An in vitro analysis of the interaction between infliximab and granulocyte–monocyte apheresis / Análisis in vitro de la interacción entre infliximab y la aféresis de granulocitos y monocitos

Objective: Primary non-response and secondary loss of response to anti-TNF agents are common in inflammatory bowel disease. Increasing drug concentrations are correlated to better clinical response and remission rates. Combination of granulocyte–monocyte apheresis (GMA) with anti-tumor necrosis factor (TNF) agents could be an option in these patients. The objective of our study was to perform an in vitro assay to determine if the GMA device can lead to infliximab (IFX) adsorption. Patients and methods: A blood sample was obtained from a healthy control. It was incubated with three concentrations of IFX (3, 6, and 9μg/ml) at room temperature for 10min. At that time, 1ml was collected to determine the IFX concentration. Then, 10ml of each drug concentration was incubated with 5ml of cellulose acetate (CA) beads from the GMA device at 200rpm for 1h at 37°C to simulate physiological human conditions. A second sample of each concentration was collected and IFX levels were determined. Results: No statistically significant differences were observed in the IFX levels in the blood samples before and after incubation with the CA beads (p=0.41) and after repeated measurements (p=0.31). Mean change was 3.8μg/ml. Conclusions: The in vitro combination of GMA and IFX did not change the circulating levels of IFX at the three concentrations tested, suggesting that there is no interaction between the drug and the apheresis device in vitro and that they might be safely combined with each other. (AU)

Objetivo: La falta de respuesta primaria y la pérdida de respuesta secundaria a los agentes antifactor de necrosis tumoral (TNF) son comunes en la enfermedad inflamatoria intestinal. El aumento de los niveles de fármaco se correlaciona con una mejor respuesta clínica y de las tasas de remisión. La combinación de la aféresis selectiva de granulocitos y monocitos (GMA) con agentes anti-TNF podría ser una opción en estos pacientes. El objetivo de nuestro estudio fue realizar un ensayo in vitro para determinar si el dispositivo de GMA puede interaccionar con infliximab (IFX). Pacientes y métodos: Se obtuvo una muestra de sangre de un control sano. Se incubó con 3 concentraciones de IFX (3, 6 y 9μg/ml) a temperatura ambiente durante 10 minutos. En ese momento, se recogió 1ml para determinar la concentración de IFX. Luego, se incubaron 10ml de cada concentración de fármaco con 5ml de cuentas de acetato de celulosa del dispositivo GMA a 200rpm durante una hora a 37°C para simular las condiciones fisiológicas humanas. Se recogió una segunda muestra de cada concentración y se determinaron los niveles de IFX. Resultados: No se observaron diferencias estadísticamente significativas en los niveles de IFX en las muestras de sangre antes y después de la incubación con las cuentas de acetato de celulosa (p=0,41) ni tras mediciones repetidas (p=0,31). La media de cambio fue de 3,8μg/ml. Conclusiones: La combinación in vitro de IFX y GMA no modificó los niveles circulantes del fármaco en las 3 concentraciones probadas, lo que indica que no existe interacción entre el fármaco y el dispositivo de aféresis in vitro y que podrían combinarse de forma segura. (AU)

Akkermansia muciniphila-induced trained immune phenotype increases bacterial intracellular survival and attenuates inflammation.

The initial exposure to pathogens and commensals confers innate immune cells the capacity to respond distinctively upon a second stimulus. This training capacity might play key functions in developing an adequate innate immune response to the continuous exposure to bacteria. However, the mechanisms involved in induction of trained immunity by commensals remain mostly unexplored. A. muciniphila represents an attractive candidate to study the promotion of these long-term responses. Here, we show that priming of macrophages with live A. muciniphila enhances bacterial intracellular survival and decreases the release of pro- and anti-inflammatory signals, lowering the production of TNF and IL-10. Global transcriptional analysis of macrophages after a secondary exposure to the bacteria showed the transcriptional rearrangement underpinning the phenotype observed compared to acutely exposed cells, with the increased expression of genes related to phagocytic capacity and those involved in the metabolic adjustment conducing to innate immune training. Accordingly, key genes related to bacterial killing and pro-inflammatory pathways were downregulated. These data demonstrate the importance of specific bacterial members in the modulation of local long-term innate immune responses, broadening our knowledge of the association between gut microbiome commensals and trained immunity as well as the anti-inflammatory probiotic potential of A. muciniphila.

