Modeling Dynamics of Human Gut Microbiota Derived from Gluten Metabolism: Obtention, Maintenance and Characterization of Complex Microbial Communities.

Western diets are rich in gluten-containing products, which are frequently poorly digested. The human large intestine harbors microorganisms able to metabolize undigested gluten fragments that have escaped digestion by human enzymatic activities. The aim of this work was obtaining and culturing complex human gut microbial communities derived from gluten metabolism to model the dynamics of healthy human large intestine microbiota associated with different gluten forms. For this purpose, stool samples from six healthy volunteers were inoculated in media containing predigested gluten or predigested gluten plus non-digested gluten. Passages were carried out every 24 h for 15 days in the same medium and community composition along time was studied via V3-V4 16S rDNA sequencing. Diverse microbial communities were successfully obtained. Moreover, communities were shown to be maintained in culture with stable composition for 14 days. Under non-digested gluten presence, communities were enriched in members of Bacillota, such as Lachnospiraceae, Clostridiaceae, Streptococcaceae, Peptoniphilaceae, Selenomonadaceae or Erysipelotrichaceae, and members of Actinomycetota, such as Bifidobacteriaceae and Eggerthellaceae. Contrarily, communities exposed to digested gluten were enriched in Pseudomonadota. Hence, this study shows a method for culture and stable maintenance of gut communities derived from gluten metabolism. This method enables the analysis of microbial metabolism of gluten in the gut from a community perspective.

Anti-miR-518d-5p overcomes liver tumor cell death resistance through mitochondrial activity.

Dysregulation of miRNAs is a hallmark of cancer, modulating oncogenes, tumor suppressors, and drug responsiveness. The multi-kinase inhibitor sorafenib is one of the first-line drugs for advanced hepatocellular carcinoma (HCC), although the outcome for treated patients is heterogeneous. The identification of predictive biomarkers and targets of sorafenib efficacy are sorely needed. Thus, selected top upregulated miRNAs from the C19MC cluster were analyzed in different hepatoma cell lines compared to immortalized liver human cells, THLE-2 as control. MiR-518d-5p showed the most consistent upregulation among them. Thus, miR-518d-5p was measured in liver tumor/non-tumor samples of two distinct cohorts of HCC patients (n = 16 and n = 20, respectively). Circulating miR-518d-5p was measured in an independent cohort of HCC patients receiving sorafenib treatment (n = 100), where miR-518d-5p was analyzed in relation to treatment duration and patient's overall survival. In vitro and in vivo studies were performed in human hepatoma BCLC3 and Huh7 cells to analyze the effect of miR-518d-5p inhibition/overexpression during the response to sorafenib. Compared with healthy individuals, miR-518d-5p levels were higher in hepatic and serum samples from HCC patients (n = 16) and in an additional cohort of tumor/non-tumor paired samples (n = 20). MiR-518d-5p, through the inhibition of c-Jun and its mitochondrial target PUMA, desensitized human hepatoma cells and mouse xenograft to sorafenib-induced apoptosis. Finally, serum miR-518d-5p was assessed in 100 patients with HCC of different etiologies and BCLC-stage treated with sorafenib. In BCLC-C patients, higher serum miR-518d-5p at diagnosis was associated with shorter sorafenib treatment duration and survival. Hence, hepatic miR-518d-5p modulates sorafenib resistance in HCC through inhibition of c-Jun/PUMA-induced apoptosis. Circulating miR-518d-5p emerges as a potential lack of response biomarker to sorafenib in BCLC-C HCC patients.

The Human Digestive Tract Is Capable of Degrading Gluten from Birth.

The human gastrointestinal system has the capacity to metabolize dietary gluten. The capacity to degrade gliadin-derived peptide is present in humans from birth and increases during the first stages of life (up to 6-12 months of age). Fecal samples from 151 new-born and adult non-celiac disease (NCD) volunteers were collected, and glutenase and glianidase activities were evaluated. The capacity of total fecal proteins to metabolize 33-mer, 19-mer, and 13-mer gliadin peptides was also evaluated by high-performance liquid chromatography (HPLC). Feces from new-borns (meconium) showed glutenase and gliadinase activities, and peptidase activity against all three gliadin peptides. Maximal gluten degradative activity was observed in fecal samples from the youngest volunteers (0-12 months old). After the age of nine months, the gluten digestive capacity of gastrointestinal tract decreases and, from ±8 years old, individuals lose the ability to completely degrade toxic peptides. The gastrointestinal proteases involved in gluten digestion: elastase 2A, elastase 3B, and carboxipeptidase A1 are present from earlier stages of life. The human digestive tract contains the proteins capable of metabolizing gluten from birth, even before starting gluten intake. Humans are born with the ability to digest gluten and to completely degrade the potentially toxic gliadin-derived peptides (33-, 19-, and 13-mer).

