Bioenergetic profiles of peripheral mononuclear cells and systemic inflammation in women with Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS).

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a condition that occurs primarily in women and consists of symptomatic pelvic and bladder pain for more than six months [ 1 ]. Approximately 3.3 to 7.9 million women are affected by IC/BPS in the United States [ 2 ]. Patients with IC/BPS often experience the need to urinate urgently and frequently. The optimal way to diagnose IC/BPS is limited and is based on the exclusion of other urological/gynecological disorders such as urinary tract infections, overactive bladder, or pelvic inflammatory disease. The exact cause of IC/BPS appears to multifactorial and is not well understood. Some factors thought to contribute to the condition include genetics, autoimmune diseases, and infection [ 3 ]. In addition, patients with IC/BPS have a higher risk of developing additional co-morbidities such as fibromyalgia, endometriosis, and irritable bowel disease [ 4 – 6 ]. Thus, it appears the pathophysiology of IC/BPS could be largely influenced by systemic factors. Inflammation has been suggested to contribute to IC/BPS and to be associated with pain in afflicted patients [ 1 , 7 ]. During chronic inflammation, the bladder is infiltrated with mononuclear cells such as macrophages, lymphocytes, eosinophils and mast cells [ 1 ]. These cells elicit an immune response within the bladder and recruit other inflammatory cells from the circulation. Pro-inflammatory cytokines and chemokines, such as interleukin-6 (IL-6) and tumor necrosis factor (TNF-ɑ), are associated with mast cell activation and are elevated in the serum of this cohort [ 8 ]. TNF-ɑ is known to play a role in apoptosis and immune system development [ 9 ]. It is stimulated by the pro-inflammatory cytokine, interferon-gamma (IFN-ɣ) [ 10 ]. Such an environment in the circulation could promote a pro-inflammatory response and negatively affect immune cell function. A number of diseases such as diabetes, atherosclerosis, Alzheimer’s disease, chronic kidney disease, and cancer have been associated with systemic oxidative stress and mitochondrial dysfunction [ 11 ]. Mitochondria play a critical role in immune cell function and their ability to respond to immune activation [ 12 ]. Several studies have evaluated metabolism in peripheral blood mononuclear cells isolated from those with a variety of conditions including non-alcoholic fatty liver disease [ 13 ], mild cognitive impairment [ 14 ], Parkinson’s disease [ 15 ], fibromyalgia [ 16 ], and human immunodeficiency virus [ 17 ]. We have previously determined that monocytes have decreased cellular bioenergetics in patients with calcium oxalate kidney stone disease compared to age and gender matched healthy subjects [ 18 ]. A noticeable similarity among the aforementioned disease processes is their association with inflammation and other systemic disorders. The ability to isolate circulating immune cells to assess disease progression or treatment is an area of research that has many potential translational implications. The objective of this study was to determine whether circulating immune cells and inflammatory markers may be affected in IC/BPS. We assessed bioenergetic profiles in circulating monocytes and lymphocytes and plasma cytokine and chemokine levels from women with IC/BPS and age-matched healthy women. Monocytes and lymphocytes are derived from myeloid and lymphoid lineage, respectively, and are involved in innate and adaptive immunity. Our results suggest that monocytes and lymphocytes may contribute to the pathogenesis of IC/BPS and that inflammatory cytokines may be linked. These findings could aid in the development of novel therapeutic avenues for these patients to improve clinical outcomes, and methods of monitoring treatment responses. To our knowledge, this is the first study to assess immunometabolism in a cohort of patients with IC/BPS.