Mitochondrial dysfunction promotes microbial composition that negatively impacts on ulcerative colitis development and progression.

Recent evidence demonstrates potential links between mitochondrial dysfunction and inflammatory bowel diseases (IBD). In addition, bidirectional interactions between the intestinal microbiota and host mitochondria may modulate intestinal inflammation. We observed previously that mice deficient in the mitochondrial protein MCJ (Methylation-controlled J protein) exhibit increased susceptibility to DSS colitis. However, it is unclear whether this phenotype is primarily driven by MCJ-/- associated gut microbiota dysbiosis or by direct effects of MCJ-deficiency. Here, we demonstrate that fecal microbiota transplantation (FMT) from MCJ-deficient into germ-free mice was sufficient to confer increased susceptibility to colitis. Therefore, an FMT experiment by cohousing was designed to alter MCJ-deficient microbiota. The phenotype resulting from complex I deficiency was reverted by FMT. In addition, we determined the protein expression pathways impacted by MCJ deficiency, providing insight into the pathophysiology of IBD. Further, we used magnetic activated cell sorting (MACS) and 16S rRNA gene sequencing to characterize taxa-specific coating of the intestinal microbiota with Immunoglobulin A (IgA-SEQ) in MCJ-deficient mice. We show that high IgA coating of fecal bacteria observed in MCJ-deficient mice play a potential role in disease progression. This study allowed us to identify potential microbial signatures in feces associated with complex I deficiency and disease progression. This research highlights the importance of finding microbial biomarkers, which might serve as predictors, permitting the stratification of ulcerative colitis (UC) patients into distinct clinical entities of the UC spectrum.

Mitochondrial dysfunction-associated microbiota establishes a transmissible refractory response to anti-TNF therapy during ulcerative colitis.

Anti-TNF therapy can induce and maintain a remission status during intestinal bowel disease. However, up to 30% of patients do not respond to this therapy by mechanisms that are unknown. Here, we show that the absence of MCJ, a natural inhibitor of the respiratory chain Complex I, induces gut microbiota changes that are critical determinants of the lack of response in a murine model of DSS-induced inflammation. First, we found that MCJ expression is restricted to macrophages in human colonic tissue. Therefore, we demonstrate by transcriptomic analysis of colon macrophages from DSS-induced mice that MCJ-deficiency is linked to the expression of genes belonging to the FcγR signaling pathway and contains an anti-TNF refractory gene signature identified in ulcerative colitis patients. The gut microbial composition changes observed upon DSS treatment in the MCJ-deficient mice revealed the increased presence of specific colitogenic members, including Ruminococcus gnavus and Oscillospira, which could be associated with the non-response to TNF inhibitors. Further, we show that the presence of a microbiota associated resistance to treatment is dominant and transmissible to responsive individuals. Collectively, our findings underscore the critical role played by macrophage mitochondrial function in the gut ecological niche that can substantially affect not only the severity of inflammation but also the ability to successfully respond to current therapies.

An in vitro analysis of the interaction between infliximab and granulocyte-monocyte apheresis.

