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.

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.

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.

Bi-directional signaling between the intestinal epithelium and type-3 innate lymphoid cells regulates secretory dynamics and interleukin-22.

Type-3 innate lymphoid cells (ILC3) respond to localized environmental cues to regulate homeostasis and orchestrate immunity in the intestine. The intestinal epithelium is an important upstream regulator and downstream target of ILC3 signaling, however, the complexity of mucosal tissues can hinder efforts to define specific interactions between these two compartments. Here, we employ a reductionist co-culture system of murine epithelial small intestinal organoids (SIO) with ILC3 to uncover bi-directional signaling mechanisms that underlie intestinal homeostasis. We report that ILC3 induce global transcriptional changes in intestinal epithelial cells, driving the enrichment of secretory goblet cell signatures. We find that SIO enriched for goblet cells promote NKp46+ ILC3 and interleukin (IL)-22 expression, which can feedback to induce IL-22-mediated epithelial transcriptional signatures. However, we show that epithelial regulation of ILC3 in this system is contact-dependent and demonstrate a role for epithelial Delta-Like-Canonical-Notch-Ligand (Dll) in driving IL-22 production by ILC3, via subset-specific Notch1-mediated activation of T-bet+ ILC3. Finally, by interfering with Notch ligand-receptor dynamics, ILC3 appear to upregulate epithelial Atoh1 to skew secretory lineage determination in SIO-ILC3 co-cultures. This research outlines two complimentary bi-directional signaling modules between the intestinal epithelium and ILC3, which may be relevant in intestinal homeostasis and disease.

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.

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.

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.

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.

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.

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.

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.

TLR2 and TLR4 interact with sulfide system in the modulation of mouse colonic motility.

BACKGROUND: H2 S is a neuromodulator that may inhibit intestinal motility. H2 S production in colon is yielded by cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) enzymes and sulfate-reducing bacteria (SRB). Toll-like receptors (TLRs) recognize intestinal microbiota. The aim of this work was to evaluate the influence of TLR2 and TLR4 on the endogenous and SRB-mediated synthesis of H2 S and its consequences on the colonic motility of mouse. METHODS: Muscle contractility studies were performed in colon from WT, Tlr2-/- , and Tlr4-/- mice. The mRNA levels of TLR2, TLR4, CBS, CSE, and SRB were measured by real-time PCR. Free sulfide levels in colon and feces were determined by colorimetric assays. RESULTS: NaHS and GYY4137, donors of H2 S, reduced the contractility of colon. Aminooxyacetic acid (AOAA), inhibitor of CBS, and D-L propargylglycine (PAG), inhibitor of CSE, increased the contractility of colon. In vivo treatment with NaHS or GYY4137 inhibited the spontaneous contractions and upregulated TLR2 expression. The in vivo activation of TLR4 with lipopolysaccharide increased the contractile response to PAG, mRNA levels of CSE, and the free sulfide levels of H2 S in colon. In Tlr2-/- and Tlr4-/-  mice, the contractions induced by AOAA and PAG and mRNA levels of CBS and CSE were lower with respect to WT mice. Deficiency of TLR2 or TLR4 provokes alterations in free sulfide levels and SRB of colon. CONCLUSIONS AND INFERENCES: Our study demonstrates interaction between TLR2 and TLR4 and the sulfide system in the regulation of colonic motility and contributes to the pathophysiology knowledge of intestinal motility disorders.

A multi-omic analysis reveals the regulatory role of CD180 during the response of macrophages to Borrelia burgdorferi.

Macrophages are cells of the innate immune system with the ability to phagocytose and induce a global pattern of responses that depend on several signaling pathways. We have determined the biosignature of murine bone marrow-derived macrophages and human blood monocytes using transcriptomic and proteomic approaches. We identified a common pattern of genes that are transcriptionally regulated and overall indicate that the response to B. burgdorferi involves the interaction of spirochetal antigens with several inflammatory pathways corresponding to primary (triggered by pattern-recognition receptors) and secondary (induced by proinflammatory cytokines) responses. We also show that the Toll-like receptor family member CD180 is downregulated by the stimulation of macrophages, but not monocytes, with the spirochete. Silencing Cd180 results in increased phagocytosis while tempering the production of the proinflammatory cytokine TNF. Cd180-silenced cells produce increased levels of Itgam and surface CD11b, suggesting that the regulation of CD180 by the spirochete initiates a cascade that increases CR3-mediated phagocytosis of the bacterium while repressing the consequent inflammatory response.