Decidual Inflammation Drives Chemokine-Mediated Immune Infiltration Contributing to Term Labor

Abstract

Infiltration of maternal peripheral leukocytes into the uterine tissues is a critical event occurring before, during, and after term labor (TL). In this article, we investigate the contribution of uterine smooth muscle (myometrium) and pregnant endometrium (decidua) to the inflammatory process during human TL. We hypothesize that labor-related physiological inflammation is orchestrated by uterine-secreted cytokines, which dually activate the uterine vascular endothelium and maternal leukocytes to promote their adhesion and infiltration into the uterus. Using Luminex and ELISA assays, we examine a full range of cytokines (45 proteins) in media conditioned by primary decidual and myometrial cells from TL and term not in labor (TNL) women. The effect of conditioned media on the activation of human uterine microvascular endothelial cells was measured by qPCR and on peripheral leukocytes by flow cytometry. Transendothelial migration of calcein-labeled primary leukocytes toward media was assessed by fluorometry. Stromal decidual cells secrete significantly higher levels of multiple cytokines compared with myometrial cells (p < 0.05) and significantly more cytokines during TL than TNL. These cytokines activate uterine microvascular endothelial cells through the upregulation of cell adhesion molecule VCAM-1 and peripheral leukocytes by upregulation of CD11b. Furthermore, multiple cytokines secreted from the TL decidua and myometrium significantly increase migration of granulocytes, monocytes, and lymphocytes compared with TNL (p < 0.05), which was blocked by a broad-spectrum chemokine inhibitor (FX125L). These data reveal the critical role for decidual- and myometrial-secreted cytokines in the activation of inflammatory pathways leading to labor. We suggest that these pathways represent targets for therapeutic intervention during preterm labor.

Figure 1.. Comparative characteristics of cytokine levels released by primary decidual and myometrial cells from pregnant women at term and during labor.

(A) Multiplex analysis of cytokine secreted by primary human decidual cells compared to primary myometrial cells from term not-in-labor patients. Culture media conditioned (CM) by primary decidual and myometrial cells isolated from myometrial biopsies and decidua samples from term not-in-labor (TNL) women was collected and analyzed by the 5-plex and 40-plex Panels of the Bio-Plex Pro Human Chemokine Assays as instructed by the manufacturer. White bars represent cytokines detected in the myometrial conditioned media (TNL MCM, N=11), whereas black bars represent cytokines detected in the decidual conditioned media (TNL DCM, N=11). Absolute concentration values (pg/ml) are presented as mean ± SD on a logarithmic scale. Statistical significance (p<0.05) was determined by unpaired t-tests between DCM and MCM groups for each cytokine (IL-6, MIF, G-CSF, GM-CSF, CXCL1, CXCL5, CXCL8, CXCL10, CXCL16, CCL1, CCL2, CCL4, CCL5, CCL21, CCL24, CCL25, CX3CL1, IL-1RA and IL-10). *p<0.05, **p<0.01, ***p<0.001 comparing DCM to MCM group. (B) Multiple analysis of cytokines secreted by human decidual cells during term labor (TL) compared to term not in labor (TNL). Conditioned media was collected from primary cells isolated from decidua samples of TNL and TL deliveries. TNL DCM was compared to TL DCM using 27-plex panels of the Bio-plex Pro Human Cytokine Assay as instructed by the manufacturer. Protein levels of CXCL8, CCL2 and IL6 were higher than the capabilities of the Luminex assay, thus they were re-analyzed using specific ELISAs. Grey bars represent cytokines detected in the TL DCM (N=6), whereas black bars represent cytokines detected in the TNL DCM (N=9). Absolute concentration values (pg/ml) are presented as mean ± SD on a logarithmic scale. Statistical significance (p<0.05) was determined by unpaired t-tests between two DCM groups for each cytokine (G-CSF, GM-CSF, VEGF, IL-1RA, IL-10, CXCL8, CCL2 and IL6). C) Changes in pro-inflammatory cytokine (IL6), and chemokine (CCL2, CXCL8) protein expression in human myometrial cells during term labor (TL) compared to term not in labor (TNL). Supernatants were collected after 48 hours and analyzed by ELISA. Protein concentrations are presented as absolute concentration values (pg/ml) are presented as mean ± SD. Statistical significance (p<0.05) was determined by unpaired t-tests, *p<0.05, **p<0.01, ***p<0.001.

Figure 2.. Cytokines secreted by laboring human decidua activates uterine microvascular endothelial cells through enhanced cell adhesion molecule mRNA expression.

UtMVEC-myo cells were treated with decidua conditioned media generated from term not-in-labor (TNL DCM, solid grey bar) or term laboring (TL DCM, solid black bar) patients (N=3/group) for 6 hours and RNA was extracted for qRT-PCR analysis. Serum free media (SFM, solid white bar) was used as a negative control, denote by dashed line. Statistical significance (*, p<0.05) was determined by unpaired t-tests between DCM groups (TNL and TL). Data presented as mean ± SD.

Figure 3.. Cytokines secreted by laboring human decidua significantly upregulate the protein expression of activation markers on peripheral leukocytes from pregnant women.

