Despite major progress in the understanding of the pathophysiology and therapeutic options for common ageing-related musculoskeletal conditions , there is still a considerable proportion of patients who respond sub optimally to available treatments or experience adverse effects. Emerging microbiome research suggests that perturbations in microbial composition, functional and metabolic capacity (i.e. dysbiosis) are associated with intestinal and extra-intestinal disorders including musculoskeletal diseases. Besides its contributions to disease pathogenesis, the role of the microbiome is further extended to shaping individuals’ responses to disease therapeutics (i.e. pharmacomicrobiomics). In this review, we focus on the reciprocal interactions between the microbiome and therapeutics for osteoporosis, sarcopenia and osteoarthritis. Specifically, we identify the effects of therapeutics on microbiome’s configurations, functions and metabolic output, intestinal integrity and immune function, but also the effects of the microbiome on the metabolism of these therapeutics, which in turn, may influence their bioavailability, efficacy and side-effect profile contributing to variable treatment responses in clinical practice. We further discuss emerging strategies for microbiota manipulation as preventive or therapeutic  approaches for improving outcomes of musculoskeletal health and disease. Understanding the factors that contribute to variability in treatment responses is critical for providing safe and effective therapies, while meeting patients’ expectations and reducing wasteful health spending.Chronic musculoskeletal conditions including osteoporosis and associated fractures, sarcopenia and osteoarthritis present major threats to healthy ageing. The microbiome is dynamic throughout the lifespan and varies greatly within and between individuals as a result of intrinsic and extrinsic factors.1012 Most microbes live symbiotically with the human host and promote health through their involvement in a vast array of functions ranging from maintaining intestinal barrier function and nutrient fermentation to endocrine and immune functions. Conversely, perturbations in microbiota composition, functional and metabolic capacity, collectively termed dysbiosis, have been associated with intestinal and extra-intestinal disorders including musculoskeletal diseases.1117 Besides its contributions to disease pathogenesis, the microbiome has an emerging role in disease therapeutics. This realization led to the development of ‘pharmacomicrobiomics’, a new discipline which investigates the interactions between drugs and the microbiome and how these affect clinical responses to drugs.1820 This field has great potential to advance microbiome-related precision medicine approaches including strategies that predict response to therapies and interventions that reshape the microbiome to enhance treatment responses.The microbiome, the diverse ecosystem of bacteria, fungi, archaea and viruses and their genetic material is now identified as an integral ‘human organ’.via direct  or indirect (e.g. modulation of host immune function or host pathways for drug/nutrient metabolism) mechanisms. In the second part of this review, based on clinical data in adults, we discuss emerging approaches for the manipulation of microbiome to improve clinical parameters related to musculoskeletal health and disease, and/or treatment efficacy and side-effect profiles.In this review, we focus on the complex interactions of the microbiome with pharmacological and non-pharmacological approaches for managing common ageing-related musculoskeletal diseases, namely osteoporosis, sarcopenia and osteoarthritis. Specifically, we identify (a) the effects of available treatments on microbiota composition and associated metabolites, intestinal integrity and immune function , and als By using a metabolomics approach, Clayton et al. demonstrated that individuals with higher urinary levels of p-cresol  prior to acetaminophen administration, had a lower post-treatment urinary ratio of acetaminophen sulphate to acetaminophen glucuronide. Acetaminophen and p-cresol compete for sulphation by the cytosolic sulphotransferase, therefore, the more p-cresol produced by the gut bacteria, the less acetaminophen is sulphated and detoxified, shifting the metabolism of acetaminophen towards the pathway that leads to N‑acetyl-p‑benzoquinone imine production. Conceivably, intra-, and inter-individual variations in the diet (i.e. substrate availability) and gut microbial capacity to form p-cresol can contribute to within- and between- individual discrepancies in acetaminophen metabolism and hepatotoxicity.23Acetaminophen’s metabolism in the liver gives rise predominantly to two inactive, non-toxic compounds (acetaminophen sulphate and acetaminophen glucuronide), and to minor amounts of N‑acetyl-p‑benzoquinone imine, which has been linked to hepatotoxicity. Animal and human studies suggest that opioid use compromises gut homeostasis contributing to impaired immune responses and susceptibility to infectious diseases and gut-origin sepsis. Specifically, treatment with opioids has been associated with reduced intestinal motility, inhibition of protective mechanisms for the gut epithelium , altered gut microflora composition (e.g. growth of Gram-positive pathogens and reduction in bacteria that metabolize bile acids) and loss of gut barrier function, allowing bacterial translocation.25 Such microbial dysbiosis might also influence the metabolism of these drugs. Morphine is deconjugated in the liver to morphine-3-glucuronide that exerts no analgesic effects. In the gut, this metabolite can be reconverted to morphine by microbial β-glucuronidases and reabsorbed into the circulation. An increased morphine-3-glucuronide-to-morphine ratio in the gut and reductions in bacteria that encode β-glucuronidases following morphine treatment suggest reduced enterohepatic recycling of this drug and potentially altered analgesic effects.Opioids are potent analgesics; their use is, however, limited by side effects including addiction, gastrointestinal symptoms and sedation.28 Evidence from animal work indicates that germ-free (GF) animals (devoid in microbiota) are protected against NSAID enteropathy, whereas the re-introduction of microbiota leads to intestinal damage. Mechanistically, the inhibition of prostaglandin synthesis by NSAIDs increases the susceptibility of the intestinal mucosa to injury and impairs its repair capacity. In parallel, NSAIDs have been associated with marked alterations in the variety and abundance of intestinal microbiota, which contribute to the development of enteropathy by compromising mucosal integrity and allowing the penetration of pathogens, but also by interfering with the metabolism of these drugs.2729 Specifically, microbial β-glucuronidases deconjugate the metabolites of several NSAIDs and release aglycones which can induce gut toxicity. Administration of β‑glucuronidase inhibitors to animals treated with NSAIDs was shown to reduce mucosal injury and enteropathy compared to animals not receiving the inhibitors. The feasibility of this strategy to reduce NSAID enteropathy remains to be explored in humans.Despite their efficacy in ameliorating pain and inflammation in osteoarthritis and inflammatory arthritis, chronic non-steroidal anti-inflammatory drug (NSAID) use is concerning due to associated upper and lower gastrointestinal (GI) side effects in a substantial proportion of patients. The gut microbiome has been lately implicated in the development of NSAID-induced enteropathy.31 In animal experiments, PPIs worsened the intestinal damage and bleeding caused by NSAIDs. These results were attributed to pronounced alterations in gut microbiota composition (i.e. reductions in Actinobacteria and Bifidobacteria spp.), while daily supply of bifidobacteria reversed dysbiosis and prevented NSAID-induced ulceration. In the same study, GF mice colonized with bacteria from healthy animals experienced mild intestinal injury following NSAID treatment, whereas colonization of GF mice with bacteria from PPI-treated rats resulted in severe NSAID-induced intestinal damage. In humans, PPI users display decreased alpha diversity  and changes in 20% of bacterial taxa compared with non-users, while an increased abundance of Bacteroides and Erysipelotrichaceae spp. can discriminate the combined use of NSAIDs and PPIs from NSAID use alone.To reduce the upper GI side effects of NSAIDs, NSAID users are often co-prescribed proton pump inhibitors (PPIs); these agents may, however, potentiate the NSAID-induced enteropathy by exacerbating dysbiosis.In vitro studies have demonstrated that bisphosphonates possess antimicrobial properties, nevertheless, it is unclear if such actions are exerted in humans or whether/how these are related to the clinical efficacy or toxicity of these agents. For example, patients with bisphosphonate-induced jaw osteonecrosis, a very rare but severe side effect of bisphosphonate use, have been reported to have different oral microbiome and increased pathogenic bacteria than non-affected patients. The microbiome may further influence host immune responses to inflammation, bacterial infections and wound healing, thus, predisposing or protecting the host to/from this side effect.36Few studies have explored the bisphosphonates–microbiome interactions.  While conventional mice lost trabecular bone, the GF mice were protected from it. The favourable bone phenotypes in the GF animals were attributed to their failure to increase bone marrow CD4+ cells and T-helper 17 (Th17) cells (an osteoclastogenic sub-population of CD4+ cells), pro-osteoclastogenic cytokines  and osteoclast numbers, as well as to their ability to maintain barrier function. Convincingly, these phenotypes were reversed following colonization of GF animals with microflora from conventional mice. Further reinforcing the idea of an oestrogen–microbiota interplay, treatment with oestrogens in animal models has been associated with significantly higher microbial diversity, reduced growth of lipopolysaccharides (LPS)-producing Gram-negative bacteria, changes in bacterial activity, tightening of gut permeability and decreased inflammation induced by high-fat feeding. Interestingly, in postmenopausal women or animal models of human menopause, treatment with antibiotics,42 probiotics4346 and prebiotics48 can blunt the bone loss induced by sex-steroid depletion by several mechanisms, including strengthening the gut barrier integrity and dampening intestinal and systemic inflammation. Although most of these findings originate from animal studies, and hence, require replication in humans, they suggest a feedback loop between oestrogens and the gut microbiome that influences the immune system. They also raise the possibility that oestrogen-based therapies might be improved by gut microbiota modulation that may work independently or synergistically to ameliorate inflammation and bone loss.Mounting evidence suggests a reciprocal relationship between oestrogens and the microbiome with implications for osteoporosis treatment with hormonal replacement therapy and selective oestrogen-receptor modulator (SERMs). A series of experiments that compared conventional with GF female mice following oestrogen depletion using the gonadotropin-releasing hormone (GnRH) agonist, leuprolide, to mimic postmenopausal status, revealed that the gut microbiota has a pivotal role in sex-deficiency-induced osteoporosis. The deconjugated oestrogens can be reabsorbed into the circulation and bind to their receptors in peripheral tissues to elicit their biological effects. In postmenopausal women who were not taking oestrogens