Priscilla Ajiji

Anil Uzunali

Emmanuelle Ripoche

Emilie Vittaz

Thierry Vial

Patrick Maison
email: patrick.maison@ansm.sante.fr

MD Pr Patrick Maison

Investigating the efficacy and safety of metronidazole during pregnancy; A systematic review and meta-analysis

Keywords: Metronidazole/adverse effects, Pregnancy, Premature Birth, Congenital Abnormalities

scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Results:

Twenty-four studies (17 observational studies and 7 RCTs) were selected. Pooled RR was 1.10 (95% CI 0.78-1.55; n=7; I 2 = 72%) for preterm birth. Subgroup analysis found RR 1.67; 95% CI 1.07-2.62; n=3; I²=32%) for treatment duration of ≤3 days among women with a previous preterm delivery. Pooled OR for spontaneous abortion was 1.72 (95% CI 1.40-2.12; n=5; I 2 =72%) and 1.15 (95% CI 0.98-1.34; n=12; I 2 = 25%) for major malformations. After exclusion of studies with critical risk of bias, pooled OR were 1.7 (1.42-2.04; n=3; I 2 = 19%) and 1.13 (0.93-1.36; n=9; I 2 = 28%) respectively. Among several specific malformations analyzed, only congenital hydrocephaly was significantly increased at 4.06 (95% CI 1.75-9.42; n=2; I²= 0%).

Introduction

Metronidazole (MET) is an imidazole derivative acting both as an antiprotozoal and anti-bacterial agent [START_REF] Wang | Pharmacokinetics of metronidazole in pregnant patients with bacterial vaginosis[END_REF]. It is commonly used to treat genitourinary tract infections in pregnant women [START_REF] Freyer | Drugs in Pregnancy and Lactation 8th Edition: A Reference Guide to Fetal and Neonatal Risk[END_REF]. A study conducted in 2010 found a prevalence of 7.1% of bacterial vaginosis among [START_REF] Carr-Hill | The repetition of spontaneous preterm labour[END_REF]193 pregnant women in France [START_REF] Desseauve | Prevalence and risk factors of bacterial vaginosis during the first trimester of pregnancy in a large French populationbased study[END_REF]. In the United States (US), Koumans et al found that 21 million women suffered from bacterial vaginosis between 2001 and 2004 [START_REF] Koumans | The Prevalence of Bacterial Vaginosis in the United States, 2001-2004; Associations With Symptoms, Sexual Behaviors, and Reproductive Health[END_REF]. Accordingly, MET is used to prevent preterm birth [START_REF] Mcdonald | Impact of metronidazole therapy on preterm birth in women with bacterial vaginosis flora (Gardnerella vaginalis): a randomised, placebo controlled trial[END_REF] in pregnant women with bacterial vaginosis or trichomoniasis.

Inconsistent data regarding the efficacy of MET but also its safety for the mother and newborn has led to conflicting guidelines on its use during pregnancy [START_REF] Schmitz | Preterm premature rupture of the membranes: Guidelines for clinical practice from the French College of Gynaecologists and Obstetricians (CNGOF)[END_REF] [START_REF] Sherrard | European (IUSTI/WHO) guideline on the management of vaginal discharge, 2011[END_REF]. Randomized Controlled Trials (RCTs) have evaluated the efficacy of MET in reducing the risk of preterm birth among pregnant women with either asymptomatic [START_REF] Klebanoff | Failure of Metronidazole to Prevent Preterm Delivery among Pregnant Women with Asymptomatic Trichomonas vaginalis Infection[END_REF] or symptomatic vaginosis, [START_REF] Mcdonald | Impact of metronidazole therapy on preterm birth in women with bacterial vaginosis flora (Gardnerella vaginalis): a randomised, placebo controlled trial[END_REF] which may be coupled with either a positive fetal fibronectin test [START_REF] Shennan | A randomised controlled trial of metronidazole for the prevention of preterm birth in women positive for cervicovaginal fetal fibronectin: the PREMET Study[END_REF] [START_REF] Goldenberg | Metronidazole treatment of women with a positive fetal fibronectin test result[END_REF] or a previous preterm birth [START_REF] Odendaal | Preterm labour--is bacterial vaginosis involved?[END_REF] [START_REF] Morales | Effect of metronidazole in patients with preterm birth in preceding pregnancy and bacterial vaginosis: a placebo-controlled, double-blind study[END_REF].

The findings give conflicting information whereby, researchers have demonstrated both reduction [START_REF] Mcdonald | Impact of metronidazole therapy on preterm birth in women with bacterial vaginosis flora (Gardnerella vaginalis): a randomised, placebo controlled trial[END_REF] [START_REF] Morales | Effect of metronidazole in patients with preterm birth in preceding pregnancy and bacterial vaginosis: a placebo-controlled, double-blind study[END_REF] or increase [START_REF] Klebanoff | Failure of Metronidazole to Prevent Preterm Delivery among Pregnant Women with Asymptomatic Trichomonas vaginalis Infection[END_REF] [START_REF] Morency | The effect of second-trimester antibiotic therapy on the rate of preterm birth[END_REF] in the incidence of preterm birth. Others found the risk is increased among women with a history of preterm birth [START_REF] Odendaal | Preterm labour--is bacterial vaginosis involved?[END_REF] which is cited as the most leading risk factor [START_REF] Carr-Hill | The repetition of spontaneous preterm labour[END_REF] [START_REF] Mercer | The preterm prediction study: effect of gestational age and cause of preterm birth on subsequent obstetric outcome. National Institute of Child Health and Human Development Maternal-Fetal Medicine Units Network[END_REF] [START_REF] Mercer | Are women with recurrent spontaneous preterm births different from those without such history?[END_REF].

The Safety of MET treatment during pregnancy has also been evaluated, whereby some studies found an association between MET and congenital malformations [START_REF] Czeizel | A population based case-control teratologic study of oral metronidazole treatment during pregnancy[END_REF] [START_REF] Muanda | Use of antibiotics during pregnancy and the risk of major congenital malformations: a population based cohort study[END_REF] or even spontaneous abortion [START_REF] Muanda | Use of antibiotics during pregnancy and risk of spontaneous abortion[END_REF]. Two meta-analyses conducted in 1995 [START_REF] Burtin | Safety of metronidazole in pregnancy: A meta-analysis[END_REF] and 1997 [START_REF] Caro-Patón | Is metronidazole teratogenic? A meta-analysis[END_REF] found no association between the risk of malformations and MET exposure. Since then, several studies have been published and some indicated an increased risk of major malformations [START_REF] Czeizel | A population based case-control teratologic study of oral metronidazole treatment during pregnancy[END_REF], congenital hydrocephaly [START_REF] Kazy | Teratogenic potential of vaginal metronidazole treatment during pregnancy[END_REF] and clubfoot [START_REF] Werler | Medication Use in Pregnancy in Relation to the Risk of Isolated Clubfoot in Offspring[END_REF]. Safety has also been discussed with regards to low birth weight, [START_REF] Koss | Investigation of Metronidazole Use during Pregnancy and Adverse Birth Outcomes[END_REF] [START_REF] Morgan | Metronidazole treatment in pregnancy[END_REF] and stillbirth [START_REF] Piper | Prenatal use of metronidazole and birth defects: no association[END_REF].

Available in oral, rectal, topical, vaginal and intravenous forms(1), MET is well absorbed, with an elimination half-life of six to eight hours in healthy subjects [START_REF] Murphy | Use of oral metronidazole in pregnancy. Risks, benefits, and practice guidelines[END_REF]. Whereas literature documents no difference in cure rates of different doses, durations of treatment [START_REF] Lugo-Miro | Comparison of different metronidazole therapeutic regimens for bacterial vaginosis. A meta-analysis[END_REF] and routes of administration [START_REF] Mitchell | Comparison of oral and vaginal metronidazole for treatment of bacterial vaginosis in pregnancy: impact on fastidious bacteria[END_REF], the impact of these parameters on adverse pregnancy outcomes remains controversial. Therefore, this meta-analysis aims to review and analyze studies focusing on the efficacy and safety of MET taking into account the different doses, duration of treatment and routes of administration.

Methods

Data sources

We conducted a systematic review and meta-analysis on literature citing MET treatment during pregnancy. Electronic databases searched were Embase, Pubmed and Cochrane Central Register of Controlled Trials (CENTRAL) up to 29 July 2019 using the keywords "metronidazole", "fetal or embryo or offspring", "adverse or risk or side or toxicity or undesirable or complication or event or outcome", and "congenital malformation or preterm or abortion or stillbirth or miscarriage or small for gestational age or growth retardation or birth weight or gestational age or birth defect or neonatal or teratogen or developmental disorders or child or neurodevelopment or cognitive or developmental disability or learning disorder or intelligence or cognition" without restrictions on language.

Other sources included: hand searching references from eligible studies, abstracts presented at conferences, communications with experts in the field of medicines and pregnancy, the International Prospective Register of Systematic Review Protocols (PROSPERO) and the Electronic Thesis Online Service (ETHOS). Corresponding authors were contacted to obtain data for publications without specific analysis when MET was studied along with other drugs.

Study selection

RCTs and observational studies i.e. case-control and retrospective or prospective cohort studies citing MET use during pregnancy were considered eligible. We included observational studies with control groups which included 1) unexposed with disease 2) unexposed, disease-free 3) exposed to other treatment with disease or 4) unexposed (unspecified). Case reports, systematic reviews, meta-analysis and letters to editors or commentaries were excluded, in addition to publications with insufficient data to reconstruct two by two contingency tables, even after having contacted corresponding authors.

Exposure was determined as randomized treatment with MET for RCTs, or prescription of MET for observational studies, whatever the route of administration, dose and duration of treatment or even period of exposure.

Selected outcomes were: Preterm births due to spontaneous or indicated preterm labour (<37 weeks) and associated delivery outcomes such as spontaneous abortions (death before 20 weeks of gestation), stillbirths (death after 20 weeks), low birth weight <2500g and caesarian delivery irrespective of the period of exposure. We also explored the risk of preterm birth among women with a history of preterm birth, as a high-risk group.

