The Effect Of Aqp4 Deficiency On Cd4~+ Cd25~+ Foxp3~+ T Regulatory Cells And The Severity Of Parkinson's Disease

Aquaporins (AQPs) are integral membrane proteins that serve as channels in the transfer of water, and in some cases, small solutes across the membrane. AQPs play important roles in regulating water homeostasis under physiological and pathological conditions. So far, at least 13 aquaporin isoforms (AQP0-AQP12) have been identified in mammalian species. Aquaporin-4 (AQP4) is mainly expressed in mammalian brains and has gained much attraction due to its involvement in the physiopathology of cerebral disorders including hydrocephalus, stroke, tumor, infection, epilepsy and traumatic brain injury. In addition, it is also present in mammalian eye, ear, skeletal muscle, stomach parietal cells, and kidney collecting ducts. AQP4 may play important roles in central osmoreception, neurotransduction, and cerebrospinal fluid formation. Recent researches report a novel class of autoimmune channelopathy--Neuromyelitis optica (NMO), which is an inflammatory demyelinating disease that selectively affects optic nerves and spinal cord. AQP4 is identified as its target antigen. This study tomards NMO is the first to implicate AQP4 in the pathogenesis of any autoimmune disorder. However, there is little research whether abnormal AQP4 levels are associated with immune system dysfunction.In this study, RT-PCR and immunohistochemistry were carried out to detect the AQP4 mRNA transcription and AQP4 expression in thymus, spleen and lymph nodes of the wild-type (WT) mice. Then we used the flow cytometry to compare the proportion of the following immune cell subsets in spleens between AQP4 knockout (AQP4 KO) and WT mice, including B cells, T cells, CD⁸⁺T cells, CD⁴⁺T cells, Th1/Th2/Th17 cells, CD⁴⁺CD²⁵⁺Foxp³⁺ Treg cells, natural killer cells (NK cells), dendritic cells (DC) and macrophages (Mφ). Finally, we used a disease model to test the possible role of AQP4 in immun system.The main results we got are as follows:1. AQP4 is expressed in mouse thymus, spleen and lymph nodes.2. The proportion and absolute number of CD⁴⁺CD²⁵⁺Foxp³⁺ Treg cells are decreased in AQP4 KO mice.There were no statistically significant differences in the proportion of B cells, T cells, CD⁸⁺ T cells, CD⁴⁺ T cells, Th1, Th2, Th17 cells, NK cells, DC and Mφbetween AQP4 WT and KO mice. However, the proportion of CD⁴⁺CD²⁵⁺Foxp³⁺ Tregs in AQP4 WT mice was nearly two-fold higher than that in AQP4 KO mice. The absolute number of Tregs in peripheral blood was also significantly reduced in AQP4 KO mice .3. AQP4 deficiency does not affect the immuno-suppressive function of Tregs.To determine whether the immuno-suppressive function of Tregs were different between AQP4 WT and KO mice, we carried out the [3H]-TdR incorporation test. There were no statistically significant differences in the suppressive function of Tregs between AQP4 WT and KO mice.4. AQP4 deficiency decreases the thymic generation of natural occuring CD⁴⁺CD²⁵⁺Foxp³⁺ Treg cells.Next we set out to understand why CD⁴⁺CD²⁵⁺Foxp³⁺ Treg cells were decreased in AQP4 KO mice. We found that AQP4 KO mice had substantially less proportion of CD⁴⁺CD²⁵⁺Foxp³⁺ thymocytes compared with wild-type mice in the thymus 3–5d after birth. No statistically significant differences in the apoptic CD⁴⁺CD²⁵⁺ Treg cells could be detected in thymus between AQP4 WT and KO mice. These observations collectively demonstrate that AQP4 deficiency decreases the thymic generation of natural occuring CD⁴⁺CD²⁵⁺Foxp³⁺ Treg cells.5. AQP4 deficiency induces hyperactive cellular immune responses.The aforementioned results demonstrated that CD⁴⁺CD²⁵⁺Foxp³⁺ Treg cells were decreased in AQP4 KO mice. If that conclusion were correct, then spleenocytes or lymphocytes from AQP4 KO mice might have stronger responses upon stimulation than their WT littermates. To test that hypothesis, we carried out the [3H]-TdR incorporation experiment to analyze the level of lymphocyte proliferation. As expected, the lymphocytes from the AQP4 KO mice showed a significantly increased proliferation upon anti-CD3 stimulation compared with the WT mice. These results suggest that the AQP4 KO mice have a hyperactive cellular immune responses in vitro due to the decrease of the CD⁴⁺CD²⁵⁺Foxp³⁺ Treg cells. Furthermore, the AQP4 KO mice may be more prone to some inflammation-associated diseases than AQP4 WT mice after some specific stimulation.6. In MPTP-induced Parkinson's disease, the losses of DA neurons in AQP4 KO mice are more severe which is partly due to the hyperactive microglial responses.Recently a large body of evidence confirms that Microglia-mediated neuroinflammation plays an important role in the pathogenesis of Parkinson's disease. And it has been reported that CD⁴⁺CD²⁵⁺Foxp³⁺ Treg cells have neuroprotective activities through suppression of microglial responses to stimuli. Therefore, in our studies, we established a PD model to test whether the microglial reactions and inflammation were different between AQP4 KO and WT mice. After MPTP injection, we found much more activated microglial cells with thicker ramifications were seen in the midbrain of AQP4 KO mice, in contrast to MPTP-injected WT mice. As a consequence, immunohistochemical analysis revealed remarkable losses of TH+ neurons in the substantia nigra pars compacta (SNc) from MPTP-intoxicated AQP4 KO mice compared with MPTP-intoxicated WT mice. Atoptively transferring of anti-CD3 activated CD⁴⁺CD²⁵⁺Foxp³⁺ Treg cells effectively decreased neuroinflammation and reliefed immunopathogensis in recipient PD model mice.In summary, our findings demonstrates that AQP4 is expressed in mouse thymus, spleen and lymph nodes. AQP4 deficiency inhibits the thymic generation of CD⁴⁺CD²⁵⁺Foxp³⁺ Tregs. Lack of AQP4 can enhance the sensitivity of DA neurons to neurotoxicity and aggravate the severity of PD via microglia inflammation mediated by the decrease of Tregs. Thus, our study indicates that AQP4 is involved in the thymic generation of natural CD⁴⁺CD²⁵⁺Foxp³⁺ Treg cells, and suggests that the therapeutic strategies target to Treg cell modulation with AQP4 may offer a great potential approach for the development of new strategies for neurodegenerative diseases or other immune-associated diseases treatment.