Macrophages that were differentiated from bone marrow cells for 7 days in the presence or absence of butyrate were collected and counted using a Coulter Counter. Cells were plated at a density of 2 × 10 5 cells per well in a 24-well plate (Costar) in complete RPMI (GIBCO) in the presence of 100 ng ml -1 recombinant mouse M-CSF (Peprotech) for 24 h at 37°C and 5% CO 2 . For M0 cultures no further cytokines were added. For M1 cultures 500 ng ml -1 IFNγ (Peprotech) was added throughout the culture period. For M2 cultures 10 ng ml -1 IL-4 (Peprotech) was added throughout the culture period. After 24 h, supernatants were removed and cells were lysed in 250 μL Tri Reagent (Molecular Research Center Inc.) and subjected to RNA extraction.

Tibia and femur from hind legs of 8-12 week-old female mice fed a control diet were extracted and bone marrow cells were flushed out into complete RPMI medium (GIBCO). Cell counts were determined using a Coulter Counter and 1 × 10 7 cells were then plated in a 100 × 20 mm cell culture Petri dish (Corning) containing 10 mL complete RPMI medium containing 10% L929 hybridoma supernatant (generated in-house) for 7 days at 37°C and 5% CO 2 . At day 2, 5 mL of fresh complete RPMI medium supplemented with 10% L929 hybridoma supernatant were added. At day 5, 5 mL were gently removed from the culture and another 5 mL of fresh complete RPMI medium containing 10% L929 hybridoma supernatant were added. During the whole culture period butyrate was mixed into the culture medium at a concentration of 0, 5, 50, and 100 μM. At day 7, supernatants were taken off, cells were washed once with complete RPMI and then scraped off the plate. Cells were then further used for polarization experiments.

Adult (8-12 week-old) female mice were anaesthetized with a mixture of ketamine and xylazine (Dr. E. Graeub AG, Bern, Switzerland) and infected intranasally with varying doses of the influenza virus strain PR8 (A/Puerto Rico8/34, H1N1) (Virapur LLC, San Diego, CA) in 50 μL sterile phosphate-buffered saline (PBS). Airway inflammation was examined at either day 1, 3, 5, 7 or 8 after infection. 1,100 PFU were used for high-dose infection of BALB/c, 4,500 PFU for high-dose infection in C57BL/6, and 100 PFU for low-dose infections. Mice were monitored daily during the course of infection and euthanized when reaching a weight loss of 15%–20% or a body temperature below 33°C.

Adult female mice were fed a low-fiber diet (KLIBA NAFAG diet 2122) for 4 weeks prior to and throughout the experiment. Mice then received sodium butyrate (Sigma-Aldrich, St. Louis, MO) in the drinking water at a final concentration of 500 mM for 2 weeks prior to influenza infection and throughout the whole study.

4-week-old BALB/c or C57BL/6 mice were purchased from Charles River Laboratories (L’Arbresle, France) and housed under specific pathogen-free conditions. Ffar3 −/− and Ffar2 −/− mice (both on a C57BL/6 background) were originally obtained from Novartis Institutes for Biomedical Research, Basel, Switzerland. Mice were fed specific diets for 4 to 6 weeks before being used for breeding or short-chain fatty acid (SCFA) supplementation experiments. Cd8 −/− mice were originally obtained from the Laboratory Animal Services Center (LASC), Zurich, Switzerland. Ly5.1 C57BL/6 mice were purchased from Charles River Laboratories. We used 8-12 week-old female mice for all experiments. All animal experiments were performed in accordance with institutional guidelines and Swiss federal and cantonal laws on animal protection.

Method Details

Cellular Infiltration of the Airways Broncho-alveolar lavage fluid (BALF) was collected to assess the cellular compartment of the airway lumen. Total cell numbers in the BALF were determined using a Coulter Counter (IG Instrumenten-Gesellschaft AG, Basel, Switzerland). Differential cell counts were performed on cytospins stained with Diff-Quik solution (Dade Behring, Siemens Healthcare Diagnostics, Deerfield, IL). Percentages of eosinophils, neutrophils, macrophages and lymphocytes were determined by counting 200 cells per sample.

