PRISTANE-INDUCED LUPUS MICE PRESENT INCREASED MEGAKARYOCYTE COUNTS ALONGSIDE OTHER HISTOPATHOLOGICAL FEATURES IN THE SPLENIC TISSUE

PV014 / #607 Poster Topic: AS02 - Animal Models Background/Purpose Pristane-induced lupus (PIL) mice develop an autoimmune response to a single intraperitoneal (i.p.) injection of pristane oil. Consequently, there is the production of autoantibodies, leading to the deposition of immune complexes in tissues. Splenic involvement is observed in this model, especially as splenomegaly and histopathological features. One of these features is the increased proliferation of hematopoietic cells such as megakaryocytes (MKs). Although its impact on lupus splenic pathology remains unclear, vitamin D (vitD) has potential immunomodulatory effects. This study examined the effects of vitD supplementation on splenic alterations in a PIL model. Methods Thirty-eight BALB/c mice were randomized into 3 groups: control (CO, n = 12), PIL (n = 13), and PIL supplemented with vitD (VD, n = 13). PIL and VD received an i.p. injection of 500 μL of pristane; VD received 2 μg/kg of 1α,25-dihydroxycholecalciferol subcutaneous injections every 2 days for 180 days. After euthanasia, spleens were weighed and paraffin embedded. Spleen index was calculated as the proportion of spleen-to-body weight (mg/g). H&E slides were produced and analyzed for histopathological features. MKs were counted in 10 random fields. IgM and IgG expressions were determined via immunofluorescence. One-way ANOVA followed by Tukey’s or Kruskal-Wallis followed by Dunn’s tests (p ≤ 0.05) were used and results expressed as mean ± SD or median (IQR). Results PIL mice demonstrated significant higher spleen index (CO: 2.63 mg/g ± 0.3; PIL: 3.42 ± 0.59; VD: 3.56 ± 0.84; PIL vs. CO: p = 0.009; VD vs CO: p = 0.002), elevated MK counts (CO: 0.15 cells/field (0.02-0.28); PIL: 0.6 (0.35-0.8); VD: 0.9 (0.3-1.7); PIL vs CO: p = 0.04; VD vs. CO: p = 0.003), and characteristic histopathological features such as the presence of foam cells, disorganization of red and white pulps, and fibrous capsule expansion. VitD supplementation did not reduce spleen size (VD vs PIL: p = 0.84), splenic MK counts (VD vs PIL: p > 0.99) nor did it prevent histological alterations. Either, it has not altered immunoglobulins expression – PIL and VD groups showed similar IgM (CO: 0.06 (0.0-0.16); PIL 0.09 (0.06-0.17); VD: 0.06 (0.01-0.18); p = 0.46) and IgG (CO: 0.55 ± 0.59; PIL: 1.6 ± 1.8; VD: 1.24 ± 1.21; p = 0.15) fluorescence intensity. Conclusions PIL led to splenomegaly and structural changes in the spleen. This highlights the spleen’s altered response to inflammation in the model. Additionally, a higher presence of MKs in the splenic tissue has been previously observed in PIL mice, but to the best of our knowledge our study is the first to quantify and attest the statistical difference in MK counts. The higher values in both PIL and VD groups imply that the inflammatory process potentially reflects an immune or compensatory response in this tissue. In this study, the similar IgM intensities across groups suggest that there is stability of early immune response markers in the late stage of the model. Also, IgG presence confirms that PIL mice develop immune complex deposition in the spleen. Finally, our results imply that while vitD might have some immunological effects, it does not appear to impact spleen involvement in the PIL model. Acknowledgments: HCPA, UFRGS, CAPES, and SRRS, for funding of this project.

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