DIFFERENTIAL ROLES OF TNFRI AND TNFRII IN THE MORPHOLOGY OF SECONDARY LYMPHOID ORGANS

Background:Tumour necrosis factor (TNF) induced signaling events are important in lymphoid organ development and function, both in health and in immune-mediated inflammatory diseases such as arthritis. Important receptors involved in this process include TNF receptors (R) I and II that exert distinct functions. Mice overexpressing transmembrane (tm)TNF develop various features of chronic inflammation, including arthritis, that are mediated via TNFRI and/or TNFRII.Objectives:To investigate the importance of TNFRI and TNFRII in spleen and peripheral lymph node (PLN) morphology in mice overexpressing tmTNF.Methods:Spleen and PLN were collected from 13 week old mice with the following genotypes: WT, tmTNF transgenic heterozygotes (tmTNF tg HET), WT x TNFRI-/-, tmTNF tg HET x TNFRI-/-, WT x TNFRII-/-and tmTNF tg HET x TNFRII-/-. Spleens were cut and stained by immunofluorescence for B and T cell markers, imaged by confocal microscopy and analyzed using FIJI software. PLN were used for whole mount tissue staining for B and T cell markers, cleared, 3D imaged by light sheet microscopy and analyzed using Imaris software.Results:Although no macroscopic differences were observed, spleens of tmTNF tg HET animals exhibited an altered morphology characterized by smaller follicles (69,73 ± 72,25 µm2) compared to spleens of WT mice (139,3 ± 71,36 µm2), which was accompanied by a decrease in central T cell areas as measured by % of the total follicle (15,22 ± 9,685; WT 26,60 ± 10,46). WT mice lacking either TNFRI or TNFRII had a morphology comparable to WT (follicle size: TNFRI 234,0 ± 166,9 µm2; TNFRII 154,2 ± 87,92 µm2; % of T cell area: TNFRI 27,81 ± 11,03; TNFRII 28,14 ± 19,66). tmTNF tg x TNFRI-/-exhibited a normal spleen architecture (follicle size: 239,2 ± 250,5 µm2; % of T cell area: 29,92 ± 11,46), whereas the spleen follicles of tmTNF tg x TNFRII-/-mice had T cell areas (17,31 ± 8,88) that were comparable to tmTNF tg HET mice.The size of PLN of tmTNF tg HET mice (3,74 ± 1,31 mm3) was increased compared to WT (2,41 ± 0,60 mm3), also in combination with deficiency of TNFRI (3,98 ± 2,07 mm3) or TNFRII (4,01 mm3). tmTNF tg HET mice had an increase in absolute B cell volume (0,86 ± 0,33 mm3) vs WT (0,51 ± 0,07 mm3) but no change in B cell area as % of total PLN volume. The increased B cell volume was critically dependent on TNF-RII (tmTNF tg HET x TNFRII-/-(14,87). Interestingly, whereas B cell area morphology of tmTNF tg HET, tmTNF tg HET x TNFRII-/-and WT x TNFRII-/-PLN was similar to WT, TNFRI deficiency caused profound alterations in the morphology of the B cell areas that appeared as one peripheral layer of B cells covering a central T cell area rather than properly developed follicles.Conclusion:The current study demonstrates that overexpression of tmTNF leads to an aberrant spleen architecture, characterized by smaller follicles and a decrease in central T cell areas, which is critically dependent on TNFRI. In addition, overexpression of tmTNF results in enlarged PLN and increased total B cell volume, which is dependent on TNFRII, whereas TNFRI is more important in the proper organization of B cell follicles. Overall, this study employing different state-of-the-art (3D) imaging techniques highlights the importance of tmTNF-TNFR-induced signaling events in secondary lymphoid organ morphology and function, and reveals distinct roles for TNFRI and II. Advancing our knowledge in this field might provide a better understanding of the pathophysiology of TNF-associated diseases such as arthritis, which may be important to develop new or improved treatment strategies.Disclosure of Interests:None declared