The seasonal burden of influenza coupled with the pandemic outbreaks of more pathogenic strains underscore a critical need to understand the pathophysiology of influenza injury in the lung. Interleukin-22 (IL-22) is a promising cytokine that is critical in protecting the lung during infection. This cytokine is strongly regulated by the soluble receptor IL-22-binding protein (IL-22BP), which is constitutively expressed in the lungs where it inhibits IL-22 activity. The IL-22/IL-22BP axis is thought to prevent chronic exposure of epithelial cells to IL-22. However, the importance of this axis is not understood during an infection such as influenza.Here we demonstrate through the use of IL-22BP-knockout mice (il-22ra2 −/− ) that a pro-IL-22 environment reduces pulmonary inflammation during H1N1 (PR8/34 H1N1) infection and protects the lung by promoting tight junction formation. We confirmed these results in normal human bronchial epithelial cells in vitro demonstrating improved membrane resistance and induction of the tight junction proteins Cldn4, Tjp1, and Tjp2. Importantly, we show that administering recombinant IL-22 in vivo reduces inflammation and fluid leak into the lung. Taken together, our results demonstrate the IL-22/IL-22BP axis is a potential targetable pathway for reducing influenza-induced pneumonia.Mucosal Immunology (2020) 13:64-74; https://doi.

Background Influenza attacks the epithelium of the lung, causing cell death and disruption of the epithelial barrier leading to fluid buildup in the lung and impairment of gas exchange. Limited treatment options for severe influenza pneumonia prioritize the need for the discovery of effective therapies. IL-22 is a cytokine that promotes tissue integrity and has strong promise as a treatment option. While research has been focused on the cytokine itself, there is limited understanding of the regulation of the IL-22 receptor (IL-22Ra1) at the epithelial surface during infection. Methods IL-22Ra1 levels were measured by qRT-PCR, western blot and immunofluorescence following H1N1 influenza infection (A/PR/8/34 H1N1) or synthetic TLR3 mimetic, Poly (I:C). Regulation of the receptor was determined using STAT inhibitors (STAT1, STAT3 and PanSTAT inhibitors), TLR3 inhibition, and neutralization of interferon alpha receptor 2 (IFNAR2). Significance was determined by a p -value of greater than 0.05. Significance between two groups was measured using unpaired t-test and significance between more than two groups was measured using one-way ANOVA with Tukey Multiple Comparison Test. Results Here we show both in vivo and in vitro that IL-22Ra1 was induced as early as 24 h after influenza (H1N1 PR8) infection. This induction was triggered by toll-like receptor 3 (TLR3) as a TLR3 mimetic [Poly (I:C)] also induced IL-22Ra1 and inhibition of endosomal formation required for TLR3 function inhibited this process. This upregulation was dependent upon IFNβ signaling through STAT1. Importantly, induction of IL-22Ra1 significantly increased IL-22 signaling as evidenced by pSTAT3 levels following IL-22 treatment. Conclusion Collectively, these data suggest epithelial cells may optimize the beneficial effects of IL-22 through the induction of the IL-22 receptor during viral infection in the lung.

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Axl is a receptor tyrosine kinase (RTK) that is activated by its ligand Gas6 and regulates proliferation, cell survival, and clearance of apoptotic cells in both normal physiology and a number of disease states. In breast cancer, Axl is overexpressed in Her2+ and triple-negative breast cancers and is associated with resistance to RTK targeted therapies. Recent studies have shown that upon kinase inhibition, the Axl intracellular domain (ICD) can translocate to the nucleus, however, the biological significance and functions for nuclear Axl are poorly understood. Furthermore, a functional role for Axl during ductal carcinoma in situ (DCIS) progression, a requisite step for invasive breast cancer, has not been established. To address whether Axl mediates localized invasion during DCIS progression, Axl was deleted in a DCIS cell line (MCF10A-DCIS.com) using a CRISPR-Cas9 approach. Deletion of Axl shows a significant decrease in progression to invasive cancer in vivo, which is accompanied by decreased proliferation, however microinvasion is unchanged as compared to wildtype. Analysis of patient samples of pure DCIS, DCIS with microinvasion, and DCIS with invasive cancer shows a significant increase in Axl expression in patients with evidence of invasion. Interestingly, there is a significant increase in nuclear Axl in all patient samples with evidence of invasion as compared to normal breast or pure DCIS, suggesting an important role for Axl-ICD in progression to invasive cancer. Soft agar assays using normal cells (MCF10A) shows that while full length Axl (Axl-FL) is sufficient to induce colonies, Axl-ICD-transduced cells do not form colonies, suggesting that Axl-ICD alone does not have oncogenic activity. However, Axl-ICD significantly increases colonies in DCIS cells, suggesting that the ICD is important in the progression of transformed cells. Ongoing studies are aimed at identifying transcriptional targets of Axl-ICD in DCIS cells. These studies will advance our understanding of how Axl mediates breast cancer progression and will have critical implications for ongoing therapeutic strategies aimed at targeting Axl. This work is supported by NIH R01CA212518 (H.L.M.) and Susan G. Komen CCR1637765 (H.L.M.). Citation Format: Darby R. Graham, Kelly D. Hebert, Emma E. Newton, Angelica M. Gomes, Fariba Behbod, Heather L. Machado. Nuclear translocation of Axl during early breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 861.