Immune Responses and Exacerbations in Severe Asthma

Abstract

Asthma as a clinical entity manifests with a broad spectrum of disease severity. Unlike milder asthma, severe disease is poorly controlled by inhaled corticosteroids, the current standard of care. Transcriptomic data, along with patient characteristics and response to biologics show that though Type 2 (T2) immune response remains an integral feature of asthma, additional molecular and immunologic factors may play important roles in pathogenesis. Mechanisms of T2 development, cellular sources of T2 cytokines and their relationship to additional immune pathways concurrently activated may distinguish several different sub-phenotypes, and perhaps endotypes of asthma, with differential response to non-specific and targeted anti-inflammatory therapies. Recent data have also associated non-T2 cytokines derived from T cells, particularly IFN-γ, and epithelial mediators with severe asthma. These topics and their relationships to acute asthma exacerbations are discussed in this review.

Asthma as a clinical entity has been long recognized for reversible airflow limitation in the setting of additional symptoms such as wheeze, dyspnea or cough. Clinical manifestations of asthma are non-uniform with 5–10% of patients displaying poor response to inhaled corticosteroids (ICS), the current standard of care, even when used at high doses or accompanied by systemic administration [1–5]. The economic toll of severe asthma on the US economy is estimated at 56 billion dollars annually, driven primarily by frequent exacerbations and need for emergency care [6]. Current guideline-based definitions of asthma utilize spirometric measurement of lung function or provocational challenge with inhaled irritants to quantify bronchoconstriction and confirm diagnosis [7]. These tests, however, do not account for heterogeneity in the disease. Divergence in factors such as age of onset, lung function and relation to allergic stimuli were first described by Francis Rackemann in the 1940s [8]. As treatment with corticosteroids (CS) became commonplace, it became clear that not all patients responded similarly to nonspecific immunosuppression [9,10]. This realization, combined with a growing appreciation for the pathological divergence of asthma patients and the observation that Th2 (now Type 2/T2)-pathway targeted biologics were negative across “all-comers” with asthma [11], drove increased interest in disease heterogeneity during the late 1990s, leading to the evolution of molecular phenotyping.

Early hypothesis-driven studies utilized association between clinical, physiological and pathobiological traits to identify subgroups of asthma patients with shared characteristics [12,13]. This work recapitulated historic findings that age of onset was an important differentiator in phenotype and shed light on the relationship between eosinophilic inflammation in the blood or sputum and exacerbation prone, CS-responsive, late-onset asthma [9,12,14]. This recognition was instrumental in the completion of the first successful precision medicine trials utilizing eosinophil targeted therapies [15,16]. As statistical and computational methods evolved, so did the complexity of these analyses. Maturation of large asthma networks such as the National Heart, Lung and Blood Institute’s Severe Asthma Research Program (SARP) network allowed for clustering of patients, first along clinical variables [17]. It was followed by use of bronchial epithelial gene expression to cluster patients in association with biomarkers such as bronchoalveolar lavage (BAL) cell differentials and fraction exhaled nitric oxide (FeNO) [18]. Validating earlier studies, the authors identified a late-onset, nasal polyposis-prone and highly eosinophilic cluster as well as several early-onset and more allergic groups. Both analyses found a very severe patient cluster with persistent airflow obstruction. The inclusion of inflammatory characteristics showed that these most severe patients had high levels of FeNO as well as concomitant neutrophilia and eosinophilia. Mixed granulocytic inflammation in the sputum of the most severe patients was independently observed, as was a link between FeNO and oral CS use [19,20]. Cluster analysis of the British Thoracic Society Severe Asthma Registry found similar patterns in clinical characteristics, laying the groundwork for subsequent molecular studies [21].

The term molecular phenotyping refers to the identification of specific pathways with relation to clinically distinguishable traits and outcomes. This concept may be extended to endotype, whereby targeting this pathway alleviates disease in the affected individual [22]. Platforms such as gene expression microarray, RNA sequencing, mass spectroscopy and high dimensional flow cytometry have been employed on multiple organ compartments, including bronchial epithelial cells, BAL fluid, sputum, blood, nasal brushings and urine to gain insight into the pathobiological underpinnings of asthma [3]. Compared to clinical phenotyping, these technologies require integration of thousands if not millions of variables, thus increasing their complexity by orders of magnitude. As such, few studies have satisfactorily combined (or harmonized) data from differing technologies or physiologic compartments, such that this remains a pressing need.

