Purpose Tertiary lymphoid structures (TLS) are organized aggregation of immune cells at sites of inflammation. The development of TLS in autoimmune settings indicate that they may be sites for both activation and regulation of immune responses, and a local site for autoantibody production.
The aim of this study was to investigate if imiquimod, an TLR7 agonist, induce TLS in kidneys, of normal mice and if IL-18 expression plays a role in the induction of TLS.
Methods The ears of 5 to 10 weeks old (wo) C57BL/6J (C57BL) and B6.129P2-Il18tm1Aki/J (IL-18 KO) mice were topically treated with 1.25 mg of 5% IMQ three times a week. Blood samples were analyzed every week using IDEXX ProCyte Dx for hematological analyses and ELISA for anti-dsDNA and anti-RNA antibodies. Immune cells isolated from kidney, spleen and LN at 10 and 14 wo were analyzed by flow cytometry. Total mRNA were isolated from kidney, spleen and LN and gene expression of TNF, IL1β, INFα, IFNγ, IL-18, and CXCL13 were analyzed by qPCR. Kidney sections from 10 and 14 wo treated and control mice were analyzed by immunohistochemistry (IHC) and immunofluorescence (IF).
Results Both anti-dsRNA and anti-dsDNA antibody (ab) production increased in treated mice at week 7. The anti-dsRNA ab production was significantly higher than the anti-dsDNA ab production at weeks 8–14. IMQ treated IL18-KO mice produced more anti-DNA antibodies at 10 wo compared to C57Bl mice. The number of reticulocytes and mean platelets volume (MPV) increased while platelets were reduced in IMQ treated C57Bl mice. TLS were observed in both 10 and 14 wo C57Bl and IL-18 KO mice treated with IMQ. Flow cytometric analyses of kidney infiltrating immune cells showed an increase in both CD4+ and CD8+ T cells in 14 wo IMQ treated C57BL mice. This increase was not observed in IL-18 KO mice. Gene expression of TNF and CXCL13 increased in IMQ treated C57Bl mice, but there were little differences in the expression of IL1β, INFα, and IFNγ. Individual differences were observed in IL-18 KO mice
Conclusion IMQ induce anti-RNA and anti-dsDNA ab production in normal and IL-18 KO mice. IL-18 deficiency does not affect the development of renal TLS.
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