Background
Infection during pregnancy is one of the leading causes of maternal mortality and morbidity worldwide, accounting for more than 10% of all deaths [
1]. Notably, both viral and bacterial infections have been linked to adverse pregnancy outcomes [
2]. Indeed, viral infection during pregnancy has been associated with increased risk of pregnancy complications such as miscarriage, stillbirth, preterm birth, pre-eclampsia, fetal growth restriction, and congenital defects, among others [
3‐
6]. Considering past and recent viral pandemics, as well as the growing knowledge of viral infection during pregnancy, it has become evident that specific viral infections can have devastating short- and long-term effects on both the mother and offspring [
7‐
17]. Thus, it is imperative to elucidate the underlying mechanisms whereby viral infection disproportionately impacts pregnant women to design novel preventative and therapeutic approaches.
Viruses are broadly classified by the type of carried genetic material (RNA or DNA) and display infection strategies that vary accordingly [
18]. Moreover, each type of virus, together with its mechanisms of replication, requires tailored mechanisms of detection and clearance by host cells [
19‐
22]. Importantly, although viral proteins typically elicit an intense initial immune response, the higher viral mutation rates make continuous surveillance by the host immune system challenging [
23,
24]. Thus, the ability to detect general patterns of viral genetic material is a critical component of the early antiviral immune response that is primarily accomplished by innate immune cells such as monocytes [
21,
25‐
27].
Monocytes are part of the first line of defense against pathogens, including viral infection [
28‐
32]. These innate immune cells are equipped to detect and kill microbes, being the primary subset of circulating mononuclear phagocytic cells, and are capable of quickly secreting pro-inflammatory cytokines in response to viral encounter [
33‐
35]. Monocytes express multiple pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), which can recognize conserved viral motifs known as pattern-associated molecular patterns (PAMPs) [
27,
36]. Intracellular PRRs include TLR3, TLR7, TLR8, and TLR9, all of which are located within the endosomal membrane [
37‐
40] and are specific for double-stranded (ds)RNA (TLR3) [
41], single-stranded (ss)RNA (TLR7, TLR8) [
42,
43], or dsDNA (TLR9) [
44‐
46]. Cells also express specific PRRs within the cytosolic space, such as Retinoic Acid-Inducible Gene I (RIG-I) and Melanoma Differentiation-Associated Protein 5 (MDA5) [
47], both of which detect dsRNA [
48]. Interestingly, some viruses can be recognized by multiple PRRs due to their replication cycle, which includes phases wherein the virus contains both dsRNA and ssRNA [
47,
49‐
52]. Thus, the host response to viruses is complex and requires the expression of multiple PRRs by sentinel cells such as monocytes. Given that monocytes are increased in number [
53‐
56] and display activated phenotypes during pregnancy [
56‐
60], such innate immune cells are likely primed to participate in maternal response to viral infection. However, the evaluation of circulating monocyte responses to different types of virus during pregnancy has not been undertaken.
Herein, we performed a comprehensive in vitro study of peripheral monocyte responses to viral genetic material mimetics in pregnant and non-pregnant women. We investigated the population distribution and expression of surface proteins (i.e., adhesion molecules and chemokine receptors) by conventional monocyte subsets (classical, intermediate, non-classical, and CD14loCD16−) using flow cytometry. In addition, we profiled specific type I, II, and III interferons released by monocytes in response to viral ligand stimulation. Together, these data provide an overview of changes in the monocyte response to viral infection during pregnancy.
Methods
Human subjects, clinical specimens, and definitions
Peripheral blood samples were obtained from August 2020 – February 2021 from healthy pregnant and non-pregnant women recruited by the Pregnancy Research Branch, an intramural program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), U.S. Department of Health and Human Services, Wayne State University (Detroit, MI, USA), and the Detroit Medical Center (Detroit, MI, USA). Blood sample collection was performed from all women after obtaining written informed consent. The collection and use of biological specimens for research purposes was approved by the respective Institutional Review Boards of Wayne State University and the Detroit Medical Center (WSU IRB 031318MP2F). The present study included pregnant women (n = 20), predominantly African American, whose peripheral blood was collected in the third trimester at a median gestational age of 39.1 (ranging from 37.4 – 41) weeks prior to the onset of labor or administration of any medication. The control study group comprised healthy non-pregnant women (n = 20) of reproductive age from the same community, of whom all except one had never been pregnant.