Asthma and Type 2 (T2) immune response

The Th2 arm of the adaptive immune system was first associated with inflammation in asthma in the early 1990s [23] following murine studies establishing the Th1/Th2 paradigm. Subsequent work identified GATA-3 as the master regulator of Th2 development and associated the transcription factor with allergic asthma in mouse models and human asthma [24–26]. Human studies also reported IL-4, −5 and −13 expression in both peripheral blood cells and airways of asthma patients, although subsequent work has shown wide variability in levels. It has also since become clear that many cells produce type 2 cytokines, including ILC2s, mast cells, basophils and even eosinophils [27]. While these observations were initially viewed with skepticism, nearly all ‘omic studies to date have identified subsets of asthma patients with evidence of T2 inflammation unrelated to Th2 cells.

The most commonly used methodology for molecular studies of asthma patients has used traits as dependent or fixed variables to detect differential levels of analytes between categories. The simplest implementations of this approach identified differentially expressed genes between asthma and health. These studies have consistently produced relationships between type 2 gene sets and traits such as airflow limitation and eosinophilic asthma phenotypes. The original identification of “Th2-High” asthma (now T2-high) based around a signature of genes upregulated by the addition of IL-13 to cultured human airway epithelial cells (HAECs) [28,29]. Around 50% of mild, CS-naïve asthma patients showed upregulation of these genes in their airways in addition to eosinophilia and more bronchial hyperreactivity. Importantly, these T2-high patients had robust improvement in forced expiratory volume in one second (FEV1) to inhaled CS, while those with low expression did not.

Origin of T2 cytokines and CS sensitivity: multiple immune cell types

Population based identification of T2-Hi patients is limited by the availability, sensitivity and specificity of biomarkers, as measurement of the cytokines themselves is hamstrung by their very low and highly variable levels. Using the approach of single cell RNA-sequencing, the airways of mild-moderate asthma patients taken off CS prior to tissue sampling revealed all of the hallmarks of a T2 immune response including GATA-3 expression in T cells [30]. While CS suppress T2 immune response in mild-moderate asthma, it is not as effective against T2 response in severe asthma. With the advent of biologicals, several clinical trials have been conducted directed against T2 cytokines or their receptors. While these severe asthma patients with elevated commonly used T2 biomarkers FeNO and blood eosinophils, used for patient stratification, do generally respond to targeted therapies, this response is not universal. Clinically meaningful responses to dupilumab, which targets the IL4Rα, may been noted in patients with low levels of FeNO or blood eosinophils, suggesting imperfect performance of these surrogate markers. Even with these limitations, consistent pathobiological processes have been associated with the T2-Hi phenotype, including eosinophilia, elevated IgE/atopy, bronchodilator responsiveness, mucus plugging and frequent exacerbations [31–34]. Yet despite these consistencies, patients under the T2-Hi umbrella may vary significantly from one another in CS and targeted therapy responsiveness, age of onset, inflammatory mediators and comorbidities. Multiple reports have demonstrated greater efficacy for IL-5 pathway inhibition in patients with adult-onset T2-Hi disease as compared to early onset asthma, supporting this heterogeneity [35,36]. Though the cause for this remains unclear, these data suggest that factors such as the cellular source of T2 cytokines, organ compartment and accompanying signaling into T2+ cells as well as target cells (airway epithelial and smooth muscle cells) from innate sources (e.g. thymic stromal lymphopoietin (TSLP), IL-33) and adaptive immune cells (e.g. IFN-γ, IL-17) may impact signal integration and physiologic effect.

Type 1 and type 17 immune responses in severe asthma

As already discussed, the currently available biomarkers, FeNO and blood eosinophil counts, are inadequate to guide therapy. While three of the currently available T2-targeted biologics have shown efficacy in CS-dependent and refractory asthma, some patients remain refractory to therapy despite elevations in traditional T2 biomarkers. Inducible nitric oxide synthase (iNOS), the primary enzyme contributing to nitric oxide (NO) generation in human airways, is increased by IFN-γ in addition to IL-4/IL-13 [37], suggesting heightened FeNO may not be attributable to T2 signaling alone. Many additional factors cause blood eosinophilia, supporting the need for more specific T2 biomarkers and a better understanding of complex inflammatory phenotypes.