Other outcomes were major malformations after first-trimester exposure, which were defined by using the Brighton Collaboration Congenital Anomalies Working Group [START_REF] Desilva | Congenital anomalies: Case definition and guidelines for data collection, analysis, and presentation of immunization safety data[END_REF] Results were pooled using Risk Ratios (RR) for RCTs and Odds Ratios (OR) for observational studies with corresponding 95% confidence interval (CI) calculated using the random-effects model, developed by DerSimonian and Laird in 1986 [START_REF] Dersimonian | Meta-analysis in clinical trials[END_REF]. Heterogeneity was assessed utilizing Q and I-square statistics. The analysis was performed using R [START_REF]The R Project for Statistical Computing[END_REF] with the packages meta (37) and metafor [START_REF] Viechtbauer | Conducting Meta-Analyses in R with the metafor Package[END_REF].

Subgroup analysis was conducted to explore differences in effect size between and across different groups. These include (1) indication for MET, (2) period of exposure, (3) dose, (4) duration, (5) route of administration and (6) study design. Sensitivity analysis was done to ascertain the robustness of our results, first by excluding studies with the critical risk of bias from the analysis and secondly, excluding studies whose overall effect was not adjusted.

Results

Twenty four studies published between 1965 and 2019 were selected for analysis i.e. 17

observational studies (5 case-control and 12 cohort studies) and 7 RCTs (Figure 1). and exposed to other treatment (2 studies). Thirteen studies reported exposure in at least the first trimester while 9 reported exposure to MET outside the first-trimester (Table 1).

Risk of bias assessment

Four observational studies had a "critical" level of bias arising from bias in reporting, assessment of exposure and non-adjustment of confounding factors. Only one RCTs evaluated with ROB2 assessment had "some concerns" due to the selection of the reported result. A summary of ROB2

or ROBINS-I assessment is provided in appendix B.

Preterm birth and associated delivery outcomes following MET exposure in utero

Figure 2A presents results for the efficacy of MET in reducing the risk of preterm birth among pregnant women. The overall RR was 1.10 (95% CI 0.78-1.55; n=7; I 2 =72%). Subgroup analysis showed significant results for dose ≤250mg (RR 0.41; 95% CI 0.20-0.85; n=1) and thirdtrimester exposure (RR 1.59; 95% CI 1.05-2.41; n=1). No significant effect on preterm birth was found after subgroup analyses for the route of administration, treatment duration, effect estimates and the type of control group (Supplementary Figure A1).

Conversely, 5 observational studies (24) (40) (42) (45) (48) evaluated the safety of MET exposure (OR 0.96; 95% CI 0.80-1.16; I2= 39%, Figure 2B). Subgroup analysis showed significant results for third trimester exposure (OR 0.81; 95% CI 0.66-0.98; n=1) but not for study design, dose, route of administration, treatment duration, effect estimates and the type of control group (Supplementary Figure A2).

Preterm birth among women with a history of preterm birth

We identified 4 RCTs (8) (11) (12) (39) evaluating the use of MET in an attempt to reduce the risk of preterm birth among women with a preceding preterm birth (128 cases and 342 controls)

(Figure 2A). The control groups consisted of unexposed sick women with a history of preterm birth. Global results were not statistically significant however, an increased effect was found for the treatment duration of ≤3 days and dose of 400mg-2g (RR 1.67; 95% CI 1.07-2.62; n=3; I²=32%) compared to a duration of 7 days and dose ≤250mg (RR 0.26; 95% CI 0.10-0.63; n=1), Supplementary Figure A3.

Regarding the safety of MET, only 1 study [START_REF] Koss | Investigation of Metronidazole Use during Pregnancy and Adverse Birth Outcomes[END_REF] was identified. Women with a history of preterm birth exposed to MET were 2.94 times more likely to have a preterm birth compared to their unexposed counterparts (Figure 2B).

Spontaneous abortions

One RCT [START_REF] Morales | Effect of metronidazole in patients with preterm birth in preceding pregnancy and bacterial vaginosis: a placebo-controlled, double-blind study[END_REF] documented spontaneous abortion as an outcome when MET was used in an attempt to reduce the risk of preterm birth (Figure 2A).

Five observational studies (19) (40) (43) (44) (46) evaluated the association of spontaneous abortion and MET exposure (Figure 2B). Subgroup analysis showed a statistically significant effect for women exposed in the first trimester (figure 3). The results of sensitivity analysis remained similar to the original result after the exclusion of two studies with a "critical" risk of bias (OR 1.7; 95% CI 1.42-2.04; n=3; I²=19%).

Figure 3: Subgroup analysis of spontaneous abortion after maternal metronidazole exposure

Stillbirths

Only 1 RCT (8) documented stillbirth as an outcome when MET was used in an attempt to reduce the risk of preterm birth (Figure 2A). Regarding safety, we included 2108 exposed and 2202 unexposed pregnant women from 3 observational studies (25) (26) [START_REF] Diav-Citrin | Pregnancy outcome after gestational exposure to metronidazole: a prospective controlled cohort study[END_REF]. No statistically significant association was found between stillbirth and MET exposure at any point during pregnancy (Figure 2B). Subgroup analysis was not conducted due to the limited number of studies.

Low birth weight

We included 4 RCTs (8) (9) (12) (39) documenting low birth weight as an outcome when MET was used to reduce the incidence of preterm birth. No statistically significant association was found between low birth weight and MET exposure during pregnancy, even after subgroup analysis. (Supplementary Figure A4).

Based on three observational studies(24)(25)(42) (2,532 cases and 38,460 controls), no statistically significant association was found between low birth weight and MET exposure during pregnancy (Figure 2B). Subgroup analysis was not conducted due to the limited number of studies.

Caesarian delivery

Based on two RCTs (8) (9), 195 pregnant patients were included in the analysis. The risk of caesarian delivery was not significantly decreased after exposure to MET at any point during pregnancy (Figure 2A).

Major congenital malformations

Twelve (10 cohort, 2 case-control) studies( 17

(48) [START_REF] Muanda | Use of antibiotics during pregnancy and the risk of major congenital malformations: a population based cohort study[END_REF] were included in the analysis. Out of 411,380 pregnant women, 57,718 were cases in which 440 were exposed at least in the first-trimester (OR 1.15: 95% CI 0.98-1.34; I²=25%, Figure 4). The odds of major malformations were increased for case-control studies (OR 1.32; 95% CI 1.02-1.70; n=3, I²=35%) but not with studies with a higher level of evidence (prospective and retrospective cohort studies). No difference was found between the systemic and local route of administration. After the exclusion of 3 studies with a "critical" level of bias, the results remained similar to the original result. Further sensitivity analysis based on adjusted effect estimates (after excluding 3 studies with crude effect estimates showed an increased result of 1.22 (95% CI 1.05-1.40; n=9; I²=7%).

Figure 4: Major malformations and subgroup analysis after maternal metronidazole exposure

Specific malformations

Results of specific malformations were grouped for visualization purposes. Among 10 types of malformations, only the odds of congenital hydrocephaly were significantly increased (OR 4.06, 95%CI 1.75-9.42, n=2; I 2 =0 %) after first-trimester exposure to MET (Figure 5).

Discussion

This systematic review and meta-analysis of 24 studies on MET exposure during pregnancy including 163,104 cases and 637,091 controls did not confirm the efficacy of MET in reducing the risk of preterm birth. Regarding other adverse pregnancy outcomes, a significant effect was observed between spontaneous abortion and MET exposure.

Preterm birth and associated delivery outcomes

No relationship was found between MET treatment during pregnancy and preterm birth, stillbirth, low birth weight and caesarian delivery. There are conflicting views on the use of MET in averting or increasing the risk of preterm birth and associated outcomes (13)(49)(50) [START_REF] Sheehy | The Use of Metronidazole During Pregnancy: A Review of Evidence [Internet[END_REF]. Recent guidelines for clinical practise and reviews stated insufficiency of data on the efficacy of MET to recommend its use (or non-use) (6) [START_REF] Okun | Antibiotics for Bacterial Vaginosis or Trichomonas vaginalis in Pregnancy: A Systematic Review[END_REF]. Based on the analysis, we cannot confirm the efficacy of MET in reducing the risk of preterm birth.

Contrary to previous studies(11) [START_REF] Koss | Investigation of Metronidazole Use during Pregnancy and Adverse Birth Outcomes[END_REF], the overall risk of preterm birth was not significantly decreased among women with previous preterm birth. Subgroup analysis suggested pregnant women who were administered MET for ≤3 days may have an increased risk of preterm birth.

The recommended guidelines for the treatment of trichmoniasis and or bacterial vaginosis among pregnant women include the single dose of 2g or 400mg-500mg for up to 7 days (52) (7) [START_REF]WHO | Trichomoniasis and bacterial vaginosis in pregnancy: inadequately managed with the syndromic approach[END_REF]. In our meta-analysis, the duration of treatment of <3 days is related to two doses of 2g administered [START_REF] Odendaal | Preterm labour--is bacterial vaginosis involved?[END_REF]. Further studies are required to assess the hypothesis that a high dose for a short duration may increase the risk of preterm birth among women with a previous preterm birth, compared to a lower dose for a longer duration. In observational studies, pregnant women with preterm birth in the preceding pregnancy had a higher likelihood of preterm birth compared to their unexposed counterparts. Unfortunately, the results are based on one study.

Whereas pregnant women infected with T.vaginalis are more likely to have a stillbirth [START_REF] Cotch | Trichomonas vaginalisAssociated With Low Birth Weight and Preterm Delivery[END_REF], the literature does not confirm the impact of MET treatment on perinatal mortality [START_REF] Menezes | Reducing stillbirths: prevention and management of medical disorders and infections during pregnancy[END_REF]. Our study did not confirm the beneficial effect of MET on stillbirth (OR 0.42; 95% CI 0.17-1.03; n=3; I²=0%).