Stimulation of peripheral blood mononuclear cells with viral ligands
Peripheral blood samples were obtained by venipuncture and collected into EDTA tubes. Peripheral blood mononuclear cells (PBMCs) were isolated using the Lymphoprep density gradient medium (Cat# 07801; StemCell Technologies Inc., Vancouver, Canada), per the manufacturer’s instructions. Isolated PBMCs were cultivated in RPMI 1640 Medium (Cat# 11875–093; Thermo Fisher Scientific, Life Technologies Limited, Paisley, UK) supplemented with 5% human serum (Cat# H3667; Sigma-Aldrich, St Louis, MO, USA) and 1% Penicillin–Streptomycin (Cat# 15140122; Thermo Fisher Scientific). The cells were plated onto cell culture plates at a density of 1 × 106 cells/mL prior to treatment. For viral ligand stimulation, PBMCs were individually incubated with 2.5 µg/mL R848 (TLR7/8-based adjuvant; Cat# vac-r848; InvivoGen, San Diego, CA, USA), 1 µM Gardiquimod (TLR7 ligand; Cat# tlrl-gdqs; InvivoGen), 10 µg/mL Poly(I:C) (HMW) VacciGrade™ (TLR3-based adjuvant; Cat# vac-pic; InvivoGen), 50 µg/mL Poly(I:C) (HMW) LyoVec™ (RIG-I/MDA-5 ligand; Cat# tlrl-piclv; InvivoGen), and 2 µg/mL ODN 2216 (TLR9 ligand; Cat# tlrl-2216; InvivoGen) at 37 °C with 5% CO2 for 24 h with the addition of protein transport inhibitor cocktail (Cat# 00-4980-03; ThermoFisher Scientific) for the last 4 h of incubation. Following incubation, the isolated PBMCs were gently collected using a cell scraper and centrifuged at 300 × g and 4 °C for 5 min. Finally, the resulting cell supernatants from PBMCs were stored at -80 °C prior to cytokine profiling, while the cell pellets were immediately processed for immunophenotyping.
Immunophenotyping
Collected PBMC pellets were resuspended in 1X phosphate-buffered saline (PBS; Life Technologies Limited, Pailey, UK) and incubated with 1 µL/mL of Fixable Viability Stain 510 (Cat# 564406; BD Biosciences, Franklin Lakes, NJ, USA) in the dark at room temperature for 15 min. Next, cells were washed and resuspended in FACS Stain Buffer (Cat# 554656; BD Biosciences). Extracellular anti-human monoclonal antibodies (Supplementary Table
1) were added to the cell suspensions, which were incubated in the dark at 4 °C for 30 min. Cells were then fixed and permeabilized using the BD Cytofix/Cytoperm Kit (Cat# 554714; BD Biosciences), according to the manufacturer’s instructions. Following permeabilization, intracellular anti-human monoclonal antibodies (Supplementary Table
1) were added to cell suspensions, which were incubated in the dark at 4 °C for 30 min. Finally, the cells were washed and resuspended in 0.5 mL of FACS Stain Buffer and acquired using the BD LSR Fortessa flow cytometer (BD Biosciences) with FACSDiva 9.0 software (BD Biosciences). FlowJo software version 10 (TreeStar, Ashland, OR, USA) was used to perform data analysis and create figures. Monocytes were identified as CD14
+ cells. As shown in Supplementary Fig.
1, monocyte subsets were classified as follows: classical monocytes (CD14
hiCD16
−), intermediate monocytes (CD14
hiCD16
+), non-classical monocytes (CD14
loCD16
+), and CD14
loCD16
− monocytes. Additional markers (Supplementary Table
1) were used to further immunophenotype cells within the identified subsets.
Interferon profile of viral ligand-stimulated PBMCs
PBMCs were isolated, cultured, and the resulting cell supernatants were collected as previously described. The concentrations of interferons were determined in cell supernatants using the U-PLEX Interferon Combo (human) (Cat# K15094K-1; Meso Scale Discovery, Rockville, MD, USA), following the manufacturer’s instructions. The following immune mediators were assayed: IFN-α2a, IFN-β, IFN-γ, and IL-29/IFN-λ1. A MESO QuickPlex SQ 120 was used to read the plates, and cytokine concentrations were calculated using the Discovery Workbench software version 4.0 (Meso Scale Discovery). The assay sensitivities were: 4 pg/mL (IFN-α2a), 3.1 pg/mL (IFN-β), 1.7 pg/mL (IFN-γ), and 1.2 pg/mL (IL-29/IFN-λ1).
