br Materials and methods br Results br Discussion

Materials and methods
Results
Discussion GPR35 expression has been identified within discrete regions of the nervous system, including the spinal cord [[43], [44], [45], [46], [47], [48]]. Importantly, GPR35 is expressed not only in neurons but also in spinal glial cells [43]. Unfortunately, changes in the GPR35 protein levels are very difficult to study because highly selective commercial antibodies are not available for Western blot analysis. Maravillas-Montero et al. [26] and Guo et al. [49] proved that CXCL17 may act via GPR35, however recent in vitro studies suggested that also another receptors for CXCL17 may exist [50,51]. The CXCL17 first, it was found to be involved in tumour angiogenesis [[52], [53], [54], [55]] and to be co-regulated with vascular endothelial growth factor [55], but its role in nociceptive transmission has not studied, to the best of our knowledge. For the first time, we have shown in these experiments that intrathecal administration of CXCL17 in naive mice led to the development of strong tactile and thermal hypersensitivity. This effect is not surprising, as previous research has shown that other chemokines from the CXC group, such as CXCL1, CXCL4, CXCL5, CXCL9, CXCL10 and CXCL12, have similar properties after intrathecal administration in mice [10]. Moreover, CXCL1 was shown to be upregulated in a spinal nerve ligation model [56] and CXCL4, CXCL9, CXCL10 and CXCL12 were upregulated after sciatic nerve injury [57]; Kwiatkowski et al. [in preparation] and CXCL13 after infraorbital and spinal nerve ligation [58,59]. The observed pronociceptive properties of CXCL17, which is a strong macrophage/microglia chemoattractant [26,60], are very important and may play a role in the development of neuropathy. Presently, it is known that peripheral nerve damage led to the influx and activation of microglia/macrophages [5,36,[61], [62], [63]] and that these cells contribute to the secretion of many pronociceptive mediators [61,64]. Kynurenic cerk is a product of the normal metabolism of the amino acid l-tryptophan and is without any doubt considered to be an endogenous agonist of GPR35 [45,46,65], however is also a noncompetitive antagonist at the glycine site of the NMDA receptor [[66], [67], [68], [69]] and an antagonist at ionotropic NMDA (as well as AMPA and kainate) glutamate receptors [41,70]. It has been shown that kynurenic acid exerts anticonvulsant effects and neuroprotective activity and is synthesized mainly in astroglial cells [[71], [72], [73], [74]]. In our present study, we have shown that intrathecal administration of kynurenic acid reduced mechanical and thermal hypersensitivity in a sciatic nerve injury model in mice. These results correspond with previous reports showing that in rat neuropathic pain models, kynurenic acid administered intrathecally reversed the tactile hypersensitivity [75]. Moreover, kynurenic acid has analgesic properties in mice/rats pain models [33,45,47,76,77]. Similar results were observed with zaprinast, which is a high-affinity synthetic ligand of GPR35 [27,44,48,[78], [79], [80], [81]]. In 2005, Yoon et al. [82] showed that zaprinast dose-dependently reduced the number of flinches in a formalin pain model in rats. Analgesic effects of zaprinast were later confirmed in other rat and mouse inflammatory pain models [33,45]. In our study, zaprinast also revealed analgesic properties with neuropathic pain, similar to others [[43], [44], [45], [46], [47], [48],83]. Our studies provide the first evidence that kynurenic acid and zaprinast injection diminished CXCL17 pronociceptive properties in naïve mice. This may be explained with the fact that the activation of GPR35 with zaprinast, kynurenic acid or CXCL17 leads to the distinct modulation of Ca2+ levels. In 2013, Southern et al. [84] showed that kynurenic acid and zaprinast reduced Ca2+ mobilization, while in 2015, Maravillas-Monetro et al. [26] provided evidence that in contrast, CXCL17 increased Ca2+ mobilization. Recently published in vitro results of Amir et al. [50] and Park et al. [51] suggest that perhaps CXCL17 exerts its effects also through the unknown yet receptor. In our experiments the pronociceptive properties of CXCL17 were not completely blocked, which may suggest that CXCL17 act via multiple receptors. However, this issue needs to be examined in the future.