The Immunomodulatory Imbalance in Patients with Ketamine Cystitis

The pathogenesis of ketamine cystitis (KC) has been recently linked with immune response to patients but the same has not yet been established. Hence, this study aims to propose a possible immune mechanism of irreversible bladder damage caused by KC. A total of 53 KC patients and 21 healthy volunteers as controls have been retrospectively assessed. The levels of serum immunoglobulin E (IgE), IL-6, and IFN-γ of KC patients were significantly higher than those of controls, whereas the TGF-β levels of KC patients substantially reduced but the IL-2 and IL-4 levels of KC patients were comparable to those of controls. Moreover, the KC patients had significantly higher counts of TH1, TH2, and TH17 cells than those of controls. The immune response of KC users may begin with the IL-6 production and differentiation of TH17 and may be followed by alternating between high expressions of TH1 and TH2. The IL-6 may further suppress the TREG cells which can aggravate chronic inflammation in KC patients and the imbalance in TH17 and TREG cells may involve the pathogenesis of KC. Further investigation is needed to define the role of IL-6 in TH1/TH2/TH17-regulated signaling pathway in ketamine-induced cystitis.


Introduction
Ketamine is a phencyclidine hydrochloride derivative which is mainly used for starting and maintaining anesthesia [1]. It induces a dissociative, trance-like state in which the patient is unable to respond to external stimuli [2]. Chronic abuse has been associated with an out-of-body experience known as the K-hole phenomenon [3]. Due to low price and availability, ketamine has been increasingly used as a recreational drug which affects the central nervous and cardiovascular systems [4]. The chronic ketamine abuse may cause ulcerative cystitis and dysfunction of the lower urinary tract, both of which have been recognized as a new disease entity, called "ketamine cystitis" (KC). The number of KC patients has been increased dramatically in the past decade [5]. The KC shares many common histopathological features with interstitial cystitis/bladder pain syndrome (IC/BPS), including urothelial ulceration, inflammatory cell infiltration, and varying degrees of bladder wall fibrosis [6]. Among them, the long-term bladder inflammation may cause fibrosis of the bladder wall and, eventually, a contracted bladder [7]. In particular, the eosinophil and mast cell infiltration are frequently seen in human bladder tissue after long-term ketamine use [6,8,9]. A recent study reported that the KC patients had higher serum immunoglobulin E (IgE) than patients with IC/BPS or acute bacterial cystitis, which may be associated with bladder pain severity and small maximal bladder capacity in KC patients [10]. Therefore, the immune response to ketamine may play a key role in the pathogenesis of KC. However, only little knowledge on the immune activity in KC patients has been established.
On the other hand, the pivotal immune events of immune diseases are the development of antigen-specific effector T helper type 2 (T H 2) cells, T H 1 cells, or the recently defined T H 17 cells that are associated with clinical features and disease progression [11]. It is known that the phenotype of T cells is influenced by the tissue microenvironment which is produced through the dominant kind of cytokines, costimulatory molecules, and the character and dose of antigen presented. The T H 1 cell development is promoted by interleukin 12 (IL-12) and gamma interferon (IFN-) and primarily involves T-bet signaling. The generation of T H 2 cells is probably induced by IL-4, IL-5, and IL-6 costimulation. The T H 17 lineage is induced by the secretion of IL-6, IL-21, and the transforming growth factor beta (TGF-), whereas the T H 17 cells secrete a profile of potent proinflammatory cytokines (IL-17, IL-21, and IL-22), potent tumor necrosis factor (TNF-), and IL-6 upon certain stimulation [12]. It is to be noted that the regulatory T (T REG ) cells secrete IL-10 and TGF-, which modulates helper T cell activity and suppresses some of their functions, inducing tolerance to antigens.
The T H 1, T H 2, and T H 17 populations, and the cytokines they release, are antagonistic to each other and one or the other subtype is dominant in response to a particular pathogen at any one time. However, it is important to know how the body determines, and which differentiation pattern and immune response are appropriate for a specific pathogen, and what the molecular mechanisms that underpin differentiation into a T H 1, a T H 2, or a T H 17 cell are [13]. Since the contribution of specific T H cell subsets and related cytokines in the pathogenesis of KC is unknown, we hypothesize that measuring circulating cytokines may elucidate the imbalance between T H cell subsets in KC patients. In this context, we conducted this case-control study in order to investigate plasma levels of T H 1 (IL-2, IFN-), T H 2 (IL-4, IL-5), T H 17 (IL-17), and T REG (TGF-) cytokines.

