Pregnancy trained decidual NK cells protect pregnancies from harmful Fusobacterium nucleatum infection

Rebecca Kotzur; Shira Kahlon; Batya Isaacson; Moriya Gamliel; Yoav Charpak-Amikam; Judy Lieberman; Gilad Bachrach; Debra Goldman-Wohl; Simcha Yagel; Ofer Beharier; Ofer Mandelboim

doi:10.1371/journal.ppat.1011923

Abstract

Natural killer cells (NKs) found during pregnancy at the maternal-fetal interface named decidual (d)NKs, show signs of education following first pregnancy, resulting in better placentation and fetus-growth, hence termed pregnancy trained dNKs (PTdNKs). Here we show that PTdNKs provide increased protection of the fetus from Fusobacterium nucleatum (FN) infection. We demonstrate that PTdNKs secrete elevated amounts of the bacteriocidal protein granulysin (GNLY) upon incubation with FN compared to dNKs derived from first pregnancies, which leads to increased killing of FN. Furthermore, we showed mechanistically that the GNLY secretion is mediated through the interaction of the FN’s Fap2 protein with Gal-GalNAc present on PTdNKs. Finally, we show in vivo, using GNLY-tg mice that enhanced protection of the fetuses from FN infection is observed, as compared to wild type and that this enhance protection is NK cell dependent. Altogether, we show a new function for PTdNKs as protectors of the fetus from bacterial infection.

Author summary

During this project, we aimed to elucidate the influence of natural killer (NK) cells in the protection of fetuses during pregnancy. At the maternal-fetal interface a special subset of NK cells can be found, the decidual NK (dNK) cells, which have been shown to be rather nurturing, in contrast to the cytotoxic peripheral blood NK cells. These cells are thought to be essential for the healthy development of placenta and fetus in the uterus. Bacterial and viral infections are quite common during pregnancy and pose a significant risk for the fetus and its development. In this manuscript, we describe how the dNK cells primed through first pregnancy show specific antibacterial properties against the bacterium Fusobacterium nucleatum via the lytic protein granulysin in contrast to the peripheral blood NK cells. Additionally, we showed that NK cells expressing granulysin in mice protect the developing fetuses from the effects of a maternal infection with the bacterium.

Citation: Kotzur R, Kahlon S, Isaacson B, Gamliel M, Charpak-Amikam Y, Lieberman J, et al. (2024) Pregnancy trained decidual NK cells protect pregnancies from harmful Fusobacterium nucleatum infection. PLoS Pathog 20(1): e1011923. https://doi.org/10.1371/journal.ppat.1011923

Editor: Steven R. Blanke, University of Illinois, UNITED STATES

Received: August 9, 2023; Accepted: December 22, 2023; Published: January 12, 2024

Copyright: © 2024 Kotzur et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: Data entailing Fig 1A may be found in the GEO database, under accession GSE79879. All other data are in the manuscript and/or Supporting information files.

Funding: RK was awarded the H2020-MSC_ITN_765104_MATURE-NK grant from the European Commision (H2020-MSC_ITN_765104_MATURE-NK to RK and OM). Furthermore, the work was funded by the Israel Innovation Authority Kamin grant(62615 to OM), the Israeli Science Foundation ISF Moked grant (442-18 to OM), the German-Israeli Foundation for Scientific Research and Development grant (1412-414.13/2017 to OM), ICRF Professorship grant Israeli Cancer Research Fund (to OM), the Israeli Science Foundation ISF China grant (2554/18 to OM), the Ministry of Science and Technology Foundation - Deutsches Krebsforschungszentrum MOST-DKFZ grant (3-14931 to OM), the Ministry of Science and Technology grant (3-14764 to OM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Natural killer (NK) cells are an important part of the innate immunity, essential in fighting infections and cancerous cells, while sparing the body’s own healthy cells [1]. The NK cell activity is tightly controlled by a complex system of activating and inhibiting receptors [2–5]. In recent years, a curious phenomenon regarding these “innate” cells has been observed, first in mice and monkeys, and later in humans, as it was demonstrated that NK cells have a certain type of memory [6–11].

NK cells are known to exist in different organs all throughout the body, specialized towards the immunological challenges specifically found at these sites [12,13]. The focus of this study are the decidua resident decidual NK cells (dNK). During the first trimester of pregnancy, 50–80% of the lymphocytes present in the decidua (the maternal uterine mucosa located directly at the maternal-fetal interface) are dNK cells characterized by being CD56^bright, CD16^neg [14]. The dNK cells display a distinctly different behavior compared with the blood circulating NK cells which only contain 5–10% of this specific CD56^brightCD16^neg subset [15]. The dNK cells are less cytotoxic and instead are able to regulate and support implantation and early pregnancy [16].

Combining the recent findings of adaptive-like memory and the unique function of dNK cells [17], Gamliel et al. discovered the existence of pregnancy trained decidual (PTd)NK cells [18,19]. Previous to that finding, subsequent pregnancies were found to have improved trophoblast invasion, as well as showing a higher degree of placental vascularization and angiogenesis than primary pregnancies [20]. The PTdNK cells were found in subsequent pregnancies and were characterized by a consistently increased expression of the receptors NKG2C and LILRB1, as well as increased secretion of IFNγ and VEGFα [18].

The PTdNK cells are hypothesized to remember first pregnancies and to enable more efficient support for succeeding pregnancies [18]. Confirming these results, a recent study characterizing the maternal-fetal interface on a single cell level revealed three different dNK cell subsets, one of them, dNK1, is shown to express the receptor LILRB1 in a higher abundance than the other two, whereas the elevated NKG2C expression is shared with dNK2 as well [21].

During pregnancy, several infections can spread from mother to child and are known to endanger the survival and development of the growing fetus. These infections are often combined under the acronym TORCH, comprising viruses, parasites and bacteria, and have been known for decades [22,23]. Despite this knowledge, these infections are still a major threat to fetuses and mothers around the globe due to the sheer variety of different pathogenic agents and transmission routes. In addition to these already well characterized and manyfold observed infections, many still unknown bacterial infections are able to induce adverse pregnancy outcomes accounting to up to 10–25% of all stillbirths [24].

The pathogenic organism of choice for this investigation is the Gram-negative anaerobic bacterium Fusobacterium nucleatum (FN). This bacterium is highly involved in periodontal infections [25], but has also been linked to adverse pregnancy outcomes despite its absence from the regular vaginal bacterial flora. This has led to the hypothesis of hematogenous transfer from the oral cavity due to gingival bleeding to the uterus followed by colonization of the decidua and placenta and subsequent spread to the fetus [24,26]. FN can act directly invasive and adherent, and indirectly pro-inflammatory [27]. Parhi et al. showed that FN’s adhesin Fap2 recognizes the trophoblasts’ Gal-GalNAc (Galβ1-3GalNAc), which expression levels are shown to rise during pregnancy, leading to binding of the bacteria to murine and human placentas [27].

Gal-GalNAc also belongs to a group of antigens presented in abundance on specific tumors [28]. But Gal-GalNAc is not the only ligand of Fap2, since the bacterium is known to bind via Fap2 to the inhibitory NK cell receptor TIGIT in order to suppress NK and T cells and therefore evade associated tumor cell killing [29]. Additionally, FN is able to bind CEACAM1, another inhibitory receptor, via the surface protein CbpF and to consequently inhibit immune cell activities [30].

Regarding the question how dNK cells are able to selectively kill intracellular bacteria without harming the fetus, the antimicrobial peptide granulysin (GNLY) seems to be the part of the answer. It was shown that GNLY on its own was able to annihilate intracellular bacteria inside of infected trophoblasts, without lysing the host cells through insertion into the infected cell via nanotubes [31]. All in all, these experiments showed that the presence of GNLY improved the pregnancy outcome in a murine model infected with Listeria monocytogenes [32].

Considering all the given information we wanted to elucidate the impact of PTdNK cells on the clearance and survival of fetuses during FN infection in pregnancy. We demonstrated that PTdNK cells’ increased secretion of GNLY is able to reduce the bacterial load in vitro, and that GNLY expression in murine model prevents further adverse pregnancy outcomes and weight differences in fetuses in vivo. Additionally, we demonstrated that the interaction between Fap2 and Gal-GalNAc is exclusively expressed on the dNK cells, leading to their activation.

Results

PTdNK cells secrete more GNLY when challenged with FN

To investigate whether PTdNKs [18] will be able to better kill bacteria as compared to non-PTdNKs we searched for effector molecules that are expressed more on PTdNKs. One of the major differences we observed was that mRNA expression of GNLY, which was higher in pooled PTdNK samples, derived from subsequent pregnancies as compared to the pooled non-PTdNK samples (Fig 1A).

