Profiling of microdissected gastric epithelial cells reveals a cell type—specific response to Helicobacter pylori infection  Anne Mueller, D. Scott Merrell, Jan Grimm, Stanley Falkow  Gastroenterology  Volume 127, Issue 5, Pages 1446-1462 (November 2004) DOI: 10.1053/j.gastro.2004.08.054 Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 1 Colonization density of H pylori at 1, 2, 4, 7, 10, 14, and 28 days after infection in BALB/c mice. Infections were performed by oral gavage with 109 bacteria, and 6 mice were infected per time point (1 morbid animal was excluded at day 4). Average colonization densities are shown for each time point. CFU, colony-forming units. Gastroenterology 2004 127, 1446-1462DOI: (10.1053/j.gastro.2004.08.054) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 2 Molecular profiles of the 3 major gastric epithelial lineages. Sixty-nine arrays corresponding to 22 chief-cell samples (blue), 24 mucus-producing cell samples (red), and 23 parietal cell samples (green) were clustered by using 912 genes whose log2 of the experimental sample/reference-normalized ratio was greater than 2 in at least 3 arrays. Samples from both infected and mock-infected animals were included (see designation of arrays). Only genes for which information was available for more than 70% of arrays were used. The entire data set is shown on the left, and 3 cell type—specific clusters are enlarged on the right. Red and green represent high and low experimental sample/reference ratios, respectively (see scale bar). Gray signifies missing data. Selected genes are designated in the cell type’s color code. A marker gene for each cell type is given in black. dehydrog., dehydrogenase; fact., factor; ind., inducible; pancr., pancreas; proprot., proprotein; prot., protein; prt., protein; relat., related; transcr., transcript. Gastroenterology 2004 127, 1446-1462DOI: (10.1053/j.gastro.2004.08.054) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 3 Schematic illustrating the 3 gastric epithelial cell types’ major functions and biochemical features as inferred from their transcriptional signatures. MHC, major histocompatibility complex. Gastroenterology 2004 127, 1446-1462DOI: (10.1053/j.gastro.2004.08.054) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 4 Verification of marker transcript localization. Adjacent cryosections were stained with H&E or HistoGene dye or probed with digoxigenin-labeled RNA probes as indicated. Pepsinogen served as a marker for chief cells, H+/K+ ATPase as a marker for parietal cells, and trefoil factor-1 as a marker for mucus-producing cells. Circles and squares in the upper right panel mark cell clusters that would typically have been selected for microdissection (red, mucus cells; blue, chief cells; green, parietal cells). Gastroenterology 2004 127, 1446-1462DOI: (10.1053/j.gastro.2004.08.054) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 5 Transcriptional changes occurring in mucus-producing cells in response to H pylori infection over time. The roughly 1000 genes that are differentially expressed between mock-infected and infected mucus-producing cell samples (as determined by the SAM algorithm; approximately 10% false-positive discovery rate) were clustered and inspected visually in time course order with respect to their induction kinetics. Various clusters representing induced genes are shown (see http://falkow.stanford.edu/whatwedo/supplementarydata/ for the complete list). Genes induced early in infection (2 days after infection) are annotated in green, intermediate genes (7 and 14 days after infection) in blue, late genes (28 days after infection) in red, and genes induced throughout the time course in black (see legend). Colored bars to the left of the clusters indicate whether clusters of genes were induced early, late, at intermediate time points, or throughout the time course. Many of the late genes were verified in a separate set of mice; these are marked with an asterisk. A comparison of the 2 studies is available on our Web site (http://falkow.stanford.edu/whatwedo/supplementarydata/). (Inset) Clustering of the 6 samples harvested from animals on day 28 after infection. The mock-infected samples were segregated from the infected samples. Ig, immunoglobulin; GTPase, guanosine triphosphatase; MHC, major histocompatibility complex; PECAM, platelet/endothelial cell adhesion molecule (CD31 atigen). Gastroenterology 2004 127, 1446-1462DOI: (10.1053/j.gastro.2004.08.054) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 6 Quantification by real time reverse-transcription PCR of select transcripts up-regulated in infected mucus-producing cells. The starting quantities of unamplified transcripts encoding IFN-γ—inducible protein (A) and small proline-rich protein 2A (B) were determined in triplicate for the 3 mock-infected and the 3 infected animals of the day 28 time point (black bars). The error bars indicate SDs. Transcript levels were normalized to GAPDH expression. A representative experiment is shown. For comparison, the gray bars indicate the expression ratios as determined by microarray analysis. Gastroenterology 2004 127, 1446-1462DOI: (10.1053/j.gastro.2004.08.054) Copyright © 2004 American Gastroenterological Association Terms and Conditions