Total dose and frequency of administration critically affect success of nasal mucosal tolerance induction

Abstract

Aims: Nasal tolerance induction with autoantigens can effectively protect against a variety of experimental models of autoimmune disease. The aims of this study were to characterise the dosage and kinetics of inhibition of experimental autoimmune uveoretinitis (EAU) via intranasal administration of the uveitogenic antigen interphotoreceptor retinal binding protein (IRBP) in the murine model of IRBP induced EAU.

Methods: B10RIII mice were tolerised by intranasal administration of IRBP either with a long term multiple low dose or a short term/high dosing regimen before subcutaneous immunisation with IRBP in complete Freund's adjuvant (CFA). On day 15 post-immunisation, mice were killed and eyes were removed for histological examination and quantification of inflammatory cell infiltration and degree of target organ (rod outer segment, ROS) destruction.

Results: Nasal administration of multiple low doses of IRBP (1 microg or 3 microg IRBP per mouse per day for 10 days) significantly protected mice from IRBP induced EAU. Short term/high dose regimens were only effective when given either as a single or, at most, as two consecutive doses (40 microg per dose). Multiple doses in the range of 45-120 microg over 3 days afforded no protection.

Conclusions: These results indicate that both dose and frequency of intranasal antigen administration are pivotal to tolerance induction and subsequent suppression of T cell mediated autoimmune disease.

Figure 1

Multiple low dose intranasal administered IRBP suppresses IRBP induced EAU. EAU was markedly suppressed in tolerising a total of 10 µg or 30 µg IRBP administered intranasally over 10 days compared with the PBS pretreated mice. Incidence (diseased/total mice), EAU histological grade mean (SD), and p value between the antigen and PBS tolerised groups are detailed.

Figure 2

A single high dose of intranasal IRBP is effective in protecting mice from EAU. EAU was suppressed significantly by a single intranasal dose of 40 µg or two doses of 80 µg, but not by a total of 120 µg IRBP administered intranasally over 3 days. Incidence (diseased/total mice), EAU histological grade mean and SD, and p value between the antigen and PBS tolerised groups are detailed.

Figure 3

Effect of frequency and dose delivery during tolerance induction. EAU was significantly suppressed by a total of 45 µg IRBP in one or two doses. A total of 45 µg IRBP intranasally administered over 3 days failed to protect against IRBP induced EAU. Incidence (diseased/total mice), EAU histological grade mean and SD, and p value between the antigen and PBS tolerised groups are detailed.

Figure 4

Histological and immunohistochemical analysis of retina from tolerised and control animals in IRBP induced EAU. (a) Normal retina structure from a mouse that received a total of 10 µg of IRBP intranasally before immunisation. ILM = inner limiting membrane layer; INL = inner nuclear layer; ONL = outer nuclear layer; ROS = photoreceptor rod outer segment layer; RPE = retinal pigment epithelial cell layer; Ch = choroid; Sc = sclera. (b) Retina from a mouse that received a total of 30 µg of IRBP intranasally before immunisation. A large number of infiltrating cells were observed in the vitreous and inner layers of retina, but not in the ROS layer. (c) Retina from control mouse that received PBS intranasally before immunisation. There was a massive leucocyte infiltrate in all layers of retina, particularly within the ROS layer. The whole structure of retina was damaged with loss of ROS. (d) and (e) Immunohistochemical staining of CD45 positive cells within retina from animals that received a single dose of 40 µg IRBP intranasally. Retina shows infiltrating leucocytes in the vitreous, some inner layers of retina (partially accounted for by activated microglia), but not in the ROS layer. (f) CD45 positive leucocytes were found largely within the ROS layer, although there was an infiltrate within inner retina in a control mouse.