The Sex-Specific Effects of Early Life Adversity and Chronic Psychosocial Stress during Adulthood on Bone Are Mitigated by Mycobacterium vaccae NCTC 11659 in Mice

Chronic stress is a significant challenge in modern life, increasing the risk for various psychosomatic health issues, including post-traumatic stress disorders (PTSD) or depression [1‒3]. Many of these disorders are accompanied by an over-reactive immune system and chronic low-grade inflammation [4, 5], and prospective human and mechanistic animal studies strengthen the hypothesis that an exaggerated immune (re)activity plays a causal role in their pathogenesis [4, 6‒8]. Notably, the impact of stress varies widely among individuals, with some being more resilient than others [9‒12]. Women, for instance, are at a higher risk of developing PTSD compared to men, consistent with the overall trend of higher prevalence of stress-related disorders in women [13]. Given the link between an over-reacting inflammatory stress response and the development of stress-associated disorders including PTSD, all genetic and environmental factors facilitating an adult’s immune (re)activity are, therefore, likely to increase their vulnerability for chronic psychosocial stress during adulthood (CAS). One such environmental factor might be early life adversity (ELA). This hypothesis is consistent with the fact that psychological trauma has particularly adverse consequences if it occurs in a cumulative manner [9, 10]. Moreover, child maltreatment causes chronic low-grade inflammation, characterized by increased levels of proinflammatory cytokines and C-reactive protein, fibrinogen, and white blood cells [14‒16]. Most interestingly, psychosocial stress has been shown repeatedly to activate peripheral inflammatory pathways [8, 17], and to do so more robustly in people with histories of early life abuse and/or neglect [18, 19], who are also at significantly heightened risk for PTSD development in response to trauma exposure in adult life [20].

Consistent with the aforementioned role of immune activation in the development of stress-related disorders, we and others have previously shown that repeated subcutaneous (s.c.) preimmunizations with a heat-killed preparation of Mycobacterium vaccae NCTC (National Collection of Type Cultures) 11659, an abundant saprophytic “Old Friend” from mud with immunoregulatory properties, are effective in (i) stabilizing the gut microbiome [21, 22], (ii) increasing the percentage of Tregs in mesenteric lymph node cells [21], (iii) preventing stress-induced colitis and proinflammatory cytokine secretion from freshly isolated mesenteric lymph node cells stimulated with anti-CD3 antibody ex vivo [21], (iv) preventing stress-induced aggravation of dextran sulfate sodium-induced colitis [21], (v) preventing stress-induced exaggeration of anxiety [21], (vi) preventing stress-induced microglial priming and neuroinflammation [23‒26], (vii) ameliorating features of age-associated microglia activation in the amygdala and hippocampus [27], (viii) preventing negative outcomes of sleep deprivation [28], and (ix) enhancing fear extinction [29]. In extension of these findings and in support of using “Old Friends” not only to prevent but also to treat stress-associated disorders, we recently showed that M. vaccae NCTC 11659 also ameliorates stress-induced anxiety when administered repeatedly via the s.c. route during chronic psychosocial stressor exposure, i.e., after the first psychosocial traumatization has occurred [30]. Our own studies confirm the stress-protective effects of M. vaccae NCTC 11659 even when administered via the noninvasive intranasal (i.n.) [31] or intragastric (i.g.) [32] route, respectively.