Mounting evidence suggests involvement of other arms of the adaptive immune system, including Th1 and Th17 cells. In patients with very severe, CS-dependent asthma, video-assisted thoracoscopic biopsy has revealed non-caseating granulomas within distal lung tissue without relation to eosinophilic granulomatosis with polyangiitis, suggesting immune signaling beyond T2 alone. IFN-γ and downstream cytokines CXCL9 and −10, as well as the macrophage/dendritic cell transcription factor IRF5, all associated with Type 1 (T1) inflammation, have been reported increased in severe asthma [38–43]. Association of neutrophilic inflammation with severe asthma spurred interest in the contribution of IL-17 in severe asthma. Although IL-17 expression and pathway genes have been identified in severe asthma [31,41], with one study even suggested complete dissociation between T2 and Th17/type 17 inflammation [44], clinical trials directed against IL-17 or its receptor in severe asthma have not been successful [45].

Mechanisms underlying development of distinct immune profiles: role of the tissue

The exact mechanisms underlying the development of distinct immune phenotypes in asthma are still unclear. While mild allergic asthma, typically characterized as early onset, is mediated by an aberrant Th2 response that can be triggered by a wide variety of allergens and is responsive to CS [23,29,46], epithelial injury is increasingly being implicated as an initial trigger with downstream engagement of immune cells [47,48]. The type of allergen or pathogen encountered at the epithelial barrier combined with genetic predisposition likely plays a role in the development of the specific immune profile in asthma. Proteases from complex allergen sources like house dust mite (HDM), cockroach and fungi (Aspergillus fumigatus, Alternaria alternata) have been shown to damage and activate airway epithelial cells [49,50] (Figure 1). Activated epithelial cells secrete a myriad of mediators, chemokines, cytokines and alarmins like IL-33, TSLP and IL-25 among others. IL-33 and TSLP play a crucial role in asthma pathophysiology including CS-refractory disease [51–53], and are known to activate ILC2s, resulting in upregulation/secretion of T2 cytokines IL-13 and IL-5 [54]. Apart from their role in activating ILC2, IL-33 and TSLP have been associated with in-situ hematopoiesis to generate effector cells-mast cells, eosinophils and basophils that further drive T2 cytokine-driven downstream responses [55]. IL-5 has been also linked to in situ eosinophilopoiesis in the airway mucosa [56]. Whether in situ hematopoiesis can lead to the generation of other innate cells types in the airways requires additional investigation.

Figure 1. Immune responses in severe asthma.

Proteases present in complex allergens and pathogens cause epithelial injury triggering secretion of the alarmins TSLP, IL-33 and IL-25. The alarmins activate ILC2s and other cell types that secrete T2 cytokines. IL-5 plays a central role in increased eosinophil differentiation in the bone marrow in turn causing eosinophilia in the periphery and in the airways. IL-5 may also induce local eosinophilopoiesis. TSLP and IL-33 have been also implicated in in situ hematopoiesis from hematopoietic stem progenitor cells (HSPC), either present in the mucosa or recruited from the circulation contributing to increased T2 response. Antigens in allergens or pathogens at the epithelial barrier can also induce an adaptive immune response with generation of effector (TE) and memory (TEM, TCM and TRM) T cells. TRM (CD4 or CD8) cells thus generated reside in the tissue. Second antigen encounter leads to TRM and TCM reactivation. Activated TRM cells undergo rapid proliferation and secretion of pro-inflammatory cytokines that have the ability to promote recruitment of other immune cells to the site of encounter. Some secondary TRM cells can shed tissue residency and migrate to secondary lymphoid organs and the periphery. These TRM cells can also differentiate into TE cells and mount a robust immune response in collaboration with newly generated effector cells. The colored arrows represent the following: green: cell migration, blue: cytokine secretion, red: cytokine milieu or cytokine effect and black: cell differentiation.