MET exposure was associated with an increased risk of spontaneous abortion. The results from sensitivity analysis remained similar to the original result with a decreased level of heterogeneity (I 2 =19%). Findings from recent studies show a relationship between bacterial vaginosis and miscarriage in the first [START_REF] Ralph | Influence of bacterial vaginosis on conception and miscarriage in the first trimester: cohort study[END_REF] and second trimester [START_REF] Donders | Relationship of bacterial vaginosis and mycoplasmas to the risk of spontaneous abortion[END_REF]. Because the analysis was based on observational studies, our results regarding spontaneous abortion may be subject to indication or protopathic bias.

Major congenital malformations

Our meta-analysis revealed no statistically significant association between major malformations and MET exposure during the first trimester of pregnancy. This is consistent with the metaanalyses conducted by Caro-Paton et al [START_REF] Caro-Patón | Is metronidazole teratogenic? A meta-analysis[END_REF] and Burtin(20)

Strengths and limitations

This is the first meta-analysis to highlight both the safety and efficacy of MET during pregnancy.

Moreover, the robustness of our results was proved using sensitivity analyses that produced findings similar to the original analyses. Compared to two previous meta-analyses of 1995(20) (7 studies) and 1997 [START_REF] Caro-Patón | Is metronidazole teratogenic? A meta-analysis[END_REF] (5 studies) on malformations, our meta-analysis is an update of 12 studies with 411,380 participants. Furthermore, this is the first meta-analysis to consider the assessment of risks related to MET exposure in light of the dose, duration, route of administration, indication and the period of exposure of the treatment. Lastly, the symmetric funnel plot (Supplementary Figure A5) does not suggest the presence of publication bias.

However, our analysis was dependent on the precision of authors regarding the dose, duration, route of administration, indication, the period of exposure of the treatment and the definition of outcomes such as stillbirth. This was not always the case. Finally, most of the observational studies included in our analysis use the prescription of MET as a proxy for exposure, which may bias the results.

Conclusion

In conclusion, data do not confirm the efficacy of MET in reducing the risk of preterm birth and associated delivery outcomes. Further research is required to confirm the effect of high dose and short duration of metronidazole treatment on preterm birth among the high-risk group. Regarding the increased odds of spontaneous abortion, RCTs are required to assess the role of the underlying infection. The need for further studies to confirm the risk of congenital hydrocephaly is paramount.

  and classified by using the European Surveillance for Congenital Anomalies(31) (EUROCAT) criteria. Two authors independently identified, screened and reviewed publications for eligibility thereby generating the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)(32) flow diagram in Figure 1. A data extraction form was developed and piloted to ensure its usefulness before data extraction commenced, which was equally conducted by two reviewers. Disagreements were resolved through consensus or discussion with a third-party. Risk of Bias was assessed using the Revised Cochrane Risk-Of-Bias tool for randomized trials (ROB 2) (33) for RCTs and Risk Of Bias In Non-randomized Studies-of Interventions (ROBINS-I) (34) for observational studies.

  Figure 1: PRISMA Flow diagram

Figure 2 :

 2 Figure 2: Delivery outcomes after maternal metronidazole exposure

  ) (22) (23) (24) (40) (41) (25) (43) (26) (46) (

  et al. Despite the increased odds after analyzing only adjusted effect estimates, we are unable to conclude on the risk of major malformations due to the quality of some studies included in the analysis, even after adjusting for confounding factors. Moreover, when studies with the critical risk of bias are excluded, the risk remains similar to the original result. Regarding specific malformations, MET exposure in the first trimester was associated with congenital hydrocephaly. The results are based on two casecontrol studies conducted byKazy et al, 2005(22) (70 cases exposed to vaginal MET treatment during the first trimester) andCzeizel et al, 1998(17) (136 cases exposed to oral MET during the first trimester) who both used the same data source with malformed cases identified from the Hungarian Congenital Abnormality Registry, matched to controls and were subject to recall bias regarding drug exposure. The possible risk of overlap between the two studies should be noted.

Table 1 .

 1 Characteristics of studies by study design, dose, duration, route of administration, indication and studied outcomes

	Ref.	Year	Study	n	Dose	Durati	Administr	Exposure	Indication	Outcomes
			design		(mg)	on (d)	ation	(trimester
								)
	(39)	Carey et al	RCT	1953	2000	48	Systemic	2	Bacterial vaginosis	Preterm birth, low
		2000				hours					birth weight
						apart
	(17) Czeizel et al	Case-	47,963	2000 or	7	Systemic	1,3	Infectious disease of the	Malformations
		1998	control		750		and local		respiratory system,
									urinary tract and genital
									organs
	(40) Diav-Citrin	Prospect.	765	973	7.9	Systemic	1	Helicobacter	pylori,	Malformations and
		et al 2001	Cohort		(483)	(3.8)	and local		genital or urinary tract	spontaneous abortion
									infection,	giardiasis,
									trichomoniasis,
									amebiasis, and pelvic
									inflammatory disease.

  The total number of participants was 800,195 included at any time during pregnancy with 5,044 METexposed cases and 158,060 unexposed cases. The systemic route of administration was cited in 13 studies and the local route in 2 studies. Whereas dose and duration were not mentioned in 10

	studies, the administered doses under systemic administration were 250mg (5 studies), 400mg (3
	studies), 200mg (2 studies) and 2 grams (2 studies) for 7 days (6 studies), 3 days (2 studies) or 2

days (2 studies). Indication of bacterial vaginosis/trichomoniasis was reported in 15 studies while others did not consider a specific indication as a criterion for participant inclusion. All RCTs used placebo in the control group while observational studies consisted of unexposed sick patients (2 studies), unexposed disease-free (8 studies), unexposed but not otherwise specified

(5 studies)

authors received funding for this review.


Serge A Van De Pavert
email: vandepavert@ciml.univ-mrs.fr

Lymphoid Tissue Inducer (LTi) cell ontogeny and functioning in embryo and adult

Innate Lymphoid Cells (ILC) are involved in homeostasis and immunity. Their dynamic differentiation and characterization depend on their tissue of residency and is adapted to their role within these tissues. Lymphoid Tissue Inducer (LTi) cells are an ILC member and essential for embryonic lymph node (LN) formation. LNs are formed at pre-defined and strategic positions throughout the body and how LTi cells are initially attracted towards these areas is under debate. Besides their role in LN formation, LTi-like and the closely related ILC type 3 (ILC3) cells have been observed within the embryonic gut. New studies have now shown more information on their origin and differentiation within the embryo. This review will evaluate the embryonic LTi cell origin from a specific embryonic hemogenic wave, which has recently been described in mouse. Moreover, I will discuss their differentiation and similarities with the closely related ILC3 cells in embryo and adult. Keywords ILC3, ILC, hemogenic endothelium, bone-marrow, hematopoietic stem cells (HSC), lymph node hematopoietic lineage than from the HSC derived lineage. During adulthood, these embryonic LTi cells are replaced by bone-marrow derived LTi cells. The role of replaced LTi cells in adult remains obscure.

In this review, I will discuss the role of the LTi cells within LN formation and evaluate the newest insights on initiation of LN formation. Furthermore, I will discuss the new developments in LTi cell ontogeny and functioning, also in relation to the closely related ILC3s.

ILC ontogeny 2.1 Hemogenic endothelium progenitors

. However, it is not clear which other specific hematopoietic lineages appear from these embryonic sites.

In vitro cultured mouse yolk-sac cells isolated at E8.5 had a potential to become NK cells [15]. To directly show a relation between the yolk-sac and NK cells lineage tracing models are required. Simic et al.[19] used the Cxcr4-CreErt2 lineage tracing model to exclude a yolk-sac contribution as

Introduction

The enigmatic Innate Lymphoid Cell (ILC) family consists of 3 main family members in which transcription factors for development, marker expression and functionality are shared [START_REF] Eberl | The brave new world of innate lymphoid cells[END_REF][START_REF] Vivier | Innate Lymphoid Cells: 10 Years On[END_REF][START_REF] Spits | Innate lymphoid cells--a proposal for uniform nomenclature[END_REF]. These are the 1) ILC1 and natural killer cell (NK), 2) ILC2 and 3) ILC3 and Lymphoid Tissue Inducer (LTi) cell members. Their role in defense and repair is versatile, as are their cues for differentiation in situ determining their action. NK cells are considered cytotoxic ILCs, while generally other ILCs are considered ILC-helper cells. The LTi cells were previously considered part of the ILC3 ontogeny, based on retinoic acid orphan receptor isoform γt (RORγt) expression plus overlap in marker and cytokine expression. However, in both human and mice, ILC3 progenitors cells require the transcription factor promyelocytic leukemia zinc finger (Plzf)(encoded by gene Zinc finger and BTB domain containing for differentiation [START_REF] Nagasawa | Human CD5+ Innate Lymphoid Cells Are Functionally Immature and Their Development from CD34+ Progenitor Cells Is Regulated by Id2[END_REF][START_REF] Constantinides | PLZF expression maps the early stages of ILC1 lineage development[END_REF], while in the mouse Zbtb16 knock-out LTi cells were still observed [START_REF] Constantinides | A committed precursor to innate lymphoid cells[END_REF].

Therefore, LTi cells are now considered to be separate from ILC3 cells [START_REF] Ishizuka | Single-cell analysis defines the divergence between the innate lymphoid cell lineage and lymphoid tissueinducer cell lineage[END_REF]. Additionally, evolutionary the LTi cells emerged later when compared to the other ILCs, and their function in LN formation was mainly associated with placentation [START_REF] Vivier | The evolution of innate lymphoid cells[END_REF][START_REF] Lane | Lymphoid tissue inducer cells: pivotal cells in the evolution of CD4 immunity and tolerance?[END_REF].

