Probiotic supplementation reduces systemic in ammation in dialysis patients: the effect on circulating regulatory T-cells and pro-in ammatory monocytes

Eunho Choi Korea University Anam Hospital Jihyun Yang Korea University Anam Hospital Geun-Eog Ji Food and Nutrition Research Institute of Human Ecology, Seoul National University Myeong Soo Park Deparment of Food and Nutrition, Research Institute of Human Ecology, Seoul National University Yeongje Seong Research Center, BIFIDO Co.Ltd Se Won Oh Korea University Anam Hospital Myung Gyu Kim Korea University Anam Hospital Won Yong Cho Korea University Anam Hospital Sang Kyung Jo (  sang-kyung@korea.ac.kr ) Korea University Anam Hospital https://orcid.org/0000-0002-0496-0258


Introduction
Maladaptive and persistent in ammation has been recognized as an important player in the development of cardiovascular diseases and also as a predictor of mortality in patients with chronic kidney disease (CKD) [1][2][3][4].
Although not completely understood, retention of uremic solutes, oxidative stress, immune dysfunction, or dialysis-related factors including repeated exposure to dialysis membranes, contaminated dialysis water, and recently, intestinal dysbiosis, have been implicated as important culprits in the development of chronic in ammation.
Signi cant alterations in the diversity and number of operational taxonomic units (OTUs) associated with increased intestinal permeability have been demonstrated in preclinical and clinical studies. Uremic milieu, slower colonic transit time, bowel edema due to volume overload, restrictions of ber-rich diets, medications including phosphate binders, and frequent use of antibiotics might be factors contributing to the generation and maintenance of dysbiosis [5][6][7][8].
Probiotics are "live microorganisms that, when administered in adequate amounts, confer a health bene t to the host" [9]. Probiotics have been shown to partially restore normal intestinal microbiota and reduce the level of uremic toxins and systemic in ammation. A recent study by Soleiman et al., showed improvement of glycemic control and reductions in C-reactive proteins (CRP) in diabetic patients undergoing HD [10]. One of the mechanisms of probiotic-induced anti-in ammatory effects might be mediated by probiotic-induced immune modulation.
CD4 + CD25 + regulatory T-cells (Tregs) are a subpopulation of T-cells with regulatory function and have been demonstrated to be effective in reducing in ammation. CD14 + CD16 + nonclassical monocytes are a subset of proin ammatory monocytes which increase in advanced CKD patients. Lee et al., previously demonstrated a positive correlation between these cells and vascular stiffness, suggesting the possible important role of this monocyte subset in increased cardiovascular risks in CKD patients [11].
The purpose of this study was to assess the effect of probiotic supplementation on chronic in ammation in maintenance HD patients. We analyzed the microbiome, fecal short chain fatty acids (SCFAs), and in ammatory responses before and 3 months after probiotic supplementation. The percentage of circulating Tregs and the number of CD14 + CD16 + monocytes were also measured by ow cytometry. The effect of probiotic supplementation on these parameters was also examined 4 months after the discontinuation of probiotics.