Measurement of Lung Function Lung resistance was quantified using the whole body–restrained plethysmograph system flexiVent (SCIREQ). Mice were anesthetized by intramuscular injection of 100 mg per kg body weight ketamine and intraperitoneal injection of 50 mg per kg body weight pentobarbital (Esconarkon, Streuli Pharma). Eight minutes later, mice were tracheotomized and mechanically ventilated at a rate of 200 breaths per min and a tidal volume of 10 mL per kg body weight. Airway hyper-responsiveness was measured by administering increasing doses of acetyl methylcholine (12.5, 25, 50 and 100 mg/ml) (Sigma).

Histology Whole lung or right lobes were fixed in 10 mL of 10% buffered formalin at 4°C and embedded into paraffin. Prepared sections (4 μm) were stained with Haemotoxylin and Eosin (H&E) reagents using standardized protocols and analyzed with an Axioskop 2 plus microscope equipped with an Axio-Cam HRc (Carl Zeiss Microimaging GMbH, Jena, Germany).

ELISAs The activity of neutrophilic myeloperoxidase was quantified in BALF of infected mice (7 days post-infection) using a mouse MPO ELISA kit (Hycult biotech, Uden, Netherlands). Vascular leakage was determined by quantification of serum albumin in the BALF seven days after influenza infection using a mouse Albumin ELISA kit (ICL, Portland, OR). Assays were performed according to manufacturer’s instructions and the colorimetric reaction was read at 450 nm on the Synergy H1 microplate reader (Biotek, Luzern, Switzerland). IL-6, IL-1β, CXCL1, CXCL2, and CCL3 protein levels were quantified in BALF using a ProcartaPlex Assay System (Thermo Fisher Scientific). Assays were performed according to manufacturer’s instructions and read on the Luminex 200 Multiplexing Instrument (Bio-Rad).

In vivo Neutrophil Reduction In order to diminish neutrophils, 8-12 week-old female BALB/c mice were injected intraperitoneally with 20 μg anti-Ly6G (clone 1A8, BioXCell) in 200 μL PBS on day −1 and 1. Control mice received 20 μg of corresponding isotype control antibody (clone 2A3, BioXCell). On day 0, mice were infected with 600 PFU of influenza A (strain PR8). Mice were monitored daily during the course of infection and euthanized when reaching a weight loss of 15%–20% or a body temperature below 33°C.

In vivo Neutrophil Chemoattraction Assay In order to test the capacity of neutrophils from mice fed a fiber-rich diet to respond to the main neutrophil chemoattractant CXCL1, 5 μg of CXCL1 (Peprotech) were instillated intranasally to 8-12 week-old female BALB/c mice fed a control or inulin-rich diet. Six hours post-administration, the presence of neutrophils in the lungs and BALF was assessed and quantified by flow cytometry.

In Vivo Killing Assay Spleens from naive 8-12 week-old female BALB/c mice were extracted, cut in small pieces and passed through a 70 μm cell strainer to obtain a single cell suspension. Splenocytes were RBC lysed, counted and resuspended at a concentration of 5 × 106 ml-1. The cell suspension was then divided into two equal fractions. One fraction was kept unpulsed, the other one was pulsed with 1 μL ml-1 of a 200 μM stock of NP-peptide (H2-Kd; TYQRTRALV) (TCF UNIL, Lausanne, Switzerland) for 1 h at 37°C in a water bath. Afterward, cells were recounted and resuspended at a concentration of 5 × 107 ml-1. Unpulsed cells were labeled with 5 μM of Carboxyfluorescein succinimidyl ester (CFSE; Enzo Life Sciences), whereas the NP-pulsed cells were labeled with 0.5 μM CFSE for 10 min at 37°C, resulting in a CSFEhigh unpulsed fraction and a CFSElow NP-pulsed fraction. Both fractions were recounted and mixed at a 1:1 ratio. The mixture was then resuspended in PBS at a concentration of 1 × 108 ml-1 and 200 μL were injected intravenously to control and HFD-fed mice 7 days after influenza infection with 100 PFU PR8. 14 hours later, mice were sacrificed and the mediastinal lymph nodes were extracted. The ratio between the CFSElow and CFSEhigh fraction was determined by flow cytometry to assess killing efficiency.