LTi cells are essential for the formation of the secondary lymphoid organs during embryogenesis. They interact with the Lymphoid Tissue organizer (LTo) cells at specific locations within the embryo to first form aggregates which are named LN anlagen. There is some discussion on which cells attract the first LTi cells involved in the initiation of LN formation when using different mouse models [START_REF] Onder | Lymphatic Endothelial Cells Control Initiation of Lymph Node Organogenesis[END_REF][START_REF] Van De Pavert | New insights into the development of lymphoid tissues[END_REF][START_REF] Bovay | Multiple roles of lymphatic vessels in peripheral lymph node development[END_REF]. ILC3s, which consists of Natural cytotoxicity triggering receptor 1 (NKp46) + and NKp46 -(LTi-like) cells, have immune modulatory and homeostatic roles within the gut. LTi cells have a unique function during embryogenesis and are the first of the ILCs to be observed during embryogenesis [START_REF] Van De Pavert | New insights into the development of lymphoid tissues[END_REF]. In later embryonic stages other ILCs were reported, mainly in gut and lung [START_REF] Bando | Identification and distribution of developing innate lymphoid cells in the fetal mouse intestine[END_REF][START_REF] Schneider | Tissue-Resident Group 2 Innate Lymphoid Cells Differentiate by Layered Ontogeny and In Situ Perinatal Priming[END_REF][START_REF] Dege | Potently Cytotoxic Natural Killer Cells Initially Emerge from Erythro-Myeloid Progenitors during Mammalian Development[END_REF], but their role during embryogenesis is unclear. ILCs were shown to be derived from the HSC lineage within the bonemarrow in adult [START_REF] Xu | An Id2RFP-Reporter Mouse Redefines Innate Lymphoid Cell Precursor Potentials[END_REF][START_REF] Walker | Polychromic Reporter Mice Reveal Unappreciated Innate Lymphoid Cell Progenitor Heterogeneity and Elusive ILC3 Progenitors in Bone Marrow[END_REF][START_REF] Seillet | Deciphering the Innate Lymphoid Cell Transcriptional Program[END_REF][START_REF] Simic | Distinct Waves from the Hemogenic Endothelium Give Rise to Layered Lymphoid Tissue Inducer Cell Ontogeny[END_REF]. However, since there is no contribution from bone-marrow within the mouse embryo, the embryonic ILCs, like embryonic LTi cells, are likely to be derived from another chemokine receptor Cxcr4 is not expressed within the yolk-sac [START_REF] Simic | Distinct Waves from the Hemogenic Endothelium Give Rise to Layered Lymphoid Tissue Inducer Cell Ontogeny[END_REF][START_REF] Mcgrath | Embryonic expression and function of the chemokine SDF-1 and its receptor, CXCR4[END_REF][START_REF] Werner | Cxcr4 distinguishes HSCderived monocytes from microglia and reveals monocyte immune responses to experimental stroke[END_REF]. Consequently, progenitors from the yolk-sac don't express Cxcr4 [START_REF] Werner | Cxcr4 distinguishes HSCderived monocytes from microglia and reveals monocyte immune responses to experimental stroke[END_REF]. Lineage-tracing in the Cxcr4-CreErt2 model only labelled embryonic derived progenitors [START_REF] Werner | Cxcr4 distinguishes HSCderived monocytes from microglia and reveals monocyte immune responses to experimental stroke[END_REF] and it was shown that LTi cell progenitors originated around E8.5 from an embryonic hemogenic source [START_REF] Simic | Distinct Waves from the Hemogenic Endothelium Give Rise to Layered Lymphoid Tissue Inducer Cell Ontogeny[END_REF]. Since the first hemogenic clusters in the AGM are observed after E8.5 [START_REF] Boisset | In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium[END_REF][START_REF] Baron | Single-cell transcriptomics reveal the dynamic of haematopoietic stem cell production in the aorta[END_REF], the LTi hemogenic source could either be the embryonic vitelline artery or the AGM [START_REF] Zovein | Vascular remodeling of the vitelline artery initiates extravascular emergence of hematopoietic clusters[END_REF]. Described as active in later stages, other embryonic regions were less likely to be the source for LTi cells [START_REF] Yzaguirre | Insights into blood cell formation from hemogenic endothelium in lesser-known anatomic sites[END_REF][START_REF] Gordon-Keylock | Mouse extraembryonic arterial vessels harbor precursors capable of maturing into definitive HSCs[END_REF][START_REF] Shigeta | Endocardially Derived Macrophages Are Essential for Valvular Remodeling Article Endocardially Derived Macrophages Are Essential for Valvular Remodeling[END_REF]. The appearance of the embryonic LTi progenitors preceded the appearance of the pre-HSCs, making it unlikely that embryonic LTi cells are derived from HSCs. On the contrary, embryonic LTi cells involved in LN formation were replaced in the adult mice by bonemarrow HSC derived LTi cells, as was also shown for ILC2s and some ILC3s [START_REF] Schneider | Tissue-Resident Group 2 Innate Lymphoid Cells Differentiate by Layered Ontogeny and In Situ Perinatal Priming[END_REF][START_REF] Sawa | Lineage relationship analysis of RORgammat+ innate lymphoid cells[END_REF]. In human, ILCs were observed in the fetal gut but it has not been shown if they are HSC derived, nor if they will be replaced in the neonate by ILCs from HSCs in the bone-marrow [START_REF] Li | Mass cytometry reveals innate lymphoid cell differentiation pathways in the human fetal intestine[END_REF]. Interestingly, thymic LTi cells in the mouse embryo around E18 were described to originate from HSC and could indicate the first LTi cells to originate from a HSC origin [START_REF] Elsaid | A wave of bipotent T/ILC-restricted progenitors shapes the embryonic thymus microenvironment in a time-dependent manner[END_REF]. How the HSC derived ILCs functionally relate to the embryonic derived ILC they replace is not yet known.

Fetal Liver

Hematopoietic progenitors migrate from the hemogenic endothelial sites towards the fetal liver (FL).

Intriguingly, in mouse embryos the FL is mostly colonized around E11.5, so it is unclear where earliest hematopoietic progenitors reside between E7.5-E11. Within the FL, progenitors expand and differentiate toward precursors stages of their respective lineages, except for the microglial precursors which differentiate within the brain [START_REF] Hoeffel | C-Myb+ Erythro-Myeloid Progenitor-Derived Fetal Monocytes Give Rise to Adult Tissue-Resident Macrophages[END_REF]. The FL plays an important role in the expansion of the progenitors as it was estimated that the progenitors multiply up to 33 times within this environment [START_REF] Ema | Expansion of hematopoietic stem cells in the developing liver of a mouse embryo[END_REF].

Common Lymphoid Progenitors (CLP) are present within the FL and express Fms related receptor tyrosine kinase 3 (Flt3) [START_REF] Ishizuka | Single-cell analysis defines the divergence between the innate lymphoid cell lineage and lymphoid tissueinducer cell lineage[END_REF][START_REF] Simic | Distinct Waves from the Hemogenic Endothelium Give Rise to Layered Lymphoid Tissue Inducer Cell Ontogeny[END_REF][START_REF] Klose | Differentiation of type 1 ILCs from a common progenitor to all helper-like innate lymphoid cell lineages[END_REF]. They are driven towards an ILC lineage by the expression of the Inhibitor of DNA binding 2 (Id2), which inhibits Single-stranded DNA-binding protein (E2A) functioning and thereby excludes commitment to a B-cell lineage [START_REF] Boos | Mature natural killer cell and lymphoid tissue-inducing cell development requires Id2-mediated suppression of E protein activity[END_REF]. At this stage, they are named α4β7-Integrin expressing Lymphoid Precursor (αLP) cells. Flt3 RNA transcripts were not observed in this stage, and protein expression was detected on a αLP subset [START_REF] Ishizuka | Single-cell analysis defines the divergence between the innate lymphoid cell lineage and lymphoid tissueinducer cell lineage[END_REF][START_REF] Simic | Distinct Waves from the Hemogenic Endothelium Give Rise to Layered Lymphoid Tissue Inducer Cell Ontogeny[END_REF][START_REF] Chea | Single-Cell Gene Expression Analyses Reveal Heterogeneous Responsiveness of Fetal Innate Lymphoid Progenitors to Notch Signaling[END_REF]. An αLP Flt3 -subset is sensitive to Notch signaling and affects its proliferation [START_REF] Chea | Single-Cell Gene Expression Analyses Reveal Heterogeneous Responsiveness of Fetal Innate Lymphoid Progenitors to Notch Signaling[END_REF] (please see the review by Golub in this issue for the role of Notch in ILC differentiation). Within the αLP Flt3 -stage, transcription factor Plzf marks differentiation into the ILC-Precursor (ILCP), which will generate ILC1, 2 and 3 cells in both human and mouse [4,5][6].

In adult mouse Plzf expression was reported to be lowered, while it remained high in human ILCs [START_REF] Nagasawa | Human CD5+ Innate Lymphoid Cells Are Functionally Immature and Their Development from CD34+ Progenitor Cells Is Regulated by Id2[END_REF].

LTi-precursors (LTiP) in mouse, on the other hand, do not require Plzf, express chemokine receptors CXCR5 and CXCR6, and appear to segregate from the other ILC populations [START_REF] Constantinides | A committed precursor to innate lymphoid cells[END_REF][START_REF] Ishizuka | Single-cell analysis defines the divergence between the innate lymphoid cell lineage and lymphoid tissueinducer cell lineage[END_REF][START_REF] Possot | Notch signaling is necessary for adult, but not fetal, development of RORγt(+) innate lymphoid cells[END_REF][START_REF] Chea | Notch signaling in group 3 innate lymphoid cells modulates their plasticity[END_REF](reviewed in [START_REF] Ishizuka | The Innate Lymphoid Cell Precursor[END_REF])(Figure 1). These chemokine receptors expressed during the LTiP stage could aid their migration to-and retention within the LN anlagen, essential for the next phase in LTi differentiation.