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.

The role of RcsA in the adaptation and survival of Escherichia coli K92.

The Rcs phosphorelay is a two-component signal transduction system that senses stressful environmental signals such as desiccation or low temperatures, which serve as natural inducers in bacteria. RcsA is an important coregulator in this system involved in some functions regulated by the Rcs system, including biofilm formation and capsule synthesis. In this sense, we previously showed that RcsA is necessary for colanic acid synthesis in Escherichia coli K92. Here, using an E. coli K92ΔrcsA mutant lacking rcsA gene we further characterize the implications of RcsA on E. coli K92 survival under osmotic and oxidative stressful conditions, and bacterial attachment and biofilm formation on both biotic and abiotic surfaces. Our results show that RcsA protects E. coli K92 against osmotic and, especially, oxidative stress at low temperatures. In addition, RcsA did not interfere in biofilm formation in any surface tested, including polystyrene, stainless steel, silicone, Teflon, aluminum and glass. By contrast, deletion of rcsA increased bacterial attachment to the caco-2 cells monolayer used as biotic surface.

MiR-873-5p acts as an epigenetic regulator in early stages of liver fibrosis and cirrhosis.

Glycine N-methyltransferase (GNMT) is the most abundant methyltransferase in the liver and a master regulator of the transmethylation flux. GNMT downregulation leads to loss of liver function progressing to fibrosis, cirrhosis, and hepatocellular carcinoma. Moreover, GNMT deficiency aggravates cholestasis-induced fibrogenesis. To date, little is known about the mechanisms underlying downregulation of GNMT levels in hepatic fibrosis and cirrhosis. On this basis, microRNAs are epigenetic regulatory elements that play important roles in liver pathology. In this work, we aim to study the regulation of GNMT by microRNAs during liver fibrosis and cirrhosis. Luciferase assay on the 3'UTR-Gnmt was used to confirm in silico analysis showing that GNMT is potentially targeted by the microRNA miR-873-5p. Correlation between GNMT and miR-873-5p in human cholestasis and cirrhosis together with miR-873-5p inhibition in vivo in different mouse models of liver cholestasis and fibrosis [bile duct ligation and Mdr2 (Abcb4)-/- mouse] were then assessed. The analysis of liver tissue from cirrhotic and cholestatic patients, as well as from the animal models, showed that miR-873-5p inversely correlated with the expression of GNMT. Importantly, high circulating miR-873-5p was also detected in cholestastic and cirrhotic patients. Preclinical studies with anti-miR-873-5p treatment in bile duct ligation and Mdr2-/- mice recovered GNMT levels in association with ameliorated inflammation and fibrosis mainly by counteracting hepatocyte apoptosis and cholangiocyte proliferation. In conclusion, miR-873-5p emerges as a novel marker for liver fibrosis, cholestasis, and cirrhosis and therapeutic approaches based on anti-miR-873-5p may be effective treatments for liver fibrosis and cholestatic liver disease.

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.

Phagocytosis Assays for Borrelia burgdorferi.

Phagocytosis of Borrelia burgdorferi, the causative agent of Lyme disease, is a poorly understood process, despite its importance during the host immune response to infection. Thus, macrophages infiltrate the infected tissues, including the base of the heart and phagocytose the spirochete, therefore contributing to their elimination from infected tissues and leading to inflammation. An impaired bacterial clearance will result in bacterial persistence that may interfere with normal physiology of the heart, such as electrical signals from the heart, resulting in an impaired coordination of the beating of the heart or "heart block." This chapter presents a protocol for establishing primary mouse macrophage cultures, a method for lentivirus silencing of primary cells, and a method for the in vitro study of macrophage phagocytosis of fluorescently labeled Borrelia burgdorferi.

The mitochondrial negative regulator MCJ is a therapeutic target for acetaminophen-induced liver injury.

