Bindloss et al Principal Coordinate(PC) analysis taxa summaries using weighted UniFrac were created through QIIME. Subsequent data transformation and analysis was performed using r open- sourcesoftware(www.r-project.org)andthepackagesPhyloseqandgggplot(31).Profili dataweredepositedattheBiosampledatabase(https://www.ncbi.nlmnihgov/biosample/) under accession numbers samno7152 105 through SaMno7152132 Within the class Clostridia, butyrate production is most common for the Lachnospiraceae (cluster XI Va)and the Ruminococcaceae (cluster IV) and oTUs belonging to these families were classified as predicted butyrate producers (32). For OTUs assigned to other classes of bacteria, the presence in available bacterial genomes of genes generating butyrate through the acetyl-CoA pathway, the glutarate pathway, the 4-aminobutyrate pathway or the lysine pathway has been described previously (9)and was used to assign each OTUs to a family containing predicted butyrate-producers or a family predicted not to contain butyrate Fluorescent microscopy A single microfluidic chamber( 8x3x0.075mm, length x width x height) was used for ulturing the Caco2 cells. The microfluidic devices were fabricated using standard soft lithography protocols by replica molding polydimethylsiloxane(PDMS)(33). Caco2 cell culture inside the microfluidic devices was modified from a previous cell culture protocol for neurons and cancer cells (34). Briefly, the microfluidic platforms were first sterilized under UV for Ih. Collagen solution(0.2mg/ml) was then coated on the glass coverslip for 4 h at 37oC inside a cell incubator. The devices were washed with lx Pbs once and then fille with culture media to provide a hospitable environment before cell seeding. At the same time of washing, the Caco2 cells grown in T25 flask(in MEM media supplemented with 10% FBS and 1% Glutamax) were harvested and re-suspended in fresh media at a density of 5,000 cells per microliter of media. After removing the excess media, a 15-20 ul of the cell suspension was loaded into the upstream reservoir. The microfluidic platforms were then placed in a cell culture incubator with 5%CO2 at 37C to allow Caco2 cells to attach to the collagen-coated glass coverslips. After 2h, the seeding solution was removed and approximately 200ul of culture media was added to each reservoir, and further cultured overnight. The following day, 400ul treatment solution containing rhIL-22(100ng/mL, R&D Systems)and rhIFNg(40 ng/mL, R&D Systems)was added for 30 minutes Incubation of cytokines with treatments, either 2mM or 5mM sodium butyrate(Sigma-Aldrich)or 5uM Rosiglitazone( Cayman Chemical) was added to the upstream reservoir and a 100ul of fresh media was added to the downstream reservoir to create a continuous flow. after 3 days of treatment, the cells in the device were immunostained for PPAR-Y Immunostaining began by washing the cells in the chambers with Ix PBS and filling the channel with4% paraformaldehyde with 0.2% Triton X-100 for 15min at room temperature. The surface was blocked by 10% Goat serum with 0. 3M Glycine for Ih at room temperature. Subsequently, the primary PPAR-y antibody (1: 100, Santa Cruz Biotechnology was incubated overnight at 4C. The secondary antibody (1: 1000, BD Pharmigen) was then incubated for Ih at room temperature. Finally, the cells were incubated with Phlloidin for labeling actin filaments Between each step, the devices were washed by lx PBs for 10min. The stained cells were aged using a Nikon Eclipse Ti-S fluorescent microscope(Melville, NY)equipped with the Science Author manuscript; available in PMC 2017 October 1Principal Coordinate (PC) analysis taxa summaries using weighted UniFrac were created through QIIME. Subsequent data transformation and analysis was performed using R open￾source software (www.r-project.org) and the packages Phyloseq and Gggplot (31). Profiling data were deposited at the BioSample database (https://www.ncbi.nlm.nih.gov/biosample/) under accession numbers SAMN07152105 through SAMN07152132. Within the class Clostridia, butyrate production is most common for the Lachnospiraceae (cluster XIVa) and the Ruminococcaceae (cluster IV) and OTUs belonging to these families were classified as predicted butyrate producers (32). For OTUs assigned to other classes of bacteria, the presence in available bacterial genomes of genes generating butyrate through the acetyl-CoA pathway, the glutarate pathway, the 4-aminobutyrate pathway or the lysine pathway has been described previously (9) and was used to assign each OTUs to a family containing predicted butyrate-producers or a family predicted not to contain butyrate￾producers. Fluorescent microscopy A single microfluidic chamber (8×3×0.075mm, length × width × height) was used for culturing the Caco2 cells. The microfluidic devices were fabricated using standard soft￾lithography protocols by replica molding polydimethylsiloxane (PDMS) (33). Caco2 cell culture inside the microfluidic devices was modified from a previous cell culture protocol for neurons and cancer cells (34). Briefly, the microfluidic platforms were first sterilized under UV for 1h. Collagen solution (0.2mg/ml) was then coated on the glass coverslip for 4 h at 37°C inside a cell incubator. The devices were washed with 1× PBS once and then filled with culture media to provide a hospitable environment before cell seeding. At the same time of washing, the Caco2 cells grown in T25 flask (in MEM media supplemented with 10% FBS and 1% Glutamax) were harvested and re-suspended in fresh media at a density of 5,000 cells per microliter of media. After removing the excess media, a 15~20 μl of the cell suspension was loaded into the upstream reservoir. The microfluidic platforms were then placed in a cell culture incubator with 5% CO2 at 37°C to allow Caco2 cells to attach to the collagen-coated glass coverslips. After 2h, the seeding solution was removed and approximately 200μl of culture media was added to each reservoir, and further cultured overnight. The following day, 400μl treatment solution containing rhIL-22 (100ng/mL, R&D Systems) and rhIFNg (40 ng/mL, R&D Systems) was added for 30 minutes. Incubation of cytokines with treatments, either 2mM or 5mM sodium butyrate (Sigma-Aldrich) or 5μM Rosiglitazone (Cayman Chemical) was added to the upstream reservoir and a 100μl of fresh media was added to the downstream reservoir to create a continuous flow. After 3 days of treatment, the cells in the device were immunostained for PPAR-γ. Immunostaining began by washing the cells in the chambers with 1× PBS and filling the channel with 4% paraformaldehyde with 0.2% Triton X-100 for 15min at room temperature. The surface was blocked by 10% Goat serum with 0.3M Glycine for 1h at room temperature. Subsequently, the primary PPAR-γ antibody (1:100, Santa Cruz Biotechnology) was incubated overnight at 4°C. The secondary antibody (1:1000, BD Pharmigen) was then incubated for 1h at room temperature. Finally, the cells were incubated with Phlloidin for labeling actin filaments. Between each step, the devices were washed by 1× PBS for 10min. The stained cells were imaged using a Nikon Eclipse Ti-S fluorescent microscope (Melville, NY) equipped with the Byndloss et al. Page 11 Science. Author manuscript; available in PMC 2017 October 16. Author Manuscript Author Manuscript Author Manuscript Author Manuscript