Optimal Isolation Protocols for Examining and Interrogating Mononuclear Phagocytes From Human Intestinal Tissue

Area of Science:

• Immunology
• Gastroenterology
• Cell Biology

Background:

• Human intestines host diverse mononuclear phagocytes (MNP) crucial for immune homeostasis.
• Isolating viable MNP from intestinal tissue is challenging due to marker degradation and cell death during extraction.
• Current methods often trigger MNP activation, altering their functional phenotype for analysis.

Purpose of the Study:

• To develop an optimized method for isolating functionally immature intestinal MNP from human jejunum, ileum, and colon.
• To enable high-parameter flow cytometry for comprehensive MNP subset identification.
• To facilitate functional assays using sorted, viable intestinal MNP.

Main Methods:

• Anatomical separation of intestinal layers (mucosa, submucosa) and lymphoid follicles.
• Optimized enzymatic digestion protocols to yield viable, immature MNP.
• Screening of antibody clones for high-parameter flow cytometry panels to identify MNP subsets (pDCs, cDC1, cDC2, DC3, monocytes, Mf1-4).
• Novel method developed to correct for autofluorescence in tissue macrophages.

Main Results:

• Successful isolation of viable, functionally immature intestinal mononuclear phagocytes.
• Development of comprehensive flow cytometry panels capable of discriminating all known human intestinal MNP subsets.
• Demonstrated utility of the method for sorting functional, immature dendritic cells for cytokine production assays.

Conclusions:

• The described method provides a robust approach for isolating and analyzing diverse intestinal MNP subsets.
• This technique preserves cellular integrity and function, enabling deeper understanding of MNP roles in intestinal immunity.
• The optimized protocols and antibody panels are valuable tools for future research in gut immunology, disease, and immunotherapy.