OBJECTIVE: Primary non-response and secondary loss of response to anti-TNF agents are common in inflammatory bowel disease. Increasing drug concentrations are correlated to better clinical response and remission rates. Combination of granulocyte-monocyte apheresis (GMA) with anti-tumor necrosis factor (TNF) agents could be an option in these patients. The objective of our study was to perform an in vitro assay to determine if the GMA device can lead to infliximab (IFX) adsorption. PATIENTS AND METHODS: A blood sample was obtained from a healthy control. It was incubated with three concentrations of IFX (3, 6, and 9µg/ml) at room temperature for 10min. At that time, 1ml was collected to determine the IFX concentration. Then, 10ml of each drug concentration was incubated with 5ml of cellulose acetate (CA) beads from the GMA device at 200rpm for 1h at 37°C to simulate physiological human conditions. A second sample of each concentration was collected and IFX levels were determined. RESULTS: No statistically significant differences were observed in the IFX levels in the blood samples before and after incubation with the CA beads (p=0.41) and after repeated measurements (p=0.31). Mean change was 3.8µg/ml. CONCLUSIONS: The in vitro combination of GMA and IFX did not change the circulating levels of IFX at the three concentrations tested, suggesting that there is no interaction between the drug and the apheresis device in vitro and that they might be safely combined with each other.

Plasma and milk metabolomics in lactating sheep divergent for feed efficiency.

Enhancing the ability of animals to convert feed into meat or milk by optimizing feed efficiency (FE) has become a priority in livestock research. Although untargeted metabolomics is increasingly used in this field and may improve our understanding of FE, no information in this regard is available in dairy ewes. This study was conducted to (1) discriminate sheep divergent for FE and (2) provide insights into the physiological mechanisms contributing to FE through high-throughput metabolomics. The ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q/TOF-MS) technique was applied to easily accessible animal fluids (plasma and milk) to assess whether their metabolome differs between high- and low-feed efficient lactating ewes (H-FE and L-FE groups, respectively; 8 animals/group). Blood and milk samples were collected on the last day of the 3-wk period used for FE estimation. A total of 793 features were detected in plasma and 334 in milk, with 100 and 38 of them, respectively, showing differences between H-FE and L-FE. The partial least-squares discriminant analysis separated both groups of animals regardless of the type of sample. Plasma allowed the detection of a greater number of differential features; however, results also supported the usefulness of milk, more easily accessible, to discriminate dairy sheep divergent for FE. Regarding pathway analysis, nitrogen metabolism (either anabolism or catabolism) seemed to play a central role in FE, with plasma and milk consistently indicating a great impact of AA metabolism. A potential influence of pathways related to energy/lipid metabolism on FE was also observed. The variable importance in the projection plot revealed 15 differential features in each matrix that contributed the most for the separation in H-FE and L-FE, such as l-proline and phosphatidylcholine 20:4e in plasma or l-pipecolic acid and phosphatidylethanolamine (18:2) in milk. Overall, untargeted metabolomics provided valuable information into metabolic pathways that may underlie FE in dairy ewes, with a special relevance of AA metabolism in determining this complex phenotype in the ovine. Further research is warranted to validate these findings.

The outcome of boosting mitochondrial activity in alcohol-associated liver disease is organ-dependent.

BACKGROUND AND AIMS: Alcohol-associated liver disease (ALD) accounts for 70% of liver-related deaths in Europe, with no effective approved therapies. Although mitochondrial dysfunction is one of the earliest manifestations of alcohol-induced injury, restoring mitochondrial activity remains a problematic strategy due to oxidative stress. Here, we identify methylation-controlled J protein (MCJ) as a mediator for ALD progression and hypothesize that targeting MCJ may help in recovering mitochondrial fitness without collateral oxidative damage. APPROACH AND RESULTS: C57BL/6 mice [wild-type (Wt)] Mcj knockout and Mcj liver-specific silencing (MCJ-LSS) underwent the NIAAA dietary protocol (Lieber-DeCarli diet containing 5% (vol/vol) ethanol for 10 days, plus a single binge ethanol feeding at day 11). To evaluate the impact of a restored mitochondrial activity in ALD, the liver, gut, and pancreas were characterized, focusing on lipid metabolism, glucose homeostasis, intestinal permeability, and microbiota composition. MCJ, a protein acting as an endogenous negative regulator of mitochondrial respiration, is downregulated in the early stages of ALD and increases with the severity of the disease. Whole-body deficiency of MCJ is detrimental during ALD because it exacerbates the systemic effects of alcohol abuse through altered intestinal permeability, increased endotoxemia, and dysregulation of pancreatic function, which overall worsens liver injury. On the other hand, liver-specific Mcj silencing prevents main ALD hallmarks, that is, mitochondrial dysfunction, steatosis, inflammation, and oxidative stress, as it restores the NAD + /NADH ratio and SIRT1 function, hence preventing de novo lipogenesis and improving lipid oxidation. CONCLUSIONS: Improving mitochondrial respiration by liver-specific Mcj silencing might become a novel therapeutic approach for treating ALD.