Statistical analyses
The R statistical programming language was used to perform all statistical analyses. Linear mixed effects models were fit for the comparison of flow cytometry data and cytokine concentrations between groups to account for repeated measurements. The data obtained by flow cytometry were modeled as frequencies. A false discovery rate adjusted
p-value (q-value) < 0.05 was considered statistically significant. Differences in proportions of monocytes subsets are represented as heatmaps, and selected significant comparisons are displayed as box and whiskers plots. GraphPad Prism version 9.5.1 for Windows (GraphPad Software, San Diego, California, USA,
www.graphpad.com) was used to conduct statistical analysis to evaluate differences in interferon concentrations using the Kruskal–Wallis test with post hoc multiple comparisons. A
p-value < 0.05 was considered statistically significant.
Discussion
Herein, we showed that the frequency of monocytes expressing the adhesion molecules CD147 and CD162 was diminished in pregnant women in response to TLR7/TLR8 stimulation. CD147, commonly termed Basigin, is a membrane receptor and member of the immunoglobulin superfamily that participates in cellular functions including migration and adhesion [
77‐
80]. Similarly, CD162, or P-selectin glycoprotein ligand-1 (PSGL-1), acts as a ligand for selectins and is also a key player in leukocyte migration/adhesion [
81‐
83]. Cellular adhesion molecules are among the primary points of cell entry for multiple viral families [
84], and the modulation of such receptors can be a mechanism for viral pathogenicity. For example, the ssRNA Zika virus was shown to upregulate integrins and other adhesion molecules in monocytes, which potentially enhanced dissemination into neural cells [
85]. Importantly, infection with ssRNA viruses during pregnancy is linked to increased risk of adverse outcomes and more severe clinical features compared to non-pregnant patients [
8,
61,
63‐
65,
86‐
88]. Indeed, while enteroviruses are largely asymptomatic in non-pregnant patients, they have been shown to induce obstetric complications [
64]. The enhanced downregulation of adhesion receptors upon TLR7/TLR8 stimulation in pregnancy-derived monocytes may thus represent a defensive strategy to slow viral entry and potentially protect the fetus at the expense of the mother.
The above concept is further supported by the distinct regulation of CCR5 and CCR2 expression in response to TLR7/TLR8 stimulation of pregnancy-derived monocytes observed herein. The chemokine receptors CCR5 and CCR2 are integral for mediating monocyte trafficking and inflammatory responses [
89,
90], and thus the distinct changes in the distribution of pregnancy-derived monocytes expressing these receptors could help to explain the differing susceptibility to viral infection. Specifically, we found that monocytes with a double-positive CCR5
+CCR2
+ phenotype were diminished in pregnant women compared to non-pregnant, while CCR5
−CCR2
+ and CCR5
−CCR2
− subsets were more abundant, suggesting a tendency for enhanced downregulation of these chemokine receptors during viral stimulation. Importantly, CCR5 has been implicated as a co-receptor in viral cell entry by the ssRNA virus HIV-1 [
91‐
95], with the expression levels of this receptor being directly associated with the rates of monocyte/macrophage infection [
96]. Together, these findings provide evidence for distinct modulation of monocyte phenotypes in response to ssRNA viral stimulation during pregnancy that may serve to protect the fetus from vertical transmission. However, the reduced abundance of cells expressing adhesion molecules and chemokine receptors in monocytes from pregnant women exposed to ssRNA viruses may also disrupt the capacity of these immune cells to migrate to sites of infection/inflammation.
Notably, we also found that stimulation of monocytes via TLR7 alone was not associated with differences between pregnant and non-pregnant individuals, suggesting that the differential effects of ssRNA viruses in monocytes from pregnant women are mediated primarily through TLR8. This concept is supported by a previous in vitro investigation of the relationship between placental growth factor-1 (PlGF-1), which increases in the maternal circulation during pregnancy and peaks in the third trimester [
97], and CD14
+ cellular responses to TLRs stimulation [
98]. It was observed that TNF release was enhanced when TLR8 stimulation occurred in the presence of PlGF-1. Moreover, while targeted TLR7 stimulation in the presence of PIGF-1 triggered a mild increase in TNF production, the combined stimulation of TLR7/TLR8 induced the strongest effect [
98]. These results suggest that pregnancy-specific physiologic changes can modulate TLR signaling pathways in monocytes, including a greater responsiveness to ssRNA-mediated TLR8 stimulation, which may contribute to an enhanced maternal response to viral infections such as HIV, influenza, and coronaviruses. It is worth mentioning that, despite their shared recognition of ssRNA viral ligands, TLR7 and TLR8 have been reported as modulating distinct signaling pathways in monocytes, resulting in the biased release of cytokines and interferons [
99]. Thus, the observed greater dependence on TLR8 signaling for pregnancy-specific responses to ssRNA may have additional implications for subsequent mediator release by maternal monocytes that were not revealed by the analysis performed herein. Moreover, the potential combined action of TLR7 and TLR8 in monocytes has not been adequately investigated and thus the stimulation of both receptors, or crosstalk between their signaling pathways, may have additional effects that are not yet understood [
99].
