Aging and longevity are complex multi-faceted biological processes which are heavily integrated with the composition of the gut microbiota. The power of the gut microbiota comes in its ability to simultaneously influence multiple age-related biological processes including inflammation, oxidative stress, metabolic regulation and energy homeostasis (Fig. 6). It was previously shown that Drosophila’s gut microbiota varied significantly with age with a strong expansion of most groups and a disproportionate rise in the pathogenic Gammaproteobacteria spp. and Enterococcus spp.28. This is a known phenomenon in Drosophila reflecting age-related immunosenescence and accumulating intestinal barrier dysfunctions from proinflammatory pathobionts29. These changes parallel the age-related variations observed in aging humans14,19. Treatment with the probiotic and/or prebiotic agents used in the present study were previously shown to reduce the bacterial load in aging Drosophila28, have immunomodulatory effects30, regulate metabolism31 and enhance longevity28 indicating the potential of developing a probiotic-based therapeutic agent for age-related disease.

There is a significant sexual dimorphism in longevity mechanisms in Drosophila. For example, dietary restriction in Drosophila larvae induced a significant increase in InR gene expression in adult males, but not females32 while sexual dimorphisms in mitochondrial maintenance mechanisms including autophagy, Akt, p53 and FOXO in mediating sex-specific differences in stress resistance and aging33. Finally, resveratrol was shown to increase longevity in Drosophila in a gender- and diet-dependent manner34 indicating the importance of distinguishing gender in longevity studies. For this matter, the present study was conducted exclusively in males to reduce the interference of hormonal regulation on typical longevity measures including InR expression, inflammation and oxidative stress.

The synbiotic formulation boosted longevity by 60% compared to Drosophila on a conventional diet while the probiotic formulation increased longevity by 55%. There have been a handful of other studies indicating the prolongevity potential of probiotic supplementation35,36,37,38 though, to the author’s knowledge this is the first study that demonstrates the simultaneous and multi-faceted action of a novel synbiotic formula on several markers of aging attributing to its prolongevity effects.

Suppression of insulin-like growth factor (IGF)-1 and insulin signaling have been identified as the main mechanisms through which calorie restriction leads to an increase in longevity39. In the present study, all levels of metabolic distress in aging control Drosophila were rescued by the synbiotic formulation including total weight, glucose and triglyceride levels. Previous studies have shown that downregulation of the dilps and the insulin receptor increases lifespan in Drosophila40 and these factors are under dietary regulation41.

Regarding the insulin-signaling pathway, elevation of dAkt and dTOR along with the reduction of dFOXO were all rescued by the synbiotic treatment, with beneficial yet variable effects by the individual probiotics and TFLA. Inhibition of TOR signaling with its natural inhibitor rapamycin has been shown to increase longevity in yeast42, nematodes43, Drosophila44 and mice45 by mimicking the effects of calorie restriction. Downstream of TOR signaling is the FOXO family of transcription factors. AMPK activation by calorie restriction is also linked to elevated FOXO expression and pro-longevity effects46 including the simultaneous inhibition of ROS production47, NF-κB induction48, senescence49 and prevention of apoptosis50 along with encouragement of the protective mechanisms of mitophagy and autophagy51. Several studies have linked polymorphisms of FOXO3 in humans to increased longevity5,52 while FOXO3 overexpression in Drosophila53 and mice54 also imparted lifespan extension. Interestingly, several studies indicated that consumption of polyphenols including green tea epigallocatenin, curcumin and resveratrol stimulate FOXO expression and consequently longevity through mechanisms involving increased SOD, GPx and sirtuin 1 expression with decrease in NF-κB, TNF-α, ROS, inflammation and oxidative stress55,56. Likewise, in the present study, FOXO expression was upregulated by the synbiotic formulation.