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Fig 1. Granulysin (GNLY) expression and secretion by pregnancy trained decidual NK cells (PTdNKs) compared to non-PTdNKs.

(A) mRNA Expression of the GNLY gene compared between pooled PTdNK and non-PTdNK cells samples, black = PTdNK cells, gray = non-PTdNK cells. (B) ATAC seq signal amplification of chromatin detected on chromosome 2 at position 85918877–85956340, encoding the GNLY gene, black = PTdNK cells, gray = non-PTdNK cells. (C-F) GNLY secretion detected in sandwich-ELISA measured at 650nm after incubation with (C-E) Wildtype (WT) Fusobacterium nucleatum (FN) and (F) Fap2 deficient FN mutant (FN Fap2 def. mut. named K50). (C) shows non-PTdNK cells’ secretion of GNLY after incubation with PBS (untreated) and WT FN, (D) pbNK cells’ GNLY secretion after incubation with PBS (untreated) and WT FN, (E) PTdNK cells’ GNLY secretion after incubation with PBS (untreated) and WT FN and (F) GNYL secretion of PTdNK cells following incubation with PBS (untreated) and FN K50. Non-PTdNK cells were obtained from first pregnancy, pb = peripheral blood NK cells. PTdNK cells were obtained from repeated pregnancies. * = p<0.05; (C-F) Depicted are 1 out of 2 representative repeats.

https://doi.org/10.1371/journal.ppat.1011923.g001

Next, we investigated whether the interaction of PTdNKs with Fusobacterium nucleatum (FN) will result in GNLY secretion. We compared GNLY secretion from non-PTdNKs derived from first pregnancies, peripheral blood (pb)NKs and PTdNKs after 72h of co-incubation. As can be seen, incubation of non-PTdNKs and pbNKs with FN resulted in reduced GNLY secretion compared to the untreated control (Fig 1B and 1C, respectively). The reason for that reduction is currently unknown. In marked contrast, when PTdNKs were incubated with FN, a significant elevation of GNLY secretion was observed (Fig 1D). This elevation of GNLY secretion was abolished when a Fap2-deficient FN mutant called K50 [33] was incubated with PTdNKs (Fig 1E). Thus, PTdNK, but not non-PTdNK or pbNK cells secrete GNLY upon interaction with FN in a Fap2-dependent manner.

FN is killed efficiently by PTdNK cells in a degranulation independent mechanism

To further assess the functional abilities of PTdNKs to fight bacterial infection, we established a bacteriocidal assay directed against FN. During this assay, we incubated non-PTdNKs and PTdNKs with a constant amount of FN for 1.5h, lysed the NK cells using water, and plated the surviving FN for 48-72h. We then compared the colony forming units (CFU) pf untreated FN with that of FN treated with the various NK cells to determine an increase or decrease. In Fig 2 we clearly see that FN incubation with non-PTdNKs did not lead to a decrease in CFU (Fig 2A), but when PTdNKs were used, a significant decrease of FN CFU was observed (Fig 2B). Furthermore, when employing the Fap2-deficient FN mutant K50, the bacteriocidal effects of the PTdNKs were abolished (Fig 2C).

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Fig 2. Bacteriocidal and degranulation potential of PTdNKs when encountering Fusobacterium nucleatum (FN).

A-C) Bacteriocidal assay performed with A+B) Wildtype (WT) FN, and C) Fap2 deficient FN mutant (FN Fap2 def. mut. K50), after incubation with A) non-PTdNK cells derived from first pregnancy, and B+C) PTdNK cells, results measured in Colony forming units (CFU) compared and normalized to untreated control of FN only; * = p<0.05; D) Degranulation assay performed on PTdNKs incubated with 721.221 and 721.221 cells overexpressing HLA-G in their surface, as well as FN WT and FN Fap2 def. mut. K50, degranulation measured in %CD107a/CD56 positive cells after incubation detected via FACS, * = p<0.05, ** = p<0.01; Depicted are 1 out of 2 representative repeats; E) IFNγ secretion after incubation of PTdNKs with FN WT. Depicted is 1 out of 2 representative repeats; * = p<0.05, ** = p<0.01.; F) Bacteriocidal assay with WT FN utilizing supernatant gathered from PTdNK cells preincubated with WT FN supplemented with (preFN + GNLYab) or without (preFN—GNLYab) GNLY blocking antibody, depicted are 1 out of 3 representative experiments, N = 6, * = p<0.05, ** = p<0.01.G) Bacteriocidal assay with RKO cell intracellularly infected with WT FN utilizing supernatant gathered from PTdNK cells preincubated with WT FN supplemented with (preFN + GNLYab) or without (preFN—GNLYab) GNLY blocking antibody, depicted are 1 out of 3 representative experiments, N = 3, * = p<0.05, ** = p<0.01.

https://doi.org/10.1371/journal.ppat.1011923.g002

To investigate the mechanisms through which PTdNKs kill FN we used the widely established degranulation assay, measuring the percentage of CD107a on NK cell surface after incubation with target cells. We were able to see that the PTdNKs degranulate when incubated with 721.221 and that degranulation is further elevated when 721.221 HLA-G transfectant are used (probably because of the interaction of LILRB1 with HLA-G, [18]). In contrast, after incubation with FN (both WT and the Fap2-deficient mutant K50), no increase degranulation was observed (Fig 2D). Thus, PTdNKs kill FN in a Fap2-mediated, degranulation-independent mechanism. Furthermore, we investigated the change in IFNγ secretion after incubation of PTdNKs with FN WT, observing a significant increase indicating an INFγ-secretion mechanism independent of degranulation (Fig 2E).

We next wanted to demonstrate that GNLY is responsible for the FN killing. for that we preincubated PTdNK cells with WT FN for 72h in the presence or absence of anti-GNLY antibody. Next, we collected the supernatants of the cultured cells and incubated it with newly added FN, for 1.5h and determined the CFU, as described above. After incubation with WT FN, but only without prior addition of GNLY blocking antibody (Fig 2F). As can be seen, reduced FN CFU was observed following incubation of FN with the supernatants and this CFU reduction was completely abolished when the anti-GNLY antibody was present in the assay (Fig 2F). We next employed the same supernatants used in Fig 2F, on cells infected with FN. We incubated RKO cells with WT FN at an MOI of 50:1 for 2h for intracellular infection, selectively extinguished the remaining extracellular bacterial cells via gentamycin treatment (which cannot permeate into the cells, before treating the cells with the supernatant) and confirmed that FN was no longer present outside the RKO cells. The infected cells were lysed using sterile UPW to extract the bacteria before quantitative cultivation. As can be seen in Fig 2G the number of colony forming units was significantly reduced only after treatment with the supernatant that were taken from NK cells incubated with FN only. Importantly, in the presence of anti-GNLY antibody, a complete abolishment of the CFU reduction was observed (Fig 2G). These combined results indicate that GNLY secreted from PTdNKs is essential for the killing of extra- and intracellular FN.

FN binds to PTdNK cells via Gal-GalNAc and not TIGIT

In the above figures we showed that the killing of FN by PTdNKs is Fap2-dependent. Fap2 interacts with two ligands: TIGIT and Gal-GalNAc [28,29]. To test which of these ligands is expressed on PTdNKs we detected Gal-GalNAc using the Gal-GalNAc binding lectin PNA, and for TIGIT we used an anti-TIGIT antibody. We compared the expression of both surface molecules between PTdNKs and pbNKs. In Fig 3A it can be appreciated that only the pbNK cells express TIGIT on their surface, but the PTdNKs do not. In contrast, pbNKs did not express a noticeable amount of Gal-GalNAc, whereas the PTdNKs show a high abundance of this carbohydrate. From these findings we can conclude Gal-GalNAc expressed on PTdNKs is the probable Fap2 ligand. To further corroborate these results, we labeled the WT and the Fap2-deficient FN mutant with FITC and determined binding to PTdNKs. In Fig 3B it can be seen that the WT FN efficiently binds to PTdNK cells, whereas only limited binding was observed with the Fap2 deficient mutant. To demonstrate that this binding is indeed Gal-GalNAc dependent, soluble GalNAc was preincubated with the labeled bacteria before the co-incubation with the PTdNKs. In Fig 3C a dose-dependent decrease in WT FN binding can be seen. Lastly, we also visualized the interaction using immunofluorescence (Fig 3D). In this figure, we can appreciate the clustering of PTdNK (blue and red) cells in the presence of FN (green) together generating an agglomeration of cells and bacteria. The observed accumulation is disrupted due to the use of GalNAc that was pre-incubated with FN before exposure to PTdNK cells (Fig 3D).

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Fig 3. Binding of Fap2 interaction partners and FN by PTdNKs.