or antibiotics, the ratio of oestrogen metabolites to their precursors and systemic oestrogen concentrations were shown to be proportional to their gut microbial diversity and/or deconjugation potential. Although the associations between the microbiome, oestrogen metabolism and bone outcomes in women with/at risk of osteoporosis on synthetic/exogenous oestrogens remain to be elucidated, the evidence of extensive oestrogen metabolism in the gut raises a number of scenarios relevant to oestrogen therapies. Differences in gut microbial composition and glucoronidase activity may contribute to the variability in oestrogen-based therapies responses. Furthermore, oestrogen-based therapies may induce changes in bacterial composition and microbial-mediated deconjugation process, with implications for the half-life, efficacy, and safety of these agents and potential benefits to be gained from their co-administration with specific nutrients or bacteria.Conversely, the gut microbiome can metabolize oestrogens, hence, regulating their circulating levels. Some bacteria possess genes (collectively termed the estrobolome) that encode β-glucuronidases, which deconjugate oestrogens into their biologically active forms. Although no study has explored the effects of denosumab on the microbiome of animals/individuals with osteoporosis, a recent animal study using an experimental model of colitis demonstrated that mice treated with denosumab experienced altered alpha and beta diversity  of faecal microflora and downregulated inflammatory responses  in the colonic mucosa. Further indirect evidence from supplementation studies in relevant animal models  suggest that treatment with probiotics reduces bone resorption and improves bone mineral density (BMD), while mechanistically, these outcomes may be partially mediated by alterations in the RANKL/RANK/OPG (osteoprotegerin) signalling pathway (e.g. downregulation of RANKL and pro-inflammatory cytokines and/or upregulation of OPG and anti-inflammatory factors). Based on these findings which require further investigations in animal and human trials, it can be speculated that denosumab may have microbiome- and immune-modulating properties, while manipulations of the microbiome may influence musculoskeletal outcomes through tipping the RANKL–OPG balance.Denosumab is a humanised monoclonal antibody that binds with high affinity to RANKL to block the development and function of osteoclasts, and thereby, it reduces bone resorption. and bone resorption. The microbiome via butyrate production  appears to be essential for PTH anabolic actions. In particular, treatment with intermittent PTH increased trabecular bone-volume fraction, trabecular thickness and number in mice with replete microbiota , but these effects were not observed in GF and antibiotic-treated mice with depleted microbiota, and thus, deficient in microbiota-derived butyrate. There were no differences between groups in parameters of cortical bone, suggesting that the microbiota is involved in the mechanism through which PTH increases trabecular, but not cortical bone mass. The authors further demonstrated that butyrate supplementation in mice treated with antibiotics reversed the ability of PTH to promote anabolism. Butyrate increased Tregs in the intestine and bone marrow and enabled PTH to differentiate naïve CD4+ T cells into Tregs, a lineage of T cells which induce CD8+ T cells to release Wnt10b. Wnt10b is a powerful activator of Wnt signalling in stromal cells and osteoblasts, and thus, it stimulates osteoblast recruitment and lifecycle, and ultimately, bone formation. Butyrate’s effects on enhancing the anabolic effects of PTH were mediated through binding to its receptor (GPR43) on dendritic cells, but also by interacting with T cells via a GPR43-independent pathway. The translation of these data to humans and their significance for treatment with PTH analogues await future study. Given that responders to teriparatide treatment experience BMD gains of different magnitudes, while a subgroup of patients show no BMD improvement after this treatment, it would be important to explore whether these variable responses are at least partially explained by inter-patient disparities in the microbiome and/or SCFA/butyrate availability. Future studies should also investigate whether the addition of probiotics, prebiotics or SCFAs could make patients’ microbiomes, especially those microflorae which are less abundant in butyrate bacterial taxa, more receptive to the anabolic effects of PTH analogues.Although no previous study has explored the interactions between the available parathyroid hormone (PTH) analogues, teriparatide and abaloparatide, and the microbiome in humans, two recent studies implicate the gut microbiome in PTH-mediated bone formation Several dietary nutrients including calcium are digested and released by intestinal bacteria, while microbiota manipulation with prebiotics  may upregulate the expression of intracellular calcium transporters. Increases in calcium absorption, reduce PTH and may have downstream effects on several tissues including bone (i.e. reduction in bone resorption), adipose tissue , and muscle (e.g. increased glucose uptake and insulin sensitivity).Given the importance of calcium for bone structure and homeostasis, an adequate calcium intake is recommended for osteoporosis prevention and treatment, while an adequate intake of dietary calcium may also be beneficial for weight management and glycaemic control. High-calcium diets or calcium supplementation in animal models of diet-induced obesity  promote the proliferation of potentially beneficial bacteria and SCFA production, attenuate the damage of intestinal mucosal and LPS translocation, prevent endotoxemia and regulate tight junction gene expression.7072 Future preclinical and clinical research is required to investigate the calcium–microbiome interactions in relation to musculoskeletal and metabolic outcomes in dysbiotic conditions .The beneficial effect of calcium ingestion on metabolic health, and possibly skeletal health, could be associated with modifications of intestinal microbiota and integrity.74 More recently, the discovery that the vitamin D receptor (VDR) and the vitamin D activating enzyme 1-α-hydroxylase (CYP27B1), are present in bone and kidneys, but also on cells in the intestine, muscle, pancreas, prostate and immune system, has unravelled numerous extra-skeletal effects of vitamin D, including immunomodulation, maintenance of intestinal homeostasis and gut microbiota eubiosis.76 Dysbiotic gut microbiota profiles with increased abundance of the phyla Bacteroidetes and associated taxa have been reported in mice fed diets low in vitamin D (versus mice fed diets high in vitamin D) and VDR or CYP27B1 knockout mice (versus wild-type mice). Additional investigations provided evidence that vitamin D deficiency compromises the mucosal barrier integrity (by disrupting junction proteins) and antimicrobial functions at epithelial surfaces.78 Such changes may allow opportunistic bacteria to outperform commensal bacteria, resulting in the upregulation or downregulation of immune responses. These findings are consistent with findings from human studies supporting that gut microbiota composition, circulating levels of LPS and inflammatory markers differ according to vitamin D intakes or vitamin D levels.80 Furthermore, vitamin D supplementation may reinstate a healthier gut microbiome profile and lessen inflammation,8082 with these effects shown to be dose dependent  and more pronounced in the upper GI tract.Vitamin D has well-established roles in calcium homeostasis, bone growth/maintenance and muscle function. Well-known regulators of vitamin D levels may be involved in the relationship between vitamin D metabolism and microbiota. A recent study demonstrated that GF mice exhibit impairments in vitamin D metabolism , which are progressively restored following reductions in FGF23; while conventionalization of GF mice induces inflammation that inhibits FGF23, resulting in increases in 1,25-dihydroxvitamin D and calcium levels. In addition to these findings, administration of probiotics in animals or humans has been shown to enhance vitamin D levels and upregulate VDR expression and activity in the host; while probiotic protection against bacterial infection and gut inflammation appear to be dependent on the VDR signalling pathway.8587The gut microbiome can also regulate vitamin D metabolism, and potentially, status and function. This is supported by preclinical data demonstrating that some intestinal bacteria possess hydroxylases that activate vitamin D.89 Studies investigating their efficacy on disease-related outcomes have yielded mixed results, with a recent meta-analysis suggesting improvements in pain intensity and physical function post-supplementation in patients with hip or knee osteoarthritis.90 Several lines of evidence suggest that the gut microbiome plays a significant role in the bioavailability of both supplements. Chondroitin is poorly absorbed in the small intestine and requires degradation to its component disaccharides by gut bacteria before it can be absorbed. In contrast to chondroitin, glucosamine is a monosaccharide largely utilized by gut bacteria, hence, the amount of the drug available for absorption is limited. Further, chondroitin degradation rates vary among individuals due to their distinct bacterial compositions, with this observation providing an explanation of inter-patient differences in the efficacy of equal doses of the drug.Chondroitin sulphate and glucosamine sulphate are slow-acting nutraceuticals  with widespread use among patients with osteoarthritis.via effects on gut mucosal immunity. A recent systematic review of animal and human studies concluded that chondroitin sulphate supplementation consistently increases the relative abundance of the genus Bacteroides, while there was insufficient evidence to draw conclusions on the effects of glucosamine sulphate on gut microbial composition. Interestingly, some species of the Bacteroides genus may use chondroitin as an energy source, thus, sparing other endogenous energy sources  and preventing gut inflammation.95 Similarly, some sulphate-reducing bacteria belonging to Bacteroides and able to cleave sulphate groups from chondroitin sulphate, appear to be involved in the production of anti-inflammatory molecules. Further indirect evidence suggests that since glucosamine and chondroitin are important constituents of the intestinal mucins, supplementation with these nutraceuticals may boost gut barrier function and intestinal immune responses.98On the other hand, glucosamine sulphate and chondroitin sulphate may exert some of their joint protective effects though modifying gut bacterial growth and metabolic activity, but also  The gut microbiome is implicated in protein metabolism and utilization. The intestinal bacteria have the capacity to break down undigested protein into amino acids, but also de novo synthesize essential amino acids from nitrogen sources.100102 In the small intestine, microbial-derived amino acids can be taken up by the host and added to amino-acid plasma pools and body proteins. Interestingly, gut microbiota may enhance the amino-acid balance in individuals with low-protein intakes and improve the bioavailability of amino acids of significance for musculoskeletal health (e.g. leucine).104 In the large intestine, the amino acids resulting from bacterial protein degradation are mainly utilized by luminal bacteria or further catabolized by the microbiota to yield a range of metabolites  which exert positive or negative effects on microbial composition, intestinal homeostasis and other host functions.100105 