Social isolation of aged mice drives dramatic release of inflammatory lipoxygenase-derived oxylipins

The inflammatory status of 7 organs (brain, heart, fat, liver, lung, muscle, spleen) and serum of adult (5 months) and aged (20 months) C57BL6/J/UKJ mice was assessed by analysis of inflammation-related cytokine and oxylipin profiles. To investigate the effect of social isolation stress at a later stage in life, we recurrently separated mice at an age of 18 months into single cages for three individual nights per week over a period of 8 weeks. Moreover, we addressed exercise as an established, anti-inflammatory metabolic intervention by providing a running wheel to a subgroup of isolated aged mice. In this context, aged mice additionally underwent a Barnes maze test to assess intervention-related changes in cognition. The detailed experimental setup is depicted in Fig. 1.

**Fig. 1: Schematic representation of the recurring social isolation and physical exercise intervention.**

Organ-specific inflammatory cytokines are differentially affected by ageing but significantly induced by recurring social isolation

Ageing significantly increased (p = 0.018) the body weight of the mice. Old mice in group-housing (O-GH) had a mean weight of 35.2 ± 0.6 g, while the mean weight of adult mice in group-housing (Ad-GH) was 32.1 ± 0.9 g. Social isolation stress in old mice due to repeated isolation into single cages (O-ISO) did not significantly alter the final body weight (35.9 ± 1.7 g, p = 0.692). However, the body weight of these isolated aged mice (O-ISO) showed a higher degree of variation versus old mice in group-housing (O-GH) (Fig. 2a).

**Fig. 2: Ageing leads to organ-specific inflammatory phenotypes that are aggravated by social isolation stress.**

To address the inflammatory status of the investigated organs, we examined the protein level of the pro-inflammatory cytokine interleukin (IL)-1β, that acts as a robust marker of a chronic inflammatory microenvironment, IL-6, IL-12 p70, tumour necrosis factor alpha (TNF-α) and the IL-1β counteractor IL-1 receptor antagonist (IL-1ra). We found that ageing did not markedly change IL-1β levels in fat, muscle, and spleen (Fig. 2b). However, IL-1β levels were significantly decreased due to ageing in brain (Ad-GH: 8.7 ± 0.2, O-GH: 6.5 ± 0.3 pg/50 mg), heart (Ad-GH: 34.5 ± 5.3, O-GH: 12.3 ± 1.5 pg/50 mg), and liver (Ad-GH: 273.7 ± 31.7, O-GH: 117.5 ± 17.6 pg/50 mg). Only in the lung, IL-1β was significantly elevated (Ad-GH: 143.5 ± 8.1, O-GH: 205.9 ± 14.6 pg/50 mg) in the aged compared to the adult mice (Fig. 2b). Similarly, the levels of the additional pro-inflammatory cytokines IL-6, IL-12 p70 and TNF-α remained largely unaltered or significantly decreased due to ageing, only muscle displayed significantly increased levels of IL-12 p70 following ageing compared to adult mice (Supplementary Fig. S1). When the aged mice underwent social isolation (O-ISO), however, IL-1β significantly increased in all studied organs except in spleen with only minor elevation versus the O-GH mice (Fig. 2b, 1.8-fold, p = 0.309). Again, other pro-inflammatory cytokines revealed comparable dynamics following isolation with strongly increased levels observed in fat, heart, liver and muscle homogenates (Supplementary Fig. S1). Overall, this translates to a distinct, organ-specific ageing-related impact on the secretion of pro-inflammatory cytokines, while stress due to isolation seems to consistently promote inflammation in most organs. A similar pattern was observed for the levels of IL-1ra, being significantly increased following isolation of aged mice in fat, heart, liver, muscle, and spleen (Fig. 2c).

Social isolation potently induces lipoxygenase-derived oxylipin formation

Since oxylipins are crucial regulators of all stages of inflammation^10,34,35, we assessed the oxylipin signature profiles of the different organs using a targeted lipidomic approach based on ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). To get an overview of age- and isolation-related changes in the complex oxylipin profiles, we performed an unbiased principal component analysis. This analysis revealed distinct age-related alterations in the oxylipin profiles for brain, fat, heart, and spleen between both group-housed adult (Ad-GH) and aged mice (O-GH) (Fig. 2d). An even more pronounced separation of the clusters for all organs was observed following isolation of aged mice (O-ISO) and only in the heart, age had a stronger effect on oxylipin profiles than isolation (Fig. 2d).