A) Expression of known Fap2 interaction partners TIGIT and Gal-GalNAc on the surface of PTdNK and peripheral blood (pb)NK cells, TIGIT detected via anti-TIGIT antibody and Gal-GalNAc detected by Gal-GalNAc-binding lectin peanut agglutin (PNA), Gray = Background staining, Red = antibody binding; B) Binding of FN to PTdNKs, measured in %CD56/FITC positive cells, comparing untreated PTdNKs and PTdNKs incubated with FN WT and FN Fap2 def. mut., depicted are 1 out of 3 representative experiments, * = p<0.05; C) Inhibition of binding of FN WT to PTdNK cells by preincubation with GalNAc 4.5μM, 22μM and 45μM, measured in %CD56/FITC positive cells compared to untreated control and PTdNK cells incubated with FN without pretreatment, * = p<0.05, ** = p<0.01, depicted are 1 out of 2 representative repeats; D) Immunofluorescence staining of FN WT interaction with PTdNK cells with (right) and without (left) preincubation with GalNAc, magnification 60x, scale bar 60μm, Blue = DAPI, Green = FN WT, Red = Actin.

https://doi.org/10.1371/journal.ppat.1011923.g003

GNLY-tg mice protect fetuses better from FN infection than WT mice

To assess the effect of FN and GNLY in vivo, we utilized humanized GNLY-tg mice [34] and a pregnancy model that was employed previously [27]. In Fig 4A a schematic experimental plan is shown. First, male and female mice were mated, and pregnancy was confirmed via vaginal mucus plug and weight gain. At day E 15.5, the pregnant mice were injected intravenously with FN or PBS, and after 72h the mice were sacrificed. After sacrificing the mice, the fetuses were weighted. The measured weights are depicted in Fig 4B. The fetal weight showed only a significant decrease in the WT mice infected with WT FN, whereas the GNLY-tg mice did not show any diminution of fetal weight compared to the PBS control (Fig 4B). This reduction in weight loss was Fap2 dependent as no reduction was observed when WT or GNLY-tg mice were infected with the Fap2-deficient mutant K50 (Fig 4B). Additionally, we characterized the immune cells’ subsets present in the mice without infection and with FN WT infection (S1A Fig). We see only a slight increase for NCR1⁺ NK cells in the GNLY-tg mice infected with FN WT, whereas the WT mice infected with FN presented an decreased NK cell amount after infection. Regarding NCR1⁺/CD3⁺ NKT cells, both mouse lines showed an increased population after infection (S1B Fig). Observing T cells in the mice after infection, we were able to discover the opposite picture. Here we see decreased CD3⁺ cells in infected GNLY-tg mice and an increased population in the WT mice. The T cells observed in these mice appeared to belong to the CD4⁺ Th cells (S1C Fig).

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Fig 4. Murine GNLY-tg pregnancy model of FN infection in vivo.

(A) Experimental scheme: GNLY-tg and Wildtype (WT) mice are coupled and day E0.5 is determined via vaginal plug discovery, establishing mating success. At day E15.5 FN or PBS is injected intravenously (i.v.). After 72h, the maternal mice are sacrificed, and fetuses were excised and weighted. Created with BioRender.com. (B) Weight of the fetuses derived from mice used in this study comparing the fetus weight between GNLY-tg and WT mice, PBS-, FN WT-, and FN Fap2 def. mut. K50 injected. Weight is given in grams each dot equals one fetus, n>15, (C) Weight of the fetuses derived from mice used in this study comparing the fetus weight between GNLY-tg and WT mice, PBS- and FN WT- injected after depletion of NK cells one day prior to injection via an anti-NK1.1 antibody. Weight is given in grams. Each dot equals one fetus, n>13, * = p<0.05, ** = p<0.01.

https://doi.org/10.1371/journal.ppat.1011923.g004

To demonstrate that the NK cells at the maternal-fetal interface are responsible for this effect, we depleted NK cells using anti-NK1.1 antibody in the pregnant mice one day prior to FN injection. The fetuses that were retained and brought to the E18.5 mark, bear a significantly lower weight compared to the fetuses in the PBS injected control mice, both in WT and GNLY-tg mice (Fig 4C), indicating that NK cells and GNLY play a significant role in mice protection from FN infection.

Discussion

Here we show that PTdNK cells secrete high levels of GNLY when incubated with FN and possess a higher bacteriolytic activity than first pregnancy dNK or pbNK cells. We also prove that the bacteriolytic abilities of PTdNKs are mediated by the GNLY secretion upon interaction with WT FN. It was demonstrated by the Lieberman group that dNK cells kill intracellular pathogens found inside trophoblasts in a process which is independent of degranulation [31]. Here, we show that the GNLY-mediated direct killing of FN by PTdNKs is also degranulation independent and that PTdNKs can kill intracellular as well as extracellular FN. The exact mechanism of killing will be investigated in the future, but we established that the bacteriocidal abilities of the PTdNKs rely on the presence and secretion of GNLY upon activation of the cells. We demonstrate that FN interacts with PTdNK via the bacterial virulence factor Fap2 and the carbohydrate moiety Gal-GalNAc present on PTdNKs but not on pbNK. Indeed, when the Fap2-deficient FN mutant strain, K50, was used a complete abolishment of the bacteriolytic, secretory and even general binding capacity to the PTdNK cells was observed. The same effect was observed when FN WT was incubated with GalNAc before interacting with the NK cells. The expression of Gal-GalNAc on PTdNKs and not on pbNKs is an interesting matter and has not been extensively investigated yet. There are two alternatives that will be investigated in the future: one is elevated levels of GalNAc synthesis in PTdNKs and the other is a removal of terminal sialic acids residues.

Our murine in vivo pregnancy model uncovered that transgene human-GNLY expressing mice are not able to prevent infection with FN of the fetuses, decidua and placenta. Nevertheless, we observed no significant differences in the weight of the fetuses undergoing this infection. In contrast, reduced fetus weights were observed when WT mice that do not express GNLY were used, suggesting that GNLY reduces the infection’s effect on the fetuses. When we depleted NK cells in the GNLY tg mice before injection with FN WT, this effect was abolished, and we were able to see a significant weight loss in the fetuses derived from infected pregnant mice. Thus, GNLY in NK cells is responsible for the observed effects. Nevertheless, we still observed a protective effect in the mice. This is congruent with the findings in S1 Fig showing an increase of T cells in the mice after infection. The maternal-fetal interface is comprised of a high percentage of immune cells, apart from NK cells, which can influence the infection of the fetus and the severity of the infection’s effects [35]. The cells’ composition changes during pregnancy progression, from a predominantly NK cell population in the first trimester, towards a more T cell dependent mixture. The T cells found at the maternal-fetal interface are only in their early stage of being further characterized and are already known to comprise a complex mixture of Th1, Th2, Th17 and Treg cells [35, 36]. GNLY is not only expressed by NK cells, but also by cytotoxic lymphocytes, explaining the remaining effect at the maternal-fetal interface in the NK cell-depleted mice [37].

To put this research into scientific context of NK cell education in pregnancy, we can say that there are definite indices for education of the dNK cells during first pregnancy regarding their reactivity towards pathogenic threats. As mentioned before, the cell population first described in Gamliel et al. termed PTdNK showed a higher abundance of NKG2C and LILRB1, and higher availability of genes involved in secretion of inflammatory molecules [18]. This dNK cells subset was further characterize by the Teichman group while mapping the maternal-fetal interface on the single cell level. Their DNK1 cell population strongly resembles the PTdNK cells [21]. Combining these recent findings with the work from the Lieberman group regarding the involvement of GNLY in the prevention of adverse pregnancy outcomes after infection with Listeria monocytogenes [31], we can position our research in the realm of further investigating and characterizing the mechanisms at play during dNK cell education. So far, the exact mechanism of education, as well as the precise pathway involved in activating the enhanced bacteriolytic properties of the trained dNK cells, have not been unveiled. Showing a concrete way of interaction between the pathogen and the dNK cells poses a major new alleyway for further investigation and characterization of downstream pathways involved in this activation.

To sum up the presented work, we can say that dNK cells get educated and trained during the first pregnancy and are functionally able to protect the fetus better from the harmful effects of maternal infection with FN during pregnancy. The findings can potentially be translated into a more clinical and cancer biologically frame since the association between FN and tumorigenesis are becoming more and more evident and researched [27,38,39]. Modifications of the PTdNK cells helping to fight the bacteria can be proven to be beneficial in other settings as well and combined with modern immunotherapy like CAR-NK cell these results of training can be used to disrupt the tumor microenvironment containing FN leading to a better outcome of immunotherapy.

