Oncostatin M promotes mucosal epithelial barrier dysfunction and is elevated in eosinophilic mucosal disease

Introduction

Mucosal epithelium maintains tissue defense and homeostasis under normal circumstances through many functions, including mucociliary action, the production of mucus and antimicrobial peptides, maintenance of air-surface liquid and ion transport¹. The epithelial barrier also serves as a first line of defense by creating a physical barrier to pathogens, allergens and other environmental factors. The integrity of the epithelial barrier is largely determined by the structure of intercellular epithelial junctions, including tight junctions, adherens junctions, and desmosomes². Upon breach of the epithelial barrier, pathogens, allergens and environmental factors can synergize with alarmins and danger associated molecular patterns (DAMP) that are released in response to cellular damage, thereby activating innate and adaptive immune responses³. The ability of the immune system to neutralize the pathogenic stimulus and repair the tissue damage is critical for the restoration of the tissue to a normal homeostatic state.

Dysfunction of epithelial maintenance and repair of barrier has been implicated in the pathogenesis of many diseases, including asthma, atopic dermatitis, eosinophilic esophagitis (EoE), and chronic rhinosinusitis (CRS). The airway epithelium of allergic asthmatic patients has been shown to have a number of defects, including decreased expression of intercellular junction proteins, increased susceptibility to injury, dysregulated repair, and increased proliferation^{4, 5, 6, 7}. Dysfunctional epithelial barrier function, shown through abnormal electrical and ion permeability, has also been demonstrated in CRS^{8, 9, 10, 11}. Furthermore, sinus epithelium in nasal polyps of patients with CRS was shown to have morphological changes consistent with epithelial dysfunction, including basal cell proliferation and goblet cell hyperplasia, in addition to increased epithelial damage, and decreased tissue resistance compared to control^{8, 12, 13, 14}.

Oncostatin M (OSM) is a member of the IL-6 family of cytokines, and has been shown to signal through 2 receptors; the type 1 receptor is a heterodimer of LIFR and gp130 and the type 2 receptor is a heterodimer of OSM receptor β chain (OSMR) and gp130. OSM has been shown to be expressed by multiple cell types of hematopoietic lineage, but it is not known to be expressed by epithelium, fibroblasts, or smooth muscle, the cells that express the different forms of the OSM receptor¹⁵. Previous studies have reported elevated levels of OSM in sinus tissue from allergic rhinitis patients, psoriatic skin and sputum of asthmatic patients with irreversible airflow obstruction^{16, 17, 18}. Moreover, intratracheal administration of adenoviral OSM to mice was shown to be sufficient to induce a type 2 immune response in the lung that was characterized by goblet cell hyperplasia, formation of bronchial associated lymphoid tissue (BALT), eosinophil infiltration, production of IL-4, IL-5, IL-13 and eotaxins, and airway hyperresponsiveness in a mouse model^{19, 20}. Studies using reconstituted epidermis have shown that OSM treatment decreased expression of filaggrin, a molecule that is important for barrier function in keratinocytes, suggesting that OSM could contribute to barrier dysfunction in the skin²¹. OSM has also been shown to decrease barrier function in endothelial cells²². Given that OSM is sufficient to induce a type 2 immune response in the lung¹⁹, and has been shown to potentially contribute to barrier dysfunction in the endothelium and the skin, we tested whether OSM was elevated in CRS and contributed to epithelial barrier dysfunction in this disease.

Methods

Results

Discussion

We have found that OSM was elevated in nasal polyp tissues derived from CRSwNP patients (Figure 1). Furthermore, studies using cultured bronchial and nasal epithelium demonstrated that stimulation with OSM at concentrations similar to those seen in vivo were sufficient to cause significant loss of barrier function and disorganization of tight junction structure (Figures 2 and 3). Additionally, OSM expression detected in nasal polyp tissue lysates correlated with nasal lavage levels of α2-macroglobulin, a marker of epithelial leak in localized nasal secretions (Figure 4). Together, these data suggest that OSM may be mediating epithelial barrier dysfunction in CRS, potentially leading to a chronic inflammatory state through increased exposure of the tissue to environmental factors including pathogens, allergens, and pollutants (figure 6). In support of this hypothesis, we found increased OSM mRNA in esophageal biopsies of EoE patients compared to controls (Figure 5), and increased OSM protein following allergen challenge in allergic asthmatics that correlated with epithelial leak in the BAL (Figure 5). Although these findings suggest that OSM may play a role in other diseases of mucosal epithelium that are also associated with type 2 inflammation, further studies are needed to verify these findings in other diseases.

