The role of liver X receptor (LXR) in intestinal inflammation and mucosal healing

Tissue repair following inflammatory damage is a critical process, essential for restoring tissue homeostasis. Nowhere is this more crucial than in the intestine, where the gut epithelium endures constant assaults from physical, chemical, and microbial threats. These challenges are ever-present, whether under normal conditions or during severe episodes of chronic intestinal diseases like inflammatory bowel disease (IBD). When an IBD flare-up occurs, the body is thrust into a dual struggle: it must urgently calm the overactive immune response while simultaneously mending the compromised epithelial barrier, a critical step toward achieving true mucosal healing.

Current treatments aim to suppress inflammation by blocking inflammatory pathways, which, while necessary to quell the immediate immune response, may also promote the regenerative processes required for healing. Yet, the reality is stark: up to 50% of IBD patients either don't respond to these therapies or suffer relapses after an initial improvement. Despite the clear link between mucosal healing and long-term remission, no therapies currently exist that specifically promote epithelial regeneration to facilitate this healing process.

We believe that the signals triggered by inflammation and tissue damage are intricately intertwined with the body's repair mechanisms. Effective communication between various cellular components-including but not limited to epithelial, immune, and stromal cells-is crucial for maintaining a healthy intestinal barrier under normal conditions and restoring it after injury and inflammation. In my thesis, I set out to unravel these complex interactions to deepen our understanding of the cellular and molecular mechanisms that drive intestinal inflammation damage and tissue repair.

In Study I, we used spatial transcriptomics to reveal a transcriptomics profile in mouse colon at steady state and during mucosal healing with a previously unachieved spatial resolution. By integrating tissue bulk RNA sequencing and single cell RNA sequencing datasets from mouse and human, we identified distinct molecular regionalization and compartmentalized transcriptional programs with human relevance. This study provides a useful methodology and resource for the followings studies to understand mechanism involved in tissue regeneration.

In Study II, B cell expansion was found to be a major event within the immune compartment during the tissue regeneration phase following withdrawal of Dextran Sulfate Sodium (DSS). Depletion of B cells from mice during recovery phase resulted in an improved outcome in experimental colitis. Mechanistic study using tissue and single cell RNA sequencing and immunostaining validation revealed that expanded B cells interfered with the interaction between stromal cells and epithelium by physically reducing their proximity. The findings emphasize the impact of cellular interactions and the local microenvironment on tissue regeneration. They suggest that targeting B cells during the regenerative phase may be a promising approach to improve the management of colitis and related intestinal disorders.

In Study III, Liver X receptor (LXR) was identified as a common pathway induced by tissue damage in the intestine. Activation of LXR by feeding a synthetic LXR ligand GW3965 in diet resulted in enhanced crypt cell proliferation in vivo with an improved histological outcome in DSS and irradiation induced damage. Mechanistically, the pro-regenerative effect of LXR is achieved by inducing amphiregulin expression in epithelial cells and regulated by the upstream endogenous ligands producing enzyme Cyp27a1. Surprisingly, the elevated regenerative capacity did not result in increased tumor burden, in contrast, activating LXR modulates the anti-tumor immunity and suppressed tumorigenesis in both AOM-DSS induced and ApcMin/+ tumor models. This study unveiled the bi- directional modulation of LXR in enhancing regeneration while controlling tumorigenesis.

In Study IV, we observed that LXR activation dampened tuft cell and Type 2 innate lymphoid cells (ILC2) abundance in the small intestine of wild type C57BL/6 mice during homeostasis. The decreased tuft cell and ILC2 number failed to be rescued by an inducer of tuft cell expansion, i.e. succinate but was rescued by rIL-25 in vitro and in vivo and. This negative regulation resulted in impaired anti-helminth response in the mouse infected by Nippostrongylus brasiliensis and Heligmosomoides polygyrus, as well as impaired mast cell activation, indicating a role of LXR in suppressing type 2 immunity. Similarly, LXR activation in BALB/c mice was found to inhibit mast cells and showed a protective role by preventing weight loss in an OVA-induced food allergy model. This study underscores the function of LXR in controlling innate type 2 immunity during mucosal homeostasis and in limiting type 2 inflammation upon challenges.

