TPN-associated intestinal epithelial cell atrophy is modulated by TLR4/EGF signaling pathways

Abstract

Recent studies suggest a close interaction between epidermal growth factor (EGF) and TLR signaling in the modulation of intestinal epithelial cell (IEC) proliferation; however, how these signaling pathways adjust IEC proliferation is poorly understood. We utilized a model of total parenteral nutrition (TPN), or enteral nutrient deprivation, to study this interaction as TPN results in mucosal atrophy due to decreased IEC proliferation and increased apoptosis. We identified the novel finding of decreased mucosal atrophy in TLR4 knockout (TLR4KO) mice receiving TPN. We hypothesized that EGF signaling is preserved in TLR4KO-TPN mice and prevents mucosal atrophy. C57Bl/6 and strain-matched TLR4KO mice were provided either enteral feeding or TPN. IEC proliferation and apoptosis were measured. Cytokine and growth factor abundances were detected in both groups. To examine interdependence of these pathways, ErbB1 pharmacologic blockade was used. The marked decline in IEC proliferation with TPN was nearly prevented in TLR4KO mice, and intestinal length was partially preserved. EGF was significantly increased, and TNF-α decreased in TLR4KO-TPN versus wild-type (WT)-TPN mice. Apoptotic positive crypt cells were 15-fold higher in WT-TPN versus TLR4KO-TPN mice. Bcl-2 was significantly increased in TLR4KO-TPN mice, while Bax decreased 10-fold. ErbB1 blockade prevented this otherwise protective effect in TLR4KO-sTPN mice. TLR4 blockade significantly prevented TPN-associated atrophy by preserving proliferation and preventing apoptosis. This is driven by a reduction in TNF-α abundance and increased EGF. Potential manipulation of this regulatory pathway may have significant clinical potential to prevent TPN-associated atrophy.

Keywords: apoptosis; epidermal growth factor; epithelial cell proliferation; mucosal atrophy; total parenteral nutrition.

Figure 1.

Gross and microscopic morphometric measurements of TLR4KO and WT mice. A) Mean small intestine lengths. B) Percentage change in small intestine length with TPN administration in WT and TLR4KO groups. C) Differences in crypt depth. D) Differences in villus length. Results represent means ± sd, n = 6/group. *P < 0.05. E) Differences in transepithelial resistance for each study group. F) Permeability of FITC-dextran for each study group. G) Representative images of ZO-1 and occludin staining for each group.

Figure 2.

Epithelial cell proliferation is measured between the groups using percentage positive BrdU and PCNA staining. A) Percentage positive BrdU IEC per crypt (mice were injected with BrdU 24 hours before the mice were killed). B) PCNA percentage positive IEC per crypt. C) Representative images of PCNA staining for each study group. Results of (A) and (B) are expressed as means ± sd of epithelial cell counts from 10 well-oriented crypt–villus complexes from each mouse, n = 6/group. *P < 0.05.

Figure 3.

Epithelial cell apoptosis results using TUNEL and caspase 3 staining. A) Mean TUNEL-positive IEC per 100 villi are shown for each study group. B) Mean TUNEL-positive IEC per 100 crypts are shown for each study group. C, D) Representative WT images. E) Mean caspase 3 positive per 100 villi IEC are shown for each study group. F) Mean caspase 3 positive per 100 crypt IEC are shown for each study group. G) Representative images of caspase 3 staining for each study group.

Figure 4.

Epithelial cell apoptosis results and contributing apoptotic pathways. A–C) Factors are measured by RT-PCR of jejunal mucosal scrapings, corrected to expression of 18S. They represent Bcl-2, Bax, and the Bax/Bcl-2 ratio, respectively. D, E) Fas ligand and Fas mRNA expression adjusted as a ratio to β-actin expression. F) Representative Western blot of phosphorylated Iκβα to Iκβα and ratio of these factors. Results represent means ± sd, n = 6/group. *P < 0.05.

Figure 5.

mRNA cytokine expression using RT-PCR of selected cytokines and receptors and TLRs. A) Changes in TNF-α. B, C) Changes in TNFR1 and TNFR2, respectively. D) changes in IFN-γ. E, F) Changes in IL-6 and IL-10, respectively. Gi–iv) show changes in selected TLRs as a result of the knockout of TLR4 by RT-PCR. mRNA expression is corrected to the expression of 18S. Results represent means ± sd, n = 6/group. *P < 0.05.

Figure 6.

EGF/EGFR and downstream signaling factors. A, B) Changes in EGF and EGFR, respectively, by RT-PCR of jejunal mucosal scrapings. C) Changes in amphiregulin by RT-PCR. D, E) Representative blots from analysis of Western immunoblots of epithelial cell lysate. D) Ratio of p-AKT to β-actin. E) Ratio of phosphorylated Erk to Erk. Results represent means ± sd, n = minimum of 5/group. *P < 0.05.

Figure 7.

Effect of ErbB1 blockade on the small intestine of TLR4KO mice. A) Percentage change between preoperative and postoperative weights for each group. B) Changes in small intestine lengths. C) Percentage change in small intestine length with TPN and ErbB1 blockade. WT is the percentage change in small intestine length between WT-sham and WT-TPN mice. TLR4KO is the percentage change in small intestine length between TLR4KO-sham and TLR4KO-TPN mice. TLR4KO-sham with EGF blockade is the percentage change in small intestine length between TLR4KO-sham and TLR4KO-sham EGF blockade mice. TLR4KO-TPN with EGF blockade is the percentage change in small intestine length between TLR4KO-sham and TLR4KO-TPN EGF blockade mice. D) Differences in crypt depth for each study group. E) Differences in villus length for each study group. F) Representative images of ZO-1 and occludin staining in ErbB1 blockade for TLR4KO sham and TLR4KO TPN groups compared to WT groups. G) Changes in proliferation by changes in PCNA staining (%PCNA⁺ crypt cells). H, I) Changes in apoptosis by TUNEL staining. Results represent means ± sd, n = 6/group. *P < 0.05.

Figure 7.

Effect of ErbB1 blockade on the small intestine of TLR4KO mice. A) Percentage change between preoperative and postoperative weights for each group. B) Changes in small intestine lengths. C) Percentage change in small intestine length with TPN and ErbB1 blockade. WT is the percentage change in small intestine length between WT-sham and WT-TPN mice. TLR4KO is the percentage change in small intestine length between TLR4KO-sham and TLR4KO-TPN mice. TLR4KO-sham with EGF blockade is the percentage change in small intestine length between TLR4KO-sham and TLR4KO-sham EGF blockade mice. TLR4KO-TPN with EGF blockade is the percentage change in small intestine length between TLR4KO-sham and TLR4KO-TPN EGF blockade mice. D) Differences in crypt depth for each study group. E) Differences in villus length for each study group. F) Representative images of ZO-1 and occludin staining in ErbB1 blockade for TLR4KO sham and TLR4KO TPN groups compared to WT groups. G) Changes in proliferation by changes in PCNA staining (%PCNA⁺ crypt cells). H, I) Changes in apoptosis by TUNEL staining. Results represent means ± sd, n = 6/group. *P < 0.05.

Figure 8.

Changes in factors mediating apoptosis and selected cytokines and TLR after ErbB1 blockade in TLR4KO mice; results measured by RT-PCR of jejunal mucosal scrapings. A, B) Changes in Bcl-2 and Bax, respectively. C, D) Changes in FasL and Fas, respectively, with ErbB1 blockade. E, F) Changes in TNF-α and IFN-γ, respectively. G) Changes in TLR2. H) Changes in EGF.