Study design
Enrolled patients included those aged > 18 years undergoing maintenance dialysis for more than 3 months at Korea University Anam Hospital, a tertiary hospital in Seoul, South Korea, from Nov to Dec 2018. Exclusion criteria were patients who: (a) were on HD for acute kidney injury; (b) were on HD less than twice per week; (c) had uncontrolled diarrhea or gastrointestinal infection; (d) were treated with oral or intravenous antibiotics within 4 weeks of enrollment; and (e) were actively being treated for cancer, or with immunosuppressive drugs. Among the 23 patients providing informed consent, 1 patient withdrew from the study and 22 patients completed the trial.
These patients were treated with sachets [2 g mixtures of probiotics containing 7.0×10 9 colony forming units (CFU)/g of Bi dobacterium bi dum BGN4 and 2.0×10 9 CFU/g of Bi dobacterium longum BORI] twice per day for 3 months. B. bi dum BGN4 and B. longum BORI were isolated from the feces of healthy breast-fed infants, and have been used as probiotics since 2000 [12][13][14][15][16]. Its complete genomic sequence was reported to GenBank [17]. B. longum BORI has been shown to shorten the duration of diarrhea in a clinical study of infants infected with rotavirus [12]. A safety assessment of B. bi dum BGN4 and B. longum BORI concerning ammonia production, hemolysis of blood cells, biogenic amine production, antimicrobial susceptibility patterns, antibiotic resistance gene transferability, polymerase chain reaction (PCR) data on antibiotic resistance genes, mucin degradation, genome stability, and the presence of virulence factors has been recently reported [18] Both strains have been considered Generally Recognized As Safe by the United States Food and Drug Administration (US FDA) (GRN813 for B. longum BORI and GRN814 for B. bi dum BGN4; https://www.accessdata.fda.gov/scripts/fdcc/? set=GRASNotices). Blood and fecal samples were obtained at baseline and at 3 and 6 months. The study protocol was approved by the Korea University Medical Center Institutional Review Board (IRB No. 2018AN0346) Written informed consent was provided by all participants.

Laboratory measurements
Routine laboratory measurements including complete blood counts with white blood cell differentials, CRP, albumin, blood urea nitrogen, creatinine, and electrolytes were obtained before the dialysis session. Patients' demographic factors and current medications were also recorded.
Blood and fecal samples were centrifuged at 2,500×g for 15 min and stored at -80 O C for calprotectin measurements. Samples were analyzed using an enzyme-linked immunosorbent assay (ELISA -LSBio, LS-F9275, Seattle, WA, USA) according to the manufacturer's instructions.
Flow cytometric detection of Tregs and pro-in ammatory monocyte subsets Blood samples were collected before the dialysis session. Whole blood (2.5 ml) was collected in a heparinized tube and 200 µl aliquots of heparinized blood were stained for 15 min at room temperature with either anti-human CD4 conjugated with allophycocyanin (CD4-APC), anti-human CD25 conjugated with phycoerythrin (CD25-PE) antibodies for detection of Tregs, anti-human CD14 conjugated with uorescein isothiocyanate (CD14-FITC), or anti-human CD16 conjugated with allophycocyanin (CD16-APC) antibodies for the detection of monocytes (BD Biosciences, San Jose, CA, USA). After red blood cell lysis and washing, ow cytometric detection of Tregs and the CD14 + CD16 + pro-in ammatory monocyte subset from among 10 6 cells were performed (FACSCalibur™; BD Biosciences, San Jose, CA, USA) and analyzed by FlowJo v8.5.2. The percentage of cells or the actual number of cells at baseline were compared to the values 3 months after initiation of probiotic supplementation as well as 4 months after the discontinuation of probiotic treatment.

Quanti cation of Cytokines before and after a Lipopolysaccharide (LPS) challenge
Fold changes of cytokine production upon an LPS challenge were compared before and after 3 months of probiotic supplementation. Quanti cation of plasma cytokines were performed using human in ammation cytometric bead array kits (BD Biosciences, San Jose, CA, USA) and cytometric bead arrays (Human in ammation kit, BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions to simultaneously detect levels of interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-12p70, and tumor necrosis factor (TNF)-α.

Measurement of fecal SCFAs
The concentrations of SCFAs in fecal samples were analyzed using high-performance liquid chromatography in the National Instrumentation Center for Environmental Management at Seoul National University (Seoul, Korea). Feces were prepared in normal saline (300 µl per 1 g of feces). The fecal slurry supernatants were obtained through centrifugation (2,500 rpm for 10 min). SCFAs, including acetic, butyric, propionic, isovaleric, and valeric acids were measured before and 3 months after probiotic supplementation.