Thus, a healthy gut microbiota further contributes to host protein metabolism indirectly by controlling immune responses and inflammation, preventing insulin resistance, modulating host gene expression and maintaining gut barrier and mitochondrial function, whereas dysbiosis due to ageing or disease may negatively affect these parameters.Adequate consumption of dietary protein is important for maintaining musculoskeletal integrity.107110 These effects are complex and depend on the quantity of the protein, but also on protein sources, several dietary factors, and host characteristics. Indeed, increases in dietary protein have been associated with modest alterations in gut microbiota composition, unless these increases are accompanied by caloric and/or carbohydrate restrictions.107109 High-protein diets consistently increase the amount of undigested protein reaching the large intestine causing a transition towards protein fermentation and production of relevant metabolites; however, the quantity and quality of this metabolic output may considerably vary according to various factors. For example, plant and animal proteins differentially affect metabolomes and gene expression in the rectal mucosa, with these effects possibly related to their different digestibility and amino-acid composition. High-protein, low-carbohydrate diets have been associated with increased production of potentially harmful metabolites  and reduced production of beneficial metabolites (butyrate and phenolic acids), whereas supplementation of complex carbohydrates during high-protein diets lowers p-cresol, a microbial metabolite associated with disease.112 Further, since distinct bacterial species have different capacities to catabolize amino acids, the same protein intervention may have variable effects in individuals with distinct gut bacteria.On the other hand, dietary protein intakes influence the gut microbiota and host health through changes in microbiota composition and metabolic activities and host gene expression.113115 mediated by gut microbiota? Is the absence of anabolic responses to protein or amino-acid supplementation reported in some studies116119 related to the counteraction of positive protein effects by the negative impact of some amino-acid-derived metabolites or intestinal inflammation? If this is the case, can we develop interventions  to reduce the negative effects associated with protein fermentation and maximize musculoskeletal benefits?These findings are relevant to musculoskeletal health research and open new avenues for future work. Are the musculoskeletal benefits often seen with habitually high protein intakes and protein supplementation in populations at risk for musculoskeletal conditions121 or an active lifestyle and participation in sports124 or novel endurance exercise has been associated with largely positive modulations in gut microbiome such as enhanced diversity, proliferation of beneficial bacteria and elevated faecal SCFAs. Some of these effects seem to occur independently of diet, and fade upon return to a sedentary lifestyle. Further, indirect evidence supports positive or negative effects of exercise on gut transit time, inflammatory markers, immune function and intestinal barrier integrity depending on exercise modality, intensity, and duration. Notably, most of this evidence comes from preclinical studies and studies in athletes/physically active or sedentary healthy individuals, while the impact of exercise in dysbiotic conditions , which predispose to musculoskeletal diseases, is understudied. To this end, animal studies suggest that exercise may partially reinstate gut dysbiosis and impairments in intestinal villi morphology induced by high-fat feeding. Lambert et al. also demonstrated interactions between exercise and diabetic status on gut microbial ecology in a mouse model of type 2 diabetes. In older individuals, two interventions involving aerobic exercise resulted in compositional changes in gut microbiota and parallel increases in cardiorespiratory fitness.131Having a high cardiorespiratory fitness Evidence from animal studies suggest that GF and antibiotic-treated mice exhibit reduced endurance exercise capacity,133136 skeletal muscle atrophy and impaired ex vivo skeletal muscle contractile function; whereas gut microbiota restoration through natural reseeding, SCFA supplementation or acetate infusion  reverses these phenotypes. These gut microbiome effects on exercise responses are likely mediated by altering SCFA availability, substrate utilization and storage, oxidative stress, neural and immune functions or by interacting with mitochondria in energy production and inflammation.132138 In humans, we have currently limited understanding on how distinct gut microbiomes affect responses to exercise. One such study demonstrated that the microbiotas of sedentary lean and obese individuals had different baseline compositions and responded differently to a 6-week endurance exercise intervention. Exercise increased SCFA-producing taxa and faecal SCFA concentrations in lean, but not obese, individuals, and these effects were independent of diet. These findings suggest that some individuals may be more responsive to a given exercise intervention than others and these discrepancies may be related to differences in their gut microbiome.The microbiome also has marked effects on exercise capacity and metabolic parameters and it has been recently proposed to contribute to the inter-individual variability in the responses to exercise training in humans. Weight loss may also improve physical function in obese frail individuals; however, it has been associated with bone and muscle loss, impaired bone microstructure and elevated fracture risk, especially among elderly.142Weight loss appears to have differential effects on different aspects of musculoskeletal health. Weight loss of at least 10% of body weight is recommended for obese patients with osteoarthritis. and conversely, weight loss interventions involving dietary restrictions,143145 bariatric surgery146 or direct microbiota modifications can affect microbial composition and associated metabolites. Nevertheless, the weight-loss–microbiome interactions have been rarely characterized in relation to musculoskeletal outcomes. A meta-analysis of 11 trials demonstrated that restrictive diets for weight loss were associated with lower total bacterial abundance and overall reductions of butyrate-producing bacteria , which were mostly related to macronutrient composition/deficiency. In the same meta-analysis, inconsistent results were observed for alpha diversity and compositional changes at phylum level. Besides alterations in microbiome composition, individual and total SCFA concentrations remain unaltered or significantly decreased, especially in the presence of low-carbohydrate/fibre intake, in response to weight-loss diets. Reductions in another gut microbial metabolite, namely trimethylamine N-oxide (TMAO), and its precursors (choline and L-carnitine) have been documented following dietary weight loss interventions; such changes may benefit cardiometabolic health, but, surprisingly, were associated with impairments in bone health among overweight/obese individuals.In a bidirectional relationship, baseline microbiota composition (e.g. richness and certain types of bacteria) can predict responsiveness to weight-loss diets,149 Several human studies suggest that smoking alters microbial communities at different sites of the body  sometimes towards a dysbiotic profile.150153 A recent review that summarized the results of observational and interventional studies on the associations between intestinal microbiota and smoking suggested that overall, the intestinal microbiota of smokers are characterized by reduced microbial diversity, and abundance  or scarcity  of certain phyla and genera. The effects of smoking of the microbiome are postulated to be due to direct contact with tobacco smoke, exposure to pathogenic micro-organisms and chemicals present in tobacco cigarettes, and/or tobacco metabolites, which, in turn, affect microbial populations via alterations in the GI microenvironment , influences on the integrity of intestinal tight junctions and the composition of intestinal mucin, and immunosuppression. Such interactions might contribute to the development of intestinal and systemic diseases; nonetheless, the associations between smoking, dysbiosis and musculoskeletal diseases are unexplored. Smoking cessation has been associated with improved microbial diversity and favourable bacterial composition, raising the question whether the skeletal benefits seen after smoking cessation in osteoporotic patients are partially attributable to augmentations in dysbiosis.Smoking is considered an important environmental risk factor in the pathogenesis of musculoskeletal diseases, while its avoidance is recommended as part of the management of patients with osteoporosis.via different mechanisms,156 and the promotion of dysbiosis has been proposed as one of them. Heavy drinking has been associated with alterations in gut microbiota composition (e.g. growth of Proteobacteria and depletion of Bacteroidetes) and metabolites (reduced butyrate levels), barrier function disruption (increased LPS levels) and intestinal inflammation (increased levels of pro-inflammatory cytokines).158Alcohol abuse and high alcohol intake have been shown to increase the risk for several diseases including osteoporosis and sarcopenia 159 Besides the amount of ethanol, it is likely that the observed associations are influenced by other factors specific to the type of alcoholic drinks. Compared with distilled alcoholic beverages , fermented alcoholic drinks such as wine and beer are lower in alcohol content  and contain nutrients (i.e. polyphenols and fibres) with potential beneficial effects on the microbiome. For example, in a randomized cross-over trial, exclusive alcohol consumption (gin) over 20 days resulted in Bacteroides and Clostridium enrichment and scarcity of Prevotellaceae compared with baseline microbiota  or in comparison with the microbiota changes seen during the 20-day periods of de-alcoholized red-wine consumption (polyphenols only) and red-wine consumption . Furthermore, the consumption of de-alcoholized red wine and red wine resulted in growth of beneficial bacteria, reduction in potentially harmful bacteria and positive changes in markers of metabolic health. In another intervention, moderate consumption of red wine over a month increased alpha diversity and levels of some minor genera capable of metabolizing polyphenols. In contrast to these results, non-alcoholic beer resulted in more pronounced changes in gut microbiota diversity and composition than alcoholic beer, and enhancements in glucose tolerance which were not seen after the consumption of alcoholic beer, with these results suggesting that the presence of ethanol in alcoholic beer may blur potential favourable health effects of other nutrients. Future studies are needed to explore the impact of ethanol with/without other nutrients on the microbiome and musculoskeletal outcomes.Less clear are the effects of low-to-moderate alcohol consumption on musculoskeletal health, with some epidemiological studies suggesting reduced risk of osteoporosis in moderate alcohol drinkers.Globally, the composition and metabolic activity of the gut microbiota could be manipulated through diet, antibiotics, probiotics , prebiotics , postbiotics  or faecal microbiota transplantation. Since there are no available clinical studies on antibiotics, postbiotics and faecal microbiota transplantation with regards to osteoporosis, sarcopenia or osteoarthritis, in this review, we focus on