By Jamshed Arslan, Pharm. D., PhD.
Toll-like receptors (TLRs) are microbe-sensing proteins that act as first responders to danger signals. TLRs help the intestinal epithelial cells (IECs) recognize commensal bacteria from the harmful/foreign ones. Polymorphisms and variants of TLRs have been linked with immune system’s attack on commensal bacteria, leading to inflammatory bowel disease and colorectal cancers. The endogenous pattern of TLR expression in IECs and the functional outcome of TLR signaling in vivo are not well defined due to various technical difficulties. Thanks to the researchers in institutes in California and Massachusetts who generated TLR-reporter mice to overcome the technical obstacles, now we have a blueprint of TLR expression and function in IECs. These scientists have discovered the spatial, temporal and cell type-specific TLR expression pattern and the IEC-gene expression in response to a variety of TLR ligands and cytokines.
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Pattern of TLR expression in the intestinal epithelium
To visualize TLR expression in IECs through cytoplasmic fluorescence, the researchers generated mice that express a TLR transcript with a fluorescent protein: TLR 2 and TLR 9 with green fluorescent protein (GFP), TLR 4 with yellow fluorescent protein (YFP), and TLR 5 and TLR 7 with red tomato. The reporter cassettes for TLR-reporter mice were designed in a way that the 3’ end of a TLR gene had FLAG or HA epitope tag, internal ribosome entry site to allow cap-independent translation, and a fluorescent protein. Using antibodies specific to the fluorescent proteins, the team revealed lower levels of TLR 2, 4 and 5 in small intestinal epithelial cells from these mice relative to the colonic IECs. No reporter expression for TLR7 or 9 was observed, possibly indicating a lack of these TLRs in the epithelium. In small intestine, TLR5 showed a unique pattern in that tomato was present only in a small subset of cells in the small intestinal crypt. When scientists co-stained small intestinal crypts with lysozyme (marker for Paneth cells), this subset of cells was identified as Paneth cells.
The next question was: what is the consequence of this unique pattern of TLR5 expression in colon IECs and Paneth cells?
TLR5 was detected in immersion fixed paraffin-embedded sections of human intestine using Mouse Anti-Human TLR5 Monoclonal Antibody(Catalog # MAB6704) at 15 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Mouse HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS002) and counterstained with hematoxylin (blue). Lower panels show a lack of labeling when primary antibodies are omitted and when tissue is stained only with secondary antibody followed by incubation with detection reagents. Specific staining was localized to cell surfaces and cytoplasm.
Distinct gene expression due to TLR5 signaling
To determine the consequence of TLR5 expression, researchers used three-dimensional epithelial organoids and found a common gene expression profile. When stimulated by bacterial component flagellin, Paneth cells and colon IECs exhibited TLR5-dependent induction of several chemokines and cytokines (Ccl20, M-CSF/Csf1, TNF) as well as increased expression of genes related to NF-kB signaling (A20/Tnfaip3, Ikb-alpha/Nfkbia, NFkb2) and oxidative stress (Duox2, iNOS/Nos2, Nox1). Stimulation with IL-18 (a cytokine that signals through TLR signaling adapter MyD88) or ligands for TLR4, 7, or 9 did not elicit any distinct gene expression in the organoids. The scientists were intrigued to see that TLR expression did not modulate antimicrobial genes. Rather, it was the inflammatory cytokines (IL-22 and IFN-gamma) that led to increased antimicrobial peptides (Reg3gamma and Reg3beta) and proteins (RELM-beta/Retnlb) in the organoids.
In sum, TLRs have a distinct pattern of expression in gut epithelium that leads to activation of specific host defense genes.
Paneth Cell Markers
||Antimicrobial protein found in Paneth cell’s secretory granules
|Cationic antimicrobial peptides present in dense core granules and secreted in response to bacterial infection
|Metalloproteinase in Paneth granules required for defensin activation
|Defensin alpha 5
|Highly expressed in secretory granules of Paneth cells
Antibody & Beyond, Paneth Cell Markers: www.antibodybeyond.com/reviews/cell-markers/paneth-cell-marker
This study is a big step forward towards understanding the biology of the innate immune system as it gives a blueprint of TLR signaling in IECs. This TLR map can be used to investigate crosstalk between microbiome in various parts of the gut. Moreover, the reporter mice generated in this research can be employed to examine TLR biology in myriad of other contexts.
Jamshed Arslan, Pharm D., PhD.
University of Alabama at Birmingham, School of Medicine
Dr. Arslan studies cell signaling in mitochondrial defects in
C. elegans and transgenic mice.
Price, April E., et al. "A Map of Toll-like Receptor Expression in the Intestinal Epithelium Reveals Distinct Spatial, Cell Type-Speciﬁc, and Temporal Patterns."
Immunity, vol. 49, no. 3, 2018, pp. 560–575. https://doi.org/10.1016/j.immuni.2018.07.016