HDAC1 and HDAC2 restrain the intestinal inflammatory response by regulating intestinal epithelial cell differentiation

Abstract

Acetylation and deacetylation of histones and other proteins depends on histone acetyltransferases and histone deacetylases (HDACs) activities, leading to either positive or negative gene expression. HDAC inhibitors have uncovered a role for HDACs in proliferation, apoptosis and inflammation. However, little is known of the roles of specific HDACs in intestinal epithelial cells (IEC). We investigated the consequences of ablating both HDAC1 and HDAC2 in murine IECs. Floxed Hdac1 and Hdac2 homozygous mice were crossed with villin-Cre mice. Mice deficient in both IEC HDAC1 and HDAC2 weighed less and survived more than a year. Colon and small intestinal sections were stained with hematoxylin and eosin, or with Alcian blue and Periodic Acid Schiff for goblet cell identification. Tissue sections from mice injected with BrdU for 2 h, 14 h and 48 h were stained with anti-BrdU. To determine intestinal permeability, 4-kDa FITC-labeled dextran was given by gavage for 3 h. Microarray analysis was performed on total colon RNAs. Inflammatory and IEC-specific gene expression was assessed by Western blot or semi-quantitative RT-PCR and qPCR with respectively total colon protein and total colon RNAs. HDAC1 and HDAC2-deficient mice displayed: 1) increased migration and proliferation, with elevated cyclin D1 expression and phosphorylated S6 ribosomal protein, a downstream mTOR target; 2) tissue architecture defects with cell differentiation alterations, correlating with reduction of secretory Paneth and goblet cells in jejunum and goblet cells in colon, increased expression of enterocytic markers such as sucrase-isomaltase in the colon, increased expression of cleaved Notch1 and augmented intestinal permeability; 3) loss of tissue homeostasis, as evidenced by modifications of claudin 3 expression, caspase-3 cleavage and Stat3 phosphorylation; 4) chronic inflammation, as determined by inflammatory molecular expression signatures and altered inflammatory gene expression. Thus, epithelial HDAC1 and HDAC2 restrain the intestinal inflammatory response, by regulating intestinal epithelial cell proliferation and differentiation.

Figure 1. Conditional intestinal epithelial HDAC1/2 loss alters small intestine and colon size.

A. Representative example of four-month-old control (Ctrl) and intestinal epithelial HDAC1/2 deficient (HDAC1/2ΔIEC) intestines. B, C. Small intestine and colon length of four-month-old (n=12-18) (B) or one-year-old (n=11-12) (C) control (Ctrl) and conditional intestinal epithelial HDAC1/2 ((HDAC1/2ΔIEC) mice was measured. Results represent the mean ± SEM (*p≤0.05; **p≤0.01; *** p≤0.005). D, E. Small intestine and colon weight of four-month-old (n=7-10) (D) or one-year-old (n=9-12) (E) control and intestinal epithelial HDAC1/2 deficient mice was measured. Results represent the mean ± SEM (*p≤0.05; **p≤0.01; ***p≤0.005).

Figure 2. Conditional intestinal epithelial HDAC1/2 loss alters intestinal architecture.

Tissue sections from four-month-old control (Ctrl) and conditional intestinal epithelial HDAC1/2 (HDAC1/2ΔIEC) jejunum (A) and colon (B) were stained with hematoxylin and eosin. A branched villus is shown in the insert. Immune cells are indicated by arrows. Magnification: 20 X or 40 X (insert). C. Four-month-old colonic crypt length was measured (n=4-9, 20 to 40 crypts each). Results represent the mean ± SEM (one-way ANOVA, **** p≤0.001).

Figure 3. Conditional intestinal epithelial HDAC1/2 loss leads to increased proliferation.

2 h after BrdU intraperitoneal injection, four-month-old jejunal (A) or colonic (B) tissue sections from control (Ctrl) or conditional intestinal epithelial HDAC1/2 (HDAC1/2ΔIEC) mice were revealed with an antibody against BrdU. The insert in A shows the absence of BrdU-labelled cells in branched villi. The average number of BrdU-labelled cells per jejunal (C) or proximal and distal colonic (D) crypts was measured (n=3; 20 to 30 crypts each). Results represent the mean ± SEM (**p≤0.01). Magnification: 20 X.

Figure 4. Conditional intestinal epithelial HDAC1/2 loss leads to increased migration.

Four-month-old control (Ctrl) or conditional intestinal epithelial HDAC1/2 (HDAC1/2ΔIEC) mice were killed 48 h after BrdU peritoneal injection to determine jejunal migration (A) and 14 h after BrdU injection for colonic migration (B). Jejunal (A) or colonic (B) tissue sections were revealed with an antibody against BrdU. The average distance of BrdU-labelled cells from jejunal (C) or colonic (D) crypts was measured (n=3; 20 to 40 villi or colonic glands each). BrdU labelled cells are indicated by arrows. Results represent the mean ± SEM (*p≤0.05). Magnification: 20 X.

Figure 5. Conditional intestinal epithelial HDAC1/2 loss leads to altered activation of cell homeostasis regulators.

