Selected Uterine Immune Events Associated With the Establishment of Pregnancy in the Dog

Abstract

In the dog, implantation takes place at approximately 17 days of embryonal life and, while exposed to relatively high circulating progesterone concentrations, embryos presence is required for the formation of decidua. Furthermore, a balance between pro- and anti-inflammatory responses in conceptus-maternal communication is crucial for the onset of pregnancy. Strikingly, the understanding of such immune mechanisms in canine reproduction is still elusive. Here, canine uterine samples from pre-implantation (day 10-12, E+) and corresponding non-pregnant controls (E-), implantation (day 17, Imp) and post-implantation (day 18-25, Post-Imp) stages of pregnancy were used to investigate the expression and localization of several immune-related factors. The most important findings indicate increased availability of CD4, MHCII, NCR1, IDO1, AIF1, CD25, CCR7, and IL6 in response to embryo presence (E+), while FoxP3 and CCL3 were more abundant in E- samples. Implantation was characterized by upregulated levels of FoxP3, IL12a, ENG, and CDH1, whereas CD4, CCR7, IL8, and -10 were less represented. Following implantation, decreased transcript levels of TNFR1, MHCII, NCR1, TLR4, CD206, FoxP3, and IL12a were observed concomitantly with the highest expression of IL6 and IL1β. MHCII, CD86, CD206, CD163, TNFα, IDO1, and AIF1 were immunolocalized in macrophages, CD4 and Nkp46 in lymphocytes, and some signals of IDO1, AIF1, and TNF-receptors could also be identified in endothelial cells and/or uterine glands. Cumulatively, new insights regarding uterine immunity in the peri-implantation period are provided, with apparent moderated pro-inflammatory signals prevailing during pre-implantation, while implantation and early trophoblast invasion appear to be associated with immunomodulatory and rather anti-inflammatory conditions.

Keywords: dog (Canis lupus familiaris); early pregnancy; embryo-maternal communication; immune system; uterus.

Figure 1

Relative gene expression of selected immune cell markers in the canine early pregnant uterus. Relative gene expression as determined by semi-quantitative real time (TaqMan) PCR (mean ± SEM). (A–E,G–I) One-way ANOVA was applied to test the effects of time (pregnancy stage) on gene expression revealing: P = 0.005 for MHCII, P < 0.0001 for CD206, P = 0.2 for CD163, P = 0.0032 for CD4, P = 0.06 for CD8, P = 0.02 for CD25, P = 0.01 for FoxP3 and P < 0.0001 for NCR1. In the case of P < 0.05, the analysis was followed by a Tukey-Kramer multiple comparisons post-test. (F) Comparison of relative gene expression between CD4 and CD8 was evaluated by applying Student's unpaired two-tailed t-test at each investigated stage. Bars with asterisks differ at: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

Immunolocalization of selected markers of macrophages and lymphocytes in the canine uterus. Immunohistochemical detection of MHCII, CD86, CD206, CD163, CD4, CD8, and NKp46 (encoded by NCR1) at selected stages during early pregnancy. MHCII (A) and CD86 (B) were localized in individual macrophages distributed in the superficial layer of the endometrium during pre-implantation. (C–E) CD206 signals were observed in macrophages localized in the superficial layer of the endometrium during pre-implantation, around uterine glands (C). At the time of implantation, CD206-positive macrophages were present around uterine glands (D, left panel), and scattered in the endometrium during implantation (D, right panel), and post-implantation period (E). Isolated cells could also be identified in the myometrium in Post-Imp samples (E). (F–H) CD163 was expressed by macrophages detected in the superficial layer of the endometrium during pre-implantation (F) and implantation (G). At post-implantation, single CD163-positive macrophages were localized in deep layers of the endometrium, close to the myometrium (H). To differentiate CD4-expressing macrophages (MHCII⁺) and lymphocytes (MHCII⁻), consecutive slides were stained against CD4 (I) and MHCII (J). Both CD4 (K) and CD8-positive lymphocytes (L) were localized in the superficial layer of the endometrium during pre-implantation. During implantation (M), individual CD4⁺ lymphocytes were identified in endometrial superficial layer (top panel) and within blood vessels (bottom panel). Nkp46 (NK cells) were localized in the superficial layer of the endometrium (N) and around blood vessels (O) during pre-implantation and implantation stages. In samples from post-implantation stage (P), NK cells were identified around superficial (left panel) and deep uterine glands (right panel). (solid arrow = macrophages; open arrow = lymphocytes; closed arrowhead = uterine gland; open arrowhead = blood vessel; asterisk = myometrium). No staining was observed in the isotype controls [inset in (A–C,F,I,L,N)].

