Modulation of cell-mediated immunity during pregnancy in wild bonobos

Abstract

During pregnancy, the mammalian immune system must simultaneously protect against pathogens while being accommodating to the foreign fetal tissues. Our current understanding of this immune modulation derives predominantly from industrialized human populations and laboratory animals. However, their environments differ considerably from the pathogen-rich, resource-scarce environments in which pregnancy and the immune system co-evolved. For a better understanding of immune modulation during pregnancy in challenging environments, we measured urinary neopterin, a biomarker of cell-mediated immune responses, in 10 wild female bonobos (Pan paniscus) before, during and after pregnancy. Bonobos, sharing evolutionary roots and pregnancy characteristics with humans, serve as an ideal model for such investigation. Despite distinct environments, we hypothesized that cell-mediated immune modulation during pregnancy is similar between bonobos and humans. As predicted, neopterin levels were higher during than outside of pregnancy, and highest in the third trimester, with a significant decline post-partum. Our findings suggest shared mechanisms of cell-mediated immune modulation during pregnancy in bonobos and humans that are robust despite distinct environmental conditions. We propose that these patterns indicate shared immunological processes during pregnancy among hominins, and possibly other primates. This finding enhances our understanding of reproductive immunology.

1. Introduction

The immune system protects the host against intruding pathogens and is key for health and survival. Yet, immune responses present a dilemma to mammals during pregnancy: the immune system must protect the mother and the developing fetus from potential infections while being simultaneously tolerant of foreign tissues within the uterine environment [1]. Therefore, precise modulation of maternal immune activity during embryonic implantation and pregnancy is necessary for a successful birth (e.g. [2–4]). The maternal immune system plays an active role during pregnancy to support fetal development: a pro-inflammatory state in the early stages of pregnancy allows implantation and placentation. The second trimester is characterized as an anti-inflammatory state, promoting fetal growth. Towards the end of pregnancy, there is renewed inflammation as the body prepares for childbirth with an influx of immune cells into the uterus [5].

Most knowledge about immune system modulation during pregnancy derives from studies on industrialized human populations [6–8], and well-established model species investigated under laboratory conditions [9,10]. These environments differ in resource availability and pathogen load from the environments in which mammalian immune systems and pregnancies co-evolved [11]. To fully understand the function of cell-mediated immune modulation and associated implications in pregnancy outcomes [12–14], it is essential to investigate whether the immunological patterns observed during pregnancy in pathogen-devoid, nutrient-rich environments are also present when pathogen pressure is higher and energy availability is limited [15]. While pathogen loads differ between industrialized and other populations [3,16], studies comparing the immunity of pregnant women from industrialized and indigenous societies do not exist to our knowledge.

Most mammalian model organisms are not ideal substitutes for human-participant research because of fundamental differences in placental features and immune system development [3]. Bonobos (Pan paniscus) are a suitable model species as duration [17], course of pregnancies [18,19] and placentation [20,21], including immunological aspects are similar to humans [22,23]. Additionally, wild bonobos inhabit pathogen-rich, resource-limited environments, a habitat likely resembling conditions under which primate pregnancies and immune systems co-evolved [24,25]. Long-term research sites allow longitudinal monitoring of individual females and frequent assessment of physiological urinary markers across pregnancies [26].

Neopterin increases when cell-mediated immune responses are activated in response to infection and inflammation in humans and bonobos [27–29] and can be reliably measured in their urine, facilitating regular, non-invasive monitoring [30,31]. In humans, neopterin is used to track maternal immune responses and shifts in immunity during pregnancy. In blood and urine samples, neopterin levels were consistently elevated in pregnant women compared to their non-pregnant peers, with highest levels observed during the third trimester, followed by a decline postpartum [32–37]. Neopterin results in pregnant non-human primates are inconsistent. Neopterin levels were significantly higher in urine of pregnant chimpanzees [38] and faeces of pregnant mandrills [39], and showed a trend in bonobo urine samples [28] but not in mandrill plasma [40].

