Pathogen recognition in the human female reproductive tract: expression of intracellular cytosolic sensors NOD1, NOD2, RIG-1, and MDA5 and response to HIV-1 and Neisseria gonorrhea

Abstract

Problem: Expression patterns and regulation of cytosolic pattern recognition receptors (PRR) NOD-1, NOD-2, RIG-1, and MDA5 have not been elucidated in the human female reproductive tract (FRT).

Method of study: Primary epithelial cells (EC) isolated from Fallopian tube (FT), endometrium (EM), cervix (Cx), and ectocervix (Ecx) were treated with estradiol, poly(I:C), Neisseria gonorrhea (GC), and HIV-1. PRR mRNA expressions were analyzed by Real-time RT-PCR. Conditioned media were analyzed for IL-8 by ELISA.

Results: EC from all FRT compartments constitutively expressed NOD1, NOD2, RIG-1, and MDA5 with highest levels expressed by FT. Stimulation with poly(I:C) resulted in upregulation of NOD2, RIG-1, and MDA5 in all FRT compartments and correlated with increased secretion of IL-8, whereas estradiol treatment had no effects. Exposure to GC and HIV-1 IIIB but not BaL resulted in selective upregulation of NOD2 and MDA5.

Conclusion: PRR are expressed throughout the FRT and differentially regulated by poly(I:C), GC and HIV-1.

Figure 1. Constitutive mRNA expression of NOD1, NOD2, RIG-1 and MDA5 by epithelial cells of the female reproductive tract in multiple patients

Real-time RT-PCR was used to determine the mRNA expression levels of NOD1, NOD2, RIG-1, and MDA5 in primary FT, EM, Cx, and Ecx epithelial cells. Values from each sample were normalized to housekeeping gene β-actin and expressed as copies of mRNA × 10⁵. An average of 3–5 different patient tissues were used from each compartment. Expression of NOD1 in FT, EM, Cx, and Ecx is shown in (A), NOD2 in (B), RIG-1 in (C) and MDA5 in (D).

Figure 2. Induction of NOD1, NOD2, RIG-1, and MDA5 mRNA by epithelial cells from EM, FT, Cx, and Ecx, upon treatment with Poly(I:C)

Primary FRT epithelial cells were treated with poly(I:C) for 24 hr. Real-time RT-PCR was used to determine mRNA expression levels of NOD1, NOD2, RIG-1, and MDA5. After normalization to endogenous control β-actin, each patient sample was further calibrated to its own untreated control and expressed as relative fold change. Data from (A) three distinct FT, (B) seven EM, (C) three Cx, and (D) four Ecx are shown. A 2-fold or greater change was considered to be significant.

Figure 3. Enhanced secretion of IL-8 by primary FRT epithelial cells following stimulation with TLR3 agonist poly(I:C)

Primary epithelial cells from FT, EM, Cx, and Ecx were treated with poly(I:C) for 24 hr. Apical and basolateral conditioned media were collected and assayed for IL-8 secretion by ELISA. Data from one representative patient sample from each compartment is shown. Apical secretions are shown in black and basolateral secretions are shown in white. The p values were calculated by comparing triplicate wells for Control and Treatment, using a two-tailed paired t test. Significance denoted by *; *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 4. No change in mRNA expression of NOD1, NOD2, RIG-1, and MDA5 by primary FRT epithelial cells upon estradiol treatment

Primary epithelial cells from FT, EM, and Cx, were treated apically and basolaterally with 5×10⁻⁸M E₂ for 24 hr and real-time RT-PCR was used to determine expression of NOD1, NOD2, RIG-1, and MDA5. After normalization to endogenous control β-actin, each sample was further calibrated to its own untreated control and expressed as relative fold change. Data from three distinct FT (black), three EM (white), and four Cx (grey) are shown. A ≥2-fold change was considered to be significant.

Figure 5. Selective upregulation of NOD2 and MDA5 by primary cervical epithelial cells upon exposure to heat-killed Neisseria gonorrhea

Primary epithelial cells from Cx, were treated with heat-killed GC for 6 hrs. (A) shows induction of NOD2 and MDA5 mRNA by Cx epithelial cells (Control, black; heat-killed, white). (B) shows significantly higher secretion of IL-8 by Cx epithelial cells upon GC treatment (apical conditioned media, black; basolateral, white). The p values were calculated by comparing triplicate wells for Control and Treatment, using a two-tailed paired t test; *, p<0.05.

Figure 6. Selective upregulation of NOD2 and MDA5 by primary endometrial epithelial cells upon exposure to HIV-1

Primary epithelial cells from EM were grown to confluence and high TER prior to exposing them apically to 10⁶ infectious units of HIV-1 for 24 hrs. RNA was extracted from cells and the expression of NOD1, NOD2, RIG-1, and MDA5 was determined by real-time RT-PCR. (A) shows induction of NOD2 (4/4 samples) and MDA5 (3/4 samples) upon treatment with HIV IIIB whereas no such induction was observed in (B) when cells were similarly treated with HIV BaL.