The role of inflammatory cytokines and ERK1/2 signaling in chronic prostatitis/chronic pelvic pain syndrome with related mental health disorders

Mental health disorders(MHD) in chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) have been widely studied. However, the underlying role of inflammatory cytokines and their associated signaling pathways have not been investigated. Here, we report the potential role of cytokines and associated signaling pathways in CP/CPPS patients with MHD and in a CP/CPPS animal model. CP/CPPS patients (n = 810) and control subjects (n = 992) were enrolled in this case-control multicenter study, and serum cytokine levels were measured. Male Sprague-Dawley rats received multiple intracutaneous injections of an immuno-agent along with a pertussis-diphtheria-tetanus triple vaccine for autoimmune CP/CPPS development. The results revealed that, in CP/CPPS patients with significant MHD, elevated IL-1α, IL-1β, IL-4, IL-13, and TNF-α serum levels were observed. The above five cytokines in CP/CPPS rats were significantly elevated in prostate tissue (p < 0.05), and IL-1β levels were elevated in serum and cerebrospinal fluid. In behavioral tests, CP/CPPS rats showed anxiety- and depression-like symptoms, and impaired spatial and associative memory performance (p < 0.05). In the CP/CPPS group, ERK1/2 phosphorylation levels were increased in the amygdala and nucleus accumbens, and decreased in the hippocampus, but not caudate nucleus. Thus, prostate-derived cytokines, especially IL-1β, cross the blood brain barrier and may lead to enhanced ERK1/2 signaling in several brain areas, possibly underlying induction of CP/CPPS-related MHD.

. Serum cytokine levels in control subjects and CP/CPPS patients, and their correlation with NIH-CPSI, SAS, and SDS. IL: interleukin; MCP: monocyte chemokine protein; TNF: tumor necrosis factor; IFN: interferon; Mean values ± SD are reported for all parameters. * CP/CPPS compared to controls when p < 0.05 (Student's t-test). * * The index correlated to cytokines when p < 0.05 (Spearman rank correlation).

Evaluation of the CP/CPPS rat model. Histological examination.
After H&E staining of the prostates, N45 and N60 control groups showed normal prostatic histology with minimal inflammatory infiltration, duct changes, or tissue damage at 50x and 200x magnifications. However, the prostates of CP/CPPS rats showed a prominent increase in inflammatory infiltration, duct ectasia, and vascular congestion in the C45 group at both magnifications indicating that the autoimmune CP/CPPS model was successfully established in the individual rats. Furthermore, inflammatory duct infiltration and gland destruction was observed in the C60 group (Fig. 1).
Increased anxiety-like behavior in the CP/CPPS rat model. In the EPM, the percentage of time spent in the open arms was significantly lower in the C45 group (10.42%) compared with the N45 group ( Fig. 2A 48.40%, respectively), but differences did not reach significance levels. During the first 5 min the percentage of ambulatory counts in the center zone in the C60 group (5.07%) was significantly lower compared with the N60 group (Fig. 2D, 9.82%, p < 0.05). Similarly, the distance traveled (5.59% vs. 8.87%) and number of entries (40.25% vs. 47.09%) into the center zone were lower, although these did not reach significance levels. During the entire test (10 min), the percentage of the distance traveled in the center zone in the C60 group (6.31%) was significantly lower compared to the N60 group (9.77%, p < 0.05). The percentage of ambulatory counts (6.51%) and entries (45.76%) in the center zone was lower compared to the N60 group (10.40% and 48.44%, respectively) without reaching significance levels. Therefore, the reduced activities in the center zone observed in the CP/CPPS groups compared to the control groups indicated depression-like behavior.
Impaired spatial memory in the CP/CPPS rat model. In the Y maze test, reduced time spent in the novel arm (34.96% vs. 38.21%) and the absolute latency (35.07% vs. 37.73%) was observed in the C45 group when compared with the N45 group, but the latency between the C45 and N45 group did not differ from each other (Fig. 2E, both 33.37%). In the novel arms of the Y maze, the percentage of the absolute latency in the C60 group (53.18%) was significantly shortened when compared with the N60 group (Fig. 2F, 27.36%, p < 0.05). Although the percentage of time spent in the novel arms (44.30%) and the latency (26.13%) in the C60 group were lower compared with the N60 group (48.12% and 46.12%, respectively), the differences were not statistically significant. Overall, the decreased activities at day 60 in the novel arms indicated that the CP/CPPS groups had difficulties in recollecting memory, which implies impaired spatial memory capacity in the CP/CPPS groups. Impaired associative memory related behavior was observed in the CP/CPPS rat model. In the SBPA test (Fig. 2G), the latency in the C45 group was significantly lower compared with the N45 group (10.78 s vs. 172.87 s, p < 0.05).
In addition, the latency in the C60 group remained significantly lower than that in the N60 group (120.28 s vs. 215.19 s, p < 0.05). Although rodents instinctively enter darkness, the previous foot shock provided a negative association with darkness. Thus, the lessened latency reflected impaired associative memory in the CP/CPPS groups. The expression of cytokines and their receptors in prostate and specific brain areas associated with anxiety, depression, and memory. In prostates, RT-qPCR showed that the levels of IL-1α , IL-1β and IL-4 in the CP/CPPS rats were significantly elevated compared with controls (p < 0.05, Fig. 3A). However, the difference in IL-13 between the N60 and C60 groups, and TNF-α between the N45 and C45 groups were not significant. Furthermore, in brain tissues the receptors of these cytokines, including IL-1R1, IL-1R2, IL-1R4, IL13R1, IL-13R2, TNFR1, and Elevated cytokines in prostate correlated to MHD related behavioral pattern in rats. Correlation analysis using the Spearman rank correlation was performed for cytokines in the prostate and MHD related behavioral pattern. The significantly elevated IL-1α , IL-1β , IL-4, IL-13, and TNF-α levels in the prostate were all negatively related to positive responses in MHD related behavioral tests, which means positive correlation to anxiety, depression, and spatial and associative memory impairment, although significance levels were not reached (Table 3).

