Macrophage gene expression associated with remodeling of the prepartum rat cervix:Microarray and pathway analyses

As the critical gatekeeper for birth, prepartum remodeling of the cervix is associated with increased resident macrophages (Mφ), proinflammatory processes, and extracellular matrix degradation. This study tested the hypothesis that expression of genes unique to Mφs characterizes the prepartum from unremodeled nonpregnant cervix. Perfused cervix from prepartum day 21 postbreeding (D21) or nonpregnant (NP) rats, with or without Mφs, had RNA extracted and whole genome microarray analysis performed. By subtractive analyses, expression of 194 and 120 genes related to Mφs in the cervix from D21 rats were increased and decreased, respectively. In both D21 and NP groups, 158 and 57 Mφ genes were also more or less up- or down-regulated, respectively. Mφ gene expression patterns were most strongly correlated within groups and in 5 major clustering patterns. In the cervix from D21 rats, functional categories and canonical pathways of increased expression by Mφ gene related to extracellular matrix, cell proliferation, differentiation, as well as cell signaling. Pathways were characteristic of inflammation and wound healing, e.g., CD163, CD206, and CCR2. Signatures of only inflammation pathways, e.g., CSF1R, EMR1, and MMP12 were common to both D21 and NP groups. Thus, a novel and complex balance of Mφ genes and clusters differentiated the degraded extracellular matrix and cellular genomic activities in the cervix before birth from the unremodeled state. Predicted Mφ activities, pathways, and networks raise the possibility that expression patterns of specific genes characterize and promote prepartum remodeling of the cervix for parturition at term and with preterm labor

Decreased expression of genes in whole cervix from prepartum D21 postbreeding versus nonpregnant rats (p<0.01; n = 3/group).

<p>Decreased expression of genes in whole cervix from prepartum D21 postbreeding versus nonpregnant rats (p<0.01; n = 3/group).</p

Increased expression of Mφ genes exclusively in prepartum D21 rat cervix (p<0.01; n = 3/group).

<p>Function designations in rat derived from DAVID (<a href="http://david.abcc.ncifcrf.gov" target="_blank">http://david.abcc.ncifcrf.gov</a>), Biolayout <i>Express</i>^3D cluster analyses, and IPA (ECM = extracellular matrix, INFL = inflammation, SIG = Signaling).</p><p>Increased expression of Mφ genes exclusively in prepartum D21 rat cervix (p<0.01; n = 3/group).</p

Subtractive approach in which macrophages were depleted from dispersed cervix from prepartum and nonpregnant mice to identify differential gene expression by macrophages.

<p><b>A</b>. Cervix from perfused adult female Sprague-Dawley rats, on prepartum day 21 post-breeding (D21) or non-pregnant (NP) was carefully trimmed and dispersed (n = 6 each). Mφs were removed by magnetic bead separation from 3 rats in each group as described in detail in Methods. <b>B</b>. Venn diagram of differentially expressed Mφ genes in the cervix from prepartum (D21) or nonpregnant (NP) rats. Genes were exclusively increased or decreased in cervices from D21 or NP rats, except in the overlap region which indicates genes that were increased or decreased in cervices from both D21 and NP rats. No gene whose expression increased or decreased in the cervix from D21 or NP groups then decreased or increased in the cervix from NP or D21 groups, respectively. Number is the average of differentially expressed Mφ genes in whole divided by Mφ -depleted cervix/group (p<0.01, fold >2 or <-2; n = 3 rats). <b>C</b>. Pearson correlation of clustering patterns of gene expression in cervix of individual rats in NP and D21 groups with or without macrophages (Mφ-). Lines connecting microarray analysis for each individual indicates R>0.9 (group specific colors), R = 0.851–0.89 (grey), or R = 0.8–0.85 (thin grey).</p

Biolayout <i>Express</i>^3D analyses of Mφ gene expression and clustering analyses.

<p>Data were transposed in an analysis to identify correlations among groups based on individual gene expression levels (Pearson’s correlation of R≥0.9 and intensity >1 of dataset with fold >2 or <-2 and p<0.01). Markov clustering (MCL, inflation = 1.7) was used to identify clusters related to Mφ genes (microarray results from whole divided by Mφ -depleted cervix). These clusters were analyzed again to create the network schema with 1418 nodes and 69761 edges. Color of nodes represents membership in clusters. The 5 largest MCL clusters had distinct patterns that represent 80% of differentially regulated Mφ genes in cervix (histogram insets, average intensity ± SE, n = 3 rats/group).</p

Proposed network of pathways that reflect expression of Mφ genes in the prepartum cervix based upon Ingenuity Pathway Analysis (IPA).

<p>Network includes Mφ genes exclusively regulated in the cervix from D21 rats and those regulated in both in D21 prepartum and NP groups, as well as known key molecules. Red signifies up-regulated genes and green indicates down-regulated expression. IPA drawn lines predicted activation (orange) or inhibition (blue) of gene expression between key molecules. Other lines indicate no known relationship (black) or uncertainty about relationship (yellow). Color intensity indicates relative expression based upon p-value or prediction intensity. Groups of genes are clustered into Inflammation, Extracellular matrix, or Signaling based on annotations provided by IPA.</p

Functional annotations from Ingenuity Pathway Analysis (IPA) of increased expression of genes in the whole cervix from prepartum (D21) versus nonpregnant (NP) rats (A) and Mφ genes exclusively up-regulated in the cervix (whole vs Mφ-depleted) from prepartum rats (B).

<p>Categories of Function (Headings) and Annotations (histogram labels) consist of IPA designations based upon Z-score rank (threshold>2 estimates proportion of genes that were increased within each annotation; p < 0.01). Categories were broadened (Subheadings combined) to eliminate redundancy in IPA assignment of genes. p value is indicated by triangles. Mφ data analysis included expression of genes that were most divergently regulated, exclusively and in common, in cervices from D21 prepartum and NP groups (ratio of fold > 2 or < 2, D21 vs NP groups).</p

Canonical annotations from Ingenuity Pathway Analysis (IPA) of increased expression of genes in the whole cervix from prepartum (D21) versus NP rats (A) and Mφ genes exclusively up-regulated in the cervix (whole vs Mφ—depleted) from prepartum rats (B).

<p>Canonical pathways are IPA designations of rankings based on the number of genes with increased or decreased expression; p < 0.01. p value is indicated by triangles. Subheadings reflect IPA assignment of genes in common for Category annotations. Mφ data analysis included expression of genes that were most divergently regulated, exclusively and in common, in cervices from D21 prepartum and NP groups (ratio of fold >2 or < 2, D21 vs NP groups).</p