Enterohemorrhagic Escherichia coli specific enterohemolysin induced IL-1β in human macrophages and EHEC-induced IL-1β required activation of NLRP3 inflammasome - PubMed (original) (raw)

Enterohemorrhagic Escherichia coli specific enterohemolysin induced IL-1β in human macrophages and EHEC-induced IL-1β required activation of NLRP3 inflammasome

Xiaoai Zhang et al. PLoS One. 2012.

Abstract

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major foodborne pathogen causing hemorrhagic colitis and hemolytic-uremic syndrome. The role of EHEC O157:H7-enterohemolysin (Ehx) in the pathogenesis of infections remains poorly defined. In this study, we used gene deletion and complement methods to confirm its putative functions. Results demonstrated that, in THP-1 cells, EHEC O157:H7-Ehx is associated with greater production of extracellular interleukin (IL)-1β than other cytokines. The data also showed that EHEC O157:H7-Ehx contributed to cytotoxicity in THP-1 cells, causing the release of lactate dehydrogenase (LDH). Although we observed a positive correlation between IL-1β production and cytotoxicity in THP-1 cells infected with different EHEC O157:H7 strains, our immunoblot results showed that the majority of IL-1β in the supernatant was mature IL-1β and not the pro-IL-1β that can be released after cell death. However, EHEC O157:H7-Ehx had no detectable effect on biologically inactive pro-IL-1β at the mRNA or protein synthesis levels. Neither did it affect the expression of apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, or NOD-like receptor family pyrin domain containing 3 (NLRP3). RNA interference experiments showed that EHEC O157:H7-induced IL-1β production required the involvement of ASC, caspase-1, and NLRP3 expression in THP-1 cells. Our results demonstrate that Ehx plays a crucial role in EHEC O157:H7-induced IL-1β production and its cytotoxicity to THP-1 cells. NLRP3 inflammasome activation is also involved in EHEC O157:H7-stimulated IL-1β release.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Construction of the mutant strains.

(A) The genes ehx and ecf were detected in EDL933 but not in ΔpO157. Lane M: marker; Lanes 1 and 3: EDL933; Lanes 2 and 4: ΔpO157 mutant. (B) Comparison of genomic DNA of EDL933 and ΔpO157 using PFGE of Xba_I-digested. Lane M: marker; Lane 1: EDL933; Lane 2: ΔpO157 mutant. (C) Using primer ehxA_-3,4, EDL933 was amplified as a ≈3.3 kb fragment. Δ_ehxA and Δ_ehxA/pehxA were amplified as a reduced fragment to ≈1.6 kb. Using primer ehxA-5,6, Δ_ehxA showed no PCR product. EDL933 and Δ_ehxA/pehxA were amplified as a ≈360 bp fragment. Lane 1: EDL933; Lane 2: Δ_ehxA_; Lane 3: Δ_ehxA_/pehxA.

Figure 2

Figure 2. Cytotoxicity of human macrophages as indicated by the release of lactate dehydrogenase (LDH).

Differentiated THP-1 cells were exposed to different bacterial strains (EDL933, ΔpO157, Δ_ehxA_, Δ_ehxA_/pehxA) for 2 and 4 h. The release of LDH was assessed at specific times during incubation. Data are shown as mean ± S.D. of experiments performed in triplicate. Significant differences (* _P<_0.05) are indicated. n.s., no significant differences (_P_>0.05).

Figure 3

Figure 3. Effects of EHEC O157:H7 enterohemolysin on the production of IL-1β.

Differentiated THP-1 cells were infected with EDL933, ΔpO157, Δ_ehxA_, Δ_ehxA_/pehxA, and LPS for 2 or 4 h. Concentrations of interleukin (IL)-1β, IL-6, IL-8, chemokine CC motif ligand 5 (RANETS/CCL5), monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), and Interferon-gamma (IFN-γ) were measured using ELISA. Values are expressed as mean ± S.D. of triplicate experiments. Significant differences (* P<0.05) were indicated. n.s., no significant differences (_P_>0.05).

Figure 4

Figure 4. Pro-IL-1β and mature IL-1β in cell extract and supernatant as visualized by Western blotting.

At 4 h after infection, pro-IL-1β and IL-1β in cell extracts (CX) and supernatants (SN) were visualized by Western blot analysis.

Figure 5

Figure 5. Roles of caspase-1, apoptosis-associated speck-like protein containing a CARD (ASC), and the NOD-like receptor family pyrin domain containing 3 (NLRP3) in EHEC O157:H7-induced IL-1β production.

THP-1 cells were transfected with control siRNA or siRNA specific to caspase-1, ASC, or NLRP3, respectively. After 48 h, cells were infected with EDL933, Δ_ehxA_, ΔpO157, and Δ_ehxA_/pehxA, respectively. (A) Knockdown of caspase-1, ASC, and NLRP3, was assayed by Western blotting. (B) Cell culture supernatants were collected 4 h after infection and subjected to IL-1β ELISA. Results represent the mean ± S.D. of three independent experiments. Significant differences (**p<0.01, *_P_<0.05) were indicated. n.s., no significant differences (_P_>0.05).

Figure 6

Figure 6. Expression of inflammasome components in differentiated THP-1 cells.

Differentiated THP-1 cells were left untreated or were infected with EDL933 or Δ_ehxA_. They were then lysed over 4 h postinfection. mRNA expression of selected genes was analyzed using RT-PCR.

Figure 7

Figure 7. Correlation between the release of LDH and concentration of IL-1β in THP-1 cells infected with EHEC O157:H7.

A significant positive correlation was observed (P<0.01).

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Grants and funding

This work was supported by grants (2011CB504901, 2008ZX10004-008, 2008ZX10004-009, 2009ZX10004-203, 2011SKLID203, and 2008SKLID106) from the Ministry of Science and Technology and State Key Laboratory for Infectious Disease Prevention and Control, People’s Republic of China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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