To obtain a more comprehensive understanding of urinary neopterin patters throughout pregnancies in non-human primates, we collected samples longitudinally from wild female bonobos before, during, and after pregnancy. We hypothesized that modulation of cell-mediated immunity during pregnancy is similar in bonobos and humans due to their close evolutionary relatedness and shared pregnancy characteristics, despite different environments. Consequently, we predicted (1) higher neopterin levels during pregnancy compared to non-pregnant phases, and (2) highest neopterin levels during the third trimester compared to earlier trimesters or non-pregnant phases.

2. Results and discussion

Model assumptions were met for all models presented below, unless indicated otherwise (for exact procedures and handling of outliers electronic supplementary material, S1). When testing model assumptions, we found that several residuals, in particular in the non-pregnant condition, were outside the interquartile range (IQR). Re-fitting the model with a dataset excluding those datapoints (electronic supplementary material, S1 and table S1) produced the same results.

We found that urinary neopterin levels decreased throughout the day (table 1), as found in other primates [32,38,41–44]. This finding emphasizes the importance of considering sampling time in future urinary neopterin studies.

Table 1.

Results of the pregnancy model. The model contained the main effects of the binary pregnancy variable (pregnant–not pregnant), the time of sample collection (z-transformed minutes since midnight), and a random intercept for the individual. Estimates (β), standard errors (SE) and p-values for individual variables are presented. For the categorical variable pregnancy, reference category is indicated. Bold indicates significance.

	β (estimate)	SE	p-value
intercept	5.184	0.075
pregnancy state: pregnant	0.295	0.086	<0.001
time (z-transformed)	−0.283	0.087	0.002
	variance	s.d. (standard deviation)
individual female	0.020	0.141
residual	0.619	0.781

(a) . Urinary neopterin levels in pregnant and non-pregnant females

First, we tested whether females had higher neopterin levels during pregnancy than in other reproductive states. Since pathogen load can be expected to vary randomly across reproductive states, we did not collect data or controlled for pathogen load in our analyses. The fitted model included a binary variable indicating pregnancy state (pregnant, not pregnant), the z-transformed sampling time, and a random intercept for individuals (table 1). A log-likelihood ratio test revealed that adding a random slope for pregnancy state (pregnant–not pregnant) to the model did not improve model fit (λ² = 4.21, d.f. = 2, p = 0.122). Comparing this model to the null model (without the two predictor variables) revealed significant differences between them (λ² = 20.63, d.f. = 2, p < 0.01). Both variables, pregnancy (λ² = 11.38, d.f. = 1, p < 0.01) and sampling time (λ² = 10.01, d.f. = 1, p = 0.002) significantly predicted urinary neopterin levels, with higher levels in samples from pregnant females (table 1). This finding is in line with results from human studies [32,33,45,46] and wild chimpanzees [38]. The consistency of this pattern across these three species indicates that immune modulation during pregnancy is a shared trait, independent of pathogen richness and energy availability. Increasing neopterin levels during pregnancy are attributed to increased immunogenic stimuli by the placenta and the fetus [36]. Macrophages secrete neopterin when activated, and macrophage numbers of maternal and fetal origin increase during pregnancy in the decidua and placenta, respectively [47,48]. Therefore, urinary neopterin levels during pregnancy originate from the mother, the placenta, and the fetus, and probably reflect regulatory, nuanced anti-inflammatory and inflammatory processes [48], rather than environmental pathogen load.

(b) . Urinary neopterin levels before, during and after pregnancies

To test if neopterin was highest during the third trimester compared to earlier trimesters or non-pregnant conditions, we divided the pregnancy state variable into five phases: before pregnancy, first, second, third trimester of a pregnancy, and after pregnancy. We fitted our second model including the time variable (z-transformed) and the five levels of pregnancy state as predictors as well as a random intercept for each female (electronic supplementary material, S1 and table S2). Adding a random slope for pregnancy state to the random effect structure improved model fit (λ² = 25.86, d.f. = 14, p = 0.027), but resulted in a singular fit warning, indicating that coefficient estimates would not be reliable [49]. Therefore, we proceeded with the initial model. This model was different from the corresponding null model only containing the random effect structure (λ² = 25.73, d.f. = 5, p < 0.001).

