Trans-Golgi protein TVP23B regulates host-microbe interactions via Paneth cell homeostasis and Goblet cell glycosylation

A key feature in intestinal immunity is the dynamic intestinal barrier, which separates the host from resident and pathogenic microbiota through a mucus gel impregnated with antimicrobial peptides. Using a forward genetic screen, we have found a mutation in Tvp23b, which conferred susceptibility to chemically induced and infectious colitis. Trans-Golgi apparatus membrane protein TVP23 homolog B (TVP23B) is a transmembrane protein conserved from yeast to humans. We found that TVP23B controls the homeostasis of Paneth cells and function of goblet cells, leading to a decrease in antimicrobial peptides and more penetrable mucus layer. TVP23B binds with another Golgi protein, YIPF6, which is similarly critical for intestinal homeostasis. The Golgi proteomes of YIPF6 and TVP23B-deficient colonocytes have a common deficiency of several critical glycosylation enzymes. TVP23B is necessary for the formation of the sterile mucin layer of the intestine and its absence disturbs the balance of host and microbe in vivo.


16S rRNA sequencing and data analysis on tissue and fecal sample
The hypervariable region V3 & V4 of bacterial 16S rRNA gene were captured using Zymo Quick 16S protocol (Zymo Quick-16S NGS Library Prep Kit (Catalog # D6400)). PCR product was cleaned using Agencourt AmpureXP beads from Beckman Counter Genomics. Illumina adapter and barcode sequences were ligated to amplicon in order to attach them to MiSeqDx flow cell and for multiplexing. Quality and quantity of each sequencing library was assessed using Bioanlyzer and picogreen measurements, respectively. About 6pM of pooled library was loaded onto a MiSeqDX flow cell and sequenced using PE300 (Paired end 300 bp) v3 kit. Raw fastq files were demultiplexed based on unique barcodes and assessed for quality. Samples with more than 50K QC pass sequencing reads were used for downstream 16S Operational Taxonomic Unit (OTU) analysis.

16S gene sequencing analysis pipeline
Taxonomic classification and OTUs abundance analysis was done using CLC Bio microbial genomics module version 12.0 (https://www.qiagenbioinformatics.com/plugins/clc-microbialgenomics-module/). Individual sample reads were annotated with Greengene database and taxonomic features were determined. Alpha and beta diversity analysis were done to measure the within-and between-sample diversity, respectively. Abundance data was used for numeric Principal Component Analysis (PCA) in SVS, Golden Helix Software. Raw fastq files from this study will be submitted to Sequence Read Archive and are also available on direct request.

In vivo NK Cell and CD8+ T cell cytotoxicity analyses.
For the CTL assay, splenocytes were harvested from B6 mice and divided in half. According to established methods, half were stained with 5 μM CFSE (CFSE hi ), and half were labeled with 0.5 μM CFSE (CFSE lo ). CFSE hi cells were loaded with SIINFEKL peptide(5 μM). CFSE lo cells were not stimulated. CFSE hi and CFSE lo cells were mixed (1:1) and 2 × 10 6 cells were administered to naïve mice and mice immunized with alum-ova through intravenous injection. Blood was collected 48 h after transfer, and CFSE intensities from each population were assessed by flow cytometry. Lysis of target (CFSE hi ) cells was calculated as: % lysis = [1 -(ratio control mice/ratio vaccinated mice)] × 100; ratio = percent CFSE lo /percent CFSE hi . To measure NK cell-mediated killing, splenocytes from control C57BL/6J (0.5 μM Violet; Violet lo ) and B2m −/− mice (5 μM Violet; Violet hi ) were stained with CellTrace Violet. Equal numbers of Violet hi and Violet lo cells were transferred to recipient mice by retro-orbital injection. Twenty-four hours after transfer, blood was collected and Violet intensity from each population was assessed by flow cytometry. % lysis = [1 -(target cells/control cells) / (target cells/control cells in B2m −/− )] × 100.

Flow cytometry
Peripheral blood cells were isolated and red blood cell (RBC) lysis buffer was added to remove RBCs. Peripheral blood cells were washed with FACS staining buffer (PBS with 1% (w/v) BSA) and then centrifuged at 500 × g for 5 minutes. Peripheral blood cells were stained for 1 hour at 4°C, in 100 μl of a 1:200 cocktail of fluorescence-conjugated antibodies to 8 cell surface markers encompassing the major immune lineages: B220, CD19, CD3ε, CD4, CD8α, CD11b, F4/80, NK 1.1 and 1:200 Fc block.
Lamina propria lymphocytes were stained at a 1:200 dilution in the presence of anti-mouse CD16/32 antibody for 30 min at 4°C with fluorochrome-conjugated antibodies against CD45, CD19, TCRβ, CD4, CD8α, MHC-II, CD11c and CD64. To analyze IL-17 production, lamina propria lymphocytes were stimulated with the Leukocyte Activation Cocktail, with BD GolgiPlug™ (BD) for 3 hours. For intracellular staining, cells were stained with surface markers and then fixed, permeabilized, and stained with antibodies specific for IL-17 or FOXP3.
ALDH activity was measured using an Aldefluor Assay kit (Stem Cell Tech) per manufacturers guidelines.

RNA sequencing library preparation
RNA was extracted using RNeasy Mini Kit (QIAGEN) according to the manufacturer's protocol. RNA quantity and purity was assessed on a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific), and integrity was measured on an Agilent Bioanalyzer 2100 (Agilent Technologies). RNA-seq libraries were prepared with the KAPA Stranded RNA-Seq Kit with RiboErase (HMR) (KAPA Biosystems) according to the manufacturer's protocol.

Supplemental Figures
Supplemental Figure S1. Colitis Screening Scheme of ENU-mutagenized mice. (a) Breeding and screening scheme of mice mutagenized with ENU to generate mice for phenotypic screening. First generation (G1) mice define the pedigree and undergo whole exome sequencing to determine ENU induced mutations. Third generation (G3) mice are genotyped according to their G1 grandsire for mutations and then screened for phenotype. G3 mice are for susceptibility to low dose dextran sodium sulfate, measuring weights on Day 0 and Day 7. (b) Distribution of relative weight loss across 55,863 G3 mice tested. Figure S1A was generated in BioRender. (e) ALDH activity as determined by Aldefluor assay of dendritic cells (CD11c + MHCII + CD64 -) in the small intestines and colons of Tvp23b +/+ and Tvp23b -/mice. Each data point represents an independent mouse for indicated genotypes. Data are expressed as means ±s.d. and significance was determined by one-way ANOVA (b-e) (*P=0.0239). ns, not significant. Data is reflective of at least 3 independent experiments.

Supplemental
Supplemental Figure S4. Normal RNA-seq Expression of Goblet cell markers. Colonocytes were isolated from the distal colon of Tvp23b +/+ and Tvp23b -/mice and mRNA was purified from these cells. A total of 3 mice were analyzed per genotype.