In the current study, we utilized poly(I:C) to induce TLR3 stimulation and thereby model dsRNA viral infection, and found that monocytes expressing CD181 (CD181
+CD182
+ and CD181
+CD182
−) were increased, while those without CD181 expression (CD181
−CD182
+ and CD181
−CD182
−) were diminished, in pregnant women compared to non-pregnant individuals. More commonly known as CXCR1 and CXCR2, these molecules act as receptors for multiple chemokines including CXCL1 and IL-8 [
100‐
102]. A prior report indicated that poly(I:C) treatment in pregnant rats resulted in elevated concentrations of multiple mediators in the circulation, including monocyte chemoattractants such as CXCL1, CCL3, and CCL20 [
103], supporting the participation of monocyte chemokine signaling pathways as part of the response to TLR3 stimulation. CXCR1 and CXCR2 have overlap in their recognized chemokines and were thought to induce similar functions that centered on neutrophil recruitment [
89,
90]. However, reports have suggested that the downstream effects mediated by these receptors may differ; for example, while both CXCR1 and CXCR2 respond to IL-8 (CXCL8), the latter receptor undergoes rapid internalization compared to the former [
104]. Thus, the biased modulation of these two chemokine receptors favoring CXCR1-expressing monocytes in response to TLR3 stimulation may indicate a pregnancy-specific program of immune regulation; yet, this concept requires further investigation.
TLR9 is an important component of host defense against dsDNA viruses [
44‐
46]. Here, we report that stimulation of TLR9 resulted in a modest but consistent increase in intermediate monocytes as well as those expressing chemokine receptors and inflammatory cytokines/chemokines in both pregnant and non-pregnant samples. Our results are consistent with studies demonstrating that TLR9 mRNA is expressed in the murine uterus, cervix, and placenta throughout gestation [
105], and that the protein expression of TLR9 in human peripheral leukocytes is unaltered by pregnancy [
106]. Given the frequency of encountering dsDNA viruses such as CMV during pregnancy [
107‐
111], the conservation of TLR9 signaling and its downstream effects may be important for ensuring a sufficient maternal immune response against such common viral threats. Interestingly, alterations in TLR9 signaling resulting from single nucleotide polymorphisms or the activation of this receptor via mtDNA have been linked to obstetrical pathologies such as preeclampsia and spontaneous preterm birth [
112‐
114]. Moreover, TLR9 has also been proposed to respond to cell-free fetal DNA (cffDNA), small DNA fragments derived from placental cells and released into maternal circulation [
115,
116], in the context of obstetrical disease [
117‐
122], as demonstrated using animal models [
123,
124], and in normal term parturition [
125,
126]. In light of the reported link between TLR9 signaling and adverse pregnancy outcomes, it is likely that the downstream inflammatory cascade is tightly regulated under steady-state conditions. Regardless, further investigation is required to mechanistically investigate the role of TLR9 responses to pathogen-derived CpGs or cffDNA in pregnancy complications.
Interferons, which represent one of the first lines of soluble defense against pathogens and are critical for an effective anti-viral response, are divided into three types that differ according to their receptor complexes and signal transduction pathways [
127]. Here, we determined the release of select Type I, II, and III interferons in response to TLR3 and TLR7/TLR8 stimulation, and noted substantial increases across all mediators in PBMCs from both pregnant and non-pregnant women. The maintenance of interferon signaling is particularly important during pregnancy to protect the fetus against potential congenital infection [
128]. Yet, it is worth mentioning that a substantial proportion of human genes can potentially be differentially regulated by interferons; indeed, the family of interferon-stimulated genes (ISGs) continues to grow as new members are identified [
129]. Thus, it is possible that, although the released IFN profile appears unchanged during pregnancy, the monocyte signature of ISGs that is modulated by viral signaling may undergo distinct regulation compared to non-pregnant individuals, resulting in a tailored immune response.
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