Chlamydia trachomatis infection modulates trophoblast cytokine/chemokine production

Abstract

It is well established that intrauterine infections can pose a threat to pregnancy by gaining access to the placenta and fetus, and clinical studies have strongly linked bacterial infections with preterm labor. Although Chlamydia trachomatis (Ct) can infect the placenta and decidua, little is known about its effects on trophoblast cell immune function. We have demonstrated that Ct infects trophoblast cells to form inclusions and completes the life cycle within these cells by generating infectious elementary bodies. Moreover, infection with Ct leads to differential modulation of the trophoblast cell's production of cytokines and chemokines. Using two human first trimester trophoblast cell lines, Sw.71 and H8, the most striking feature we found was that Ct infection results in a strong induction of IL-1beta secretion and a concomitant reduction in MCP-1 (CCL2) production in both cell lines. In addition, we have found that Ct infection of the trophoblast results in the cleavage and degradation of NF-kappaB p65. These findings suggest that the effect of a Chlamydia infection on trophoblast secretion of chemokines and cytokines involves both activation of innate immune receptors expressed by the trophoblast and virulence factors secreted into the trophoblast by the bacteria. Such altered trophoblast innate immune responses may have a profound impact on the microenvironment of the maternal-fetal interface and this could influence pregnancy outcome.

Figure 1

Inclusion formation and infection rates in trophoblast cells infected with C. trachomatis. (A) Trophoblast cells (Sw.71) were exposed to either No infection (NI), Ct serovar D (Ct-D) or Ct serovar L1 (Ct-L1) at a MOI of 1, by rocking/resting at room temperature for 2 hour. Inclusion formation was evaluated by light microscopy at 24 and 36 hours post-infection for Ct-L1 and Ct-D, respectively. Inclusions are highlighted by arrow heads (Mag. ×40). (B) After 36 hours of infection with or without Ct-L1 or Ct-D, inclusion formation in the Sw.71 and H8 cells was evaluated by by staining cells intracellularly with a FITC-conjugated mouse anti-Ct LPS mAb. Infection rates were then determined by both immmunofluorescent microscopy and flow cytometry. The flow cytometry histograms show two distinct populations: the left hand peak being the uninfected cells, and the right hand peak with the marker representing the infected trophoblast population.

Figure 2

Infection rates of trophoblast cells infected with C. trachomatis by different techniques. (A) H8 and Sw.71 cells were infected with Ct (serovar D) at an MOI of 1 by either rocking or by centrifugation. After 36 hours, the cells were collected and stained intracellularly with a FITC-conjugated mouse anti-Ct LPS mAb. Centrifugation resulted in a higher rate of infection in both cell lines (B) H8 and Sw.71 cells were infected with or without Ct (serovar D) at an MOI of 1 by centrifugation. After 48 and 72 hours inclusion formation was visualized by light microscopy. Inclusions contained within the cells are highlighted by arrow heads, while extruded inclusions are highlighted by asterisks (Mag. ×40).

Figure 3

Chlamydia-infected trophoblast cells produce viable EBs. H8 and Sw.71 cells were infected with Ct (serovar D) at an MOI of 1 by centrifugation. After 36 hours, the cells were collected and stained intracellularly with a FITC-conjugated mouse anti-Ct LPS mAb. The infection levels were then determined by flow cytometry (i & iv). In parallel, lysates were prepared from Ct-infected H8 and Sw.71 cells and these were then immediately applied to a culture of uninfected HeLa cells. After 48 hours, the HeLa cells exposed to infected Sw.71 or H8 lysates were collected and the infection levels determined by flow cytometry. Histograms (ii & v) show the levels of HeLa cell Ct infection (solid line), when compared to the uninfected HeLa cells (dotted line). The HeLa cells exposed to infected Sw.71 or H8 lysates were also evaluated for inclusion formation by light microcopy (iii & vi). Inclusions contained within the cells are highlighted by arrow heads, while extruded inclusions are highlighted by asterisks (Mag. ×40) (iii & iv).

Figure 4

Cytokine profile of trophoblast cells infected with C. trachomatis. First trimester trophoblast cells were treated with either media, or infected with Ct serovar D at a MOI of 1 by centrifugation. After 24, 36, 48, 60 and 72 hours, cell-free/EB-free supernatants were collected and evaluated for cytokine/chemokines by multiplex analysis. Barcharts show the levels of IL-8, IL-1β, IL-6, MCP-1, GROα and RANTES secreted by (A) H8 cells and (B) Sw.71 cells, after treatment with either media or Ct (n=3; *p<0.05). A representative experiment of three is presented.

Figure 5

C. trachomatis modulates trophoblast cytokine/chemokine production in a dose-dependent manner. Trophoblast cells (H8 and Sw.71) were infected with Ct at a MOI of 0 (uninfected), 0.5, 1 or 2. (A) After 36 hours infection levels were determined by flow cytometry. Histograms show the percentage of infected cells (solid line) when compared to the uninfected cells (dotted line). After 48 and 72 hours, cell-free/EB-free supernatants were collected and evaluated for cytokines/chemokines by multiplex analysis. (B & C) Line charts show the levels of IL-8, IL-1β, IL-6, MCP-1, GROα and RANTES secreted by (B) H8 cells and (C) Sw.71 cells, after infection with or without Ct (n=3; *p<0.05). A representative experiment of three is presented.

Figure 6

Infection of trophoblast cells with C. trachomatis reduces NFκB p65 expression levels. Trophoblast cells (H8 and Sw.71) were either non-infected (NI), or infected with C. trachomatis (serovar D) at a MOI of 1. After 48 hours, cell lysates were prepared and Western blot analysis for NFκB p65 (65kDa) and Ct MOMP (45kDa) was performed.