Blocking mitochondrial pyruvate import in brown adipocytes induces energy wasting via lipid cycling.

Combined fatty acid esterification and lipolysis, termed lipid cycling, is an ATP-consuming process that contributes to energy expenditure. Therefore, interventions that stimulate energy expenditure through lipid cycling are of great interest. Here we find that pharmacological and genetic inhibition of the mitochondrial pyruvate carrier (MPC) in brown adipocytes activates lipid cycling and energy expenditure, even in the absence of adrenergic stimulation. We show that the resulting increase in ATP demand elevates mitochondrial respiration coupled to ATP synthesis and fueled by lipid oxidation. We identify that glutamine consumption and the Malate-Aspartate Shuttle are required for the increase in Energy Expenditure induced by MPC inhibition in Brown Adipocytes (MAShEEBA). We thus demonstrate that energy expenditure through enhanced lipid cycling can be activated in brown adipocytes by decreasing mitochondrial pyruvate availability. We present a new mechanism to increase energy expenditure and fat oxidation in brown adipocytes, which does not require adrenergic stimulation of mitochondrial uncoupling.

Assessment of mitochondrial physiology of murine white adipose tissue by mechanical permeabilization and lipid depletion.

White adipose tissue (WAT) represents a major site of triacylglycerol energy storage and is directly associated with metabolic disorders. Mitochondria regulate cellular energy expenditure and are active in WAT. Although isolated mitochondria have been classically used to assess their functions, several artifacts can be introduced by this approach. Furthermore, important limitations exist in the available methods to determine mitochondrial physiology in permeabilized WAT. Here, we established and validated a method for functional evaluation of mice mesenteric WAT (mWAT) mitochondria by using MEchanical Permeabilization and LIpid DEpletion (MEPLIDE) coupled to high-resolution respirometry. We observed that mild stirring of mWAT for 20 min at room temperature with 4% fatty acid-free albumin (FAF-BSA) followed by 50 min without FAF-BSA selectively permeabilized white adipocytes plasma membrane. In these conditions, mWAT mitochondria were intact, exhibiting succinate-induced respiratory rates that were sensitive to classical oxidative phosphorylation modulators. Finally, the respiratory capacity of mWAT in female mice was significantly higher than in males, an observation that agrees with reported data. Therefore, the functional assessment of mWAT mitochondria through MEPLIDE coupled to high resolution respirometry proposed here will contribute to a better understanding of WAT biology in several pathophysiological contexts.

Gut microbial metabolite butyrate protects against proteinuric kidney disease through epigenetic- and GPR109a-mediated mechanisms.

Butyrate is a short-chain fatty acid derived from the metabolism of indigestible carbohydrates by the gut microbiota. Butyrate contributes to gut homeostasis, but it may also control inflammatory responses and host physiology in other tissues. Butyrate inhibits histone deacetylases, thereby affecting gene transcription, and also signals through the metabolite-sensing G protein receptor (GPR)109a. We produced an mAb to mouse GPR109a and found high expression on podocytes in the kidney. Wild-type and Gpr109a-/- mice were induced to develop nephropathy by a single injection of Adriamycin and treated with sodium butyrate or high butyrate-releasing high-amylose maize starch diet. Butyrate improved proteinuria by preserving podocyte at glomerular basement membrane and attenuated glomerulosclerosis and tissue inflammation. This protective phenotype was associated with increased podocyte-related proteins and a normalized pattern of acetylation and methylation at promoter sites of genes essential for podocyte function. We found that GPR109a is expressed by podocytes, and the use of Gpr109a-/- mice showed that the protective effects of butyrate depended on GPR109a expression. A prebiotic diet that releases high amounts of butyrate also proved highly effective for protection against kidney disease. Butyrate and GPR109a play a role in the pathogenesis of kidney disease and provide one of the important molecular connections between diet, the gut microbiota, and kidney disease.-Felizardo, R. J. F., de Almeida, D. C., Pereira, R. L., Watanabe, I. K. M., Doimo, N. T. S., Ribeiro, W. R., Cenedeze, M. A., Hiyane, M. I., Amano, M. T., Braga, T. T., Ferreira, C. M., Parmigiani, R. B., Andrade-Oliveira, V., Volpini, R. A., Vinolo, M. A. R., Mariño, E., Robert, R., Mackay, C. R., Camara, N. O. S. Gut microbial metabolite butyrate protects against proteinuric kidney disease through epigenetic- and GPR109a-mediated mechanisms.