interventions with prebiotics and probiotics supplemented in considerable quantities  in patients with, or at risk of, these musculoskeletal diseases. Notably, most of the available studies have explored whether these strategies can prevent/protect against musculoskeletal diseases, while studies on the effects of prebiotics/probiotics as treatment strategies  remain very limited.Clinical studies evaluating the effects of prebiotics on outcomes related to musculoskeletal health and disease in adults are summarized in 6066 Although overall beneficial effects have been observed with different types of prebiotics  provided at different doses (8–20 g), treatment effects are likely larger with greater doses and specific prebiotic types. For example, in postmenopausal women, an increase in calcium absorption was seen after a 9-day supply of lactulose at 10 g/day, but not after its provision at 5 g/day. Providing further evidence on dose-response effects, Jakeman et al. showed that the consumption of soluble corn fibres at doses of 10 and 20 g/day resulted in increases in skeletal calcium retention by 5% and 7%, respectively. Notably, most of the available studies reflect the short-term effects of prebiotics on calcium absorption (9 days to 8 weeks), while the long-term effects necessitate further investigation.Administration of prebiotics has been shown to enhance calcium absorption in the majority of studies in adults including older individuals. or kefir-fermented milk on BMD in populations at increased risk for bone loss . It is uncertain if these results reflect true absence of treatment effects or whether these interventions were too short (3–6 months) to capture small changes in bone mass. A study of longer duration (24 months) assessed bone turnover markers (BTMs) and BMD  changes in response to short-chain FOS plus calcium (CaFOS), calcium only or maltodextrin (placebo–control) in postmenopausal women. Treatment with CaFOS decreased serum C-telopeptide of type 1 collagen at 12 months and serum osteocalcin at 24 months to a larger extent than control. While these results indicated that CaFOS reduced bone turnover compared with other treatments, significant protection of total body BMD was only seen in the CaFOS group compared with the calcium-only group. Further, this study suggested that, among women with osteopenia, those who consumed CaFOS experienced lower reductions in spine BMD compared with those who were provided Ca only or placebo–control, with these results suggesting that this type of intervention may benefit more women at higher risk for osteoporosis. In accordance with the BTM findings of this study, several other investigations of shorter duration (2–12 weeks) have reported decreases in BTMs,67 although some others have shown increases or no effects. In summary, current evidence based on few investigations suggests modest benefits of prebiotics on skeletal health.Three randomized placebo-controlled trials have reported the effects of prebiotics on BMD. Two of them showed no significant effects of FOS The evidence on the effects of prebiotics in sarcopenia/frailty-related outcomes is, however, limited. In a randomized controlled trial (RCT) exploring the effects of a mixture of inulin and FOS (versus placebo) in ambulatory elderly residing in nursing homes, the intervention group experienced significant improvements in handgrip strength and self-reported feeling of exhaustion after 13 weeks of supplementation, while the overall rate of frailty remained unchanged. By using a similar intervention (inulin + FOS versus placebo over a 13-week period), Theou et al. demonstrated a modest reduction in a 62-item frailty index in older individuals receiving the prebiotic mixture. Improvements in physical function, frailty degree, nutritional status and quality of life were also shown following a 12-week intervention with an oral nutritional supplementation  plus physical exercise in frail elderly ; nevertheless, it was not possible to disentangle the contributions of the individual components of this multimodal intervention to the observed favourable changes. Collectively, the available studies suggest that prebiotics as stand-alone therapy or as complement to other treatments may positively influence some aspects of physical performance in frail individuals.Prebiotic supplementation in frail individuals has been shown to increase the abundance of certain bacterial taxa with less pronounced effects on alpha and beta diversity.Clinical studies evaluating the effects of probiotics on outcomes related to musculoskeletal health and disease in adults are summarized in 165167 These have been conducted exclusively in postmenopausal women and have used several probiotic strains in variable doses over 6- to 12-month supplementation periods. Most of available studies (four out of five studies) support significantly attenuated bone loss in response to probiotic treatments.166 In one trial, probiotics co-administered with isoflavones mitigated BMD reductions at the lumbar spine, femoral neck and trochanter. Due to the study design, it was not, however, possible to tease out the individual effects of probiotics or isoflavones. The three other studies which demonstrated skeletal benefits, utilized distinct probiotic strains  and overall showed reduced bone loss at the distal tibia, lumbar spine or the hip. It should be noted that the magnitude of the effects observed with probiotics (versus placebo at 12 months) is comparable with that achieved with calcium and/or vitamin D supplementation, but rather small compared with the magnitude of BMD improvements following treatment with osteoporosis medications. Mechanisms that may be driving the favourable skeletal influences of probiotics include reductions in bone resorption in some studies,166 modulations in gut microbiota and/or promotion of favourable oestrogen metabolite profiles. Although none of the aforementioned investigations has tested the effects of probiotics on fracture risk, a study conducted in older patients who sustained a fracture  revealed that treatment with probiotics (skimmed milk containing Lactobacillus casei Shirota) significantly improved indicators of the fracture healing process including pain, disabilities of the arm, shoulder and hand, active range of motion and grip strength during the first 4 months after the injury compared with placebo.Few RCTs have addressed the effects of probiotics on BTMs and bone mass.L. casei Shirota compared with those receiving placebo. In another recent investigation, supplementation with another probiotic strain (Streptococcus thermophiles versus placebo) resulted in reductions in CRP and markers indicating degradation of type II collagen, but had no effect on WOMAC. Additional research is needed to enhance our understanding of the effects of probiotics in patients with different types of arthritis, while considering potential confounding factors, including age, sex, diet, medications and individual microbial signatures.We identified two probiotic interventions in patients with osteoarthritis. In one study, Western Ontario and McMaster Universities Arthritis index (WOMAC) and Visual Analogue Scale (VAS) scores improved and serum high-sensitivity C-reactive-protein (CRP) levels were significantly lower in patients administered  Interestingly, patients with musculoskeletal conditions often feel ambivalently about drugs and pharmaceutically manufactured supplements and may be more receptive towards nutritional changes complementary to their therapeutic regimens.In addition to interventional studies exploring the impact of prebiotic and probiotic administration in substantial amounts on bone, muscle and joint health, it is interesting to envisage whether foods and dietary patterns naturally rich in these ingredients could prevent or ameliorate musculoskeletal conditions. If effective, a food approach to manipulate the microbiome is worth considering for a number of reasons. It is affordable, promotes adequacy and variety and allows flexibility and personalized solutions. Furthermore, there is increasing interest in food matrices, which consider that whole foods/food groups are more than simply the sum of their components. favourable bone microstructure, lower bone loss and fracture rates; nevertheless, the potential links to changes in gut microbiota were not studied. Overall, the Mediterranean diet and other plant-based diets  have been associated with benefits for the prevention and/or management of musculoskeletal diseases,175 with the exception of vegan diets that exclude animal proteins and dairy products and have been associated with harmful effects on skeletal health. In separate investigations, such dietary patterns have also been linked to proliferation of beneficial bacteria, increases in SCFA synthesis, lower intestinal inflammation and limited bacterial translocation.178 A recent prospective investigation was the first to simultaneously assess the association between diet, microbiota patterns and frailty-related outcomes. They suggested that older individuals adhering to the Mediterranean diet had favourable changes in the gut microbiome, which were further associated with lower levels of inflammatory markers, greater walking speed and handgrip strength, and better cognitive function independent of major confounders . Future prospective studies and clinical trials are required to verify these findings and provide additional insights into the complex diets/microbiome/musculoskeletal health interactions.To this end, several epidemiological studies have investigated the associations between diet and musculoskeletal outcomes or the association between dietary patterns and gut microbiome characteristics, but the three-way interplay has rarely been explored. Our group and others have shown that consumption of fermented dairy products  were associated with higher BMD,Over the past decade, experiments using different approaches and human investigations have provided exciting novel insights into the complex interplay between microbiome and treatment options in ageing-related musculoskeletal diseases. Nevertheless, current evidence is limited and often indirect. For example, our understanding of the interactions between the microbiome and drugs for osteoporosis treatment largely derives from animal experiments that have explored the contribution of the microbiome to key metabolic pathways involved in bone metabolism , rather than the microbial metabolism of the available drugs exploiting these pathways  or the effects of these drugs on microbiota characteristics. Furthermore, several studies have looked at the two-way interactions between lifestyle factors/drugs and musculoskeletal health outcomes or between lifestyle factors/drugs and microbiome; however, the three-way interactions have only rarely been assessed. Differences between conventional-raised, antibiotic-treated, and GF animals have provided preliminary data that the microbiome is involved in the metabolism of drugs in musculoskeletal diseases, nevertheless, well-controlled clinical trials are needed to explore how differences in microbiota composition affect outcomes in response to disease therapeutics. Further understanding of these aspects could aid the development of tools that consider patients’ gut microbiome and predict treatment responses, and drive advances in therapeutics based on, or targeted at, the gut microbiome, which may have the potential to improve therapeutics’ efficacy or reduce drug side effects. To this end, future studies are required to investigate the therapeutic use of prebiotic and probiotic supplementation as monotherapy or as adjunctive to conventional treatments for musculoskeletal diseases.


