The Effects of Shiga Toxin 1, 2 and Their Subunits on Cytokine and Chemokine Expression by Human Macrophage-Like THP-1 Cells

Abstract

Infection by Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC) results in severe diarrhea, hemorrhagic colitis, and, occasionally, hemolytic-uremic syndrome (HUS). HUS is associated with an increase in pro-inflammatory cytokines and chemokines, many of which are produced by macrophages in the kidneys, indicating that localized host innate immunity likely plays a role in renal pathogenesis. EHEC serotypes may express one or two classes of serologically defined but structurally and functionally-related Shiga toxins called Stx1 and Stx2. Of these, Stx2 appears to be linked to higher rates of HUS than Stx1. To investigate a possible reason for this, we exposed human macrophage-like THP-1 cells to Stx1 or Stx2 and then used the Luminex multiplex system to assess cytokine/chemokine concentrations in culture supernatant solutions. This analysis revealed that, relative to Stx1, Stx2 significantly caused increased expression of GRO, G-CSF, IL-1β, IL-8 and TNFα in macrophage-like THP-1 cells. This was determined to not be due to a difference in cytotoxicity since both Stx1 and Stx2 displayed similar cytotoxic activities on macrophage-like THP-1 cells. These observations indicate that, in vitro, Stx2 can provoke a greater pro-inflammatory response than Stx1 in macrophages and provides a possible partial explanation for higher rates of HUS in patients infected with EHEC strains expressing Stx2. To begin to determine a mechanism for Shiga toxin-mediated cytokine production, we exposed macrophage-like THP-1 cells to Stx1 or Stx2 A and B subunits. Luminex analysis of cytokines in cell culture supernatant solutions demonstrated that neither subunit alone induced a cytokine response in THP-1 cells.

Role of Shiga Toxins in Cytotoxicity and Immunomodulatory Effects of Escherichia coli O157:H7 during Host-Bacterial Interactions in vitro

“…Previous studies revealed that macrophages derived from THP-1 express Gb3 on their membrane [33,34]. As consequence, these cells are able to respond to Stx1/Stx2 [35,36], inducing cytokine/chemokine production, ribotoxic stress, and death of macrophages [22]. Thus, to further elucidate the Stx2-dependent mechanism responsible for macrophage death, we treated cultures with an anti-Stx2 neutralizing antibody since the beginning of 125/99 infection [37].…”

“…Cytokine secretion by macrophages is one of the major inflammatory responses upon bacterial stimulus. In this regard, TNF-α and IL-1β are the earliest secreted ones with special impact on EHEC pathogenesis [22], because they significantly enhance the toxin-specific glycolipid receptor (Gb3) on the endothelium and Stx2-sensitivity [23]. Thus, we assayed IL-1β levels in the supernatants of macrophages infected with 125/99.…”

Augmenting the Detection of Verotoxin-Producing Escherichia coli Through use of Fluorescently-labeled DNA Oligonucleotides

“…It has been suggested that VT2 induces a greater expression of signalling molecules, leading to more severe disease progression in humans. VT1 and VT2 were shown to have the same level of toxicity and that A and B subunits did not induce the expression on their own (Brandelli et al, 2015). However, in macrophage-like THP-1 cells treated with VT2, 14 of the 40 cytokines or chemokines monitored showed significantly increased levels including pro-inflammatory, immune cell proliferating, growth-promoting factors and other chemokines (Brandelli et al, 2015).…”

“…However, in macrophage-like THP-1 cells treated with VT2, 14 of the 40 cytokines or chemokines monitored showed significantly increased levels including pro-inflammatory, immune cell proliferating, growth-promoting factors and other chemokines (Brandelli et al, 2015). These factors have been associated with HUS suggesting that VT2 may play a greater role than VT1 in enhancing macrophage infiltration and fibrin deposition (Brandelli et al, 2015;Fu et al, 2004). Also, both TNF-ɑ and IL-1β, as well as a few other cytokines to a lesser extent, induce Gb3 expression and may contribute to greater impact of the toxin at target organs (van der Kar et al, 1992).…”

“…VT1 and VT2 were shown to have the same level of toxicity and that A and B subunits did not induce the expression on their own (Brandelli et al, 2015). However, in macrophage-like THP-1 cells treated with VT2, 14 of the 40 cytokines or chemokines monitored showed significantly increased levels including pro-inflammatory, immune cell proliferating, growth-promoting factors and other chemokines (Brandelli et al, 2015). These factors have been associated with HUS suggesting that VT2 may play a greater role than VT1 in enhancing macrophage infiltration and fibrin deposition (Brandelli et al, 2015;Fu et al, 2004).…”