Facilitation of IL-22 production from innate lymphoid cells by prostaglandin E2 prevents experimental lung neutrophilic inflammation

Acute lung injury is a neutrophil-dominant, life-threatening disease without effective therapies and better understanding of the pathophysiological mechanisms involved is an urgent need. Here we show that interleukin (IL)-22 is produced from innate lymphoid cells (ILC) and is responsible for suppression of experimental lung neutrophilic inflammation. Blocking prostaglandin E2 (PGE2) synthesis reduces lung ILCs and IL-22 production, resulting in exacerbation of lung neutrophilic inflammation. In contrast, activation of the PGE2 receptor EP4 prevents acute lung inflammation. We thus demonstrate a mechanism for production of innate IL-22 in the lung during acute injury, highlighting potential therapeutic strategies for control of lung neutrophilic inflammation by targeting the PGE2/ILC/IL-22 axis.


ABsTrACT
Acute lung injury is a neutrophil-dominant, lifethreatening disease without effective therapies and better understanding of the pathophysiological mechanisms involved is an urgent need. Here we show that interleukin (IL)-22 is produced from innate lymphoid cells (ILC) and is responsible for suppression of experimental lung neutrophilic inflammation. Blocking prostaglandin E 2 (PGE 2 ) synthesis reduces lung ILCs and IL-22 production, resulting in exacerbation of lung neutrophilic inflammation. In contrast, activation of the PGE 2 receptor EP4 prevents acute lung inflammation. We thus demonstrate a mechanism for production of innate IL-22 in the lung during acute injury, highlighting potential therapeutic strategies for control of lung neutrophilic inflammation by targeting the PGE 2 /ILC/ IL-22 axis.

InTroduCTIon
Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a life-threatening neutrophil-dominant disease with a significant morbidity and mortality for which effective therapies are currently lacking. 1 Excessive pulmonary neutrophil recruitment mediates lung tissue damage, despite the beneficial role of neutrophils in promoting tissue repair. 2 Therefore, there is a need for more effective medicinal agents for use in these severe and often lethal lung injury syndromes. 1 Interleukin (IL)-22 is an IL-10 family member cytokine that is produced by both adaptive and innate immune cells. Recently, type 3 innate lymphoid cells (ILC3s) have been identified as the main source of innate IL-22. 3 IL-22 is expressed in healthy human lung tissue, and patients with sarcoidosis and ARDS have decreased IL-22 levels. 3 Innate IL-22 is protective against acute epithelial damage and inflammation in the lung as neutralisation of IL-22 exacerbates bacterial and viral infections and exogenous IL-22 attenuates bacterial pneumonias. 4 Non-steroidal anti-inflammatory drugs (NSAID) inhibit cyclooxygenase activity and subsequent production of prostaglandins and are very widely used to manage many inflammatory conditions. Use of NSAIDs worsens lung bacterial infections and is a risk factor for severe sepsis. 5 In contrast, prostaglandins including prostaglandin E 2 (PGE 2 ) are used for treating critical lung diseases with improved oxygenation and decreased pulmonary artery pressures. 6 PGE 2 exerts its biological actions through engagement of its four receptors, namely EP1-4. We have recently found that PGE 2 promotes IL-22 production from both T cells and ILC3s. 7 8 In this study, we have investigated the hypothesis that PGE 2 inhibits acute lung neutrophilic inflammation through modulating lung ILC3 production of IL-22.

MeThods
Wild-type C57BL/6 mice were purchased from Harlan UK. Rag1 -/mice and mice with selective EP4 deficiency in T cells (Lck Cre EP4 fl/fl mice by crossing LckCre mice to EP4-flox mice) 9 were maintained under specific pathogen-free conditions in accredited animal facilities. Mice were aged >7 weeks old at the beginning of use. All experiments were conducted in accordance with the UK Scientific Procedures Act of 1986 and had local institutional ethical approval. The ALI mouse model was induced by intratracheal injection of 10 µg of lipopolysaccharide (LPS) in combination with recombinant IL-22, EP2 and/or EP4 agonists, or indomethacin when indicated. After 24 hours, bronchoalveolar lavage (BAL) fluid and lung tissues were collected. Immune cells and cytokine production were measured by flow cytometry or ELISA. All data were expressed as mean±SD. The Student's t-test or Mann-Whitney U test was used for statistical analyses by Prism V.6 (GraphPad) and p<0.05 was considered statistically significant.

resulTs
To study the cellular sources of innate IL-22 in the lung, we administered a low dose (10 µg) of LPS to Rag1 -/mice, which have no adaptive T and B cells, and analysed IL-22 production from various lung immune cells 24 hours later using flow cytometry. In naïve mice, very few (~0.1%) CD11b + CD-11c + Ly-6G − mononuclear phagocytes (MNP) or CD11b + Ly-6G + neutrophils produced IL-22, while ~0.4% CD11b − CD11c − Ly6G -CD90. suggesting that IL-22 production was also increased at the single cell level ( figure 1C). Consistently, in response to appropriate stimulus lung innate immune cells produced IL-22 ( figure 1D). IL-22 has been demonstrated to protect against inflammation at mucosal sites including the lung. 3 To test whether IL-22 protects against ALI, we administered recombinant IL-22 intratracheally into mice immediately before LPS challenge. Exogenous IL-22 significantly suppressed LPS-induced acute lung neutrophilic inflammation (p=0.006) but had little effect on BAL total protein levels ( figure 1E, n=3-5).

dIsCussIon
Numerous experimental and clinical observations have shown that IL-22 plays a critical role in the control of mucosal injury and inflammation including pneumonia and ALI. 3 This study further demonstrates that lung ILC3s are the major contributors of innate IL-22, which displays protective actions against neutrophilic inflammation in the airway. IL-22 has been reported to either protect against or promote allergic lung inflammation in the absence or presence of IL-17, respectively, suggesting that IL-22 functions in the lung in context-dependent manners. 10 To augment IL-22 production or signalling, several approaches could be used, for example, application of IL-22 in protease-resistant forms via oral or intrapulmonary delivery or application of small chemical molecules that promote IL-22 production. 3 Our findings show that PGE 2 and its analogues (eg, EP4 agonists) could be used to catalyse the ILC3-IL-22 cascade and to facilitate the control of acute lung inflammation ( figure 2H). Many lung cell types (eg, macrophages, damaged epithelial cells and neutrophils) can produce PGE 2 , which is essential to maintain ILC3 homeostasis and activation. 7 Our findings highlight potential therapeutic strategies for control of lung neutrophilic inflammation by targeting the PGE 2 /ILC3/IL-22 pathway. It is envisioned that targeting this pathway by pharmacological intervention could have potential for the development of novel strategies for the treatment of lung inflammation especially in diseases where the neutrophil plays a prominent role.
Despite these important novel findings, we acknowledge this report has several limitations. First, the numbers of animals used in experiments were limited, so further validation of the findings may be needed by increasing mice numbers. Second, therapeutic effects of PGE 2 analogues and rIL-22 on established neutrophilic lung inflammation induced by LPS or by other stimuli were not examined. Third, we have shown that exogenous IL-22 prevented ALI and that blocking PGE 2 signalling reduced numbers of IL-22 + ILC3s in the lung. However, given the multiple roles of IL-22 in lung homeostasis as well as injury/ inflammation, further studies to determine direct roles of endogenous IL-22 in ALI are warranted. Fourth, the protective actions of the PGE 2 /ILC3/IL-22 axis have not been examined in human patients with ALI/ARDS, although findings from our mouse studies could have important translational implications.