Acetaminophen (APAP) is the active component of many medications used to treat pain and fever worldwide. Its overuse provokes liver injury and it is the second most common cause of liver failure. Mitochondrial dysfunction contributes to APAP-induced liver injury but the mechanism by which APAP causes hepatocyte toxicity is not completely understood. Therefore, we lack efficient therapeutic strategies to treat this pathology. Here we show that APAP interferes with the formation of mitochondrial respiratory supercomplexes via the mitochondrial negative regulator MCJ, and leads to decreased production of ATP and increased generation of ROS. In vivo treatment with an inhibitor of MCJ expression protects liver from acetaminophen-induced liver injury at a time when N-acetylcysteine, the standard therapy, has no efficacy. We also show elevated levels of MCJ in the liver of patients with acetaminophen overdose. We suggest that MCJ may represent a therapeutic target to prevent and rescue liver injury caused by acetaminophen.

Metabolomic Identification of Subtypes of Nonalcoholic Steatohepatitis.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is a consequence of defects in diverse metabolic pathways that involve hepatic accumulation of triglycerides. Features of these aberrations might determine whether NAFLD progresses to nonalcoholic steatohepatitis (NASH). We investigated whether the diverse defects observed in patients with NAFLD are caused by different NAFLD subtypes with specific serum metabolomic profiles, and whether these can distinguish patients with NASH from patients with simple steatosis. METHODS: We collected liver and serum from methionine adenosyltransferase 1a knockout (MAT1A-KO) mice, which have chronically low levels of hepatic S-adenosylmethionine (SAMe) and spontaneously develop steatohepatitis, as well as C57Bl/6 mice (controls); the metabolomes of all samples were determined. We also analyzed serum metabolomes of 535 patients with biopsy-proven NAFLD (353 with simple steatosis and 182 with NASH) and compared them with serum metabolomes of mice. MAT1A-KO mice were also given SAMe (30 mg/kg/day for 8 weeks); liver samples were collected and analyzed histologically for steatohepatitis. RESULTS: Livers of MAT1A-KO mice were characterized by high levels of triglycerides, diglycerides, fatty acids, ceramides, and oxidized fatty acids, as well as low levels of SAMe and downstream metabolites. There was a correlation between liver and serum metabolomes. We identified a serum metabolomic signature associated with MAT1A-KO mice that also was present in 49% of the patients; based on this signature, we identified 2 NAFLD subtypes. We identified specific panels of markers that could distinguish patients with NASH from patients with simple steatosis for each subtype of NAFLD. Administration of SAMe reduced features of steatohepatitis in MAT1A-KO mice. CONCLUSIONS: In an analysis of serum metabolomes of patients with NAFLD and MAT1A-KO mice with steatohepatitis, we identified 2 major subtypes of NAFLD and markers that differentiate steatosis from NASH in each subtype. These might be used to monitor disease progression and identify therapeutic targets for patients.

Cross-Species Interferon Signaling Boosts Microbicidal Activity within the Tick Vector.

Evolution of hematophagy in blood-sucking parasites likely involves communication with their hosts. We find that Ixodes ticks are responsive to IFNγ acquired in a blood meal from mice infected with the Lyme disease-causing bacteria Borrelia burgdorferi, leading to induction of antimicrobial responses. Ixodes ticks parasitizing B. burgdorferi-infected mice upregulated an I. scapularis Rho-like GTPase (IGTPase). IGTPase knockdown enhanced B. burgdorferi levels in post-fed ticks, suggesting this protein controls spirochete survival. Notably, IGTPase was only induced during pathogen acquisition from mice and not upon transmission to naive hosts. Microinjection of ticks with IFNγ induced IGTPase, and ticks parasitizing IFNγ knockout mice, failed to upregulate IGTPase. Additionally, ticks lacking the transcription factor STAT, which signals downstream of IFNγ, did not induce IGTPase. IGTPase expression induced antimicrobial peptides, including Dae2, previously shown to inhibit B. burgdorferi. These results identify an interspecies signaling cascade allowing ticks to detect invading bacteria and mount microbicidal responses.

Ikaros mediates the DNA methylation-independent silencing of MCJ/DNAJC15 gene expression in macrophages.

