TERTIARY LYMPHOID STRUCTURES (TLS) EXPANSION PARALLELS ILEITIS PROGRESSION IN TNFΔARE MICE, REQUIRES β7 INTEGRIN AND CCR7, AND DECREASES IN SIZE AFTER GLUCOCORTICOID ADMINISTRATION

Abstract BACKGROUND Tertiary lymphoid structures (TLS) arise at sites of chronic inflammation, including the chronically inflamed terminal ileum in Crohn’s disease (CD), but their developmental progression, molecular requirements and responsiveness to anti-inflammatories are not completely understood. TNFΔARE mice develop extensive TLS as their ileitis worsens. Here, we characterized TLS development in TNFΔARE mice with chronic ileitis, examined their requirement for integrin β7 and CCR7 and assessed the effects of glucocorticoids on TLS. METHODS TLS formation was quantified (number, area) and maturation classified (Classes I–VI) over the time course of ileitis (4- to 20-weeks-of-age), compared with Peyer patches (PP). To assess the roles of β7 integrin and CCR7, heterozygous TNFΔARE (+/–) mice were crossed with β7-/- or CCR7-/- mice and assessed disease severity and TLS induction through the disease time course. Histopathologic disease severity in ileum, colon, and cecum was assessed by blinded pathological scoring. TLS architecture and cellular composition was assessed via light microscopy and immunofluorescence. Response to corticosteroids was assessed by 7-day I.P. dexamethasone (DEX) administration followed, followed by TLS quantification and histologic analysis. RESULTS TLS architecture in TNFΔARE ilea was consistent with previous descriptions in other tissues and models, including follicle-associated epithelium, B-cell follicles, organized myeloid cell networks and TRANCE signal. TLS were infrequent at 4-weeks-of-age, increased by 8 weeks and were abundant by 20 weeks, with a marked increase in number and area (p< 0.0001, 20 vs. 4 weeks), whereas Peyer’s patch (PP) metrics remained largely unchanged (not shown). At 20 weeks, β7–deficient TNFΔARE mice (TNFΔARE/β7-/-) displayed significantly reduced TLS number and area compared with CCR7-deficient TNFΔARE mice (TNFΔARE/CCR7-/-), indicating a critical requirement for β7 integrin for TLS formation. TLS maturation shifted progressively toward higher classes (IV–VI) between 4 and 20 weeks. Ileitis severity increased in TNFΔARE and TNFΔARE/β7-/- mice closely paralleling TLS progression. Dex attenuated ileitis (n = 5, p < 0.05) and shifted TLS toward less mature classes. CONCLUSIONS TLS induction (number, maturity) correlates with ileitis progression in TNFΔARE mice. Their distinctive architecture and cellular composition resemble single follicle PP. TLS formation requires β7 integrin and CCR7 whereas glucocorticoids attenuate ileitis severity and shifts TLS maturation towards its earlier stages. Given the parallel cellular composition between TLS and PP, they may act as inducible sites for IgA class switching and luminal IgA upregulation in response to inflammatory signals and associated changes in luminal microbiota composition.