Background Alterations to epithelial tight junctions can compromise the ability of the epithelium to act as a barrier between luminal contents and the underlying tissues, thereby increasing intestinal permeability, an early critical event in inflammatory bowel disease (IBD). Tofacitinib (Xeljanz), an orally administered pan-Janus kinase (JAK) inhibitor, was recently approved for the treatment of moderate to severe ulcerative colitis. Nevertheless, the effects of tofacitinib on intestinal epithelial cell functions are largely unknown. The aim of this study was to determine if JAK inhibition by tofacitinib can rescue cytokine-induced barrier dysfunction in intestinal epithelial cells (IECs). Methods T84 IECs were used to evaluate the effects of tofacitinib on JAK-signal transducer and activator of transcription (STAT) activation, barrier permeability, and expression and localization of tight junction proteins. The impact of tofacitinib on claudin-2 promoter activity was assessed in HT-29 IECs. Tofacitinib rescue of barrier function was also tested in human colonic stem cell-derived organoids. Results Pretreatment with tofacitinib prevented IFN-γ-induced decreases in transepithelial electrical resistance (TER) and increases in 4 kDa FITC-dextran permeability (FD4), partly due to claudin-2 transcriptional regulation and restriction of ZO-1 rearrangement at tight junctions. Although tofacitinib administered after IFN-γ challenge only partially normalized TER and claudin-2 levels, FD4 permeability and ZO-1 localization were fully recovered. The IFN-γ-induced FD4 permeability in primary human colonoids was fully rescued by tofacitinib. Conclusions These data suggest differential therapeutic efficacy of tofacitinib in the rescue of pore vs leak-tight junction barrier defects and indicate a potential contribution of improved epithelial barrier function to the beneficial effects of tofacitinib in IBD patients.
BACKGROUND: Exposure to plastic-associated endocrine disrupting chemicals (EDCs) has been associated with an increased risk of cardiovascular disease (CVD) in humans. However, the underlying mechanisms for this association are unclear. Many EDCs have been shown to function as ligands of the nuclear receptor pregnane X receptor (PXR), which functions as xenobiotic sensor but also has pro-atherogenic effects in vivo. OBJECTIVE: We sought to investigate the contribution of PXR to the adverse effects dicyclohexyl phthalate (DCHP), a widely used phthalate plasticizer, on lipid homeostasis and CVD risk factors. METHODS: Cell-based assays, primary organoid cultures, and PXR conditional knockout and PXR-humanized mouse models were used to investigate the impact of DCHP exposure on PXR activation and lipid homeostasis in vitro and in vivo. Targeted lipidomics were performed to measure circulating ceramides, novel predictors for CVD. RESULTS: DCHP was identified as a potent PXR-selective agonist that led to higher plasma cholesterol levels in wild-type mice. DCHP was then demonstrated to activate intestinal PXR to elicit hyperlipidemia by using tissue-specific PXR-deficient mice. Interestingly, DCHP exposure also led to higher circulating ceramides in a PXR-dependent manner. DCHP-mediated PXR activation stimulated the expression of intestinal genes mediating lipogenesis and ceramide synthesis. Given that PXR exhibits considerable species-specific differences in receptor pharmacology, PXR-humanized mice were also used to replicate these findings. DISCUSSION: Although the adverse health effects of several well-known phthalates have attracted considerable attention, little is known about the potential impact of DCHP on human health. Our studies demonstrate that DCHP activated PXR to induce hypercholesterolemia and ceramide production in mice. These results indicate a potentially important role of PXR in contributing to the deleterious effects of plastic-associated EDCs on cardiovascular health in humans. Testing PXR activation should be considered for risk assessment of phthalates and other EDCs.
