Cyclophosphamide inhibits the progression of Meniere's disease by reducing the generation of circulating immune complex

Abstract

Endolymphatic hydrops is a characteristic pathological manifestation of Meniere's disease (MD) that has been previously associated with autoimmunity. Interest in the circulating immune complex (CIC) has increased due to its reported role in the occurrence of MD. The present study aimed to investigate the potential value of serum CIC concentration in the diagnosis of MD and the therapeutic potential of cyclophosphamide (CTX) for the treatment of MD. In the present study, guinea pigs were immunized with isologous crude inner ear antigens to establish an autoimmune MD model. Pure tone audiometry, Vestibular-evoked myogenic potential test, electrocochleography test and auditory brainstem response was applied in this study for assessing the severity of MD in guinea pigs. ELISA was applied to measure CIC, tumor necrosis factor α (TNF-α) and heat shock protein 70 (HSP70) expression levels in the serum samples of different groups of patients. Western blotting was applied to detect the protein expression of HSP70 in inner ear tissues in guinea pigs. Hematoxylin and eosin staining was applied to visualize the spiral ganglions in spiral ganglions models. CIC expression in the inner ear was detected by immunohistochemistry. In vivo experiments were performed to confirm the therapeutic effects of CTX in MD. Serum concentrations of CIC, TNF-α and HSP70 were found to be significantly higher in patients with MD, which were also associated with increases in hearing classification and the severity of endolymphatic hydrops. Using a guinea pig MD model, ELISA results revealed significantly increased serum CIC, TNF-α and HSP70 concentrations compared with those in the control group. ABR results showed that the thresholds in the CTX group were notably decreased compared with that in the dexamethasone group, whereas CIC concentrations in the serum were reduced following dexamethasone and CTX treatments compared with those after saline treatment. In the inner ear tissues, the CIC concentration in CTX group was lower than that in the dexamethasone group. Similarly, reductions in HSP70 and TNF-α concentrations was also observed in a similar manner. Immunohistochemistry staining found notably lower CIC deposition in the inner ear tissues following CTX treatment than that in dexamethasone group. Taken together, higher CIC expression can be used as a biomarker for MD diagnosis. The efficacy of CTX in MD was found to be higher compared with that in dexamethasone, which may be associated with the effective inhibition of CIC, HSP70 and TNF-α generation.

Keywords: Meniere's disease; circulating immune complex; cyclophosphamide; heat shock protein 70; tumor necrosis factor-α.

Figure 1

Expression of the immune parameters in the serum of patients with MD. ELISA was applied to detect the concentration of (A) CIC, (B) TNF-α and (C) HSP70 was upregulated in the serum of patients with MD compared with that in the control group. (D) CIC, (E) TNF-α and (F) HSP70 levels in the serum of patients with MD were increased in a manner that was dependent on the phases of pure tone audiometry tested by ELISA. The concentration of (G) CIC, (H) TNF-α and (I) HSP70 in the serum of patients with MD was increased in a manner that was dependent on the severity of endolymphatic hydrops via ELISA. ^*P<0.05, ^**P<0.01 and ^***P<0.001. CIC, circulating immune complex; TNF, tumor necrosis factor, HSP70, heat shock protein 70; MD, Meniere's disease.

Figure 2

CIC expression level in the autoimmune MD guinea pig model. (A) ELISA was conducted to detect the ICIEAg concentration in guinea pigs in early immunization, after systemic booster immunization and local lymph sac immunization was gradually increased after each stage of immunization. The auditory brainstem response threshold at (B) 4, (C) 8 and (D) 16 kHz threshold was notably higher 2 weeks after immunization compared with that before immunization through auditory brainstem response. (E) Hematoxylin and eosin staining results indicated that 2 weeks after immunization, spiral ganglions were fewer in number in guinea pigs with extensive endolymphatic hydrops. Magnification x400. (F) CIC, (G) TNF-α and (H) HSP70 concentration in serum was significantly increased in guinea pigs with MD compared with that in the control group via ELISA. ^**P<0.01 and ^***P<0.001. GP, guinea pigs; dBSPL, Decibel Sound Pressure Level; CIC, circulating immune complex; TNF, tumor necrosis factor; HSP70, heat shock protein 70; MD, Meniere's disease.

Figure 3

CTX suppresses MD progression by reducing CIC generation. A total of 1-week post-treatment, the ABR threshold at (A) 4, (B) 8 and (C) 16 kHz was the highest in the NaCl group, followed by the dexamethasone group and then the CTX group. After 2 weeks of treatment, the CTX group exhibited the lowest ABR threshold at (D) 4, (E) 8 and (F) 16 kHz and guinea pigs in the NaCl group had the highest ABR threshold. (G) ICIEAg expression in the dexamethasone group was significantly lower compared with that in the NaCl group, whilst ICIEAg expression in the CTX group was significantly decreased compared with that in the dexamethasone group tested by ELISA. The CIC concentration in the (H) serum or (I) inner ear tissues was highest in the NaCl group, followed by the dexamethasone group and then the CTX group detected by ELISA. (J-L) Pearson's correlation analysis showed the CIC concentration in the serum was positively correlated with that in the inner ear tissue in the (J) NaCl, (K) CTX and (L) Dexamethasone groups. ^*P<0.05, ^**P<0.01 and ^***P<0.001. CTX, cyclophosphamide; MD, Meniere's disease; ICIEAg, isologous crude inner ear antigens; ABR, auditory brainstem response.

Figure 4

Effects of CTX on HSP70 and TNF-α concentration levels. (A) ELISA revealed that HSP70 concentration in the circulation of the dexamethasone group was significantly lower compared with that in the NaCl group, whilst HSP70 concentration in the CTX group was significantly reduced compared with that in the dexamethasone group. (B) Western blotting showed that HSP70 protein expression in the inner ear of the dexamethasone group was significantly lower compared with that in the NaCl group, whilst HSP70 concentration in the CTX group was significantly reduced compared with that in the dexamethasone group. ELISA showed that the TNF-α concentration in the (C) serum or (D) the inner ear tissues was also decreased in the NaCI, dexamethasone compared with that in the NaCl group, whilst TNF-α concentration was lower in the CTX group compared with that in the dexamethasone group. (E) Immunohistochemical staining showed that the highest CIC deposition was in the NaCl group, followed by the dexamethasone group and then the CTX group. Magnification, x400. ^**P<0.01 and ^***P<0.001. CIC, circulating immune complex; TNF, tumor necrosis factor; HSP70, heat shock protein 70; CTX, cyclophosphamide.