Of particular importance in the context of the current study, we showed in a recent study that (i) the negative behavioral, immunological, and physiological consequences of ELA (i.e., maternal separation, MS) in both sexes, although relatively mild, are to a great extent prevented by subsequent s.c. M. vaccae NCTC 11659 administrations, (ii) male mice seem to be more susceptible to CAS (i.e., males: chronic subordinate colony housing, CSC; females: social instability paradigm, SIP) than females, with only females benefitting slightly from the stress-protective effects of s.c. M. vaccae NCTC 11659 administrations when given prior to CAS alone, and (iii) female mice seem to be more vulnerable to the cumulative effects of ELA and CAS than male mice, with both sexes benefitting from s.c. M. vaccae NCTC 11659 administrations subsequent to ELA but prior to CAS [33]. However, effects on bone have not been investigated in this study, although an overshooting inflammatory response to CAS has pronounced negative effects on bone growth as well as bone regeneration after fracture in male mice [34, 35], with stress-activated myeloid immune cells producing and secreting catecholamines and inflammatory cytokines locally in the bone marrow and fracture hematoma, playing a critical role [36]. Therefore, the main aims of the current study were to investigate in a subset of male and female mice of the above referenced study [33], (i) if ELA itself affects the bone and/or potentiates the negative effects of CAS on bone metabolisms and regeneration, and (ii) if repeated s.c. M. vaccae NCTC 11659 administrations following ELA but prior to CAS are protective against the effects of ELA and CAS on bone.

Breeding male and female C57BL/6N mice obtained from Charles River (Sulzfeld, Germany) were used to generate in-house bred C57BL/6N offspring used as experimental mice. Male CD-1 mice (30–35 g; Charles River, Sulzfeld, Germany) were used as dominant aggressors during CSC exposure. All mice were kept in standard polycarbonate mouse cages (16 cm width × 22 cm length × 14 cm height) under standard specific pathogen-free laboratory conditions (12-h light-dark cycle, lights on: 06:00 a.m. [winter time], 07:00 a.m. [summer time]; which is referred to ZT time 0; 22°C, 60% humidity) and had free access to tap water and standard mouse diet. Reporting of the animal study was carried out in accordance with the ARRIVE guidelines 2.0 [37] and the study was approved by the Federal Animal Care and Use Committee (Regierungspräsidium Tübingen, Germany). All efforts were made to minimize the number of animals used and their suffering.

The experimental timelines are identical to [33] and are shown in Figure 1a (male mice) and Figure 1b (female mice). In detail, pregnant females were observed daily and, if litters were found before 02:00 p.m., the same day was assigned to postnatal day (PND) 0, if litters were found after 02:00 p.m., the following day was assigned to PND 0. At PND 1, litters were randomly assigned to the MS or “no maternal separation” (noMS) group and exposed to MS or noMS daily until PND 14. Afterward, all litters were housed together with their dams until weaning at PND 21. Following weaning, experimental MS and noMS mice were housed according to their treatment and sex in groups of 3–4 per cage for 1–2 weeks before receiving repeated (×3) weekly s.c. administrations with sterile borate-buffered saline (BBS) vehicle or M. vaccae NCTC 11659 (0.1 mg/0.1 mL per s.c. injection) in sterile BBS. Approximately 1–2 weeks after the last administration, all experimental mice were exposed to the first round of behavioral testing (open field/novel object [OF/NO1] test followed by social preference/avoidance test [SPAT1]) on consecutive days; results of behavioral tests have been published previously [33]. The day after SPAT1, experimental male mice were exposed to the CSC paradigm (3 weeks), while experimental female mice were exposed to the social instability paradigm (SIP) (7 weeks). Controls were housed singly (SHC; males) or in groups of 3–4 (GHC; females). Litter effects were controlled by assuring that every group was composed of no more than N = 3 pups from the same litter. On days 19/20 of CSC and on days 48/49 of SIP, all experimental mice were exposed to the second round of behavioral testing (OF/NO2 test followed by the SPAT2 on consecutive days; results of behavioral tests have been published previously [33]), before being euthanized the respective following days between 07:00 a.m. and 10:00 a.m. (ZT time 1–4). Following decapitation, right femurs of a subset of mice from each group were removed and stored in 4% formalin until µCT analysis. Physiological and immunological parameters assessed in each group are reported in [33].