Enhanced mitochondrial activity reshapes a gut microbiota profile that delays NASH progression.

BACKGROUND AND AIMS: Recent studies suggest that mitochondrial dysfunction promotes progression to NASH by aggravating the gut-liver status. However, the underlying mechanism remains unclear. Herein, we hypothesized that enhanced mitochondrial activity might reshape a specific microbiota signature that, when transferred to germ-free (GF) mice, could delay NASH progression. APPROACH AND RESULTS: Wild-type and methylation-controlled J protein knockout (MCJ-KO) mice were fed for 6 weeks with either control or a choline-deficient, L-amino acid-defined, high-fat diet (CDA-HFD). One mouse of each group acted as a donor of cecal microbiota to GF mice, who also underwent the CDA-HFD model for 3 weeks. Hepatic injury, intestinal barrier, gut microbiome, and the associated fecal metabolome were then studied. Following 6 weeks of CDA-HFD, the absence of methylation-controlled J protein, an inhibitor of mitochondrial complex I activity, reduced hepatic injury and improved gut-liver axis in an aggressive NASH dietary model. This effect was transferred to GF mice through cecal microbiota transplantation. We suggest that the specific microbiota profile of MCJ-KO, characterized by an increase in the fecal relative abundance of Dorea and Oscillospira genera and a reduction in AF12 , Allboaculum , and [ Ruminococcus ], exerted protective actions through enhancing short-chain fatty acids, nicotinamide adenine dinucleotide (NAD + ) metabolism, and sirtuin activity, subsequently increasing fatty acid oxidation in GF mice. Importantly, we identified Dorea genus as one of the main modulators of this microbiota-dependent protective phenotype. CONCLUSIONS: Overall, we provide evidence for the relevance of mitochondria-microbiota interplay during NASH and that targeting it could be a valuable therapeutic approach.

Restoring cellular magnesium balance through Cyclin M4 protects against acetaminophen-induced liver damage.

Acetaminophen overdose is one of the leading causes of acute liver failure and liver transplantation in the Western world. Magnesium is essential in several cellular processess. The Cyclin M family is involved in magnesium transport across cell membranes. Herein, we identify that among all magnesium transporters, only Cyclin M4 expression is upregulated in the liver of patients with acetaminophen overdose, with disturbances in magnesium serum levels. In the liver, acetaminophen interferes with the mitochondrial magnesium reservoir via Cyclin M4, affecting ATP production and reactive oxygen species generation, further boosting endoplasmic reticulum stress. Importantly, Cyclin M4 mutant T495I, which impairs magnesium flux, shows no effect. Finally, an accumulation of Cyclin M4 in endoplasmic reticulum is shown under hepatoxicity. Based on our studies in mice, silencing hepatic Cyclin M4 within the window of 6 to 24 h following acetaminophen overdose ingestion may represent a therapeutic target for acetaminophen overdose induced liver injury.

Mitochondrial complex I dysfunction alters the balance of soluble and membrane-bound TNF during chronic experimental colitis.