STAR Methods

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Ethics statement

This study was carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals and the protocol was approved by the Ethics Committee of the Hebrew University of Jerusalem under the approval number: MD-20-16096-3. All efforts were made to reduce the number of animals, refine their living conditions and minimize their suffering.

For human samples the institutional review board of the Hadassah Hospital Medical Center approved the use of decidual material, according to the principles of the Helsinki Declaration (0423-10-HMO). All women were healthy, and pregnancies were terminated due to social reasons. Written informed consent was obtained from the women.

Mice

GNLY-Tg C57BL/6 were described previously [32,34]. Mice were bred and maintained in specific-pathogen free (SPF) conditions within the animal facility of the Hebrew University, Jerusalem until infection with Fusobacterium nucleatum. The experiment was approved by the Ethics Committee of the Hebrew University of Jerusalem under the approval number: MD-20-16096-3. Virgin female mice (6–8 weeks old) were mated with male mice and the appearance of a vaginal plug and weight gain was used to mark E 0.5 and to establish maintenance of pregnancy. Pregnant animals were infected on E 15.5. All pregnant animals were included in the final analysis.

Bacteria

FN strains (WT ATCC 23726 and Fap2-deficient mutant K50) were grown on anaerobic blood agar supplemented with 5% defibrinated sheep blood (Novamed) under anaerobic conditions generated using Oxoid AnaeroGen 2.5L Jars and Sachets (Thermo Fisher).

dNK cells preparation

Preparation of decidual sample was previously described in Gamliel et al. 2018 [18] and were obtained from healthy women who underwent elective first trimester terminations of normal pregnancies. The institutional review board of the Hadassah Hospital Medical Center approved the use of decidual material, according to the principles of the Helsinki Declaration (0423-10-HMO) and informed consent was obtained from all women. All women were healthy, and pregnancies were terminated due to social reasons. Written consent was obtained from the women. A fetal ultrasound was performed prior to all elective terminations to establish the fetal age by means of CRL (crown-rump length) and fetal viability. Decidual tissues were rinsed of blood clots and trimmed to 1mm pieces in 1xPBS supplemented with 1mM of penicillin- streptomycin. Tissue pieces were then transferred to a 50 ml tube with 15 ml warm RPMI-1640 and subjected to enzymatic digestion with 0.1 mg per 1 ml DNase type I (Roche) and 1 mg per 1 ml Collagenase IV (Warthington). Digestion was performed for 20 min in a 37°C water bath with vigorous shaking every 5 min. After collection of the supernatant, the remaining tissue pieces were treated with two additional rounds of digestion. The supernatants obtained from three digestion rounds were passed through a 40-mm cell strainer. Cells were then subjected to sterile density gradient separation by Ficoll and collected at the interface.

Bacteriocidal assay

dNK cells were isolated and prepared as described above and maintained overnight in the IL-15 supplemented NK medium without additions of antibiotics. FN was prepared as described above and distributed in 2.5x10³ CFU per 100μl per well in antibiotic-deprived NK medium. The activated dNK cells were added in various ratios per 100μl per well to the bacteria. This suspension was incubated for 1.5h at 37°C and 700μl UPW were added to dissolve effector cells. The suspension was dropped on anaerobic blood agar plates in triplicates and incubated for 72h at 37°C under anaerobic conditions generated using Oxoid AnaeroGen 2.5L Jars and Sachets (Thermo Fisher). Grown and visible CFUs were counted and normalized to the untreated bacterial control.

For the intracellular infection model, 1x10⁵ RKO cells were incubated with WT FN at MOI 50:1 for 2h at 37°C in DMEM medium without antibiotics. After the infection and washes, the infected cells were incubated with medium containing Gentamicin 50 μg/ml for 1h to kill remaining extracellular bacterial cells. Following this, the cells were incubated with the supernatant gathered from the incubation of PTdNK cells with FN supplemented with and without 5μg GNLY-blocking antibody (Biolegend) for 2h at 37°C. The RKO cells were lysed with sterile UPW and the surviving intracellular bacteria were plated and incubated as described before.

Secretion assay

dNK cells were isolated and prepared as described above and 1 x 10⁵ dNK cells were incubated with various ratios of FN K50 lacking Fap2 [33] and WT strains; 1:1, 1:50, 1:100 and 1:500, for at least 72 h in DMEM medium containing 10% human serum and rhIL-15 (Biolegend) (0.1μ/ml). The resulting supernatants were used for an ELISA against GNLY and IFNg after centrifugation. For assessment of GNLY dependency on the bacteriocidal effect of PTdNKs, additional secretion assays were performed in a similar fashion utilizing the incubation in a ration 1:50 while supplementing 5μg of GNLY-blocking antibody (Biolegend) for 72 h at 37°C.

ELISA

High absorbent ELISA 96 flat plates were precoated with purified anti granulysin and anti IFNγ antibody (Biolegend) 1ug/ml in 50 ul PBS x 1 for 2h at 37°C and blocked by 200 ul of blocking buffer (1% BSA in PBS x 1) for 2h at room temperature. Following 3 times washing with PBS x 1/tween, 100μl of the supernatant from the secretion assay were added and incubated over night at 4°C. After discarding the supernatant and 4 times wash with PBS x 1/tween, the plate was incubated with biotinylated antibody against granulysin and IFNg (Biolegend) 1 ul/ml in 100 ul PBS x 1/tween + 1% BSA for 1h at room temperature. Incubation with Streptavidin HRP (Jackson ImmunoResearch) 1 ul/ml in PBS x 1/tween + 1% BSA for 30min at room temperature was preceded by and followed by wash with PBS x 1/tween, before ultimate incubation with 100μl TMB solution immediately before measuring the fluorescence in the plate reader.

Bacterial binding/FACS stainings/blocking

FN (1x10⁹ CFU/ml) was labeled with fluorescein isothiocyanate (FITC) (Sigma-Aldrich) (0.1 mg/ml in PBS; Sigma-Aldrich) for 30 min at room temperature and washed three times in PBS. FITC labeled bacteria were incubated with dNK cells at various bacteria to cell ratios for 2h at 37°C. Additionally, FN was preincubated with various amounts of GalNAc (Sigma-Aldrich) after labeling, but before incubation with dNK cells. Cells were washed and bacterium binding was detected using flow cytometry.

For immune cells’ characterization in maternal mice, the cells were harvested from the spleen and washed with PBS before staining. For the staining 5x10⁴ cells were resuspended in FACS medium and incubated with various antibodies (NCR1-PE, CD3-APC, CD4-PE and CD8a-PE (Biolegend)) for 30 min at 4°C. After the staining, the cells were washed twice and resuspended in FACS medium for reading. Reading was done in the Cytoflex (Beckman & Coulter) and analysis was performed via FCS Express 6/7.

Immunofluorescence

FN was grown and labeled with FITC as described above. After labeling, 1x10⁶ CFU of the bacteria were fixated onto an 8-chamber slide (Bar-Naor) via 4%PFA over night at 4°C. dNK cells were prepared as described above and after permeabilization of the fixated bacteria, co-incubated on the chamber slide for 2h at 37°C. After co-incubation, the slides were washed with ice-cold 1xPBS (3000 G, 4°C, 5 min) and then blocked using CAS-Block (ThermoFisher Scientific) for 1.5h at 4°C. The cells were washed again in 1xPBS 3 times and then incubated with various antibodies for 2h at 4°C: APC-CD56 (Biolegend); Rhodamine Phalloidine (ThermoFisher Scientific). Then the cells were washed twice in ice-cold 1xPBS. The cells were incubated in the presence of DAPI (Sigma-Aldrich) (0.1 mg/ml, diluted in 1xPBS) for 25 min, washed twice in ice-cold 1xPBS and fixated using 4% PFA (Bar-Naor Ltd) overnight. Finally, the cells were mounted on an 8-chamber slide (Bar-Naor) and visualized using an Olympus Fluoview FV1000 confocal microscope.

Degranulation assay

dNK cells were prepared as described above and incubated with 721.221 cells, 721.221 HLA-G transfectants in a 1:1 ratio, and with FN, both WT and K50 mutant in a 1:50 ratio for 2h at 37°C. dNK degranulation was assessed by FACS using CD56-PE (0.5μl/100μl, BD Biosciences) and CD107a-APC (0.5μl/100μl, Biolegend). For analysis of degranulation, NK cells were gated according to their appearance in the forward/side scatter and for CD56 expression. CD107a positive percentage was determined on the gated NK cell population.

Pregnancy model

C57BL/6 (both WT and GNLY-tg) mice were coupled, and mating was determined by the presence of a white vaginal plug and consecutive weight gain. The day when the plug was detected was termed E0.5 of gestation. The pregnant mice were infected by FN ATCC 23726 wild type (WT) and the FN Fap2 mutant K50 by tail vein injection as follows. An aliquot of 100 mL of the bacterial suspension (5x10⁷ CFU) or sterile PBS was injected into the tail vein. After 72 hours, the placentas, fetuses, deciduas and maternal spleens were harvested from each pregnant mouse and homogenized under sterile conditions.