The role of mature ILC3 and LTi cells within the mouse FL is unclear. Several studies reported on the RORγt expressing (mature) ILC3 and LTi cells within the mouse FL [START_REF] Ishizuka | Single-cell analysis defines the divergence between the innate lymphoid cell lineage and lymphoid tissueinducer cell lineage[END_REF][START_REF] Sawa | Lineage relationship analysis of RORgammat+ innate lymphoid cells[END_REF][START_REF] Cherrier | Notch, Id2, and RORγt sequentially orchestrate the fetal development of lymphoid tissue inducer cells[END_REF]. On the other hand, the number of LTi0 and LTi4 cells within the FL was very low compared to LN anlagen enriched tissue [START_REF] Simic | Distinct Waves from the Hemogenic Endothelium Give Rise to Layered Lymphoid Tissue Inducer Cell Ontogeny[END_REF][START_REF] Mebius | Developing lymph nodes collect CD4+CD3-LTbeta+ cells that can differentiate to APC, NK cells, and follicular cells but not T or B cells[END_REF]. Single cell sequencing of the FL and LN anlagen enriched tissue revealed that there was no direct connection in ontogeny between LTiP and LTi cells within the FL, while this was evident within the peripheral studied LTi cells [START_REF] Simic | Distinct Waves from the Hemogenic Endothelium Give Rise to Layered Lymphoid Tissue Inducer Cell Ontogeny[END_REF]. Therefore, the final maturation would be most likely to occur within the LN anlagen, and in this scenario LTi cells present within the FL migrated back from the embryo.

2.3 LTi and ILC3s in LN anlagen and embryonic gut αLP-LTiP cells migrate out from the FL towards the LN anlagen to differentiate into LTi0 and finally the mature LTi4 cells. During this transition, RORγt expression is induced by retinoic acid which leads to LTi lineage commitment. Lack of retinoic acid signaling within these cells halts LTi differentiation [START_REF] Van De Pavert | Maternal retinoids control type 3 innate lymphoid cells and set the offspring immunity[END_REF].

RORγt drives the transcriptional program for LTi cells in both human [START_REF] Okada | Impairment of immunity to Candida and Mycobacterium in humans with bi-allelic RORC mutations[END_REF] and mice [START_REF] Eberl | An essential function for the nuclear receptor RORgamma(t) in the generation of fetal lymphoid tissue inducer cells[END_REF], although there have been RORγt deficient LTi cells observed [START_REF] Eberl | An essential function for the nuclear receptor RORgamma(t) in the generation of fetal lymphoid tissue inducer cells[END_REF][START_REF] Van De Pavert | Chemokine CXCL13 is essential for lymph node initiation and is induced by retinoic acid and neuronal stimulation[END_REF]. However, these are not sufficient for continuation of LN formation, as Rorc (RORγt) knock-out mice are devoid of LN [START_REF] Eberl | An essential function for the nuclear receptor RORgamma(t) in the generation of fetal lymphoid tissue inducer cells[END_REF][START_REF] Sun | Requirement for RORgamma in thymocyte survival and lymphoid organ development[END_REF] and RORc deficient humans did not have palpable LNs [START_REF] Okada | Impairment of immunity to Candida and Mycobacterium in humans with bi-allelic RORC mutations[END_REF]. Notch was shown to be involved in proliferation of the αLP population [START_REF] Chea | Single-Cell Gene Expression Analyses Reveal Heterogeneous Responsiveness of Fetal Innate Lymphoid Progenitors to Notch Signaling[END_REF], but Notch signaling is not necessary for LTi commitment nor differentiation [START_REF] Possot | Notch signaling is necessary for adult, but not fetal, development of RORγt(+) innate lymphoid cells[END_REF]( please see the review by Golub in this issue for the role of Notch in ILC differentiation). The LTi0 cells do not yet express the molecules necessary for interaction with mesenchymal cell within the lymph node anlagen niche [START_REF] Van De Pavert | Maternal retinoids control type 3 innate lymphoid cells and set the offspring immunity[END_REF][START_REF] Kim | Regulation of peripheral lymph node genesis by the tumor necrosis factor family member TRANCE[END_REF]. Only after differentiation into the mature LTi4 stage, these cells interact and amplify the synthesis of cytokines and adhesion factors (Figure 2). This leads to the attraction and retention of more LTiP and LTi cells.

It is unclear whether there is only one LTi4 cell population. Mosaic CD11b and MHCII expressing LTi cells were observed within the embryo [START_REF] Mebius | Developing lymph nodes collect CD4+CD3-LTbeta+ cells that can differentiate to APC, NK cells, and follicular cells but not T or B cells[END_REF][START_REF] Eberl | An essential function for the nuclear receptor RORgamma(t) in the generation of fetal lymphoid tissue inducer cells[END_REF][START_REF] Mebius | A developmental switch in lymphocyte homing receptor and endothelial vascular addressin expression regulates lymphocyte homing and permits CD4+ CD3-cells to colonize lymph nodes[END_REF]. Using single cell sequencing of the LN anlagen enriched tissue, two separate LTi4 clusters were observed. The first LTi4 population expressed Il22, a cytokine involved in gut immunity. Also, Arg1 is expressed within this LTi4 population, which was also associated with fetal gut ILC precursors (ftILCP) and ILC2s [START_REF] Bando | Identification and distribution of developing innate lymphoid cells in the fetal mouse intestine[END_REF]. Specifically, in the same study, the fetal gut contained Arginase 1(Arg1)YFP + LTi fate mapped cells and a smaller Arg1YFP -population [START_REF] Bando | Identification and distribution of developing innate lymphoid cells in the fetal mouse intestine[END_REF],

confirming two separate LTi4 populations also within the intestines. The ftILCPs could generate ILC1, 2 and 3 and were not fate mapped by the RORγt-Cre reporter, indicating that they are likely analogous to αLP and ILCP cells. Arg1 transcripts were not observed within fetal liver progenitors but only in the LN anlagen enriched tissue [START_REF] Simic | Distinct Waves from the Hemogenic Endothelium Give Rise to Layered Lymphoid Tissue Inducer Cell Ontogeny[END_REF], indicating that Arg1 could be expressed after leaving the FL and entering peripheral LN anlagen or fetal gut. The second LTi4 population observed in the single cell sequencing study was segregated based on genes associated with Mayor histocompatibility complex II (MHCII), and hence named LTi4-II. Within the LTi4-II population, Cfp (Complement Factor Properdin) was observed, a ligand for binding to NKp46 [START_REF] Narni-Mancinelli | Complement factor P is a ligand for the natural killer cell-activating receptor NKp46[END_REF]. This could indicate that the LTi4 -II population produce the ligand for NKp46 + NK, ILC1, ILC3 cells, but this has not yet been shown within the adult. A relation between the two embryonic LTi populations in the LN anlagen enriched tissue and the Arg1 + or MHCII + ILC3 and LTi-like cells observed in the gut remains to be established.

Origin of ILCs and LTi cells in adult

Hematopoietic lineages within the adult are almost all derived from HSC residing within the bonemarrow. Bone-marrow derived ILC precursors in the mouse do not express Arg1, while embryonic ILCPs express Arg1 [START_REF] Bando | Identification and distribution of developing innate lymphoid cells in the fetal mouse intestine[END_REF]. This is an indication that the differentiation pathway towards embryonic derived ILCs is likely different than those derived from HSCs in the adult bone-marrow. The mouse embryonic LTi cells, ILC3s and ILC2s were shown to be replaced by bone-marrow HSC derived ILCs [START_REF] Schneider | Tissue-Resident Group 2 Innate Lymphoid Cells Differentiate by Layered Ontogeny and In Situ Perinatal Priming[END_REF][START_REF] Simic | Distinct Waves from the Hemogenic Endothelium Give Rise to Layered Lymphoid Tissue Inducer Cell Ontogeny[END_REF][START_REF] Sawa | Lineage relationship analysis of RORgammat+ innate lymphoid cells[END_REF]. Also, it was shown that ILCs can be generated from the bone-marrow using elegant reporter mouse models and in vitro differentiation [START_REF] Bando | Identification and distribution of developing innate lymphoid cells in the fetal mouse intestine[END_REF][START_REF] Xu | An Id2RFP-Reporter Mouse Redefines Innate Lymphoid Cell Precursor Potentials[END_REF][START_REF] Walker | Polychromic Reporter Mice Reveal Unappreciated Innate Lymphoid Cell Progenitor Heterogeneity and Elusive ILC3 Progenitors in Bone Marrow[END_REF][START_REF] Seillet | Deciphering the Innate Lymphoid Cell Transcriptional Program[END_REF], although only 1 study reported the generation of LTi cells from bone marrow, while the other reporter mouse models could not follow LTi cells in their fate-mapping models [START_REF] Klose | Differentiation of type 1 ILCs from a common progenitor to all helper-like innate lymphoid cell lineages[END_REF]. The discrepancy between these studies can be explained by the differentiation stage that is used as source for the in vitro-or in vivo differentiation studies. Similar as their differentiation within the mouse embryo, LTi cells follow a slightly different pathway for their formation as other ILCs, and the precursors used in the studies could have lost their LTi cell potential.

In immunodeficient patients who received bone-marrow transfer lymphocytes were regained, but not ILCs [START_REF] Vély | Evidence of innate lymphoid cell redundancy in humans[END_REF]. This observation could reflect the inability of some studies to observe LTi cell differentiation within the adult from HSCs. Similar as for LTi cells, it is important to understand from which hematopoietic precursor exactly ILCs originate in (human) fetus. Possibly, other human HSCs or progenitors need to be isolated which still have the potential to become ILC after transfer, as was shown for circulating and tissue resident ILCp cells [START_REF] Lim | Systemic Human ILC Precursors Provide a Substrate for Tissue ILC Differentiation[END_REF]. The presence of different types of ILC1, 2, 3 or LTi cells in different tissues could also reflect their difference in origin, i.e. embryonic resident ILCs vs. bone-marrow derived ILCs, and thus affect their functioning. More studies are required to understand the precise ILC progenitors and their differentiation pathways and what the functional difference is between the embryonic vs. adult ILCs.