Flow Cytometry Characterization and phenotyping of the various cell types in lung were performed by flow cytometry. Lung tissue was digested using collagenase IV (Thermo Fisher Scientific) in Iscove’s modified Dulbecco’s medium for 45 min at 37°C. Samples were then filtered through a 70 μm cell strainer, washed and resuspended in PBS supplemented with 0.2% bovine serum albumin (BSA). Total cell counts were determined using a Coulter Counter and cells were then stained for flow cytometry. Neutrophils were identified using antibodies to CD11c-allophycocyanin (APC)/Cy7 (117324, 1:600), CD11b-pacific blue (101223, 1:600), and Gr-1-phycoerythrin (PE)/Cy5 (15-5931, Thermo Fisher Scientific, 1:800). Activation and frequency of pulmonary conventional and inflammatory dendritic cells (DCs) and those of the monocyte and macrophage subsets were assessed using antibodies to CD11c-APC/Cy7 (117324, 1:600), CD11b-PerCP/Cy5.5 (101228, 1:600), F4/80-Alexa Fluor (AF) 647 (123122, 1:500), I-A/I-E-AF700 (107622, 1:1,000), Ly6c-pacific blue (128014, 1:600), CCR2-PE/Cy7 (150611, 1:400), CX3CR1-PE (149005, 1:400), Gr-1-PE/Cy5 (15-5931, Thermo Fisher Scientific, 1:800), CD40-PE (124610, 1:400), CD70-PE (104605, 1:400), CD206-PE (141705, 1:400), PD-L2-PE (107205, 1:400), CD86-biotin (105003, 1:400), and streptavidin-PE/Cy7 (405206, 1:1,000). CXCL1 intracellular expression was assessed after 4h incubation with Brefeldin A (Biolegend) using an unlabelled primary antibody against murine CXCL1 (R&D Systems, MAB4532, 1:100) and a goat-anti-rabbit-PE secondary antibody (R&D Systems, F0110, 1:50) to visualize the signal. To determine its expression in CD45neg cells, lungs were stained with CD45.2-AF700 (109822, 1:400). CD8+ T cell numbers and activation were characterized by staining with antibodies to CD3-pacific blue (100214, 1:400), CD4-APC (100412, 1:600) or CD4-PerCP/Cy5.5 (100434, 1:600), CD8α-PE/Cy7 (100722, 1:800), CD62L-PerCP/Cy5.5 (104432, 1:600), and CD107α-APC/Cy7 (121615, 1:400), and intracellular staining using a 0.5% solution of saponin from Quillaja bark (Sigma-Aldrich) and antibodies to Granzyme B-FITC (515403, 1:100), IFNγ-APC (505810, 1:200), and TNFα-AF488 (506313, 1:200). Influenza A-specific CD8+ T cells were identified using a MHC Multimer (PE-labeled) staining the nucleoprotein (NP) sequence TYQRTRALV (TCF UNIL, Lausanne, Switzerland). CD8+ T cell metabolic state was determined using extra- or intracellular staining with an antibody to Glut-1-PE (NB110-39113PE, 1:400, Novus Biologicals) or -AF488 (NB110-39113AF488, 1:400, Novus Biologicals), and intracellular staining of c-myc-AF647 (NB600-302AF647, 1:400, Novus Biologicals). MitoTracker® Green FM dye (Life technologies) was used to mark mitochondria. For identification of progenitor cells, bone marrow was isolated from the left leg of mice. To distinguish different progenitor populations bone marrow cells were stained with antibodies to Ly-6A/E-PE/Cy7 (108113, 1:800), CD117-AF647 (105817, 1:400), CD16/32-APC/Cy7 (101327, 1:400), CD34-PerCP/Cy5.5 (128607, 1:300), CD135-PE (135305, 1:400), CD11c-FITC (11-0114-81, 1:400, Thermo Fisher Scientific), I-A/I-E–AF700 (107622, 1:1,000), and a Lineage Cocktail-pacific blue (133310, 1:50). If not indicated otherwise, antibodies were purchased from Biolegend (San Diego, CA). Fluorescence minus one (FMO) internal staining controls were set up by pooling cells from all the different experimental groups. Cells were acquired on BD Fortessa or LSR-II (BD Biosciences, San Jose, CA). Samples were analyzed using FlowJo 10.4.2 software (Tree Star Inc., Ashland, OR).