Collectively, this thesis contributes to spatially resolved transcriptomic profile of murine intestine in naïve and challenged conditions, underscoring the novel finding of LXR modulation in intestinal inflammation and mucosal healing. The findings in thesis further our understanding of restoring balance, enhancing tissue resilience, and paving the way for novel treatments that could transform the management of inflammatory diseases in the intestine, such as IBD.

List of scientific papers

I. Sara M. Parigi#, Ludvig Larsson#, Srustidhar Das#, Ricardo O. Ramirez Flores, Annika Frede, Kumar P. Tripathi, Oscar E. Diaz, Katja Selin, Rodrigo A. Morales, Xinxin Luo, Gustavo Monasterio, Camilla Engblom, Nicola Gagliani, Julio Saez-Rodriguez, Joakim Lundeberg & Eduardo J. Villablanca*

The spatial transcriptomic landscape of the healing mouse intestine following damage Nature Communication. 13, 828 (2022). https://doi.org/10.1038/s41467-022-28497-0

II. Annika Frede#, Paulo Czarnewski#, Gustavo Monasterio#, Kumar P Tripathi, David A Bejarano, Ricardo O Ramirez Flores, Chiara Sorini, Ludvig Larsson, Xinxin Luo, Laura Geerlings, Claudio Novella-Rausell, Chiara Zagami, Raoul Kuiper, Rodrigo A Morales, Francisca Castillo, Matthew Hunt, Livia Lacerda Mariano, Yue O O Hu, Camilla Engblom, Ana-Maria Lennon- Duménil, Romy Mittenzwei, Astrid M Westendorf, Nadine Hövelmeyer, Joakim Lundeberg, Julio Saez-Rodriguez, Andreas Schlitzer, Srustidhar Das, Eduardo J Villablanca*

B cell expansion hinders the stroma-epithelium regenerative cross talk during mucosal healing Immunity. 2022 Dec 13;55(12):2336-2351.e12. https://doi.org/10.1016/j.immuni.2022.11.002

III. Srustidhar Das*#, S. Martina. Parigi#, Xinxin Luo#, Jennifer Fransson, Bianca Carola Kern, Ali Okhovat, Oscar E. Diaz, Chiara Sorini, Paulo Czarnewski, Anna T. Webb, Rodrigo A. Morales, Sacha Lebon, Gustavo Monasterio, Francisca Castillo, Kumar P. Tripathi, Ning He, Penelope Pelczar, Nicola Schaltenberg, Marjorie De la Fuente, Francisco López-Köstner, Susanne Nylén, Hjalte List Larsen, Raoul Kuiper, Per Antonson, Marcela A. Hermoso, Samuel Huber, Moshe Biton, Sandra Scharaw, Jan-Åke Gustafsson, Pekka Katajisto, Eduardo J. Villablanca*

Liver X receptor unlinks intestinal regeneration and tumorigenesis. Nature. [Accepted]

IV. Xinxin Luo, Qilin Zhu, Jennifer Fransson, Tilde Andersson, Ning He, Marta Campillo Poveda, Claire Ciancia, Ricardo O. Ramírez Flores, Ludvig Larsson, Sara Martina Parigi, Rodrigo A. Morales Castro, Bianca C. Kern, Francisca Castillo, Joakim Lundeberg, Julio Saez-Rodriguez, Srustidhar Das, Rick M. Maizels, Christoph Schneider, and Eduardo J. Villablanca*

Liver X Receptor modulates type 2 immune responses in parasitic infection and food allergy. [Manuscript]

# Equal contribution

* Corresponding author