Modulation of mitochondrial metabolism as a biochemical trait in blood feeding organisms: the redox vampire hypothesis redux.

Hematophagous organisms undergo remarkable metabolic changes during the blood digestion process, increasing fermentative glucose metabolism, and reducing respiratory rates, both consequence of functional mitochondrial remodeling. Here, we review the pathways involved in energy metabolism and mitochondrial functionality in a comparative framework across different hematophagous species, and consider how these processes regulate redox homeostasis during blood digestion. The trend across distinct species indicate that a switch in energy metabolism might represent an important defensive mechanism to avoid the potential harmful interaction of oxidants generated from aerobic energy metabolism with products derived from blood digestion. Indeed, in insect vectors, blood feeding transiently reduces respiratory rates and oxidant production, irrespective of tissue and insect model. On the other hand, a different scenario is observed in several unrelated parasite species when exposed to blood digestion products, as respiratory rates reduce and mitochondrial oxidant production increase. The emerging picture indicates that re-wiring of energy metabolism, through reduced mitochondrial function, culminates in improved tolerance to redox insults and seems to represent a key step for hematophagous organisms to cope with the overwhelming and potentially toxic blood meal.

Gut Bacteria Products Prevent AKI Induced by Ischemia-Reperfusion.

Short-chain fatty acids (SCFAs) are fermentation end products produced by the intestinal microbiota and have anti-inflammatory and histone deacetylase-inhibiting properties. Recently, a dual relationship between the intestine and kidneys has been unraveled. Therefore, we evaluated the role of SCFA in an AKI model in which the inflammatory process has a detrimental role. We observed that therapy with the three main SCFAs (acetate, propionate, and butyrate) improved renal dysfunction caused by injury. This protection was associated with low levels of local and systemic inflammation, oxidative cellular stress, cell infiltration/activation, and apoptosis. However, it was also associated with an increase in autophagy. Moreover, SCFAs inhibited histone deacetylase activity and modulated the expression levels of enzymes involved in chromatin modification. In vitro analyses showed that SCFAs modulated the inflammatory process, decreasing the maturation of dendritic cells and inhibiting the capacity of these cells to induce CD4(+) and CD8(+) T cell proliferation. Furthermore, SCFAs ameliorated the effects of hypoxia in kidney epithelial cells by improving mitochondrial biogenesis. Notably, mice treated with acetate-producing bacteria also had better outcomes after AKI. Thus, we demonstrate that SCFAs improve organ function and viability after an injury through modulation of the inflammatory process, most likely via epigenetic modification.

A short-term high-sugar diet is an aggravating factor in experimental allergic contact dermatitis.

Allergic contact dermatitis (ACD) is an inflammatory skin reaction whose incidence has increased and has been associated with a dietary pattern rich in saturated fats and refined sugars. Considering the increased incidence of ACD and the lack of research about the influence of a short-term high-sugar diet on dermatitis, our aim is to improve understanding of the influence of a high-sugar diet on ACD. We introduced a diet rich in sugar fifteen days before inducing contact dermatitis with oxazolone, in mice, and maintained it until the end of the experiment, which lasted three weeks in total. The dermatitis model increased cholesterol and triglycerides in the liver, and the combination of diet and dermatitis increased weight and worsened liver cholesterol measurements. Furthermore, the high-sugar diet increased the production of IL-6, IFN-γ and TNF-α in the skin, which may be involved in the increase in epithelial skin thickness observed in experimental ACD.

Preservation of thrombin generation in cirrhosis despite abnormal results of international normalized ratio: implications for invasive procedures.