Figure 3

Relative gene expression of selected cytokines in the canine uterus. (A–L) Relative gene expression as determined by semi-quantitative real time (TaqMan) PCR (mean ± SEM). One-way ANOVA was applied, revealing: P < 0.0001 for IL1β, P < 0.0001 for IL6, P < 0.0001 for IL8, P = 0.01 for IL10, P = 0.02 for IL12a, P = 0.16 for TGFβ, P = 0.19 for TNFα, P = 0.02 for TNFR1, P = 0.19 for TNFR2, P = 0.006 for CCL3, P = 0.083 for CCL13 and P = 0.01 for CCR7. In the case of P < 0.05, this was followed by a Tukey-Kramer multiple comparisons post-test. Bars with asterisks differ at: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Relative gene expression and localization of TLR4, IDO1 and AIF1 in the canine uterus. (A–C) Relative gene expression as determined by semi-quantitative real time (TaqMan) PCR (mean ± SEM). One-way ANOVA was applied to test the variation among the investigated groups, revealing: P = 0.0033 for TLR4, P = 0.03 for IDO1, and P = 0.04 for AIF1. In the case of P < 0.05, this was followed by Tukey-Kramer multiple comparisons post-test. Bars with asterisks differ at: *P < 0.05, **P < 0.01. (D–M) Immunohistochemical localization of members of the TNF-system, IDO1, and AIF1 in the canine uterus at selected stages of early pregnancy. Signals of TNFα were present in macrophages during the pre-implantation (D) and implantation (E) periods. Similarly, both TNFR1 (F) and TNFR2 (G,H) were present in macrophages. In addition, weaker signals for both receptors were also observed in epithelial cells of uterine glands during pre-implantation (F,G) and in endothelial cells (F,H). Positive signals of IDO1 were detected in macrophages between superficial glands during pre-implantation (I) and were also detected in endothelial cells at the time of implantation (J). At post implantation, weaker signals were observed in macrophages localized in deep endometrium layers, as well as in endothelial cells (K). AIF1 positive signals were identified in macrophages close to superficial uterine glands in pre-implantation (L) and implantation (M) stages, while at post-implantation they were localized in the deep layer of the endometrium (N). Some weak signals were also observed in uterine glands at the pre-implantation period (L), while apparently stronger signals were observed in the same glands at implantation (M) and post-implantation (N). Weak positive signals were also detected in endothelial cells (M, N) (solid arrow = macrophages; closed arrowhead = uterine gland; open arrowhead = blood vessel). No staining is observed in the isotype controls [inset in (D,F,G,I,L)].

Figure 5

Relative gene expression of selected factors involved in tissue remodeling in the canine uterus. (A–F) Relative gene expression as determined by semi-quantitative real time (TaqMan) PCR (mean ± SEM). One-way ANOVA was applied, revealing: P = 0.0007 for IGF1, P = 0.0032 for IGF2, P < 0.0001 for ENG, P = 0.0002 for CDH1, P < 0.0001 for ECM2, P = 0.0174 for MM2. In the case of P < 0.05, this was followed by a Tukey-Kramer multiple comparisons post-test. Bars with asterisks differ at: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

Schematic presentation of the proposed model of immune system–mediated events in the canine uterus during establishment of pregnancy. Arrows (↑ and ↓) indicate increased or decreased expression, respectively, of different selected factors during establishment of pregnancy: pre-implantation (days 10–12), implantation (day 17 of embryonal life), and post-implantation (day 25 of pregnancy). The presence of the free-floating embryo during pre-implantation is associated with increased inflammatory activity, marked by upregulated expression of markers of M1 macrophages (MHCII) and Th lymphocytes (CD4, CD25). This reaction appears to be moderated by the increased expression of immunomodulatory factors like IDO1, AIF1, and IL10. There is a shift in the immune milieu at the time of implantation, with decreased transcripts of MHCII and increased transcripts of Treg markers (FoxP3). Both pre-implantation and implantation stages show increased expression of the NK cell marker NCR1. During implantation, concomitantly, the first morphological signs of decidualization are observed in subepithelial endometrial layers. The contribution of the immune system-derived factors to the decidualization process needs further clarification. In the post-implantation period, the expression of several factors investigated is decreased at the implantation sites, indicating a decreased local immune activity in response to embryo invasion.