NK cells were depleted one day before FN injection via i.v. injection of 25μg InVivoMAb anti mouse NK1.1, clone PK136 (Bio X Cell). The injection was applied i.v. due to the progressed pregnancy of the mice.

RNAseq data

Detailed results of the Transcriptome analysis comparing NKG2C^hi and NKG2C^neg cells from multigravid dNK cells is accessible in the GEO database, under accession GSE79879 [18].

Statistical analysis

Statistical analysis was performed by pairwise two-sided Student’s T-test and one-sided Mann-Whitney U test, significance indicated in the figure legends.

Supporting information

S1 Fig. Characterization of immune cells in the maternal mouse before and after infection with FN.

(A) Histograms describing the maternal spleenocytes of GNLY-tg and WT mice, injected with PBS (not infected) and FN. Depicted are double positive stainings with NCR1, CD3; CD4, CD3 and CD8, CD3. The arrows indicate the antibodies. 1 out of 3 representative repeats shown. B) Graph quantifying the percent of positive cells for NCR1 and NCR1/CD3; * = p<0.05, ** = p<0.01. C) Graph quantifying CD3, CD4, CD4/CD3, CD8 and CD8/CD3 positive cells; * = p<0.05, ** = p<0.01.

https://doi.org/10.1371/journal.ppat.1011923.s001

(TIF)

S1 Data. Raw data.

https://doi.org/10.1371/journal.ppat.1011923.s002

(XLSX)

