Rapid Isolation and Flow Cytometry Analysis of Murine Intestinal Immune Cells after Chemically Induced Colitis

Background

An intricate network of various immune cells and secreted cytokines and chemokines function in cooperative ways to protect the host from adverse environmental conditions and microbial infections to maintain or restore homeostasis (Hooper and Macpherson, 2010). The GI tract is constantly exposed to commensal, opportunistic, and pathogenic microbes, creating perturbations that can lead to life-threatening infections and chronic inflammation. The host defense system in the gastrointestinal (GI) tract is comprised of a single layer epithelium fortified with adjoining cells and lymphoid tissues that operate efficiently to maintain barrier homeostasis. This barrier strikes a balance of remaining tolerant to commensal microbes and dietary antigens while preserving the capacity to mount an inflammatory response against pathogens.

IBD, a general term for chronic inflammatory conditions of the GI tract, is comprised of two main clinical entities: Crohn’s disease and ulcerative colitis. A broadly compromised or dysregulated immune system due to environmental changes, genetic background, or a qualitative and quantitative abnormal gut microbiota is a hallmark of IBD pathogenesis (de Souza and Fiocchi, 2016). Despite several decades of research, our understanding of onset, recovery, and recurrent flares in IBD remains tenuous. Recruitment and activation of immune cells at inductive sites [Peyer’s patches (PP); isolated lymphoid follicles (IELs); and mesenteric lymph nodes (MLN)] and initiation of effector functions in the lamina propria and epithelium is a sequential and complex process. In chronic disease conditions, constant activity of immune cells (i.e., ROS generation, chemokine, and cytokine secretion) in conjunction with architectural damage of tissues further induces immune cell recruitment, hence forming a perpetual destructive loop (Brandtzaeg et al., 2008). To fully comprehend the spatiotemporal dynamics of transmigrated cells and their crosstalk with other cell types, efficient isolation and characterization of distinct immune cell populations is essential. A relatively simple and rapid protocol for isolating immune cells from intestinal tissue will be highly enabling as intestinal immunity is at the center stage in IBD, colorectal cancer (CRC), or host-microbe interactions. Isolating healthy and viable cells from inflamed gut, including hyperactivated neutrophils and macrophages in early acute inflammation or various T lymphocytes at later stages of the disease, is challenging but crucial for immunophenotyping and functional assays of selected cell populations. The majority of the existing protocols describes isolation of cells from normal (non-inflamed) tissues during homeostasis (Weigmann et al., 2007; Geem et al., 2012; Reißig et al., 2014); however, those that isolate from inflamed tissues have extended digestion period and often lack in viability data (Drakes et al., 2004; Bowcutt et al., 2015; Li et al., 2016, Gui et al., 2018).

This protocol describes a straightforward method for rapid isolation of highly viable immune cells from murine intestinal tissues such as colonic and ileal lamina propria, PP, and MLN using Medimachine II (Syntec International, Ireland). This protocol has also been used to isolate viable cells from the murine spleen and can easily be adapted to phenotype diverse cell populations from human tissues. Here, we used chemically induced models of acute colitis in mice, such as 2,4,6-trinitrobenzene sulfonic acid (TNBS) and dextran sodium sulfate (DSS) treatment, for isolating cells from colonic lamina propria at the peak of disease.