IL-18 associated with lung lymphoid aggregates drives IFNγ production in severe COPD

The pathogenesis of Chronic Obstructive Pulmonary Disease (COPD) is associated with epithelial dysfunction and the excessive recruitment of inflammatory cells in response to chronic insult of the lung [1]. Among tissue infiltrating leukocytes are effector cells of both innate and adaptive immune responses, such as neutrophils, macrophages, NK cells, T cells and B cells [2]. Apart from classic tissue infiltration, accumulation of immune cells in COPD lungs is also manifested as formation of bronchial/bronchiolar-associated as well as alveolar lymphoid aggregates (LA), and the number of these structures increases with disease severity [2, 3]. This de novo formation of lymphocyte and macrophage-rich lymphoid aggregates, referred to as ectopic lymphoid tissue, is a well-recognised example of immunological remodelling in COPD. Whilst intact immune responses to pathogens are required to protect vulnerable COPD patients from infection, an inappropriate response in a chronically inflamed lung may trigger exacerbations of symptoms and contribute to accelerated decline in lung function [4].

Underlying mechanisms responsible for lung tissue destruction are not well understood but an aberrant response of NK cells and T cells, CD8⁺ T cells in particular, is thought to play an important role [5, 6]. The dysregulation of the cytotoxic function of NK cells and CD8⁺ T cells may lead to the destruction of the lung parenchyma through excessive response to pathogens and bystander damage to surrounding tissues. Production of interferon gamma (IFNγ) by inflammatory T cells, NK and NKT cells is an important component of the host response, inhibiting viral replication, promoting and perpetuating antigen presentation via increased expression of MHC molecules and priming other leukocytes, notably macrophages [7]. In lymph nodes that drain infected tissues, prompt lymphocyte-macrophage communication, involving rapid production of IFNγ from resident lymphocytes and release of interleukin-18 from proximally associated macrophages, is crucial to limit the spread of infection [8]. This response is too rapid to be explained by conventional adaptive T cell function or plasticity. Kastenmuller et al. [8] demonstrated the critical role of IL-18, and also an additional unidentified second signal, in this innate IFNγ response, and established the requirement for spacial organisation of the macrophages and lymphocytes within the lymph node to facilitate it.

There is ample clinical evidence of IFNγ being a significant component of the immunopathology in COPD. IFNγ is increased in the airways of COPD patients [9, 10] and associated with viral exacerbations of disease [11, 12]. Furthermore, IFNγ-positive T cells are increased in COPD patients [13] where disease severity correlates with IFNγ release by peripheral patient-derived CD8⁺ cells [14]. The fact that excessive IFNγ in the lungs of mice is associated with development of airspace enlargement [15, 16] further underscores the pathogenic potential of IFNγ. Therefore, better understanding of the cellular and molecular regulation of IFNγ in the lungs of COPD patients is an essential step towards determining whether this mechanism plays a part in the link between excessive chronic inflammation, exacerbation and increase in the rate of decline.

We and others showed previously that IL-1α, IL-1β and IL-18 were increased in the lung of COPD patients [17, 18] and that these mediators were released by epithelial cells on infection with rhinovirus [19]. Consequently, we hypothesised that these cytokines could chronically stimulate an innate IFNγ response via local lymphocyte-macrophage communication in inflamed lungs as well as during exacerbation of disease. In this regard, ectopic lymphoid follicle formation in COPD is of particular interest. The present study investigates which mediators most closely correlate with IFNγ in sputum in stable and exacerbating disease, and seeks to shed light on the spatial requirements for innate production of IFNγ, as reported in mouse lymph nodes, to observe whether such microenvironmental cellular organisation [8] is relevant to IFNγ production in COPD lung.

IL-1 family members play an important role downstream of pattern recognition receptors in the maintenance of mucosal surface integrity. IL-1 and IL-18 in particular are associated with the recruitment and activation of myeloid and lymphoid effector cells. In this study, we have demonstrated that, in the context of lung inflammation involving a complex mix of mediators and despite disease heterogeneity, IL-1 and IL-18 are statistically significantly correlated with IFNγ in the sputum of COPD patients. Both mediators, released from epithelial cells or macrophages, likely are important in driving an IFNγ response in NK cells in the lung, with different spatial requirements. In agreement with recent observations in the human gastro-intestinal tract [22] we further revealed that IL-18⁺ macrophages and dendritic cells are in COPD foremost localized to lung lymphoid aggregates. Increased lymphoid aggregates in the lung are a histological hallmark of severe COPD status and this study is the first to highlight that IL-18- expressing myeloid cells within these lung lymphoid aggregates may significantly influence IFNγ release from closely-situated lymphocytes, such as NK cells, thereby contributing to tissue destruction in severe COPD pathology.