Similar to the glucose-regulating factors, many of the underlying lipogenic factors were also positively affected by the probiotic and synbiotic formulations. There was an improvement of lipogenesis dysregulation, indicated by the rescued expression of FAS, SREBP and LSD2 in Drosophila supplemented with the synbiotic formulation. The inherent increase in lipogenic ACC and FAS genes in aging control flies was downregulated most significantly by the probiotic and synbiotic formulations, as was the gluconeogenic PEPCK factor. These changes may be attributed to the transcriptional regulation by SREBP, whose expression was reduced to the level of young flies by both the probiotic and synbiotic formulations. Importantly, SREBP regulation is under the control of the IlS pathway, being activated by both Akt and TOR signaling57. The regulation of fatty acid lipogenesis is an important consideration to many aspects of age and age-related conditions, particularly inflammation as obesity is inherently linked with a proinflammatory state58 which is known to be preventable with adequate probiotic and prebiotic treatment59,60.

It was previously shown that PPARγ mediated responses are central to the synbiotic’s action in metabolic stress models in Drosophila melanogaster31. PPARγ is highly expressed in the adipose tissue and a key regulator of lipogenesis and adipogenesis61 as well as a major insulin sensitizer62. Further PPARγ expression in other tissues is thought to regulate their metabolism and the inflammatory response63 making it at the hub of many aging hypotheses64. In the present study, the Drosophila PPARγ target E75 was significantly downregulated in aging control Drosophila, an effect that was improved only in the probiotic and synbiotic groups with the latter actually increasing PPARγ expression over time. This effect would explain the beneficial action on the lipogenesis factors and insulin sensitivity supporting the notion that the synbiotic treatment is regulating metabolic stress in aging Drosophila at a high level and explaining the broad metabolic effects.

Aging is associated with immunosenescence caused by an exhaustion of stem cells reducing the immune system’s regenerative capacity, accumulation of antigens and thymic atrophy65. Dysfunctional immune cells disable the body from mounting an appropriate immune response leading to the accumulation of damaged cells that release proinflammatory cytokines. Chronic low-grade inflammation (inflammaging) is associated with many age-related diseases such as neurodegeneration, cardiovascular disease, insulin resistance, diabetes, osteoporosis, cognitive decline, dementia, frailty, cancer and importantly, mortality (rev. in66).

The gut microbiota of elderly persons reflects a pro-inflammatory constitution enriched in Proteobacteria spp. and lacking butyrate producing bacteria2. With age, the integrity of the GIT epithelial lining becomes compromised allowing the infiltration of bacteria and bacterial products into the host’s bloodstream contributing to inflammaging67. All of these factors involve the gut microbiota68, and age-related variations in the microbiota reduce gut epithelial integrity and induce intestinal dysplasia69. The proinflammatory environment also encourages NF-κB activation through LPS-TLR4 interaction as well as the differentiation of naïve T cells into proinflammatory Th17 cells70. The gradual accumulation of chronic low-grade inflammaging could be the source of many age-related chronic diseases as inflammation is comorbid with elevated ROS production, mitochondrial dysfunction and metabolic abnormalities.

As previously shown, there is an upregulation of proinflammatory pathobionts in aging Drosophila, which were downregulated by the synbiotic formulation28. Indeed, aging Drosophila have been shown to be more prone to infection and have an impaired immune system, such as phagocytosis and melanization71. A general increase in immune-related genes, increased bacterial loads and more persistent AMP activation after infection resembling the chronic-inflammation state observed in humans has also been observed in Drosophila72. In the present study, there was an age-related decline in innate immune functionality in control Drosophila. In particular, there was a decline in the active immunological agents against both gram-positive (S. aureus) and gram-negative (E. coli) challenges, an effect that was significantly impacted by all the probiotic treatments, but to the greatest extent, by the probiotic and synbiotic formulations. This could reflect the decreased ability of aging Drosophila to mount an immune attack against invading pathogens as previously noted73. In contrast to other studies that observed an increase in immune gene expression71, a decrease in the expression of Duox and IMD was observed in aging control flies indicating a weakening of the innate immune response and supporting the weakened immunity to a pathogenic insult as observed in the agar diffusion assay. Duox and IMD are among the first line of defense of the Drosophila innate immune system and regulated directly by antigen recognition of invading pathogens, so, it is possible that the response of the core immune-modulating cells in the fat body is compromised by age affecting the production of Duox and IMD. This could include the Janus Kinase/Signal Transducer Activator of Transcription (JNK/STAT) pathway which has been shown to have competing or cooperative action on the systemic immune response in Drosophila74.