The five-level pregnancy state variable (λ² = 16.47, d.f. = 4, p = 0.003; figure 1a) as well as time of day (λ² = 11.17, d.f. = 1, p < 0.001) predicted urinary neopterin levels (electronic supplementary material, S1 and table S2). Post-hoc pairwise comparisons revealed significantly higher marginal means for the third trimester compared to after pregnancy (β = 0.53, SE = 0.14, p = 0.001, electronic supplementary material, S1 and table S3). Marginal means of the third trimester were higher than before a pregnancy, although not significantly (β = 0.37, SE = 0.14, p = 0.055, electronic supplementary material, S1 and table S3). Other pairwise comparisons did not indicate differences between conditions (electronic supplementary material, S1 and table S3). Likewise, human studies noted highest neopterin levels in urine and blood during the last trimester of pregnancy [32–35,37]. This pattern has been attributed to different inflammatory states in pregnancy. The first trimester is characterized by proinflammatory processes associated with implantation, transitioning to an anti-inflammatory state in the second trimester. Proinflammatory processes rise again shortly before parturition, which probably facilitates birth [1,5,48]. Cellular immunity plays a dominant role at the end of pregnancy [33] possibly causing increasing neopterin levels in the third trimester.

Figure 1.

Urinary neopterin levels of female bonobos before, during and after pregnancy. Boxes in blue indicate non-pregnant (before and after pregnancy) states, while boxes showing measurements from pregnant females (first, second and third trimester) are coloured in orange. The boxplot shows the distribution of urinary neopterin levels (log-transformed) before, during and after pregnancy as estimated by the models (for population level effects) see (a), for within-individual patterns see (b). Each box represents the interquartile range (IQR), with the horizontal line inside the box indicating the median. Whiskers extend to the minimum and maximum values within 1.5 times the IQR. Black dots show actual datapoints.

The production of neopterin during pregnancy may serve an adaptive function similar to its role during infections and inflammation [50–52]. It may counteract hyperinflammation during the processes of implantation and childbirth and reduce oxidative stress during the development of the embryo, fetus and placenta [53–55]. In this context, low neopterin levels after pregnancy, but not before, might indicate shifts in immune modulation between pregnant and lactational phenotypes. Higher neopterin levels before pregnancy might be related to immunological changes of the endometrium during each menstrual cycle [56]. Another explanation is that our method of dating pregnancies from the day of birth may have led to misassignments, potentially categorizing some samples as ‘before pregnancy’ when the female was already pregnant, leading to higher mean neopterin levels before pregnancy. In this context it is difficult to compare with humans, because usually participants are not sampled before pregnancy, and studies use distinct cohorts of non-pregnant and pregnant females [33,45,57]. Despite differences in study design, one human study found that neopterin levels did not differ in samples from women in the first and second trimester from levels measured in samples from non-pregnant women [33], which is in line with our own results where neopterin levels before pregnancy were not significantly different from levels in the first and second trimester of pregnancy.

We found that on the individual level, patterns of urinary neopterin levels across pregnancy phases differed substantially (figure 1b). Seven out of nine females (for one female we lacked post-pregnancy samples) had higher neopterin levels in the third trimester as compared to after the pregnancy, but only five females had higher third trimester neopterin levels than before the pregnancy. In human studies, substantial variation in neopterin level has also been reported in both blood and urine [32,33,35]. One study provided individual trajectories of neopterin levels within women [45], revealing similar patterns as in our results. These differences may reflect differences in lifetime immunity, as cell-mediated immune responses to malaria antigens were found to be suppressed in pregnant Gambian women [58].