Healthy women (n = 18) and those diagnosed with IC/BPS (n = 18) were enrolled in the study ( Table 1 ). The mean ages were 43.9 ± 10.3 years and 49.6 ± 15.3 years in the HS and IC/BPS cohort respectively, which were not significantly different (p = 0.202). The body mass index (BMI) was similar (p = 0.575) among the two groups (28.8 ± 7.0 kg/m 2 HS vs. 30.3 ± 8.2 kg/m 2 IC/BPS). Of the patients with IC/BPS, sixteen (88.9%) had a non-ulcerative endoscopic diagnosis and two (11.1%) had an ulcerative endoscopic diagnosis. Some patients also had active or a history of hypertension (7; 38.8%), hypothyroidism (4; 22.2%), fibromyalgia (2; 11.1%), or endometriosis (1; 5.5%) at the time of the blood draw. These co-morbidities and phenotypic differences did not impact responses subsequently profiled. —Values are expressed in Mean ± SD. —HS = Healthy Subjects; IC/BPS = Interstitial cystitis/bladder pain syndrome —BMI = body mass index. —IC/PBS compared to HS. There are no statistically significant group differences for age (p = 0.202) or BMI (p = 0.575). *Ulcerative indicates Hunner’s patch observed on cystoscopy. (—), not applicable. Fig 1 shows the distribution of basal, ATP-linked, proton leak, maximal, reserve capacity, and non-mitochondrial OCR in monocytes from HS and IC/BPS patients. Basal respiration was significantly decreased in IC/BPS compared to HS (3.18 ± 0.83 vs 2.28 ± 0.82 pmol/min/10,000 cells; p = 0.004) ( Fig 1A ). Consistent with the basal OCR responses, ATP-linked OCR (2.65 ± 0.78 vs 1.86 ± 0.71 pmol/min/10,000 cells; p = 0.005) ( Fig 1B ), Proton leak (0.55 ± 0.18 vs 0.41 ± 0.16 pmol/min/10,000 cells; p = 0.014) ( Fig 1C ), maximal respiration (8.63 ± 3.98 vs 5.14 ± 3.51 pmol/min/10,000 cells; p = 0.012) ( Fig 1D ), and reserve capacity (5.45 ± 3.46 vs 2.86 ± 3.04 pmol/min/10,000 cells; p = 0.030) ( Fig 1E ) were all significantly reduced in patients with IC/BPS compared to HS. However, non-mitochondrial respiration was not significantly different between the two cohorts (1.43 ± 0.43 vs 1.31 ± 0.37 pmol/min/10,000 cells; p = 0.388) ( Fig 1F ). The XF analyzer also provided extracellular acidification rate (ECAR) data shown in Fig 1G and 1H . Basal ECAR (4.43 ± 1.48 vs 3.52 ± 1.00 mpH/min/10,000 cells; p = 0.033) and Oligo-sensitive ECAR (1.15 ± 0.52 vs 0.76 ± 0.38 mpH/min/10,000 cells; p = 0.030) were significantly decreased in patients with IC/BPS compared to HS ( Fig 1G and 1H ). Distribution of (A) basal, (B) ATP-linked, (C) Proton Leak, (D) Maximal, (E) Reserve Capacity, and (F) Non-mitochondrial OCR parameters as well as (G) Basal ECAR and (H) Oligo-sensitive ECAR parameters in monocytes from study participants. Data expressed as mean ± SD, n = 5–6 replicates; up to n  = 17 HS, n = 16 IC/BPS. *p<0.05, different from HS. Fig 2 shows the distribution of basal, ATP-linked, proton leak, maximal, reserve capacity, and non-mitochondrial respiration in lymphocytes from HS and patients with IC/BPS. Basal (0.88 ± 0.28 vs 1.19 ± 0.44 pmol/min/10,000 cells; p = 0.018, Fig 2A ), ATP-linked (0.82 ± 0.23 vs 1.01 ± 0.33 