B Cell Immunosenescence.

Innate and adaptive immune responses decline with age, leading to greater susceptibility to infectious diseases and reduced responses to vaccines. Diseases are more severe in old than in young individuals and have a greater impact on health outcomes such as morbidity, disability, and mortality. Aging is characterized by increased low-grade chronic inflammation, so-called inflammaging, that represents a link between changes in immune cells and a number of diseases and syndromes typical of old age. In this review we summarize current knowledge on age-associated changes in immune cells with special emphasis on B cells, which are more inflammatory and less responsive to infections and vaccines in the elderly. We highlight recent findings on factors and pathways contributing to inflammaging and how these lead to dysfunctional immune responses. We summarize recent published studies showing that adipose tissue, which increases in size with aging, contributes to inflammaging and dysregulated B cell function.

Age-related factors that affect B cell responses to vaccination in mice and humans.

Aging significantly changes the ability to respond to vaccinations and infections. In this review, we summarize published results on age-related changes in response to infection with the influenza virus and on the factors known to increase influenza risk infection leading to organ failure and death. We also summarize how aging affects the response to the influenza vaccine with a special focus on B cells, which have been shown to be less responsive in the elderly. We show the cellular and molecular mechanisms contributing to the dysfunctional immune response of the elderly to the vaccine against influenza. These include a defective interaction of helper T cells (CD4+) with B cells in germinal centers, changes in the microenvironment, and the generation of immune cells with a senescence-associated phenotype. Finally, we discuss the effects of aging on metabolic pathways and we show how metabolic complications associated with aging lead to immune dysfunction.

Transcriptomic assessment of dietary fishmeal partial replacement by soybean meal and prebiotics inclusion in the liver of juvenile Pacific yellowtail (Seriola lalandi).

BACKGROUND: Seriola lalandi is an important species for aquaculture, due to its rapid growth, adaptation to captivity and formulated diets, and high commercial value. Due to the rise in fishmeal (FM) price, efforts have been and still are made to replace it partially or entirely with vegetable meals in diets for carnivorous fish. The use of prebiotics when feeding vegetable meals has improved fish health. METHODS: Four experimental diets were assessed in juveniles, the control diet consisted of FM as the main protein source, the second diet included 2% of GroBiotic®-A (FM-P), in the third diet FM was partially replaced (25%) by soybean meal (SM25), and the fourth consisted of SM25 with 2% of GroBiotic®-A (SM25-P). Growth was evaluated and RNA-seq of the liver tissue was performed, including differential expression analysis and functional annotation to identify genes affected by the diets. RESULTS: Growth was not affected by this level of FM replacement, but it was improved by prebiotics. Annotation was achieved for 59,027 transcripts. Gene expression was affected by the factors: 225 transcripts due to FM replacement, 242 due to prebiotics inclusion, and 62 due to the interaction of factors. The SM25-P diet showed the least amount of differentially expressed genes against the control diet. CONCLUSION: The replacement of FM (25%) by soybean meal combined with prebiotics (2%) represents a good cost-benefit balance for S. lalandi juveniles since the fish growth increased and important metabolic and immune system genes in the liver were upregulated with this diet.

Hyper-metabolic B cells in the spleens of old mice make antibodies with autoimmune specificities.

BACKGROUND: Aging is associated with increased intrinsic B cell inflammation, decreased protective antibody responses and increased autoimmune antibody responses. The effects of aging on the metabolic phenotype of B cells and on the metabolic programs that lead to the secretion of protective versus autoimmune antibodies are not known. METHODS: Splenic B cells and the major splenic B cell subsets, Follicular (FO) and Age-associated B cells (ABCs), were isolated from the spleens of young and old mice and left unstimulated. The RNA was collected to measure the expression of markers associated with intrinsic inflammation and autoimmune antibody production by qPCR. B cells and B cell subsets were also stimulated with CpG and supernatants collected after 7 days to measure autoimmune IgG secretion by ELISA. Metabolic measures (oxygen consumption rate, extracellular acidification rate and glucose uptake) were performed using a Seahorse XFp extracellular flux analyzer. RESULTS: Results have identified the subset of ABCs, whose frequencies and numbers increase with age and represent the most pro-inflammatory B cell subset, as the cell type mainly if not exclusively responsible for the expression of inflammatory markers and for the secretion of autoimmune antibodies in the spleen of old mice. Hyper-inflammatory ABCs from old mice are also hyper-metabolic, as compared to those from young mice and to the subset of FO B cells, a feature needed not only to support their higher expression of RNA for inflammatory markers but also their higher autoimmune antibody secretion. CONCLUSIONS: These results identify a relationship between intrinsic inflammation, metabolism and autoimmune B cells and suggest possible ways to understand cellular mechanisms that lead to the generation of pathogenic B cells, that are hyper-inflammatory and hyper-metabolic, and secrete IgG antibodies with autoimmune specificities.