Microbiome analysis
Sample DNA extraction and next generation sequencing (NGS) Bacterial genomic DNA was extracted from stool samples using a QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany). DNA extraction was performed after homogenization at 30 s for 1 minute using a TissueLyser system (Qiagen) and quanti ed using a QUBIT 3.0 Fluorometer (Thermo Fisher Scienti c, Waltham, MA, USA). For NGS, 16S rRNA gene ampli cations and index PCRs were performed following the Illumina 16S metagenomic Sequencing Library preparation guide (Illumina, San Diego, CA, USA). The V3 and V4 regions of 16S rRNA were ampli ed using the following primer pair (forward 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3', reverse 5'-TCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3'). Nextera XT index kits (Illumina, San Diego, California, USA), using 8 cycles, were then used to fragment DNA and add adapter sequences onto the DNA template. Each PCR product was puri ed using AMPure XP beads (Beckman Coulter, Pasadena, CA, USA). The amplicon library was sequenced by the 2×300 bp paired-end method on a Miseq instrument (Illumina) according to the Illumina protocol.

Statistical analysis
All the analyses were performed using SPSS software, version 25.0 (IBM Corporation, USA). Data are expressed as medians [interquartile ranges] according to the distribution. Continuous variables (baseline, after 3 months) were compared using the Wilcoxon signed-rank test. A p-value < 0.05 was considered signi cant.

Results
Baseline patient characteristics Twenty-two patients completed the study. The mean age was 68.1 years and 16 patients (73%) were male. The average time for receiving HD was 8 years and the prevalences of diabetes, hypertension, or histories of gastrointestinal surgeries were 18%, 81.5%, and 22%, respectively (Table 1). Eighty-six percent of patients were taking calcium polystyrene sulfonate and the percentage of patients on various medications, including phosphate binders, antacids, iron, and stool softeners, are reported in Table 2. During the study period, 2 patients received systemic antibiotics: one for community-acquired pneumonia, and the other for calculous cholecystitis.
The effect of probiotic supplementation on the microbiome and microbial metabolites Faith's phylogenetic diversity showed no signi cant changes in richness and evenness after probiotic supplementation (p=0.222 and 0.597) (Fig1 A, B). However, at the genus level, the relative abundance of Bacteroides, Faecalibacterium, Oscillospira, Parabacteroides, Lachnospira, and Akkermansia decreased whereas Bi dobacterium, Ruminococcaceae and Lachnospiraceae increased after probiotic supplementation. (Fig1 C).

Short chain fatty acids
SCFAs from gut bacteria are an important nutrient source for enterocytes and are key mediators of the bene cial effects elicited by the gut microbiome. Probiotic supplementation for 3 months resulted in a signi cant increase of SCFAs such as acetic, butyric, propionic, and valeric acids in feces (Fig. 2).

Effect of probiotic supplementation on in ammatory responses
Serum levels of CRP, albumin, calcium, phosphate, and intact parathyroid hormone (iPTH) were similar before and after 3 months of probiotic supplementation. Hemoglobin, total white blood cell and monocyte counts were also comparable before and after probiotic supplementation (Table 3). Despite comparable fecal calprotectin levels (data not shown), serum calprotectin, a marker of acute in ammation, decreased signi cantly after 3 months of probiotic supplementation (Fig. 3A).
We also compared the cytokine production upon LPS stimulation and found that fold increase of IL-6 after LPS stimulation signi cantly decreased 3 months after probiotic supplementation (Fig. 3B).
Effect of probiotic supplementation on CD14 + CD16 + pro-in ammatory monocyte subset We assessed the impact of probiotic supplementation on circulating pro-in ammatory monocytes. Using ow cytometry, monocyte subpopulations were divided into 3 different subsets: classical, intermediate, and nonclassical monocytes according to CD14 CD16 positivity (Fig. 4A). Although the percent monocytes did not change in complete blood count, the actual number of CD14 + CD16 + proin ammatory, non classical monocytes decreased signi cantly after 3 months of probiotic supplementation (310 vs. 194/mm 2 , p < 0.05) (Fig. 4B).