Cholesterol is an essential molecule in cellular membranes, but too much cholesterol can be toxic. Therefore, mammalian cells have developed complex mechanisms to remove excess cholesterol. In this review article, we discuss what is known about such efflux pathways including a discussion of reverse cholesterol transport and formation of high-density lipoprotein, the function of ABC transporters and other sterol efflux proteins, and we highlight their role in human diseases. Attention is paid to the biophysical principles governing efflux of sterols from cells. We also discuss recent evidence for cholesterol efflux by the release of exosomes, microvesicles, and migrasomes. The role of the endo-lysosomal network, lipophagy, and selected lysosomal transporters, such as Niemann Pick type C proteins in cholesterol export from cells is elucidated. Since oxysterols are important regulators of cellular cholesterol efflux, their formation, trafficking, and secretion are described briefly. In addition to discussing results obtained with traditional biochemical methods, focus is on studies that use established and novel bioimaging approaches to obtain insight into cholesterol efflux pathways, including fluorescence and electron microscopy, atomic force microscopy, X-ray tomography as well as mass spectrometry imaging. Cholesterol is a small lipid molecule consisting of a steroid ring system, a short alkyl-chain and a hydroxy group at position 3 as the only polar constituent. These properties, together with the asymmetric orientation of its methyl groups, give cholesterol a unique ability to interact with phospholipids in cellular membranes. Cholesterol reduces the propensity of gauche configurations in carbon chains, thereby ordering the fatty acid acyl chains, decreasing the distance between phospholipid head groups and condensing the lipid bilayer . This abAll carbon atoms in cholesterol originate from acetate, and cholesterol-specific biosynthesis starts with the reduction of the activated ketone body hydroxymethylglutaryl-Coenzyme A into mevalonate by the cytoplasmic enzyme hydroxymethylglutaryl-Coenzyme A reductase (HMG-CoA reductase) . This isLow density lipoprotein (LDL) provides cholesterol for uptake into cells. Upon binding to the LDL-receptor at the plasma membrane (PM), the LDL/LDL receptor complex can be internalized by clathrin-dependent endocytosis . AdaptorSince several important regulators of cholesterol homeostasis reside in the ER and the sterol concentration in this organelle is low under physiological conditions, the ER is an excellent control center for cholesterol homeostasis. Cells respond to a drop in cholesterol levels with increased expression of HMG-CoA reductase and other biosynthetic enzymes, which is an important feedback mechanism. . This feIn addition to the SREBP/SCAP/Insig system, nuclear receptors play a major role in cholesterol regulation. For example, the liver X receptors (LXRs) function as complementary and independent sterols sensors, which are particularly important for the control of cellular cholesterol efflux. LXRs are activated by oxysterols, which are generated when cellular cholesterol levels are high . Upon acCholesterol is most abundant in the PM, the endocytic pathway, and the TGN with lower concentrations in the ER, mitochondria, and other organelles. Since cholesterol can exchange between organelle membranes by non-vesicular transport without apparent free energy consumption, a homogeneous distribution would be expected, if the chemical potential of cholesterol in all membranes would be equal . To prevBiophysical studies in binary and ternary model membranes have shown that cholesterol can cause the formation of a liquid-ordered phase coexisting with a fluid lipid phase at physiologically relevant temperatures . Above aA variety of mechanisms including the formation of stoichiometric complexes, specific hydrogen bonding and lipid-specific shielding of cholesterol underneath phospholipid headgroups have been invoked to explain specific interactions of cholesterol with other lipids in cellular membranes . NotablyEarly observations made more than 50 years ago showed that cholesterol can be partly removed from mammalian cells by incubation with serum and particularly the high-density lipoprotein (HDL) fraction of serum . Later sCholPM, . In the to cells .How can cells regulate the efflux of cholesterol based on this physicochemical principle? There are primarily two mechanisms mammalian cells use to ensure efflux of cholesterol by lowering its chemical potential in the acceptor particles; 1) the fraction of phospholipids in acceptor particles is increased, providing interaction partners for cholesterol, thereby lowering its chemical potential, and 2) by preventing cholesterol from being recaptured by cells via its esterification in the acceptor particle . Both printained . Electrointained . Importaintained . This isintained . Thus, bs (VLDL) . A thirds (VLDL) . Unfortus (VLDL) . Howevers (VLDL) . Anacetrs (VLDL) . Other ds (VLDL) . Based os (VLDL) . In somes (VLDL) .Since cholesterol is very hydrophobic its water solubility is very low , and there is a high free energy cost of moving it between membranes through the water phase . ConsequMutations in ABCA1 have been associated with Tangier disease, a rare genetic disorder characterized by very low levels of HDL and apoA1 and high accumulation of CEs, primarily in blood-derived macrophages but also in tissue macrophages in various organs, such as tonsils, liver, spleen, and lymph nodes . AdditioLive-cell imaging and correlative microscopy have contributed greatly to our current understanding of proteins involved in cholesterol export from cells. For example, by combining EM with light microscopy using diaminobenzidine induced photooxidation, Stangl and co-workers studied subcellular trafficking of SR-BI and its lipid substrates with high resolution . Recent Several hypotheses have been put forward to explain the molecular mechanisms underlying ABCA1’s function: 1) ABCA1 acts as a receptor for apoA1 on the cell surface thereby catalyzing the direct transfer of lipids onto apoA1 . 2) ABCAPhospholipid asymmetry in the PM has a regulatory function in cholesterol efflux, and there is a large body of evidence that ABCA1 affects the transverse distribution of phospholipids and cholesterol in the PM, thereby increasing the propensity of these lipids to efflux from cells. Translocation of negatively charged phospholipids to the outer PM leaflet by ABCA1 causes altered surface membrane potential and reduced rate of endocytosis . ExposinBinding experiments with fluorescence-labeled PFO, which detects membrane cholesterol above a characteristic threshold concentration, found strong binding in cells expressing ABCA1 . The choInterestingly, a very similar vesiculation mechanism to that described above has been proposed to mediate biliary secretion of cholesterol and PC. This process depends on the ABCG5/8 heterodimer and the PC-translocating ABC transporter ABCB4, also named multidrug resistance protein 2 (MDR2), which are both expressed in the canalicular membrane of hepatocytes . ABCG5/8Extracellular vesicles formed independently of the HDL/ABC transporter system have started to be recognized as cholesterol efflux routes, eventually complementing the classical efflux pathways . Based oUsing an antibody named mAb 58B1, which is supposed to bind specifically to two-dimensional arrays of 10–20 cholesterol molecules, Using a combination of proteomics, tracer experiments, super-resolution microscopy, SEM, live cells imaging, and nanoSIMS, Young and co-workers showed that the membrane-derived particles, they described earlier, were released from the macrophages during cell migration . The autUsing a combination of fluorescence and cryo-soft X-ray tomography (cryo-SXT), we have recently shown that human fibroblasts can shed cholesterol-rich microvesicles from the cell surface without chemical pretreatment . These vSeveral organelles can provide cholesterol for efflux, including the cholesterol-rich recycling endosomes, LE/LYSs, the Golgi apparatus, and LDs . Since m−/−) mice have reduced cholesterol efflux capacity, which could be overcome by stimulating ABCA1 transporters with LXR agonists. Heterozygous NPC1+/− mice show less apoptosis and lesional necrosis, likely because cholesterol transport from lysosomes to the ER is impaired, as this pathway is essential for inflammosome activation and the UPR stress response  . LBPA isresponse . Similarresponse . TogetheTo understand the role of lysosomes in the trafficking of lipoprotein-derived cholesterol, it is important to realize that cellular efflux of LDL-derived cholesterol differs between native LDL and oxidized, acetylated, or aggregated LDL, as such atherogenic LDL is differently processed in endo-lysosomes . For exaOxysterols are oxidized derivatives of cholesterol bearing additional hydroxy-, keto- or epoxy groups which significantly increase their water solubility compared to the parent cholesterol molecule . Since iThe lack of appropriate tools to follow oxysterol transport in living cells has limited our knowledge about intracellular trafficking of oxysterols. Given that oxysterols, such as 25- or 27-hydroxycholesterol, differ from cholesterol only by having one additional hydroxy group, additional modifications to introduce fluorescent moieties for live-cell imaging must be kept to an absolute minimum. One approach is two add an alkyne group to the oxysterol, load this analogue into cells and covalently link it to a fluorescent group by click chemistry . This ap• What is the nature of active cholesterol, and how is the cholesterol pool accessible to bacterial toxins related to cholesterol efflux and precipitation from membranes? While being a powerful concept, the molecular picture of “active cholesterol” is very vague at the moment. New techniques, including novel biophysical and imaging approaches, are required to substantiate this concept in living cells. Also, a better understanding of the molecular mechanisms underlying the binding of bacterial toxins to cellular membranes is needed to ensure that the observed threshold phenomena indeed solely reflect different cholesterol pools in the membrane.• Are there other mechanisms of controlling the chemical potential of cholesterol and thereby its fugacity in cellular membranes and lipoproteins? Our current understanding of the mechanisms regulating cholesterol’s chemical potential in cellular membranes and lipoproteins are based on a large body of physico-chemical data about specific phospholipid-cholesterol interactions in model membrane systems. Such systems are necessarily much simpler than cellular membranes, and we might miss an important piece of the puzzle by relying too much on model membrane studies. Thus, more biophysical studies in living cells and development of more realistic model systems are needed to answer this question.• How important are membrane contact sites for cellular efflux of cholesterol? It is well known that close, protein-mediated contact between organelles are involved in intracellular cholesterol transport, but it is little understood about whether the organelle interactome also regulates cellular cholesterol efflux and how sterol transporters, such as GramD1/Aster or oxysterol binding proteins function at those sites during export of excess cholesterol from cells.