Thrombin generation is normal or elevated in patients with cirrhosis when tested in the presence of thrombomodulin, the activator of the main natural anticoagulant protein C. However, the relationship between thrombin generation with bleeding has been little explored in literature. 97 Consecutive patients with cirrhosis were prospectively included (58 men; 54 ±â€Š10 years) and divided into two groups international normalized ratio (INR) less than 1.5 (n = 72) or INR at least 1.5 (n = 25). 46 Healthy individuals were tested as controls. Endogenous thrombin potential (ETP) was measured without and with the addition of thrombomodulin. ETP measured without thrombomodulin was reduced in patients with cirrhosis when compared with controls, but no significant difference was found between the INR less than 1.5 and INR at least 1.5 groups (1250 ±â€Š315.7 versus 1186 ±â€Š238 nmol/l × min; P = 0.3572). After the addition of thrombomodulin, both groups generated thrombin comparable with controls (INR ≥ 1.5: 965.9 ±â€Š232.3; INR < 1.5: 893.0 ±â€Š368.6; controls: 915.0 ±â€Š458 nmol/l × min). 80% of patients had high ETP without/with thrombomodulin ratio, demonstrating the resistance to the anticoagulant action of thrombomodulin for both groups. This was more marked in the INR at least 1.5 group (0.81 ±â€Š0.1 versus 0.69 ±â€Š0.2; P = 0.0042). Postligation of esophageal varices bleeding occurred in 5.2% of patients (INR < 1.5, n = 3; INR ≥ 1.5, n = 2), all of them with ETP without/with thrombomodulin ratio ranging from 0.72 to 0.90 (controls 0.57 ±â€Š0.21). This study confirms that thrombin generation in the presence of thrombomodulin was normal in most patients with cirrhosis, including those with high INR value, but did not correlate with postligation of esophageal varices bleeding.

A probiotic has differential effects on allergic airway inflammation in A/J and C57BL/6 mice and is correlated with the gut microbiome.

The phenotypes of allergic airway diseases are influenced by the interplay between host genetics and the gut microbiota, which may be modulated by probiotics. We investigated the probiotic effects on allergic inflammation in A/J and C57BL/6 mice. C57BL/6 mice had increased gut microbiota diversity compared to A/J mice at baseline. Acetate producer probiotics differentially modulated and altered the genus abundance of specific bacteria, such as Akkermansia and Allistipes, in mouse strains. We induced airway inflammation followed by probiotic treatment and found that only A/J mice exhibited decreased inflammation, and the beneficial effects of probiotics in A/J mice were partially due to acetate production. To understand the relevance of microbial composition colonization in the development of allergic diseases, we implanted female C57BL/6 mice with A/J embryos to naturally modulate the microbial composition of A/J mice, which increased gut microbiota diversity and reduced eosinophilic inflammation in A/J. These data demonstrate the central importance of microbiota to allergic phenotype severity. Video Abstract.

Bifidobacterium Strains Present Distinct Effects on the Control of Alveolar Bone Loss in a Periodontitis Experimental Model.

Periodontitis is an inflammatory disease induced by a dysbiotic oral microbiome. Probiotics of the genus Bifidobacterium may restore the symbiotic microbiome and modulate the immune response, leading to periodontitis control. We evaluated the effect of two strains of Bifidobacterium able to inhibit Porphyromonas gingivalis interaction with host cells and biofilm formation, but with distinct immunomodulatory properties, in a mice periodontitis model. Experimental periodontitis (P+) was induced in C57Bl/6 mice by a microbial consortium of human oral organisms. B. bifidum 1622A [B+ (1622)] and B. breve 1101A [B+ (1101)] were orally inoculated for 45 days. Alveolar bone loss and inflammatory response in gingival tissues were determined. The microbial consortium induced alveolar bone loss in positive control (P + B-), as demonstrated by microtomography analysis, although P. gingivalis was undetected in oral biofilms at the end of the experimental period. TNF-α and IL-10 serum levels, and Treg and Th17 populations in gingiva of SHAM and P + B- groups did not differ. B. bifidum 1622A, but not B. breve 1101A, controlled bone destruction in P+ mice. B. breve 1101A upregulated transcription of Il-1ß, Tnf-α, Tlr2, Tlr4, and Nlrp3 in P-B+(1101), which was attenuated by the microbial consortium [P + B+(1101)]. All treatments downregulated transcription of Il-17, although treatment with B. breve 1101A did not yield such low levels of transcripts as seen for the other groups. B. breve 1101A increased Th17 population in gingival tissues [P-B+ (1101) and P + B+ (1101)] compared to SHAM and P + B-. Administration of both bifidobacteria resulted in serum IL-10 decreased levels. Our data indicated that the beneficial effect of Bifidobacterium is not a common trait of this genus, since B. breve 1101A induced an inflammatory profile in gingival tissues and did not prevent alveolar bone loss. However, the properties of B. bifidum 1622A suggest its potential to control periodontitis.