Tregs and pro-in ammatory monocytes after discontinuation of probiotic supplementation
The increased percentage of Tregs at 3 months after initiation of probiotic supplementation showed a complete return to baseline levels 4 months after the discontinuation of probiotics (Fig. 6A). The number of proin ammatory monocytes that decreased after probiotic supplementation also showed a trend to return to baseline after discontinuation of probiotic supplementation (p = 0.061) (Fig. 6B). However, decreased serum calprotectin level was maintained after discontinuation of probiotics (Fig. 6C).

Discussion
In this study, we demonstrated that the probiotic combination of B. bi dum BGN4 and B. longum BORI had an anti-in ammatory and immunomodulatory effect in patients undergoing maintenance HD. Supplementation of probiotics for 3 months resulted in increased SCFA levels in feces. Serum calprotectin levels as well as IL-6 response upon an LPS challenge decreased signi cantly 3 months after initiation of probiotic supplementation, and this anti-in ammatory effect was associated with a decreased number of circulating pro-in ammatory, nonclassical monocytes and an increased percentage of immunomodulatory Tregs. However, this effect on circulating immune cells was only transient and returned to baseline after discontinuation of the probiotics.
Emerging evidence shows that intestinal microbiota play an important role in both normal physiology as well as in acute or chronic in ammatory conditions. Both qualitative and quantitative changes in microbiomes have also been reported in uremic animals as well as in patients. Chen et.al. reported the increase of relative abundance of Enterobacteriaceae, Ruminococcaceae, and Lachnospiraceae families in CKD patients and Sampaio-Maia et.al. showed the decrease of butyrate producing bacteriae such as Roseburia, Faecalibacterium, Clostridium, Coprococcus, and Prevotella in end-stage kidney disease (ESKD) (23,24). However, it is not clear whether the perturbation of microbiomes has a causal relationship with CKD. Although a recent meta-analysis of 7 studies enrolling 8-60 dialysis patients showed the potential bene cial effects of probiotics on in ammation, uremic toxins, and GI symptoms, the mechanisms underlying the bene cial effects remains unclear [23].
CKD has emerged as a major cardiovascular risk factor and chronic in ammation is recognized as an important player. The intestine is the single largest organ of the immune system, harboring the largest number of T-cells and macrophages in our body and thus considered to be potent regulator of systemic immunity. Therefore, it can be hypothesized that pre or probiotics induced bene cial effect might be mediated by effects on immune cells.
We rst observed that probiotic supplementation resulted in decreased numbers of pro-in ammatory monocytes. Monocytes are cells of the innate immune system and have heterogeneous phenotypes according to surface expression of CD14 or CD16 proteins [24]. Among them, CD14 + CD16 + are nonclassical monocytes that have been known to invade the endothelium and cause plaque formation in the general population and in patients with CKD [25]. We also demonstrated the expansion of these subsets and also their associations with CRP and vascular stiffness in patients with advanced CKD [26]. Signi cant decrease of actual number of CD14 + CD16 + nonclassical monocytes after probiotic supplementation could suggest that the anti-in ammatory effect of probiotics could be partially mediated by inhibitory effects on the pro-in ammatory monocyte subset. In addition to monocytes, we compared the percentage and number of circulating Tregs before and after probiotic supplementation and observed that the percentage of CD4 + CD25 + cells in blood showed a signi cant increase after 3 months of probiotic supplementation. Mahajan et al., showed that Tregs protect against constant macrophage in ammation and reduce pro-in ammatory cytokine production in murine CKD models [27]. Impaired Treg function was also observed in CKD patients that led to chronic in ammation and subsequent atherosclerosis and cardiovascular diseases [27,28]. Wang et al., also demonstrated that CKD patients with cognitive dysfunction who had increased percentages of Tregs showed signi cantly higher mini-mental state examination scores than those with reduced percentages of Tregs [29]. Given that potent immune suppressive function, expansion of circulating Tregs after probiotic supplementation is likely contribute to anti-in ammatory effect in our patients. However, we observed that the increased percentage of Tregs returned to baseline in all patients 4 months after the discontinuation of probiotics. Although statistically insigni cant, the number of nonclassical monocytes which decreased after 3 months of probiotics also returned to baseline, showing that the anti-in ammatory, immune modulatory effect of probiotics is only transient.
