Role of the intestinal microbiota in contributing to weight disorders and associated comorbidities.

SUMMARYThe gut microbiota is a major factor contributing to the regulation of energy homeostasis and has been linked to both excessive body weight and accumulation of fat mass (i.e., overweight, obesity) or body weight loss, weakness, muscle atrophy, and fat depletion (i.e., cachexia). These syndromes are characterized by multiple metabolic dysfunctions including abnormal regulation of food reward and intake, energy storage, and low-grade inflammation. Given the increasing worldwide prevalence of obesity, cachexia, and associated metabolic disorders, novel therapeutic strategies are needed. Among the different mechanisms explaining how the gut microbiota is capable of influencing host metabolism and energy balance, numerous studies have investigated the complex interactions existing between nutrition, gut microbes, and their metabolites. In this review, we discuss how gut microbes and different microbiota-derived metabolites regulate host metabolism. We describe the role of the gut barrier function in the onset of inflammation in this context. We explore the importance of the gut-to-brain axis in the regulation of energy homeostasis and glucose metabolism but also the key role played by the liver. Finally, we present specific key examples of how using targeted approaches such as prebiotics and probiotics might affect specific metabolites, their signaling pathways, and their interactions with the host and reflect on the challenges to move from bench to bedside.

Involvement of the gut microbiota in cancer cachexia.

Cancer cachexia, or the unintentional loss of body weight in patients with cancer, is a multiorgan and multifactorial syndrome with a complex and largely unknown etiology; however, metabolic dysfunction and inflammation remain hallmarks of cancer-associated wasting. Although cachexia manifests with muscle and adipose tissue loss, perturbations to the gastrointestinal tract may serve as the frontline for both impaired nutrient absorption and immune-activating gut dysbiosis. Investigations into the gut microbiota have exploded within the past two decades, demonstrating multiple gut-tissue axes; however, the link between adipose and skeletal muscle wasting and the gut microbiota with cancer is only beginning to be understood. Furthermore, the most used anticancer drugs (e.g. chemotherapy and immune checkpoint inhibitors) negatively impact gut homeostasis, potentially exacerbating wasting and contributing to poor patient outcomes and survival. In this review, we 1) highlight our current understanding of the microbial changes that occur with cachexia, 2) discuss how microbial changes may contribute to adipose and skeletal muscle wasting, and 3) outline study design considerations needed when examining the role of the microbiota in cancer-induced cachexia.

Skeletal Muscle Proteome Modifications following Antibiotic-Induced Microbial Disturbances in Cancer Cachexia.

Cancer cachexia is an involuntary loss of body weight, mostly of skeletal muscle. Previous research favors the existence of a microbiota-muscle crosstalk, so the aim of the study was to evaluate the impact of microbiota alterations induced by antibiotics on skeletal muscle proteins expression. Skeletal muscle proteome changes were investigated in control (CT) or C26 cachectic mice (C26) with or without antibiotic treatment (CT-ATB or C26-ATB, n = 8 per group). Muscle protein extracts were divided into a sarcoplasmic and myofibrillar fraction and then underwent label-free liquid chromatography separation, mass spectrometry analysis, Mascot protein identification, and METASCAPE platform data analysis. In C26 mice, the atrogen mafbx expression was 353% higher than CT mice and 42.3% higher than C26-ATB mice. No effect on the muscle protein synthesis was observed. Proteomic analyses revealed a strong effect of antibiotics on skeletal muscle proteome outside of cachexia, with adaptative processes involved in protein folding, growth, energy metabolism, and muscle contraction. In C26-ATB mice, proteome adaptations observed in CT-ATB mice were blunted. Differentially expressed proteins were involved in other processes like glucose metabolism, oxidative stress response, and proteolysis. This study confirms the existence of a microbiota-muscle axis, with a muscle response after antibiotics that varies depending on whether cachexia is present.

Analysis of mouse faecal dysbiosis, during the development of cachexia, induced by transplantation with Lewis lung carcinoma cells.