Looking at the changes of the individual screened oxylipins due to ageing, we found distinct organ-specific ageing signatures with liver, muscle, and spleen exhibiting an overall decrease in oxylipins, whereas brain, fat, and lung showed divergent oxylipin profiles with age (Fig. 2e, left). In the heart, ageing led to increased amounts of oxylipins, particularly pronounced for PGs (Fig. 2e, left). Social stress in aged mice due to isolation, however, drives a remarkable increase of a broad spectrum of oxylipins, especially derived from 5-, 12-, and 15-LOX across various organ systems (Fig. 2e, right). Interestingly, in lung and spleen, the inflammation-resolving SPMs RvD5 and PDX produced by 12/15-LOX were elevated along with pro-inflammatory LTs derived from 5-LOX. In the brain and heart, the oxylipin levels were mostly unaffected by isolation of the aged mice (Fig. 2e, right).

In order to further characterise the age- and isolation-related effects on the oxylipin formation, we classified the oxylipins according to their rate-limiting biosynthetic enzymes, i.e., COX, 5-LOX, 12-LOX, and 15-LOX. In detail, for COX products, age-related increases were most pronounced in the heart (3.71-fold) with minor changes in other organs (Fig. 3a, Supplementary Fig. S2). Isolation did not affect COX products in most organs (fat, heart, lung, and muscle), except for brain (0.71-fold), liver (2.79-fold) and spleen (1.41-fold; Fig. 3a). Taken together, the effect size and its statistical significance indicate that age and isolation only have a moderate impact on COX-derived oxylipins in most organs.

**Fig. 3: Social isolation stress leads to induction of LOX pathways.**

5-LOX products were much more affected by age and isolation. Thus, 5-LOX product levels strongly decreased in fat, liver, muscle, and spleen of aged mice compared to adult controls (Fig. 3b, Supplementary Fig. S2). Conversely, ageing markedly increase 5-LOX products in the heart, which is surprisingly not further aggravated by isolation (Fig. 3b, Supplementary Fig. S2). For all other organs – except muscle where 5-LOX products were not detectable in aged mice – isolation led to a marked increase of 5-LOX-derived oxylipins (Fig. 3b, brain: 2.0-fold, fat: 2.1-fold, liver: 18.2-fold, lung: 8.8-fold, spleen: 17.1-fold). The same trend was observed for oxylipins produced by 12- and 15-LOX (Fig. 3c, d, Supplementary Fig. S2). Thus, ageing caused only moderate changes in the levels of 12-LOX and 15-LOX products, while social isolation caused a dramatic increase for both 12-LOX (brain: 5.7-fold, fat: 7.1-fold, liver: 30.1-fold, lung: 4.5-fold, muscle: 22.2-fold, spleen: 4.3-fold) and 15-LOX products (brain: 1.6-fold, fat: 7.5-fold, liver: 33.4-fold, lung: 7.9-fold, muscle: 2.8-fold, spleen: 15.6-fold) in all organs except for the heart (no elevation). In more detail, the radar charts showing individual representative oxylipins for each enzyme class (COX: PGE₂; 5-LOX: LTB₄ and 5-HETE; 12-LOX: 12-HEPE, 12-HETE and 14-HDHA; 15-LOX: PDX, 15-HEPE, 15-HETE and 17-HDHA) confirm the massive elevation of LOX-derived oxylipins in all organs caused by social stress due to isolation, except for heart, with minor or even opposite impact on COX products (e.g., PGE₂). Together, social isolation stress consistently upregulates LOX-derived oxylipins in organs of aged mice while only the heart and, to a lesser extent, the brain exhibit a lower susceptibility to social stress-related oxylipin alterations (Fig. 3e). These increases in LOX-derived oxylipins were evident for conversion of all PUFAs as substrates, i.e. AA, EPA and DHA.