The repair of epithelial damage occurs in two basic steps. First, basal cells proliferate and migrate into the wound to cover the damaged area, and second, the progenitor cells stop proliferating and differentiate into ciliated or goblet cells, and intercellular junctions are then formed to reestablish barrier function in the repaired tissue³³. While the mechanism of OSM mediated barrier dysfunction in mucosal epithelium in vitro is currently unknown, elevated OSM may be one signal that initiates the first step of repair following cellular damage. However, in vivo in a diseased state, high levels of OSM, or the absence of a signal to initiate the second step of the repair process, could potentially prevent the induction of the second step, leading to a chronically proliferative state in which differentiation and establishment of barrier function does not occur. OSM might achieve its barrier disrupting effects in vitro via a number of signaling pathways, including STAT1, STAT3, STAT5, phosphatidylinositol-3-kinase (PI3K) and the MAPK pathway^{34, 35, 36, 37, 38}. Future studies will be aimed at determining which of these pathways is responsible for the in vitro effects of OSM. Whether OSM disrupts barrier in vivo, and the molecular pathway by which it might do so, will be challenging to assess. The STAT3 pathway is of particular interest since it mediates both host defense responses as well as barrier maintenance and repair. In vivo studies of STAT3 phosphorylation in fresh primary epithelial cells from either healthy subjects or patients with CRS have proven to be technically difficult. Although we have previously shown a decrease in phosphorylation of STAT3 in whole nasal polyp tissue, it is quite possible that the level of STAT3 activation in epithelial cells differs from that of the whole polyp tissue^{39, 40}. The results of in vitro studies of possible signaling pathways for the OSM effects will hopefully point the way toward the pathways that should be the focus of future in vivo studies.

A recent study by Soyka et al investigated whether ex vivo sinus tissue from CRS patients had decreased resistance compared to control tissue. Large sinus tissue biopsies were obtained and resistance of the tissue was measured in an Ussing chamber. They found that tissue from CRSwNP patients had profoundly decreased resistance compared to control tissue. This is an important finding because it showed that the in vivo barrier function in CRSwNP sinus tissue was indeed impaired. The Soyka, et al study also showed decreased TEER in ex vivo cultured epithelia grown at air liquid interface from CRSwNP patients compared to epithelia from control patients¹³.

While the in vivo findings of Soyka et al. are in agreement with the present findings, the in vitro studies using epithelial cells are discordant with our results. As shown in figure 4A, we detected no differences in TEER among cells derived from CRS groups and control subjects, suggesting that barrier dysfunction in CRS is a cell extrinsic event. Many factors may explain the discrepancy between these findings, including differences in culture technique, nasal scraping technique, the anatomical location of the scrapings, timing of the measurements, or differences in levels of OSM in vivo, among others.

It is not yet clear which cells are responsible for the elevated production of OSM found in diseased mucosal tissues, but ongoing studies in our lab are aimed at identifying these cells in CRS. Many types of hematopoietic cells that are known to be present in CRS tissue have been shown to make OSM in vitro, including activated T cells, dendritic cells, mast cells, eosinophils, neutrophils, and macrophages^{41, 42, 43, 44}. It would be particularly interesting if M2 macrophages produce OSM in CRS. Our laboratory and others have previously shown that M2 macrophages are increased in nasal polyps compared to control UT, and these cells produced factor XIII-A and contributed to excessive fibrin deposition in nasal polyps^{45, 46, 47, 48}. Interestingly, OSM also has been shown to upregulate the production of fibrinogen, which is cleaved into fibrin by thrombin to provide substrate for fibrin deposition⁴⁹. Thus, future studies are needed to determine if OSM also plays a role in the fibrin deposition and tissue remodeling that occurs in nasal polyps.

