The pathogenesis of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), such as granulomatosis with polyangiitis, is not well understood. These diseases cause significant inflammation in the upper airway. The nares and upper airway are host to many commensal microbes as well as a frequent site of exposure to pathogenic microbes. This review explores the association between upper airway microbial dysregulation and AAV. The role of Staphylococcus aureus colonization as a possible driver of disease is discussed, as well as recent work exploring how fluctuations in the abundance and diversity of commensal microbes are related to vasculitis and risk of flare.
Key points
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The upper airway microbiome plays an important role in regulating inflammation and immune responses, and dysregulation of these microbes may drive autoimmunity.
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Antineutrophil cytoplasmic antibody-associated vasculitis has been frequently associated with Staphylococcus aureus colonization, although the exact role these bacteria may play is not fully understood.
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Patients with granulomatosis with polyangiitis have been observed to carry lower levels and decreased diversity of commensal bacteria in their nares.
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Changes in microbial composition in the nares of patients with granulomatosis with polyangiitis have been associated with risk of flare.
Introduction
Respiratory tract involvement is common in patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, which includes granulomatosis with polyangiitis (GPA), eosinophilic granulomatosis with polyangiitis (EGPA), and microscopic polyangiitis (MPA). In particular, inflammation of the upper respiratory tract is a distinguishing feature of ANCA-associated vasculitis (AAV) and can be severe and difficult to treat. Upper airway involvement can lead to deformity, atrophy, and stenosis of structures resulting in significant suffering and morbidity.
The underlying cause of vasculitis is unknown but likely polyfactorial, including genetic predisposition as well as environmental triggers. , Microbes are commonly implicated environmental factors of AAV. Because of the frequent involvement of the upper respiratory system, the role of upper respiratory microbes in driving vasculitis has been an ongoing area of study. This review will explore the association between the upper respiratory microbial milieu, immune system dysregulation, and AAV, to piece together what has been uncovered thus far and provide a look toward future research.
The mucosal barrier of the upper respiratory tract
The human respiratory tract is divided into 2 segments: upper and lower. The upper respiratory tract includes the nasal passages, paranasal sinuses, naso- and oro-pharynx, and larynx, while the lower respiratory tract includes the trachea, bronchial tubes, and lung parenchyma. The upper respiratory tract is therefore an initial site of entry and serves as both an immune surveillance region as well as a site for microbial colonization.
The upper respiratory tract is lined by a pseudostratified columnar epithelium that provides a physical barrier as well as immune surveillance against infection ( Fig. 1 ). The epithelium is primarily composed of basal cells, club cells, goblet cells, and ciliated cells. Of note, there are also rare cell types recently identified by immunofluorescence as well as single cell sequencing that play roles in chemosensing and inflammatory pathway modulation. Basal cells are undifferentiated multipotent stem cells and thereby play an essential role in replenishing the epithelium that is subject to a frequent onslaught of toxins and infections. Basal cells can differentiate into club cells, which can further differentiate into both goblet cells and ciliated cells. , Club and goblet cells secrete proteins into the airway lumen that become important components of mucus. Club cells are well known for their secretion of the protein secretoglobin 1A1 (also known as Club Cell Secretory Protein, Club Cell Protein 16, and uteroglobin, among other names), which has been shown to have an anti-inflammatory effect, including inhibition of neutrophil recruitment, prostaglandin metabolism, and cytokine release. , Goblet cells secrete mucins, notably MUC5AC and MUC5B, in the upper airway; mucins are glycosylated proteins that trap microbes and block access to the epithelium through dynamic conformational changes and polymerization. Finally, the ciliated cells evacuate microbes and debris caught within the mucus. Altogether, these cells maintain the composition and effective clearance of mucus, which requires tight control of low salt content, sufficiently alkaline pH, and adequate hydration. , Mucus, the airway surface liquid atop the cells, is a hydrogel that can be thought of in 2 layers—an upper secreted mucus layer that contains the mucin proteins, and the periciliary layer which contains cell-bound mucins and the cilia. The biochemical and biophysical differences between these layers allow proper function of the secreted mucins and the ciliary escalator.
There is growing evidence to support the possibility of disturbed airway epithelium in AAV. Prior studies demonstrated altered transcriptional signatures associated with the epithelial barrier and innate immune response in the nasal mucosa of patients with GPA. Impaired ciliary function in the nasal epithelium in GPA has also been observed. In addition to cilia, abnormal mucin composition results in failure of mucus clearance and innate immune dysfunction. For example, in chronic obstructive pulmonary disease, higher levels of mucins MUC5AC relative to MUC5B increase mucus viscosity, which may aggravate inflammation. In idiopathic pulmonary fibrosis, a polymorphism in the promoter of MUC5B is associated with a higher MUC5B expression in lung tissue ; this same MUC5B polymorphism was subsequently found to be associated with interstitial lung disease in AAV and in rheumatoid arthritis (RA), suggesting a shared mechanism of disease. , Together these studies support the possibility of an altered respiratory epithelium, which potentially may impact the regulation of resident microbes.