Metabolite screening Metabolite analysis by LC–tandem mass spectrometry was performed using a LCMS-8050 by Shimadzu triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source and operated in multiple reaction mode (MRM). For graphical representation in a heatmap, the data is arranged in a matrix, in which each metabolite is depicted in one row, each group is depicted in one column, and mean metabolite abundances in a specific group are illustrated by color. Each metabolite’s abundances are centered and scaled. First, centring is done by subtracting the total metabolite’s mean. Second, scaling is done by dividing the centered metabolite by its standard deviation. The resulting values are in a similar range for all metabolites.

SCFA Quantification Organic acids - acetate, propionate and butyrate - present in the feces of 8-12 week-old female BALB/c mice fed our different diets were analyzed by gas chromatography (GC) on DB-FFAP column, from Agilent Technologies 6890 series GC System with a FID detection. Briefly, feces were first homogenized in a solution of ortho-phosphoric acid 0.1%, Mercury chloride 0.1%, and 2,2 Dimethyl-Butyrate as internal standard, corresponding to 4 times the weight of feces. Samples were then homogenized with 2 mm glass beads for 20 min with a multivortex. Preparations were centrifuged at 2,000 g for 15 min at 4°C. Supernatants were collected and weighed. Subsequently, 10 μL HCl 37% and 3 mL of chloroform were added and homogenized for 20 min. After centrifugation for 10 min at 1,800 g and 4°C, the upper layer was eliminated. After addition of 10 μL T-butyldimethylsilylimidazole, samples were heated at 60°C for 30 min then cooled down before being injected. Freeze-drying to determine humidity rate and humidity content was used for the calculation of SCFA per gram of dry material. Serum samples were analyzed using the HPLC Ultimate 3000 (Dionex, Sunnyvale, CA) equipped with an U3000 RS diode array detector (Dionex). Sera were diluted in a 1:6 ratio with 25% metaphosphoric acid, incubated on ice for 30 min before being centrifugated at 12,000 g for 15 min at 4°C. Supernatants were then applied onto a Ultrafree MC filtering unit (Merck Millipore) and spun down at 12,000 g for 4 min at 4°C. Eluates were analyzed by HPLC. SCFAs were separated on a Hi-Plex H column (8 mm; 300 × 7.7 mm; Polymer Laboratories (Varian), Shropshire, UK) run isocratic with 5 mM sulfuric acid at a flow rate of 0.6 mL per min and a temperature of 55°C. Organic acids were detected at a wavelength of 210 nm and quantified using external standard curves from 0.5 to 100 mM of the respective authentic organic acids (Sigma-Aldrich). Dionex Chromeleon software version 6.8 (Thermo Fisher Scientific) was employed to pilot the HPLC and determine the concentrations of each individual organic acid.

Bacterial DNA Isolation from Mouse Feces Feces from 8-12 week-old female BALB/c mice housed under specific pathogen-free conditions were harvested under sterile conditions in a 2 mL Biopure tube (Eppendorf, Hamburg, Germany) and immediately snap-frozen in liquid nitrogen. Samples were stored at −80°C until processing for DNA isolation. Total bacterial DNA from 1-2 fecal pellets were isolated using the QiaAMP Fast DNA Stool Mini Kit (QIAGEN) according to manufacturer’s instructions. DNA was eluted in 80 μL DNase/RNase-free water (QIAGEN) and its concentration was determined using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Asheville, NC). DNA was stored at 4°C until being used for sequencing.