ERK1/2 activation in BLA and NAc and inactivation in Hippo and Cau in the CP/CPPS rat model.
Western blot analysis revealed that the phosphorylation level of the ERK1/2 was significantly increased in BLA and NAc of CP/CPPS rats compared with controls (p < 0.05), indicating enhanced ERK1/2 activation. In contrast, ERK1/2 phosphorylation levels were significantly reduced in the Hippo in CP/CPPS rats compared with controls (p < 0.05), indicating ERK1/2 inactivation in these brain regions. However, the phosphorylation level of ERK1/2 did not show alterations in Cau between CP/CPPS and control rats (Fig. 4A,B).
Elevated IL-1β in serum and CSF of the CP/CPPS rat models. In serum, the levels of IL-1β in both the C45 and C60 group were significantly increased compared with controls (p < 0.05). Similarly, in the CSF the level of IL-1β was elevated although the differences were not significant (Fig. 5).

Discussion
The present study comprising 810 CP/CPPS patients with MHD and 992 control subjects showed significantly more anxiety and depression with higher SAS and SDS scores observed in CP/CPPS patients compared with normal control subjects, which was consistent with the previous reports [32][33][34] . Next, we investigated potential mechanisms that may facilitate CP/CPPS-related MHD. Five cytokines, IL-1α , IL-1β , IL-4, IL-13 and TNF-α , were significantly elevated in the serum of CP/CPPS patients. Among them, both IL-1β and TNF-α were positively correlated with SAS and SDS scores, which hint to the potential role of IL-1β and TNF-α in CP/CPPS-related MHD.
To further investigate the role of cytokines in CP/CPPS-related MHD, an established autoimmune rodent animal model of CP/CPPS was employed. Multiple intracutaneous injections at the neck, the tail, and the pelvic limbs with immuno-agent and an intraperitoneal injection with pertussis-diphtheria-tetanus triple vaccine for 45 and 60 days generated CP/CPPS in rats. CP/CPPS-related MHD in rats was evaluated by tests for anxiety in EPM, depression-related activity levels in OF, spatial memory impairment in the Y maze, and associative memory impairment in SBPA [35][36][37][38] . Similar to the finding in human subjects, anxiety-like and depression-like behavior was more common in the CP/CPPS rat model compared with control animals. Moreover, significant spatial and associative memory impairment was observed in the CP/CPPS group, what supports a possible link between disturbances in anxiety-and depression-like behavior and memory.
Similar to the findings in CP/CPPS patients, elevated levels of IL-1α , IL-1β , IL-4, IL-13, and TNF-α were observed in CP/CPPS rat prostates, and these elevated cytokine levels showed a positive trend in their correlation with anxiety, depression, and memory impairment. Even though significant levels were not achieved, there was a consistent trend for a correlation between elevated cytokine patterns and the behavioral performance of MHD, which suggested a potential valuable link between cytokines and CP/CPPS-related MHD. In the present study, we found that not only cytokine expression showed significant changes in specific brain areas, but that also the receptors of IL-1α , IL-1β , IL-4, and IL-13 showed decreased expression profiles, some of them reaching statistical significance levels, in BLA, NAc, and Hippo in CP/CPPS rats compared with controls. Interestingly, the changes of these receptors in Cau between CP/CPPS rats and controls seemed not predictable from the changes observed for their ligands. Thus, it is likely that rather than one kind of cytokine probably the integration of multiple cytokines and their receptors is important for the behavioral impact. Previous studies have demonstrated that cytokines by interacting with their specific receptors may modulate the MAPK/ERK downstream signaling pathway 39,40 . Through stabilizing structural changes in dendritic spines, MAPK/ERK regulates a wide variety  of forms of synaptic plasticity. Synaptic plasticity is a crucial cellular mechanism underlying a variety of brain functions, including learning and memory, but also depressive and anxiety behavior. Interestingly, in anxiety and depression patients, ERK1/2 has been found activated in BLA and NAc [41][42][43][44] . However, during impairment of memory functions ERK1/2 has been shown to be inactivated in the Hippo and Cau 45,46 . Briefly, when ERK1/2 was inhibited synapse formation was reduced leading to the inhibition of synaptic plasticity 26 . Consistently, by using the 3T morphometric system, Mordasini et al. described reduction in relative gray matter volume in the anterior cingulate cortex, a core structure of emotional pain processing, correlating with bother of CP/CPPS. Even though the disaccord on brain area alterations were found compared to the demonstration of Farmer et al., both of these  . * Indicate p < 0.05 vs. controls. The data are presented as means ± SD.