MCJ (DNAJC15) is a mitochondrial protein that regulates the mitochondrial metabolic status of macrophages and their response to inflammatory stimuli. CpG island methylation in cancer cells constitutes the only mechanism identified for the regulation of MCJ gene expression. However, whether DNA methylation or transcriptional regulation mechanisms are involved in the physiological control of this gene expression in non-tumor cells remains unknown. We now demonstrate a mechanism of regulation of MCJ expression that is independent of DNA methylation. IFNγ, a protective cytokine against cardiac inflammation during Lyme borreliosis, represses MCJ transcription in macrophages. The transcriptional regulator, Ikaros, binds to the MCJ promoter in a Casein kinase II-dependent manner, and mediates the repression of MCJ expression. These results identify the MCJ gene as a transcriptional target of IFNγ and provide evidence of the dynamic adaptation of normal tissues to changes in the environment as a way to adapt metabolically to new conditions.

Regulation of oxidative stress by methylation-controlled J protein controls macrophage responses to inflammatory insults.

Mitochondria contribute to macrophage immune function through the generation of reactive oxygen species, a byproduct of the mitochondrial respiratory chain. MCJ (also known as DnaJC15) is a mitochondrial inner membrane protein identified as an endogenous inhibitor of respiratory chain complex I. Here we show that MCJ is essential for the production of tumor necrosis factor by macrophages in response to a variety of Toll-like receptor ligands and bacteria, without affecting their phagocytic activity. Loss of MCJ in macrophages results in increased mitochondrial respiration and elevated basal levels of reactive oxygen species that cause activation of the JNK/c-Jun pathway, lead to the upregulation of the TACE (also known as ADAM17) inhibitor TIMP-3, and lead to the inhibition of tumor necrosis factor shedding from the plasma membrane. Consequently, MCJ-deficient mice are resistant to the development of fulminant liver injury upon lipopolysaccharide administration. Thus, attenuation of the mitochondrial respiratory chain by MCJ in macrophages exquisitely regulates the response of macrophages to infectious insults.

Transcriptional control of RfaH on polysialic and colanic acid synthesis by Escherichia coli K92.

The transcriptional antiterminator RfaH promotes transcription of long operons encoding surface cell components important for the virulence of Escherichiacoli pathogens. In this paper, we show that RfaH enhanced kps expression for the synthesis of group 2 polysialic acid capsule in E. coli K92. In addition, we demonstrate for the first time that RfaH promotes cps expression for the synthesis of colanic acid, a cell wall component with apparently no role on pathogenicity. Finally, we show a novel RfaH requirement for growth at low temperatures.

Polysialic and colanic acids metabolism in Escherichia coli K92 is regulated by RcsA and RcsB.

We have shown previously that Escherichia coli K92 produces two different capsular polymers known as CA (colanic acid) and PA (polysialic acid) in a thermoregulated manner. The complex Rcs phosphorelay is largely related to the regulation of CA synthesis. Through deletion of rscA and rscB genes, we show that the Rcs system is involved in the regulation of both CA and PA synthesis in E. coli K92. Deletion of either rcsA or rcsB genes resulted in decreased expression of cps (CA biosynthesis cluster) at 19°C and 37°C, but only CA production was reduced at 19°C. Concerning PA, both deletions enhanced its synthesis at 37°C, which does not correlate with the reduced kps (PA biosynthesis cluster) expression observed in the rcsB mutant. Under this condition, expression of the nan operon responsible for PA catabolism was greatly reduced. Although RcsA and RcsB acted as negative regulators of PA synthesis at 37°C, their absence did not reestablish PA expression at low temperatures, despite the deletion of rcsB resulting in enhanced kps expression. Finally, our results revealed that RcsB controlled the expression of several genes (dsrA, rfaH, h-ns and slyA) involved in the thermoregulation of CA and PA synthesis, indicating that RcsB is part of a complex regulatory mechanism governing the surface appearance in E. coli.

MCJ/DnaJC15, an endogenous mitochondrial repressor of the respiratory chain that controls metabolic alterations.

