Vitamin D₃and carbamazepine protect againstClostridioides difficileinfection in mice by restoring macrophage lysosome acidification

Abstract

Clostridioides difficile infection (CDI) is a common cause of nosocomial diarrhea. TcdB is a major C. difficile exotoxin that activates macrophages to promote inflammation and epithelial damage. Lysosome impairment is a known trigger for inflammation. Herein, we hypothesize that TcdB could impair macrophage lysosomal function to mediate inflammation during CDI. Effects of TcdB on lysosomal function and the downstream pro-inflammatory SQSTM1/p62-NFKB (nuclear factor kappa B) signaling were assessed in cultured macrophages and in a murine CDI model. Protective effects of two lysosome activators (i.e., vitamin D 3 and carbamazepine) were assessed. Results showed that TcdB inhibited CTNNB1/β-catenin activity to downregulate MITF (melanocyte inducing transcription factor) and its direct target genes encoding components of lysosomal membrane vacuolar-type ATPase, thereby suppressing lysosome acidification in macrophages. The resulting lysosomal dysfunction then impaired autophagic flux and activated SQSTM1-NFKB signaling to drive the expression of IL1B/IL-1β (interleukin 1 beta), IL8 and CXCL2 (chemokine (C-X-C motif) ligand 2). Restoring MITF function by enforced MITF expression or restoring lysosome acidification with 1α,25-dihydroxyvitamin D 3 or carbamazepine suppressed pro-inflammatory cytokine expression in vitro . In mice, gavage with TcdB-hyperproducing C. difficile or injection of TcdB into ligated colon segments caused prominent MITF downregulation in macrophages. Vitamin D 3 and carbamazepine lessened TcdB-induced lysosomal dysfunction, inflammation and histological damage. In conclusion, TcdB inhibits the CTNNB1-MITF axis to suppress lysosome acidification and activates the downstream SQSTM1-NFKB signaling in macrophages during CDI. Vitamin D 3 and carbamazepine protect against CDI by restoring MITF expression and lysosomal function in mice. Abbreviations: ATP6V0B: ATPase H+ transporting V0 subunit b; ATP6V0C: ATPase H+ transporting V0 subunit c; ATP6V0E1: ATPase H+ transporting V0 subunit e1; ATP6V1H: ATPase H+ transporting V1 subunit H; CBZ: carbamazepine; CDI: C. difficile infection; CXCL: chemokine C-X-X motif ligand; IL: interleukin; LAMP1: lysosomal-associated membrane protein 1; LC3: microtubule-associated protein 1 light chain 3; LEF: lymphoid enhancer binding factor 1; MITF: melanocyte inducing transcription factor; NFKB: nuclear factor kappa B; PMA: phorbol 12-myristate 13-acetate; TcdA: Clostridial toxin A; TcdB: Clostridial toxin B; TFE3: transcription factor E3; TFEB: transcription factor EB.

Glucose deprivation-reperfusion induces labile iron overload and ferroptosis in renal tubules through V-ATPase-mTOR axis-mediated ferritinophagy and iron release by TPC2

“…Next, to directly determine the relationship between LIP levels and V-ATPase function, we tested the labile iron in HK2 cells after GD-R, with or without carbamazepine (CBZ), a lysosomal acidi cation activator which can restore V-ATPase function (29). As expected, CBZ improved lysosomal acidi cation after GD-R, con rming the restoration of V-ATPase function after treatment of CBZ (Figure 2I and 2J).…”

A Study of the Resistance of Hu Sheep Lambs to Escherichia coli F17 Based on Whole Genome Sequencing

“…TcdB is an exotoxin of Clostridioides difficile, which induces macrophage inflammation and epithelial damage, and destroys the normal function of lysosomes in macrophages. TcdB inhibits CTNNB1/β-catenin activity and lysosomal acidification in macrophages; the resulting lysosomal dysfunction affects the inflammatory response and causes tissue damage through autophagy [ 31 ]. It has been reported that mutations at the upper site of CTNNB1 are associated with tuberculosis caused by a conjugated divergent bacterium; this study also showed that CTNNB1 can affect host susceptibility to a conjugated divergent bacterium and this can be used as a marker gene to identify tuberculosis [ 32 ].…”

Rethinking of Alzheimer's disease: Lysosomal overloading and dietary therapy

“…However it is also an immunosuppressant and may increase a risk of infection for the old people. Carbamazepine an mTOR (mechanistic target of rapamycin kinase)-independent autophagy stimulator, prevented the compromised autophagic flux in septic mouse liver (Lin et al, 2014 ) and restored the mitophagic flux caused by a decline in lysosomal acidification (Ebrahimi-Fakhari et al, 2016 ; Chan et al, 2022 ). However, it is an antiepileptic drug and may not be applicable to all AD patients.…”

“…Vitamins may also be supplied in the ketogenic diet. Vitamin D 3 (VD 3 ) promoted lysosomal degradation through inducing the nuclear translocation of PDIA3 (protein disulfide isomerase family A member 3) - STAT3 (signal transducer and activator of transcription 3) protein-complex and up-regulated the MCOLN3 (mucolipin 3) channel subsequently, which resulted in Ca 2+ releasing from the lysosome and the normal lysosomal acidification (Hu et al, 2019 ; Chan et al, 2022 ). VD 3 and VE and their combination improved memory and learning deficit, and decreased neuronal loss and oxidative stress in a rat model of AD (Mehrabadi and Sadr, 2020 ).…”