Fas ligand expression on T cells is sufficient to prevent prolonged airway inflammation in a murine model of asthma.

Our previous studies revealed that, in a murine model of asthma, mice that received Fas-deficient T cells developed a prolonged phase of airway inflammation, mucus production, and airway hyperreactivity that failed to resolve even 6 weeks after the last challenge. To investigate how Fas-Fas ligand (FasL) interaction occurs between T cells and other cells in vivo, Gld mice with abnormalities of the FasL signaling pathway were used. The reconstituted mice were made by transferring T cells from B6 or Gld mice to Rag(-/-) or FasL-deficient Rag(-/-) mice. We found that Rag(-/-) mice that received B6 T cells resolved the airway inflammation, whereas FasL-deficient Rag(-/-) mice that received Gld T cells developed a prolonged airway inflammation at Day 28, with decreased IFN-gamma production. Both FasL-deficient Rag(-/-) mice that received B6 T cells and Rag(-/-) mice that received Gld T cells also had completely resolved their airway inflammation by Day 28 after challenge. Interestingly, FasL-deficient Rag(-/-) mice that received Gld T cells eventually resolved airway inflammation at Day 42, with a similar level of IFN-gamma production to that of control group. These results demonstrate that FasL expression on either T cells only or non-T cells only was sufficient for the eventual resolution of airway inflammation, and the prolonged airway inflammation in FasL-deficient Rag(-/-) mice that received Gld T cells was correlated with decreased IFN-gamma production by Gld T cells.

OVA-Induced Allergic Airway Inflammation Mouse Model.

Asthma is a worldwide public health issue, affecting the sufferer's quality of life. Many researchers are extensively studying the cellular processes involved in the affected airways. Experimental asthma using animals has been performed for a long time, mainly applying murine models due to well-known advantages. The aim of this study is to present an allergic airway inflammation protocol in mice. Basically, the allergic airway inflammation is induced by intraperitoneal sensitization and intratracheal challenge with ovalbumin (OVA). The model provided here mimics acute asthma characteristics including excessive mucus production, airway hyperresponsiveness, and eosinophilic airway inflammation.

Description of Ovariectomy Protocol in Mice.

Estrogen and ovarian function decline are relevant characteristics of menopause period. Numerous physiological, metabolic and immunological alterations in the female body occur in the menopause period and some of these changes remain uncertain. The animal model that mimics menopause phase is an important approach to better comprehend the biological process involved in this period of women life. Ovariectomy is a procedure where ovaries are surgically excised and have been a valuable tool for understanding estrogen deficiency through animal experiments. Despite the diversity of ovariectomy protocols, the aim of this chapter is to provide a comprehensive guideline in performing ovariectomy in mice. Furthermore, isoflurane anesthesia system, postoperative care and surgery success evaluation will be described. We highlight that all procedures must be carried out by a qualified and trained professional, respecting ethical and safety principles.

Allergen Exposure in Lymphopenic Fas-Deficient Mice Results in Persistent Eosinophilia Due to Defects in Resolution of Inflammation.

Asthma is characterized by chronic airway type-2 inflammation and eosinophilia, yet the mechanisms involved in chronic, non-resolving inflammation remain poorly defined. Previously, our group has found that when Rag-deficient mice were reconstituted with Fas-deficient B6 LPR T cells and sensitized and challenged, the mice developed a prolonged type-2-mediated airway inflammation that continued for more than 6 weeks after the last antigen exposure. Surprisingly, no defect in resolution was found when intact B6 LPR mice or T cell specific Fas-conditional knockout mice were sensitized and challenged. We hypothesize that the homeostatic proliferation induced by adoptive transfer of T cells into Rag-deficient mice may be an important mechanism involved in the lack of resolution. To investigate the role of homeostatic proliferation, we induced lymphopenia in the T cell-specific Fas-conditional knockout mice by non-lethal irradiation and sensitized them when T cells began to repopulate. Interestingly, we found that defective Fas signaling on T cells plus antigen exposure during homeostatic proliferation was sufficient to induce prolonged eosinophilic airway inflammation. In conclusion, our data show that the combination of transient lymphopenia, abnormal Fas-signaling, and antigen exposure leads to the development of a prolonged airway eosinophilic inflammatory phase in our mouse model of experimental asthma.