Despite the decrease in IMD expression in aging Drosophila, an increase in AMP expression, particularly Attacin A, Defensin and Diptercin, was observed and dramatically benefitted by both individual probiotic, prebiotic and the probiotic and synbiotic formulation supplementation. This has been previously observed75 and directly linked to intestinal barrier dysfunction, which is correlated to lifespan in Drosophila76. The reason for the discrepancy between the IMD signaling and AMP expression could be due to the different levels of regulation of AMP expression. Elevated AMP expression in aging flies is correlated to an increase in oxidative stress77. Some of the AMPs including Attacin A are co-regulated by inflammatory elements such as the AP-1 and NF-κB proteins as well as HDAC activity75. Also, the AMPs are influenced by hormonal signaling, namely 20-hydroxyecdysone and juvenile hormone78, which are differentially affected by aging. AMP expression is also impacted by IlS71 which could explain the dramatic impact of probiotic treatment on AMP expression in aging Drosophila. Indeed, dFOXO was shown to regulate AMP expression, especially in conditions when the Toll and IMD pathways are defective79.

Mitochondria progressively lose their energetic capacity with age80 along with morphological changes, reduction in numbers, loss of protein quantity and mtDNA mutations81. Dysregulation of mitophagy and the mitochondrial fission-fusion cycles also compromises the mitochondrial integrity leading to elevated ROS production and consecutive mitochondrial damage82.

A decline in mitochondrial functionality was confirmed in the present study as the activity of each of the ETC complexes in control flies was reduced over time. The mitochondria play a key role in aging and Drosophila have been identified as powerful model for studying mitochondrial activity in age83. A similar phenomena was observed in various tissues in mice and rats, though decline in activities of only complexes I and IV were observed84 while the activity of complexes II and III remained relatively unchanged85. TFLA, the probiotic and synbiotic formulations were able to increase ETC complex 1, 3 and 4 activities at day 30 compared to controls, which is very significant in demonstrating how a probiotic treatment can influence mitochondrial complex integrity and consequently the production of ROS particles.

One of the key regulators of mitochondrial biogenesis is the PGC-1 family of transcriptional coactivators, whose expression declines with age86. The loss of PGC-1α is associated with a reduction in mitochondrial biogenesis, reduced fission-fusion cycles and dysfunctions in mitophagy87. Nutrient deprivation is a key modulator of mitochondrial dynamics as calorie restriction will lower the AMP/ATP and NADH/NAD + ratios which ultimately stimulates mitochondrial biogenesis and activity through PGC-1α and SIRT1, respectively. It was shown in Drosophila that overexpression of the PGC-1α homology (dPGC-1/spargel) was sufficient to increase mitochondrial activity and that tissue-specific expression of dPGC-1 in the digestive tract extends longevity86. Supporting this, E75, the Drosophila PPARγ target, was shown to decline with age in the present study, likely representing the decline in PGC-1α equivalents. The individual probiotic treatment offered little benefit to E75 expression; however, treatment with either the probiotic or synbiotic formulations elevated E75 expression at day 30. This indicates that the management of PPARγ can be one of the critical mechanisms through which the gut microbiota is managing longevity through mitochondrial complex integrity.

ROS production is an essential part of health, though in excess promotes disease. Immunosenescence aggravates redox stress and vice versa stimulating a positive-feedback loop between ROS production and inflammation88. ROS levels were significantly elevated in the current aging model as control Drosophila saw an increase in total oxidants and LPO levels with significant decreases in SOD and GPx activity. The probiotic and synbiotic formulations had a mild impact on the anti-oxidant enzyme activities, however significantly reduced the levels of total oxidant and LPO, with the synbiotic formulation being more significant in the latter. This is very significant in the context of neurodegeneration as there are a high level of PUFAs in neuronal membranes and the level of LPO in Alzheimer’s disease is correlated with the degree of cognitive impairment89.