Rationale: Macrophages are the frontline immune cells in response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Angiotensin-converting enzyme 2 (ACE2) serves as the binding receptor to SARS-CoV-2 Spike glycoprotein for fusion and internalization into the human host cells. However, the mechanisms underlying SARS-CoV-2-elicited macrophage inflammatory responses remain elusive. Neutralizing SARS-CoV-2 by human ACE2 (hACE2) decoys has been proposed as a therapeutic approach to ameliorate SARS-CoV-2-stimulated inflammation. This study aims to investigate whether an engineered decoy receptor can abrogate SARS-CoV-2-induced macrophage inflammation. Methods: hACE2 was biotinylated to the surface of nano-liposomes (d = 100 nm) to generate Liposome-human ACE2 complex (Lipo-hACE2). Lentivirus expressing Spike protein (D614G) was also created as a pseudo-SARS-CoV-2 (Lenti-Spike). Liposome-hACE2 was used as a decoy receptor or competitive inhibitor to inhibit SARS-CoV-2 or Lenti-Spike-induced macrophage inflammation in vitro and in vivo . Results: Both SARS-CoV-2 and Lenti-Spike stimulated strong inflammatory responses by inducing the expression of key cytokine and chemokines, including IL-1β, IL-6, TNFα, CCL-2, and CXCL-10, in murine and human macrophages in vitro , whereas Lipo-hACE2 decoy abolished these effects in macrophages. Furthermore, intravenous injection of Lenti-Spike led to increased macrophage and tissue inflammation in wild type mice, which was also abolished by Lipo-hACE2 treatment. Mechanistically, Spike protein stimulated macrophage inflammation by activating canonical NF-κB signaling. RNA sequencing analysis revealed that Lenti-Spike induced over 2,000 differentially expressed genes (DEGs) in murine macrophages, but deficiency of IκB kinase β (IKKβ), a key regulator for NF-κB activation, abrogated Lenti-Spike-elicited macrophage inflammatory responses. Conclusions: We demonstrated that the engineered Lipo-hACE2 acts as a molecular decoy to neutralize SARS-CoV-2 or Spike protein-induced inflammation in both murine and human macrophages, and activation of the canonical IKKβ/NF-κB signaling is essential for SARS-CoV-2-elicited macrophage inflammatory responses.
The intracellular distribution of Auger electron-emitting radiopharmaceuticals is a determinant of cytotoxicity. However, the mechanisms by which these agents are routed through the cell are ill understood. The aim of this study was to investigate how trafficking of 111 In-labeled human epidermal growth factor ( 111 In-DTPA-hEGF) relates to that of the EGF receptor (EGFR) and whether coadministration of agents that modulate EGFR signaling alters the efficacy of 111 In-DTPA-hEGF. Methods: The spatiotemporal interaction between AlexaFluor488-EGF (AF488-EGF) and Cy3-conjugated anti-EGFR antibody (Cy3-anti-EGFR) was studied in the breast cancer cell line MDA-MB-468 using fluorescence resonance energy transfer and 2-photon fluorescence lifetime imaging. 111 In internalization and nuclear fractionation assays were performed to investigate the effect of the ErbB-2-blocking antibody trastuzumab and a prenyltransferase inhibitor, L-778,123, on the subcellular localization of 111 In-DTPA-hEGF in MDA-MB-468 (1.3 · 10 6 EGFR per cell; ErbB-2 negative) and 231-H2N (0.2 · 10 6 EGFR per cell; 0.4 · 10 5 ErbB-2 per cell) cell lines. The cytotoxicity of 111 In-DTPA-hEGF (0-64 nM) plus trastuzumab (0-50 mg/mL) or L-778,123 (0-22.5 mM) was measured using clonogenic assays in a panel of breast cancer cell lines that express different levels of EGFR and ErB-2. Clonogenic survival data were used to calculate combination indices. Tumor growth inhibition was measured in vivo in 231-H2N xenograft-bearing mice treated with 111 In-DTPA-hEGF plus trastuzumab or L-788,123. Results: Using fluorescence resonance energy transfer, we showed that EGF interacts with EGFR in the cytoplasm and nucleus after internalization of the ligand-receptor complex in MDA-MB-468 cells. Nuclear localization of 111 In-DTPA-hEGF is enhanced by trastuzumab and L-788,123. Trastuzumab and L-788,123 sensitized 231-H2N cells to 111 In-DTPA-hEGF. Nuclear localization and cytotoxicity of 111 In-DTPA-hEGF were significantly increased in 231-H2N xenografts by cotreatment with L-788,123 (P , 0.0001). Conclusion: The therapeutic efficacy of 111 In-DTPA-hEGF is increased through the coadministration of selected molecularly targeted drugs that modulate EGFR signaling and trafficking.
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