Late-life exercise cannot rescue isolation effects on inflammatory state

Since voluntary exercise yields a multitude of positive outcomes prolonging the life and health span³⁰ with impact on cytokines and oxylipins^31,32, we further investigated whether exercise can mitigate the effects on the inflammatory mediators induced by social isolation stress. We employed voluntary wheel running as a late-life exercise intervention in socially isolated aged mice (runners: O-EX, non-runners: O-ISO) by providing running wheels to O-EX mice during the isolated nights of the 8-week-long repeated social isolation period. After the intervention period, the group of O-ISO mice (non-runners) showed a mean weight of 35.9 ± 1.7 g, whereas the group of O-EX mice (runners) showed a slight decrease in body weight (33.9 ± 1.1 g), yet this difference was not significant (Fig. 4a, p = 0.357). However, the weight loss seems to be connected to the individual running performance of the mice, as the mouse with the lowest running performance across the 8-week-long experimental period showed the highest weight and vice versa (Fig. 4b).

**Fig. 4: Exercise cannot ameliorate increased oxylipin secretion in aged mice affected by social isolation stress.**

We then investigated the levels of pro-inflammatory cytokines IL-1β, IL-6, IL-12 p70, TNF-α and anti-inflammatory IL-1ra and assessed the oxylipin signature profiles of organs from aged mice during isolation that were either allowed to exercise (O-EX) or not (O-ISO). Only in the lung, exercise led to a significant decrease in IL-1β levels (0.72-fold) (Fig. 4c) and a small decrease by trend was observed in heart (0.72-fold, p = 0.241) and spleen (Fig. 4c, 0.62-fold, p = 0.151). For all other organs, the IL-1β levels were essentially unchanged. Although no significant reductions in the other pro-inflammatory cytokines were observed in the organs of O-EX mice, most organs, except for the brain, showed a decrease by trend (Supplementary Fig. S3). In contrast, IL-6 levels in the brain, were significantly elevated. Similarly, IL-1ra levels remained essentially unaltered between O-EX and O-ISO, without significant impact of exercise in any organ (Fig. 4d). Principal component analysis of the overall oxylipin profiles showed no marked differences (no separation of clusters) for all organs comparing O-ISO versus O-EX, with both the position of mean and 95% confidence intervals being similar (Fig. 4e). Yet, when looking at individual oxylipins in detail (Fig. 4f), oxylipin levels in fat (increased) and liver (decreased) were most affected by exercise, particularly pronounced for monohydroxylated fatty acids like 5-HETE, 4-HDHA, 12-HETE, 12-HEPE etc. (Fig. 4f). Interestingly, in the lung, exercise decreased PG and RvD5 formation but did not affect other oxylipins (Fig. 4f). Analysis of oxylipin groups according to biosynthetic enzymes (i.e., COX, 5-, 12-, 15-LOX) reveals no significant alterations due to exercise in any organ (Supplementary Fig. S4), and only in fat and liver a tendency for changes was observed (Fig. 4g, h). Thus, the most notable change by trend upon exercise is the increase of 5- and 15-LOX-derived products in fat tissue (5-LOX: 2.02-fold, p = 0.131; 15-LOX: 1.52-fold, p = 0.082) while COX- and 12-LOX-derived products remain at a similar level to sedentary mice (Fig. 4g). Furthermore, exercise led to a small decrease in LOX-derived products (5-LOX: 0.72-fold, p = 0.214; 12-LOX: 0.52-fold, p = 0.140; 15-LOX: 0.67-fold, p = 0.188) in the liver, while the level of COX-derived products was unaltered (Fig. 4h). Together, for aged mice undergoing social isolation stress, the variable of voluntary exercise has only a minor impact on the massive alterations of oxylipins and cytokine levels caused by isolation.

Cognitive function does not benefit from voluntary exercise in isolation

In a previous study²⁷, we addressed the relation between age and cognition in male, group-housed mice by subjecting them to a Barnes maze test, revealing a cognitive decline that occurs between the ages of 15 and 24 months. It was also shown that for aged mice, constant social isolation further impairs cognitive function. To evaluate whether voluntary wheel running can mitigate cognitive decline in ageing and social stress through repeated isolation and resocialisation, we tested isolated aged mice in the Barnes maze to assess spatial learning, as well as short- and long-term memory. The timeline of the implementation of the Barnes maze test is shown in the schematic in Fig. 1. During the training period for the Barnes maze test, both cohorts with recurring social stress (O-ISO and O-EX) showed a similar, improved performance over time.

