1. Volume 139, Issue 5, Pages 1642-1653.e6 (November 2010)
Altered Macrophage Function Contributes to Colitis in Mice Defective in the Phosphoinositide-3 Kinase Subunit p110δ 
Jennifer K. Uno, Kavitha N. Rao, Katsuyoshi Matsuoka, Shehzad Z. Sheikh, Taku Kobayashi, Fengling Li, Erin C. Steinbach, Antonia R. Sepulveda, Bart Vanhaesebroeck, R. Balfour Sartor, Scott E. Plevy 
Gastroenterology 
Volume 139, Issue 5, Pages e6 (November 2010)
DOI: /j.gastro
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2. Figure 1
PI3K p110δD910A/D910A mice develop colitis. (A and B) Histologic scores of colonic sections from WT and PI3K p110δD910A/D910A mice at different ages. Results are represented as (A) percentage of microscopic fields in each age group with score 0, 1 to 2, or 3 to 4, or (B) mean colitis scores. *P < .05 vs WT 25- to 45-week-old mice. (C) Colonic sections from 10-week-old PI3K p110δD910A/D910A mice show leukocytic infiltration of the lamina propria (white circle) and intraepithelial lymphocytes (white arrows) in the crypts. Focal crypt abscesses were observed (black arrow). (D) Colonic explants from WT (black bars) and PI3K p110δD910A/D910A (gray bars) mice were assayed for spontaneous secretion of cytokines. *P < .05 vs WT explants. Error bars represent mean ± SEM of 3 independent experiments.
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3. Figure 2
PI3K p110δD910A/D910A mice display enhanced expression of IL-12 p40. (A) Splenocytes from WT (black bars) and PI3K p110δD910A/D910A (gray bars) mice were untreated or stimulated with LPS (1 μg/mL) alone or LPS and IFN-γ (10 ng/mL) for 24 hours. IL-12 p40 was measured by ELISA. (B and C) Colonic macrophages from WT or PI3K p110δD910A/D910A mice were stimulated with heat-killed E coli (mode of multiplicity 10) for 24 hours. ELISAs were performed to assess (B) IL-10 and (C) IL-12 p40 levels. (D and E) BMMs from WT (dashed lines) and PI3K p110δD910A/D910A (black lines) mice were unstimulated or stimulated with LPS (1 μg/mL) and supernatants analyzed for IL-12 p40 (D). PI3K p110δD910A/D910A BMMs were harvested at each time point and (E) IL-12 p40 (Il12b) mRNA levels were assessed by real-time reverse-transcription PCR. Results are expressed as fold induction normalized to β-actin. Error bars represent mean ± SEM of 3 independent experiments. *P < .05 vs WT cells.
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4. Figure 3
PI3K p110δD910A/D910A macrophages show heightened sensitivity to TLR stimulation. BMMs from WT (black bars) and PI3K p110δD910A/D910A (gray bars) mice were stimulated with TLR9 (CpG), TLR2 (sBLP), or TLR5 (Flagellin) ligands for 24 hours. Supernatants were analyzed for IL-12 p40, IL-12 p70, or IL-23 secretion by ELISA and NO secretion by Greiss reaction. Error bars represent mean ± SEM of 3 independent experiments.
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5. Figure 4
PI3K p110δD910A/D910A macrophages show altered kinetics and magnitude of MAP kinase activation. (A) BMMs from WT or PI3K p110δD910A/D910A mice were stimulated with (right) LPS (100 ng/mL) or (left) sBLP (100 ng/mL) for the indicated periods and phosphorylation of Akt (p-Akt) was assayed by ELISA. Results are presented as a ratio of p-Akt to total Akt. (B) BMMs from WT and PI3K p110δD910A/D910A mice were stimulated with LPS (1 μg/mL) for the indicated times. Whole cell extracts were analyzed for phosphorylation of MAP kinase (JNK, ERK, p38) by Western blot. Results represent mean ± SEM of 3 independent experiments (*P < .05 vs WT).