pmol/min/10,000 cells; p = 0.035, Fig 2B ), and maximal respiration (1.47 ± 0.63 vs 1.99 ± 0.87 pmol/min/10,000 cells; p = 0.037, Fig 2D ) were significantly higher in lymphocytes from patients with IC/BPS than HS. However, proton leak (0.24 ± 0.21 vs 0.21 ± 0.13 pmol/min/10,000 cells; p = 0.379, Fig 2C ), reserve capacity (0.58 ± 0.37 vs 0.82 ± 0.69 pmol/min/10,000 cells; p = 0.121, Fig 2E ), and non-mitochondrial OCR (0.64 ± 0.27 vs 0.73 ± 0.28, Fig 2F ) were not significantly different between the two groups. In addition, both basal ECAR (0.50 ± 0.18 vs 0.82 ± 0.27 mpH/min/10,000 cells; p = 0.001) ( Fig 2G ) and Oligo-sensitive ECAR (0.18 ± 0.15 vs 0.31 ± 0.22 mpH/min/10,000 cells; p = 0.040) ( Fig 2H ) were significantly higher in patients with IC/BPS compared to healthy subjects. Distribution of (A) basal, (B) ATP-linked, (C) Proton Leak, (D) Maximal, (E) Reserve Capacity, and (F) Non-mitochondrial OCR parameters as well as (G) Basal ECAR and (H) Oligo-sensitive ECAR parameters in lymphocytes from study participants. Data expressed as mean ± SD, n = 5–6 replicates; up to n  = 16 HS, n = 13 IC/BPS. *p<0.05, ***p<0.001 different from HS. Several cytokine and chemokine analytes were measured in the plasma of both cohorts using MSD Technology ( Fig 3 ). IFN-ɣ, TNF-ɑ, TGF-β, IL-6, IL-8, IL-10, and VEGF plasma levels were significantly different in patients with IC/BPS compared to HS ( Fig 3 ). IFN-ɣ is a cytokine secreted by the innate and adaptive immune systems and is known to activate macrophages [ 21 ]. IFN-ɣ plasma levels were significantly elevated in patients with IC/BPS compared to HS (2.12 ± 0.63 vs 2.88 ± 1.05 pg/ml; p = 0.029, Fig 3A ). Plasma TNF-ɑ levels were also significantly elevated in patients with IC/BPS compared to HS (1.25 ± 0.38 vs 1.81 ± 0.44 pg/ml; p = 0.001, Fig 3B ). TNF-ɑ is mainly secreted by macrophages and is involved in regulating a number of cellular processes such as cell proliferation and cell death [ 22 ]. In contrast, plasma TGF-β levels were significantly reduced in the individuals with IC/BPS (0.19 ± 0.06 vs 0.13 ± 0.06 pg/ml; p = 0.034, Fig 3C ). TGF-β is an important regulatory cytokine for maintenance of immune homeostasis by controlling the proliferation, differentiation and survival of lymphocytes [ 23 ]. Other pro-inflammatory cytokines, IL-6 and IL-8, were also significantly upregulated (0.69 ± 0.35 vs 1.18 ± 0.68 pg/ml (p = 0.022) and 4.42 ± 1.70 vs 6.62 ± 3.33 pg/ml (p = 0.035) respectively, Fig 3D and 3E ); whereas, IL-10, an anti-inflammatory cytokine, was reduced in patients with IC/BPS compared to healthy subjects (0.72 ± 0.38 vs 0.31 ± 0.15 pg/ml; p = 0.002, Fig 3F ). Vascular endothelial growth factor (VEGF) is another pro-inflammatory cytokine that was elevated in the plasma of individuals with IC/BPS (9.99 ± 4.72 vs 17.09 ± 8.76 pg/ml; p = 0.042, Fig 3G ). VEGF plays an important role in vascularization and the infiltration of monocytes and macrophages to inflammatory sites [ 24 ]. Lastly, IL-12/IL-23p40 and GM-CSF were not significantly different between the two