Hemorrhagic cystitis is one of the adverse effects of treatment with CYP, which is widely used as both an antineoplastic agent and an immunosuppressor for autoimmune disorders such as systemic lupus erythematosus and rheumatoid arthritis . Severe inflammation in the bladder occurs in both mice and rats receiving CYP [2, 3]. Hence, a CYP-induced mouse model can be used to clarify the mechanism of disease progression of HC.
Inflammatory responses are triggered by tissue-resident innate immune cells after recognition of pathogen-associated molecular patterns and DAMPs through TLRs and CLRs [4-6]. The interactions via TLRs and CLRs elicit different downstream pathways through MyD88 and CARD9, respectively, and subsequently induce various inflammatory mediators [5, 7]. It has previously been shown that pretreatment of CYP-induced cystitis with plant-derived glucose–mannose binding lectin reduces leucocyte infiltration and ameliorates tissue damage . In the bladders of mouse models of CYP-induced cystitis, marked increases in cytokines such as γ–interferon, TNF-α, IL-1β and IL-6 and chemokines such as CXCL9, CXCL10 and CXCL11 reportedly occur [9-13]. Neutralization of CXCL10 reduces the severity of CYP-induced cystitis in mice . However, it is still unclear what mechanism triggers the array of inflammatory responses involved in the development of acute CYP-induced cystitis.
γδ T cells are naturally occurring IL-17A-producing cells that are generated within the fetal thymus . In naive mice, they are localized in the periphery, including the bladder, as long-lived effector cells . IL-17A-producing γδ T cells constitutively express large amounts of IL-23R, which induce prompt IL-17A production after binding of IL-23 . Recently, several lines of evidence have suggested that vesical IL-17A-producing γδ T cells are potent inflammatory cells that are directly involved in host defense and anti-tumor activity [17, 18]. In a murine model of urinary tract infection with E. coli, IL-17A-producing γδ T cells reportedly contribute to clearance of the pathogens by mediating neutrophil infiltration and macrophage activation . In bladder cancer, IL-17A-producing γδ T cells contribute to the antitumor effect in the context of adjuvant therapy with bacillus Calmette–Guérin . In fact, accumulation of γδ T cells has been observed in the bladder in human cystitis but not in healthy controls ; however, the role of vesical γδ T cells in human cystitis remain unknown.
In this study, we found that the pathogenesis of acute CYP-induced cystitis in mice depends, at least in part, on CARD9, which elicits an inflammatory cascade of IL-1β, IL-6 and IL-23-induced IL-17A production by vesical γδ T cells.
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- MATERIALS AND METHODS
Systemic administration of CYP induces chemical cystitis in both mice and rats and is used as an experimental model of HC [2, 3]. While the precise cause of HC is not known, a number of researchers have suggested that non-infectious urinary bladder inflammation is caused by innate immune responses involving pro-inflammatory cytokines and cell infiltration [9-13]. In the present study, we found that FcRγKO mice and CARD9KO mice are less susceptible to acute CYP-induced cystitis than WT mice. To our knowledge, this is the first evidence that the FcRγ-CARD9 pathway, which is an important CLR signal pathway, is involved at an early stage of acute CYP-induced cystitis.
Caspase recruitment domain-containing protein 9 is an adaptor protein that has an essential role in NF-κB activation and pro-inflammatory cytokine and chemokine production in myeloid cells through CLRs . Several CLRs, such as Mincle (also known as clec4e) and CLEC9a (also known as DNGR-1) have been reported as recognizing self-antigens derived from damaged cells as DAMPs [24, 32]. Mincle associates with ITAM-containing adaptor molecular FcRγ chain and recognizes TDM and spliceosome-associated protein 130 (SAP130), which is released from necrotic cells . TDM is an abundant mycobacterial cell wall glycolipid. Acrolein, a toxic CYP-derived metabolite, triggers HC by damaging the layer of proteoglycan and glycoprotein that covers the epithelium of the bladder in humans and mice [1, 33]. CLEC9a recognizes an unidentified ligand on necrotic cells and contains cytoplasmic ITAM-like motif. In this study, FcRγ which has ITAM, was responsible for proinflammatory cytokine production. Taken together, we speculate that DAMPs such as SAP130 may be released from acrolein-induced damaged apoptotic bladder cells after CYP administration and thence involved in the pathogenesis of acute CYP-induced cystitis.