Of the mucosal diseases analyzed in this manuscript, the pathogenesis of asthma has been most studied. Asthma is classically thought to involve a predisposition to the development of allergic or type 2 immune responses, leading to persistent inflammation of the airways and subsequent tissue remodeling. However, this allergic/type 2 immunity model fails to fully explain many clinical features of asthma, including the heterogeneity of asthma phenotypes and the lack of association between airway eosinophilia and airway hyper-responsiveness^{50, 51, 52, 53, 54}. An alternative model of asthma pathogenesis has been proposed, in which the generation of allergy and type 2 immunity is the result of epithelial barrier dysfunction⁵⁵. This barrier hypothesis is perhaps best supported by the finding that single nucleotide polymorphisms in the skin barrier gene, filaggrin, are associated with the development of atopic dermatitis and asthma, suggesting that barrier dysfunction precedes development of atopy, or at least sensitization⁵⁶. Epithelial dysfunction has been characterized in asthma patients compared to control patients. Asthma patients have decreased expression of intercellular junction proteins including occludin, ZO-1, and E-cadherin and have decreased barrier function assessed by TEER and dextran permeability^{7, 57, 58}. Additionally, reduced caveolin-1 expression in airway epithelium from asthma patients was shown to associate with decreased E-cadherin expression, and promoted epithelial expression of TSLP and IL-33, suggesting that barrier dysfunction may initiate type 2 immune responses⁵⁷. Thus, therapeutic intervention focused on either preventing barrier dysfunction, or restoring barrier once it is lost, may be effective in the treatment of mucosal diseases including asthma, CRS, and EoE.

Importantly, we have shown that the OSM-mediated decrease in barrier function in cultured NEC from CRS patients was reversible (Figure E2C), which suggests that therapeutic intervention targeting OSM has the potential to be beneficial in the treatment of CRS through the restoration of epithelial barrier function. OSM has been therapeutically targeted in rheumatoid arthritis (RA) through the neutralization of OSM, and the inhibition of the JAK/STAT pathway^{59, 60}. JAK inhibitors CP-690,550 and INCB028050, which inhibit JAK-1, JAK-2 and JAK-3, were shown to effectively block the activation of STAT1, STAT3 and STAT5 in OSM-treated fibroblasts in vitro⁶⁰. In contrast, a phase II trial in rheumatoid arthritis (RA) using an anti-OSM monoclonal antibody, GSK315234, did not show any benefit of blocking OSM, potentially due to low affinity OSM binding. However, the drug was well tolerated in patients and the study investigators concluded that a high-affinity anti-OSM antibody could be beneficial in the treatment of RA⁵⁹. Other potential strategies for the therapeutic neutralization of OSM include using soluble gp130 (sgp130), or receptor fusion proteins. Sgp130 is a natural inhibitor of IL-6 family cytokines, and treatment with this protein could potentially be effective in preventing the downstream effects of OSM. However, sgp130 is not specific to OSM and would also prevent downstream signaling of other IL-6 cytokine family members including IL-6, LIF, IL-11 and CNTF⁶¹. A receptor fusion protein (RFP) for the inhibition of OSM has been recently described in a murine model. The RFP consists of OSM ligand binding subunits of OSMR and gp130 connected by a flexible linker domain, and it was shown to be a highly potent and specific inhibitor of OSM⁶².

In summary, we have shown that OSM was elevated in the nasal polyps of patients with CRS, profoundly decreased barrier function of airway epithelium in vitro, and correlated with epithelial leak in nasal secretions in vivo. In addition, our data suggest that OSM may be increased in EoE and allergic asthma. Thus, OSM may play a role in barrier dysfunction in mucosal disease, and inhibition of OSM and/or its downstream effects may be beneficial in the treatment of diseases associated with type 2 inflammation and loss of barrier function by potentially restoring the barrier function of mucosal epithelial cells.

Supplementary Material