B Cells with a Senescent-Associated Secretory Phenotype Accumulate in the Adipose Tissue of Individuals with Obesity.

Senescent cells accumulate in the adipose tissue (AT) of individuals with obesity and secrete multiple factors that constitute the senescence-associated secretory phenotype (SASP). This paper aimed at the identification of B cells with a SASP phenotype in the AT, as compared to the peripheral blood, of individuals with obesity. Our results show increased expression of SASP markers in AT versus blood B cells, a phenotype associated with a hyper-metabolic profile necessary to support the increased immune activation of AT-derived B cells as compared to blood-derived B cells. This hyper-metabolic profile is needed for the secretion of the pro-inflammatory mediators (cytokines, chemokines, micro-RNAs) that fuel local and systemic inflammation.

Single cell analyses reveal specific distribution of anti-bacterial molecule Perforin-2 in human skin and its modulation by wounding and Staphylococcus aureus infection.

Perforin-2 (P-2) is a recently described antimicrobial protein with unique properties to kill intracellular bacteria. We investigated P-2 expression pattern and cellular distribution in human skin and its importance in restoration of barrier function during wound healing process and infection with the common wound pathogen Staphylococcus aureus. We describe a novel approach for the measurement of P-2 mRNA within individual skin cells using an amplified fluorescence in situ hybridization (FISH) technique. The unique aspect of this approach is simultaneous detection of P-2 mRNA in combination with immune-phenotyping for cell surface proteins using fluorochrome-conjugated antibodies. We detected P-2 transcript in both hematopoietic (CD45+ ) and non-hematopoietic (CD45- ) cutaneous cell populations, confirming the P-2 expression in both professional and non-professional phagocytes. Furthermore, we found an induction of P-2 during wound healing. P-2 overexpression resulted in a reduction of intracellular S. aureus, while infection of human wounds by this pathogen resulted in P-2 suppression, revealing a novel mechanism by which S. aureus may escape cutaneous immunity to cause persistent wound infections.

Increased IL-17-expressing γδ T cells in seborrhoeic dermatitis-like lesions of the Mpzl3 knockout mice.

Seborrhoeic Dermatitis (SD) is a common inflammatory skin disorder, but its molecular pathogenesis remains elusive. Previously, we have established the Mpzl3 knockout (-/-) mice as a model for SD. In this study, we focused on early phases of skin inflammation and determined the cytokine profiles and identified immune cell types in the lesional skin in the Mpzl3 -/- mice. Using flow cytometry, we detected a significant increase of CD45+ leucocytes, CD3+ T lymphocytes and especially γδ T cells but not αß T cells in the lesional skin compared to control. We also detected high levels of IL-17 and determined that the γδ T cells were a major contributing source. CD3+ and γδ T cell localization in the skin was verified by indirect immunofluorescent staining. Since neither γδ T cells nor IL-17 had been implicated in SD, our study provides novel insights into the role of MPZL3 in the pathogenesis of SD-like skin inflammation.

Adipocyte-derived inflammatory molecules induce senescent B cells through metabolic pathways.