• What are the molecular mechanisms underlying shedding of cholesterol-rich vesicles from the PM? Secretion of micro-vesicles as an export mode for cholesterol from cells is an exciting new field that will complement and expand our understanding of cholesterol efflux in the future. Understanding the underlying mechanisms of vesicle secretion and their regulation will be possible by combining live-cell and correlative super-resolution microscopy with genetic knockdown techniques in different cell types and under various cholesterol loading conditions.• How is the recruitment of endo-lysosomes to the PM for cholesterol efflux regulated at a molecular level? Exciting new findings have revealed the importance of intracellular location and dynamics for lysosome function, and lysosomal cholesterol appears to be an important regulating factor in this context. Reallocation of LE/LYSs to the PM has been found to be linked to cholesterol efflux, and determining the underlying molecular mechanisms will enable us to understand, how intracellular cholesterol is mobilized for efflux from cells.• Is there a quantitative contribution of the release of entire LE/LYSs to net cholesterol efflux, and if so, what are the underlying mechanisms? Shedding of vesicles containing entire endo-lysosomes and other organelles has been observed during cholesterol efflux, but also in other cellular processes and various cell types. Whether this indeed plays a physiological role or is exaggerated under cell culture conditions needs to be explored in the future.• What are the molecular mechanisms and biophysical principles of oxysterol transport and efflux from cells and how can oxysterols control cholesterol efflux at the cellular and molecular level? Oxysterols play an important role in cholesterol efflux; they are efficiently exported from cells due to their higher water solubility compared to cholesterol, and they regulate cellular cholesterol efflux as ligands of sterol-metabolizing enzymes and transcription factors. Future studies could combine novel imaging technologies with analytical approaches, such as mass spectrometry to study trafficking and metabolism of oxysterols at a cellular and tissue level.Since mammalian cells cannot degrade cholesterol, they have developed intricate mechanisms for its disposal. Here, we have summarized our current understanding of the cellular mechanisms of cholesterol efflux. A particular challenge is the extremely low water solubility of cholesterol, which cells overcome by employing several mechanisms. In this review we discussed in detail; 1) how cells use a variety of transporters to shuttle cholesterol between membrane and lipoproteins; 2) how cells generate close protein-mediated membrane contacts to accelerate sterol exchange and 3) how they couple cholesterol export to the formation of extracellular carrier vesicles and lipoproteins. In addition to the classical pathways of reverse cholesterol transport and formation of HDL, we highlighted novel findings of the concerted action and molecular function of a variety of ABC transporters. We also provided an overview of recently described vesicular secretion pathways for cholesterol, ranging from the secretion of exosomes over shedding of ectosomes from the PM to the formation of migrasomes, and we explained, how those mechanisms might contribute to net cholesterol efflux from cells. A particular focus was set on human diseases related to disturbed cholesterol efflux, either at the cell surface or due to defective mobilization from intracellular sites, such as endo-lysosomes and LDs. Finally, we explained the important link between cholesterol efflux and the formation and export of oxysterols from cells. However, many questions remain and future studies could focus on the following:In the past, mechanisms of cellular cholesterol efflux were explored primarily by biochemical approaches, and this has generated a large amount of data on the formation and clearance of HDL. With the increasing use of quantitative live-cell imaging, super-resolution microscopy and correlative imaging, elucidation of the spatiotemporal orchestration of cholesterol efflux at a subcellular level becomes possible. We expect this trend to continue in the future with novel imaging technologies further contributing to our understanding of this important process.


Neural firing patterns are critical for specific information coding and transmission, and abnormal firing is implicated in a series of neural pathologies. Recent studies have indicated that enhanced burst firing mediated by T-type voltage-gated calcium channels (T-VGCCs) in specific neuronal subtypes is involved in several mental or neurological disorders such as depression and epilepsy, while suppression of T-VGCCs relieve related symptoms. Burst firing consists of groups of relatively high-frequency spikes separated by quiescence. Neurons in a variety of brain areas, including the thalamus, hypothalamus, cortex, and hippocampus, display burst firing, but the ionic mechanisms that generating burst firing and the related physiological functions vary among regions. In this review, we summarize recent findings on the mechanisms underlying burst firing in various brain areas, as well as the roles of burst firing in several mental and neurological disorders. We also discuss the ion channels and receptors that may regulate burst firing directly or indirectly, with these molecules highlighted as potential intervention targets for the treatment of mental and neurological disorders. Many Kv channels also contribute to the regulation of burst firing, and their activation can hyperpolarize membranes and re-activate T-VGCCs  receptors can affect adenylyl cyclase via the Gsα protein. In rats, 5-HT application in the adrenal glands activate 5-HT7 receptors and enhances adenylyl cyclase activity, which increases T-type calcium currents  and phospholipase C (PLC) inhibits Cav3.2 channels in cultured cells, which rely on tyrosine phosphatase/kinase to enhance Cav3.1 conductance  in response to reward-predictive stimuli or reward better than expected significantly increases terminal DA phasic release in downstream targets like the nucleus accumbens and prefrontal cortex, and therefore promotes the motivation of reward-seeking and reward-driven learning Schultz, . In thisIncreasing evidence indicates that certain neuronal subpopulations display abnormal burst firing or shift firing patterns from tonic to burst firing in several nuclei during pathophysiological progress of certain neurological diseases.Anxiety is an emotion state defined as the anticipation of future or potential threat and stress. Normal adaptive anxiety can promote individual survival, however, excessive pathological anxiety will have detrimental effects on the mind and body and requires clinical treatment  and GAERS model rats, and its anti-epileptic effects mainly act via inhibition of T-VGCCs  is a progressive neurodegenerative disease manifested with core motor symptoms like tremor. Oscillation due to central neuronal pacemakers is considered an important mechanism underlying rhythmic oscillatory activity in such tremors  is thought to be involved in the pathology of several neurological and physical diseases, including anxiety, depression, and epilepsy. Importantly, antagonists and GPCRs affecting those ion channels can regulate neuronal burst firing, suggesting potential application for the clinical treatment of anxiety, depression, epilepsy, and other burst firing-induced syndromes. Moreover, neurons in many brain regions exhibit burst firing (Qiu et al., JS and YL wrote the draft of the manuscript. FY designed, wrote, checked, and finalized the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A 48-year-old woman submitted to anterior exenteration plus ileal-cutaneous conduit for metastatic cervical cancer during the change of the ureteral stent showed massive bleeding in the left ureter. A selective intra-arterial angiography showed a fistula between the ureter and the left common iliac artery that the interventional radiologist quickly repaired by inserting a vascular endoprosthesis. Six months later, gross hematuria secondary to right ureter-iliac fistula occurred again and a second endoprosthesis was inserted. Asynchronous bilateral ureteric stent-related vascular fistula is an uncommon scenario, but it should be suspected in the presence of hematuria following ureteral stent replacement. The ureteric-arterial fistula (UAF) is a rare condition characterized by a direct fistulous communication between the ureter and the iliac artery resulting in bleeding into the ureter that was described for the first time by Moschcowitz in 1908 , 3. UAF In this study, we report the clinical management of non-synchronous bilateral ureteric-iliac fistula.In 2009, a 48-year-old woman underwent radical hysterectomy for cervical cancer followed by adjuvant radiotherapy and chemotherapy. After two years, the patient was submitted to ureter-ileal-cutaneous conduit because of pelvic metastatic disease. During a check-up two years from anterior exenteration, a fibrosis plate in the lower pelvis that compressed both ureters and subsequently caused a hydronephrosis on both sides was diagnosed by abdominal computed tomography (CT). Therefore, at first, the kidneys were drained by percutaneous nephrostomy those later replaced by bilateral ureteral stent.During a programmed change of the ureteral stent in September 2019, a massive bleeding in the left ureter was showed; through the quick change of the stent, a spontaneous tamponade was made, and after stabilizing the cardiovascular system and correcting the heavy loss of blood, a CT scan followed by a selective intra-arterial angiography in digital technique was carried out. Both examinations showed an ureteroarterial fistula between the left common iliac artery and the ureter . During Ureteral stent-related UAF are reported in a small number of cases; the causative factor has been attributed to prolonged stent placement varying from 5 months to 15 years before the onset of hematuria . The etiMultiple treatment options, such as open repair with ligation, embolization of the hypogastric artery or endovascular stenting, are available with various success rates; endovascular stenting has been proven to be effective and provides a faster resolution especially in a setting of massive hemorrhage , 5. To oIn our case, the patient presented with intermittent hematuria following the ureteral stent replacement; although the initial diagnosis of left ureteric-iliac fistula was delayed because the unusual clinical presentation, conversely, the presence of recurrent hematuria allowed to quickly diagnose the contralateral ureteroiliac fistula that was restored by an interventional radiologist.Ureteric stent-related vascular fistula is an uncommon scenario, but it should be suspected in the presence of early and/or delayed hematuria following ureteral stent replacement; in addition, a multidisciplinary approach  is mandatory to achieve the best clinical results.


