We identified genes with differential expression within the human colon during acute and convalescent amebic colitis. REG 1A and REG1B were the most upregulated genes during acute amebiasis, and may provide protection for the intestinal epithelium. Evidence suggests REG 1 is involved in tissue repair [13
], cell growth and differentiation [9
]. In addition, REG 1 expression occurs in areas of cell renewal, including intestinal crypt epithelial cells [7
We confirmed the significant differences between acute and convalescent REG 1A and REG 1B expression in colonic biopsy samples by real-time PCR. These findings are in keeping with other studies which have found REG 1 expression is increased in the colon in the setting of inflammation. One study found upregulation of REG1A mRNA in epithelium from inflamed colonic tissue compared to inactive inflammatory bowel disease (IBD) or normal colonic tissue [17
]. Other studies have also found increased REG1A mRNA expression in colonic tissue affected by ulcerative colitis [12
] or Crohn's disease [10
] compared to normal colonic tissue, with the level of expression positively correlated with the severity of inflammation [10
Studies evaluating REG1A expression by immunohistochemistry have found that REG1A protein expression in normal colonic tissue is detectable only among epithelial cells in the basal portion of crypts. The intensity and number of positively stained crypt cells increased within ulcerative colitis tissue [10
]. A study using in situ hybridization showed REG1A to be localized to epithelial cells of the intestinal crypts in the setting of active colitis, with minimal expression in mesenchymal cells, and no expression in normal colonic tissue [17
In the current study, REG 1A and REG 1B staining was intense during the acute stage of E. histolytica
colitis and present throughout the intestinal cells of the crypt epithelium. In contrast, both REG 1A and REG 1B staining were limited to the basal portion of the colonic crypts after recovery. These findings are similar to other studies assessing REG 1A expression in IBD or normal colon [10
], and also correspond with our microarray and PCR data showing markedly increased REG 1A and REG 1B expression during acute E. histolytica
colitis. REG 1A, but not REG 1B, staining was also detectable in the lamina propria of both acute and convalescent samples. REG 1A and REG 1B staining were not detected in the negative controls when primary antibody was excluded, or with the IgG isotype controls.
REG1A has been shown to have anti-apoptotic effects in gastric and colon cancer lines [8
] through activation of the Akt/Bad/Bcl-xL pathway [18
]. In contrast, E. histolytica
is able to induce apoptosis of the intestinal epithelium [4
]. Host intestinal cell apoptosis by E. histolytica
occurs by activation of host caspase-3 in a caspase-8 and -9 independent manner [19
]. To test the hypothesis that REG 1 may be protective in E. histolytica
disease due to its anti-apoptotic effects, we evaluated E. histolytica
-induced apoptosis of intestinal epithelial cells from REG 1 -/- or REG 1 +/+ mice. We observed increased apoptosis of intestinal epithelial cells from REG 1 -/- mice compared to intestinal epithelial cells from REG1 +/+ mice. E. histolytica
lysate further enhanced apoptosis in the REG 1-/- epithelium.
This study is the first to assess differential gene expression during acute and convalescent amebic colitis outside of an animal model. Of the ten genes most significantly changed in the human colon between acute and convalescent E. histolytica
disease, including REG 1A and REG 1B, eight were upregulated during acute disease. Matrix metalloproteinases-1 and -3 (MMP-1, MMP-3) are involved in tissue injury and repair, S100 calcium binding protein A8 (S100A8) is expressed in neutrophil and monocyte cytosol and increased with inflammation [20
], Deleted in malignant brain tumors 1 (DMBT1) is involved in epithelial cell differentiation [21
] and innate immunity [22
], and Olfactomedin 4 (OLFM4) has a role in cell adhesion [23
] and anti-apoptosis [24
]. The apical water channel, Aquaporin 8 (AQP8), and Solute carrier family 26, member 2 (SLC26A2), were decreased during acute disease.
Many of the genes which were differentially expressed during amebiasis are similarly involved in other inflammatory intestinal diseases. The top 5 most upregulated genes during acute amebiasis in the current study (REG1A, REG1B, MMP-1, S100A8, and MMP-3) were found, in a study by Dieckgraefe et al which used genome wide expression analysis, to be more abundant in colonic tissue during active ulcerative colitis as compared to non-inflamed control tissue [25
]. In another study, DMBT1 expression was found to be increased in inflammatory bowel disease tissue compared to controls, correlated with disease activity, and predominately occurred within intestinal epithelial and Paneth cells [26
]. Aquaporin 8 was also shown to be downregulated in three experimental models of colitis in mice, and in affected colonic tissue of patients with inflammatory bowel disease compared to nonaffected colon [27
A mouse model has been used to compare transcriptional responses within human colonic xenografts at four and twenty-four hours post-infection with either Shigella flexneri
or E. histolytica
]. Several of the genes elevated post-infection with either organism, similar to the current study, encoded proteins involved in tissue injury and repair including matrix metalloproteinases, tissue inhibitor of metalloproteinase 1, and cystine-rich angiogenic inducer. Unlike the current study, increased expression of several leukotrienes (1β, -6, -8, -11) and monocyte chemotactic proteins (-1, -2, -3) were also found. Notably, a mouse model of Citrobacter rodentium
, a murine attaching and effacing pathogen similar to enteropathogenic and enterohaemorrhagic Escherichia coli
, found that genes differentially expressed between susceptible and resistant strains of mice were greatly enriched for transport activity [29
]. More transport genes than immune-related genes were demonstrated to determine susceptibility to infection. We also found differential expression of several transport molecules during acute and convalescent amebic colitis, including seven solute family members and aquaporin 8.
There were limitations in this study. The increased spontaneous apoptosis in REG 1 -/-epithelial cells found in vitro requires confirmation in vivo in uninfected and E. histolytica-infected mice. In addition, the incremental increase in apoptosis seen with amebic lysate should be tested to see if it is specific to E. histolytica, since lysate from other parasites or pathogens was not tested. Other potential mechanisms of REG 1 protection during amebiasis, such as its pro-proliferative effects, should also be evaluated. Further studies in vivo are planned to assess the susceptibility and severity of amebic disease in REG 1 -/- and wild type mice.
In summary, we found that REG 1A and REG 1B mRNA and protein expression were significantly increased in the human intestine during acute E. histolytica colitis. We also found that apoptosis was increased in REG 1 -/- intestinal epithelial cells compared to REG 1 +/+ cells, and E. histolytica lysate further enhanced apoptosis. Therefore, we hypothesize that increased REG 1 expression may be beneficial during E. histolytica colitis through a protective role against E. histolytica-induced apoptosis.