Inflammatory bowel disease (IBD) is a complex, chronic, relapsing and heterogeneous disease induced by environmental, genomic, microbial and immunological factors. MCJ is a mitochondrial protein that regulates the metabolic status of macrophages and their response to translocated bacteria. Previously, an acute murine model of DSS-induced colitis showed increased disease severity due to MCJ deficiency. Unexpectedly, we now show that MCJ-deficient mice have augmented tumor necrosis factor α converting enzyme (TACE) activity in the context of chronic inflammation. This adaptative change likely affects the balance between soluble and transmembrane TNF and supports the association of the soluble form and a milder phenotype. Interestingly, the general shifts in microbial composition previously observed during acute inflammation were absent in the chronic model of inflammation in MCJ-deficient mice. However, the lack of the mitochondrial protein resulted in increased alpha diversity and the reduction in critical microbial members associated with inflammation, such as Ruminococcus gnavus, which could be associated with TACE activity. These results provide evidence of the dynamic metabolic adaptation of the colon tissue to chronic inflammatory changes mediated by the control of mitochondrial function.

Resazurin-based high-throughput screening method for the discovery of dietary phytochemicals to target microbial transformation of L-carnitine into trimethylamine, a gut metabolite associated with cardiovascular disease.

Nowadays, there is great interest in the discovery of food compounds that might inhibit gut microbial TMA production from its methylamine precursors. In this work, an innovative novel screening strategy capable of rapidly determining the differences in the metabolic response of Klebsiella pneumoniae, a bacteria producing TMA under aerobic conditions, to a library of extracts obtained from food and natural sources was developed. The proposed high-throughput screening (HTS) method combines resazurin reduction assay in 384-well plates and Gaussian Processes as a machine learning tool for data processing, allowing for a fast, cheap and highly standardized evaluation of any interfering effect of a given compound or extract on the microbial metabolism sustained by L-carnitine utilization. As a proof-of-concept of this strategy, a pilot screening of 39 extracts and 6 pure compounds was performed to search for potential candidates that could inhibit in vitro TMA formation from L-carnitine. Among all the extracts tested, three of them were selected as candidates to interfere with TMA formation. Subsequent in vitro assays confirmed the potential of oregano and red thyme hexane extracts (at 1 mg mL-1) to inhibit TMA formation in bacterial lysates. In such in vitro assay, the red thyme extract exerted comparable effects on TMA reduction (∼40%) as 7.5 mM meldonium (∼50% TMA decrease), a reported L-carnitine analogue. Our results show that metabolic activity could be used as a proxy of the capacity to produce TMA under controlled culture conditions using L-carnitine to sustain metabolism.

A structurally unique Fusobacterium nucleatum tannase provides detoxicant activity against gallotannins and pathogen resistance.

Colorectal cancer pathogenesis and progression is associated with the presence of Fusobacterium nucleatum and the reduction of acetylated derivatives of spermidine, as well as dietary components such as tannin-rich foods. We show that a new tannase orthologue of F. nucleatum (TanBFnn ) has significant structural differences with its Lactobacillus plantarum counterpart affecting the flap covering the active site and the accessibility of substrates. Crystallographic and molecular dynamics analysis revealed binding of polyamines to a small cavity that connects the active site with the bulk solvent which interact with catalytically indispensable residues. As a result, spermidine and its derivatives, particularly N8 -acetylated spermidine, inhibit the hydrolytic activity of TanBFnn and increase the toxicity of gallotannins to F. nucleatum. Our results support a model in which the balance between the detoxicant activity of TanBFnn and the presence of metabolic inhibitors can dictate either conducive or unfavourable conditions for the survival of F. nucleatum.

Mitochondrial bioenergetics boost macrophage activation, promoting liver regeneration in metabolically compromised animals.