As the immune response to a primary infection wanes, the number of effector T cells diminishes via apoptosis while that of memory T cells increases [57]. These memory T cells can be further classified into three subtypes viz., TEM (T effector memory), TCM (T central memory) and TRM (T resident memory), based on their phenotype, expression of tissue homing receptors and migratory properties [58]. TRM cells reside in non-lymphoid tissues and play a crucial role in mounting optimal responses during reinfections at mucosal barriers. TRM cells patrol the mucosal barriers and are responsible to mount a fast and robust response during second encounter with the pathogen. Upon reinfection TRM cells undergo local proliferation to generate secondary TRM cells, rapidly secrete effector cytokines that promote recruitment of other immune cells to the affected sites. Further, secondary TRM cells shed tissue residency phenotype and are capable of migration to secondary lymphoid organs and differentiation into circulating TEM cells [59]. Although, studies have attempted to gain insight into role of TRM cells in asthma, most of our understanding about origination, development and functioning of TRM cells stem from CD8+TRM dominant murine models of viral infection. Recent studies have shown generation and development of CD4+ TRM cells in HDM allergen sensitized murine models. These, CD4+ TRM cells remain in mouse lungs for extended periods and provide for life long Th2 memory [60]. The CD4+/CD8+ TRM dynamics and/or their interactions in the context of asthma is unclear. A recent study showed that although HDM exposure leads to infiltration of both CD4+ and CD8+ T cells only HDM specific CD4+ TRMs are retained in the murine lungs [61]. The role of CD4+ and CD8+ TRM cells in CS-refractory severe asthma remains to be determined. Upon reactivation TRM cells actively proliferate, rapidly secrete effector cytokines and induce airway hyperresponsiveness [62]. The attributes of TRM cells- a) persistence b) self-renewal after subsequent activation c) rapid activation and secretion of effector cytokines and d) ability to be activated non-specifically [63] may contribute to recurrent asthma exacerbations triggered by viral infections.

Asthma exacerbations, immune response and the role of eosinophils and neutrophils

Asthma exacerbations are defined as episodes of increased airway symptoms such as shortness of breath, wheezing, and cough which may be accompanied by decreased lung function assessed by pulmonary function testing. Exacerbations can occur secondary to exposure to environmental pollutants or infection with bacteria and fungi, but much more commonly by viruses which are associated with as much as 80% of adverse events [64]. While multiple viral upper respiratory tract infections can cause exacerbations [65], human rhinoviruses are by far responsible for most exacerbations, being found in up to 70–80% of pediatric and adult cases [66].

Of note, in the present environment of the COVID-19 pandemic, initial studies suggested that SARS-CoV-2 infection had minimal impact on asthma exacerbation [67,68]. However, more recent studies suggest that asthma in certain individuals may adversely impact COVID-19 disease [69] and there is a report of worse COVID-19 disease in severe asthmatic subjects receiving biologic therapy, which could be a confounding factor [70]. Susceptibility in subsets of patients may be related to relative expression of molecules such as the cellular receptor for SARS-CoV-2, angiotensin-converting enzyme-2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) which aids viral binding to ACE2 and subsequent cell entry [69,71].

In the case of the most common virus-induced exacerbations, it has been suggested that T2 immune response present in many forms of asthma impairs anti-viral Type I and III interferon (IFN) response to infection [72]. Additionally, infected epithelial cells can stimulate the secretion of the alarmins IL-25, IL-33 and TSLP, all of which are associated with severe asthma and T2 immune responses [51–53]. Normally, eosinophils can become infected with viruses resulting in their degranulation thereby contributing to antiviral immunity. However, this antiviral activity appears to be defective in asthmatic individuals [73]. An example of a dramatic induction of asthma exacerbation induced by environmental exposure with associated T2 immune response and rapid eosinophilic inflammation is response to aerosolized pollen during severe thunderstorms, which is known as thunderclap asthma [74]. Consistent with this theory of pathogenesis, biologics targeted against eosinophils (anti-IL-5) [75,76] and general T2 inflammation (anti-IL-4Rα) [77] have shown their greatest benefit in exacerbation reduction.

