Background: Eosinophilic esophagitis (EoE) is a chronic immune-mediated inflammatory disorder characterized by persistent inflammation, decreased esophageal distensibility, and esophageal stricture. We have previously shown that lysyl oxidase (LOX), a collagen cross-linking enzyme, is upregulated in the esophageal epithelium in EoE, however its role beyond collagen cross-linking is unknown. Herein, we propose a non-canonical epithelial-specific mechanism of LOX in the maintenance of epithelial homeostasis using 3-D organoid and murine models.
Methods and Results: Short-hairpin RNA (shRNA) was used to create a doxycycline-inducible LOX knockdown model in immortalized non-transformed human esophageal epithelial cells (EPC2-hTERT). RNA sequencing was then performed on doxycycline-treated shSCR and shLOX organoids. We identified enrichment of Hallmark cell cycle pathways in the setting of LOX knockdown. In addition, markers of terminal differentiation including NOTCH3 (p < 0.001), desmoglein-1 (DSG1) (p < 0.0001), and filaggrin (FLG) (p < 0.05) were all significantly decreased in doxycycline-treated shLOX organoids. Because cell cycle-related pathways were enriched, we first assessed cell proliferation in vitro. We found that LOX knockdown decreased proliferative capacity (p < 0.0001) (Figure 1A), dramatically reduced organoid formation rate (OFR) (p<0.0001) (Figure 1B-C), and decreased Ki67 staining (Figure 2B). We then used qRT-PCR, immunohistochemistry, and immunofluorescence to assess the role of LOX in differentiation. LOX knockdown decreased mRNA expression of differentiation markers filaggrin (p<0.0001), and loricrin (p<0.0001) in doxycycline-treated shLOX organoids (Figure 2A). Further, LOX knockdown resulted in increased basaloid content assessed by H&E, as well as decreased involucrin (IVL) expression (Figure 2B). Global LOX deletion is embryonically lethal, therefore we created a murine model with a tamoxifen-induced LOX knockout specific to the stratified epithelium (K5-CreERT2). We then used dermal sensitization to induce EoE in the tamoxifen/vehicle control-treated mice using OVA-albumin. LOX KO mice demonstrated decreased Ki67 staining compared to vehicle-treated mice, suggesting reduced proliferation in the LOX-deficient epithelium in vivo.
Discussion: Our results demonstrate that LOX may regulate epithelial homeostasis in the esophagus through modulation of epithelial proliferation and differentiation. Understanding mechanisms of perturbation in epithelial proliferation and differentiation in EoE could lead to the development of novel treatments that promote epithelial healing and restore homeostasis.

<b>Figure 1: LOX inhibition reduces proliferative ability in the esophageal epithelium. </b>(A) LOX inhibition significantly reduced proliferative ability, as measured by cell count for shSCR control and shLOX cells cultured with continuous doxycycline exposure on passage 2 (P2) and passage 3 (P3). (B-C) Organoid formation rate (OFR) was dramatically reduced in the absence of LOX expression, with no organoids formed following shLOX induction (<i>p</i><0.0001).

Figure 1: LOX inhibition reduces proliferative ability in the esophageal epithelium. (A) LOX inhibition significantly reduced proliferative ability, as measured by cell count for shSCR control and shLOX cells cultured with continuous doxycycline exposure on passage 2 (P2) and passage 3 (P3). (B-C) Organoid formation rate (OFR) was dramatically reduced in the absence of LOX expression, with no organoids formed following shLOX induction (p<0.0001).

<b>Figure 2: LOX silencing alters markers of cell proliferation and differentiation. </b>(A) Organoids grown in the absence of LOX demonstrated decreased mRNA expression of differentiation markers filaggrin (<i>p</i><0.0001) and loricrin (<i>p</i><0.0001). (B) Histologically, organoids showed increased basaloid content, as well as decreased Ki67 and IVL expression.

Figure 2: LOX silencing alters markers of cell proliferation and differentiation. (A) Organoids grown in the absence of LOX demonstrated decreased mRNA expression of differentiation markers filaggrin (p<0.0001) and loricrin (p<0.0001). (B) Histologically, organoids showed increased basaloid content, as well as decreased Ki67 and IVL expression.

Ritsu Nakagawa

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