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6. Figure 5
PI3K p110δD910A/D910A BMMs show differences in defective bactericidal activity. (A) WT and p110dD910A/D910A BMMs were cultured with K12 E coli, NC101 E coli, or S typhimurium. No significant bacterial recoveries were seen 1 hour after bacterial infection (left panel). P13K p110δD910A/D910A BMMs show decreased bactericidal activity relative to WT BMMs at 8 hours postinfection (right panel) (*P < .05 vs WT BMMs). (B) IL-12 p40 production by ELISA was assessed in P13K p110δD910A/D910A BMMs infected with K12 E coli. (C) BMMs were treated with LPS (100 ng/mL) or IFN-γ (10 ng/mL) before bacterial infection, and bacteria were recovered from lysed cells 8 hours postinfection (*P < .05 vs WT BMMs). (D) Total bacterial DNA in spleen and mesenteric lymph nodes were detected by real-time PCR using primers for total 16S ribosomal RNA genes. Primers for GAPDH were used to show loading control of host genomic DNA. Error bars represent mean ± SEM of 3 independent experiments (*P < .05 vs WT).
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7. Figure 6
The enteric microbiota induces colonic PI3K p110δ expression in WT but not colitis-prone IL-10−/− mice. GF WT and IL-10−/− mice were transitioned to an SPF microbiota. Colonic mRNA was isolated and expression of PI3K p110δ, p85a, and p55a mRNA was assessed by real-time reverse-transcription PCR. (A) Colonic PI3K p110δ was determined in WT and IL-10−/− mice at 0, 3, 7, and 14 days after colonization of GF mice with SPF microbiota (*P < .05 vs WT). (B) Colonic expression of PI3K p110δ, p85a, and p55a mRNA was examined 14 days after transition of GF mice to SPF microbiota. Results are expressed as fold induction normalized to β-actin. Error bars represent mean ± SEM of 3 independent experiments. (C) BMMs from WT and IL-10−/− mice were stimulated with LPS (100 ng/mL) for the indicated times. PI3K p110δ mRNA levels were assessed by real-time reverse-transcription PCR (left panel). Results are expressed as fold induction normalized to β-actin and represent mean ± SEM of 3 independent experiments (*P < .05 vs WT). BMMs from WT and IL-10−/− mice were stimulated with LPS (100 ng) for 16 hours. Whole cell extracts were analyzed for PI3K p110δ by Western blot (right panel). Results are representative of 3 independent experiments.
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8. Figure 7
IL-10−/− PI3K p110δD910A/D910A mice exhibit severe colitis at an early age. (A) Colitis scores were determined for 4-week-old IL-10−/−/PI3K p110δD910A/D910A (DK), WT, IL-10−/−, and PI3K p110δD910A/D910A (PI3Km) mice using criteria established for IL-10−/− mice13 by a pathologist blinded to genotype (*P < .05 vs WT). (B) IL-12 p40, (C) IL-23, and (D) IL-12 p70 protein in supernatants from colon explant cultures from IL-10−/−/PI3K p110δD910A/D910A (DK), WT, IL-10−/−, and PI3K p110δD910A/D910A (PI3Km) mice were analyzed by ELISA. Error bars represent mean ± SEM of 3 independent experiments (*P < .05 vs WT).
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9. Supplementary Figure 1
Increases in CD3+ intraepithelial lymphocytes in the colonic crypts in PI3K p110δD910A/D910A mice. Representative immunohistochemical analysis of paraffin-embedded colonic sections from WT and PI3K mutant mice for CD3. A marked increase in CD3-positive intraepithelial lymphocytes prominent at the base of the crypts (black arrows) is observed in PI3K p110δD910A/D910A colonic sections compared with WT colons.