Mitochondria are the main engine that generates ATP through oxidative phosphorylation within the respiratory chain. Mitochondrial respiration is regulated according to the metabolic needs of cells and can be modulated in response to metabolic changes. Little is known about the mechanisms that regulate this process. Here, we identify MCJ/DnaJC15 as a distinct cochaperone that localizes at the mitochondrial inner membrane, where it interacts preferentially with complex I of the electron transfer chain. We show that MCJ impairs the formation of supercomplexes and functions as a negative regulator of the respiratory chain. The loss of MCJ leads to increased complex I activity, mitochondrial membrane potential, and ATP production. Although MCJ is dispensable for mitochondrial function under normal physiological conditions, MCJ deficiency affects the pathophysiology resulting from metabolic alterations. Thus, enhanced mitochondrial respiration in the absence of MCJ prevents the pathological accumulation of lipids in the liver in response to both fasting and a high-cholesterol diet. Impaired expression or loss of MCJ expression may therefore result in a "rapid" metabolism that mitigates the consequences of metabolic disorders.

Identification of synthetic host defense peptide mimics that exert dual antimicrobial and anti-inflammatory activities.

A group of synthetic antimicrobial oligomers, inspired by naturally occurring antimicrobial peptides, were analyzed for the ability to modulate innate immune responses to Toll-like receptor (TLR) ligands. These synthetic mimics of antimicrobial peptides (SMAMPs) specifically reduced cytokine production in response to Staphylococcus aureus and the S. aureus component lipoteichoic acid (LTA), a TLR2 agonist. Anti-inflammatory SMAMPs prevented the induction of tumor necrosis factor (TNF), interleukin 6 (IL-6), and IL-10 in response to S. aureus or LTA, but no other TLR2 ligands. We show that these SMAMPs bind specifically to LTA in vitro and prevent its interaction with TLR2. Importantly, the SMAMP greatly reduced the induction of TNF and IL-6 in vivo in mice acutely infected with S. aureus while simultaneously reducing bacterial loads dramatically (4 log(10)). Thus, these SMAMPs can eliminate the damage induced by pathogen-associated molecular patterns (PAMPs) while simultaneously eliminating infection in vivo. They are the first known SMAMPs to demonstrate anti-inflammatory and antibacterial activities in vivo.

Macrophage p38 mitogen-activated protein kinase activity regulates invariant natural killer T-cell responses during Borrelia burgdorferi infection.

The interaction of macrophages with infectious agents leads to the activation of several signaling cascades, including mitogen-activated protein (MAP) kinases, such as p38. We now demonstrate that p38 MAP kinase-mediated responses are critical components to the immune response to Borrelia burgdorferi. The pharmacological and genetic inhibition of p38 MAP kinase activity during infection with the spirochete results in increased carditis. In transgenic mice that express a dominant negative form of p38 MAP kinase specifically in macrophages, production of the invariant natural killer T (iNKT) cell-attracting chemokine MCP-1 and of the antigen-presenting molecule CD1d are significantly reduced. The expression of the transgene therefore results in the deficient infiltration of iNKT cells, their decreased activation, and a diminished production of interferon γ (IFN-γ), leading to increased bacterial burdens and inflammation. These results show that p38 MAP kinase provides critical checkpoints for the protective immune response to the spirochete during infection of the heart.

Multiserotype protection elicited by a combinatorial prime-boost vaccination strategy against bluetongue virus.

Bluetongue virus (BTV) belongs to the genus Orbivirus within the family Reoviridae. The development of vector-based vaccines expressing conserved protective antigens results in increased immune activation and could reduce the number of multiserotype vaccinations required, therefore providing a cost-effective product. Recent recombinant DNA technology has allowed the development of novel strategies to develop marker and safe vaccines against BTV. We have now engineered naked DNAs and recombinant modified vaccinia virus Ankara (rMVA) expressing VP2, VP7 and NS1 proteins from BTV-4. IFNAR((-/-)) mice inoculated with DNA/rMVA-VP2,-VP7-NS1 in an heterologous prime boost vaccination strategy generated significant levels of antibodies specific of VP2, VP7, and NS1, including those with neutralizing activity against BTV-4. In addition, vaccination stimulated specific CD8(+) T cell responses against these three BTV proteins. Importantly, the vaccine combination expressing NS1, VP2 and VP7 proteins of BTV-4, elicited sterile protection against a lethal dose of homologous BTV-4 infection. Remarkably, the vaccine induced cross-protection against lethal doses of heterologous BTV-8 and BTV-1 suggesting that the DNA/rMVA-VP2,-VP7,-NS1 marker vaccine is a promising multiserotype vaccine against BTV.