LTi cells in embryonic lymph node formation

Secondary lymphoid organs, like mesenteric and peripheral lymph nodes (LN) and Peyer's patches, are formed during embryogenesis in which the LTi cells are critical. Consequently, deleting or mutation of RORc in both mice [START_REF] Eberl | An essential function for the nuclear receptor RORgamma(t) in the generation of fetal lymphoid tissue inducer cells[END_REF] and human [START_REF] Okada | Impairment of immunity to Candida and Mycobacterium in humans with bi-allelic RORC mutations[END_REF] results in loss of LTi cells and concomitantly loss of LNs (reviewed in [START_REF] Van De Pavert | New insights into the development of lymphoid tissues[END_REF]). RORc deficient humans lacked palpable LNs but still had tonsils, but are surprisingly only more sensitive to Mycobacteria and Candida due to defective γδ-T cells and CD4 + CCR6 + CXCR3 +αβ T cells [START_REF] Okada | Impairment of immunity to Candida and Mycobacterium in humans with bi-allelic RORC mutations[END_REF]. The presence of the tonsils, and other lymphoid structures like spleen which do not rely on LTi cells could be sufficient to drive the adaptive immune response. In mouse, initiation of most lymph nodes occurs between E12.5 until E15.5 (Figures 2 and3). The aggregates of LTi-and mesenchymal cells, called lymph node anlagen, are present until birth in mouse. Final organization of the LN due to differentiation of the stromal subsets and attraction of the specific lymphocytes to their respective regions take place in the first weeks after birth. On the contrary, in human, LN organogenesis takes place around the 12-17 th week of pregnancy and humans are born with a fully organized LN at birth [START_REF] Hoorweg | Development of human lymph nodes and Peyer's patches[END_REF].

The first event which can be visualized in embryonic LN formation is the aggregation of the LTi cells at fixed positions within the embryo (Figure 2). The chemokine CXCL13 is essential for the aggregation of the LTi cells in most LN anlagen, except the mesenteric LN [START_REF] Ansel | A chemokine-driven positive feedback loop organizes lymphoid follicles[END_REF][START_REF] Luther S A | Overlapping roles of CXCL13, interleukin 7 receptor alpha, and CCR7 ligands in lymph node development[END_REF]. CXCR5 + LTiP cells are present within the LN anlagen, to be retained by local CXCL13 expression within the niches where LN are formed [START_REF] Ishizuka | Single-cell analysis defines the divergence between the innate lymphoid cell lineage and lymphoid tissueinducer cell lineage[END_REF][START_REF] Simic | Distinct Waves from the Hemogenic Endothelium Give Rise to Layered Lymphoid Tissue Inducer Cell Ontogeny[END_REF][START_REF] Chea | Single-Cell Gene Expression Analyses Reveal Heterogeneous Responsiveness of Fetal Innate Lymphoid Progenitors to Notch Signaling[END_REF]. The role of CXCR6 [START_REF] Chea | Single-Cell Gene Expression Analyses Reveal Heterogeneous Responsiveness of Fetal Innate Lymphoid Progenitors to Notch Signaling[END_REF][START_REF] Possot | Notch signaling is necessary for adult, but not fetal, development of RORγt(+) innate lymphoid cells[END_REF] on these cells remains unknown in the attraction toward the LN niche. Only CCR7 was shown to be very potent in attracting LTi cells, but only when lymphatic endothelial cells secrete the ligand CCL21 [START_REF] Bovay | Multiple roles of lymphatic vessels in peripheral lymph node development[END_REF][START_REF] Van De Pavert | Chemokine CXCL13 is essential for lymph node initiation and is induced by retinoic acid and neuronal stimulation[END_REF][START_REF] Luther S A | Overlapping roles of CXCL13, interleukin 7 receptor alpha, and CCR7 ligands in lymph node development[END_REF]. In later LN formation stages, after lymphotoxin signaling and when the mesenchymal cells have differentiated into lymphoid tissue inducer (LTo) cells, the other CCR7 ligand CCL19 is expressed by LTo cells within the LN anlagen. This occurs around the time when the lymphatic endothelial cells have circumvented the LN anlagen. Some rescue could be observed by the lymphatic endothelial cell (LEC) expressed chemokine CCL21 [START_REF] Luther S A | Overlapping roles of CXCL13, interleukin 7 receptor alpha, and CCR7 ligands in lymph node development[END_REF], although these are likely to be aggregations within lymphatic vessels [START_REF] Bovay | Multiple roles of lymphatic vessels in peripheral lymph node development[END_REF]. The presence of LECs and thus expression of CCL21 occurred after initial LTi cell aggregation and initial CXCL13 expression within the LN anlagen [START_REF] Bovay | Multiple roles of lymphatic vessels in peripheral lymph node development[END_REF][START_REF] Van De Pavert | Chemokine CXCL13 is essential for lymph node initiation and is induced by retinoic acid and neuronal stimulation[END_REF]. Moreover, lack of all lymphatic endothelial cells in the Prox1 knock-out mouse model did not affect the initial aggregation of the LTi cell clusters [START_REF] Vondenhoff | Lymph sacs are not required for the initiation of lymph node formation[END_REF]. Initial Cxcl13 expression, but not other chemokines like Ccl21 and Ccl19, is induced by retinoic acid. Cxcl13 expression is required to attract the first precursor LTi cells towards the LN anlagen niche. It was proposed that nearby neurons synthesize the retinoic acid, thus inferring to a neuronal influence on LN formation [START_REF] Van De Pavert | Chemokine CXCL13 is essential for lymph node initiation and is induced by retinoic acid and neuronal stimulation[END_REF]. However, it is unclear which type of neurons are involved in LN formation. After the initial LTi cell differentiation, Receptor activator of nuclear factor κ B Ligand (RANK-L), also known as TNFSF11A, either on LTi cells [START_REF] Kim | Regulation of peripheral lymph node genesis by the tumor necrosis factor family member TRANCE[END_REF][START_REF] Yoshida | Different Cytokines Induce Surface Lymphotoxin-αβ on IL-7 Receptor-α Cells that Differentially Engender Lymph Nodes and Peyer's Patches[END_REF] or mesenchymal cells [START_REF] Sugiyama | Expression pattern changes and function of RANKL during mouse lymph node microarchitecture development[END_REF] activates RANK on LTi cells. RANK signaling induced expression of Lymphotoxin-α1ß2 ligand on the LTi cells, which is the latest maturation step of the LTi cell. Thus loss of this signaling resulted in the loss of LTi4 cells and consequently loss of lymph nodes [START_REF] Kim | Regulation of peripheral lymph node genesis by the tumor necrosis factor family member TRANCE[END_REF][START_REF] Sugiyama | Expression pattern changes and function of RANKL during mouse lymph node microarchitecture development[END_REF].

Lymphotoxin-α1ß2 will subsequently interact with its LTß-receptor on the mesenchymal cells. This interaction triggers the expression of cytokines like IL7, chemokines like Cxc13 and adhesion molecules like Madcam-1 [START_REF] Yoshida | Different Cytokines Induce Surface Lymphotoxin-αβ on IL-7 Receptor-α Cells that Differentially Engender Lymph Nodes and Peyer's Patches[END_REF][START_REF] Vondenhoff | LTbetaR signaling induces cytokine expression and up-regulates lymphangiogenic factors in lymph node anlagen[END_REF], which attract and retain more (precursor) LTi cells. These increasing interactions results in an amplification of the process and hence a strong increase in the size of the aggregate.

There is currently a discussion on the definition of the initial phase of LN formation. It was suggested that embryonic lymphatic vessels interacted with LTi cells through lymphotoxin signaling and that this delineated the initial event of LN formation [START_REF] Onder | Lymphatic Endothelial Cells Control Initiation of Lymph Node Organogenesis[END_REF]. Indeed, lymphatic vessels play an important role in lymph node formation but notably during the expansion phase. During the earliest clustering of LTi cells, well before E14.5, lymphatic vessels were not present nor required [START_REF] Bovay | Multiple roles of lymphatic vessels in peripheral lymph node development[END_REF][START_REF] Van De Pavert | Chemokine CXCL13 is essential for lymph node initiation and is induced by retinoic acid and neuronal stimulation[END_REF][START_REF] Vondenhoff | Lymph sacs are not required for the initiation of lymph node formation[END_REF]. Starting at E14.5 in the anterior lymph node anlagen and later in the posterior located anlagen, LECs circumvent the LN anlagen and connect the lymphatic vasculature to the LN anlagen. This allows the migration of LTi cells from the skin towards the LN anlagen [START_REF] Bovay | Multiple roles of lymphatic vessels in peripheral lymph node development[END_REF][START_REF] Vondenhoff | Lymph sacs are not required for the initiation of lymph node formation[END_REF][START_REF] Wang | Differential Roles of LTβR in Endothelial Cell Subsets for Lymph Node Organogenesis and Maturation[END_REF]. Notably, an elegant study by the Petrova lab showed that flow from the lymphatic vasculature was important for Cxcl13 expression within the inguinal lymph node anlagen. Blocking lymphangiogenesis by administering vascular endothelial growth factor receptor 3 (VEGFR3)-blocking antibodies resulted in an arrest of the small, initial, LTi cell aggregate and significant fewer LTi4 cells within the inguinal LN anlagen [START_REF] Bovay | Multiple roles of lymphatic vessels in peripheral lymph node development[END_REF].