Immune surveillance in the upper airway is performed by immune cells such as dendritic cells as well as the nasal epithelial cells themselves. This process is also enhanced by M cells, which are associated with mucosal lymphoid tissue and facilitate direct transfer of luminal antigens to antigen-presenting cells (APCs). , Toll-like receptors (TLRs) on the surface of APCs recognize pathogen-associated molecular patterns to activate the innate immune system. Activation of TLRs results in release of cytokines including interleukin (IL)-1, IL-6, and tumor necrosis factor alpha. Ultimately this results in a recruitment of an adaptive immune response. Nasal epithelial cells specifically have been shown to produce thymic stomal lymphopoietin, IL-33, and IL-25 leading to T helper 2 cell stimulation. The innate immune response plays an important role in the upper airway as well. Neutrophils and macrophages will be recruited to the tissue primarily by damage-associated molecular patterns, which are breakdown components of cells often associated with pathogen activity. Neutrophils themselves also have TLRs and thereby further modulate adaptive immunity. Thus, stimulation of the upper airway immune system by foreign molecules results in immune system recruitment and proliferation.
The upper respiratory microbiome in humans
Although the upper airway contains a robust system to identify and attack microbiota, there are many commensal bacteria that colonize this area. Even within the upper respiratory tract itself there exists variation of bacterial subpopulations. In the nose and nasopharynx, bacterial genera found include Staphylococcus , Corynebacteria , Moraxella , Streptococcus , Cutibacterium (formerly Propionibacterium ), Dolosigranulus , and Haemophilus , while further down in the oropharynx are other species such as Rothia , Veillonella , Prevotella , and Leptotrichia . The bacterial composition of the microbiome varies throughout life depending on age, and is also influenced by environmental factors such as cigarette smoking. ,
Commensal microbes may mediate their immune tolerance through lowered inflammatory response. Upper airway commensals induce lower levels of proinflammatory molecules including IL-8, IL-10, and IL-23. , Some bacteria also express proteins that combat the immune response through degrading antibacterial peptides or targeting immunoglobulins. Additionally, the common commensal bacteria can play a role in modulating other bacteria that enter the upper airway; for example, Corynebacterium have been observed to transform S aureus into a more commensal and less virulent state. Introduction of commensal bacteria has been shown to counteract pathologic infection, possibly by disrupting biofilms and producing antibiotics. The microbiome of the upper airway acts alongside the immune system to manage new intruders; this symbiotic relationship both prevents infection of the human host and preserves resources for the commensals.
While the immune system and bacteria may at times work hand in hand, their interaction is also thought to be a possible driver of immune dysregulation leading to disease. Variation in levels of Streptococcus , Moraxella, and Haemophilus has been associated with development, progression, and flare of asthma. Patients with chronic rhinosinusitis have alterations in the upper respiratory microbiome, notably reduction of Corynebacterium and decrease in microbial diversity, although it is unclear whether these changes are causal or downstream of the pathology. The role of the upper airway microbiome in pathologic disease states continues to be an active area of research. The remainder of this review will discuss the roles of S aureus and other microbes within the upper airway associated with vasculitis.
A role for Staphylococcus aureus in antineutrophil cytoplasmic antibody-associated vasculitis
Nasal colonization with Staphylococcus aureus is common: a total of 10% to 30% of people are estimated to be permanent carriers, while 10% to 47% of people are noncarriers and the remainder intermittent carriers. The presence and persistence of S aureus within the nose is likely due to multiple factors, including production of antimicrobial compounds by S aureus against other bacteria, as well as through activation of toll-like receptors by S aureus lipoproteins resulting in increased immune activity that culls other microbes but does not impact S aureus . Overall, colonization with S aureus , though asymptomatic, is associated with morbidity including severe infections.
Colonization with S aureus has been identified in some studies as a risk factor for relapse in AAV, especially GPA. Patients with GPA have been noted to have higher rates of S aureus colonization, and chronic carriage of S aureus in these patients has been associated with higher rates of relapse. Studies demonstrated mucociliary dysfunction in GPA patients, which is postulated to contribute to chronic nasal colonization. , S aureus superantigens have been observed to increase rate of relapse, with the superantigen toxic shock syndrome 1 (TSST-1) found to be the most proinflammatory. Other proteins in S aureus including leukocidins have also been implicated as drivers of AAV.