OBJECTIVE: The aim of this study was to demonstrate that an adipocyte tissue-derived conditioned medium (ACM) contains inflammatory molecules that induce senescence in B cells. METHODS: We incubated blood-derived B cells from lean donors with ACM obtained from the adipose tissue of adult female donors with obesity undergoing weight reduction surgery or with medium as control. After 24 h, cells were harvested, and the expression of transcripts for proinflammatory cytokines (TNF/IL-6), chemokines (IL-8), and for markers of the senescence-associated secretory phenotype (SASP) was measured by quantitative polymerase chain reaction. B cells were also stained with the marker of immunosenescence ß-galactosidase, and their metabolic status was evaluated in Seahorse using a Mito Stress Test. RESULTS: We show that the incubation of B cells from lean donors with ACM induces the expression of transcripts for inflammatory and SASP transcripts, increases the amount of ß-galactosidase staining, and induces a metabolic phenotype characterized by higher basal and maximal oxygen consumption, spare respiratory capacity (difference between maximal and basal respiration), nonmitochondrial oxygen consumption, ATP production, and proton leak. CONCLUSIONS: These results demonstrate that B cells from lean individuals, after incubation with ACM, become inflammatory and senescent, and this occurs through metabolic pathways needed to support their secretory phenotype.

Immunometabolic effects of lactate on B cell function in healthy individuals of different ages.

Aging is characterized by chronic systemic inflammation and metabolic changes. When we compared B cells from young and elderly donors, we found that aging induces higher oxygen consumption rates, and especially higher extracellular acidification rates, measures of oxidative phosphorylation and of anaerobic glycolysis, respectively. Importantly, this higher metabolic status, which reflects the age-associated expansion of pro-inflammatory B cell subsets, was found associated with higher secretion of lactate and autoimmune antibodies after in vitro stimulation. B cells from elderly individuals, induce in vitro generation of pro-inflammatory CD4+ T cells from young individuals through metabolic pathways mediated by lactate secretion. Lactate also induces immunosenescent B cells that are glycolytic and express transcripts for multiple pro-inflammatory molecules. These results altogether may have relevant clinical implications and suggest novel targets for therapeutic interventions in patients with inflammatory conditions and diseases.

B cells from old mice induce the generation of inflammatory T cells through metabolic pathways.

We have measured the capacity of B cells from young and old mice to induce the differentiation of naïve CD4 + T cells from young mice into pro-inflammatory subsets. We found that only B cells from old mice are inflammatory and induce in vitro secretion of the pro-inflammatory cytokines IL-17A and IFN-γ by T cells. In co-culture experiments, B cells from old mice showed a strong helper function on T cells from young mice, making them pro-inflammatory, and this effect is regulated by metabolic pathways, mainly anaerobic glycolysis, leading to increased RNA expression of the enzyme lactate dehydrogenase (LDHA) and increased secretion of lactate. These results have indicated that lactate is a crucial player of the B cell-induced polarization of T cells. When we measured the effects of lactate on isolated CD4 + T cells from young mice, we found that lactate increases RNA expression of LDHA, secretion of pro-inflammatory cytokines and NF-kB activation. Moreover, lactate effects in culture can be abrogated in the presence of the specific inhibitor of LDHA, FX11. These results altogether may have relevant clinical implications and suggest novel targets for therapeutic interventions in patients with inflammatory conditions and diseases.

Phenotypic and functional features of B cells from two different human subcutaneous adipose depots.

In this study, we have compared frequencies, phenotype, function and metabolic requirements of B cells isolated from the breast and abdominal subcutaneous adipose tissue (AT) of women with obesity who underwent weight reduction surgeries. Results show that B cells from the abdominal AT are more inflammatory than those from the breast, characterized by higher frequencies of inflammatory B cell subsets and higher expression of RNA for inflammatory markers associated with senescence. Secretion of autoimmune antibodies is also higher in the abdominal AT as compared to the breast, and is associated with higher frequencies of autoimmune B cells with the membrane phenotype CD21lowCD95+ B cells expressing the transcription factor T-bet. Moreover, glucose uptake is higher in B cells from the abdominal AT as compared to the breast, thereby suggesting a better capacity to perform glycolysis, needed to support intrinsic B cell inflammation and autoimmune antibody secretion.