Patient and Sample Collection.
A total of 53 KC patients and 21 healthy volunteers were included in this retrospective study. Each of the KC patients showed symptoms of lower urinary tract irritation, such as urgency, nocturia, and frequency. The urine and blood samples of each patient were collected for analysis. The urine test panel was conducted for pH and sediments which include urine red blood cells (RBCs), urine white blood cells (WBCs), and epithelial cells. Blood analysis included WBC counts, RBC counts, and differential count (neutrophils, lymphocytes, monocytes, and eosinophils). The paraffin-embedded urothelial tissues were obtained by urothelial biopsy from six patients under sterile conditions after one week of the urine and blood sample collection.

Statistical Analysis.
All the data were evaluated by analysis of variance (Sigma plot, 2001). The values are reported as mean ± SD of at least three experiments. Paired -test and chi-square test were employed for comparison between two groups, and values ≤ 0.05 were considered statistically significant. The correlation coefficient ( ) was applied between the serum cytokine levels.

Results
A total of 74 subjects (53 KC users and 21 nonusers) were enrolled in this study. The chronic ketamine users are 31 males and 22 females in which their age was 23.84 ± 4.74 years (mean ± SD). The average duration of ketamine usage was 50.44 ± 4.99 months for KC patients (Table 1). Among the 53 KC patients, 26 patients underwent full clinical examination and cystoscopy with hydrodistention, and 3 patients refused bladder biopsy. Table 2 lists the clinical information and serum Ig levels. The laboratory studies revealed an elevated creatinine (1.25 ± 0.42 mg/dL) and IgE levels (261.59 ± 56.03 IU/mL) with positive RBCs and WBCs in urine analysis. Further, the urothelial biopsies were performed a week after urine and blood sample collection. And, the tissue histology analyzed using hematoxylin and eosin (H&E) stain. The urinary bladder mucosa revealed denuded bladder epithelium, hemorrhage (Figures 1(a) and 1(c)), acute and chronic inflammation as well as the presence of  Flow-cytometric analysis of KC patients showed considerably higher T H 1 (IFN-, 10.94 ± 1.81%), T H 2 (IL-4, 10.72 ± 2.79% , and IL-5, 11.09 ± 4.17%), and T H 17 (IL-17A, 7.11 ± 2.12%; = 0.0001) cells and also gave some representative fluorescence-activated cell sorting (FACS) plots ( Figure 2). The univariate comparison of 6 cytokines in the normal and KC groups revealed a two-to ninefold increase in serum IL-6 (2-tailed = 0.05), IL-1 , IL-5, and IL-17 expression. The serum levels of the remaining proteins, namely, IL-2 and IL-4, were not significantly increased in the KC group relative to control groups (Figure 3(a)). The distribution of all serum cytokines tested passed the Mann-Whitney test for normality. In addition, the serum cytokine assays were used to determine the effects of ketamine on inflammatory cytokine levels which showed a remarkably increased level of TNF-(KC versus control; 193.59 ± 282.17 versus 98.43 ± 85.48 pg/mL) and IFN-(369.78 ± 564.56 versus 0 pg/mL; < 0.001) in KC cases (Figure 3(b)). Patients with KC had significant decrease in TGF-1 (KC versus control; = 25429.14 ± 25940.15 versus 38857.60 ± 13482.19 pg/mL; = 0.008) and TGF-3 (950.01 ± 498.500 versus 1236.30 ± 218.88 pg/mL; = 0.046; Mann-Whitney test) levels ( Figure 3(c)). The TGF-2 level was also found to lower in KC patients comparing to the controls (1068.22 ± 695.280 versus 1380.99 ± 387.45 pg/mL; = 0.093) although there was no significance. Since the KC group revealed that the levels of TGF-1, TGF-2, TGF-3 were below the level of detection in the control group, these results lend support to our hypothesis that an increase in cytokines is linked to inflammatory cells and regulation between T H 1, T H 2, and T H 17.
The basis of several statistical tests that result in a correlation coefficient is defined as a numerical representation of the strength and direction of a relationship. On moving to the correlation of cytokines of KC patients (Figure 4)

