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

Abstract

Introduction. Clostridioides difficile 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. Hypothesis statement. 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. Aim. 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. Methodology. 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. Results. 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. Conclusion. Our results showed that C. difficile toxins, including CDT, enhanced macrophage-mediated CXCL2 and TNF-α production via TLR2 and TLR4, indicating that CDT affects host immune responses.

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

“…CDI typically occurs after the use of antibiotics owing to the disruption of the normal gut microbiota, allowing for the overgrowth of C. difficile . Toxin A (TcdA) and toxin B (TcdB) are major exotoxins secreted by C. difficile and they play a key role in the pathogenesis of CDI by inducing macrophage activation, pro-inflammatory cytokine release, neutrophil infiltration, and epithelial cell damage [ 4–6 ]. An inflammatory cascade is responsible for many aspects of the pathology associated with C. difficile- associated diarrhea [ 7 ].…”

Adopting orphan receptors: group IX/X transition metals as potential ligands of zebrafish Tlr4 homologs

“…HEK293T cells do not normally express TLR4 but can be rendered responsive to TLR4 agonists when transfected with human TLR4 and MD-2 – they have been shown to upregulate proinflammatory gene expression in response to known TLR4 agonists (Babolmorad et al, 2021; Chow et al, 1999; Domingo et al, 2023; Medvedev & Vogel, 2003; Yang et al, 2000). IL-8 has been established by numerous studies as a marker for TLR4 activation making HEK293T cells an appropriate in vitro cell model to assess TLR4 function (Kurt-Jones et al, 2000; McKee et al, 2021; Oblak et al, 2015; Potnis et al, 2013; Quevedo-Diaz et al, 2010; Schmidt et al, 2010).…”

Effect of Doxycycline in Decreasing the Severity of Clostridioides difficile Infection in Mice

“…Doxycycline had been regarded as an antibiotic with anti-inflammatory, antioxidant, anti-apoptotic, and immunomodulatory effects [ 13 ]. Doxycycline may be beneficial for C. difficile -induced inflammation, which was evidenced by reports that C. difficile toxin could enhance the production of MIP-2 and TNF-α in macrophages to drive colonic inflammation, and the anti-inflammatory effect of doxycycline can enhance its therapeutic role in acute CDI [ 38 , 39 , 40 , 41 ]. In the polymicrobial infection sepsis mice model, doxycycline could ameliorate systemic and pulmonary inflammation through reducing the plasma and lung pro-inflammatory cytokines [ 40 ].…”