In vivo chronic exposure to inorganic mercury worsens hypercholesterolemia, oxidative stress and atherosclerosis in the LDL receptor knockout mice.

Heavy metal exposure leads to multiple system dysfunctions. The mechanisms are likely multifactorial and involve inflammation and oxidative stress. The aim of this study was to evaluate markers and risk factors for atherosclerosis in the LDL receptor knockout mouse model chronically exposed to inorganic mercury (Hg) in the drinking water. Results revealed that Hg exposed mice present increased plasma levels of cholesterol, without alterations in glucose. As a major source and target of oxidants, we evaluated mitochondrial function. We found that liver mitochondria from Hg treated mice show worse respiratory control, lower oxidative phosphorylation efficiency and increased H2O2 release. In addition, Hg induced mitochondrial membrane permeability transition. Erythrocytes from Hg treated mice showed a 50% reduction in their ability to take up oxygen, lower levels of reduced glutathione (GSH) and of antioxidant enzymes (SOD, catalase and GPx). The Hg treatment disturbed immune system cells counting and function. While lymphocytes were reduced, monocytes, eosinophils and neutrophils were increased. Peritoneal macrophages from Hg treated mice showed increased phagocytic activity. Hg exposed mice tissues present metal impregnation and parenchymal architecture alterations. In agreement, increased systemic markers of liver and kidney dysfunction were observed. Plasma, liver and kidney oxidative damage indicators (MDA and carbonyl) were increased while GSH and thiol groups were diminished by Hg exposure. Importantly, atherosclerotic lesion size in the aorta root of Hg exposed mice were larger than in controls. In conclusion, in vivo chronic exposure to Hg worsens the hypercholesterolemia, impairs mitochondrial bioenergetics and redox function, alters immune cells profile and function, causes several tissues oxidative damage and accelerates atherosclerosis development.

Microglial phagolysosome dysfunction and altered neural communication amplify phenotypic severity in Prader-Willi Syndrome with larger deletion.

Prader-Willi Syndrome (PWS) is a rare neurodevelopmental disorder of genetic etiology, characterized by paternal deletion of genes located at chromosome 15 in 70% of cases. Two distinct genetic subtypes of PWS deletions are characterized, where type I (PWS T1) carries four extra haploinsufficient genes compared to type II (PWS T2). PWS T1 individuals display more pronounced physiological and cognitive abnormalities than PWS T2, yet the exact neuropathological mechanisms behind these differences remain unclear. Our study employed postmortem hypothalamic tissues from PWS T1 and T2 individuals, conducting transcriptomic analyses and cell-specific protein profiling in white matter, neurons, and glial cells to unravel the cellular and molecular basis of phenotypic severity in PWS sub-genotypes. In PWS T1, key pathways for cell structure, integrity, and neuronal communication are notably diminished, while glymphatic system activity is heightened compared to PWS T2. The microglial defect in PWS T1 appears to stem from gene haploinsufficiency, as global and myeloid-specific Cyfip1 haploinsufficiency in murine models demonstrated. Our findings emphasize microglial phagolysosome dysfunction and altered neural communication as crucial contributors to the severity of PWS T1's phenotype.

Impact of Microbiota Depletion by Antibiotics on SARS-CoV-2 Infection of K18-hACE2 Mice.

Clinical and experimental data indicate that severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infection is associated with significant changes in the composition and function of intestinal microbiota. However, the relevance of these effects for SARS-CoV-2 pathophysiology is unknown. In this study, we analyzed the impact of microbiota depletion after antibiotic treatment on the clinical and immunological responses of K18-hACE2 mice to SARS-CoV-2 infection. Mice were treated with a combination of antibiotics (kanamycin, gentamicin, metronidazole, vancomycin, and colistin, Abx) for 3 days, and 24 h later, they were infected with SARS-CoV-2 B lineage. Here, we show that more than 80% of mice succumbed to infection by day 11 post-infection. Treatment with Abx had no impact on mortality. However, Abx-treated mice presented better clinical symptoms, with similar weight loss between infected-treated and non-treated groups. We observed no differences in lung and colon histopathological scores or lung, colon, heart, brain and kidney viral load between groups on day 5 of infection. Despite some minor differences in the expression of antiviral and inflammatory markers in the lungs and colon, no robust change was observed in Abx-treated mice. Together, these findings indicate that microbiota depletion has no impact on SARS-CoV-2 infection in mice.

STING regulates metabolic reprogramming in macrophages via HIF-1α during Brucella infection.

