Abstract Background Intestinal macrophages display remarkable plasticity1, yet the mechanisms driving their tissue specialization and response to inflammation remain poorly understood. Among the diverse macrophages, a PLA2G2D+ macrophage population has been described in mice, where it promotes immunoregulation through anti-inflammatory lipid mediators2. Methods We performed single-cell RNA-sequencing (scRNA-seq) of 6 healthy controls (HC), 36 ulcerative colitis (UC), and 50 Crohn’s Disease (CD) biopsies. Markers from each cellular subset were mapped onto a spatial transcriptomics (ST) dataset1 (CosMx-SMI, 3 HC, 3 UC, and 3 CD). Human monocyte-derived macrophages were cultured under different conditions and analysed by qPCR. Macrophage markers were detected by in situ hybridization (ISH), anti-CD68 staining, or Molecular Cartography in additional intestinal samples. Results Macrophage subsets (markers identified by scRNA-seq) were mapped to intestinal sections (Fig. 1A). In addition to resident macrophage populations (FOLR2+ and M0) or M1 macrophages (INHBA, IL1B), we identified a PLA2G2D-expressing population that was spatially restricted to isolated lymphoid follicles (ILFs) in HC and IBD samples, and present in CD-granuloma (GAs) (Fig. 1B). PLA2G2D+ macrophages were adjacent to B cells, T cells, and fibroblastic reticular cells, consistent with a canonical lymphoid niche localization3. In contrast, inflammatory SPP1+ macrophages (co-expressing S100A8/9 and MT1X) appear in ST as an abundant population in CD-GAs and areas of ulcerated mucosa, but not in ILFs. This was further confirmed using ISH and molecular cartography in additional patient samples (Fig. 1C and D). The distinct environments where each macrophage subset were found suggested different signals may drive their in vivo differentiation. LTα1β2, LIGHT and TNF, which are required for the formation of lymphoid tissues4, induced the expression of PLA2G2D in monocyte-derived macrophages in vitro, while IL-1β and hypoxic conditions drove SPP1 expression (Fig. 1E). Within ILFs, PLA2G2D+ macrophages interacted with B cells (the most abundant source of LTα1β2 in ILFs), while GA-SPP1 macrophages were found to interact with inflammatory fibroblasts (MMP1, CHI3L1, MMP3). Conclusion We demonstrate that PLA2G2D+ cells are a distinct macrophage population, driven by lymphoid tissue-promoting signals that may play a role in maintaining tolerance in gut-associated tissues. Importantly, PLA2G2D+ macrophages persist in IBD and are associated with tertiary lymphoid structures, including ILFs, and CD-GAs in a role that remains only partially understood. SPP1+ macrophages represent an independent subset polarized in GAs through IL-1β/hypoxia-driven pathways. References: 1.Garrido-Trigo A, Corraliza AM, Veny M, et al. Macrophage and neutrophil heterogeneity at single-cell spatial resolution in human inflammatory bowel disease. Nat Commun. 2023;14(1):4506. Published 2023 Jul 26. doi:10.1038/s41467-023-40156-6 2.Miki Y, Yamamoto K, Taketomi Y, et al. Lymphoid tissue phospholipase A2 group IID resolves contact hypersensitivity by driving antiinflammatory lipid mediators. J Exp Med. 2013;210(6):1217-1234. doi:10.1084/jem.20121887 3.Krausgruber T, Redl A, Barreca D, et al. Single-cell and spatial transcriptomics reveal aberrant lymphoid developmental programs driving granuloma formation. Immunity. 2023;56(2):289-306.e7. doi:10.1016/j.immuni.2023.01.014 4.Lorenz RG, Chaplin DD, McDonald KG, McDonough JS, Newberry RD. Isolated lymphoid follicle formation is inducible and dependent upon lymphotoxin-sufficient B lymphocytes, lymphotoxin beta receptor, and TNF receptor I function. J Immunol. 2003;170(11):5475-5482. doi:10.4049/jimmunol.170.11.5475 Conflict of interest: Sanzo Machuca, Angela: No conflicts. Moro Buendia, Marc: None Sabate, Sofia: No conflict of interest Veny, Marisol: Nothing to declare Gudiño, Victoria: No conflicts. Acera, Mario: No conflict of interest Dotti, Isabella: I declare no conflicts of interest Melón-Ardanaz, Elisa: Grant: This is an independent study promoted by IDIBAPS and financed by a Pfizer Independent Medical Grant (grant 54549477). Elisa Melón-Ardanaz is funded by grant RH042155 (RTI2018-096946-B-I00) from Ministerio de Ciencia e Innovacion. Garrido Trigo, Alba: Alba currently works as Posdoctoral Researcher at Roche Lopez-Prades, Sandra: No conflict of interest Carrasco, Antonio: No conflict of interest Fernandez Clotet, Agnes: None Ordás Jiménez, Ingrid: I have received financial support for travel and educational activities, and have served as a speaker or advisory board member for the following companies AbbVie, MSD, Pfizer, Takeda, Janssen, Kern Pharma, Chiesi, Falk Pharma, and Faes Farma. I have also received research funding from AbbVie, Faes Farma, and Takeda. Martin Cardona, Albert: No conflict of interest Loras, Carmen: No conflict of interest Esteve Comas, Maria: No conflict of interest Mereu, Elisabetta: No conflict of interest Cuatrecasas, Miriam: No conflict of interest Ricart Gomez, Elena: E. Ricart has received support for congress and conference attendance, speaker fees, research support or consulting fees from MSD, Abbvie, Ferring, Janssen, Otsuka, Pfizer, Takeda, Faes Farma, Galapagos/Alphasigma, Kern Pharma, Lilly, J & J, Dr Falk Pharma, and Fresenius-Kabi. Salas, Azucena: Grant: Takeda, Roche, Nestle, Pfizer, Genentech, AbbVie, GSK, Scipher Medicine, Alimentiv, Inc, Boehringer Ingelheim and Agomab. Personal Fees: Lilly, Johnson & Johnson, Genentech, GSK, Pfizer, Galapagos, AdBio Partners, HotSpot Therapeutics, Alimentiv, Nestle, GoodGut and Agomab.
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