S aureus is proposed to not just be a risk factor but play an active role in the initiation and persistence of the autoimmune inflammatory pathways. A 6-phosphogluconate dehydrogenase peptide found in some S aureus has been shown to contain an epitope that cross-reacts with myeloperoxidase (MPO), inducing MPO-ANCA in mice with glomerulonephritis. This study provides evidence that bacterial proteins with homology to the native proteins targeted by ANCA could possibly serve as instigators in directing the immune system for generation of these antibodies; however, whether S aureus induces MPO-ANCA in humans is still unclear. Senapati and colleagues found that the human leukocyte antigen (HLA) allele DPB1∗04:01, which is strongly associated with GPA, shows a strong affinity for TSST-1 peptide, suggesting a combined genetic predilection and environmental exposure as a driver of dysregulated inflammatory pathways in AAV patients. Beyond a potential role in providing epitopes for self-recognition, S aureus may also drive AAV through inducing neutrophil extracellular trap proliferation. Interestingly, patients with GPA have lower levels of anti- S aureus IgG as compared to healthy controls, suggesting that they are less able to mount a sufficient immune response against this instigating microbe. While these studies raise intriguing hypotheses regarding the potential mechanism of S aureus -induced relapse of AAV, definitive evidence demonstrating causality is still lacking. Additionally, there is debate regarding the extent of the role of S aureus in AAV, as multiple studies that have not found a difference in disease severity for patients colonized with S aureus . ,
The most compelling evidence to suggest nasal bacteria may be important in AAV relapse came from clinical trials. In 2 randomized controlled trials, treatment with trimethoprim-sulfamethoxazole (TMP-SMX) reduced the frequency of flares in patients with GPA, although it is unclear if the benefit of TMP-SMX was due to its antimicrobial versus anti-inflammatory properties. , In contrast, an observational study by Salmela and colleagues found that while patients with chronic S aureus carriage within the nasal cavity experience higher rates of relapse, use of prophylactic TMP-SMX did not reduce relapse even though rates of chronic S aureus carriage was reduced. The conflicting data as well as high rate of adverse events associated with TMP-SMX have resulted in limited clinical use of TMP-SMX for the purpose of preventing relapse of AAV. A deeper understanding of the potential mechanisms that links nasal bacteria to disease processes and the specific subpopulations in which this occurs may eventually lead to better targeted therapy in AAV.
Other microbes implicated in antineutrophil cytoplasmic antibody-associated vasculitis
Nasal colonization with S aureus, though frequently explored in AAV, may not tell the whole story. The relationship between AAV and the nasal microbiome may be more complex, and specific patterns of bacterial populations may be associated with disease and flares. Furthermore, it’s possible that alterations in the microbiome are epiphenomena and reacting to proinflammatory responses. Most likely the relationship between the human microbiome and host immune response is bidirectional and unraveling these complex relationships is an active area of investigation.
In a study by Rhee and colleagues, the nasal microbiome of patients with GPA was evaluated using bacterial 16S ribosomal RNA gene sequencing of nasal swabs. This study found that nasal bacterial composition is significantly different in patients with GPA versus healthy controls. Specifically, the relative abundance of Propionibacterium acnes and Staphylococcus epidermidis was lower in GPA patients versus healthy controls. More dysbiosis was observed in patients off immunosuppressive therapy compared to those with GPA on immunosuppressives or healthy controls. There were also lower levels of the fungi Malassezia in patients with active GPA as compared to patients in remission and healthy controls. These microorganisms are all considered commensals which may possibly play a role in preventing colonization by other bacteria; for example, S epidermidis has been shown to prevent colonization with S aureus. However, in this study, no difference in S aureus levels was observed between patients. Alternatively, differences in bacterial composition may simply be a result of inflammatory activity or damage in the nasal mucosa.
This disruption of commensal bacteria in the nares of patients with active or unsuppressed GPA was further explored in a study by Lamprecht and colleagues. When comparing GPA patients to both healthy controls, it was identified that patients with GPA demonstrated relatively decreased diversity of microbes. Interestingly, they identified that patients with GPA and patients with RA both had decreased Propionibacterium and Staphylococcus, suggesting that the lack of commensal representation is not necessarily specific to GPA but to a dysregulated inflammatory state. In their study, they did find there was a significantly higher colonization with S aureus in GPA patients versus healthy controls and RA patients. They suggested this difference between the findings of Rhee and colleagues was potentially due to lack of antibiotic treatment in their cohort.
Further research has also suggested that the nasal commensal microbiome patterns may be harbingers of disease flare in the months prior to a relapse in GPA patients. Alterations in the ratio of nasal Staphylococcus -to- Corynebacterium were observed at the visit several months prior to relapse while this ratio remained stable in patients with quiescent disease. Exploratory analyses at the species-level found an association between increasing relative abundance of nasal Corynebacterium tuberculostearicum , a bacterium implicated in chronic rhinosinusitis, and relapse as well as a correlation between C tuberculostearicum abundance and PR3 (proteinase-3)-ANCA titers. Further studies are needed to validate these findings and determine if changes in these bacteria are driving versus reacting to upper airway inflammation.