Cachexia (CC) is a complex wasting syndrome that significantly affects life quality and life expectancy among cancer patients. Original studies, in which CC was induced in mouse models through inoculation with BaF and C26 tumour cells, demonstrated that CC development correlates with bacterial gut dysbiosis in these animals. In both cases, a common microbial signature was observed, based on the expansion of Enterobacteriaceae in the gut of CC animals. However, these two types of tumours induce unique microbial profiles, suggesting that different CC induction mechanisms significantly impact the outcome of gut dysbiosis. The present study sought to expand the scope of such analyses by characterizing the CC-associated dysbiosis that develops when mice are inoculated with Lewis lung carcinoma (LLC) cells, which constitutes one of the most widely employed mechanisms for CC induction. Interestingly, Enterobacteriaceae expansion is also observed in LLC-induced CC. However, the dysbiosis identified herein displays a more complex pattern, involving representatives from seven different bacterial phyla, which were consistently identified across successive levels of taxonomic hierarchy. These results are supported by a predictive analysis of gene content, which identified a series of functional/structural changes that potentially occur in the gut bacterial population of these animals, providing a complementary and alternative approach to microbiome analyses based solely on taxonomic classification.

Molecular characterization of skin microbiota between cancer cachexia patients and healthy volunteers.

Systemic inflammation contributes to both the development of cancer and of cachexia. The microenvironment of bacterial habitats might be changed during the progression of cancer cachexia. The aim of this study was to quantitatively and qualitatively compare the composition of the skin microbiota between cancer cachexia patients and healthy volunteers. Cutaneous bacteria were swabbed at the axillary fossa of 70 cancer cachexia patients and 34 healthy individuals from China. Nested-PCR-denaturing gradient gel electrophoresis (PCR-DGGE) with primers specifically targeting V3 region and quantitative PCR (qPCR) for total bacteria, Corynebacterium spp., Staphylococcus spp., and Staphylococcus epidermidis were performed on all samples. Barcoded 454 pyrosequencing of the V3-V4 regions was performed on 30 randomly selected samples. By comparing diversity and richness indices, we found that the skin microbiome of cachectic cancer patients is less diverse than that of healthy participants, though these differences were not significant. The main microbes that reside on human skin were divided into four phyla: Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes. Staphylococcus spp. and Corynebacterium spp. were the dominant bacteria at the genus level. Significantly fewer Corynebacterium spp. had been observed in cachexia patients compared to healthy subjects. These results suggest that the presence of cancer and cachexia alters human skin bacterial communities. Understanding the changes in microbiota during cancer cachexia may lead to new insights into the syndrome.

Bacterial translocation contributes to cachexia from locally advanced gastric cancer.

BACKGROUND/AIMS: Studies have indicated that cancer cachexia patients have high cytokines levels and worse prognosis and bacterial translocation can increase cytokines production. So we aimed to investigate the association of BT with cachexia and prognosis of cachectic patients. METHODOLOGY: The locally advanced gastric cancer patients enrolled in this study were divided into cachectic and non-cachectic. Polymerase chain reaction was performed to detect bacterial DNA Cytokines levels were tested by enzyme-linked immunosorbent assay. Flow cytometry was used to detect immunological indicators. RESULTS: BT ratio was significantly higher in cachectic patients than in non-cachectic patients and healthy volunteers (p=0.019, p=0.000). BT positive cachectic patients had significantly higher levels of IL-1a, IL-6, TNF-α and IFN-γ and worse survival than BT negative cachectic patients. The levels of CD3⁺T, CD4⁺T, NK cell and CD4⁺T /CD8⁺T in gastric cancer patients were lower as compared to healthy volunteers. The level of CD8⁺T-cell was significantly higher in gastric cancer patients than that in healthy volunteers. CONCLUSIONS: This study for the first time suggested that bacterial translocation may contribute to cancer cachexia and impact prognosis of cachectic patients with locally advanced gastric cancer.

Superior mesenteric artery syndrome secondary to tuberculosis induced cachexia.

Superior mesenteric artery (SMA) syndrome is a rare cause of obstruction of 3rd part of duodenum between abdominal aorta and the overlying superior mesenteric artery caused by decrease in the angle between the two vessels as a result of rapid loss of retroperitoneal fat. It is seen in conditions causing severe weight loss and catabolic states. We report a case of pulmonary tuberculosis leading to superior mesenteric artery syndrome.

