Interleukin-10 prevents epithelial cell apoptosis by regulating IFNγ and TNFα expression in rhesus macaque colon explants

Abstract

Interleukin-10 (IL-10) is an important immunomodulatory cytokine that plays an obligate role in regulating inflammatory responses. Here we demonstrated the role of IL-10 in regulating crypts length and breadth as well as maintaining the survival of epithelial cells using rhesus colon explant cultures. Anti-IL-10 antibody treatment of colon explant cultures induced increased production of inflammatory cytokines/molecules like IFNγ, TNFα, CD107a and perforin as well as increased epithelial cell apoptosis compared to media controls tested. Our results suggest that IL-10 plays a crucial role in maintaining mucosal homeostasis by regulating mucosal IFNγ and TNFα cytokine production.

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

“…We have recently demonstrated the role of IL-10 in maintaining the survival of ECs and regulating crypt breadth using colon explant cultures (14). Our study suggested that IL-10 played an obligate role in maintaining mucosal homeostasis by regulating the production of mucosal gamma interferon (IFN-␥) and tumor necrosis factor alpha (TNF-␣) cytokines.…”

“…Colon specimens ϳ8 cm in length were collected in ice-cold Hanks balanced salt solution and were processed as described previously (14,25). 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).…”

“…Labeled tissue sections were mounted using Prolong Gold antifade medium (Invitrogen) and imaged using a TCS SP2 confocal laser scanning microscope (Leica, Germany) equipped with an argon-krypton laser at 488 nm (green), a krypton laser at 568 nm (red), and a helium-neon laser at 633 nm (blue). Negativecontrol slides were incorporated in each experiment either by omitting the primary antibody or using isotype IgG1 and IgG(HϩL) controls (9,14). ImageJ (version 1.49d; National Institutes of Health [NIH], Bethesda, MD) and Adobe Photoshop CS5 Extended (USA) were used to assign colors to the channels collected.…”

“…Colon specimens ϳ8 cm in length were collected in ice-cold Hanks balanced salt solution and were processed as described previously (14,25). 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).…”

“…Conversely, IL-10 polymorphisms associated with increased IL-10 production might have a protective role against rapid disease progression by suppressing chronic immune activation and reducing CD4 T-cell loss (21). Despite the role of IL-10 as an immunomodulatory cytokine in maintaining the EC barrier in colon explants (14), the main source of IL-10 in intestinal tissues has not been well defined. The role of IL-10 in maintaining intestinal homeostasis during SIV infection, as well as its effect on mucosal IFN-␥ and TNF-␣ cytokines, is also not well understood.…”