16S rRNA Library Preparation and Sequencing 16S rRNA gene amplicon library was generated using modified 27F and 338R universal primers targeting the V1-V2 hypervariable region of the 16S rRNA gene. Primers were designed as follows: 27F-5′-AATGATACGGCGACCACCGAGATCTACACTATGGTAATTCCAGMGTTYGATYMTGGCTCAG-3′ and 338R-5′-CAAGCAGAAGACGGCATACGAGATNNNNNN NNNNNNAGTCAGTCAGAAGCTGCCTCCCGTAGGAGT-3′ where Illumina adaptor sequences are bold, linkers italicized and NNNNNNNNNNNN sequences represents the sample-specific MID tag barcodes. Each PCR reaction was performed in duplicates in 20 μL total volume using the AccuPrime Taq DNA polymerase high fidelity kit (Invitrogen) with 4 μL of DNA template and 0.44 μL of each 27F and 338R primers at 10 μM. Amplification conditions consisted of 3 min of initial denaturation at 94°C, followed by 30 cycles of 30 s of denaturation at 94°C, 30 s of annealing at 56°C, and 90 s of extension with 5 min of final extension at 72°C. Amplicons size and quantity was assessed on the LabChip GX (Perkin Elmer) before combining PCR products in equimolar amounts of amplicons. Pooled library was purified using Agencourt AMPure XP magnetic beads (Beckman Coulter) and sequencing was performed on an Illumina MiSeq platform with MiSeq reagent kit V2-500 (pair-end, 2x250).

16S rRNA Gene Sequencing Data Analysis Caporaso et al., 2010 Caporaso J.G.

Kuczynski J.

Stombaugh J.

Bittinger K.

Bushman F.D.

Costello E.K.

Fierer N.

Peña A.G.

Goodrich J.K.

Gordon J.I.

et al. QIIME allows analysis of high-throughput community sequencing data. Edgar, 2010 Edgar R.C. Search and clustering orders of magnitude faster than BLAST. DeSantis et al., 2006 DeSantis T.Z.

Hugenholtz P.

Larsen N.

Rojas M.

Brodie E.L.

Keller K.

Huber T.

Dalevi D.

Hu P.

Andersen G.L. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. 16S rRNA sequences were processed and analyzed using Quantitative Insights Into Microbial Ecology (QIIME, v.1.9.0) software (). Merging of paired forward and reverse reads was performed using fastq-join before demultiplexing and quality filtering (quality Phred score Q > 20, < 3 low quality base calls). Operational taxonomic units (OTUs) were assigned using a closed OTU picking strategy with Uclust () at 97% identity against the 97% Greengenes reference database (v13.5) (). Beta diversity was estimated using Bray-Curtis distance matrix generated in QIIME followed by principal component analysis using cmdscale in the R software. Statistical significance of differences in community composition was assessed by analysis of similarity (ANOSIM) with 100000 permutations using adonis command (vegan package) in the R software. Alpha diversity was assessed using chao1 and Shannon indexes calculated in QIIME. Unpaired Student’s t tests were used to test for differences in alpha diversity between the two experimental groups. Significant differences in OTU abundance among experimental groups were assessed using Wilcoxon statistical procedure (runWilcox in EMA package) on filtered OTU table (> 0.001% of relative abundance) with P-value correction using Benjamini-Hochberg false discovery rate (FDR) with a corrected alpha value of 0.05. A heatmap was generated using heatmap.2 function (gplots package) in R with data representing row-scaled z-scores. Ward’s hierarchical clustering (ward.D) and corresponding dendrogram were performed using hclust algorithm on Bray-Curtis dissimilarity matrix.

Quantitative Polymerase Chain Reaction (qPCR) Relative lung Csf3, Il-4, Il-10, Ifnγ, Arg1, Nos2, and Cxcr2 mRNA expression as well as viral load was assessed by quantitative RT-PCR using the following primer sets: Csf3 forward 5′-TGACACAGCTTGTAGGTGGC-3′ and reverse 5′-TCCTGCTTAAGTCCCTGGAG-3′; Il-4 forward 5′-TGTCATCCTGCTCTTCTTTCTC-3′ and reverse 5′-GCACCTTGGAAGCCCTAC-3′; Il-10 forward 5′-GCATGGCCCAGAAATCAAGG-3′ and reverse 5′-AGAAATCGATGACAGCGCCTC-3′; Ifnγ forward 5′-GCTCTGAGACAATGAACGCTAC-3′ and reverse 5′-TTCTAGGCTTTCAATGACTGTGCC-3′; influenza matrix protein (to determine viral load) forward 5′-CCCTGAAGTACCCCATTGAAC-3′ and reverse 5′-CTTTTCACGGTTGGCCTTAG-3′; Arg1 forward 5′-GCAACCTGTGTCCTTTCTCC-3′ and reverse 5′-TCTACGTCTCGCAAGCCAAT-3′; Nos2 forward 5′-CTGCCTCATGCCATTGAGTT-3′ and reverse 5′-TGAGCTGGTAGGTTCCTGTTG-3′; Cxcr2 forward 5′-ATCCACCTTGAATTCTCCCAT-3′ and reverse 5′-GTCACAGAGAGTTGGGACCC-3′; β-Actin forward 5′-GATCAAGATCATTGCTCCTCC TGA-3′ and reverse 5′-CAGCTCAGTAACAGTCCGCC-3′. Ten microliter PCR reactions were set up containing 2 μL of template cDNA at a concentration of 50 ng ml−1, 5 μL SsoAdvanced SYBR Green reaction mix (Bio-Rad, Hercules, CA), 0.25 μL of each primer at a concentration of 20 μM and 2.5 μL of nuclease-free water. All reactions were performed in duplicates and each gene was normalized to β-actin. Quantitative PCR was performed on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad) using the following conditions: one cycle at 95°C for 2 min and then 40 cycles at 95°C for 15 s and 60°C for 30 s, followed by a dissociation stage at 65°C for 31 s and cycles of 5 s starting at 65°C, raising 0.5°C per cycle, to obtain melting curves for specificity analysis. Following amplification, C q values were obtained using the CFX ManagerTM software 2.1 (Bio-Rad).

