Nod1, but not the ASC inflammasome, contributes to induction of IL-1β secretion in human trophoblasts after sensing of Chlamydia trachomatis - PubMed (original) (raw)

Nod1, but not the ASC inflammasome, contributes to induction of IL-1β secretion in human trophoblasts after sensing of Chlamydia trachomatis

P B Kavathas et al. Mucosal Immunol. 2013 Mar.

Abstract

Chlamydia trachomatis (Ct) is an obligate intracellular bacterial pathogen. Previously, we showed that infection of human trophoblast cells by Ct triggers the secretion of the pro-inflammatory cytokine, interleukin (IL)-1β. The aim of this study was to understand the innate immune pathways involved in trophoblast production of IL-1β after Ct infection. The approach we took was to inhibit the expression or function of the key Toll-like receptors (TLRs), Nod-like receptors, and inflammasome components that have been associated with chlamydia infection. In this study, we report that Ct-induced trophoblast IL-1β secretion is associated with the transcription of IL-1β mRNA, the translation and processing of pro-IL-1β, and the activation of caspase-1. In addition, we demonstrate that Ct-induced IL-1β production and secretion by the trophoblast is independent of TLR2, TLR4, MyD88, and the Nalp3/ASC inflammasome. Instead we report, for the first time, the importance of Nod1 for mediating trophoblast IL-1β secretion in response to a Ct infection.

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Conflict of interest statement

The authors have no financial conflicts of interest.

Figures

Figure 1

Figure 1. Chlamydia infection of human trophoblast cell lines leads to induction of IL-1β expression and IL-1β processing

Trophoblast cells were either non-infected (NI) or infected with Chlamydia (Ct), after which supernatants were collected and either RNA or protein extracted from cells. (a) Ct infection of HTR8 cells significantly induced IL-1β mRNA expression after 24hrs as determined by qRT-PCR; and (b) significantly induced IL-1β secretion after 48hrs as determined by ELISA. (c) Ct infection of Sw.71 cells significantly induced IL-1β secretion after 36hrs (n=3; *p<0.05 relative to the NI control). Cell lysates from (d) HTR8 and (e) Sw.71 cells after either NI or infection with Ct were evaluated for pro-IL-1β (31kDa), active IL-1β (17kDa) and active caspase-1 (20kDa) expression by Western blot (representative blots are shown). Barcharts show quantification of protein expression as determined by densitometry and normalized to β-actin (n=3; *p<0.05 relative to the NI control). (f) Cell supernatants from Sw.71 cells either NI or infected with Ct for 36hrs were evaluated for pro- and active IL-1β and active caspase-1 expression by Western blot. Barcharts show quantification of protein expression as determined by densitometry and normalized to β-actin (n=4; *p<0.05 relative to the NI control).

Figure 2

Figure 2. _Chlamydia_-induced trophoblast pro-IL-1β expression and secretion of IL-1β and IL-8 is independent of TLR2 and TLR4

Wildtype trophoblast (3A and HTR8) and trophoblast cells transfected to express either a TLR2-dominant negative (DN) (in 3A cells) or a TLR4-DN (in HTR8 cells) were either NI or infected with Ct for 48hrs. (a & b) Cell lysates were evaluated for pro-IL-1β expression by Western blot (representative blots are shown). Barcharts show quantification of pro-IL-1β levels, as determined by densitometry and normalized to β-actin. Culture supernatants were analyzed for (c & d) IL-1β and (e & f) IL-8 levels by ELISA. Data are pooled from three independent experiments and no significance was observed between the response of wildtype and TLR2-DN or TLR4-DN cells to Ct infection.

Figure 3

Figure 3. _Chlamydia_-induced trophoblast IL-8 secretion, but not IL-1β expression or secretion is dependent on MyD88

Wildtype HTR8 trophoblast and HTR8 cells transfected to express a MyD88-DN were either NI or infected with Ct for 48hrs. (a) RNA was analyzed for IL-1β mRNA levels by qRT-PCR; (b) cell lysates were analyzed for pro-IL-1β expression levels by Western blot (representative blots are shown) and densitometry; and culture supernatants were measured for (c) IL-1β and (d) IL-8 by ELISA. Data are pooled from three independent experiments; *p<0.05 relative to the wildtype cells.

Figure 4

Figure 4. _Chlamydia_-induced trophoblast IL-1β secretion is not dependent on ASC or Nalp3

(a–b) Sw.71 trophoblast cells were transfected to express either shRNA for ASC (sh-ASC) or a control sequence (sh-control) (a) Western blot of lysates from the Sw.71 trophoblast cells expressing either sh-control or sh-ASC for ASC expression. β-actin served as a loading control. (b) 36 hrs after either NI or Ct infection, levels of secreted IL-1β in the supernatants of sh-control and sh-ASC cells were measured by ELISA. n=3; *p<0.05 relative to the NI control. (c–d) Sw.71 trophoblast cells were transfected to express either shRNA for Nalp3 (sh-Nalp3) or a control sequence (sh-control). (c) RNA from the Sw.71 trophoblast cells expressing either sh-control or sh-NAlp3 was analyzed for Nalp3 mRNA expression by qRT-PCR. n=3; *p<0.05 relative to the sh-control. (d) 72hrs after either no treatment (NT) or treatment with MSU (100µg/ml) levels of secreted IL-1β in the supernatants of sh-control and sh-Nalp3 cells were measured by ELISA. n=3; *p<0.05 relative to the NT control unless otherwise indicated. (e) 36 hrs after either NI or Ct infection, levels of secreted IL-1β in the supernatants of sh-control and sh-Nalp3 cells were measured by ELISA. n=3; *p<0.05 relative to the NI control.

Figure 5

Figure 5. C_hlamydia_-induced trophoblast IL-1b processing and secretion is dependent on Nod1

Sw.71 trophoblast cells were transfected to express either shRNA for Nod1 (sh-Nod1) or a control sequence (sh-control) and were either NI or infected with Ct for 36hrs. (a) Expression of Nod1 protein in the sh-control and sh-Nod1 cells was determined by Western blot analysis, and barchart shows densitometry with Nod1 expression normalized to β-actin levels. (b–d) Barcharts show levels of secreted IL-1β, IL-8 and G-CSF in the supernatants of the sh-control and sh-Nod1 cells. (e) Supernatants were evaluated for pro- and active IL-1β and active caspase-1 expression by Western blot. Barcharts show quantification of protein expression in the sh-control and sh-Nod1 cells as determined by densitometry and normalized to β-actin. n=4; *p<0.05; **p<0.001 relative to the NI control unless otherwise indicated.

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