NAD(P)H Oxidase Activity in the Small Intestine Is Predominantly Found in Enterocytes, Not Professional Phagocytes

Abstract

The balance between various cellular subsets of the innate and adaptive immune system and microbiota in the gastrointestinal tract is carefully regulated to maintain tolerance to the normal flora and dietary antigens, while protecting against pathogens. The intestinal epithelial cells and the network of dendritic cells and macrophages in the lamina propria are crucial lines of defense that regulate this balance. The complex relationship between the myeloid compartment (dendritic cells and macrophages) and lymphocyte compartment (T cells and innate lymphoid cells), as well as the impact of the epithelial cell layer have been studied in depth in recent years, revealing that the regulatory and effector functions of both innate and adaptive immune compartments exhibit more plasticity than had been previously appreciated. However, little is known about the metabolic activity of these cellular compartments, which is the basic function underlying all other additional tasks the cells perform. Here we perform intravital NAD(P)H fluorescence lifetime imaging in the small intestine of fluorescent reporter mice to monitor the NAD(P)H-dependent metabolism of epithelial and myeloid cells. The majority of myeloid cells which comprise the surveilling network in the lamina propria have a low metabolic activity and remain resting even upon stimulation. Only a few myeloid cells, typically localized at the tip of the villi, are metabolically active and are able to activate NADPH oxidases upon stimulation, leading to an oxidative burst. In contrast, the epithelial cells are metabolically highly active and, although not considered professional phagocytes, are also able to activate NADPH oxidases, leading to massive production of reactive oxygen species. Whereas the oxidative burst in myeloid cells is mainly catalyzed by the NOX2 isotype, in epithelial cells other isotypes of the NADPH oxidases family are involved, especially NOX4. They are constitutively expressed by the epithelial cells, but activated only on demand to ensure rapid defense against pathogens. This minimizes the potential for inadvertent damage from resting NOX activation, while maintaining the capacity to respond quickly if needed.

Coregistered Spectral Optical Coherence Tomography and Two‐Photon Microscopy for Multimodal Near‐Instantaneous Deep‐Tissue Imaging

“…In the last three decades, two‐photon microscopy (2PM) has proven to be the method of choice to perform deep‐tissue imaging in tissue slices, explanted organs and in living animals . Especially in immunology, 2PM has brought unique insights into cellular motility patterns, cellular communication, and tissue functions, in health and disease, and helped to decipher key pathophysiologic phenomena involving the immune system . In order to acquire information on immune cell subsets from 2PM, fluorescent labeling of cells within organisms, either using viable dyes or using fluorescent reporter constructs, is needed.…”

“…In order to acquire information on immune cell subsets from 2PM, fluorescent labeling of cells within organisms, either using viable dyes or using fluorescent reporter constructs, is needed. The endogenous tissue fluorescence (autofluorescence), while providing valuable information about the tissue state , is not specific enough to reveal the orchestration of phenomena governing organ and tissue functions on a cellular level. Additionally, 2PM requires complex devices such as femtosecond‐pulsed lasers and dedicated optomechanics and optoelectronics.…”

From the Cradle to the Grave of an Infection: Host‐Pathogen Interaction Visualized by Intravital Microscopy

“…A recent in vitro study established a MP‐IVM‐based FLIM approach that allows for the visualization of the dynamics of NADPH oxidase activation and its requirement in triggering NETosis . This concept is also applicable for MP‐IVM, as has been shown for imaging of the small intestine , and has the potential to become a powerful tool for investigating the interactions between pathogens and the host immune system.…”

A deeper understanding of intestinal organoid metabolism revealed by combining fluorescence lifetime imaging microscopy (FLIM) and extracellular flux analyses

“…Indeed the glucose-dependent NADPH production is important for activity of enterocyte glutathione reductase, the principal supplier of GSSH in the intestine 59 . Activities of intestinal enterocyte-specific enzymes such as cytochrome P450 oxidase, reductase and NADPH oxidase 4 (NOX4) also demand for high levels of NADPH 31,60,61 . GFP + stem cell niche regions displayed [NADPH]/[NADH] ratio sensitive to presence of glucose and pyruvate, but their protein-bound NAD(P)H fraction tended to remain constant (Fig.…”

“…PLIM also enables an estimation of O2 consumption -a primary marker of mitochondrial activity 30 . Although not previously applied to the analysis of stem cellderived organoids, two-photon excited NAD(P)H-FLIM allowed for direct assessment of the balance between cell redox and energy production pathways within the intestinal crypt 31,32 . FLIM and PLIM, individually and/ or combined, can be used in various multi-parameter assays, such as live and real-time tracing of cell proliferation with oxygenation and measurement of extracellular pH fluxes by the biosensor scaffolds 25,29,33,34 ; these methods are therefore highly attractive for imaging of metabolism in stem cell-based and tissue engineered constructs.…”