References

1. Koch J, Steinle A, Watzl C, Mandelboim O. Activating natural cytotoxicity receptors of natural killer cells in cancer and infection. Vol. 34, Trends in Immunology. 2013. p. 182–91. pmid:23414611
- View Article
- PubMed/NCBI
- Google Scholar
2. Lanier LL, Le AM, Civin CI, Loken MR, Phillips JH. The relationship of CD16 (Leu-11) and Leu-19 (NKH-1) antigen expression on human peripheral blood NK cells and cytotoxic T lymphocytes. J Immunol [Internet]. 1986 Jun 15;136(12):4480–6. Available from: http://www.ncbi.nlm.nih.gov/pubmed/3086432
- View Article
- Google Scholar
3. Long EO. REGULATION OF IMMUNE RESPONSES THROUGH INHIBITORY RECEPTORS. Annu Rev Immunol [Internet]. 1999 Apr;17(1):875–904. Available from: https://www.annualreviews.org/doi/10.1146/annurev.immunol.17.1.875 pmid:10358776
- View Article
- PubMed/NCBI
- Google Scholar
4. Colonna M, Nakajima H, Navarro F, López-Botet M. A novel family of Ig-like receptors for HLA class I molecules that modulate function of lymphoid and myeloid cells. J Leukoc Biol [Internet]. 1999 Sep;66(3):375–81. Available from: https://onlinelibrary.wiley.com/doi/10.1002/jlb.66.3.375 pmid:10496306
- View Article
- PubMed/NCBI
- Google Scholar
5. Cerwenka A, Lanier LL. Natural killer cells, viruses and cancer. Nat Rev Immunol [Internet]. 2001 Oct;1(1):41–9. Available from: http://www.nature.com/articles/nri35095564 pmid:11905813
- View Article
- PubMed/NCBI
- Google Scholar
6. Gumá M, Angulo A, Vilches C, Gómez-Lozano N, Malats N, López-Botet M. Imprint of human cytomegalovirus infection on the NK cell receptor repertoire. Blood. 2004 Dec 1;104(12):3664–71. pmid:15304389
- View Article
- PubMed/NCBI
- Google Scholar
7. Paust S, Gill HS, Wang BZ, Flynn MP, Moseman EA, Senman B, et al. Critical role for the chemokine receptor CXCR6 in NK cell-mediated antigen-specific memory of haptens and viruses. Nat Immunol. 2010 Dec;11(12):1127–35. pmid:20972432
- View Article
- PubMed/NCBI
- Google Scholar
8. Reeves RK, Li H, Jost S, Blass E, Li H, Schafer JL, et al. Antigen-specific NK cell memory in rhesus macaques. Nat Immunol. 2015 Aug 19;16(9):927–32. pmid:26193080
- View Article
- PubMed/NCBI
- Google Scholar
9. Rölle A, Pollmann J, Cerwenka A. Memory of Infections: An Emerging Role for Natural Killer Cells. Racaniello V, editor. PLoS Pathog [Internet]. 2013 Sep 26;9(9):e1003548. Available from: https://dx.plos.org/10.1371/journal.ppat.1003548 pmid:24086127
- View Article
- PubMed/NCBI
- Google Scholar
10. Sun JC, Beilke JN, Lanier LL. Adaptive immune features of natural killer cells. Nature. 2009 Jan 29;457(7229):557–61. pmid:19136945
- View Article
- PubMed/NCBI
- Google Scholar
11. Gumá M, Budt M, Sáez A, Brckalo T, Hengel H, Angulo A, et al. Expansion of CD94/NKG2C+ NK cells in response to human cytomegalovirus-infected fibroblasts. Blood [Internet]. 2006 May 1;107(9):3624–31. Available from: https://ashpublications.org/blood/article/107/9/3624/133486/Expansion-of-CD94NKG2C-NK-cells-in-response-to pmid:16384928
- View Article
- PubMed/NCBI
- Google Scholar
12. Dogra P, Rancan C, Ma W, Toth M, Senda T, Carpenter DJ, et al. Tissue Determinants of Human NK Cell Development, Function, and Residence. Cell [Internet]. 2020 Feb;180(4):749–763.e13. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0092867420301033 pmid:32059780
- View Article
- PubMed/NCBI
- Google Scholar
13. Freud AG, Mundy-Bosse BL, Yu J, Caligiuri MA. The Broad Spectrum of Human Natural Killer Cell Diversity. Immunity [Internet]. 2017 Nov;47(5):820–33. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1074761317304624 pmid:29166586
- View Article
- PubMed/NCBI
- Google Scholar
14. Hanna J, Wald O, Goldman-Wohl D, Prus D, Markel G, Gazit R, et al. CXCL12 expression by invasive trophoblasts induces the specific migration of CD16- human natural killer cells. Blood. 2003 Sep 1;102(5):1569–77. pmid:12730110
- View Article
- PubMed/NCBI
- Google Scholar
15. Roberts J, Cooper D. Pathogenesis and genetics of pre-eclampsia. Lancet [Internet]. 2001 Jan;357(9249):53–6. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0140673600035777 pmid:11197372
- View Article
- PubMed/NCBI
- Google Scholar
16. Hanna J, Goldman-Wohl D, Hamani Y, Avraham I, Greenfield C, Natanson-Yaron S, et al. Decidual NK cells regulate key developmental processes at the human fetal-maternal interface. Nat Med [Internet]. 2006 Sep 6;12(9):1065–74. Available from: http://www.nature.com/articles/nm1452 pmid:16892062
- View Article
- PubMed/NCBI
- Google Scholar
17. King A, Jokhi PP, Burrows TD, Gardner L, Sharkey AM, Lore YW. Functions of Human Decidual NK Cells. Am J Reprod Immunol [Internet]. 1996 Mar;35(3):258–60. Available from: https://onlinelibrary.wiley.com/doi/10.1111/j.1600-0897.1996.tb00041.x pmid:8962657
- View Article
- PubMed/NCBI
- Google Scholar
18. Gamliel M, Goldman-Wohl D, Isaacson B, Gur C, Stein N, Yamin R, et al. Trained Memory of Human Uterine NK Cells Enhances Their Function in Subsequent Pregnancies. Immunity [Internet]. 2018 May;48(5):951–962.e5. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1074761318301286 pmid:29768178
- View Article
- PubMed/NCBI
- Google Scholar
19. Netea MG, Joosten LAB, Latz E, Mills KHG, Natoli G, Stunnenberg HG, et al. Trained immunity: A program of innate immune memory in health and disease. Vol. 352, Science. American Association for the Advancement of Science; 2016. p. 427. pmid:27102489
- View Article
- PubMed/NCBI
- Google Scholar
20. Prefumo F, Ganapathy R, Thilaganathan B, Sebire NJ. Influence of parity on first trimester endovascular trophoblast invasion. Fertil Steril. 2006 Apr;85(4):1032–6. pmid:16580391
- View Article
- PubMed/NCBI
- Google Scholar
21. Vento-Tormo R, Efremova M, Botting RA, Turco MY, Vento-Tormo M, Meyer KB, et al. Single-cell reconstruction of the early maternal–fetal interface in humans. Nature [Internet]. 2018 Nov 15;563(7731):347–53. Available from: http://www.nature.com/articles/s41586-018-0698-6 pmid:30429548
- View Article
- PubMed/NCBI
- Google Scholar
22. Nahmias AJ, Walls KW, Stewart JA, Herrmann KL, Flynt WJ. The ToRCH complex-perinatal infections associated with toxoplasma and rubella, cytomegol- and herpes simplex viruses. Pediatr Res [Internet]. 1971 Aug;5(8):405–6. Available from: http://www.nature.com/doifinder/10.1203/00006450-197108000-00144
- View Article
- Google Scholar
23. Stegmann BJ, Carey JC. TORCH Infections. Toxoplasmosis, Other (syphilis, varicella-zoster, parvovirus B19), Rubella, Cytomegalovirus (CMV), and Herpes infections. Curr Womens Health Rep [Internet]. 2002 Aug;2(4):253–8. Available from: http://www.ncbi.nlm.nih.gov/pubmed/12150751
- View Article
- Google Scholar
24. Han YW, Fardini Y, Chen C, Iacampo KG, Peraino VA, Shamonki JM, et al. Term Stillbirth Caused by Oral Fusobacterium nucleatum. Obstet Gynecol [Internet]. 2010 Feb;115(2):442–5. Available from: https://journals.lww.com/00006250-201002001-00018 pmid:20093874
- View Article
- PubMed/NCBI
- Google Scholar
25. Han YW, Shi W, Huang GT-J, Kinder Haake S, Park N-H, Kuramitsu H, et al. Interactions between Periodontal Bacteria and Human Oral Epithelial Cells: Fusobacterium nucleatum Adheres to and Invades Epithelial Cells. O’Brien AD, editor. Infect Immun [Internet]. 2000 Jun;68(6):3140–6. Available from: https://journals.asm.org/doi/10.1128/IAI.68.6.3140-3146.2000 pmid:10816455
- View Article
- PubMed/NCBI
- Google Scholar
26. Figuero E, Han YW, Furuichi Y. Periodontal diseases and adverse pregnancy outcomes: Mechanisms. Periodontol 2000 [Internet]. 2020 Jun 8;83(1):175–88. Available from: https://onlinelibrary.wiley.com/doi/10.1111/prd.12295
- View Article
- Google Scholar
27. Parhi L, Abed J, Shhadeh A, Alon-Maimon T, Udi S, Ben-Arye SL, et al. Placental colonization by Fusobacterium nucleatum is mediated by binding of the Fap2 lectin to placentally displayed Gal-GalNAc. Cell Rep [Internet]. 2022 Mar;38(12):110537. Available from: https://linkinghub.elsevier.com/retrieve/pii/S2211124722002789 pmid:35320712
- View Article
- PubMed/NCBI
- Google Scholar
28. Abed J, Emgård JEM, Zamir G, Faroja M, Almogy G, Grenov A, et al. Fap2 Mediates Fusobacterium nucleatum Colorectal Adenocarcinoma Enrichment by Binding to Tumor-Expressed Gal-GalNAc. Cell Host Microbe [Internet]. 2016 Aug;20(2):215–25. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1931312816303055 pmid:27512904
- View Article
- PubMed/NCBI
- Google Scholar
29. Gur C, Ibrahim Y, Isaacson B, Yamin R, Abed J, Gamliel M, et al. Binding of the Fap2 Protein of Fusobacterium nucleatum to Human Inhibitory Receptor TIGIT Protects Tumors from Immune Cell Attack. Immunity [Internet]. 2015 Feb;42(2):344–55. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1074761315000369 pmid:25680274
- View Article
- PubMed/NCBI
- Google Scholar
30. Galaski J, Shhadeh A, Umaña A, Yoo CC, Arpinati L, Isaacson B, et al. Fusobacterium nucleatum CbpF Mediates Inhibition of T Cell Function Through CEACAM1 Activation. Front Cell Infect Microbiol [Internet]. 2021 Jul 15;11. Available from: https://www.frontiersin.org/articles/10.3389/fcimb.2021.692544/full pmid:34336716
- View Article
- PubMed/NCBI
- Google Scholar
31. Crespo ÂC, Mulik S, Dotiwala F, Ansara JA, Sen Santara S, Ingersoll K, et al. Decidual NK Cells Transfer Granulysin to Selectively Kill Bacteria in Trophoblasts. Cell [Internet]. 2020 Sep;182(5):1125–1139.e18. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0092867420308849 pmid:32822574
- View Article
- PubMed/NCBI
- Google Scholar
32. Walch M, Dotiwala F, Mulik S, Thiery J, Kirchhausen T, Clayberger C, et al. Cytotoxic Cells Kill Intracellular Bacteria through Granulysin-Mediated Delivery of Granzymes. Cell [Internet]. 2014 Jun;157(6):1309–23. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0092867414005923 pmid:24906149
- View Article
- PubMed/NCBI
- Google Scholar
33. Coppenhagen-Glazer S, Sol A, Abed J, Naor R, Zhang X, Han YW, et al. Fap2 of Fusobacterium nucleatum Is a Galactose-Inhibitable Adhesin Involved in Coaggregation, Cell Adhesion, and Preterm Birth. Camilli A, editor. Infect Immun [Internet]. 2015 Mar;83(3):1104–13. Available from: https://journals.asm.org/doi/10.1128/IAI.02838-14 pmid:25561710
- View Article
- PubMed/NCBI
- Google Scholar
34. Huang LP, Lyu S-C, Clayberger C, Krensky AM. Granulysin-Mediated Tumor Rejection in Transgenic Mice. J Immunol [Internet]. 2007 Jan 1;178(1):77–84. Available from: http://www.jimmunol.org/lookup/doi/10.4049/jimmunol.178.1.77 pmid:17182542
- View Article
- PubMed/NCBI
- Google Scholar
35. Yang F, Zheng Q, Jin L. Dynamic function and composition changes of immune cells during normal and pathological pregnancy at the maternal-fetal interface. Frontiers in immunology. 2019 Oct 18;10:2317. pmid:31681264
- View Article
- PubMed/NCBI
- Google Scholar
36. Huang Y, Zhu XY, Du MR, Li DJ. Human trophoblasts recruited T lymphocytes and monocytes into decidua by secretion of chemokine CXCL16 and interaction with CXCR6 in the first-trimester pregnancy. The Journal of Immunology. 2008 Feb 15;180(4):2367–75. pmid:18250446
- View Article
- PubMed/NCBI
- Google Scholar
37. Tewary P, Yang D, De La Rosa G, Li Y, Finn MW, Krensky AM, Clayberger C, Oppenheim JJ. Granulysin activates antigen-presenting cells through TLR4 and acts as an immune alarmin. Blood, The Journal of the American Society of Hematology. 2010 Nov 4;116(18):3465–74. pmid:20660289
- View Article
- PubMed/NCBI
- Google Scholar
38. Parhi L, Alon-Maimon T, Sol A, Nejman D, Shhadeh A, Fainsod-Levi T, et al. Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression. Nat Commun [Internet]. 2020 Dec 26;11(1):3259. Available from: https://www.nature.com/articles/s41467-020-16967-2 pmid:32591509
- View Article
- PubMed/NCBI
- Google Scholar
39. Kostic AD, Chun E, Robertson L, Glickman JN, Gallini CA, Michaud M, et al. Fusobacterium nucleatum Potentiates Intestinal Tumorigenesis and Modulates the Tumor-Immune Microenvironment. Cell Host Microbe [Internet]. 2013 Aug;14(2):207–15. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1931312813002552 pmid:23954159
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Koch J, Steinle A, Watzl C, Mandelboim O. Activating natural cytotoxicity receptors of natural killer cells in cancer and infection. Vol. 34, Trends in Immunology. 2013. p. 182–91. pmid:23414611
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Lanier LL, Le AM, Civin CI, Loken MR, Phillips JH. The relationship of CD16 (Leu-11) and Leu-19 (NKH-1) antigen expression on human peripheral blood NK cells and cytotoxic T lymphocytes. J Immunol [Internet]. 1986 Jun 15;136(12):4480–6. Available from: http://www.ncbi.nlm.nih.gov/pubmed/3086432
View Article
Google Scholar

[6] View Article

[7] Google Scholar

[ref3] 3. Long EO. REGULATION OF IMMUNE RESPONSES THROUGH INHIBITORY RECEPTORS. Annu Rev Immunol [Internet]. 1999 Apr;17(1):875–904. Available from: https://www.annualreviews.org/doi/10.1146/annurev.immunol.17.1.875 pmid:10358776
View Article
PubMed/NCBI
Google Scholar