Fecal Microbiota Transplantation from Overweight or Obese Donors in Cachectic Patients with Advanced Gastroesophageal Cancer: A Randomized, Double-blind, Placebo-Controlled, Phase II Study.

PURPOSE: Cachexia is a multifactorial syndrome, associated with poor survival in patients with cancer, and is influenced by the gut microbiota. We investigated the effects of fecal microbiota transplantation (FMT) on cachexia and treatment response in patients with advanced gastroesophageal cancer. EXPERIMENTAL DESIGN: In a double-blind randomized placebo-controlled trial performed in the Amsterdam University Medical Center, we assigned 24 cachectic patients with metastatic HER2-negative gastroesophageal cancer to either allogenic FMT (healthy obese donor) or autologous FMT, prior to palliative chemotherapy (capecitabine and oxaliplatin). Primary objective was to assess the effect of allogenic FMT on satiety. Secondary outcomes were other features of cachexia, along with disease control rate (DCR), overall survival (OS), progression-free survival (PFS), and toxicity. Finally, exploratory analyses were performed on the effect of FMT on gut microbiota composition (metagenomic sequencing) and metabolites (untargeted metabolomics). RESULTS: Allogenic FMT did not improve any of the cachexia outcomes. Patients in the allogenic group (n = 12) had a higher DCR at 12 weeks (P = 0.035) compared with the autologous group (n = 12), longer median OS of 365 versus 227 days [HR = 0.38; 95% confidence interval (CI), 0.14-1.05; P = 0.057] and PFS of 204 versus 93 days (HR = 0.50; 95% CI, 0.21-1.20; P = 0.092). Patients in the allogenic group showed a significant shift in fecal microbiota composition after FMT (P = 0.010) indicating proper engraftment of the donor microbiota. CONCLUSIONS: FMT from a healthy obese donor prior to first-line chemotherapy did not affect cachexia, but may have improved response and survival in patients with metastatic gastroesophageal cancer. These results provide a rational for larger FMT trials.

A clinical correlate of the dysregulated immunoendocrine response in human tuberculosis.

Wasting is a prominent feature in tuberculosis (TB), but its underlying mechanisms are incompletely understood. Immunoendocrine disturbances may be linked to the consumption state of TB patients, since hormones and cytokines can affect energy expenditure and metabolism. To approach this possibility, we have determined leptin, IL-18, and adrenal steroid plasma levels and body mass index (BMI) in newly diagnosed patients with mild, moderate and severe pulmonary TB, household contacts (HHC), and healthy controls (HCO). HHC displayed higher levels of leptin than HCO and TB patients. TB patients showed a gradual decrease in BMI and leptin concentrations with increasing disease severity, whereas a positive correlation between this hormone and BMI was found in the HCO group. Cortisol concentrations tended to be higher in TB patients. DHEA levels were decreased in TB patients and to a lesser extent in HHC, whereas IL-18 concentration was significantly increased in patients with severe disease. Since HHC are known to cause a latent subclinical infection, it seems clear that controlled tuberculous infection and manifested TB disease are accompanied by a dissimilar profile of immunoendocrine markers.

The Effects of Prebiotic Supplementation with OMNi-LOGiC® FIBRE on Fecal Microbiome, Fecal Volatile Organic Compounds, and Gut Permeability in Murine Neuroblastoma-Induced Tumor-Associated Cachexia.