Topical advances in studying molecular and cellular mechanisms responsible for regeneration in the peripheral nervous system have highlighted the ability of the nervous system to repair itself. Still, serious injuries represent a challenge for the morphological and functional regeneration of peripheral nerves, calling for new treatment strategies that maximize nerve regeneration and recovery. This review presents the canonical view of the basic mechanisms of nerve regeneration and novel data on the role of exosomes and their transferred microRNAs in intracellular communication, regulation of axonal growth, Schwann cell migration and proliferation, and stromal cell functioning. An integrated comprehensive understanding of the current mechanistic underpinnings will open the venue for developing new clinical strategies to ensure full regeneration in the peripheral nervous system. The peripheral nervous system (PNS) exhibits a limited capacity for functional and morphological repair and regeneration. Peripheral nerve recovery is a multistep process with a complex molecular and cellular regulatory circuitry. Severe injury of peripheral nerves often results in a loss of motor, sensory, and autonomic functions of innervated organs and tissues, therefore calling for novel treatment strategies to ensure effective regeneration.This review aims to augment the classical view on the basic mechanisms of nerve regeneration with the recent experimental data on exosome-transferred microRNAs contributing to axonal growth and Schwann cell functioning. First, we overview the role of neurotrophins, guidance receptors, extracellular matrix, and adhesion proteins as well as immune system contribution to nerve regeneration. The recently emerging data expand the well-known players in nerve regeneration, thus opening new avenues in tackling this problem efficiently.Since mechanisms orchestrating axon regeneration are complicated and diverse, it is important to take into consideration all of them before developing new strategies to improve regeneration and reinnervation. At the site of injury, a subset of cellular and biochemical responses in neurons occur, both in the cell somas and axons, and in their innervated organs. The damage to the axonal membrane after nerve lesion leads to the rapid sodium and calcium influx into the axoplasm and generates a high-frequency burst of action potentials at the proximal part of the axons . As a reRAG programs ensure the change of neuron phenotype from neuro-transmitter state conducting the action potential to the pro-regenerative one . In partAlong with that, the distal stump of the injured nerve undergoes a set of molecular and cellular changes described as Wallerian degeneration. Immediately after injury, both axons and myelin at the distal stump degenerate. The surviving Schwann cells as well as macrophages migrating into the injury site clear the debris. Schwann cells engulf the degrading myelin contributing to debris clearance and repurpose it for future remyelination . Loss ofThe joint action of Schwann cells and macrophages results in debris removal after Wallerian degeneration. Schwann cells actively proliferate at the distal stump and align in columns called bands of Büngner. These columns create a permissive environment rich in trophic factors and ensuring guided axonal growth .Inflammation is another well-recognized aspect of nerve regeneration, since the extent and duration of inflammation affects the regeneration ultimate outcome. The balance between M1 and M2 polarization is extremely important for peripheral nerve regeneration, since the shift towards M1 state and the lack of M2 macrophages lead to a chronic inflammation and impaired regeneration . M2 macrElongation of the injured axon in the proximal stump of the regenerating nerve occurs due to the transformation of the axon tip into a highly mobile structure with a so-called “growth cone” at the end. Growth cones guide the axons towards their target organs along the gradient of signals produced by the microenvironment, such as neurotrophic growth factors, extracellular matrix (ECM) proteins, guidance receptors, and cell adhesion molecules. Schwann cells, macrophages, and target organs act as a source of neurotrophic and growth factors . In partOverall, the molecular mechanisms implicated in axonal regeneration and pathfinding after injury are multifactorial. Successful regeneration is dependent upon a crosstalk between the growing axons, Schwann cells and other cells, timely revascularization, and stimuli produced by the target organs. This interaction is coordinated by signals derived from transmembrane receptors, guidance molecules, ECM components, and cell adhesion molecules, which are expressed by all neural cells and act in a paracrine mode. In addition to these canonical mechanisms of intercellular interaction, the possibility of communication through secreted exosomes has been recently explored. The pertinent literature points to the exosome-mediated lateral transfer of regulatory molecules between Schwann cells and neurons important for axonal growth and regeneration . The rolPeripheral nerve repairment relies on the ability of regenerating axons to navigate through a structurally altered ECM, myelin, and cell debris, infiltrating inflammatory cells. During early Wallerian degeneration, not only do the axons degenerate and Schwann cells adopt transdifferentiation, but ECM undergoes a substantial degradation thus removing the inhibitory influence of chondroitin-sulfate proteoglycans and myelin-associated glycoprotein, predominantly expressed in the matrix of intact axons, and facilitating the new axon sprouting . The spaFollowing ECM degradation, Schwann cells and fibroblasts rapidly up-regulate the production of new ECM and basement membrane proteins at the site of injury, with laminin, fibronectin, and collagen being predominant types promoting nerve regeneration. This change in the ECM composition promotes the axon growth-cone formation and axon elongation with new integrin-mediated adhesive contacts playing an important role in regenerating peripheral axons. In particular, cytoskeleton proteins undergo qualitative and quantitative changes characterized by an increased expression of actin, tubulin, and peripherin, and a decrease in neurofilaments regulating the axon caliber . The mobUsing an in vivo model, we have demonstrated that the increase in α5β1 integrin expression is a key feature of peripheral nerve regeneration after injury. Our in vitro experiments revealed that the increase in α5β1 integrin expression stimulates neuritogenesis, while α5β1 integrin inhibition disrupts neurite outgrowth on fibronectin matrix . The intIn addition to cell adhesion molecules, the elongation of the growth cone and the trajectory of axon growth regeneration are controlled by guidance receptors being expressed on both neurons and Schwann cells.Recent decades have witnessed substantial progress in describing the guidance molecules and related mechanisms required to transmit their signals in embryogenesis. Studies on peripheral nerve regeneration in adults have revealed a key role of signaling cascades downstream of the guidance receptors, such as EphrinB2, Slit3, and Netrin1 . After tSchwann cells migrating from the injured nerve stumps and expressing EphB2 come into a direct contact with fibroblasts expressing EphrinB2. This interaction results in Ephrin-B/EphB2-mediated cell sorting of these two types, which in turn coordinates migration of Schwann cells as multicellular Büngner bands and guides the axonal growth across the injury site .Macrophages secrete high levels of Slit3, while the migrating Schwann cells and fibroblasts inside the nerve bridge as well as the regenerating axons express Robo1 receptor on their surface. The underlying mechanism of Slit3 action, being a restrictive signal to stay inside the nerve bridge for Robo1-expressing growing axons and migrating Schwann cells, reveals an important function of Slit3-Robo1 signaling axis for axon regeneration. Aligning with this pattern of Slit3 and Robo1 expression, numerous axon regeneration and cell migration defects have been observed in recovering nerves of Sox2-, Slit3-, and Robo1-deficient mice .Semaphorins are well-described chemorepulsive proteins in the developing nervous system regulating axon pathfinding, yet they fall short of studies as far as their role in PNS recovery after injury is concerned. Sema3A is produced by fibroblasts in the epineurial space and appears to be secreted into the extracellular matrix. Sema3A binds NRP-1 (neuropilin-1 receptor) on the membrane of axon growth cones, which leads to their collapse, the change of the trajectory of outgrowing axons and impaired reinnervation of the target organs . Along with the canonical guidance molecules, literature provides abundant data on glycosylphosphatidylinositol (GPI)-anchored proteins also playing a regulatory role in axon pathfinding. Peering into the functioning of urokinase receptor (uPAR), a (GPI)-anchored protein, in nerve regeneration, we obtained solid evidence for a unique role of uPAR in stimulating axonal growth and proliferation of Schwann cells in response to injury in PNS . Using aNeurotrophic factors are a heterogeneous group of signaling molecules that play a pivotal role in PNS development, maintenance, and plasticity. The group comprises three multigene families—the neurotrophins , neuropoetic cytokines , oncostatin M (OSM), CNTF), and the glial cell line-derived neurotrophic factor ligands  .Previous studies have shown that growth factors and cytokines are intricately involved in the axon regeneration, formation of Büngner bands, and neuronal survival . When seMultiple studies indicate that the absence of endogenous BDNF impedes the growth of sciatic nerve axons and their myelination after injury ,43, wherSeveral papers reported that the sustained delivery of GDNF, rather than NGF, to the site of nerve injury enabled efficient in vivo regeneration of both sensory and motor axons . Using aNon-neurotrophic growth factors also actively participate in peripheral nerve regeneration. VEGF secreted by Schwann cells promotes the ingrowth of newly formed blood vessels into the area of nerve injury , while bSchwann cells are the main source of neuropoetic cytokines such as LIF, CNTF and IL6 at the site of injury . SpecifiLIF has a pattern similar to CNTF activation in sympathetic neurons. LIF and CNTF employ comparable signaling pathways, and their roles in reinnervation is considered overlapping. Following the nerve transection, LIF secretion is