CD8+ T Cell Adoptive Transfer Splenic CD8+ T cells from 8-12 week-old wild-type (Ly5.1) and Ffar3−/− (Ly5.2) female mice were obtained by negative selection using magnetic beads (Miltenyi Biotec), mixed in a 1:1 ratio and 6 × 106 CD8+ T cells were transferred intravenously to Cd8−/− recipient mice, given water or butyrate orally. The purity of the selection, as well as the accuracy of the 1:1 ratio were determined by flow cytometry before being injected into the recipient mice. The day following the adoptive transfer, Cd8−/− mice were infected intranasally with influenza PR8. Mice were sacrificed eight days post-infection to evaluate the frequency and effector function of influenza-specific Ly5.1+ and Ly5.2+ CD8+ T cells present in the lung by flow cytometry.

Mixed Bone Marrow Chimeras Bone marrow cells were obtained from 8-12 week-old wild-type (Ly5.1) and Ffar3−/− (Ly5.2) female mice, mixed in a 1:1 ratio and 5 × 106 cells were injected intravenously into lethally γ-irradiated 12 week-old C57BL/6 recipient mice (2 doses of 450 Rads spaced by a 4 hour interval between irradiations). Five weeks post-transfer, engraftment of donor cells was verified by flow cytometry by assessing the presence of Ly5.1+ and Ly5.2+ cells in the blood of recipient mice. Six weeks post-transfer, mice were infected intranasally with influenza PR8 and sacrificed three days post-infection. The proportions of Ly5.1+ and Ly5.2+-derived Ly6cneg patrolling monocytes and interstitial macrophages present in the blood and lung of recipient mice were determined by flow cytometry.

In Vitro CD8+ T Cell Assays CD8+ T cells were isolated from the spleens of naive 8-12 week-old female mice fed a low-fiber diet either supplemented with 30% inulin or 30% cellulose by negative selection using magnetic beads (Miltenyi Biotech, Germany). Cells were plated in the presence of 5 μg ml-1 plate-coated anti-CD3 and 2 μg ml-1 soluble anti-CD28 and incubated for 1.5 h at 37°C and 5% CO 2 . Cells were then starved for an additional hour by removing glucose from the medium. At last, glucose-deprivation was reversed by re-adding medium containing 100 μM 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-DG) (Sigma-Aldrich) for 1 h prior to analysis. Metabolic state and activation of CD8+ T cells from mice fed the different diets was compared by flow cytometry. To determine the direct effect of the SCFA butyrate on CD8+ T cells, cells obtained from cellulose-fed mice were pooled and plated in the presence of 5 μg ml-1 plate-coated anti-CD3, 2 μg ml-1 soluble anti-CD28, and increasing doses of sodium butyrate (Sigma-Aldrich) for 24 h at 37°C and 5% CO 2 . 500 μM etomoxir (Sigma-Aldrich) was added to the culture to block fatty acid oxidation. Metabolic state and activation of CD8+ T cells in the presence or absence of sodium butyrate was compared using Flow cytometry.