Experimental mice received three s.c. immunizations with either 0.1 mg whole heat-inactivated M. vaccae NCTC 11659 suspension (10 mg/mL solution; strain NCTC 11659, batch ENG 1, provided by BioElpida [Lyon, France] diluted to 1 mg/mL in 100 μL sterile BBS) or injections of 100 μL of the vehicle (BBS) using 21-gauge needles within 2 consecutive weeks (see Fig. 1 for details), respectively. Of note, this is the same dose that our group has used previously for s.c. [21, 30], i.n. [31], and i.g. [38] administrations of M. vaccae NCTC 11659 in mice.

MS was performed as previously described [33, 39, 40]. Briefly, pups were separated from their dams daily for 3 h (08:00 a.m.–11:00 a.m. during wintertime; 09:00 a.m.–12:00 p.m. during summertime, ZT time 2–5) between PND 1 and 14. Therefore, dams were removed from the maternity cage and placed into separate individual cages before pups were transferred as a whole litter into a small box and placed onto a heating pad (30–33°C) in an adjacent room. After the 3-h separation period, the pups were placed back into the home cage, before being unified with the respective dam. Unseparated litters (noMS) served as controls and were left undisturbed, except for daily handling. Bedding was not changed for the entire duration of the MS paradigm.

The CSC paradigm was conducted as previously described [30, 33, 34, 41, 42]. Briefly, mice were weighed and assigned to either the single-housed control (SHC) or the CSC group. To induce chronic psychosocial stress, four CSC mice were housed together with a dominant male CD-1 aggressor mouse for 20 consecutive days. To avoid habituation, each dominant male was replaced by a novel dominant male on days 8 and 15 of the CSC paradigm. Before the CSC procedure, the future dominant males were tested for their aggressive behavior and mice that injured their opponents by excessive aggression were excluded. Notably, the number of bite wounds received by the residents could thereby be reduced, but not totally prevented. SHC mice remained undisturbed in their home cages except for weighing twice a week and a change of bedding once a week. In a previous study, we convincingly demonstrated that single housing is the adequate control group for the CSC paradigm, as group housing itself was shown to be stressful and to affect parameters assessed routinely in studies employing the CSC paradigm [43].

The SIP was performed as previously described [33]. Briefly, the cage composition of experimental mice was changed every third day over a period of seven consecutive weeks. To minimize the chance of repeatedly encountering the same cage mates, group rotation was randomized using GraphPad “Assign subjects to groups” (GraphPad Software, San Diego, CA) online version. Body weight and possible bite wounds were assessed every time the cage composition was changed. Respective GHCs were housed in groups of 3–4 mice for 7 weeks.

To assess the effects of M. vaccae NCTC 11659 and/or stress on anxiety-related behavior, the OF/NO test was conducted as previously described [30, 44, 45]. Schematic representation of the test arena and the experimental settings are reported in Figure 1. The test took place in a dedicated behavioral box between 07:00 a.m. and 10:00 a.m. (ZT time 1–3) inside the animal room where all animals were housed for the whole duration of the experiment. Briefly, during OF exposure, the test arena (length: 45 cm; width: 27 cm; height: 27 cm; 350 lx) was subdivided into an inner zone (9 cm × 27 cm) and an outer zone. The mouse was placed into the inner zone and allowed to explore the arena for 5 min. After 5 min of OF exploration, a plastic round object (diameter: 3.5 cm; height: 1.5 cm) was placed in the middle of the arena. The mouse was then allowed to explore the arena containing the unfamiliar NO for 5 min. In the OF test, the distance moved and the time spent in the inner zone by the animals were analyzed. Furthermore, in the NO test, the time spent in direct contact with the NO, indexed by the time spent in a 1.6-cm broad contact zone around the novel object, was analyzed. The test arena was cleaned thoroughly with water after each test. All parameters were analyzed using EthoVision XT (v11.5.1022; Noldus Information Technology, Wageningen, the Netherlands).