Discussion
In the current study, we have illustrated the different profiles of inflammatory cytokines and T H cell expression in KC patients versus healthy controls. The differing cytokine characteristics between normal control and chronic ketamine users may allude to a possible mechanism of KC ( Figure 5). In this regard, we hypothesize that the acute inflammation seen in the bladder mucosal and submucosal layer may be caused by the acute immune response of ketamine through the T H 1 cell pathway. Ketamine may enhance the production of IL-6 which can further result in the apoptosis and shedding of epithelial and interstitial cells. In KC patients, T H 1 and T H 2 cells may be produced in a positive feedback loop from naïve T cells and then inhibit each other; the immune response oscillates between T H 1 and T H 2 cells, triggering varying symptoms and eosinophil infiltration among chronic ketamine users.
Further, we found significantly increased levels of IL-6, TNF-, and IFN-with comparably higher levels of IL-1 , IL-5, and IL-17 in the serum of KC patients than those of the healthy controls. Since the cytokine, IL-6, promotes the development of T H 17 cells, the higher level of IL-6 in KC patients suggests that the balance between T H 17 and T H 1 responses as well as IL-6 production is dysregulated in KC, which resulted in the reinforced feedback that increases the IL-1 and IL-17 production in CD4+ T cells. This increase may contribute to disease pathogenesis ( Figure 5). Therefore, the function of IL-6 might assist in the knowledge of its complex role in inflammation: throughout acute inflammation, the IL-6 might favor the resolution from neutrophilic infiltrate as well as favor the initiation from the immune response in chronic inflammation. Further, the IL-6 induces the development of T H 17 cells from naïve T cells and, together with TGF-, may boost the mononuclear-cell infiltrate and take part in disease pathogenesis [14].
T REG cells have been recognized as a suppressor of effector T cells and, thus, are thought to control the T H 1, T H 2, and T H 17 responses. Our results showed low concentration of T REG related serum cytokines, IL-2 and TGF-, which may explain the persistent inflammation in the KC patients (Figures 3(a) and 3(c)). It is to be noted that the TGF-3 regulates molecules involved in cellular adhesion and extracellular matrix (ECM) formation in tissue [15]. So, the lower concentration of TGF-3 in the KC group suggests the impaired healing capability of bladder epithelial lining in chronic KC patients. The elevated IL-17 level and reduced TGF-level showed that the CD4+ T cell differentiation is biased away from T REG cells toward the T H 17 cell phenotype [16]. The differentiation of CD4 + cells generates the T H 1 and T H 2 phenotypes with the same receptor specificity, indicating the competition for antigenic stimulation, mediated by antigenpresenting cells (APCs), combined with cytokine-mediated cross-suppression between phenotypes to yield a response that is eventually dominated by T helper cells that are uniform in both receptor specificity and cytokine secretion phenotype. We found a different T helper cell phenotype distribution that the T H 1, T H 2, and T H 17 receptors were all expressed in PBMCs (Figure 2), consistent with recent data [17]. We subsequently analyzed the serum cytokine secreted from the T helper cells in KC patients.
Previous studies have shown that many, but not all, of the effects of IFN-paracrine can be stimulated to T H 1 cells to secrete lymphokines [18]. The increase in IFN-  paracrine may stimulate the T H 1 cells to secrete their own lymphokines TNF-, IFN-, and IL-6 ( Figure 3(b)). The IL-6, combined with TGF-and IL-1 , induces the differentiation of T H 17 from naïve cells and then enhances the IL-17 secretion (Figure 3(a)). IL-6 is an important switch that determines whether T H 17 or T REG cells are generated when TGF-is present; TGF-1 has been known to inhibit the differentiation of T H 1 and T H 2 cells [19,20]. Hence, increases in IL-6 and IL-1 noted in KC patients may cause an offsetting response to the increase in TGF-1, TGF-2, and TGF-3 (Figures 3(a)  and 3(c)). IL-6 and IL-1 have been found to be increased in inflammatory urinary tract disease, indicating that IL-6 may also play an essential role in bladder dysfunction related to urinary tract infection [21].