Total protein extracts from three to four one-year-old control (Ctrl) or conditional intestinal epithelial HDAC1/2 (HDAC1/2ΔIEC) colons were separated on a 10% SDS-PAGE gel, transferred to a PVDF membrane and analysed by Western blot for expression of (A) Cyclin D (Ccnd1, MW: 33.4 kD; Ccnd2, MW: 32.9 kD), cleaved Notch1 (MW: 110 kD) and actin (MW: 41.7 kD) as a loading control; (B) phosphorylated and total ribosomal protein S6 (MW: 28.7 kD); (C) cleaved caspase 3 (MW: 17 kD), with actin as a loading control. The histograms indicate the ratio of band intensities normalized to actin (A, C) or total ribosomal protein S6 (B). Quantification of band intensity was performed with the Quantity One software. Results represent the mean ± SEM (*p≤0.05; **p≤0.01). D. Cyclin D1 (Ccnd1) increased expression was confirmed by qPCR analysis of total RNAs isolated from control or conditional intestinal epithelial HDAC1/2 colons. Results represent the mean ± SEM (*p≤0.05).

Figure 6. Conditional intestinal epithelial HDAC1/2 loss deregulates goblet cell differentiation.

Jejunal (A, B) and colonic (C, D) tissue sections from one-year-old control (Ctrl, left panels) or conditional intestinal epithelial HDAC1/2 mice (HDAC1/2ΔIEC, right panels) mice, were stained with Alcian blue (A, C) or Periodic Acid Schiff (B, D). Magnification: 20 X.

Figure 7. Conditional intestinal epithelial HDAC1/2 loss disrupts cell lineage commitment.

A. Jejunal tissue sections from four-month-old control (Ctrl, left panel) or conditional intestinal epithelial HDAC1/2 (HDAC1/2ΔIEC, right panel) mice were stained with Best’s Carmine. Arrows indicate stained Paneth cells. Magnification: 20 X. B. Jejunal tissue sections from four-month-old control (Ctrl, left panel) or conditional intestinal epithelial HDAC1/2 (HDAC1/2ΔIEC, right panel) mice were stained with an antibody against lysozyme, a Paneth cell marker. Arrows indicate stained Paneth cells. Magnification: 20 X. C. Total RNAs were isolated from control and HDAC1/2 IEC-specific jejunum (n=4-6). Expression levels of lysozyme and Defa1 (cryptdin), two Paneth cell markers, were determined by qPCR, with Pbgd as a control. Results represent the mean ± SEM (* p≤0.05). D. Total RNAs were isolated from control and HDAC1/2 IEC-specific colons (n=4-5). Expression levels of Cdx2 and Sucrase-isomaltase (Sis), a small intestine enterocyte marker, were determined by qPCR, with Pbgd as a control. Results represent the mean ± SEM (* p≤0.05). E. Colon tissue sections from four-month-old control (Ctrl, left panel) or conditional intestinal epithelial HDAC1/2 (HDAC1/2ΔIEC, right panel) mice were stained with an antibody against Sucrase isomaltase (Sis), a small intestine enterocyte marker. Magnification: top panels: 20 X; bottom panels: 40 X.

Figure 8. Conditional intestinal epithelial HDAC1/2 loss disrupts epithelial barrier function.

A. Total protein extracts from three to five control (Ctrl) or conditional intestinal epithelial HDAC1/2 (HDAC1/2ΔIEC) colons were separated on a 10% SDS-PAGE gel, transferred to a PVDF membrane and analysed by Western blot for expression of Claudin 3 (MW: 23.3 kD) and actin as a loading control. The histograms indicate the ratio of band intensities normalized to actin. Quantification of band intensity was performed with the Quantity One software. Results represent the mean ± SEM (*p≤0.05). B. To measure intestinal permeability, blood was recovered 3 h after gavage of 4-kDa FITC-labeled dextran (n=6). FITC serum concentrations were determined with a RF-5301PC spectrofluorometer (Shimadzu Scientific Instruments, Columbia, MD, USA). Results represent the mean ± SEM (*p≤0.05). C. Total protein extracts from four to five control (Ctrl) or conditional intestinal epithelial HDAC1/2 (HDAC1/2ΔIEC) colons were separated on a 10% SDS-PAGE gel, transferred to a PVDF membrane and analysed by Western blot for expression of Phospho-Stat3 and total Stat3 (MW: 88 kD). The histogram indicates the ratio of Phospho-Stat3 band intensities normalized to Stat3. Quantification of band intensity was performed with the Quantity One software. Results represent the mean ± SEM (*p≤0.05).

Figure 9. Conditional intestinal epithelial HDAC1/2 loss leads to modifications of inflammatory and differentiation-specific gene expression patterns.

Total RNAs were isolated from four-month-old control and HDAC1/2 IEC invalidated colons (n=6, 4). Expression levels of selected highly induced genes, namely Reg3b, Reg3g, Alpi, Fabp1, Fabp6, Lcn2, Ccl8 and Cxcl5 were verified by semi-quantitative RT-PCR, with Gapdh as a loading control. The amplified products were separated on 2% agarose gels.