Non-apoptotic Fas (CD95) Signaling on T Cells Regulates the Resolution of Th2-Mediated Inflammation.

Fas (CD95/APO-1) and its ligand (FasL/CD95L) promote the resolution of type 2 lung inflammation and eosinophilia. We previously found that Fas-deficiency on T cells, but not eosinophils, delayed resolution of inflammation. However, Fas can signal both cell death and have a positive signaling function that can actually activate cells. In this study, we investigated whether Fas-induced death or Fas-activated signaling pathways promote resolution of allergic lung inflammation. By increasing T cell survival through two Fas-independent pathways, using Bim-deficient T cells or Bcl-xL overexpressing T cells, no differences in resolution of Th2-mediated inflammation was observed. Furthermore, Th2 cells were inherently resistant to Fas-mediated apoptosis and preferentially signaled through non-apoptotic pathways following FasL treatment. Utilizing Fas-mutant mice deficient in apoptotic but sufficient for non-apoptotic Fas signaling pathways, we demonstrate that non-apoptotic Fas signaling in T cells drives resolution of Th2-mediated airway inflammation. Our findings reveal a previously unknown role for non-apoptotic Fas signaling on Th2 cells in the induction of resolution of type 2 inflammation.

The Metabolic Sensor GPR43 Receptor Plays a Role in the Control of Klebsiella pneumoniae Infection in the Lung.

Pneumonia is one of the leading causes of death and mortality worldwide. The inflammatory responses that follow respiratory infections are protective leading to pathogen clearance but can also be deleterious if unregulated. The microbiota is known to be an important protective barrier against infections, mediating both direct inhibitory effects against the potential pathogen and also regulating the immune responses contributing to a proper clearance of the pathogen and return to homeostasis. GPR43 is one receptor for acetate, a microbiota metabolite shown to induce and to regulate important immune functions. Here, we addressed the role of GPR43 signaling during pulmonary bacterial infections. We have shown for the first time that the absence of GPR43 leads to increased susceptibility to Klebsiella pneumoniae infection, which was associated to both uncontrolled proliferation of bacteria and to increased inflammatory response. Mechanistically, we showed that GPR43 expression especially in neutrophils and alveolar macrophages is important for bacterial phagocytosis and killing. In addition, treatment with the GPR43 ligand, acetate, is protective during bacterial lung infection. This was associated to reduction in the number of bacteria in the airways and to the control of the inflammatory responses. Altogether, GPR43 plays an important role in the "gut-lung axis" as a sensor of the host gut microbiota activity through acetate binding promoting a proper immune response in the lungs.

Heme crystallization in a Chagas disease vector acts as a redox-protective mechanism to allow insect reproduction and parasite infection.

Heme crystallization as hemozoin represents the dominant mechanism of heme disposal in blood feeding triatomine insect vectors of the Chagas disease. The absence of drugs or vaccine for the Chagas disease causative agent, the parasite Trypanosoma cruzi, makes the control of vector population the best available strategy to limit disease spread. Although heme and redox homeostasis regulation is critical for both triatomine insects and T. cruzi, the physiological relevance of hemozoin for these organisms remains unknown. Here, we demonstrate that selective blockage of heme crystallization in vivo by the antimalarial drug quinidine, caused systemic heme overload and redox imbalance in distinct insect tissues, assessed by spectrophotometry and fluorescence microscopy. Quinidine treatment activated compensatory defensive heme-scavenging mechanisms to cope with excessive heme, as revealed by biochemical hemolymph analyses, and fat body gene expression. Importantly, egg production, oviposition, and total T. cruzi parasite counts in R. prolixus were significantly reduced by quinidine treatment. These effects were reverted by oral supplementation with the major insect antioxidant urate. Altogether, these data underscore the importance of heme crystallization as the main redox regulator for triatomine vectors, indicating the dual role of hemozoin as a protective mechanism to allow insect fertility, and T. cruzi life-cycle. Thus, targeting heme crystallization in insect vectors represents an innovative way for Chagas disease control, by reducing simultaneously triatomine reproduction and T. cruzi transmission.