Recently, it was reported that p2X7 receptor, a purinergic receptor, is involved in inflammatory and nociceptive changes in CYP-induced cystitis in mice . P2X7R is activated extracellularly in an ATP-dependent manner when ATP is released from dying cells, resulting in K+ efflux as a danger signal. K+ efflux activates NLRP3 inflammasome, which contain NALP3, the adapter ASC and pro-caspase-1, to result in caspase-1 activation. Activated caspase-1 converts pro-IL-1β to IL-1β. These findings suggest that extracellular ATP from damaged bladder cells may contribute to IL-1β production in acute CYP-induced cystitis. Indeed, we found that IL-1β production is significantly decreased in CARD9KO and FcRγKO mice after CYP administration. These findings suggest that activation of caspase-1 by ATP may convert pro-IL-1β produced via a FcRγ-CLR pathway to IL-1β, which would induce edema.
A notable finding of the present study is that the MyD88 pathway is not involved in the pathogenesis of acute CYP-induced cystitis. In a previous study, administration of acrolein caused more severe edema in the bladders of TLR4-deficient mice than in those of TLR4-sufficient mice  and acrolein inhibited lipopolysaccharide-induced homodimerization of TLR4, which resulted in down-regulation of NF-κB and interferon regulatory factor 3 activation . In addition, TLR4 activates two distinct signaling pathways: the “MyD88-dependent” and “TIR-domain-containing adapter-inducing interferon-β-dependent” pathways . S100A8/9 is known as DAMPs and interacts with TLR4. Strong expression of S100A8/9 has been detected in the bladder and kidney in acute urinary tract infection. However, S100A8/9 does not contribute to an effective host response against E. coli in the urinary tract . In this regard, further study is necessary to elucidate the role of the TLR4 pathway in acute CYP-induced cystitis.
Vesical interstitial infiltration of neutrophils is one of the earliest events induced by CYP administration. IL-17A enhances neutrophil infiltration by promoting IL-6, granulocyte colony-stimulating factor and CXCL8 production . In this study, we found that vesical γδ T cells in the neutrophil infiltrations in the bladder after CYP administration are involved in IL-17A production. Recent report suggests that innate lymphoid cells have C-type lectin receptors and produce IL-17A. Therefore it is possible that IL-17A produced by innate lymphoid cells is also involved in acute CYP-induced cystitis . However, our data showing that IL-17A production is almost completely abolished in TCRδKO mice indicate that γδ T cells are the main source of IL-17A production in acute CYP-induced cystitis. IL-23, which induces IL-17A production, was produced in the bladder 24 hr after CYP administration in a FcRγ-CARD9 dependent manner but not in a MyD88-dependent manner, as were IL-1β and IL-6. Interestingly, we found that IL-17A is not involved in vesical edema. The bladder weights of IL-17AKO mice were not significantly different from those of WT mice after CYP administration (data not shown). These data suggest that edema and neutrophil infiltration are induced by different cytokines.
Although IL-23 production was particularly dependent on the FcRγ-CARD9 pathway, production of IL-17A was not completely stopped. γδ T cells were able to produce IL-17A not only in the standard manner with IL-23, but also via TCR signaling. These results suggest that γδ T cells may recognize an unknown ligand in the bladder and produce IL-17A upon TCR stimulation. At present, the specificity of γδ T cells remains unknown. Therefore, it will be of interest to ascertain whether γδ T cells in the murine bladder recognize any such unique antigen.
In conclusion, we found that the CARD9-dependent pathway, which induces IL-17A production by γδ T cells, contributes at least in part to the pathogenesis of acute CYP-induced cystitis. In clear contrast, we have previously reported that the TLR2-MyD88 pathway induces IL-17A production by γδ T cells, which are involved in host defense against extracellular pathogens such as E. coli and Candida albicans [20, 39]. These findings clearly indicate the importance of innate immune cells, which regulate not only host defense but also autoimmune disorders by activating distinct signaling pathways. These responses are primarily regulated by ligand–receptor interactions. In this regard, further studies of the molecular mechanisms responsible for the initiation of HC are required. Nevertheless, the FcRγ-CARD9 pathway could be a novel target for the management of acute CYP- induced cystitis.