Scientific RepoRts | 6:28608 | DOI: 10.1038/srep28608 research units discovered the specific alteration in brain areas, which supported by the theory that dysfunctional central plasticity and functional reorganization lead to peripheral discomfort, such as chronic pain 5,11 . The functional magnetic resonance imaging and identification of different phases of CP/CPPS would probably be helpful in solving current divergence. In the present study, ERK1/2 phosphorylation levels were increased in the BLA and NAc and were reduced in the Hippo of CP/CPPS rats. The changes of this signaling pathway downstream of cytokines were consistent with the observed changes in behavioral test. We also noted that CP/CPPS rats showing reduced associative memory behavioral performance compared with controls, did not show changes in ERK1/2 activity. Therefore, these significant behavioral differences must be correlated with several other factors, besides ERK1/2, such as glucocorticoids and dopamine-dependent memory retrieval signaling pathways, which likely contribute to the associative memory impairment. Cytokine levels showed similarly elevated trend in humans and in the CP/CPPS rat models, hence the PPE combined FCA induced autoimmune CP/CPPS appears to mimic the inflammatory status of CP/CPPS patients. Importantly, Leonard et al. have suggested that IL-1β could be transported from peripheral blood into the brain by an active transport mechanism 47 . Our results showed elevated IL-1β in CSF of the CP/CPPS rat model. It appears likely that these elevated CSF cytokines interact through some of the identified receptors for these cytokines in specific brain areas. Therefore, IL-1β derived from prostate may pass through the blood brain barrier, be transported by CSF, and interact with its receptors in specific brain areas.
Previous reports have shown that IL-1β may lead to anxiety by interacting with the endocannabinoid system and modulate neuronal function of the BLA 48,49 . Through participating in hypothalamic-pituitary-adrenal axis hyperactivity, IL-1β has been shown to cause depression 50,51 . In addition, IL-1β has been closely correlated with spatial and associative memory injury in previous studies 52, 53 . In our study, IL-1β was significantly elevated in the CP/CPPS group and was positively correlated with SAS and SDS. Thus, the cytokine IL-1β may be one of the major molecular factors responsible for CP/CPPS-related MHD. Altogether, results in an animal model and in CP/CPPS patients confirm that anxiety and depression are associated with CP/CPPS. This study also confirms that elevated peripheral cytokine levels in the prostate and serum are likely involved in the molecular pathophysiology of CP/CPPS-related MHD. Taken together, it is a possibility that the prostate-derived cytokines, especially IL-1β , cross the blood brain barrier and can interact with their respective receptors in specific brain areas, which may lead to changes in ERK1/2 activity, and in turn lead to CP/CPPS related anxiety, depression, and spatial and associative memory impairment.
The present study has several limitations. The lack of significant differences in the activity of ERK1/2 made it difficult to assign a specific mechanism of action for prostate-derived cytokines in CP/CPPS-related MHD. The downstream ERK1/2 signaling pathway in neurons is conditioned by various kinds of factors, such cytokines interaction, growth factors, and ligands of G protein coupled receptors. The current results are probably an account of the balance of these effects. Further studies on the mechanism of action, such as perfusion with ILs, polyamines, and their antagonists by stereotaxic techniques and by in vitro chronic stimulation in primary cultures of specific brain areas are needed to address those issues. However, CP/CPPS-related MHD in patients as well as in rats and changes of peripheral inflammatory cytokines and downstream cerebral ERK1/2 signaling activity observed in the present study will undoubtedly give additional opportunity to explore uro-neurology diseases in this direction. Clinically, possibly blockage or depletion of inflammation cytokines in peripheral blood and CSF would be helpful not only in reducing inflammation, but also in developing available strategy in relieving CP/CPPS related MHD.

