Intestinal myofibroblasts in innate immune responses of the intestine Jan-Michel Otte, Ian M Rosenberg, Daniel K Podolsky  Gastroenterology  Volume 124, Issue 7, Pages 1866-1878 (June 2003) DOI: 10.1016/S0016-5085(03)00403-7

Figure 1 TLR expression profiling of intestinal and lung myofibroblasts using a custom microarray. The 52 genes constituting the TLR chip were spotted in duplicates on a single membrane as described in the Materials and Methods section. Scatter plots confirmed a high level of reproducibility between the replicates. (A ) Orientation of genes spotted in duplicates on custom-made microarrays. These include TLRs 1–9, accessory molecules, and signaling molecules. Arrays were hybridized with 33P-labeled cDNA from unstimulated CCD-18 and CCD-37 cells (control) or cells, which were either stimulated with LPS (2 μg/mL) or LTA (10 μg/mL). (B) Typical result of a hybridization with RNA obtained from unstimulated controls (left), LPS- (middle), or LTA- stimulated cells (right). Local background was subtracted from the readout of each spot. Results were then normalized to housekeeping genes (β-actin, GAPDH). Finally, values for each gene were compared between controls and LPS- or LTA-stimulated cells. (C ) Incubation of myofibroblasts cell lines CCD-18 (upper panel) and CCD-37 (lower panel) with LPS, LTA differentially alters the expression of TLRs and signaling molecules of the pathway downstream of TLR. Shown is the expression of TLRs 1–9 and the 3 accessory molecules MyD88, TIRAP, and Tollip in comparison with unstimulated controls. Expression of the genes in stimulated cells is shown in percentage of the expression of the same gene in unstimulated cells. An increase or decrease greater than 2-fold was considered significant. Results shown are means of n = 4 independent experiments. Although there were some differences, expression patterns from these 2 cell lines showed similar trends on stimulation with LPS or LTA. Alterations are given as percentage of the expression levels of the specific genes in unstimulated controls, which were set as 1; black bars indicate results from LPS-stimulated cells and gray bars show results from LTA-stimulated cells. Gastroenterology 2003 124, 1866-1878DOI: (10.1016/S0016-5085(03)00403-7)

Figure 2 Expression of TLRs and accessory molecules. mRNA expression of TLRs and accessory molecules were analyzed by PCR after RT of total RNA from cultured cells (CCD-18 and CCD-37) or freshly isolated primary colonic myofibroblasts. (A ) CCD-18 and (B) CCD-37 cells express mRNA encoding for TLRs 1–9. (C ) TLR mRNA also was detected in primary intestinal myofibroblasts isolated from colonic biopsy specimens. (D) mRNA encoding accessory molecules TIRAP, Tollip, MyD88, MD-2, NOD1, and NOD2 were amplified from cultured and primary myofibroblasts. Shown are representative results from 4 independent experiments. SM, size marker; HPCF, human primary colonic fibroblasts. GAPDH was analyzed to verify equal loading (not shown). Gastroenterology 2003 124, 1866-1878DOI: (10.1016/S0016-5085(03)00403-7)

Figure 3 Regulation of TLR and accessory molecule mRNA expression on stimulation with TLR2 and 4 ligands. Northern blot analysis of TLR 4, 6, and TIRAP expression in CCD-18 and CCD-37 cells after stimulation with LPS (2 μg/mL) or LTA (10 μg/mL) for the indicated times (in hrs). Regulation of gene expression was assayed in 3 experiments. (A ) TLR4 in LPS-stimulated CCD-18; (B) TLR4 in LPS-stimulated CCD-37; (C ) TLR6 in LTA-stimulated CCD-18; (D) TLR6 in LTA-stimulated CCD-37; (E ) TIRAP in LPS- (lanes 2–4) or LTA- (lanes 5–7) stimulated CCD-18; (F ) TIRAP in LPS- (lanes 2–4) or LTA- (lanes 5–7) stimulated CCD-37. Similar results were obtained in these experiments. Gastroenterology 2003 124, 1866-1878DOI: (10.1016/S0016-5085(03)00403-7)