Macrophages metabolic reprogramming in response to microbial insults is a major determinant of pathogen growth or containment. Here, we reveal a distinct mechanism by which stimulator of interferon genes (STING), a cytosolic sensor that regulates innate immune responses, contributes to an inflammatory M1-like macrophage profile upon Brucella abortus infection. This metabolic reprogramming is induced by STING-dependent stabilization of hypoxia-inducible factor-1 alpha (HIF-1α), a global regulator of cellular metabolism and innate immune cell functions. HIF-1α stabilization reduces oxidative phosphorylation and increases glycolysis during infection with B. abortus and, likewise, enhances nitric oxide production, inflammasome activation and IL-1ß release in infected macrophages. Furthermore, the induction of this inflammatory profile participates in the control of bacterial replication since absence of HIF-1α renders mice more susceptible to B. abortus infection. Mechanistically, activation of STING by B. abortus infection drives the production of mitochondrial reactive oxygen species (mROS) that ultimately influences HIF-1α stabilization. Moreover, STING increases the intracellular succinate concentration in infected macrophages, and succinate pretreatment induces HIF-1α stabilization and IL-1ß release independently of its cognate receptor GPR91. Collectively, these data demonstrate a pivotal mechanism in the immunometabolic regulation of macrophages during B. abortus infection that is orchestrated by STING via HIF-1α pathway and highlight the metabolic reprogramming of macrophages as a potential treatment strategy for bacterial infections.

Prostate immunology: A challenging puzzle.

Mucosal immunity defines the relationship of surfaces in contact with the environment and integrates diverse tissues such as epidermis, gum, nose, gut, uterus and prostate with the immune system. Although considered part of a system, each mucosa presents specific immune features beyond the barrier and secretory functions. Information regarding the mucosal immunology of the male reproductive tract and the prostate gland in particular is scarce. In this review, we approach the prostate as an epithelial barrier and as part of the mucosal immune system. Finally, we also raise a series of questions that will improve the understanding of this gland, its role in reproduction and its sensitivity/resistance to disease.

Using flow cytometry for mitochondrial assays.

The understanding of how different cell types adapt their metabolism in the face of challenges has been attracting the attention of researchers for many years. Recently, immunologists also started to focus on how the metabolism of immune cells can impact the way that immunity drives its responses. The presence of a pathogen or damage in a tissue changes severely the way that the immune cells need to respond. When activated, immune cells usually shift their metabolism from a high energy demanding status using mitochondria respiration to a glycolytic based rapid ATP production. The diminished amount of respiration leads to changes in the mitochondrial membrane potential and, consequently, generation of reactive oxygen species. Here, we show how flow cytometry can be used to track changes in mitochondrial mass, membrane potential and superoxide (ROS) production in live immune cells. ● This protocol suggests a quick way of evaluating mitochondrial fitness using flow cytometry. We propose using the probes MitoTraker Green and MitoTracker Red/ MitoSOX at the same time. This way, it is possible to evaluate different parameters of mitochondrial biology in living cells. ● Flow cytometry is a highly used tool by immunologists. With the advances of studies focusing on the metabolism of immune cells, a simplified application of flow cytometry for mitochondrial studies and screenings is a helpful clarifying method for immunology.

Ghrelin effects on mitochondrial fitness modulates macrophage function.

Over the past years, systemic derived cues that regulate cellular metabolism have been implicated in the regulation of immune responses. Ghrelin is an orexigenic hormone produced by enteroendocrine cells in the gastric mucosa with known immunoregulatory roles. The mechanism behind the function of ghrelin in immune cells, such as macrophages, is still poorly understood. Here, we explored the hypothesis that ghrelin leads to alterations in macrophage metabolism thus modulating macrophage function. We demonstrated that ghrelin exerts an immunomodulatory effect over LPS-activated peritoneal macrophages, as evidenced by inhibition of TNF-α and IL-1ß secretion and increased IL-12 production. Concomitantly, ghrelin increased mitochondrial membrane potential and increased respiratory rate. In agreement, ghrelin prevented LPS-induced ultrastructural damage in the mitochondria. Ghrelin also blunted LPS-induced glycolysis. In LPS-activated macrophages, glucose deprivation did not affect ghrelin-induced IL-12 secretion, whereas the inhibition of pyruvate transport and mitochondria-derived ATP abolished ghrelin-induced IL-12 secretion, indicating a dependence on mitochondrial function. Ghrelin pre-treatment of metabolic activated macrophages inhibited the secretion of TNF-α and enhanced IL-12 levels. Moreover, ghrelin effects on IL-12, and not on TNF-α, are dependent on mitochondria elongation, since ghrelin did not enhance IL-12 secretion in metabolic activated mitofusin-2 deficient macrophages. Thus, ghrelin affects macrophage mitochondrial metabolism and the subsequent macrophage function.

Mitoimmunity-when mitochondria dictates macrophage function.

In the past decade, several reports have appointed the importance of mitochondria in the immune response. Our understanding of mitochondria evolved from a simple supplier of energy into a platform necessary for immunorregulation. Proinflammatory responses are associated with enhanced glycolytic activity and breakdown of the TCA cycle. Mitochondrial reactive species of oxygen (mROS) are key regulators of classically activated macrophages, with substantial impact in the anti-microbicidal activity and pro-inflammatory cytokine secretion of macrophages. The inflammasome activation in macrophages is dependent on mROS production and mitochondrial regulation and mitochondrial dynamics and functionality direct impact inflammatory responses. Alternative activated macrophage metabolism relies on fatty acid oxidation, and the mechanism responsible for this phenotype is not fully elucidated. Thus, cellular metabolism and mitochondria function is a key immunoregulatory feature of macrophage biology. In this review, we will provide insights into recently reported evidences of mitochondria-related metabolic nodes, which are important for macrophage physiology.