groups (90.27 ± 29.58 vs 92.54 ± 26.44 pg/ml (p = 0.858) and 0.043 ± 0.052 vs 0.051± 0.056 pg/ml (p = 0.743), respectively, Fig 3H and 3I ). Distribution of plasma (A) IFN-ɣ (B) TNF-ɑ, (C) TGF-β, (D) IL-6, (E) IL-8, (F) IL-10, (G) VEGF, (H) IL-23/23 p40, and (I) GM-CSF levels in study participants; Results are presented as mean ± SD; up to n  = 15 HS, n = 16 IC/BPS *p<0.05 compared to HS. To determine whether plasma cytokine values correlated with monocyte and lymphocyte mitochondrial parameters, Pearson correlation coefficients were determined. The unadjusted correlation coefficients of mitochondrial maximal and reserve capacity OCR with plasma cytokines for both monocytes and lymphocytes are shown in Fig 4 and S1 Table . IFN-ɣ was inversely associated with maximal OCR (R = -0.7132, p = 0.021) and reserve capacity (R = -0.7041, p = 0.023) in monocytes from HS. IFN-ɣ did not have a significant relationship with either mitochondrial parameter in IC/PBS patients. IL-8 also had an inverse correlation with both maximal respiration (R = -0.723; p = 0.043) and reserve capacity (R = -0.709, p = 0.048) in monocytes from HS, while IL-8 was inversely correlated only with reserve capacity (R = -0.699, p = 0.049) in monocytes from IC/PBS patients. TGF-β was positively correlated with maximal respiration (R = 0.748, p = 0.042) in HS monocyte only. In lymphocytes, TNF-ɑ was positively correlated with maximal OCR (R = 0.818, p = 0.024) in HS, whereas IL-8 was inversely correlated (R = -0.787, p = 0.02) in HS. No significant associations were observed with any of the studied cytokines in lymphocytes from IC/PBS patients. The parametric correlation coefficients of (A) monocyte or (B) lymphocyte maximal respiration/reserve capacity oxygen consumption rates vs various plasma cytokine levels are shown. Red shades represent positive correlations and blue shades represent negative correlations. Data are from n = 18 HS and n = 18 IC/BPS.

Monocyte and lymphocyte metabolism is altered to varying degrees in women with IC/BPS compared to healthy women. The results of this study suggest that lymphocytes may be more bioenergetically active than monocytes in IC/BPS patients. In addition, TNF-ɑ plasma levels correlated positively with both maximal respiration and reserve capacity parameters in monocytes in IC/BPS. In contrast, IL-8 plasma levels correlated negatively with reserve capacity in monocytes from patients with IC/BPS. No statistically significant correlations were observed in lymphocytes in the IC/BPS group compared to HS. These data suggest that inflammation and metabolism in monocytes and lymphocytes could be important in understanding IC/BPS pathogenesis. To our knowledge, this is the first study to report these associations. Additional studies are needed to evaluate a larger cohort of patients and to understand the cross talk between inflammatory cytokines and immune cells from patients with IC/BPS. Findings from more planned studies could be useful to help understand IC/BPS epidemiology and guide therapeutic treatment in this cohort.