To assess the effects of M. vaccae NCTC 11659 and/or stress on social preference/avoidance, the SPAT was conducted as described previously [46]. The test took place in a dedicated behavioral box between 07:00 a.m. and 10:00 a.m. (ZT time 1–3) inside the animal room where all animals were housed for the whole duration of the experiment. Briefly, an experimental mouse was placed in the SPAT arena (length: 45 cm; width: 27 cm; height: 27 cm; 20 lux) for 30 s to habituate to the environment before a small empty wire mesh cage (length: 8.5 cm; width: 7.5 cm; height: 6.5 cm) was introduced for 150 s. Afterward, the empty cage was replaced by an identical cage containing an unfamiliar male (CD-1) or female (C57BL/6 N) stimulus mouse for another 150 s. The 4.5 cm broad zone around the nonsocial or social wire mesh was assigned as direct contact zone. Time spent in the direct contact zone was recorded using EthoVision XT (v11.5.1022; Noldus Information Technology, Wageningen, the Netherlands) during both 150 s trials. The box was cleaned thoroughly with water before each trial.

Following brief CO₂ inhalation, mice were euthanized by decapitation within 3 min after removing the cage from the animal room.

Right femurs were dissected, cleaned, and stored in 4% formalin. Femur lengths were assessed using a digital precision caliper. The Skyscan 1172 scanning device (Skyscan, Kontich, Belgium), operating at a voltage of 50 kV and 200 mA and a voxel size of 8 µm, was used to perform µCT measurement. Analysis was conducted using Skyscan software (NRecon, DataViewer, CTAn, and CTVox) according to ASBMR guidelines [47]. To assess tissue mineral density (TMD), two phantoms with a defined hydroxyapatite (HA) density (250 and 750 mg/cm³) were included within each scan. Regions of interest regarding trabecular and cortical bone parameters were chosen as described previously [34]. To distinguish between mineralized and nonmineralized tissue, a threshold of 642 mg HA/cm³ was used for cortical bone and 395 mg HA/cm³ for trabecular bone.

In order to determine osteoblast and osteoclast number and surface, right femurs were subjected to decalcified histology after the µCT scans. Femurs were fixed in 4% formalin for 48 h and decalcified using 20% ethylenediaminetetraacetic acid for 10–14 days. After dehydration using an ascending ethanol series, the bones were embedded in paraffin. Longitudinal sections of 4 µm thickness were stained using tartrate-resistant acid phosphatase for osteoclast and Safranin O/Fast Green for osteoblast parameters. Slices were analyzed using the Osteomeasure system (Osteometrics, Decatur, USA) as described previously [48].

For gene expression analysis, three 15 µm sections were cut from the paraffin blocks of the respective bone and transferred to an RNAse-free reaction tube. Only the proximal part of the femur was sectioned to ensure enrichment of genes related to the growth plates and metaphyseal bone. FFPE sections were processed according to the manufacturer’s instructions from the Qiagen RNeasy FFPE RNA Extraction Kit. Isolated RNA was transcribed into cDNA using the Omniscript Reverse Transcriptase Kit (Qiagen, Hilden, Germany). Quantitative PCR was performed using the Brilliant SYBR Green QPCR Master Mix Kit (Stratagene, Amsterdam, the Netherlands) according to the manufacturer’s protocol in a total volume of 25 µL using the following cycling conditions: 50°C for 2 min, 95°C for 2 min, 50 cycles each consisting of 95°C for 15 s and 60°C for 1 min. Then melting curve acquisition was performed (95°C for 15 s, 60°C for 1 min, 95°C for 15 s). B2M was used as the housekeeping gene (F: 5′-CCC GCC TCA CAT TGA AAT CC-3′, R: 5′-TGC TTA ACT CTG CAG GCG TAT-3′). The primers for Runx2 were 5′-CCA CCA CTC ACT ACC ACA CG-3′ and 5′-CAC TCT GGC TTT GGG AAG AG-3′, and for tyrosine hydroxylase (TH) 5′-AAC​CCT​CCT​CAC​TGT​CTC​GGG​C-3′ and 5′-TCA​GAC​ACC​CGA​CGC​ACA​GAA​CT-3′. Relative gene expression was calculated using the delta-delta CT method with the noMS SHC vehicle group as control.