Additionally, anti-TSLP antibody has shown potential efficacy in both eosinophilic and non-eosinophilic asthma, suggesting that alarmins may play a role in exacerbations [78]. As recently reported, the phase 3 trial of Tezepelumab (anti-TSLP) met its primary endpoint in reducing exacerbations in severe asthma patients [79,80].

Despite T2 inflammation being described in nearly 50% of asthma patients [29], exacerbations are not widespread in the asthma population. A small number of asthma patients typically account for the majority of exacerbations, and the most consistent predictor of future exacerbations has consistently been a prior history of exacerbations, suggesting that while environmental exposures play a role, intrinsic pathophysiology is also critical [81–83]. Eosinophils do, however, remain a strong predictor of exacerbation prone disease [84] although parameters such as serum IgE and FeNO, as well as comorbid conditions such as obesity, gastroesophageal reflux disease (GERD), smoking and psychological issues also have predictive value [82].

Whereas eosinophils are a cardinal feature of allergic asthma, neutrophils can also be involved particularly in severe forms of the condition and the interplay between the two cell types can be complex with regard to exacerbations [85]. The data with regard to neutrophils are more variable and predictive biomarkers for exacerbation in neutrophilic inflammation are largely lacking although there are published reports suggesting an association at least in a subset of asthmatic subjects [81,86]. A meta-analysis of older published studies determined that there is a significant increase in blood neutrophil to lymphocyte ratio in individuals with asthma exacerbations compared to those with stable disease [87], although this assessment may be confounded by CS use in exacerbation prone asthma. Similarly, high numbers of exacerbations and hospitalizations was documented in a recent study of asthmatic patients with severe disease, highlighted by irreversible airway obstruction and evidence of increased blood neutrophils in addition to eosinophils [88].

There is a paucity of information regarding direct mechanistic aspects of neutrophils in exacerbations but indirect evidence suggests that they could be responsible for at least some effects, especially considering that their generally pro-inflammatory nature in asthma is well described [85]. Anti-IL-5 biologics effectively reduce exacerbation frequency in eosinophilic severe asthma, but in the setting of coincidental neutrophilia, exacerbations can still occur [89]. In this setting, elevated T1 immune response (IFN-γ) in the airways of severe asthma patients [41] may contribute to neutrophil chemotaxis via upregulation of CCR1 and CCR3 expression on neutrophils [90]. Furthermore, chronic neutrophilia is usually steroid insensitive such that novel targeted therapeutic modalities will be required. It has also been reported that Type 1 interferon production is defective in neutrophilic asthma and in individuals requiring high dose inhaled CS [91]. While the exact mechanisms of contribution in exacerbations is unclear, current avenues of investigation include necrostatin-1 (Nec-1), which disrupts neutrophil extracellular traps in the lung [92] and modulation of CCR5, the experimental absence of which leads to enhanced neutrophilic exacerbation of rhinovirus infection [93]. Similarly, targeting CCR1 and CCR3 may also ameliorate neutrophilic inflammation with reduction in asthma exacerbation in severe asthma patients.

Concluding remarks

For many years, severe asthma has been recognized as a subset of asthma that is poorly managed by standard therapy for asthma. Analysis of transcriptomic data of BAL and epithelial cells combined with patient characteristics and response to biologics show that T2 immune response is induced in severe CS-refractory severe disease as in milder CS-responsive disease. However, while in milder disease, the T2 immune response in 50% of patients is largely mediated by Th2 cells, the T2 immune response in more severe disease is more complex accompanied by a Th1 immune response in a smaller subset with presence of Th17 cells in some [94]. Part of the complexity also lies in the cellular source of T2 cytokines and upstream epithelial cell-derived cytokines that instigate these multitude of cells potentially driving their differentiation in situ in the airways along with induction of one or more T2 cytokines [94]. As is evident in the use of multiple T2-directed biologics, no biologic shows similar efficacy in all subjects despite similarity in levels of biomarkers in the patients. This is because the current biomarkers fail to report the complexity of the underlying immune response. Use of high dimensional multi-omics approaches are needed for improved understanding of the dysregulated immune response locally in the airways of these patients as also the imprint of the local immune response on the periphery to enable the development of next generation biomarkers.