Figure 4 Expression of TLR2 and 4 protein and protein of signaling molecules in intestinal myofibroblasts. Using specific antibodies, constitutive expression of (A ) TLR2 and 4 and of (B) signaling molecules downstream of TLRs including MyD88, IRAK, and tumor necrosis factor receptor-associated factor 6 (TRAF 6) was detected in intestinal and lung myofibroblasts. Lanes 1 and 2, CCD-18 cells; lanes 3 and 4, CCD-37 cells; lanes 5 and 6, primary myofibroblasts. Original blots from a representative experiment are shown. Gastroenterology 2003 124, 1866-1878DOI: (10.1016/S0016-5085(03)00403-7)

Figure 5 Cellular localization of TLRs. CCD-18 and CCD-37 cells were cultured on glass coverslips without any stimuli, fixed, and stained with a monoclonal Ab specific for human TLR2 or 4 or an irrelevant isotype-matched Ab (controls) as indicated. Nuclear staining was performed with DAPI. (A ) Isotype control for TLR4 control on CCD-18 cells; (B) isotype control for TLR4 control on CCD-37 cells; (C ) isotype control for TLR2 on CCD-18 cells; (D) isotype control for TLR2 on CCD-37 cells; (E ) staining of CCD-18 cells with α-TLR4; (F ) staining of CCD-37 cells with α-TLR4; (G) staining of CCD-18 cells with α-TLR2 (H ) staining of CCD-37 cells with α-TLR2 (all magnification 200×); (I ) staining of CCD-18 cells with α-TLR4 (magnification 400×); ( J) staining of CCD-37 cells with α-TLR4 (magnification 400×). Human lung and intestinal myofibroblasts show specific cell surface staining, as well as some cytoplasmic staining for TLR2 and 4. Staining was intense at the leading edge of the cells Gastroenterology 2003 124, 1866-1878DOI: (10.1016/S0016-5085(03)00403-7)

Figure 6 LPS and LTA activation of signaling pathways in intestinal myofibroblasts. Stimulation of CCD-18 and CCD-37 cells with LPS (2 μg/mL); or LTA- (10 μg/mL) induced time-dependent activation of p42/44 ERK1/2, p38, and SAPK/JNK. (A–B) LPS-induced activation of p42/44 MAPK was detected as early as 5 minutes, with maximal activation seen after 30 minutes of stimulation. (C–D) Stimulation with LPS or LTA also induced phosphorylation of p38 and SAPK/JNK. Downstream events on stimulation include degradation of IκB-α ([E] positive control: tumor necrosis factor α 20 ng/mL for 5 minutes), and (F ) nuclear translocation of the NFκB-subunit p65. Blots were reprobed with antibodies specific for total p42/p44 MAPK, p38, and SAPK/JNK to verify equal loading. c, unstimulated control; BSA, bovine serum albumin; WCL, Salmonella enteritidis whole-cell lysate. Shown are representative blots with comparable results obtained in n = 4 experiments Gastroenterology 2003 124, 1866-1878DOI: (10.1016/S0016-5085(03)00403-7)

Figure 7 Functional response of intestinal myofibroblasts to TLR ligands. Intestinal and lung myofibroblasts were stimulated with LPS (2 μg/mL) or LTA (10 μg/mL) for the indicated times and supernatants were harvested for measurement of IL-8 secretion by enzyme-linked immunosorbent assay. Values are expressed as means ± SD (n = 4). (A–B) Stimulation of CCD-18 or (C–D) CCD-37 cells with LPS or LTA or with live bacteria (E. coli; S. aureus) for 6 hours induced a significant increase in IL-8 secretion into culture supernatants. Maximal increase was detected within the first 24 hours of stimulation with LPS or LTA. Total amounts of secreted IL-8 were comparable between the 2 cell lines. ∗∗P < 0.01. Values represent means ± SD (n = 6). Gastroenterology 2003 124, 1866-1878DOI: (10.1016/S0016-5085(03)00403-7)

Figure 8 Effects of MAPK inhibitors on LPS- or LTA-induced IL-8 secretion in colonic (A) and lung (B) myofibroblasts. LPS and LTA induced a significant increase in IL-8 secretion. Preincubation for 15 minutes with 20 μmol/L of a p38 MAPK inhibitor (SB-203580) or a p42/44 MAPK inhibitor (PD-98059 or U-0126) significantly decreased IL-8 secretion in LPS- or LTA-stimulated CCD-18 and CCD-37 cells. ∗∗P < 0.01; ∗P < 0.05. Values represent means ± SD (n = 4). Gastroenterology 2003 124, 1866-1878DOI: (10.1016/S0016-5085(03)00403-7)