3. Conclusion

Our data, consistent with previous human neopterin studies, indicate that elevated neopterin levels in pregnant female bonobos compared to non-pregnant individuals are primarily due to increased neopterin levels during the last trimester, followed by a post-pregnancy decline. Our study implies that cell-mediated immunomodulatory processes during pregnancy are a shared trait in humans' closest living relatives and are robust in resource-limited environments with high pathogen loads. This emphasizes the critical role of immune modulation in ensuring the successful development of the fetus while protecting both mother and offspring from pathogens.

Although the extent to which our findings can be generalized and provide insights into mechanisms is limited, we propose that these mechanisms represent a shared trait among hominins. Ultimately, comparative studies will provide insights into the mechanistic specificities of hominin pregnancy and related disease states and our conceptualization of pregnancy as an evolved relational novelty between mother and embryo [59].

4. Material and methods

(a) . Study population

Data were collected from 10 wild female bonobos in the Bompusa West and East communities, LuiKotale, Democratic Republic of the Congo. Urine samples (n = 377) were collected between 2011 and 2022 from habituated bonobos, stored in liquid nitrogen and transported to research institutes in Germany (detailed information in electronic supplementary material and methods).

(b) . Neopterin measurement

Neopterin was measured with a commercially available, competitive neopterin ELISA (Neopterin ELISA, ref. RE59321, IBL International GmbH, Hamburg, Germany). Detailed information in electronic supplementary material, S2.

(c) . Data preparation and statistical analyses

Pregnancy periods were determined based on gestation length, with trimesters defined accordingly. Samples collected before, during, or after pregnancies were categorized. Data from 344 samples across 10 females were used for analysis, with one female contributing data from two pregnancies. Data from a female with a miscarriage (33 samples) were excluded. Detailed information in electronic supplementary material, S2.

Linear mixed models with log-transformed neopterin levels were fitted, accounting for time of sample collection and individual variation. Model 1 assessed the effect of pregnancy on neopterin levels. Model 2 additionally considered the condition (before, during, after pregnancy) as a predictor. Likelihood ratio tests were used for model comparison, and assumptions were checked visually. R and relevant packages were used for analysis. Further information in electronic supplementary material, S2.

Ethics

Ethical review and approval were not required for the animal study because all samples were collected non-invasively and with permission of the Institut Congolais pour la Conservation de la Nature (0683/ICCN/DG/ADG/014/ KV/2012). Permits for exporting the urine samples from the Democratic Republic of Congo were issued by the ICCN (0521/ICCN/DG/CWB/05/01/2014).

Data accessibility

CSV file, ReadMe, source data and R-code for statistics and figures in the paper are permanently stored at GRO.data, ‘Replication data for: Modulation of cell mediated immunity during pregnancy in wild bonobos’: https://doi.org/10.25625/VTL0UL [60].

Source data and R-code for statistics and figures in the paper are permanently stored at GRO.

Supplementary material is available online [61].

Declaration of AI use

We have not used AI-assisted technologies in creating this article.

Authors' contributions

V.B.: conceptualization, funding acquisition, investigation, writing—original draft, writing—review and editing; C.D.: conceptualization, writing—original draft, writing—review and editing; J.O.: funding acquisition, writing—review and editing; B.F.: funding acquisition, project administration, resources, writing—review and editing; R.S.: formal analysis, investigation, software, visualization, writing—original draft, writing—review and editing.

All authors gave final approval for publication and agreed to be held accountable for the work performed therein.

Conflict of interest declaration

We declare we have no competing interests.

Funding

Field research was funded by the Centre for Research and Conservation of the Royal Zoological Society of Antwerp, the Max Planck Institute (MPI) of Animal Behaviour, the MPI of Evolutionary Anthropology, the Ouwehand Zoo and the Leakey Foundations. This study was funded by Deutsche Forschungsgemeinschaft (grant no. DFG BE 5511/4-1) to V.B. and by Audacity Fund, (grant no. LSC-AF2023_08) to V.B. and J.O. Further support was provided by the German Primate Center.