Abstract
Purpose :
We have previously demonstrated that CD25 knock-out (KO) mice develop worse lacrimokeratoconjunctivitis when raised in germ-free environment. The purpose of this study was to investigate the role of IL-12 in the onset and severity of dacryoadenitis in this model in germ-free conditions.
Methods :
Four-week (W) old germ-free CD25 knock-out (KO) mice of both sexes (n = 20) were randomized to receive intra-peritoneal injections of anti-IL-12 (200μg/mouse) or isotype antibody twice a week for four weeks while housed in sterile isolators. Mice were euthanized at eight weeks of age upon isolator exit. Lacrimal glands (LG) were excised and embedded in paraffin for histologic evaluation. LG severity score (range 1-5) was investigated in H&E stained LG sections by two masked observers. Single cell suspensions of cervical lymph nodes (CLN) and lacrimal glands were stained and frequency of CD4+IFN-gamma+, CD4+IL-17+ and CD4+IL-13+ cells was determined by flow cytometry and intracellular staining. Conjunctiva and LG biopsies were lysed, and real-time q-PCR investigated the expression of IFN-gamma and tumor necrosis alpha (TNF-α) and MHC II.
Results :
Eight-week old IL-12 depleted germ-free CD25KO LG had significantly decreased infiltration score and areas with normal acini, while isotype-control treated LG displayed shrunken acini and massive lymphocytic infiltration (3.6 ± 0.54 vs. 4.75 ± 0.5 severity score, P = 0.03, respectively). This was accompanied by a 50% decrease in the generation of CD4+IFN-gamma+ cells in cervical lymph nodes and a 60% decrease of infiltrating cells in LG of IL-12- depleted compared to isotype control germ-free CD25KO animals (P < 0.05 for both CLN and LG). There was no change in the frequency of CD4+IL-17+ and CD4+IL-13+ cells in either tissue. A significant 50% decrease in IFN-gamma in LG and a 60% reduction of MHC II and TNF-α mRNA transcripts levels in the conjunctiva was observed in the IL-12 depleted germ-free CD25KO mice compared with isotype control (P < 0.05 for all three genes).
Conclusions :
Depletion of IL-12 in the germ-free CD25KO model ameliorates the onset and severity of autoimmunity by decreasing generation of CD4+ IFN-g+ cells.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.