The first visible LN anlagen during human fetal development are found after week 12 [START_REF] Cupedo | Human lymph node development: An inflammatory interaction[END_REF], although the initial clustering of LTi cells occurs most likely earlier. In the mouse, the first LTi cell aggregate was observed at E12.5, either in hCD2 GFP embryos [START_REF] Veiga-Fernandes | Tyrosine kinase receptor RET is a key regulator of Peyer's patch organogenesis[END_REF] or analysis by flow-cytometry and (3D) immunofluorescence of RORyt GFP embryos [START_REF] Simic | Distinct Waves from the Hemogenic Endothelium Give Rise to Layered Lymphoid Tissue Inducer Cell Ontogeny[END_REF][START_REF] Eberl | An essential function for the nuclear receptor RORgamma(t) in the generation of fetal lymphoid tissue inducer cells[END_REF](Figure 3). In time, other lymph node anlagen appear, notably in an anterior to posterior chronological manner, with the popliteal lymph nodes appearing the latest after E14.5 [START_REF] Simic | Distinct Waves from the Hemogenic Endothelium Give Rise to Layered Lymphoid Tissue Inducer Cell Ontogeny[END_REF][START_REF] Rennert | Lymph Node Genesis Is Induced by Signaling through the Lymphotoxin β Receptor[END_REF]. During initiation at E12.5, the anterior situated mandibular and cervical LTi cell clusters are very closely situated near each other within the cervical region.

Separation of the cervical and mandibular LN anlagen occur clearly at E13.5. The brachial lymph node anlagen is present after E14.5 and located near the axillary LN anlagen. In the posterior region, initially at E12.5-E13.5, a large LTi cell cluster was observed next to mesonephros. The renal, inguinal and popliteal LN anlagen could be separately identified after E14.5 [START_REF] Simic | Distinct Waves from the Hemogenic Endothelium Give Rise to Layered Lymphoid Tissue Inducer Cell Ontogeny[END_REF]. Since in the adult renal LNs are not bigger than the inguinal LNs and LTi aggregation in the inguinal LN anlagen dramatically increases in size within a day while the number of LTi cells within the renal LN anlagen uncharacteristically plateau at the same stage [START_REF] Simic | Distinct Waves from the Hemogenic Endothelium Give Rise to Layered Lymphoid Tissue Inducer Cell Ontogeny[END_REF], it could be that the large aggregation of the initial renal LN anlagen in the mesonephros region could aid formation of other LN anlagen nearby by 'donating' LTi cells to the new niche nearby.

ILC3 and LTi-like in adult lymph nodes and gut.

In the adult mouse, RORγt + CCR6 + CD4 + LTi4 cells are mainly observed in lymph nodes and in gut. Lymph nodes are important for the functioning of the adaptive immune system, as they organize and facilitate the interaction between antigen presenting cells and the lymphocytes. The antigens are transported towards the lymph nodes by the afferent lymphatic vessels either unbound or bound to antigen presenting cells. The lymphatic vessels are connected to the lymphatic subcapsular sinus circumventing the lymph node. The antigen and cells are spread over the subcapsular sinus and migrate into the B-cell follicles or into the T-cell areas, depending on the antigen size or type of antigen presenting cell [START_REF] Roozendaal | Conduits mediate transport of low-molecular-weight antigen to lymph node follicles[END_REF][START_REF] Braun | Afferent lymph-derived T cells and DCs use different chemokine receptor CCR7-dependent routes for entry into the lymph node and intranodal migration[END_REF][START_REF] Martens | Efficient homing of T cells via afferent lymphatics requires mechanical arrest and integrin-supported chemokine guidance[END_REF](reviewed in [START_REF] Jalkanen | Lymphatic endothelial cells of the lymph node[END_REF]). Each immune reaction is taking place in a specialized region within the lymph node, thus compartmentalizing the immune response. These regions are meticulously organized and maintained by specialized stromal cells. Initially, only T-cell zone stromal cells (fibroblastic reticular cells) in the paracortex, B-cell stromal zone cells (follicular dendritic cells) and endothelial cells were described (reviewed in [START_REF] Gentek | Lymph Node Stroma Dynamics and Approaches for Their Visualization[END_REF]). This was later amended by the marginal reticular cells (MRC), positioned in the paracortex, adjacent to the B-cell follicles and just below the subcapsular sinus [START_REF] Katakai | Organizer-like reticular stromal cell layer common to adult secondary lymphoid organs[END_REF]. However, the use of single cell sequencing has allowed the identification of nine stromal cell clusters within the lymph node [START_REF] Rodda | Single-Cell RNA Sequencing of Lymph Node Stromal Cells Reveals Niche-Associated Heterogeneity[END_REF], each with specific expression profiles and functions (reviewed in [START_REF] Gentek | Lymph Node Stroma Dynamics and Approaches for Their Visualization[END_REF]). These stromal cells differentiate from mesenchymal precursor cells, or LTo cells, in the first weeks after birth in the mouse [START_REF] Jarjour | Fate mapping reveals origin and dynamics of lymph node follicular dendritic cells[END_REF][START_REF] Koning | Development of follicular dendritic cells in lymph nodes depends on retinoic acid mediated signaling[END_REF][START_REF] Denton | Embryonic FAP + lymphoid tissue organizer cells generate the reticular network of adult lymph nodes[END_REF]. Although there are indications that lymphotoxin signaling, retinoic acid or microbiota mediate mesenchymal differentiation [START_REF] Van De Pavert | Chemokine CXCL13 is essential for lymph node initiation and is induced by retinoic acid and neuronal stimulation[END_REF][START_REF] Koning | Development of follicular dendritic cells in lymph nodes depends on retinoic acid mediated signaling[END_REF][START_REF] Pezoldt | Neonatally imprinted stromal cell subsets induce tolerogenic dendritic cells in mesenteric lymph nodes[END_REF], the specific cues for differentiation towards a specific stromal cell subset are yet unclear [START_REF] Cupedo | Cellular interactions in lymph node development[END_REF]. Embryonic LTi cells, which are involved in embryonic LN formation, are replaced in the adult by LTi cells derived from the bone-marrow [START_REF] Simic | Distinct Waves from the Hemogenic Endothelium Give Rise to Layered Lymphoid Tissue Inducer Cell Ontogeny[END_REF][START_REF] Sawa | Lineage relationship analysis of RORgammat+ innate lymphoid cells[END_REF]. Thus, since their origin is different, the LTi cells in adult are likely to be functionally different compared to the embryonic LTi cells. Within the lymph node, LTi cells were found adjacent to the MRC without an apparent function. Since the MRCs were assumed being a precursor for other stromal cells during expansion of the lymph node during inflammation [START_REF] Jarjour | Fate mapping reveals origin and dynamics of lymph node follicular dendritic cells[END_REF], the MRC-LTi cell communication might reflect the interaction between embryonic LTi cells with LTo cells occurring during embryonic lymph node formation. Besides the obvious role for LTi cells during embryonic lymph node formation, there is not much known on their role in the adult. There is phenotypically not much difference, although adult LTi cells endogenously express OX40 and CD30 ligands, involved in clonal expansion and memory of lymphocytes, while the fetal LTi cells need DR3 stimulation to express these ligands [START_REF] Kim | Neonatal and adult CD4+ CD3-cells share similar gene expression profile, and neonatal cells up-regulate OX40 ligand in response to TL1A (TNFSF15)[END_REF]. It was described that adult LTi cells were involved in the formation of tertiary lymphoid structures during cancer [START_REF] Shields | Induction of lymphoidlike stroma and immune escape by tumors that express the chemokine CCL21[END_REF]. They have been described in the restoration of lymphoid tissues, as they were involved in spleen restoration after viral infection [START_REF] Scandella | Restoration of lymphoid organ integrity through the interaction of lymphoid tissue-inducer cells with stroma of the T cell zone[END_REF] and rescue of mucosal associated lymphoid tissue (MALT) in an Id2-knock out model [START_REF] Fukuyama | Initiation of NALT Organogenesis Is Independent of the IL-7R, LTβR, and NIK Signaling Pathways but Requires the Id2 Gene and CD3-CD4+CD45+ Cells[END_REF].

A very related LTi cell subset within the ILC3 branch is the LTi-like population, present in high numbers within the adult gut mucosa. These cells are different than the closely related NKp46 + CCR6 -RORγt + ILC3 cells, which originate from NKp46 -CCR6 -progenitors driven by T-Bet which upregulates IFN-y and NKp46 expression [START_REF] Klose | A T-bet gradient controls the fate and function of CCR6-RORγt+ innate lymphoid cells[END_REF][START_REF] Rankin | Complementarity and redundancy of IL-22-producing innate lymphoid cells[END_REF]. LTi-like cells are characterized as NKp46 -CCR6 + (CD4 +/-) ILC3 cells, and found to be involved in antigen presentation and immune modulation and secrete IL22 [START_REF] Chen | IL-23 activates innate lymphoid cells to promote neonatal intestinal pathology[END_REF][START_REF] Marchesi | CXCL13 expression in the gut promotes accumulation of IL-22-producing lymphoid tissue-inducer cells, and formation of isolated lymphoid follicles[END_REF]. They are guided by CCR7 expression [START_REF] Mackley | CCR7-dependent trafficking of RORγ + ILCs creates a unique microenvironment within mucosal draining lymph nodes[END_REF] and play an important role in interaction between follicular Thcells and IgA producing B-cells [START_REF] Melo-Gonzalez | Antigen-presenting ILC3 regulate T cell-dependent IgA responses to colonic mucosal bacteria[END_REF]. An embryonic LTi cell subset (LTi4 -II) expressed MHCII related genes [START_REF] Simic | Distinct Waves from the Hemogenic Endothelium Give Rise to Layered Lymphoid Tissue Inducer Cell Ontogeny[END_REF], and it is interesting to establish whether there is a lineage and functional relationship between embryonic LTi4-II and LTi-like cells in the adult gut.