Leptin deficiency impairs maturation of dendritic cells and enhances induction of regulatory T and Th17 cells.

Leptin is an adipose-secreted hormone that plays an important role in both metabolism and immunity. Leptin has been shown to induce Th1-cell polarization and inhibit Th2-cell responses. Additionally, leptin induces Th17-cell responses, inhibits regulatory T (Treg) cells and modulates autoimmune diseases. Here, we investigated whether leptin mediates its activity on T cells by influencing dendritic cells (DCs) to promote Th17 and Treg-cell immune responses in mice. We observed that leptin deficiency (i) reduced the expression of DC maturation markers, (ii) decreased DC production of IL-12, TNF-α, and IL-6, (iii) increased DC production of TGF-ß, and (iv) limited the capacity of DCs to induce syngeneic CD4(+) T-cell proliferation. As a consequence of this unique phenotype, DCs generated under leptin-free conditions induced Treg or TH 17 cells more efficiently than DCs generated in the presence of leptin. These data indicate important roles for leptin in DC homeostasis and the initiation and maintenance of inflammatory and regulatory immune responses by DCs.

Exercise and caloric restriction alter the immune system of mice submitted to a high-fat diet.

As the size of adipocytes increases during obesity, the establishment of resident immune cells in adipose tissue becomes an important source of proinflammatory mediators. Exercise and caloric restriction are two important, nonpharmacological tools against body mass increase. To date, their effects on the immune cells of adipose tissue in obese organisms, specifically when a high-fat diet is consumed, have been poorly investigated. Thus, after consuming a high-fat diet, mice were submitted to chronic swimming training or a 30% caloric restriction in order to investigate the effects of both interventions on resident immune cells in adipose tissue. These strategies were able to reduce body mass and resulted in changes in the number of resident immune cells in the adipose tissue and levels of cytokines/chemokines in serum. While exercise increased the number of NK cells in adipose tissue and serum levels of IL-6 and RANTES, caloric restriction increased the CD4+/CD8+ cell ratio and MCP-1 levels. Together, these data demonstrated that exercise and caloric restriction modulate resident immune cells in adipose tissues differently in spite of an equivalent body weight reduction. Additionally, the results also reinforce the idea that a combination of both strategies is better than either individually for combating obesity.

Immune regulatory properties of allogeneic adipose-derived mesenchymal stem cells in the treatment of experimental autoimmune diabetes.

Adipose-derived mesenchymal stem cells (ADMSCs) display immunosuppressive properties, suggesting a promising therapeutic application in several autoimmune diseases, but their role in type 1 diabetes (T1D) remains largely unexplored. The aim of this study was to investigate the immune regulatory properties of allogeneic ADMSC therapy in T cell-mediated autoimmune diabetes in NOD mice. ADMSC treatment reversed the hyperglycemia of early-onset diabetes in 78% of diabetic NOD mice, and this effect was associated with higher serum insulin, amylin, and glucagon-like peptide 1 levels compared with untreated controls. This improved outcome was associated with downregulation of the CD4(+) Th1-biased immune response and expansion of regulatory T cells (Tregs) in the pancreatic lymph nodes. Within the pancreas, inflammatory cell infiltration and interferon-γ levels were reduced, while insulin, pancreatic duodenal homeobox-1, and active transforming growth factor-ß1 expression were increased. In vitro, ADMSCs induced the expansion/proliferation of Tregs in a cell contact-dependent manner mediated by programmed death ligand 1. In summary, ADMSC therapy efficiently ameliorates autoimmune diabetes pathogenesis in diabetic NOD mice by attenuating the Th1 immune response concomitant with the expansion/proliferation of Tregs, thereby contributing to the maintenance of functional ß-cells. Thus, this study may provide a new perspective for the development of ADMSC-based cellular therapies for T1D.

Preserving the B-cell compartment favors operational tolerance in human renal transplantation.

Transplanted individuals in operational tolerance (OT) maintain long-term stable graft function after completely stopping immunosuppression. Understanding the mechanisms involved in OT can provide valuable information about pathways to human transplantation tolerance. Here we report that operationally tolerant individuals display quantitative and functional preservation of the B-cell compartment in renal transplantation. OT exhibited normal numbers of circulating total B cells, naive, memory and regulatory B cells (Bregs) as well as preserved B-cell receptor repertoire, similar to healthy individuals. In addition, OT also displayed conserved capacity to activate the cluster of differentiation 40 (CD40)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in Bregs, in contrast, with chronic rejection. Rather than expansion or higher activation, we show that the preservation of the B-cell compartment favors OT.