BACKGROUND AND AIMS: Hepatic ischemia-reperfusion injury (IRI) is the leading cause of early posttransplantation organ failure as mitochondrial respiration and ATP production are affected. A shortage of donors has extended liver donor criteria, including aged or steatotic livers, which are more susceptible to IRI. Given the lack of an effective treatment and the extensive transplantation waitlist, we aimed at characterizing the effects of an accelerated mitochondrial activity by silencing methylation-controlled J protein (MCJ) in three preclinical models of IRI and liver regeneration, focusing on metabolically compromised animal models. APPROACH AND RESULTS: Wild-type (WT), MCJ knockout (KO), and Mcj silenced WT mice were subjected to 70% partial hepatectomy (Phx), prolonged IRI, and 70% Phx with IRI. Old and young mice with metabolic syndrome were also subjected to these procedures. Expression of MCJ, an endogenous negative regulator of mitochondrial respiration, increases in preclinical models of Phx with or without vascular occlusion and in donor livers. Mice lacking MCJ initiate liver regeneration 12 h faster than WT and show reduced ischemic injury and increased survival. MCJ knockdown enables a mitochondrial adaptation that restores the bioenergetic supply for enhanced regeneration and prevents cell death after IRI. Mechanistically, increased ATP secretion facilitates the early activation of Kupffer cells and production of TNF, IL-6, and heparin-binding EGF, accelerating the priming phase and the progression through G1 /S transition during liver regeneration. Therapeutic silencing of MCJ in 15-month-old mice and in mice fed a high-fat/high-fructose diet for 12 weeks improves mitochondrial respiration, reduces steatosis, and overcomes regenerative limitations. CONCLUSIONS: Boosting mitochondrial activity by silencing MCJ could pave the way for a protective approach after major liver resection or IRI, especially in metabolically compromised, IRI-susceptible organs.

The commensal bacterium Lactiplantibacillus plantarum imprints innate memory-like responses in mononuclear phagocytes.

Gut microbiota is a constant source of antigens and stimuli to which the resident immune system has developed tolerance. However, the mechanisms by which mononuclear phagocytes, specifically monocytes/macrophages, cope with these usually pro-inflammatory signals are poorly understood. Here, we show that innate immune memory promotes anti-inflammatory homeostasis, using as model strains of the commensal bacterium Lactiplantibacillus plantarum. Priming of monocytes/macrophages with bacteria, especially in its live form, enhances bacterial intracellular survival and decreases the release of pro-inflammatory signals to the environment, with lower production of TNF and higher levels of IL-10. Analysis of the transcriptomic landscape of these cells shows downregulation of pathways associated with the production of reactive oxygen species (ROS) and the release of cytokines, chemokines and antimicrobial peptides. Indeed, the induction of ROS prevents memory-induced bacterial survival. In addition, there is a dysregulation in gene expression of several metabolic pathways leading to decreased glycolytic and respiratory rates in memory cells. These data support commensal microbe-specific metabolic changes in innate immune memory cells that might contribute to homeostasis in the gut.

Peripheral blood mononuclear cells (PBMC) microbiome is not affected by colon microbiota in healthy goats.

BACKGROUND: The knowledge about blood circulating microbiome and its functional relevance in healthy individuals remains limited. An assessment of changes in the circulating microbiome was performed by sequencing peripheral blood mononuclear cells (PBMC) bacterial DNA from goats supplemented or not in early life with rumen liquid transplantation. RESULTS: Most of the bacterial DNA associated to PBMC was identified predominantly as Proteobacteria (55%) followed by Firmicutes (24%), Bacteroidetes (11%) and Actinobacteria (8%). The predominant genera found in PBMC samples were Pseudomonas, Prevotella, Sphingomonas, Acinetobacter, Corynebacterium and Ruminococcus. Other genera such as Butyrivibrivio, Bifidobacterium, Dorea and Coprococcus were also present in lower proportions. Several species known as blood pathogens or others involved in gut homeostasis such as Faecalibacterium prausnitzii were also identified. However, the PBMC microbiome phylum composition differed from that in the colon of goats (P ≤ 0.001), where Firmicutes was the predominant phylum (83%). Although, rumen liquid administration in early-life altered bacterial community structure and increased Tlr5 expression (P = 0.020) in colon pointing to higher bacterial translocation, less than 8% of OTUs in colon were also observed in PBMCs. CONCLUSIONS: Data suggest that in physiological conditions, PBMC microbiome differs from and is not affected by colon gut microbiota in small ruminants. Although, further studies with larger number of animals and covering other animal tissues are required, results point to a common circulating bacterial profile on mammals being phylum Proteobacteria, and genera Pseudomonas and Prevotella the most abundants. All suggest that PBMC microbiome in healthy ruminants could be implicated in homeostatic condition. This study expands our knowledge about PBMC microbiome contribution to health in farm animals.

