RESUMO
BACKGROUND: Clostridium difficile infection (CDI) is a debilitating nosocomial infection. C. difficile produces toxins A and B, which cause inflammation. Existing therapies have issues with recurrence, cost, and safety. We aim to discover a safe, effective, and economical non-microbiological therapeutic approach against CDI. METHODS: We included human primary peripheral blood mononuclear cells (PBMCs), fresh human colonic explants, and humanized HuCD34-NCG mice. Toxin A+B+ VPI10463 and A-B+ ribotype 017 C. difficile strains were used. We used single-cell RNA profiling and high-throughput screening to find actionable toxin B-dependent pathways in PBMCs. RESULTS: Histamine 1 receptor-related drugs were found among the hit compounds that reversed toxin-mediated macrophage inflammatory protein one alpha (MIP-1α) expression in PBMCs. We identified Loratadine as the safest representative antihistamine for therapeutic development. Loratadine inhibited toxin B-induced MIP-1α secretion in fresh human colonic tissues. Oral Loratadine (10 mg/kg/day) maintained survival, inhibited intestinal Ccl3 mRNA expression, and prevented vancomycin-associated recurrence in the VPI10463-infected mice and ribotype 017-infected hamsters. Splenocytes from Loratadine-treated mice conferred anti-inflammatory effects to the VPI10463-infected T/B cell-deficient Rag-/- mice. Oral Loratadine suppressed human MIP-1α expression in monocytes/macrophages in toxin B-expressing ribotype 017-infected humanized HuCD34-NCG mice. CONCLUSIONS: Loratadine may be repurposed to optimize existing therapies against CDI.
Loratadine is an FDA-approved antihistamine that inhibits toxin B-mediated pro-inflammatory macrophage inflammatory protein one alpha secretion from immune cells. The anti-inflammatory effect of Loratadine ameliorates intestinal inflammation in C. difficile-infected animals. Loratadine may be repurposed to optimize existing therapies.
RESUMO
BACKGROUND: Clostridioides difficile infection (CDI) is a debilitating nosocomial disease. Postmenopausal women may have an increased risk of CDI, suggesting estrogen influence. Soybean products contain a representative estrogenic isoflavone, genistein. METHODS: The anti-inflammatory and antiapoptotic effects of genistein were determined using primary human cells and fresh colonic tissues. The effects of oral genistein therapy among mice and hamsters were evaluated. RESULTS: Within 10 days of CDI, female c57BL/6J mice in a standard environment (regular diet) had a 50% survival rate, while those with estrogen depletion and in an isoflavone-free environment (soy-free diet) had a 25% survival rate. Oral genistein improved their 10-day survival rate to 100% on a regular diet and 75% in an isoflavone-free environment. Genistein reduced macrophage inflammatory protein-1α (MIP-1α) secretion in fresh human colonic tissues exposed to toxins. Genistein inhibited MIP-1α secretion in primary human peripheral blood mononuclear cells, abolished apoptosis and BCL-2-associated X (BAX) expression in human colonic epithelial cells, and activated lysine-deficient protein kinase 1 (WNK1) phosphorylation in both cell types. The anti-inflammatory and antiapoptotic effects of genistein were abolished by inhibiting estrogen receptors and WNK1. CONCLUSIONS: Genistein reduces CDI disease activity by inhibiting proinflammatory cytokine expression and apoptosis via the estrogen receptor/G-protein estrogen receptor/WNK1 pathways.
Assuntos
Infecções por Clostridium , Isoflavonas , Feminino , Humanos , Camundongos , Animais , Genisteína/farmacologia , Receptores de Estrogênio/metabolismo , Lisina , Quimiocina CCL3 , Leucócitos Mononucleares/metabolismo , Isoflavonas/farmacologia , Estrogênios , Infecções por Clostridium/tratamento farmacológico , Proteínas QuinasesRESUMO
Epithelial cell apoptosis and compromised gut barrier function are features of inflammatory bowel disease. ADS024 is a single-strain live biotherapeutic product (LBP) of Bacillus velezensis under development for treating ulcerative colitis (UC). The cytoprotective effects of the sterile filtrate of ADS024's secreted products on UC patient-derived colonic tissues, human primary colonic epithelial cells (HPEC), and human colonic epithelial T84 cells were evaluated. ADS024 filtrate significantly inhibited apoptosis and inflammation with reduced Bcl-2 Associated X-protein (BAX) and tumor necrosis factor (TNF) mRNA expression in fresh colonic explants from UC patients. Exposure to UC patient-derived serum exosomes (UCSE) induced apoptosis with increased cleaved caspase 3 protein expression in HPECs. ADS024 filtrate diminished the UCSE-mediated apoptosis by inhibiting cleaved caspase 3. TNFα and interferon-gamma (IFNγ) damaged epithelial barrier integrity with reduced transepithelial electrical resistance (TEER). ADS024 filtrate partially attenuated the TEER reduction and restored tight junction protein 1 (TJP1) expression. Oral live ADS024 treatment reduced weight loss, disease activity, colonic mucosal injury, and colonic expression of interleukin 6 (IL-6) and TNFα in dextran sodium sulfate (DSS)-treated mice with colitis. Thus, ADS024 may protect the colonic epithelial barrier in UC via anti-inflammatory, anti-apoptotic, and tight-junction protection mechanisms.