New insight into the immunomodulatory mechanisms of Tretinoin in NMRI mice

Abstract

Objectives: Recent evidence have proposed that Tretinoin produced in the gut preferentially promote differentiation of FoxP3+Treg cells but inhibits Th17 lymphocytes, and this may be the main immunomdulatory mechanism of Tretinoin in vivo. This study was done to investigate the effects of Tretinoin in outbred white mice after challenge with sheep red blood cells (SRBC).

Materials and methods: Twenty male NMRI-mice randomly allocated in two equal groups. Mice were treated with 1×10(9) SRBCs emulsified in CFA intraperitoneally twice with one weak interval. Animals were bled 5 days after last injection. Moreover, 48 hr before bleeding time, 1×10(9) SRBCs were injected into the left hind foot pad of mice. Tretinoin (25 mg/kg-every other day) were intraperitoneally injected into the treatment group from the beginning of the study and continued throughout the study. The levels of anti-SRBC antibody and the specific cellular immune responses were measured by microhemagglutination test and footpad thickness, respectively. Moreover, splenocytes were checked for proliferation rate, respiratory burst, cytokine production and FoxP3+Treg cells frequency.

Results: Tretinoin markedly alleviated cellular immunity and concurrently potentiated humoral immunity after mice challenge with SRBCs. Furthermore, aside from reducing NBT reduction and lymphocyte proliferation, Tretinoin markedly suppressed the secretion of interleukin-17 and conversely, increased the production of interleukin-10. However, the level of IFN-γ and the frequency of FoxP3+Treg cells did not alter significantly.

Conclusion: The in vivo immunomudlatoty effects of Tretinoin may be partly due to immune deviation from pro-inflammatory cytokine interleukin-17 to anti-inflammatory cytokine interleukin-10, but not absolutely depend on the expansion of FoxP3(+)Treg cells.

Keywords: Immunomodulator; NMRI-mice; Tretinoin.

Figure 1.

Effects of administration of Tretinoin on cellular immunity. Mice were intraperitoneally immunized twice with one weak interval by 1×10⁹ sheep red blood cells (SRBC) emulsified in CFA. 3 days after last intraperitoneal immunization, 1×10⁹ SRBCs in 50 µl of PBS were administered subcutaneously into the left hind foot pad of each mouse and the same volume of PBS was injected into the right foot pad as a negative control.Footpad thickness was measured before bleeding time with a dial caliper and the mean percentage increase in footpad thickness was measured. The values were presented as mean ± S.D. (*P<0.001 versus control mice)

Figure 2.

Cytokines production assay after treatment with Tretinoin. Spleen cells isolated from immunized mice with SRBC were cultured with 50 µl of PHA (1 mg/ml) for 72 hr. The levels of IFN-γ, IL-17 and IL-10 in culture supernatants were determined after 72 hr by ELISA. The results were shown as mean ± SD (*P<0.01, ** P<0.001 versus control mice)

Figure 3.

Effects of Tretinoin administration on lymphocytes proliferation and respiratory burst in phagocytic cells. Splenocytes were isolated from sensitized mice with SRBC. A) Splenocytes cultured with 50 µl PHA solution (1mg/ml) for 72 hr. Then, lymphocytes proliferation were evluated by MTT test. B) splenocytes with Staphylococcus aureus suspension and NBT were mixed and incubated for 30 min as detailed under materials and methods. The reduced dye was extracted in dioxan and quantitated at 520 nm. The values were presented as mean ± SD (*P<0.01, ** P<0.001 versus control mice)

Figure 4.

Evaluation of Tretinoin administration on FoxP3+Treg cells frequency. At the bleeding time, spleens were removed from immunized mice with SRBC. A single-cell suspension was prepared, and the cells were stained as detailed under materials and methods. Representative dot plots illustrated the regions and gates for immune phenotypic analysis. Lymphocytes were gated on a forward vs. side scatter dot plot (A) and CD25+ T cells were gated by CD25/side scatter futures (B). Then, we analyzed CD4+ FoxP3+on CD25+ gated cells (C). (FL1, CD4+; FL2, CD25+; FL3, FoxP3+). Flow cytometry analysis demonstrated that. No significant change in the expression of CD4+CD25+FoxP3+ was observed in the treatment group compared to the control mice (D). Data were shown as mean±SD