[9] View Article

[10] PubMed/NCBI

[11] Google Scholar

[ref4] 4. Colonna M, Nakajima H, Navarro F, López-Botet M. A novel family of Ig-like receptors for HLA class I molecules that modulate function of lymphoid and myeloid cells. J Leukoc Biol [Internet]. 1999 Sep;66(3):375–81. Available from: https://onlinelibrary.wiley.com/doi/10.1002/jlb.66.3.375 pmid:10496306
View Article
PubMed/NCBI
Google Scholar

[13] View Article

[14] PubMed/NCBI

[15] Google Scholar

[ref5] 5. Cerwenka A, Lanier LL. Natural killer cells, viruses and cancer. Nat Rev Immunol [Internet]. 2001 Oct;1(1):41–9. Available from: http://www.nature.com/articles/nri35095564 pmid:11905813
View Article
PubMed/NCBI
Google Scholar

[17] View Article

[18] PubMed/NCBI

[19] Google Scholar

[ref6] 6. Gumá M, Angulo A, Vilches C, Gómez-Lozano N, Malats N, López-Botet M. Imprint of human cytomegalovirus infection on the NK cell receptor repertoire. Blood. 2004 Dec 1;104(12):3664–71. pmid:15304389
View Article
PubMed/NCBI
Google Scholar

[21] View Article

[22] PubMed/NCBI

[23] Google Scholar

[ref7] 7. Paust S, Gill HS, Wang BZ, Flynn MP, Moseman EA, Senman B, et al. Critical role for the chemokine receptor CXCR6 in NK cell-mediated antigen-specific memory of haptens and viruses. Nat Immunol. 2010 Dec;11(12):1127–35. pmid:20972432
View Article
PubMed/NCBI
Google Scholar

[25] View Article

[26] PubMed/NCBI

[27] Google Scholar

[ref8] 8. Reeves RK, Li H, Jost S, Blass E, Li H, Schafer JL, et al. Antigen-specific NK cell memory in rhesus macaques. Nat Immunol. 2015 Aug 19;16(9):927–32. pmid:26193080
View Article
PubMed/NCBI
Google Scholar

[29] View Article

[30] PubMed/NCBI

[31] Google Scholar

[ref9] 9. Rölle A, Pollmann J, Cerwenka A. Memory of Infections: An Emerging Role for Natural Killer Cells. Racaniello V, editor. PLoS Pathog [Internet]. 2013 Sep 26;9(9):e1003548. Available from: https://dx.plos.org/10.1371/journal.ppat.1003548 pmid:24086127
View Article
PubMed/NCBI
Google Scholar

[33] View Article

[34] PubMed/NCBI

[35] Google Scholar

[ref10] 10. Sun JC, Beilke JN, Lanier LL. Adaptive immune features of natural killer cells. Nature. 2009 Jan 29;457(7229):557–61. pmid:19136945
View Article
PubMed/NCBI
Google Scholar

[37] View Article

[38] PubMed/NCBI

[39] Google Scholar

[ref11] 11. Gumá M, Budt M, Sáez A, Brckalo T, Hengel H, Angulo A, et al. Expansion of CD94/NKG2C+ NK cells in response to human cytomegalovirus-infected fibroblasts. Blood [Internet]. 2006 May 1;107(9):3624–31. Available from: https://ashpublications.org/blood/article/107/9/3624/133486/Expansion-of-CD94NKG2C-NK-cells-in-response-to pmid:16384928
View Article
PubMed/NCBI
Google Scholar

[41] View Article

[42] PubMed/NCBI

[43] Google Scholar

[ref12] 12. Dogra P, Rancan C, Ma W, Toth M, Senda T, Carpenter DJ, et al. Tissue Determinants of Human NK Cell Development, Function, and Residence. Cell [Internet]. 2020 Feb;180(4):749–763.e13. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0092867420301033 pmid:32059780
View Article
PubMed/NCBI
Google Scholar

[45] View Article

[46] PubMed/NCBI

[47] Google Scholar

[ref13] 13. Freud AG, Mundy-Bosse BL, Yu J, Caligiuri MA. The Broad Spectrum of Human Natural Killer Cell Diversity. Immunity [Internet]. 2017 Nov;47(5):820–33. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1074761317304624 pmid:29166586
View Article
PubMed/NCBI
Google Scholar

[49] View Article

[50] PubMed/NCBI

[51] Google Scholar

[ref14] 14. Hanna J, Wald O, Goldman-Wohl D, Prus D, Markel G, Gazit R, et al. CXCL12 expression by invasive trophoblasts induces the specific migration of CD16- human natural killer cells. Blood. 2003 Sep 1;102(5):1569–77. pmid:12730110
View Article
PubMed/NCBI
Google Scholar

[53] View Article

[54] PubMed/NCBI

[55] Google Scholar

[ref15] 15. Roberts J, Cooper D. Pathogenesis and genetics of pre-eclampsia. Lancet [Internet]. 2001 Jan;357(9249):53–6. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0140673600035777 pmid:11197372
View Article
PubMed/NCBI
Google Scholar

[57] View Article

[58] PubMed/NCBI

[59] Google Scholar

[ref16] 16. Hanna J, Goldman-Wohl D, Hamani Y, Avraham I, Greenfield C, Natanson-Yaron S, et al. Decidual NK cells regulate key developmental processes at the human fetal-maternal interface. Nat Med [Internet]. 2006 Sep 6;12(9):1065–74. Available from: http://www.nature.com/articles/nm1452 pmid:16892062
View Article
PubMed/NCBI
Google Scholar

[61] View Article

[62] PubMed/NCBI

[63] Google Scholar

[ref17] 17. King A, Jokhi PP, Burrows TD, Gardner L, Sharkey AM, Lore YW. Functions of Human Decidual NK Cells. Am J Reprod Immunol [Internet]. 1996 Mar;35(3):258–60. Available from: https://onlinelibrary.wiley.com/doi/10.1111/j.1600-0897.1996.tb00041.x pmid:8962657
View Article
PubMed/NCBI
Google Scholar

[65] View Article

[66] PubMed/NCBI

[67] Google Scholar

[ref18] 18. Gamliel M, Goldman-Wohl D, Isaacson B, Gur C, Stein N, Yamin R, et al. Trained Memory of Human Uterine NK Cells Enhances Their Function in Subsequent Pregnancies. Immunity [Internet]. 2018 May;48(5):951–962.e5. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1074761318301286 pmid:29768178
View Article
PubMed/NCBI
Google Scholar

[69] View Article

[70] PubMed/NCBI

[71] Google Scholar

[ref19] 19. Netea MG, Joosten LAB, Latz E, Mills KHG, Natoli G, Stunnenberg HG, et al. Trained immunity: A program of innate immune memory in health and disease. Vol. 352, Science. American Association for the Advancement of Science; 2016. p. 427. pmid:27102489
View Article
PubMed/NCBI
Google Scholar

[73] View Article

[74] PubMed/NCBI

[75] Google Scholar

[ref20] 20. Prefumo F, Ganapathy R, Thilaganathan B, Sebire NJ. Influence of parity on first trimester endovascular trophoblast invasion. Fertil Steril. 2006 Apr;85(4):1032–6. pmid:16580391
View Article
PubMed/NCBI
Google Scholar

[77] View Article

[78] PubMed/NCBI

[79] Google Scholar

[ref21] 21. Vento-Tormo R, Efremova M, Botting RA, Turco MY, Vento-Tormo M, Meyer KB, et al. Single-cell reconstruction of the early maternal–fetal interface in humans. Nature [Internet]. 2018 Nov 15;563(7731):347–53. Available from: http://www.nature.com/articles/s41586-018-0698-6 pmid:30429548
View Article
PubMed/NCBI
Google Scholar

[81] View Article

[82] PubMed/NCBI

[83] Google Scholar

[ref22] 22. Nahmias AJ, Walls KW, Stewart JA, Herrmann KL, Flynt WJ. The ToRCH complex-perinatal infections associated with toxoplasma and rubella, cytomegol- and herpes simplex viruses. Pediatr Res [Internet]. 1971 Aug;5(8):405–6. Available from: http://www.nature.com/doifinder/10.1203/00006450-197108000-00144
View Article
Google Scholar

[85] View Article

[86] Google Scholar

[ref23] 23. Stegmann BJ, Carey JC. TORCH Infections. Toxoplasmosis, Other (syphilis, varicella-zoster, parvovirus B19), Rubella, Cytomegalovirus (CMV), and Herpes infections. Curr Womens Health Rep [Internet]. 2002 Aug;2(4):253–8. Available from: http://www.ncbi.nlm.nih.gov/pubmed/12150751
View Article
Google Scholar