Interferon γ expression is expected within a pro-inflammatory lung environment and indeed, in 82 out of 105 COPD sputum from GOLD II and GOLD III patients the IFNγ level was detectable. Reports of the direct measurement of IFNγ at the protein level in the lung of COPD patients are scarce, most of the time relying on the isolation of NK or T cells from bronchoalveolar lavage or lung parenchyma followed by assessment of interferon response to stimulation ex-vivo [5, 23, 24], and where IFNγ levels have been measured in sputum, they are consistent with those reported here [12]. Interferon γ is a challenging cytokine to measure in sputum samples at least in part due to its relative sensitivity to sputum processing [25]. Dithiothreitol (DTT) used in standardized sputum processing could affect detection of specific soluble mediators, in addition to IFNγ, and although we did not systematically analyse the effects of DTT on our detection limits, it is likely that the presence of DTT could have uniformly affected select analytes across all samples. In addition, factors such as the severity of the disease, the triggers of exacerbation and the timing of sampling may influence the mediators that can be measured. We also note that sputum was obtained spontaneously, which confines the sputum analysis to a more chronic bronchitic patient phenotype, whereas the surgical samples used for IHC reflect a broader COPD population. In our sputum cohort, higher levels of IFNγ were associated with higher levels of IL-1α, IL-1β and IL-18, as well as general inflammation markers such as CRP and fibrinogen. Interleukin-18 receptor is primarily expressed on T cells, NK cells and subsets of innate lymphoid cells, and IL-18 is associated with the induction of IFNγ production by these cell types [26, 27]. Whilst IL-1 is generally linked to a neutrophilic response and Th17 cell differentiation, like IL-18, it has been reported to promote IFNγ release from cytotoxic lymphocytes [28, 29]. Although other inflammatory mediators may contribute to IFNγ production, we did not observe any correlation with IL-12, IL-15, or other molecules previously reported to be associated with IFNγ and/or viral infections such as IP-10, MIG, I-TAC, MCP-1, RANTES [12]. These observations re-inforce the likelihood of IL-18 and IL-1 being the strongest drivers of IFNγ.

In order to further understand the relationship between IL-18 and IFNγ in the lung tissue of COPD patients, we developed a protocol for the detailed phenotyping of IL-18-producing cells. Consistent with the previous observation that the bronchial epithelium is an important source of IL-18 following viral infection [19], we found IL-18 staining associated with bronchial epithelial cells. Given the rapid release of IL-18 following viral infection of NHBEs [19], it is of particular interest that we observed a novel and characteristic apical staining of IL-18 in airway epithelium in COPD, which is of stronger intensity than the general staining in epithelium, suggesting IL-18 is concentrated at the apical surface of differentiated epithelium, possibly localized for rapid release. Given that the extent of this apical staining correlated with disease severity, further studies to evaluate the contribution of IL-18 in viral exacerbations in COPD patients are warranted. We have not explored here whether IL-18 released from the epithelium can activate lymphocytes in the airway lumen, or whether the IFNγ measured in sputum is purely as a result of cytokine production within the tissue. This could warrant further analysis since we suggest locally high production of IFNγ within the tissue is more likely to invoke detrimental effects to tissue structure, whilst epithelial release of IL-18 may be beneficial to pathogen clearance.

In addition to moderate alveolar macrophage expression, we saw very strong staining in macrophages and dendritic cells associated with lymphoid aggregates in the lung, which was not seen for IL-1 expression, despite observing IL-1⁺ macrophages in the tissue [17]. We demonstrated that the overall expression of IL-18 in the lung increased as a direct consequence of the enhanced number of macrophage and dendritic cell-enriched alveolar-lymphoid interfaces in severe disease. It has been demonstrated that not only bronchiolar associated lymphoid structures, but also alveolar aggregates, correlate with disease severity and may represent remodelled antigen presentation in the COPD lung [3]. This is the first description of IL-18 expression associated with myeloid cells specifically in tertiary lymphoid structures in COPD, and was not reported in a previous study of IL-18 staining in COPD lung [18]. We note that the staining in lymphoid aggregates is strikingly similar to IL-18 staining reported by Bombadieri et al. in tertiary follicles found in salivary glands in Sjörgren’s Syndrome [30], and the presence of IL-18 producing cells in lymphoid aggregates in Crohn’s disease [22]. Given elastin autoreactive T cells isolated from COPD blood have been shown to produce significant quantities of IFNγ [31] and lymphoid follicles in more severe COPD have been linked to an autoimmune-like phenotype (reviewed in [32]) this is a particularly striking parallel.