up-regulated in Schwann cells. In vivo studies imply LIF in promoting axon regeneration of motor neurons, since LIF exogenous delivery after nerve injury augments muscle mass and muscle contraction force. Specifically, regeneration of motor neuron axons is impaired in LIF knockout mice suggesting an essential role of this cytokine in neuronal survival and recovery after injury ,51. In aDespite the well-documented stimulatory effects of exogenously delivered neurotrophic factors and cytokines on nerve regeneration obtained through experimental models in vivo and in vitro, their clinical application still has certain limitations. First, their elevated local concentration can impede nerve regeneration through potentially triggering neuron apoptosis. It has been reported that exogenously administrated BDNF in high doses of (20 μg versus 2 μg) can induce neuronal apoptosis at the site of peripheral nerve injury. The underlying molecular mechanisms reside in the preferential binding of BDNF to p75 receptor, but not to TrkB, inducing apoptosis . In lineRecent advances in cell and molecular biology highlighted a novel mechanism that ensures intercellular communication via exosomes known to be secreted by all cell types ,54. As oEmerging data point to the unique role of exosomes in maintaining tissue and organ homeostasis, as well as recovery after injury. In the nervous system, exosomes are produced by a diversity of cells such as neurons, Schwann cells, microglia, oligodendrocytes, astrocytes, macrophages, and mesenchymal stem cells (MSCs), and they can impact a variety of functions, such as survival and maturation .Mounting evidence points to a putative role of exosomes, secreted by Schwann cells, macrophages, and MSCs in the enhanced regeneration of the peripheral nervous system after injury. In particular, Schwann cell-derived exosomes markedly increased the rate of axon regeneration in vitro and in explant culture of DRG ex vivo, whereas daily administration of the same exosomes into the distal segment of the injured sciatic nerve in vivo resulted in a two-fold increase in axonal growth. Exosomes can change the axon growth-cone morphology to a pro-regenerating phenotype and decrease the activity of the GTPase RhoA inducing the growth-cone collapse and axon retraction. Importantly, this effect was cell-type-specific and was related particularly to Schwann cells, since fibroblast-derived exosomes failed to exert any beneficial actions on axon growth . The exoThe abovementioned data point to an active role of exosomes in cell communication and trafficking of a huge variety of biologically active molecules. The molecular content and the ratio of stimulatory and inhibitory molecules inside the exosomes is strictly regulated as well as their targeted delivery to the recipient cells. In addition to biologically active proteins and lipids, DNA and mRNA, exosomes comprise microRNA, which are the well-known regulators of the transcriptional landscape.MicroRNAs (miRNAs) are short ~22 bp) single-stranded noncoding RNAs which function by a partial binding  to mRNA. MiRNAs are currently considered to be “master regulators” of gene expression that orchestrate protein expression at the posttranscriptional level by binding to the 3′UTR of target messenger RNA, either hindering mRNA translation or inducing mRNA degradation. For almost 30 years since miRNAs were discovered, they have received widespread attention due to their potential role in a wide variety of physiological and pathological processes, including neurogenesis, neuronal maturation, embryogenesis, and regeneration of the nervous system among other complicated biological processes. MiRNAs can affect the phenotype of both the recipient and donor cells 2 bp sing. Using microarray analysis, Yu et al. demonstrated the dynamic changes in miRNA expression following rat sciatic nerve injury. This group identified 77 regulatory miRNAs obtained from the proximal nerve stumps, which showed significant alteration at four time points after nerve transection. The elevated expression of the miR-221 and miR-222 cluster positively correlated with the injury-induced Schwann cell phenotype. It has been suggested that up-regulated expression of miR-221/222 can stimulate Schwann cell proliferation and migration in vitro by targeting LASS2 (longevity assurance homolog 2), which is a suppressor of cell proliferation; miR-221/222 silencing exerts an opposite effect. In situ hybridization using nerve tissue sections revealed that the increase of miR-221/222 in Schwann cells occurred on the 4th day after the sciatic nerve injury, which is in agreement with the proliferative activity of Schwann cells reaching a peak in 4–11 days after injury in vivo .Tissue plasminogen activator (tPA), a serine protease, is actively involved in nerve regeneration via degrading ECM and cell adhesions and therefore facilitating Schwann cell migration and axon regrowth. It has been found that exosomal miR-340 and tPA were differentially expressed after sciatic nerve injury and negatively correlated with each other. miR-340 regulated tPA through a direct targeting of the 3′UTR of tPA mRNA. The augmented or reduced expression of miR-340 resulted in an increased or decreased tPA secretion, correspondingly, by cultured Schwann cells, their down-regulated fibrinolytic activity and migration. Dysregulation of miR-340 expression at the injury site disrupted cell debris removal and axonal growth .The role of miRNA-1 in nerve regeneration has been clearly demonstrated in vivo, using a rat sciatic nerve injury model in mice. It appears that miRNA-1 directly targets BDNF, resulting in both BDNF mRNA degradation and translation inhibition. Analysis of miRNA-1 expression pattern showed its dynamic time-dependent expression after nerve injury: a rapid decrease within the first days after injury and increase after the 14th day . SimilarExploring a possibility of exosome transfer from Schwann cells to regenerating axons, Lopez-Verrilli et al. demonstrated that Schwann cell-secreted exosomes can be specifically internalized by the axons boosting their regeneration in vitro and enhancing regeneration after sciatic nerve injury in vivo. Exosomes modified the growth-cone morphology inducing pro-regenerating phenotype with active filopodia and reduced the activity of the GTPase RhoA, which stimulates the growth-cone collapse and axon retraction .Tissue macrophages can also function as a source of exosomes promoting nerve regeneration. Delivery of M2 macrophage-derived exosomes stimulated the production of NGF and laminin by Schwann cells and enhanced their migration and proliferation in vitro. Exogenous administration of the exosomes to the site of sciatic nerve injury in rats resulted in a 15-fold increase in the number of regenerating axons and up-regulated proliferation of Schwann cells in vivo. The relative expression level of miR-223 was augmented in M2 macrophages and M2-derived exosomes, providing the fundamental molecular mechanisms of nerve regeneration in vivo and in vitro. The transfected M2 macrophages with miR-223 inhibitor exerted an inhibitory effect on Schwann cell migration and proliferation and down-regulated their NGF and laminin production . In addiMSCs are self-renewing multipotent progenitors that can be found in various tissues and organs: bone marrow, adipose tissue, dental pulp, umbilical cord blood, etc. An extensive body of literature indicates that MSCs of different origin have the capacity to promote tissue repair and neuroprotection in vivo and in vitro. Specifically, MSCs promote axonal growth, maintain neuronal survival, and significantly improve functional nerve recovery after injury ,69,70. In vitro experiments have shown that exosomes harvested from MSCs of menstrual fluids or bone marrow promote neurite outgrowth in cortical neuron and sensory neuron cultures. Moreover, the exosomes derived from amniotic MSCs (hAMSCs) exert neuroprotective effects against neuron damage induced by glutamate in vitro, which may be mediated through activating the PI3/K-Akt signaling pathway . The stiBesides well-known growth factors such as IGF-1, NGF, HGF (hepatocyte growth factor), VEGF etc. that are incapsulated into exosomes derived from MSCs , these eAs described above, inflammation plays an important role in PNS regeneration. It is well-known that MSCs have immunomodulatory properties mostly mediated through paracrine secretion of interferon (IFN)-γ, TGF-β1, HGF, heme oxygenase-1, IL-6, IL-10, prostaglandin E2 (PGE2) and others . ImmunolDespite the well-documented regenerative capacity of peripheral nerves to recuperate after injury, the question of full and efficient morphological and functional recovery is far from being resolved. It is especially true after severe injuries and after injuries that are sustained far from the denervated target organs and tissues. Recent decades have witnessed remarkable progress towards understanding the mechanistic underpinnings of molecular and cellular processes underlying peripheral nerve regeneration. PNS injury triggers a complex cascade of events inside neuron somas, axons, and the surrounding cells. Schwann cells, fibroblasts, inflammatory cells, trophic and growth factors as well as their receptors, guidance receptors and signaling molecules, exosomes produced by nerve tissue—all these are involved in the nerve regeneration process. The neurons quickly respond to axon injury by altering their transcription program and shifting their phenotype to a regenerative one. In the distal nerve stump, Schwann cells transdifferentiate and function as repair cells. Resident and infiltrating macrophages as well as Schwann cells degrade the debris, facilitating regenerating axons to grow and reinnervate their target organs and tissues. Schwann cells proliferate and migrate to form Büngner bands that guide the growing axons, and subsequently contribute to axon remyelination.Emerging evidence indicates that miRNA-containing exosomes originating from MSCs, Schwann cells, or macrophages can either stimulate the axonal regeneration directly or do so indirectly by regulating the inflammation response at the site of injury. The identified possibilities of developing novel therapies through making the most of modified exosomes to either activate or regulate the key events in nerve regeneration may open up new avenues in PNS regeneration. Designing new strategies to improve nerve regeneration remains an issue of paramount importance in fundamental research and clinical applications; it may improve patient survival rate and recovery in the near future.