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SINGLE-CELL RNA PROFILING AND HIGH-THROUGHPUT SCREENING DISCOVERED MACROPHAGE INFLAMMATORY PROTEIN 1 ALPHA AND HISTAMINE 1 RECEPTOR AS KEY PATHWAYS IN C. DIFFICILE INFECTION.

Date
May 18, 2024

Background: Clostridium difficile infection (CDI) is a debilitating nosocomial infection. C. difficile produces toxins A and B, which cause inflammation, leading to diarrhea and intestinal injury. Toxin B was found to be a crucial pathogenic mediator in CDI. Existing therapies have various concerns regarding efficacy, cost, and safety. The aim was to discover new key pathways and non-microbiological therapeutic approaches against CDI.
Methods: Human primary peripheral blood mononuclear cells (PBMC), fresh human colonic explants, humanized HuCD34-NCG mice, and hamsters were treated with toxin A+B+ VPI10463 and toxin A-B+ ribotype 017 C. difficile strains. Single-cell RNA profiling and high-throughput screening were used to analyze key pathways against toxin B-mediated inflammatory responses in PBMCs.
Results: Single-cell RNA sequencing discovered that PBMCs showed increased mRNA expression of pro-inflammatory cytokines, including macrophage inflammatory protein 1 alpha (MIP-1α), after 6-hour incubation with toxin B with PBMCs. Through high-throughput screening, Histamine 1 receptor-related drugs were found to reduce toxin B-mediated MIP-1α secretion in PBMCs compared to positive controls. Through the literature review, Loratadine was identified as the antihistamine with the least adverse effects. Loratadine (30µM) inhibited toxin B-induced MIP-1α secretion from fresh human colonic tissues and PBMCs. Loratadine did not affect toxin A-induced MIP-1α secretion from PBMCs. Comparatively, two common H1R-related antihistamines, Cetirizine and Fexofenadine, did not inhibit toxin B-mediated MIP-1α secretion in PBMCs. Loratadine inhibited toxin B-mediated MIP-1α secretion by inhibiting phosphorylation of nuclear factor kappa B (NFkB). Oral Loratadine (10 mg/kg/day) maintained survival, inhibited colonic Ccl3 mRNA expression, and ameliorated acute colitis in VPI10463-infected mice. Splenocyte transplantation from Loratadine-treated mice conferred anti-inflammatory effects on the VPI10463-infected T/B cell-deficient Rag-/- mice, suggesting the involvement of immune cells. Oral Loratadine administration suppressed human MIP-1α expression in colonic monocytes/macrophages in toxin B-expressing ribotype 017-infected humanized HuCD34-NCG mice. Oral Loratadine also increased circulating CD8 CD45RA naïve cytotoxic T cells in the infected HuCD34-NCG mice. Similarly, oral Loratadine protected ribotype 017-infected hamsters with reduced cecal MIP-1α levels. Loratadine prevented vancomycin-associated CDI recurrence in infected mice and hamsters without affecting C. difficile abundance and toxin production.
Conclusions: Loratadine is a potential therapeutic for CDI by inhibiting toxin B-dependent MIP-1α secretion from PBMCs via NF-κB phosphorylation inhibition. It promotes anti-inflammatory effects and increases in naïve cytotoxic T cells in CDI animal models.

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