Study patients and design. Between July 2012 and August 2013, 1000 patients at seven hospitals (Ren Ji
Hospital and Shanghai General Hospital affiliated with the Shanghai Jiao Tong University, Zhongshan Hospital affiliated with the Fudan University, East Hospital affiliated with the Tongji University, Shanghai Eighth Hospital affiliated with the Jiangsu Hospital, and Longhua and Yueyang Hospital affiliated with the Shanghai University of Traditional Chinese Medicine) who complained of discomfort or pain in the pelvic region (penis, testes, scrotum, perineum, pubic area, or lower back) for at least three of six months, were interviewed and examined by experienced urologists. Eight hundred and ten patients (aged , who were diagnosed with CP/CPPS based on the National Institute of Health (NIH) criteria, were enrolled in this case-control study 1 . Age matched healthy men (n = 1000) without any discomfort in the pelvic region were interviewed and examined by the urologists, and 992 were recruited as healthy controls.
All patients and healthy controls underwent physical and rectal examinations, hemanalysis, midstream urine analysis for microbe growth, and quantification of the prostate specific antigen (PSA). All patients and control subjects completed the Chinese versions of the NIH Chronic Prostatitis Symptom Score (NIH-CPSI), the Self Anxiety Scale (SAS) and the Self Depression Scale (SDS) 54,55 . Both SAS and SDS set scores of 50 as cut-off value with higher values being considered diagnostic for anxiety or depression. Within both groups, the serum samples of 56 individuals (28 patients and 28 controls) were randomly selected for analysis of inflammatory cytokine levels.

Protein extraction from prostate tissue in rats.
Four-month-old male Sprague-Dawley (SD) rats (n = 7, specific pathogen-free) received anesthesia with sodium pentobarbital (30 mg/kg, i.p.) under aseptic conditions. A lower abdominal incision was made and the prostate was identified and extracted. For protein analysis, the prostate was homogenized manually with 0.5% Triton X-100 lysis buffer and centrifuged (13,500 rpm, 30 min) twice at 4 °C. The supernatant was then collected as prostate protein extract (PPE). The PPE concentration was quantified and normalized to 40 mg/ml by spectrophotometry (NanoDrop ND-100, Wilmington, DE, USA).