Statistical analysis was performed (GraphPad Prism 9) separate for males and females as well as for vehicle and M. vaccae NCTC 11659-treated groups by two-way ANOVA with Dunn’s post hoc corrections for all BBS-treated or M. vaccae NCTC 11659-treated groups (in case of females: noMS-GHC, MS-GHC, noMS-SIP, MS-SIP; in case of males: noMS-SHC, MS-SHC, noMS-CSC, MS-CSC). Results were marked as significantly different if the p value was below 0.05. Data are displayed as single data points with mean ± standard deviation (SD). Behavioral, physiological, and immunological data from respective groups have been published previously [33]. Due to technical reasons, not all mice from the previous study could be used for bone analysis; therefore, individual n-numbers are different for the different groups. Because the two CAS paradigms used for male and female mice were different in nature and duration, we did not compare males and females directly, but rather draw indirect conclusions from the obtained data on the effects on bone. By comparing detected or nondetected stress effects (CAS or ELA effects), we concluded indirectly about potential stress-protecting effects of the treatment.

We first analyzed the effects of ELA and CAS alone or in combination on the bone phenotype of vehicle-treated female mice. In female mice, ELA was induced by MS and CAS by SIP. In the trabecular part of the distal femur, neither MS, SIP, nor the combination of both did affect bone volume (Fig. 2a), tissue mineral density (Fig. 2b), trabecular number (Fig. 2e), and separation (Fig. 2f). Only MS alone significantly decreased the trabecular thickness (Fig. 2d). This effect was not visible in the MS + SIP group (Fig. 2d). Furthermore, cortical thickness (Fig. 2g), cortical tissue mineral density (Fig. 2h), and length of the femur (Fig. 2i) were unaffected. This indicates a minor osteopenic phenotype in female mice exposed to ELA with no aggravating but rather ameliorating effects of additional CAS.

We also analyzed the effects of ELA and CAS alone or in combination on the bone phenotype of vehicle-treated male mice. In male mice, ELA was also induced by MS and CAS by CSC. MS alone did not affect trabecular (Fig. 3a–f) and cortical bone parameters in the femur (Fig. 3g–h), or femoral length (Fig. 3i). CSC alone increased bone volume (Fig. 3a), trabecular tissue mineral density (Fig. 3b), trabecular thickness (Fig. 3d), trabecular number (Fig. 3e), and decreased trabecular separation (Fig. 3f) in the distal part of the femur. Furthermore, CSC increased cortical thickness (Fig. 3g), while decreasing femoral length (Fig. 3i). MS in combination with CSC induced almost the same phenotype, with the exception that trabecular number and trabecular separation were not significantly changed. This indicates that ELA does not have a significant impact on bone health in male mice, while CAS induced thicker trabecular and cortical bone accompanied by disturbed long-bone growth.

We further analyzed the cellular composition of the trabecular bone in the distal femur by measuring osteoblast and osteoclast parameters. In female mice (Table 1), the only significant effect was that MS alone induced an increased osteoclast number and surface. This effect was not visible in MS mice exposed additionally to SIP. In male mice (Table 2), none of the measured parameters were affected by ELA, CSC, or the combination of both.

Because the second aim of our study was to analyze possible protective effects of repeated M. vaccae NCTC 11659 administrations on the consequences of stress on bone, we next investigated the bone phenotype of mice subjected to either ELA, CAS, or a combination of both, which have been administered repeatedly with M. vaccae NCTC 11659 following MS or noMS exposure (males and females), but prior to SIP (females) or CSC (males) exposure. In female mice administered with M. vaccae NCTC 11659, no differences regarding all measured bone parameters were found between the groups (Fig. 4), indicating that M. vaccae NCTC 11659 was able to ameliorate the negative effect of MS on trabecular thickness reported above in respective vehicle-treated mice. In line with the latter, all assessed osteoblast and osteoclast parameters did not differ between the groups (Table 3). In male mice administered with M. vaccae NCTC 11659, CSC alone or in combination with MS still induced an increased trabecular tissue mineral density (Fig. 5b) and trabecular thickness (Fig. 5d) in the distal part of the femur. Furthermore, cortical thickness was still increased (Fig. 5g). However, all other parameters did not differ between the groups, indicating that M. vaccae NCTC 11659 was able to at least in part prevent the negative bone consequences of CAS reported in respective vehicle-treated male mice. Again, osteoblast and osteoclast parameters did not differ between the groups (Table 4).

