Isolation and Characterization of Intestinal Epithelial Cells from Normal and SIV-Infected Rhesus Macaques

Abstract

Impairment of intestinal epithelial barriers contributes to the progression of HIV/SIV infection and leads to generalized HIV-induced immune-cell activation during chronic infection. Rhesus macaques are the major animal model for studying HIV pathogenesis. However, detailed characterization of isolated rhesus epithelial cells (ECs) from intestinal tissues is not well defined. It is also not well documented whether isolated ECs had any other cell contaminants from intestinal tissues during the time of processing that might hamper interpretation of EC preparations or cultures. In this study, we identify and characterize ECs based on flow cytometry and immunohistochemistry methods using various enzymatic and mechanical isolation techniques to enrich ECs from intestinal tissues. This study shows that normal healthy ECs differentially express HLA-DR, CD23, CD27, CD90, CD95 and IL-10R markers. Early apoptosis and upregulation of ICAM-1 and HLA-DR in intestinal ECs are thought to be the key features in SIV mediated enteropathy. The data suggest that intestinal ECs might be playing an important role in mucosal immune responses by regulating the expression of different important regulatory and adhesion molecules and their function.

Lack of Interleukin-10-Mediated Anti-Inflammatory Signals and Upregulated Interferon Gamma Production Are Linked to Increased Intestinal Epithelial Cell Apoptosis in Pathogenic Simian Immunodeficiency Virus Infection

“…Epithelial cells and lymphocytes from jejunum and colon were isolated as described earlier (9,(22)(23)(24). Both intraepithelial (IE) and lamina propria (LP) lymphocytes were isolated from intestinal biopsy specimens, resection surgery, or necropsy tissues.…”

“…Mucosal explants were treated with either anti-IL-10 monoclonal antibodies (MAbs; 5 g/ml; Biolegend), recombinant IL-10 (rIL-10) protein (50 ng/ml; Biolegend), or isotype control (5 g/ml; Biolegend) for 6 h as reported earlier (14). After incubation, explant cultures were fixed in 2% paraformaldehyde and cryopreserved in 30% sucrose (Sigma, St. Louis, MO) in phosphate-buffered saline (PBS), and frozen in cryomolds containing optimal cutting temperature (OCT) compound (Sakura Fintek, Inc., Torrance, CA) as previously described (9).…”

“…Tissue sections were processed for immunofluorescent and immunohistochemistry staining with one or a combination of primary antibodies (see Table S1 in the supplemental material) as reported earlier (9). Nuclear staining was performed with ToPro3 (1 M; Life Technologies).…”

“…Furthermore, studies have shown that mucosal ECs are impacted by HIV/simian immunodeficiency virus (SIV) infection and respond directly to HIV envelope glycoproteins by upregulating inflammatory cytokines that lead to impairment of barrier functions (6)(7)(8). We have recently shown the presence of early EC apoptosis and upregulation of ICAM-1 and HLA-DR by intestinal ECs, which may be key features in SIV-mediated enteropathy (9). Intestinal permeability allows nutrients to pass through the gut, while maintaining a barrier against gut microbiota from leaving the intestine and migrating to the body.…”

“…Increased permeability due to compromised barrier function could facilitate gut microbiota crossing the mucosal epithelium and entering circulation (microbial translocation) (10). Epithelial injury and impaired epithelial regeneration are considered key factors in the pathogenesis of AIDS contributing to generalized HIV-induced immune cell activation (9,11,12).…”