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Clostridioides difficile toxins enhanced the in vitro production of CXC chemokine ligand 2 and tumor necrosis factor-α via Toll-like receptors in macrophages

McKee, HK;Kajiwara, C;Yamaguchi, T;Ishii, Y;Shimizu, N;Ohara, A;Tateda, K;

 infection (CDI) causes toxin-mediated enteropathy, such as antibiotic-associated diarrhoea and pseudomembranous colitis. Rho-glucosylating toxin A (TcdA) and toxin B (TcdB) have been clearly implicated in pathogenesis, whereas the virulence of binary toxin (CDT) is still debated.

 We hypothesized that CDT is involved in the host immune response and plays a pivotal role in establishing virulence by modulating pro-inflammatory cytokine production; this is achieved through the integral Toll-like receptor (TLR) signalling pathways.

 The aim of the present study was to determine whether and how CDT impacts macrophages compared to TcdA or TcdB by examining the induction of CXC chemokine ligand 2 (CXCL2) and tumour necrosis factor-α (TNF-α), both of which are crucial in mediating local and systematic inflammatory responses.

 RAW264.7 cells or transfected human embryonic kidney (HEK) 293 T cells were incubated with TcdA, TcdB, or CDT. In some experiments, a neutralizing antibody against TLR2 or TLR4, or myeloid differentiation 88 inhibitory peptide were added. The amount of CXCL2 and TNF-α secreted was then measured.

 In RAW264.7 macrophages, CXCL2 and TNF-α were produced via the Toll-like receptor 2 (TLR2) or Toll-like receptor 4 (TLR4) pathway in a TcdA, TcdB, or CDT dose-dependent manner. Interleukin-8 secretion was induced in TLR4/MD2/CD14-transfected, but not in TLR2-transfected, HEK 293 T cells following TcdB or CDT exposure.

 Our results showed that toxins, including CDT, enhanced macrophage-mediated CXCL2 and TNF-α production via TLR2 and TLR4, indicating that CDT affects host immune responses.