Due to the only moderate osteopenic bone phenotype detected in female mice following ELA, we conducted molecular analyses only in male mice. We analyzed gene expression of Runt-related transcription factor 2 (Runx2) in the proximal part of the femur of male mice as a surrogate parameter for chondrocyte-to-osteoblast transdifferentiation (Fig. 6a). We found that all males subjected to the CSC paradigm displayed significantly decreased expression of Runx2. Additional ELA did not further downregulate Runx2 expression (Fig. 6b). However, M. vaccae NCTC 11659 administrations were able to prevent the CSC-induced reduction in Runx2 gene expression (Fig. 6d). Expression analysis of TH, which is the rate-limiting enzyme in catecholamine synthesis, revealed that CSC significantly increased TH expression, an effect that was not affected by prior ELA (Fig. 6c) but prevented by repeated M. vaccae NCTC 11659 administrations (Fig. 6e). These findings are in line with our own earlier studies [34, 36, 42], showing that CSC disturbs long bone growth and increases bone mass by altering chondrocyte-to-osteoblast transdifferentiation via catecholamine signaling. In extension, we here provide evidence that prior ELA was not aggravating these negative CSC effects, but repeated M. vaccae NCTC 11659 administrations prior to CSC were able to prevent these.

In this study, we found that female mice display only a moderate osteopenic bone phenotype after exposure to ELA alone, with decreased trabecular thickness and increased osteoclast parameters. These ELA effects were not seen in animals which have been injected with M. vaccae NCTC 11659, indicating a protective effect. Chronic psychosocial stress during adulthood induced by SIP did neither aggravate the negative consequences of ELA nor did it affect bone on its own. In male mice, ELA alone did not affect the bone phenotype, while CAS induced by the CSC induced thicker, but shorter, bones, as we have shown in several previous studies [34, 36, 42]. Prior ELA did not aggravate the CAS effects on bone in male mice, while repeated M. vaccae NCTC 11659 administrations prior to CAS were at least in part protective against the negative consequences of CAS on bone. Together, our results support the hypotheses that (i) the negative effects of ELA and CAS on bone are highly sex-dependent, with females being more susceptible to ELA while males to CAS, (ii) ELA and CAS neither in males nor in females have cumulative negative effects on bone, and (iii) developing immunoregulatory approaches, such as repeated s.c. administrations with immunoregulatory microorganisms, have potential for prevention/treatment of stress-related bone defects.