There are numerous reports stating the importance of ILC3s in the immune defense. They are involved in immune cells interactions to induce IgA production on B-cells [START_REF] Mackley | CCR7-dependent trafficking of RORγ + ILCs creates a unique microenvironment within mucosal draining lymph nodes[END_REF][START_REF] Melo-Gonzalez | Antigen-presenting ILC3 regulate T cell-dependent IgA responses to colonic mucosal bacteria[END_REF]. They secrete IL17 and notably IL22 secretion upon IL23R activation, shown in mice and human and in different disease settings [START_REF] Chen | IL-23 activates innate lymphoid cells to promote neonatal intestinal pathology[END_REF][START_REF] Lee | AHR drives the development of gut ILC22 cells and postnatal lymphoid tissues via pathways dependent on and independent of Notch[END_REF][START_REF] Ciccia | Type 3 innate lymphoid cells producing IL-17 and IL-22 are expanded in the gut, in the peripheral blood, synovial fluid and bone marrow of patients with ankylosing spondylitis[END_REF][START_REF] Bernink | Interleukin-12 and -23 Control Plasticity of CD127+ Group 1 and Group 3 Innate Lymphoid Cells in the Intestinal Lamina Propria[END_REF]. Besides their role in immune reactions, IL22 secreted by ILC3s was shown to be also important for maintenance of epithelial integrity. IL22 from ILC3/LTi-like cells induced the fucosyltransferase 2 (Fut2) expression in gut epithelial cells, which is required for fucosylation and resistance to Salmonella typhimurium infection [START_REF] Goto | Innate lymphoid cells regulate intestinal epithelial cell glycosylation[END_REF].

There is discussion on the possible redundancy of NKp46 + ILC3s in the gut-immune response. A Ncr1 (NKp46)-Cre model was used to delete ILC3 cells or specifically knock-out Il22 within NKp46 + ILC3s during gut infection. In these models, the NKp46 + ILC3 population was shown to be redundant during an infection with Citrobacter rodentium and ILC3 deletion only affected cecal homeostasis [START_REF] Rankin | Complementarity and redundancy of IL-22-producing innate lymphoid cells[END_REF].

However, Il22 expression in the NKp46 -CCR6 + LTi-like cell population was not affected in these models [START_REF] Rankin | Complementarity and redundancy of IL-22-producing innate lymphoid cells[END_REF] and it remains unclear whether this population could (partially) rescue the loss of IL22 from the NKp46 + ILC3 population. Indeed, it was shown that the ILC3 are partially redundant but play an essential role in T-cell deficient mice [START_REF] Song | Unique and redundant functions of NKp46+ ILC3s in models of intestinal inflammation[END_REF]. In another example of possible redundancy it was observed that in patients with severe combined immunodeficiency (SCID), the restoration of the hematopoietic compartment did not restore the ILC family without any obvious susceptibility to a disease [START_REF] Vély | Evidence of innate lymphoid cell redundancy in humans[END_REF]. Lastly, in human patients with a RORc loss of function mutation, lacking RORγt functionality and thus all ILC3 and LTi-like cells, were only more susceptible to Mycobacterium and Candida infections. This effect was attributed to defective γδ-and αβ-T-cells but not ILC3's [START_REF] Okada | Impairment of immunity to Candida and Mycobacterium in humans with bi-allelic RORC mutations[END_REF]. Therefore, it remains unclear what the unique roles of ILC3 and LTi-like cells are within the gut. Is there indeed redundancy, as was proposed before [START_REF] Vély | Evidence of innate lymphoid cell redundancy in humans[END_REF][START_REF] Rankin | Complementarity and redundancy of IL-22-producing innate lymphoid cells[END_REF], or is there a rescue mechanism which arises during fetal development and neonatal stages? Better mouse models, in which specifically ILC3 and/or LTi like cells can be deleted by induction at a specific time point to prevent a rescue mechanism during development, are required to answer this question. Additionally, the use of a unique gene for each population, but not genes such as Ncr1 or Rorc as drivers, are required to establish the specific effect of a specific ILC3 cell during disease.

Neuropilin-1 (NRP-1) was found to be expressed by ILC3 and LTi cells in mouse and human and involved in the formation of ectopic lymphoid organs, especially in the lungs of smokers and COPD patients [START_REF] Shikhagaie | Neuropilin-1 Is Expressed on Lymphoid Tissue Residing LTi-like Group 3 Innate Lymphoid Cells and Associated with Ectopic Lymphoid Aggregates[END_REF]. Recently, Neuropilin-1 was also associated as host factor for Sars-Cov2 entry and the virus was associated with NRP-1 positive cells within the nasal cavity [START_REF] Cantuti-Castelvetri | Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity[END_REF][START_REF] Daly | Neuropilin-1 is a host factor for SARS-CoV-2 infection[END_REF]. If Sars-Cov2 specifically transfects ILC3 and LTi cells is still unknown. It will be important to establish whether during this infection LTi cells could mediate ectopic lymphoid formation in lungs, which possibly exacerbates the immune reaction.

In general, ILC differentiation in embryo and adult is very flexible in order to adapt to the niche they reside in. Also, ILCs can be derived from different progenitors (reviewed in [START_REF] Bal | Plasticity of innate lymphoid cell subsets[END_REF][START_REF] Gronke | Innate lymphoid cells, precursors and plasticity[END_REF]). Therefore, marker expression for the ILC family members can vary and depend on their origin and differentiation program. This complicates the determination of the ILC members and induces discussion on their nomenclature. Especially for the characterization of enigmatic ILC1 subset this has been problematic [START_REF] Björklund | The heterogeneity of human CD127+ innate lymphoid cells revealed by single-cell RNA sequencing[END_REF] and based on CyTOF analysis it was even suggested that there is no specific human ILC1 population [START_REF] Simoni | Human Innate Lymphoid Cell Subsets Possess Tissue-Type Based Heterogeneity in Phenotype and Frequency[END_REF]. Other testimonies on ILC dynamicity are that the ILC3 cells within the gut could differentiate into ILC1 cells [START_REF] Rankin | Complementarity and redundancy of IL-22-producing innate lymphoid cells[END_REF], fetal human intermediate-ILC1 were observed to become NK and ILC3s [START_REF] Li | Mass cytometry reveals innate lymphoid cell differentiation pathways in the human fetal intestine[END_REF] progenitors in the gut differentiate to ILC2 cells which migrate to the lung [START_REF] Huang | S1P-dependent interorgan trafficking of group 2 innate lymphoid cells supports host defense[END_REF](reviewed in [START_REF] Bal | Plasticity of innate lymphoid cell subsets[END_REF]). Since the tissue residency and its immune status is of importance to the phenotype of the ILC, it is therefore of importance to isolate different tissues to characterize the ILC in question. The use of tonsil alone will skew characterization or provide a partial marker expression for human ILCs [START_REF] Bar-Ephraim | Cross-Tissue Transcriptomic Analysis of Human Secondary Lymphoid Organ-Residing ILC3s Reveals a Quiescent State in the Absence of Inflammation[END_REF].

The origin of the ILCs, being embryonic or adult, is important to understand their eventual functioning.

Therefore, it remains of much interest to pinpoint the embryonic hemogenic source of the ILCs and when they are replaced in the neonate by bone-marrow derived ILCs. This knowledge will aid our understanding of their miscellaneous roles in immunity and how to possibly dampen them in unwanted immunological reactions.

Acknowledgements

The group was supported by the FRM Amorçage de jeunes équipes (AJE20150633331), ANR ACHN (ANR-16-ACHN-0011), ANR PRCI (ANR-17-CE13-0029-01) and institutional grants from INSERM, CNRS and Aix-Marseille University to the CIML. I would like to thank Ana Cumano for the discussions on the hematopoietic progenitors and Lionel Spinelli for his help on the transcriptional data. I am grateful to Milesa Simic, Shuaiwei Wang and especially Rachel Golub for critically reading the manuscript.

Legends

Figure 1; Embryonic LTi cell ontogeny. LTi ontogeny and associated markers from the origin in the hemogenic endothelium to their presence within the LN anlagen. Progenitors originate from the embryonic hemogenic endothelium between E7.5-9 as observed by the use of Cxcr4 and Cdh5 lineage tracing models [START_REF] Simic | Distinct Waves from the Hemogenic Endothelium Give Rise to Layered Lymphoid Tissue Inducer Cell Ontogeny[END_REF]. Subsequent hematopoietic precursors reside, proliferate and differentiate within the fetal liver towards the ILC lineage [START_REF] Ishizuka | Single-cell analysis defines the divergence between the innate lymphoid cell lineage and lymphoid tissueinducer cell lineage[END_REF][START_REF] Simic | Distinct Waves from the Hemogenic Endothelium Give Rise to Layered Lymphoid Tissue Inducer Cell Ontogeny[END_REF][START_REF] Chea | Single-Cell Gene Expression Analyses Reveal Heterogeneous Responsiveness of Fetal Innate Lymphoid Progenitors to Notch Signaling[END_REF][START_REF] Berthault | Asynchronous lineage priming determines commitment to T cell and B cell lineages in fetal liver[END_REF]. The αLP/LTiP population migrate towards the LN anlagen, where these cells differentiate into LTi cells, marked by the expression of Rorc. Two clusters of LTi cell populations were observed within the LN anlagen enriched tissue, mainly segregated based on MHCII related genes and hence named LTi4 and LTi4-II cells. Genes (in italics) and proteins upregulated at a specific stage are shown below the populations, while genes enriched in the two LTi4 cell populations are shown to their right [START_REF] Ishizuka | Single-cell analysis defines the divergence between the innate lymphoid cell lineage and lymphoid tissueinducer cell lineage[END_REF][START_REF] Simic | Distinct Waves from the Hemogenic Endothelium Give Rise to Layered Lymphoid Tissue Inducer Cell Ontogeny[END_REF][START_REF] Chea | Single-Cell Gene Expression Analyses Reveal Heterogeneous Responsiveness of Fetal Innate Lymphoid Progenitors to Notch Signaling[END_REF][START_REF] Possot | Notch signaling is necessary for adult, but not fetal, development of RORγt(+) innate lymphoid cells[END_REF][START_REF] Berthault | Asynchronous lineage priming determines commitment to T cell and B cell lineages in fetal liver[END_REF][START_REF] Bennion | STING Gain-of-Function Disrupts Lymph Node Organogenesis and Innate Lymphoid Cell Development in Mice[END_REF].

Figure 2 ;

 2 Figure 2; Overview of embryonic lymph node formation. Earliest phases in lymph node formation. 1)

Figure 3 ;

 3 Figure 3; Overview of the timing of initiation for LTi cell dependent lymphoid organs including Peyer's