No strong exercise-mediated improvements were observed during training (Fig. 5a). The probe trial (short-term memory) and the retention test (long-term memory) did not reveal cognitive improvement following exercise, but instead a prolonged primary latency in the retention test of the O-EX group (Fig. 5b, c). During the reversal test (cognitive flexibility), the O-EX mice showed a shorter primary latency and an increased cognitive score compared to O-ISO mice, suggesting a potential facilitation in task adaptation (Fig. 5d). Overall, exercise intervention in late life does not seem to improve spatial learning, short and long-term memory in aged mice experiencing social isolation stress, but O-EX showed an improved cognitive flexibility.

**Fig. 5: Cognitive function assessment of mice undergoing recurring social isolation with and without physical exercise.**

Reduced age-associated inflammatory repertoire of oxylipins and proteins in serum is maintained after isolation and exercise

To determine whether the observed organ-specific effects due to ageing and isolation as well as exercise are reflected in the systemic circulation, we characterised the inflammatory secretome (oxylipins and proteins) in the serum of the mice cohorts from above. Based on both, the principal component analysis of the oxylipin profiles and the detailed analysis of the individual oxylipins, age but not isolation or exercise is the main driver for the observed alterations (Fig. 6a, b). While in the PCA all aged cohorts (O-GH, O-ISO, O-EX) cluster tightly together, the oxylipin profile of the adult cohort (Ad-GH) shows a distinct shift (Fig. 6a). The majority of the screened oxylipins in serum are decreased due to age (O-GH/Ad-GH, Fig. 6b) with significant changes for 5- and 15-LOX-derived products (5-LOX: 0.55-fold, 15-LOX: 0.54-fold, Fig. 6c). To our surprise and in contrast to organs (see above), isolation only had a minor impact on oxylipin levels in serum, reflected by the lack of changes for COX and 12-LOX products, and only moderate increases in 5- and 15-LOX-derived products in serum from O-ISO versus O-GH mice (5-LOX: 1.63-fold, p = 0.087; 15-LOX: 1.44-fold, p = 0.250, Fig. 6c). These serum oxylipin levels of O-ISO did not markedly change due to exercise, apart from a 4-fold increase in 5-LOX-derived products (O-ISO: mean = 822.2 ± 124.0 pg/mL, O-EX: mean = 3341.3 ± 1661.3 pg/mL; p = 0.350) which was, however, not significant and attributed to only 2 out of the 6 biological replicates (Fig. 6c).

**Fig. 6: Impact of age and social isolation on inflammatory oxylipins and proteins in serum.**

We next employed an unbiased analysis of the repertoire of inflammation-related cytokines, chemokines, enzymes, and growth factors in the serum of Ad-GH, O-GH, O-ISO, and O-EX mice, using a commercially available Proteome Profiler, allowing us to screen for the relative abundance of 111 candidates. In line with the reduced amounts of most of the oxylipins (Fig. 6b), ageing led to an overall downregulation (82.0%) of the investigated proteins in serum (Fig. 6d). Isolation, however, led to an increased expression of 82.9% of the proteins (Fig. 6d). Despite the seemingly contradictory dynamics, the proportions of proteins being down-regulated with age and up-regulated due to isolation are not congruent (Supplementary Fig. S5d). The exercise intervention (O-EX) led to less pronounced and more balanced alterations for aged mice under isolation (O-ISO) with 59.5% up- and 38.7% downregulated proteins (Fig. 6d). Acute phase proteins (APPs, e.g., complement factors and pentraxins) and chemokines (particularly of the CCL and CXCL classes) were impaired due to age versus adult mice (Fig. 6e, f). Such overarching class effects could not be observed for the cohorts O-ISO and O-EX under isolation. Interestingly, the pro-inflammatory cytokine IL-6, the chemokine CXCL1, and the lipopolysaccharide (LPS) receptor cluster of differentiation (CD)14 were increased in O-ISO versus O-GH mice (Fig. 6e, f). Exercise of aged mice under isolation, however, induced higher levels of murine C-reactive protein (CRP), CCL11, CCL21, and vascular endothelial growth factor (VEGF) versus O-ISO mice (Fig. 6e, f). These alterations in the abundance of inflammation-related serum markers indicate a reduced immune competence with age, and distinct pro-inflammatory responses due to isolation and exercise.

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