Whole peripheral blood collected from healthy women in their 2^nd trimester of pregnancy was incubated with media conditioned by primary decidual cells from TNL and TL deliveries. Light gray bars denote TNL DCM (N=6), dark gray bars denote TL DCM (N=6). Blood cells were stained with fluorophore-conjugated Abs specific for leukocyte subpopulations: CD14 (monocyte), CD15 (granulocyte), CD4 (T-helper lymphocytes) and CD8 (cytotoxic T-lymphocytes), activation markers CD11b, CD44 and CD55, and analyzed by flow cytometry. Serum free media (SFM, solid white bar) was used as a negative control, denote by dashed line. Statistical significance (*, p<0.05) was determined by unpaired t-tests between DCM groups (TNL and TL). Data presented as mean ± SEM.

Figure 4:. Term laboring conditioned media facilitates trans-endothelial migration of peripheral human leukocytes as compared to term not-in-labor samples.

Confluent hUtMVEC-Myo cells grown on membrane inserts were stimulated for 2 hours with either decidua or myometrium conditioned media (DCM/MCM) generated from term not-in-labor (TNL, solid grey bar) and term laboring (TL, solid black bar) patients (N=3–7/group). Primary human neutrophils, monocytes, and lymphocytes pre-treated with (A) either TNL DCM or TL DCM, (B) either TNL MCM or TL MCM, were labeled with calcein-AM, loaded into endothelial-coated membrane inserts (diameter of pore 3 μm and 8 μm, 200,000 leukocytes/well) and allowed to transmigrate for 1–16 hours towards corresponding DCM/MCM. Results are shown as number of primary leukocytes transmigrated through membrane inserts /well. Serum free media (SFM, solid white bar) was used as a negative control. Values are presented shown in a box plot as median with the interquartile range. Data was analyzed by one-way ANOVA. * p<0.05, ** p<0.01, *** p<0.001 compared to TNL group or SFM control.

Figure 5:. Trans-endothelial migration of primary human neutrophils is inhibited by the BSCI (FX125L).

(A) The effective dose of FX125L was determined after stimulation of 2 hours of human primary neutrophils and human uterine microvascular endothelial cells (hUtMVEC-Myo) with media conditioned by decidual cells isolated from term laboring patients (TL DCM, solid black bar, N=4) and TL DCM supplemented with increasing concentrations of FX125L (100–500nM, patterned grey bars). Neutrophils were loaded into the upper well of endothelial-coated inserts (200,000 cells/well, diameter of pore 3 μm), and allowed to transmigrate towards TL DCM ± FX125L for 1 hour. (B) To establish the inhibitory specificity of FX125L effect we pre-incubated for 2 hours primary neutrophils (NEU) and/or monolayers of uterine microvascular endothelial cells (hUtMVEC-Myo, ENDO) with TL DCM (solid black bar, N=4) ± FX125L (400nM, patterned grey bars). Neutrophils (200,000 cells/well) were loaded into the upper well of ENDO-coated inserts, and allowed to transmigrate towards media for 1 hour. Results are shown as number of primary neutrophils transmigrated through membrane inserts/well. All data was standardized to the negative control (serum free media). Pre-stimulation of neutrophils alone was as effective in inhibiting leukocyte migration as pre-stimulation of both neutrophils and endothelial cells, however the pre-stimulation of endothelial cells alone had no effect on the decrease of migration. Statistical significance was determined by one-way ANOVA. *p<0.05,**p<0.01 compared to TL DCM.

Figure 6:. The BSCI (FX125L) inhibits labor-associate leukocyte migration.

Isolated primary human neutrophils, monocytes, and lymphocytes, and uterine microvascular endothelial cells (UtMVEC) monolayers were pre-incubated for 2 hours prior to trans-endothelial migration assay with either decidua or myometrium conditioned media (DCM/MCM) generated from TL patients (solid black bar, N=4–7 per group) or DCM/MCM supplemented with FX125L (400nM, grey oblique box). Leukocytes (200,000 cells/well) were loaded into endothelial-coated inserts, and allowed to transmigrate for 1 hour (neutrophils) or 16 hours (monocytes and lymphocytes) towards TL DCM/MCM. Results are shown as number of primary leukocytes transmigrated through membrane insert/well. All data was standardized to negative control (serum free media). Statistical significance was determined by paired t-tests, *p<0.05, **p<0.01, ***p<0.001.

Figure 7:. Model of decidual/myometrial activation of the inflammatory pathway during human term labor.

(1) The laboring decidua significantly secretes a milieu of pro-inflammatory cytokines, chemokines, and growth factors (IFNy, G-CSF, MCP1, CXCL1, TNFa, IL1B, IP10 etc.) (2) The laboring myometrium significantly secretes pro-inflammatory cytokines, chemokines (IL6, CXCL8, and CCL2). (3) Secreted cytokines/chemokines enter the maternal peripheral microvasculature and activate the expression of CAMs (ICAM, VCAM-1, and E-selectin) on the uterine vascular endothelium, and increase the expression of integrins (CD11b, CD55, and CD44) on maternal peripheral leukocytes (monocytes, granulocytes and lymphocytes), allowing increased leukocyte adhesion to the uterine vascular endothelium, (4) which subsequently promote their trans-endothelial migration into the uterine tissue, amplifying the inflammatory signal through cytokine/chemokine secretion to further the progression of labor. (5) The Broad-Spectrum Chemokine Inhibitor (FX125L), a possible anti-inflammatory therapeutic, acts on maternal leukocytes to prevent their activation and trans-endothelial migration, and subsequent infiltration into the uterine tissue, thus preventing labor onset. Illistration created using Biorender.com.