In line with our recent findings that male, relative to female, mice seemed to be more susceptible for CAS with respect to behavioral, physiological, and immunological parameters [33], males also in the present study showed more severe negative bone effects as a consequence of CAS than their female counterparts. In detail, besides thicker and shorter bones reported in the present study, CAS-exposed male mice develop social deficits, chronic HPA axis activation, splenomegaly accompanied by increased in vitro splenocyte reactivity, GC resistance, increased counts of splenic total myeloid cells, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), and neutrophils together with increased plasma levels of various pro- but not anti-inflammatory cytokines compared with respective SHC mice [33]. Note that MDSCs are a heterogeneous immune cell population with myeloid origin and immunosuppressive (i.e., adaptive immunity) and proinflammatory (i.e., innate immunity) capacities [49]. In contrast, the bone phenotype of CAS-exposed female mice was not affected, as were most of the behavioral, physiological, and immunological parameters reported earlier [33]. Noteworthy, although a direct comparison of CSC effects in males and SIP effects in females is not possible due to the different nature and duration of the two CAS paradigms, increased splenic total myeloid cells, PMN-MDSCs and neutrophils together with increased plasma interleukin (IL)-4 levels in CAS-exposed female mice clearly indicate that SIP as a model to induce CAS in female mice works reliably. However, given that SIP-exposed females in contrast to CSC-exposed males do not develop adrenal hypertrophy [33], which resembles a typical consequence of chronic stressor exposure [50], SIP in comparison to CSC seems to be a rather mild CAS, possibly explaining the lack of detectable negative bone effects in SIP females. Importantly, ELA was induced by MS in both sexes, allowing reliable conclusions to be drawn with respect to sex-specific differences in the vulnerability for ELA. We earlier reported that behavioral, physiological, and immunological ELA effects were surprisingly mild in both sexes, with by-trend increased systemic plasma keratinocyte chemoattractant and monocyte chemoattractant protein (MCP)-1 levels despite reduced spleen weight in females and transient social deficits, increased splenic myeloid counts, and decreased plasma IL-4 levels in males [33]. In contrast, the current findings indicate that the bone was only affected by ELA in females, but not in males, with the effects again being rather mild. In line with the latter, ELA is associated with elevated CRP levels in young women but not men [51] and the association between ELA and the risk of developing obesity over the life course was stronger in human samples including more women [52], all together supporting the hypothesis that female sex in general is more susceptible to ELA than male sex.

Once more in contrast to the earlier study assessing the behavioral, physiological, and immunological consequences of ELA, CAS, and the combination of both [33] and revealing mild cumulative effects of ELA and CAS on plasma keratinocyte chemoattractant and MCP-1 or G-CSF and MCP-1 levels in males and females, respectively, as well as on splenic LPS-induced in vitro cell viability in females only, cumulative negative effects on the bone were not detected in the present study. With respect to the underlying mechanisms, previous studies have described the importance of the interactions between the immune system and the skeletal system, a field of research known as osteoimmunology. A disbalance of this cross talk, for example due to excessive inflammation seen in rheumatoid arthritis, can lead to increased bone resorption and, subsequently, osteoporosis [53]. In line with the latter, a series of own recent studies have shown that an overshooting inflammatory stress response has pronounced negative effects on bone growth as well as on bone regeneration after fracture in male mice [34, 35], with stress-activated myeloid immune cells producing and secreting catecholamines and inflammatory cytokines locally in the bone marrow and fracture hematoma, playing a critical role [36].

It is well known that immune cells interact with bone cells. For example, T lymphocytes can secrete cytokines like tumor necrosis factor α, which on the one hand leads to increased osteoblast apoptosis and, on the other hand, induces osteoclastogenesis by increasing RANKL expression, both pathways leading to bone loss characteristic for various inflammatory diseases [54]. Since depletion of CD25⁺ Tregs after the last s.c. M. vaccae NCTC 11659 administration and prior to stressor exposure revealed that M. vaccae NCTC 11659 mediates its immunoregulatory properties and, thus, stress-protective effects at least partly via induction of Tregs and IL-10 secretion [21], it is not surprising that, in the present study, we were able to see an osteoprotective effect in our M. vaccae NCTC 11659-treated groups. The latter is also in line with an increased number of CD4⁺CD25⁺Foxp3⁺CD62L⁻ mesenteric lymph node (mesLN) cells in all M. vaccae NCTC 11659-treated experimental groups of an earlier study, compared with BBS-treated groups, clearly indicating that s.c.-administered M. vaccae NCTC 11659 is able to increase the percentage of Tregs [21]. Furthermore, the interaction between MDSCs and bone could also be involved in the osteoprotective effects of M. vaccae NCTC 11659 found in the present study. Our group recently showed that CAS not only influences MDSCs, but that i.g. administration of M. vaccae NCTC 11659 is also able to modulate these effects [55, 56]. Interestingly, MDSCs can influence bone homeostasis either directly by differentiating into osteoclasts or indirectly by modulating other immune cells [57]. However, more research is required to further explore this connection. Altogether, the relevance of the immune system for bone homeostasis supports our hypothesis that a dysregulation of bone-immune cross talk is likely one of the causes for bone alterations in stressed mice.