Borrelia burgdorferi infection induces long-term memory-like responses in macrophages with tissue-wide consequences in the heart.

Lyme carditis is an extracutaneous manifestation of Lyme disease characterized by episodes of atrioventricular block of varying degrees and additional, less reported cardiomyopathies. The molecular changes associated with the response to Borrelia burgdorferi over the course of infection are poorly understood. Here, we identify broad transcriptomic and proteomic changes in the heart during infection that reveal a profound down-regulation of mitochondrial components. We also describe the long-term functional modulation of macrophages exposed to live bacteria, characterized by an augmented glycolytic output, increased spirochetal binding and internalization, and reduced inflammatory responses. In vitro, glycolysis inhibition reduces the production of tumor necrosis factor (TNF) by memory macrophages, whereas in vivo, it produces the reversion of the memory phenotype, the recovery of tissue mitochondrial components, and decreased inflammation and spirochetal burdens. These results show that B. burgdorferi induces long-term, memory-like responses in macrophages with tissue-wide consequences that are amenable to be manipulated in vivo.

The mitochondrial negative regulator MCJ modulates the interplay between microbiota and the host during ulcerative colitis.

Recent evidences indicate that mitochondrial genes and function are decreased in active ulcerative colitis (UC) patients, in particular, the activity of Complex I of the electron transport chain is heavily compromised. MCJ is a mitochondrial inner membrane protein identified as a natural inhibitor of respiratory chain Complex I. The induction of experimental colitis in MCJ-deficient mice leads to the upregulation of Timp3 expression resulting in the inhibition of TACE activity that likely inhibits Tnf and Tnfr1 shedding from the cell membrane in the colon. MCJ-deficient mice also show higher expression of Myd88 and Tlr9, proinflammatory genes and disease severity. Interestingly, the absence of MCJ resulted in distinct microbiota metabolism and composition, including a member of the gut community in UC patients, Ruminococcus gnavus. These changes provoked an effect on IgA levels. Gene expression analyses in UC patients showed decreased levels of MCJ and higher expression of TIMP3, suggesting a relevant role of mitochondrial genes and function among active UC. The MCJ deficiency disturbs the regulatory relationship between the host mitochondria and microbiota affecting disease severity. Our results indicate that mitochondria function may be an important factor in the pathogenesis. All together support the importance of MCJ regulation during UC.

Regulation of macrophage activity by surface receptors contained within Borrelia burgdorferi-enriched phagosomal fractions.

Macrophages mediate the elimination of pathogens by phagocytosis resulting in the activation of specific signaling pathways that lead to the production of cytokines, chemokines and other factors. Borrelia burgdorferi, the causative agent of Lyme disease, causes a wide variety of pro-inflammatory symptoms. The proinflammatory capacity of macrophages is intimately related to the internalization of the spirochete. However, most receptors mediating this process are largely unknown. We have applied a multiomic approach, including the proteomic analysis of B. burgdorferi-containing phagosome-enriched fractions, to identify surface receptors that are involved in the phagocytic capacity of macrophages as well as their inflammatory output. Sucrose gradient protein fractions of human monocyte-derived macrophages exposed to B. burgdorferi contained the phagocytic receptor, CR3/CD14 highlighting the major role played by these proteins in spirochetal phagocytosis. Other proteins identified in these fractions include C-type lectins, scavenger receptors or Siglecs, of which some are directly involved in the interaction with the spirochete. We also identified the Fc gamma receptor pathway, including the binding receptor, CD64, as involved both in the phagocytosis of, and TNF induction in response to B. burgdorferi in the absence of antibodies. The common gamma chain, FcγR, mediates the phagocytosis of the spirochete, likely through Fc receptors and C-type lectins, in a process that involves Syk activation. Overall, these findings highlight the complex array of receptors involved in the phagocytic response of macrophages to B. burgdorferi.