Beyond bacterial associations with AAV, there are limited data implicating viruses. AAV is associated with expansion of memory (CD28-) T cells, which was found in a study by Kerstein and colleagues to be associated with co-occurring Epstein-Barr virus (EBV) or cytomegalovirus (CMV) infection. Multiple case studies have also suggested EBV as a trigger for development of AAV. More recently, there are case reports suggesting that severe acute respiratory syndrome coronavirus-2 can serve as a trigger for AAV. There are also limited data exploring possible roles for parvovirus and viral hepatitis. While viral infections or persistence are enticing explanations for disease recurrence in AAV, more conclusive studies are needed to understand these relationships. Similarly, there are limited studies on the role for fungal infection in AAV, and more data would be needed to purport any significant associations.
While these studies showed associations between the nasal microbiome and AAV, it is still yet to be understood the significance of these changes. Further research is needed to delineate the triggers and consequences of upper airway dysbiosis and whether host-microbial interactions could be targeted for treatment of AAV.
Future research and treatments
There are a growing number of studies supporting a potential role for the upper airway microbiome in the pathogenesis and perpetuation of vasculitis. Most research thus far has focused on S aureus , though there have been conflicting data on its significance and potential mechanism. Intriguingly, data based on high-throughput sequencing show an association of a decreased microbial diversity as well as decreased levels of commensal bacteria in patients with AAV. Further studies are needed to determine if upper airway dysbiosis is causally-related or a consequence of a hyperactivated immune response in AAV.
If upper airway microbes are playing a role in driving vasculitis, it is not only important to determine which pathogens may be contributing, but also how they are doing so and how they could be modulated. It is also possible that immune dysregulation may be inappropriately responding to common commensals and pathogens or that multiple microbes may be playing a significant role. It should also be considered that the pathogenic bugs may not always be the same between patients.
Regarding treatment, while antibiotics have already been studied in AAV, chronic antibiotic therapies are nonspecific and have long-term risks including antibiotic resistance. As we develop a deeper understanding of which nasal microbes are involved in vasculitis, more targeted treatments may be developed. One possibility is the role for microbiota transplantation, which is more well-known for the treatment of gastrointestinal disease. A recent study by Mårtensson and colleagues found that microbiome of healthy individuals transplanted to patients with chronic rhinosinusitis resulted in reduction of symptoms along with increased microbial diversity and abundance of flora. Even if dysbiosis is a reflection of underlying inflammation (rather than a cause), alterations in the microbiome may also be used as a prognostic biomarker to identify patients at high risk of relapse.
Summary
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AAV, most commonly GPA, affects the upper airway and leads to significant morbidity and mortality.
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The nares serve as an interface between the body and the outside world. The upper airway epithelium is equipped with a robust physical and immunologic barrier.
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The bacterial composition of the nares is governed by resident commensal species as well as the innate and adaptive immune systems. Interplay between bacteria and the immune system has been suggested to have a role in autoimmunity.
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S aureus has been heavily implicated in the pathogenesis and persistence of AAV. Multiple studies have raised possible mechanisms through which S aureus induces disease in AAV but definitive data demonstrating causality are still lacking.
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There is debate as to whether the beneficial effect of antibiotics in GPA is related to an effect on S aureus. Additionally, some studies have not found a significant difference in disease severity in AAV patients who are and are not colonized with S aureus.
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The upper airway microbiome of patients with GPA has been observed to have lower levels of commensal bacteria and decreased microbial diversity. Changes in nasal bacterial composition have been found to correlate with risk of GPA relapse.
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Further research should aim to identify the mechanism behind the microbial changes associated with AAVs and flares to inform the development of novel therapies and prognostic biomarkers.
Clinics care points
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Rhinosinusitis is a major manifestation of ANCA-associated vasculitis and epidemiologic data suggest that alterations of the microbial composition of the upper airway are associated with relapse of disease. However, there is debate as to what degree and how upper airway microbes, including S aureus , may contribute to disease pathogenesis.
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Due to concerns that longstanding treatment with oral antibiotics could result in adverse effects and have a detrimental effect on the microbiome, clinical guidelines do not recommend routine use of oral antibiotics for prevention of relapse in ANCA-associated vasculitis. However, antibiotics may occasionally still be an option in specific situations.
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Future research may determine if and how evaluation and modulation of the upper airway microbiome composition can be used for diagnostics and treatments of ANCA-associated vasculitis.
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