disease pathogenesis [29]. However, neutrophils and their production of IL-17A has been implicated in S. rectivirgula FLD as well [110]. When mice are exposed to S. rectivirgula over the course of 6 weeks and during the final two weeks of exposure neutrophils were depleted using anti-GR-1 antibody, it was found that neutrophil depletion reduces neutrophils, lymphocytes, and collagen levels in the airways when compared to isotype controls (although more recent work suggest that neutrophils may not be important for the development of inflammation in the lung [102]). This group also utilized an IL-17A enrichment kit to determine which cells produced IL-17A secreting in lung homogenates of mice exposed S. rectivirgula for 3 weeks. They found that GR1^int and GR1^high populations were the most significant populations in the IL-17A positive fraction compared to B cells, CD4⁺ or CD8⁺ T cells. Stimulating these cells in vitro followed by flow cytometric analysis also revealed the expression of intracellular IL-17A in lung neutrophils and not lymphocytes, along with expression of Il-17A mRNA. While these data suggest neutrophils, monocytes, and macrophages are the major source of IL-17A, more recent work using IL-17A cytokine reporter mice suggest instead that CD4+ T cells are the major producers of this cytokine, and that neutrophils do not produce IL17A during development of inflammation in the lung [102]).

2.2 Anti-inflammatory response during S. rectivirgula induced FLD

The anti-inflammatory responses of S. rectivirgula induced FLD remains largely unexplored. The anti-inflammatory cytokine IL-10 can reduce inflammatory responses [57]. IL-10^−/− mice have been shown to have increased inflammatory cells in response to S. rectivirgula exposure, and histological evidence suggested an increase in granuloma formation [82]. This suggest that IL-10 may be important for reducing the inflammatory responses in FLD, but are unable to completely neutralize the inflammation under usual circumstances. Immune cells that produce IL-10 include T regulatory cells, such as forkhead box 3 (Foxp3⁺ Tregs) and Foxp3⁻ type 1 T regulatory cells (Tr1). These T regulatory cells are critical for controlling inflammatory responses including allergic responses by producing the anti-inflammatory cytokines IL-10 and TGF-β. In humans, Tregs can be identified as CD4⁺CD25⁺CD127⁻Foxp3⁺ [111]. Subjects that are asymptomatic but have farmer’s lung might have functional T regulatory cells [36]. Analysis of isolated Tregs from blood or BAL from healthy, asymptomatic patients or diagnosed with FLD (n = 6) found no differences in the proportion of Tregs among the patient populations [112]. However, when Treg suppression assays were performed, they found that Tregs from patients that had FLD were unable to suppress the proliferation of activated T cells. These data suggest patients that have FLD may have impaired ability to suppress effector T cell responses, resulting in unresolved inflammation with the potential to develop irreversible lung damage. More studies need to be conducted to fully elucidate the role of T regulatory cells in this disease.

3. Conclusion

Actinobacteria are a source of natural metabolites that can be important for human health. However, some species cause disease such as HP, a collection of rare interstitial lung disease caused by the inhalation of (in)organic agents including S. rectivirgula leading to farmer’s lung, one of the most well studied forms of HP. It is unclear why some individuals develop farmer’s lung upon exposure to S. rectivirgula, and others do not. Environmental factors and genetics may play a role in the development of disease, and polymorphisms in the immune genes including TNF-α and MHC II have been found in patients diagnosed with HP [60]. However, genetic studies have not been able to fully identify specific mutations in patients predisposed to develop FLD. The pathogenesis of HP including FLD is challenging to fully elucidate. S. rectivirgula exposure results in the development of an immune response that includes macrophage TLRs recognition of S. rectivirgula, leading to the production of chemokines and cytokines that recruit neutrophils or induce the differentiation of T_H1, T_H2, or T_H17 cells (Figure 1) [20, 57]. In addition, B cell recognition of S. rectivirgula antigens lead to the differentiation of plasma cells that secrete IgG that form antigen-antibody complexes with S. rectivirgula antigens [77]. Thus neutrophils, T_H2, T_H1, T_H17 and Tregs along with the cytokines IFN-γ, TNF-α, IL-10, or IL-17A have all been considered critical for disease progression [74, 77]. While there are conflicting reports on the main cellular source of IFN-γ or IL-17A, neutrophils and T_H17 cells have been implicated as sources of IL-17A production which has been shown to be most critical for the development of farmer’s lung in response to exposure to S. rectivirgula [74, 110]. Furthermore, understanding the role of IL-10, and T regulatory cells that produce this cytokine to suppress inflammation may provide an avenue to treat disease. Newer technologies and techniques will also help gain a better understanding of disease pathogenesis in S. rectivirgula induced FLD. For instance, genome-wide association studies can help determine what genetic components may be associated with developing symptoms. Next generation sequencing such as RNA sequencing, or immune cell profiling could help identify which cells are present and important for disease pathogenesis. Advances in techniques such as multi-color flow cytometry allow for identification of many different immune cells than ever before [113, 114].

It is important to gain a better understanding of rare diseases such as HP. Global climate change caused by human activities are anticipated to affect respiratory diseases with increases in rainy, humid, hot weather which can lead to humid environments which may make environments suitable for the growth of actinobacteria such as S. rectivirgula [115, 116, 117, 118]. Understanding how S. rectivirgula causes HP such as FLD will lend to better therapeutics for patients.