With respect to the pronounced sex differences in the interaction between stress and the bone found in the present study, we can only speculate. Sex hormones and, therefore, sex have a great influence on bone development, remodeling, and healing. Their effects on bone are multifaceted and vary across different stages of life. Estrogen is essential for maintaining bone density. It inhibits bone resorption by decreasing the lifespan and activity of osteoclasts and promotes bone formation by extending the lifespan and activity of osteoblasts [58, 59]. During puberty, the surge in estrogen levels significantly contributes to the pubertal growth spurt. Estrogen promotes the proliferation of chondrocytes in the growth plate of long bones, leading to increased bone length. High levels of estrogen toward the end of puberty cause these growth plates to fuse, thereby determining final adult height [60, 61]. In women, estrogen levels decline significantly after menopause, leading to increased bone resorption and decreased bone formation [62]. Testosterone has both direct and indirect effects on bone [63, 64]. Directly, it stimulates bone formation by promoting osteoblast activity. Indirectly, testosterone can be converted into estrogen by aromatase in bone tissue, contributing to the regulation of bone resorption and formation. Similar to estrogen, testosterone does also play a significant role in bone development and epiphyseal closure during growth [64]. In men, testosterone levels gradually decline with age, which can contribute to a decrease in bone density and increased risk of fractures, though the process is generally slower and less pronounced compared to the effects of menopause in women. In summary, sex hormones are vital for bone development, maintenance, and overall health. Besides the different levels of sex hormones in females and males, also their effects might differ due to the different expression of their receptors in bone of men and women. Furthermore, especially estrogen is also known to influence inflammatory reactions, with women being more susceptible, for example, to allergic diseases [65]. Therefore, general hormonal differences between males and females might interfere with stress effects on bone, leading to the sex-specific effects of different stressors we found in the present study, although we did not investigate that in more molecular detail in this work.

Taken together and considering the limitation that CAS in males and females was induced by different paradigms, our findings are consistent with the hypotheses that the vulnerability of murine bones to stress during different phases of life is strongly sex-dependent and that developing immunoregulatory approaches, such as repeated s.c. administrations with immunoregulatory microorganisms, have potential for prevention/treatment of stress-related bone disorders.

The authors thank P. Hornischer, U. Binder, A. Böhmler, T. Hieber, B. Herde, J. Pawlak-Wurster, and I. Baum for their technical assistance and help in performing the experiments. Furthermore, the authors would also like to thank Dr. S. Ott, E. Merkel, S. Brämisch and S. Hummel (local animal research center) for their excellent support in terms of animal housing.

This study protocol was reviewed and approved by the Federal Animal Care and Use Committee (Regierungspräsidium Tübingen, Germany) (Approval No. 1392).

The authors have no conflict of interest.

This study was supported by the Research Grant RE 2911/21-1 provided by the German Research Foundation and in part by the Collaborative Research Centre CRC1149 (funded by the Deutsche Forschungsgemeinschaft, German Research Foundation, Project No. 251293561, B06). The funder had no role in the design, data collection, data analysis, and reporting of this study.

S.O.R., D.L., and M.H.-L. planned the study; D.G., T.S., G.M., B.T.K., M.Z., and D.L. performed the experiments; M.H.-L. did the statistical analysis; S.O.R., M.H.-L., D.G., D.L., and T.S. wrote the manuscript. S.O.R., M.H.-L., and A.I. acquired funding for the study. All authors approved the final version of the manuscript.

Melanie Haffner-Luntzer and Stefan O. Reber contributed equally to this work.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.