Malignant diseases can cause tumor-associated cachexia (TAC). Supplementation with prebiotic non-digestible carbohydrates exerts positive metabolic effects in experimental oncologic diseases. The aim of this project was to assess the effect of prebiotic supplementation with OMNi-LOGiC® FIBRE on intestinal microbiome, bacterial metabolism, gut permeability, and inflammation in a murine model of neuroblastoma (NB)-associated TAC. For this study, 2,000,000 NB cells (MHH-NB11) were implanted into athymic mice followed by daily supplementation with water or 200 mg prebiotic oligosaccharide (POS) OMNi-LOGiC® FIBRE (NB-Aqua, n = 12; NB-POS, n = 12). Three animals of each tumor group did not develop NB. The median time of tumor growth (first visibility to euthanasia) was 37 days (IQR 12.5 days) in the NB-Aqua group and 37 days (IQR 36.5 days) in the NB-POS group (p = 0.791). At euthanasia, fecal microbiome and volatile organic compounds (VOCs), gut permeability (fluorescein isothiocyanate-dextran (FITC-dextran), and gut barrier markers were measured. Values were compared to sham animals following injection of culture medium and gavage of either water or OMNi-LOGiC® FIBRE (SH-Aqua, n = 10; SH-POS, n = 10). Alpha diversity did not differ significantly between the groups. Principal coordinate analysis (PCoA) revealed clustering differences between Aqua and POS animals. Both NB and POS supplementation led to taxonomic alterations of the fecal microbiome. Of 49 VOCs, 22 showed significant differences between the groups. NB animals had significantly higher gut permeability than Aqua animals; POS did not ameliorate these changes. The pore and leak pathways of tight junctions did not differ between groups. In conclusion, our results suggest that NB-induced TAC causes increased gut permeability coupled with compositional changes in the fecal microbiome and VOC profile. Prebiotic supplementation with OMNi-LOGiC® FIBRE seemed to induce modifications of the fecal microbiome and VOC profile but did not improve gut permeability.

The gut and intestinal bacteria in chronic heart failure.

Chronic heart failure (CHF) is now recognized as a multisystem disorder with increased sympathetic tone, hormonal derangements, an anabolic/catabolic imbalance, endothelial dysfunction, and systemic low-grade inflammation affecting various organ systems. Pro-inflammatory cytokines appear to play important roles in that context. There is increasing evidence for the gut to have a pathophysiological role for both chronic inflammation and malnutrition in CHF. Indeed, disturbed intestinal microcirculation and barrier function in CHF seem to trigger cytokine generation, thereby contributing to further impairment in cardiac function. On the other hand, myocardial dysfunction can induce microcirculatory injuries leading to a disruption in the intestinal barrier. This amplifies the inflammatory response. Furthermore, alterations of specific absorption functions of the intestinal mucosa in CHF may aggravate symptoms of cachexia. The increased number of adherent bacteria seen in patients with CHF and elevated systemic levels of anti-lipopolysaccharide immunoglobulin A underscore this fact. Therefore, the gut poses an interesting target for therapeutic interventions in patients with CHF.

Marked cachexia in probable invasive pulmonary aspergillosis with bronchopleural fistula.

A 49-year-old man with a medical history of diabetes and heavy smoking was admitted to intensive care with severe bilateral pneumonia associated with marked cachexia. He developed a complex right-sided bronchopleural fistula and was transferred to our tertiary centre for consideration of surgical intervention.Despite escalation of antibiotic therapy, he did not improve and further investigations led to a diagnosis of invasive pulmonary aspergillosis. Definitive treatment plans required a right pneumonectomy; however, given the severity of cachexia, he remained unable to undergo such a large operation. This case demonstrates an atypical presentation of invasive pulmonary aspergillosis in a mildly immunodeficient individual. It highlights the challenges in assessment and management of critically ill patients' nutrition as well as optimal timing for surgical intervention.

Klebsiella oxytoca expands in cancer cachexia and acts as a gut pathobiont contributing to intestinal dysfunction.

Cancer cachexia is a complex multi-organ syndrome characterized by body weight loss, weakness, muscle atrophy and fat depletion. With a prevalence of 1 million people in Europe and only limited therapeutic options, there is a high medical need for new approaches to treat cachexia. Our latest results highlighted microbial dysbiosis, characterized by a bloom in Enterobacteriaceae and altered gut barrier function in preclinical models of cancer cachexia. They also demonstrated the potential of targeting the gut microbial dysbiosis in this pathology. However, the exact mechanisms underlying the gut microbiota-host crosstalk in cancer cachexia remain elusive. In this set of studies, we identified Klebsiella oxytoca as one of the main Enterobacteriaceae species increased in cancer cachexia and we demonstrated that this bacteria acts as a gut pathobiont by altering gut barrier function in cachectic mice. Moreover, we propose a conceptual framework for the lower colonization resistance to K. oxytoca in cancer cachexia that involves altered host gut epithelial metabolism and host-derived nitrate boosting the growth of the gut pathobiont. This set of studies constitutes a strong progression in the field of gut microbiota in cancer cachexia, by dissecting the mechanism of emergence of one bacterium, K. oxytoca, and establishing its role as a gut pathobiont in this severe disease.