[88] View Article

[89] Google Scholar

[ref24] 24. Han YW, Fardini Y, Chen C, Iacampo KG, Peraino VA, Shamonki JM, et al. Term Stillbirth Caused by Oral Fusobacterium nucleatum. Obstet Gynecol [Internet]. 2010 Feb;115(2):442–5. Available from: https://journals.lww.com/00006250-201002001-00018 pmid:20093874
View Article
PubMed/NCBI
Google Scholar

[91] View Article

[92] PubMed/NCBI

[93] Google Scholar

[ref25] 25. Han YW, Shi W, Huang GT-J, Kinder Haake S, Park N-H, Kuramitsu H, et al. Interactions between Periodontal Bacteria and Human Oral Epithelial Cells: Fusobacterium nucleatum Adheres to and Invades Epithelial Cells. O’Brien AD, editor. Infect Immun [Internet]. 2000 Jun;68(6):3140–6. Available from: https://journals.asm.org/doi/10.1128/IAI.68.6.3140-3146.2000 pmid:10816455
View Article
PubMed/NCBI
Google Scholar

[95] View Article

[96] PubMed/NCBI

[97] Google Scholar

[ref26] 26. Figuero E, Han YW, Furuichi Y. Periodontal diseases and adverse pregnancy outcomes: Mechanisms. Periodontol 2000 [Internet]. 2020 Jun 8;83(1):175–88. Available from: https://onlinelibrary.wiley.com/doi/10.1111/prd.12295
View Article
Google Scholar

[99] View Article

[100] Google Scholar

[ref27] 27. Parhi L, Abed J, Shhadeh A, Alon-Maimon T, Udi S, Ben-Arye SL, et al. Placental colonization by Fusobacterium nucleatum is mediated by binding of the Fap2 lectin to placentally displayed Gal-GalNAc. Cell Rep [Internet]. 2022 Mar;38(12):110537. Available from: https://linkinghub.elsevier.com/retrieve/pii/S2211124722002789 pmid:35320712
View Article
PubMed/NCBI
Google Scholar

[102] View Article

[103] PubMed/NCBI

[104] Google Scholar

[ref28] 28. Abed J, Emgård JEM, Zamir G, Faroja M, Almogy G, Grenov A, et al. Fap2 Mediates Fusobacterium nucleatum Colorectal Adenocarcinoma Enrichment by Binding to Tumor-Expressed Gal-GalNAc. Cell Host Microbe [Internet]. 2016 Aug;20(2):215–25. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1931312816303055 pmid:27512904
View Article
PubMed/NCBI
Google Scholar

[106] View Article

[107] PubMed/NCBI

[108] Google Scholar

[ref29] 29. Gur C, Ibrahim Y, Isaacson B, Yamin R, Abed J, Gamliel M, et al. Binding of the Fap2 Protein of Fusobacterium nucleatum to Human Inhibitory Receptor TIGIT Protects Tumors from Immune Cell Attack. Immunity [Internet]. 2015 Feb;42(2):344–55. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1074761315000369 pmid:25680274
View Article
PubMed/NCBI
Google Scholar

[110] View Article

[111] PubMed/NCBI

[112] Google Scholar

[ref30] 30. Galaski J, Shhadeh A, Umaña A, Yoo CC, Arpinati L, Isaacson B, et al. Fusobacterium nucleatum CbpF Mediates Inhibition of T Cell Function Through CEACAM1 Activation. Front Cell Infect Microbiol [Internet]. 2021 Jul 15;11. Available from: https://www.frontiersin.org/articles/10.3389/fcimb.2021.692544/full pmid:34336716
View Article
PubMed/NCBI
Google Scholar

[114] View Article

[115] PubMed/NCBI

[116] Google Scholar

[ref31] 31. Crespo ÂC, Mulik S, Dotiwala F, Ansara JA, Sen Santara S, Ingersoll K, et al. Decidual NK Cells Transfer Granulysin to Selectively Kill Bacteria in Trophoblasts. Cell [Internet]. 2020 Sep;182(5):1125–1139.e18. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0092867420308849 pmid:32822574
View Article
PubMed/NCBI
Google Scholar

[118] View Article

[119] PubMed/NCBI

[120] Google Scholar

[ref32] 32. Walch M, Dotiwala F, Mulik S, Thiery J, Kirchhausen T, Clayberger C, et al. Cytotoxic Cells Kill Intracellular Bacteria through Granulysin-Mediated Delivery of Granzymes. Cell [Internet]. 2014 Jun;157(6):1309–23. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0092867414005923 pmid:24906149
View Article
PubMed/NCBI
Google Scholar

[122] View Article

[123] PubMed/NCBI

[124] Google Scholar

[ref33] 33. Coppenhagen-Glazer S, Sol A, Abed J, Naor R, Zhang X, Han YW, et al. Fap2 of Fusobacterium nucleatum Is a Galactose-Inhibitable Adhesin Involved in Coaggregation, Cell Adhesion, and Preterm Birth. Camilli A, editor. Infect Immun [Internet]. 2015 Mar;83(3):1104–13. Available from: https://journals.asm.org/doi/10.1128/IAI.02838-14 pmid:25561710
View Article
PubMed/NCBI
Google Scholar

[126] View Article

[127] PubMed/NCBI

[128] Google Scholar

[ref34] 34. Huang LP, Lyu S-C, Clayberger C, Krensky AM. Granulysin-Mediated Tumor Rejection in Transgenic Mice. J Immunol [Internet]. 2007 Jan 1;178(1):77–84. Available from: http://www.jimmunol.org/lookup/doi/10.4049/jimmunol.178.1.77 pmid:17182542
View Article
PubMed/NCBI
Google Scholar

[130] View Article

[131] PubMed/NCBI

[132] Google Scholar

[ref35] 35. Yang F, Zheng Q, Jin L. Dynamic function and composition changes of immune cells during normal and pathological pregnancy at the maternal-fetal interface. Frontiers in immunology. 2019 Oct 18;10:2317. pmid:31681264
View Article
PubMed/NCBI
Google Scholar

[134] View Article

[135] PubMed/NCBI

[136] Google Scholar

[ref36] 36. Huang Y, Zhu XY, Du MR, Li DJ. Human trophoblasts recruited T lymphocytes and monocytes into decidua by secretion of chemokine CXCL16 and interaction with CXCR6 in the first-trimester pregnancy. The Journal of Immunology. 2008 Feb 15;180(4):2367–75. pmid:18250446
View Article
PubMed/NCBI
Google Scholar

[138] View Article

[139] PubMed/NCBI

[140] Google Scholar

[ref37] 37. Tewary P, Yang D, De La Rosa G, Li Y, Finn MW, Krensky AM, Clayberger C, Oppenheim JJ. Granulysin activates antigen-presenting cells through TLR4 and acts as an immune alarmin. Blood, The Journal of the American Society of Hematology. 2010 Nov 4;116(18):3465–74. pmid:20660289
View Article
PubMed/NCBI
Google Scholar

[142] View Article

[143] PubMed/NCBI

[144] Google Scholar

[ref38] 38. Parhi L, Alon-Maimon T, Sol A, Nejman D, Shhadeh A, Fainsod-Levi T, et al. Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression. Nat Commun [Internet]. 2020 Dec 26;11(1):3259. Available from: https://www.nature.com/articles/s41467-020-16967-2 pmid:32591509
View Article
PubMed/NCBI
Google Scholar

[146] View Article

[147] PubMed/NCBI

[148] Google Scholar

[ref39] 39. Kostic AD, Chun E, Robertson L, Glickman JN, Gallini CA, Michaud M, et al. Fusobacterium nucleatum Potentiates Intestinal Tumorigenesis and Modulates the Tumor-Immune Microenvironment. Cell Host Microbe [Internet]. 2013 Aug;14(2):207–15. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1931312813002552 pmid:23954159
View Article
PubMed/NCBI
Google Scholar

[150] View Article

[151] PubMed/NCBI

[152] Google Scholar

Figures

Abstract

Author summary

Introduction

Results

PTdNK cells secrete more GNLY when challenged with FN

FN is killed efficiently by PTdNK cells in a degranulation independent mechanism

FN binds to PTdNK cells via Gal-GalNAc and not TIGIT

GNLY-tg mice protect fetuses better from FN infection than WT mice

Discussion

STAR Methods

Ethics statement

Mice

Bacteria

dNK cells preparation

Bacteriocidal assay

Secretion assay

ELISA

Bacterial binding/FACS stainings/blocking

Immunofluorescence

Degranulation assay

Pregnancy model

RNAseq data

Statistical analysis

Supporting information

S1 Fig. Characterization of immune cells in the maternal mouse before and after infection with FN.

S1 Data. Raw data.

References