RDP58 inhibits T cell-mediated bladder inflammation in an autoimmune cystitis model

Abstract

Interstitial cystitis (IC) is a chronic inflammatory condition of the urinary bladder with a strong autoimmune component. Currently, the major challenge in IC treatment is the development of effective therapies. RDP58 is a novel d-amino acid decapeptide with potent immunosuppressive activity. In this study, we investigated whether RDP58 was effective as an intravesical agent for treating bladder autoimmune inflammation in a transgenic mouse model (URO-OVA mice). URO-OVA mice were adoptively transferred with syngeneic activated splenocytes of OT-I mice transgenic for the OVA-specific CD8(+) TCR for cystitis induction and treated intravesically with RDP58 at days 0 and 3. Compared with controls, the RDP58-treated bladders showed markedly reduced histopathology and expressions of mRNAs and proteins of TNF-alpha, NGF and substance P. To determine whether the inhibition of bladder inflammation by RDP58 was due to the interference with effector T cells, we treated the cells with RDP58 in vitro. Cells treated with RDP58 showed reduced production of TNF-alpha and IFN-gamma as well as apoptotic death. Collectively, these results indicate that RDP58 is effective for treating T cell-mediated experimental autoimmune cystitis and may serve as a useful intravesical agent for the treatment of autoimmune-associated bladder inflammation such as IC.

Fig. 1

Intravesical RDP58 treatment schedule. URO-OVA mice (Thy1.2) were transferred i.v. with in vitro pre-activated OT-I/Thy1.1 splenocytes (1 × 10⁷ cells/mouse) for cystitis induction at day 0, and treated intravesically with RDP58 at days 0 and 3. The bladders were collected for analysis at day 7. SIINFEKL: OVA_257–264 peptide.

Fig. 2

Intravesical RDP58 treatment improves histopathology of bladder inflammation. After cystitis induction and treatment, the bladders were collected, prepared for histological cross-sections, and stained with H&E. (A) a normal bladder showing unremarkable mucosa and muscularis. (B) the bladder of a mouse treated with PBS showing inflammation with urothelial hyperplasia, cellular infiltration and edema. (C) the bladder of a mouse treated with RDP58 at 2.5 μg/dose showing a relative dense inflammatory cell infiltration. (D) the bladder of a mouse treated with RDP58 at 25 μg/dose showing scattered inflammatory cells within the urothelium and lamina propria. (E) the bladder of a mouse treated with RDP58 at 250 μg/dose showing minimal inflammation. The cross-sections shown are representative of 5 animals in each group. Magnification (for all panels): ×40 for the left and ×400 for the right.

Fig. 3

Intravesical RDP58 treatment reduces effector T cell infiltration and production of neuroinflammatory factors in the inflamed bladders. (A) Decrease of infiltrating effector CD8⁺ T cells in the RDP58-treated bladders. Bladder single-cell suspensions were prepared, stained with anti-Thy1.1 and anti-CD8 antibodies, and analyzed by flow cytometry. Gate was set on lymphocytes according to scatter criteria. The total numbers of double positive T cells are presented for each bladder. Statistical analysis (compared with PBS group): * = p<0.05 and ** = p<0.001. The results are representative of three separate experiments consisting of 3 to 5 animals per group. (B) Reduction of TNF-α, NGF and SP precursor (pre-SP) mRNAs in the RDP58-treated bladders. The bladder total RNAs were extracted and amplified by RT-PCR for the indicated inflammatory mediators. GAPDH was used as an internal control. Three bladders for each group are presented. The normal bladders expressed no detectable mRNAs under the same conditions (data not shown). (C) Reduction of TNF-α, NGF and SP proteins in the RDP58-treated bladders. The bladder lysates were prepared and analyzed by ELISA for the indicated inflammatory mediators. The concentration of the proteins is presented for each bladder. Statistical analysis (compared with PBS group): * = p<0.05 and ** = p<0.001. The normal bladders showed no or minimal expression of the proteins (data not shown). The results are representative of three separate experiments consisting of 3 to 5 animals per group.

Fig. 4

RDP58 inhibits effector T cells for TNF-α and IFN-γ production in vitro. Splenocytes of OT-I/Thy1.1 mice were prepared and cultured in the absence or presence of PMA (100 ng/ml) plus ionomycin (1500 ng/ml) (for panel A) or OVA_257–264 peptide (10 μg/ml) (for panel B) for 3 days. To evaluate the effects of RDP58, cells were also cultured in the presence of the stimulators plus indicated doses of RDP58 (5 μg/ml, 50 μg/ml and 500 μg/ml) for 3 days. Production of TNF-α and IFN-γ in culture supernatants were then analyzed by ELISA. Data are presented as mean ± SD from two determinations. Statistical analysis (compared with PMA plus ionomycin or OVA_257–264 peptide group): * = p<0.05 and ** = p<0.001. The results are representative of two separate experiments.

Fig. 5

RDP58 induces effector T cell apoptotic death in vitro. Splenocytes of OT-I/Thy1.1 mice were prepared and incubated with RDP58 or a control peptide (5 μg/ml, 50 μg/ml and 500 μg/ml) for 12 and 24 h. After incubation, cells were double stained with FITC-Annexin V and propidium iodide (PI), and analyzed by flow cytometry. Gate was set on lymphocytes according to scatter criteria. The percentage of stained cells is indicated. The results are representative of two separate experiments.