Establishment of CP/CPPS rat model.
To establish a CP/CPPS animal model, two-month-old male SD rats were randomly divided into four groups, two CP/CPPS groups (C45 and C60) and two normal control groups (N45 and N60). The C45 (n = 10) and C60 (n = 10) groups were treated by the autoimmune method for 45 days or 60 days, respectively, to induce development of CP/CPPS. Briefly, the animals underwent multiple intracutaneous injections at the neck (0.4 ml), the end of the tail (0.3 ml), and the pelvic limbs (0.15 ml) with immuno-agent (1:1 mixed PPE and Freud's complete adjuvant) and an intraperitoneal injection with pertussis-diphtheria-tetanus triple vaccine (0.5 ml). The corresponding normal control groups N45 (n = 8) and N60 (n = 8) received the same injection regimen for 45 and 60 days, respectively, but using saline. The autoimmune method was conducted at the 1st day and repeated at the 30th day.
Histological examination. The inflammation status of the prostate was evaluated by visual examination of gland integrity, white blood cell infiltration, and structure of prostatic ducts through hematoxylin and eosin (H&E) staining. Briefly, the prostates from each group were immersed in 10% neutral buffered formalin, embedded in paraffin, and cut into 5 μ m sections. Slides were cleared in xylene and dehydrated, followed by standard H&E staining procedure. Open field (OF). The potential effects of CP/CPPS in rats on depression-like behavior were assessed in the OF paradigm. A single rat was placed in the center of the OF apparatus. The distance traveled, ambulatory (walking) counts, and entries into the center zone of the OF were recorded and analyzed for a period of 10 min with a video camera system and analysis software (Med-Associates, St. Albans, VT, USA). The apparatus was cleaned thoroughly with 95% ethanol and water after each test session. Low locomotor activity levels in the center zone were considered depressive behavioral condition 57 .

Behavioral analysis of anxiety, depression, and memory. Elevated plus maze (EPM
Y maze. The potential effects of CP/CPPS in rats on spatial memory were assessed in the Y maze. On the first trial, rats were habituated individually to a randomly selected "start arm" for 5 min, one of the two remaining arms was randomly blocked as "novel arm", whereas on the second trial, lasting for 5 min, all the arms of the maze were open. The two trials were separated by a 2 min break, during which the rat was returned to its home cage. Travel times into the novel arms, latency (i.e., percentage of time in novel arms compared to total time), and absolute latency (i.e., percentage of time in novel arms compared to total time without the time staying in the center zone) before moving in the novel arms were measured with a video camera system and analysis software (ANY-maze, Wood Dale, IL, USA). Between trials, the maze was cleaned using 95% ethanol and water and then dried. It was assumed that spatial memory was impaired when the activity in the novel arms was reduced 58 .
Shuttle box passive avoidance (SBPA). The potential effects of CP/CPPS in rats on associative memory related behavior were assessed in the shuttle box (Med-Associates, St. Albans, VT, USA). During the training session, rats were accustomed to the behavioral apparatus in the light box and were free to move into the dark box. Afterwards, the rats were given a foot shock (1 mA, 2 s) whenever they entered the dark box. During the test session, the rats were placed in the light box again and no electric foot shock was applied. The session ended after the animal stayed in the light box for more than 300 s. All animals were tested for associative memory retention 24 h after the training session. The latency change between training and test session was recorded and the apparatus was cleaned thoroughly with 95% ethanol and water after each session. Previous reports have shown that associative memory was impaired when the latency in the light box on the 2 nd day was decreased 59 .
CSF and brain tissue collection. CSF of each rat was collected by fine needle puncture at the foramen magnum. Under anesthesia (sodium pentobarbital, 30 mg/kg, i.p.), rats were kept in a lateral position to allow the back of the neck to be fully exposed. The puncture was made at the level of the foramen magnum and CSF was collected and stored at − 80 °C. After euthanasia, the rat brain was excised and the BLA, NAc, Hippo, and Cau were dissected according to The Rat Brain instruction and stored at − 80 °C 60 .
Enzyme-linked immunosorbent assays (ELISA). The level of IL-1β in serum and CSF was determined using the rat IL-1β Platinum Kit (eBioscience, San Diego, CA, USA) according to the manufacturer's instructions. The optical density of each well was determined at 450 nm using a Bio-Rad spectrophotometer (BioRad, Hercules, CA, USA).
Statistical analysis. Quantitative data are presented as means ± standard deviations (SD). Student's t-test was used for comparison between two samples. The chi-square test was used to compare the difference between  groups. Differences among more than two groups were assessed by one-way ANOVA with conservative Bonferroni's test. Correlation analysis was achieved by Spearman rank correlation. All statistical analyses were performed on SAS for Windows 8.0 (SAS Institute Inc, Cary, NC, USA). Statistical significance was considered at a p < 0.05 level for all parameters.