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10. Supplementary Figure 2
Colonic explants from PI3K p110δD910A/D910A secrete elevated chemokines. Intestinal explants from WT (black bars) and PI3K p110δD910A/D910A (gray bars) mice were cultured for 24 hours and cell-free supernatants were assayed for spontaneous secretion of the indicated chemokines by ELISA. Results are representative of a Luminex multiplex array analysis replicated independently 3 times.
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11. Supplementary Figure 3
PI3K p110δD910A/D910A splenocytes secrete elevated levels of TNF. Splenocytes from WT (black bars) and PI3K p110δD910A/D910A (gray bars) mice were untreated (un) or stimulated with LPS (1 μg/mL) or LPS and IFN-γ (10 ng/mL) for 24 hours. TNF levels were measured by ELISA in cell-free supernatants. Error bars represent mean ± SEM of 3 independent experiments (*P < .05 vs WT cells).
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12. Supplementary Figure 4
WT and PI3K p110δD910A/D910A BMMs and LPMC phenotypic and activation marker expression. Colonic (A) BMMs or (B) LPMCs from 8- to 10-week-old WT and PI3K p110δD910A/D910A mice were isolated and labeled with antibodies against macrophage lineage and activation markers (CD11b, F4/80, CD80, CD86, MHCII, TLR4, and CD14) and analyzed by flow cytometry. Macrophages were gated using forward scatter and side scatter to exclude contaminating cells. Black histograms show profiles of the labeled antibody, and gray histograms represent staining with isotype-matched controls. Representative patterns from 3 independent experiments are shown.
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13. Supplementary Figure 5
Further characterization of signal transduction pathways in PI3K p110δD910A/D910A macrophages. (A) BMMs from WT and PI3K p110δD910A/D910A mice were stimulated with LPS for the indicated periods. Whole cell extracts were analyzed for phospho-p65 and total p65 by Western blot. Results are representative of 3 independent experiments. (B) BMMs from WT and MyD88−/− mice were stimulated with LPS (100 ng) for 4 hours. PI3K p110δ mRNA levels were assessed by real-time reverse-transcription PCR at the indicated time points. Results are expressed as fold induction normalized to β-actin. Results represent mean ± SEM of 3 independent experiments (*P < .05 vs WT BMMs). BMMs from WT and MYD88−/− mice were stimulated with LPS (100 ng) for 16 hours. Whole cell extracts were analyzed for PI3K p110δ by Western blot. Results are representative of 3 independent experiments.
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14. Supplementary Figure 6
Bacterial uptake/phagocytosis is intact in PI3K p110δD910A/D910A BMMs. (A) WT and (B) PI3K p110δD910A/D910A BMMs were infected for 1 hour with K12 E coli, extensively washed to remove adherent bacteria, and permeabilized and then immunostained with anti–E coli LPS antibodies. Results are representative of 3 independent experiments. (C) Infected cells were quantitated in 12 fields from 3 independent experiments and expressed as percent cells infected per total number of cells counted. P = not significant.
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15. Supplementary Figure 7
IL-10/PI3K p110δD910A/D910A mice exhibit severe colitis at an early age. Colons from 1-month-old IL-10/PI3K p110δD910A/D910A are shorter and thicker relative to age-matched WT and PI3K p110δD910A/D910A mice (top). IL-10/PI3K p110δD910A/D910A mice were significantly smaller in size (bottom).
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16. Supplementary Figure 8
Pathogenesis of IBD in PI3K p110δD910A/D910A mice. (1) PI3K p110δ dampens TLR signaling, suggesting that dysregulation of innate immune responses may contribute to the development of colitis in PI3K p110δD910A/D910A mice. This is opposed to TLR function on intestinal epithelial cells, which has been postulated to be protective against inflammation. Furthermore, IL-10 contributes to induction of p110δ expression. (2) Defective bactericidal activity is shown in PI3K p110δD910A/D910A macrophages. The inability of PI3K p110δD910A/D910A macrophages to efficiently kill and clear microbes may contribute to prolonged inflammatory responses.
Gastroenterology  , e6DOI: ( /j.gastro )
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