This study was approved by the University of Alabama at Birmingham Institutional Review Board. Informed written consent was obtained from all study participants prior to beginning the study. Age and BMI matched healthy women were recruited from the Birmingham, AL area and denied any urological disorders or symptoms. Women were only studied since IC/BPS primarily occurs in females and the ability to recruit men with this condition was limited. Detailed clinical characteristics of patients with IC/BPS were abstracted from electronic medical records. All IC/BPS subjects reported abdominal/pelvic pain, pressure, or discomfort associated with either bladder filling or frequent emptying, and did not have signs or symptoms which met the exclusionary criteria such as an active urinary tract infection. These symptoms met the IC/BPS definition generated by the Society of Urodynamics and Female Urology which continues to be endorsed by the American Urological Association’s IC/BPS guidelines panel [ 19 ]. Blood samples were collected from patients with IC/BPS prior to receiving pain management treatment at the UAB Medical Center between 7/1/2014 to 10/1/2016. Healthy subjects (HS) had their blood drawn at the UAB Center for Clinical and Translational Science Clinical Research Unit. All blood (15–25 mL) was centrifuged to collect the buffy coat/red blood cell layers [ 20 ]. The cells were diluted with RPMI media 1640 (Life Technologies, Grand Island, NY) and separated by Ficoll-paque density gradient (Sigma Aldrich, St. Louis, MO) to collect the mononuclear cell layer. Magnetic bead antibodies (anti-CD14, anti-CD65, and anti-CD235a) were used to isolate monocytes and lymphocytes as recommended by Miltenyi Biotec Inc. (San Diego, CA). Cell count was determined using the Bio-Rad TC20 Automated Cell Counter (Bio-Rad, Hercules, CA). The Seahorse XFe96 Analyzer (Agilent Technologies, Santa Clara, CA) was used to assess cellular bioenergetics. This technology assesses mitochondrial respiration and glycolysis simultaneously in cells in real-time. Monocytes and lymphocytes were plated (150,000 cells/well) on Cell-Tak coated Seahorse XF96 plates. After establishing baseline readings, the “Mitochondrial Stress Test” (MST) was initiated by injecting oligomycin (0.5 μg/mL), FCCP (0.6 μM), and antimycin A (10 μM) over time into the cellular media. The oxygen consumption rates (OCR) generated from the MST assay was used to determine several parameters including basal respiration, ATP-linked respiration, Proton Leak, Maximal respiration, Reserve Capacity, and Non-mitochondrial respiration. Basal respiration is representative of the energetic demand of cells at baseline levels. ATP-linked OCR represents the respiration used to produce ATP from the mitochondria. Proton leak is the respiration not coupled to ATP generation. Maximal OCR represents the highest level of respiration cells can generate. Reserve capacity is the ability of the cells to respond to an energetic demand. Non-mitochondrial respiration is indicative of cellular respiration from other sources outside of the mitochondria. In addition, we assessed glycolysis by determining the flux of protons in the cellular media and calculated the basal extracellular acidification rates (ECAR) and oligo-sensitive ECAR. All data were analyzed using the Wave software (Santa Clara, CA). All OCR and ECAR were normalized to cell count. Individual mitochondrial and glycolytic parameters were determined as previously described [ 18 ]. Plasma was collected from study participants and frozen at -80°C until evaluation. IFN-ɣ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNF-ɑ analyte levels were measured using a human V-Plex Plus Pro-Inflammatory 10-plex Panel; GM-CSF, IL-12/IL-23P40, IL-15, IL-16, IL-17A, IL-1ɑ, IL-5, IL-7, TNF-β, and VEGF-A analyte levels were measured using a human V-Plex Plus Cytokine Panel (Meso Scale Discovery-MSD, Gaithersburg, MD). All samples were diluted 2-fold in assay diluent according to the manufacturer’s instructions. Samples were analyzed on a multi-array plate by electrochemiluminescence using the Sector Imager 2400 (Meso Scale Discovery-MSD, Gaithersburg, MD). Data were analyzed using the MSD Workbench software. Descriptive statistics, including means and standard deviations for continuous variables and frequencies and proportions for categorical variables, were calculated. Comparisons of means between the two groups of interest (HS vs IC/BPS) were performed using the two-group t-test, or the adjusted (Satterthwaite) t-test as needed. Correlation analyses were performed using Pearson correlation coefficients; these coefficients were tested for statistical significance. Distributions of continuous variables were examined for normality using stem-and-leaf plots, box plots, normal probability plots, and the Kolmogorov-Smirnov test, and it was determined that these variables follow at least an approximate normal distribution. Statistical tests were two-sided and were performed using a 5% significance level (i.e., α = 0.05). Statistical analyses were performed using SAS software (version 9.4; SAS Institute, Inc., Cary, NC).

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