Goat milk with and without increased concentrations of lysozyme improves repair of intestinal cell damage induced by enteroaggregative Escherichia coli

Abstract

Background: Enteroaggregative Escherichia coli (EAEC) causes diarrhea, malnutrition and poor growth in children. Human breast milk decreases disease-causing bacteria by supplying nutrients and antimicrobial factors such as lysozyme. Goat milk with and without human lysozyme (HLZ) may improve the repair of intestinal barrier function damage induced by EAEC. This work investigates the effect of the milks on intestinal barrier function repair, bacterial adherence in Caco-2 and HEp-2 cells, intestinal cell proliferation, migration, viability and apoptosis in IEC-6 cells in the absence or presence of EAEC.

Methods: Rat intestinal epithelial cells (IEC-6, ATCC, Rockville, MD) were used for proliferation, migration and viability assays and human colon adenocarcinoma (Caco-2, ATCC, Rockville, MD) and human larynx carcinoma (HEp-2, ATCC, Rockville, MD) cells were used for bacterial adhesion assays. Goats expressing HLZ in their milk were generated and express HLZ in milk at concentration of 270 μg/ml. Cells were incubated with pasteurized milk from either transgenic goats expressing HLZ or non-transgenic control goats in the presence and absence of EAEC strain 042 (O44:H18).

Results: Cellular proliferation was significantly greater in the presence of both HLZ transgenic and control goat milk compared to cells with no milk. Cellular migration was significantly decreased in the presence of EAEC alone but was restored in the presence of milk. Milk from HLZ transgenic goats had significantly more migration compared to control milk. Both milks significantly reduced EAEC adhesion to Caco-2 cells and transgenic milk resulted in less colonization than control milk using a HEp-2 assay. Both milks had significantly increased cellular viability as well as less apoptosis in both the absence and presence of EAEC.

Conclusions: These data demonstrated that goat milk is able to repair intestinal barrier function damage induced by EAEC and that goat milk with a higher concentration of lysozyme offers additional protection.

Figure 1

HLZ and control goat milk enhance IEC-6 proliferation. Proliferation of IEC-6 cells after 24 hours of incubation with milk from HLZ transgenic or non-transgenic control goats at dilutions of 1:5, 1:20 and 1:40. HLZ and control milk was significantly different from glutamine free DMEM medium (DMEM Gln-), *P < 0.05; *** P < 0.001.

Figure 2

HLZ and control goat milk improve cellular migration in the presence of EAEC. Migration of IEC-6 cells after 24 hours of incubation with milk from HLZ transgenic or non-transgenic control goats at dilutions of 1:5, 1:20 and 1:40 in the absence (A) and presence (B) of EAEC-042 (2.5 x 10⁵ CFU/ml) (**P < 0.01; *** P < 0.001).

Figure 3

HLZ and control goat milk decrease bacterial adhesion. Number of colony forming units of EAEC 042 (2.5 x 10⁵ CFU/ml starting concentration) that adhered to Caco-2 cells after 1.5 hours of incubation with milk from HLZ transgenic and non-transgenic control goats at dilutions of 1:5, 1:20 and 1:40 (*** P < 0.001).

Figure 4

Bacterial adhesion in HEp-2 cells or in the media their self as indicated by the black arrow. (A) EAEC 042 (0.2 x 10² UFC/mL); EAEC 042 plus milk from non-transgenic control goats at a (B) 1:5, (C) 1:20 and (D) 1:40 dilution; EAEC 042 plus milk from HLZ transgenic goats at a (E) 1:5, (F) 1:20 and (G) 1:40 dilution. 1000X magnification.

Figure 5

HLZ and control goat milk improve cellular viability. Flow cytometry analysis was conducted to determine the percentage effect of HLZ or control milk on (A) living IEC-6 cells in the absence and presence of EAEC-042; (B) apoptotic IEC-6 cells in the absence and presence of EAEC-042; and (C) necrotic IEC-6 cells in the absence and presence of EAEC-042. The IEC-6 cells were incubated for 24 hours in HLZ transgenic or non-transgenic control goat milk at dilutions of 1:5, 1:20 and 1:40 in the absence or presence of EAEC-042 (2.5 x10⁵ CFU/ml) (*** P < 0.001).