Effects of a gut pathobiont in a gnotobiotic mouse model of childhood undernutrition.

To model how interactions among enteropathogens and gut microbial community members contribute to undernutrition, we colonized gnotobiotic mice fed representative Bangladeshi diets with sequenced bacterial strains cultured from the fecal microbiota of two 24-month-old Bangladeshi children: one healthy and the other underweight. The undernourished donor's bacterial collection contained an enterotoxigenic Bacteroides fragilis strain (ETBF), whereas the healthy donor's bacterial collection contained two nontoxigenic strains of B. fragilis (NTBF). Analyses of mice harboring either the unmanipulated culture collections or systematically manipulated versions revealed that ETBF was causally related to weight loss in the context of its native community but not when introduced into the healthy donor's community. This phenotype was transmissible from the dams to their offspring and was associated with derangements in host energy metabolism manifested by impaired tricarboxylic acid cycle activity and decreased acyl-coenzyme A utilization. NTBF reduced ETBF's expression of its enterotoxin and mitigated the effects of ETBF on the transcriptomes of other healthy donor community members. These results illustrate how intraspecific (ETBF-NTBF) and interspecific interactions influence the effects of harboring B. fragilis.

Beneficial bacteria inhibit cachexia.

Muscle wasting, known as cachexia, is a debilitating condition associated with chronic inflammation such as during cancer. Beneficial microbes have been shown to optimize systemic inflammatory tone during good health; however, interactions between microbes and host immunity in the context of cachexia are incompletely understood. Here we use mouse models to test roles for bacteria in muscle wasting syndromes. We find that feeding of a human commensal microbe, Lactobacillus reuteri, to mice is sufficient to lower systemic indices of inflammation and inhibit cachexia. Further, the microbial muscle-building phenomenon extends to normal aging as wild type animals exhibited increased growth hormone levels and up-regulation of transcription factor Forkhead Box N1 [FoxN1] associated with thymus gland retention and longevity. Interestingly, mice with a defective FoxN1 gene (athymic nude) fail to inhibit sarcopenia after L. reuteri therapy, indicating a FoxN1-mediated mechanism. In conclusion, symbiotic bacteria may serve to stimulate FoxN1 and thymic functions that regulate inflammation, offering possible alternatives for cachexia prevention and novel insights into roles for microbiota in mammalian ontogeny and phylogeny.

Synbiotic approach restores intestinal homeostasis and prolongs survival in leukaemic mice with cachexia.

Cancer cachexia is a multifactorial syndrome that includes muscle wasting and inflammation. As gut microbes influence host immunity and metabolism, we investigated the role of the gut microbiota in the therapeutic management of cancer and associated cachexia. A community-wide analysis of the caecal microbiome in two mouse models of cancer cachexia (acute leukaemia or subcutaneous transplantation of colon cancer cells) identified common microbial signatures, including decreased Lactobacillus spp. and increased Enterobacteriaceae and Parabacteroides goldsteinii/ASF 519. Building on this information, we administered a synbiotic containing inulin-type fructans and live Lactobacillus reuteri 100-23 to leukaemic mice. This treatment restored the Lactobacillus population and reduced the Enterobacteriaceae levels. It also reduced hepatic cancer cell proliferation, muscle wasting and morbidity, and prolonged survival. Administration of the synbiotic was associated with restoration of the expression of antimicrobial proteins controlling intestinal barrier function and gut immunity markers, but did not impact the portal metabolomics imprinting of energy demand. In summary, this study provided evidence that the development of cancer outside the gut can impact intestinal homeostasis and the gut microbial ecosystem and that a synbiotic intervention, by targeting some alterations of the gut microbiota, confers benefits to the host, prolonging survival and reducing cancer proliferation and cachexia.

Superior mesenteric artery (Wilkie's) syndrome as a result of cardiac cachexia.