IL-6 is a multifunctional cytokine which is produced at sites of tissue inflammation. It has been demonstrated that IL-6 derived from APCs is able to initiate IL-4 production in naïve CD4 + T cells, thereby polarizing these cells into T H 2 cells [22]. IL-4 targets B cells and mast cells, resulting in the production of IgE and inhibits IL-6 production; IL-5 targets eosinophils. The increased IFN-may induce differentiation of CD4 + cells into T H 1 cells and suppress the proliferation of T H 2 cells, causing apoptosis of eosinophils and inhibiting IgE production [23]. Our results showed that both the eosinophil counts (Table 1) and IgE (Table 2) in KC patients were higher than that of normal laboratory values. Increased T H 2 related cytokines, IL-4 and IL-5, were also found in KC patients, although there was no significant difference when compared to the normal control group (Figure 3(a)). The T H 2 cells also inhibit the development and function of T H 17 and T H 1 cells by means of their signature cytokine IL-4 [22]. These results demonstrated that IL-6 signaling has the ability to enhance the production of T H 2 related cytokines (i.e., IL-4 and IL-5) via CD4+ T cell pathway. As regards KC disease, a strong correlation between the frequencies of IL-17, TNF-, and IFN-in KC patients has been observed ( Figure 4). Previous studies have indicated that TNF-and IL-17 have synergistic effects with many other factors, including IL-1 , IL-22, and IFN- [24]. The infiltrations of T H 1 and T H 17 cells have been associated with the pathogenesis of autoimmune disorders.  T H 17 effector cells are induced in parallel to T H 1, and, like T H 1, polarized T H 17 cells have the capacity to induce inflammation and autoimmune disease [16]. The main function of IL-17-secreting T cells is to mediate inflammation, by stimulating production of inflammatory cytokines that promote the recruitment of neutrophils and macrophages [25]. Although we observed elevated IL-1 levels, the reduction of TGF-is consistent with the elevation of IL-6 and IL-17 and may reflect T H 17 cell activity rather than T REG cell activity. In addition, the elevated levels of T H 2 cells and their associated effector cytokines, IL-5, were correlated with IL-1 and IL-17, indicating a central role for T H 17-mediated immune responses associated with IL-6; IL-6 promotes T H 17 but suppresses T REG differentiation in KC disease [26]. Since the IL-5 and IL-17A regulate the production of specific-IgE in vivo [27], these results may explain the reason of increased IgE level and also can convince the eosinophil infiltration in the bladder suburothelium that was found in KC patients.
Study limitations include small sample size and the single time point serum measurement. The increased cytokine levels in KC patients reflect that the ongoing bladder inflammation noted at the bladder ulcer site is to be confirmed by further investigation. Further investigation is necessary to clarify the role of IL-6 in T H 1/T H 2/T H 17-regulated signaling pathway in ketamine-induced cystitis. These preliminary results pieced together from data collected from ketamine patients offer a possible mechanism of ketamine-associated cystitis. However, further in vivo tests would confirm the effects of various cytokine concentrations on T cell differentiation, particularly the initial concentrations of TGF-and IL-6. In vivo experiments would also allow us to control the daily amount of ketamine and the bladder could be analyzed at various stages of the inflammatory response to confirm our theory.

Conclusion
Ketamine may cause acute inflammation through T H 1 pathway and elicit secondary mediator IL-6 generation which links to the dynamic balance of T H 17/T REG cell effector functions. The IL-6 may cause tissue destruction and collagen deposition via T H 17 cells activation and suppress T REG cells resulting in sustained activation of T H 1 and T H 2 cells. Further, the IL-6 may promote the differentiation of T H 2 cells by enhancing endogenous IL-4 production and increase the production of IgE in KC patients. This is associated with T H 2-mediated chronic inflammatory response. In addition, the low concentration of TGF-3 in KC patients implied a suppression of T REG function, which may cause the tissue damage and immune-mediated inflammation. This will subsequently impair the healing of urothelium in KC patients.

Ethical Approval
This study was approved by the Institutional Review Board, Ministry of Science and Technology, National Defense Medical Center, Veterans General Hospital, and Tri-Service General Hospital, Taipei, Taiwan. There was no patient identifying information included in this manuscript. All the study subjects were strictly adhered to the Declaration of Helsinki 2012.