Prevention of changes in airway function facilitates Strongyloides venezuelensis infection in rats.

Alterations in lung function and pulmonary symptoms have been described in patients infected with helminths with a lung cycle. We have previously shown that infection with the nematode Strongyloides venezuelensis induced a significant increase in airway hyperreactivity in infected rats. The aim of the present study was to test the hypothesis that bronchodilation during the lung phase of parasite migration would favor completion of the life cycle and infection indices. For this purpose, S. venezuelensis infected rats were treated with salbutamol during the first 48 h after the nematode infection. At the dose used (0.25 mg/mL for 10 min every 4 h), treatment with salbutamol prevented changes in lung function during the parasite migration. This was accompanied by a significant increase in parasite burden, as assessed in the lung and the small intestine. Parasite infected and salbutamol-treated animals also showed a significant increase in concentration of IL-10 and IL-4 in homogenates of lungs during the worm migration that was followed by stronger lung eosinophilic inflammation at 5 dpi, after the larvae had left the host lung. Our data indicates that airway hyperactivity reduce parasite progression through the lung, facilitating the action of innate or adaptive immune mechanisms.

Influence of Oral and Gut Microbiota in the Health of Menopausal Women.

Sex differences in gut microbiota are acknowledged, and evidence suggests that gut microbiota may have a role in higher incidence and/or severity of autoimmune diseases in females. Additionally, it has been suggested that oral, vaginal, and gut microbiota composition can be regulated by estrogen levels. The association of vaginal microbiota with vulvovaginal atrophy at menopause is well described in the literature. However, the relevance of oral and gut microbiota modulation in the immune system during estrogen deficiency and its effect on inflammatory diseases is not well explored. Estrogen deficiency is a condition that occurs in menopausal women, and it can last approximately 30 years of a woman's life. The purpose of this mini- review is to highlight the importance of alterations in the oral and gut microbiota during estrogen deficiency and their effect on oral and inflammatory diseases that are associated with menopause. Considering that hormone replacement therapy is not always recommended or sufficient to prevent or treat menopause-related disease, we will also discuss the use of probiotics and prebiotics as an option for the prevention or treatment of these diseases.

Prophylactic Supplementation of Bifidobacterium longum 51A Protects Mice from Ovariectomy-Induced Exacerbated Allergic Airway Inflammation and Airway Hyperresponsiveness.

Asthma is a chronic inflammatory disease that affects more females than males after puberty, and its symptoms and severity in women change during menstruation and menopause. Recently, evidence has demonstrated that interactions among the microbiota, female sex hormones, and immunity are associated with the development of autoimmune diseases. However, no studies have investigated if therapeutic gut microbiota modulation strategies could affect asthma exacerbation during menstruation and menopause. Here we aimed to examine the preventive effects of a probiotic, Bifidobacterium longum 51A, on airway inflammation exacerbation in allergic ovariectomized mice. We first evaluated the gut microbiota composition and diversity in mice 10 days after ovariectomy. Next, we examined whether re-exposure of ovariectomized allergic mice to antigen (ovalbumin) would lead to exacerbation of lung inflammation. Finally, we evaluated the preventive and treatment effect of B. longum 51A on lung inflammation and airway hyperresponsiveness. Our results showed that whereas ovariectomy caused no alterations in the gut microbiota composition and diversity in this animal model, 10 days after ovariectomy, preventive use administration of B. longum 51A, rather than its use after surgery was capable of attenuate the exacerbated lung inflammation and hyperresponsiveness in ovariectomized allergic mice. This prophylactic effect of B. longum 51A involves acetate production, which led to increased fecal acetate levels and, consequently, increased Treg cells in ovariectomized allergic mice.