Superior mesenteric artery (SMA) syndrome is a rare acquired disorder in which acute angulation of SMA causes compression of the third part of the duodenum between the SMA and the aorta, leading to obstruction. Loss of fatty tissue as a result of a variety of debilitating conditions is believed to be the etiologic factor causing the acute angulation. We report a case of an 86-year-old man with prolonged congestive heart failure and aortic stenosis in which SMA syndrome developed as a result of cardiac cachexia. Because of poor functional status and comorbidities, he was not a suitable candidate for decompressive surgery. Conservative treatment using a gastrostomy tube with jejunal extension led to improvement in nutritional status and resolution of symptoms.

Alterations of gut barrier and gut microbiota in food restriction, food deprivation and protein-energy wasting.

Increasing evidence shows that gut microbiota composition is related to changes of gut barrier function including gut permeability and immune function. Gut microbiota is different in obese compared to lean subjects, suggesting that gut microbes are also involved in energy metabolism and subsequent nutritional state. While research on gut microbiota and gut barrier has presently mostly focused on intestinal inflammatory bowel diseases and more recently on obesity and type 2 diabetes, this review aims at summarizing the present knowledge regarding the impact, in vivo, of depleted nutritional states on structure and function of the gut epithelium, the gut-associated lymphoid tissue (GALT), the gut microbiota and the enteric nervous system. It highlights the complex interactions between the components of gut barrier in depleted states due to food deprivation, food restriction and protein energy wasting and shows that these interactions are multidirectional, implying the existence of feedbacks.

Non Digestible Oligosaccharides Modulate the Gut Microbiota to Control the Development of Leukemia and Associated Cachexia in Mice.

We tested the hypothesis that changing the gut microbiota using pectic oligosaccharides (POS) or inulin (INU) differently modulates the progression of leukemia and related metabolic disorders. Mice were transplanted with Bcr-Abl-transfected proB lymphocytes mimicking leukemia and received either POS or INU in their diet (5%) for 2 weeks. Combination of pyrosequencing, PCR-DGGE and qPCR analyses of the 16S rRNA gene revealed that POS decreased microbial diversity and richness of caecal microbiota whereas it increased Bifidobacterium spp., Roseburia spp. and Bacteroides spp. (affecting specifically B. dorei) to a higher extent than INU. INU supplementation increased the portal SCFA propionate and butyrate, and decreased cancer cell invasion in the liver. POS treatment did not affect hepatic cancer cell invasion, but was more efficient than INU to decrease the metabolic alterations. Indeed, POS better than INU delayed anorexia linked to cancer progression. In addition, POS treatment increased acetate in the caecal content, changed the fatty acid profile inside adipose tissue and counteracted the induction of markers controlling ß-oxidation, thereby hampering fat mass loss. Non digestible carbohydrates with prebiotic properties may constitute a new nutritional strategy to modulate gut microbiota with positive consequences on cancer progression and associated cachexia.

Bacterial translocation and wasting in stressed mice.

The effects of stress on immunity and on the bacterial translocation from intestine to mesenteric lymph nodes, liver, and spleen were studied in a group of newborn CD1 mice. Animals were separated into three experimental groups. Mice from group I were stressed by intraperitoneal (i.p.) injections of heat-killed staphylococci for 4 weeks. Mice from group II were i.p. injected with saline solution only. The remaining mice, group III, were not injected. The clinical condition, presence of bacteria in abdominal organs, mitochondrial activity in splenic cells, lymphocyte proliferative response to Concanavalin-A and in vitro antibody production were evaluated in each mouse. Results showed that prolonged i.p. stressor challenge causes severe weight loss and immunodeficiency. The splenic lymphocytes from stressed mice exhibited a significant depression of both proliferative response to Concanavalin-A stimulation and anti-erythrocytes antibody synthesis. Instead, cultured in basal conditions, the splenic cells from stressed mice have an increased capacity to reduce the tetrazolium salts. Bacterial dissemination from intestine to mesenteric lymphoid nodes was also confirmed in the same group of mice. In contrast, mice in groups II and III presented no weight loss and no immunodeficiency. Results suggest that chronic biological stress induced in newborn mice could facilitate the translocation of Gram-negative bacteria. Probable pathogenic mechanisms are commented upon and a correlation is proposed between the bacterial dissemination and the wasting development.