Unlike other studies, serum CRP level or fecal calprotectin did not show signi cant difference in our study. Instead, we observed that serum calprotectin levels as well as fold increases of IL-6 after the LPS challenge decreased signi cantly after probiotic supplementation. Calprotectin is a heterodimeric complex of 2 S100 calcium-binding proteins: myeloid-related protein (MRP)-8 and MRP-14, which are mainly expressed in neutrophils and monocytes. Although calprotectin has been originally known to be a novel biomarker of disease activity in patients with in ammatory bowel disease when measured in feces, recent data suggests that serum calprotectin levels can be a useful biomarker of disease activity in several acute and chronic in ammatory conditions [30][31][32][33]. Serum and urine calprotectin have been demonstrated to be associated with the severity of renal injuries and endothelial dysfunction in Henoch-Schönlein purpura as well as anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis even if CRP levels are not elevated [34][35][36]. Although we observed that median calprotectin levels decreased signi cantly after 3 months of probiotic supplementation, the usefulness of serum calprotectin levels as a sensitive marker of in ammation in patients with CKD or HD requires further examination.
IL-6 has been demonstrated to be an important player in the progression of CKD [37]. It exacerbates in ammation as well as endothelial injury by reducing endothelial nitric oxide synthase (eNOS) and injection of recombinant IL-6 exacerbates atherosclerosis in mice [38]. Our data showing that the fold increase of IL-6 upon LPS challenge signi cantly decreased after 3 months of probiotic supplementation also supports its anti-in ammatory effect.
Various metabolites produced by intestinal microbiota are important in both normal physiology and in pathological conditions. SCFAs are known to be important in colonocyte survival, maintaining barrier integrity and immune modulation. In contrast, excessive production of trimethylamine (TMA) due to the consumption of an animal protein-rich diet has been shown to be associated with atherosclerosis and cardiovascular diseases. In our study, we observed that the levels of acetic, butyric, propionic, and valeric acids increased signi cantly after 3 months probiotic supplementation. SCFAs are bacterial fermentation products with pleiotropic functions including lipid regulation, energy metabolism, and immune regulation via interaction with G protein-coupled receptors, histone deacetylases, or direct humoral effects. Given that SCFAs have immunomodulatory effects, it is possible that signi cantly elevated levels of SCFAs during the probiotic supplementation might be partially responsible for decreased proin ammatory monocytes and expansion of Tregs.
Even though our data showed that probiotics elicited a very potent anti-in ammatory effect , 2 of the patients in our study required hospitalization. One patient was diagnosed with calculous cholecystitis, underwent a laparoscopic cholecystectomy, and was administered systemic antibiotics. The second patient was diagnosed with community acquired pneumonia 1 month of probiotic supplementation and was discharged after 5 days of systemic antibiotic therapy. Although a causal relationship between probiotic use and serious infections in this study was not clear, one should always be aware that excessive suppression of in ammatory responses might increase the risk of infections.

Con ict of interest
The researcher claims no con icts of interest.

Ethics approval
The study protocol was approved by the Korea University Medical Center Institutional Review Board (IRB No. 2018AN0346).

Consent to participate
Written informed consent was provided by all participants.

Consent for publication
Consent for pulication was obtained from all authors Availability of data and material (data transparency) All data support published claims and comply with eld standards.  Effect of probiotic supplementation on fecal short chain fatty acids level. *p<0.05 compared to baseline.