Effect of Feeding Cold-Pressed Sunflower Cake on Ruminal Fermentation, Lipid Metabolism and Bacterial Community in Dairy Cows.

Cold-pressed sunflower cake (CPSC), by-product of oil-manufacturing, has high crude fat and linoleic acid concentrations, being a promising supplement to modulate rumen fatty acid (FA) profile. This trial studied CPSC effects on ruminal fermentation, biohydrogenation and the bacterial community in dairy cows. Ten cows were used in a crossover design with two experimental diets and fed during two 63-day periods. The cows were group fed forage ad libitum and the concentrate individually. The concentrates, control and CPSC, were isoenergetic, isoproteic and isofat. The ruminal samples collected at the end of each experimental period were analyzed for short-chain fatty acid, FA and DNA sequencing. CPSC decreased butyrate molar proportion (4%, p = 0.005). CPSC decreased C16:0 (28%, p < 0.001) and increased C18:0 (14%, p < 0.001) and total monounsaturated FA, especially C18:1 trans-11 (13%, p = 0.023). The total purine derivative excretion tended to be greater (5%, p = 0.05) with CPSC, resulting in a 6% greater daily microbial N flow. CPSC did not affect the diversity indices but increased the relative abundances of Treponema and Coprococcus, and decreased Enterococcus, Ruminococcus and Succinivibrio. In conclusion, the changes in ruminal fermentation and the FA profile were not associated with changes in microbial diversity or abundance of dominant populations, however, they might be associated with less abundant genera.

Gut microbiome and serum metabolome analyses identify molecular biomarkers and altered glutamate metabolism in fibromyalgia.

BACKGROUND: Fibromyalgia is a complex, relatively unknown disease characterised by chronic, widespread musculoskeletal pain. The gut-brain axis connects the gut microbiome with the brain through the enteric nervous system (ENS); its disruption has been associated with psychiatric and gastrointestinal disorders. To gain an insight into the pathogenesis of fibromyalgia and identify diagnostic biomarkers, we combined different omics techniques to analyse microbiome and serum composition. METHODS: We collected faeces and blood samples to study the microbiome, the serum metabolome and circulating cytokines and miRNAs from a cohort of 105 fibromyalgia patients and 54 age- and environment-matched healthy individuals. We sequenced the V3 and V4 regions of the 16S rDNA gene from faeces samples. UPLC-MS metabolomics and custom multiplex cytokine and miRNA analysis (FirePlex™ technology) were used to examine sera samples. Finally, we combined the different data types to search for potential biomarkers. RESULTS: We found that the diversity of bacteria is reduced in fibromyalgia patients. The abundance of the Bifidobacterium and Eubacterium genera (bacteria participating in the metabolism of neurotransmitters in the host) in these patients was significantly reduced. The serum metabolome analysis revealed altered levels of glutamate and serine, suggesting changes in neurotransmitter metabolism. The combined serum metabolomics and gut microbiome datasets showed a certain degree of correlation, reflecting the effect of the microbiome on metabolic activity. We also examined the microbiome and serum metabolites, cytokines and miRNAs as potential sources of molecular biomarkers of fibromyalgia. CONCLUSIONS: Our results show that the microbiome analysis provides more significant biomarkers than the other techniques employed in the work. Gut microbiome analysis combined with serum metabolomics can shed new light onto the pathogenesis of fibromyalgia. We provide a list of bacteria whose abundance changes in this disease and propose several molecules as potential biomarkers that can be used to evaluate the current diagnostic criteria.