Given the clear association of IL-18 with lymphoid aggregates, with a pattern of distribution of IL-18⁺ cells which closely represents that described in lymph nodes in infection (including sub-capsular like IL-18⁺ cells) [8], we hypothesised that the same mechanisms which relate to cellular positioning described in lymph nodes to regulate IFNγ may be important in these ectopic follicle-like structures in the lung, and therefore become increasingly dominant as COPD progresses. However, quantifying IFNγ expression by IHC or ISH in tissues is technically challenging and we were not able to perform this analysis to our satisfaction in formalin-fixed paraffin embedded COPD lung tissues that were available for this study. Kastenmuller et al. [8] described how myeloid cells, acting as sentinels in the earliest detection of pathogens in the lymph node, were closely localised with diverse lymphoid cells in order to rapidly facilitate an innate immune amplification loop to minimise systemic pathogen spread. Their data suggested that IL-18 with a second signal was responsible for a rapid increase in lymphoid cell IFNγ production driven by cellular proximity. We therefore tried to understand the relationship between endogenous IL-18 release and IFNγ response in pathologically relevant primary cell in vitro model systems. Whilst the contribution to NK IFNγ production by IL-1 was immediately apparent, the dependence on IL-18 for this response was crucially only revealed when IL-18-producing cells were cultured in close proximity to the IFNγ-producing cells. Co-staining for IL-18 and CD56⁺ in COPD tissue, the close association of CD56⁺ NK/NKT cells with IL-18⁺ cells could be observed in the lymphoid aggregates, reminiscent of what has been reported in secondary lymphoid organs.

Although less frequent in control samples, or mild disease, lymphoid aggregates are occasionally observed, and this is likely as a normal response to a previous infection. The increase in lymphoid aggregates and concomitant increase in IL-18 may therefore be a physiological response to increased bacterial colonisation and bacterial and viral infection frequency in the vulnerable lungs of severe COPD patients. However, they may also contribute to emphysema progression, particularly since the localised effects of IL-18 on tissue resident responder cells may be to increase IFNγ without the requirement for cognate antigen. Both IL-18 and IFNγ have been reported to contribute to emphysema pathogenesis in mouse smoke [15, 16, 33, 34], and increases in lymphoid aggregates are reported in mouse smoke models in a similar timeframe to emphysema progression [35]. IL-18 has been shown to stimulate increased IFNγ production by lymphocytes isolated from COPD lung compared to smoking control tissue [5], and the cytotoxic potential of CD56⁺ cells increased with disease severity [6], therefore future studies should attempt to determine whether this phenotype is indeed evident within lymphoid follicles in COPD lung tissue with IL-18 as a key driver. Recent data also showed that CD4⁺ cells expressing IL-18Rα at mucosal surfaces have innate immune functionality and co-localise with IL-18-producing cells in lymphoid aggregates in chronic inflammation in the gut [22]. Additionally, Kearley et al. have described an altered phenotype and responsiveness of NK and ILC1s in smoked mice, which could also be relevant to plasticity of the innate cell responders and production of IFNγ in COPD [36].

We have shown that IL-18 released in pathologically relevant situations, such as from epithelium after infection or from myeloid cells in lymphoid aggregates, has a spatially limited function with respect to its rapid innate regulation of IFNγ. In contrast to its physiological function to control infection spread in lymphoid organs which is tightly regulated, this local interface between IL-18 expressing cell and responder cell is increasingly supported in the lung tissue as the severity of disease increases, and this may directly impact its potential to contribute to tissue damage via augmented IFNγ release. This may have been underappreciated in previous studies of COPD if the localisation of IL-18 and its functions has not been taken into account, and may also be relevant in other chronic inflammatory diseases.