Induction of antigen specific intrahepatic CD8+ T cell responses by a secreted heat shock protein based gp96-Ig-PfCA malaria vaccine.

Introduction: A highly efficacious and durable vaccine against malaria is an essential tool for global malaria eradication. One of the promising strategies to develop such a vaccine is to induce robust CD8+ T cell mediated immunity against malaria liver-stage parasites. Methods: Here we describe a novel malaria vaccine platform based on a secreted form of the heat shock protein, gp96-immunoglobulin, (gp96-Ig) to induce malaria antigen specific, memory CD8+ T cells. Gp96-Ig acts as an adjuvant to activate antigen presenting cells (APCs) and chaperone peptides/antigens to APCs for cross presentation to CD8+ T cells. Results: Our study shows that vaccination of mice and rhesus monkeys with HEK-293 cells transfected with gp96-Ig and two well-known Plasmodium falciparum CSP and AMA1 (PfCA) vaccine candidate antigens, induces liver-infiltrating, antigen specific, memory CD8+ T cell responses. The majority of the intrahepatic CSP and AMA1 specific CD8+ T cells expressed CD69 and CXCR3, the hallmark of tissue resident memory T cells (Trm). Also, we found intrahepatic, antigen-specific memory CD8+ T cells secreting IL-2, which is relevant for maintenance of effective memory responses in the liver. Discussion: Our novel gp96-Ig malaria vaccine strategy represents a unique approach to induce liver-homing, antigen-specific CD8+ T cells critical for Plasmodium liver-stage protection.

Obesity Accelerates Age-Associated Defects in Human B Cells Through a Metabolic Reprogramming Induced by the Fatty Acid Palmitate.

We have measured the secretion of autoimmune antibodies in plasma samples and in culture supernatants of blood-derived B cells from four groups of individuals: young lean (YL), elderly lean (EL), young obese (YO) and elderly obese (EO). We found secretion comparable in YO and EL individuals, suggesting that obesity accelerates age-associated defects in circulating B cells. To define at least one possible molecular pathway involved, we used an in vitro model in which B cells from YL and EL individuals have been stimulated with the Fatty Acid (FA) palmitate, the most common saturated FA in the human body. The rationale to use palmitate is that there is a chronic increase in circulating levels of palmitate, due to increased spontaneous lipolysis occurring during aging and obesity, and this may induce autoimmune B cells. Results herein show that in vitro incubation of B cells from YL and EL individuals with the FA palmitate induces mRNA expression of T-bet, the transcription factor for autoimmune antibodies, as well as secretion of autoimmune IgG antibodies, with B cells from YL individuals looking similar to B cells from EL individuals, confirming our initial hypothesis. The generation of autoimmune B cells in the presence of the FA palmitate was found to be associated with a metabolic reprogramming of B cells from both YL and EL individuals. These results altogether show the critical role of the FA palmitate in inducing human B cell immunosenescence and show for the first time the importance of metabolic pathways in this process.

Effects of cellular senescence on metabolic pathways in non-immune and immune cells.

Many cellular stresses induce cellular senescence and the irreversible arrest of cell proliferation in different cell types. Although blocked in their capacity to divide, senescent cells are metabolically active and are characterized by a different metabolic phenotype as compared to non-senescent cells. Changes observed in senescent cells depend from the cell type and lead to an adaptative flexibility in the type of metabolism. This metabolic reprogramming is needed to cope with survival and with the energetic demands of the senescent program that include the increased secretion of senescence-associated secretory phenotype factors.

Corrigendum: Staphylococcus epidermidis Boosts Innate Immune Response by Activation of Gamma Delta T Cells and Induction of Perforin-2 in Human Skin.

[This corrects the article DOI: 10.3389/fimmu.2020.550946.].

Identification and Characterization of Adipose Tissue-Derived Human Antibodies With "Anti-self" Specificity.

We have previously shown that the human obese adipose tissue (AT) contributes to increased secretion of adipocyte-specific IgG antibodies in individuals with obesity. This occurs without any exogenous stimulation, because the ongoing process of cell death in the obese AT leads to the release of "self" antigens able to induce chronic stimulation of B cells. We have identified several mechanisms responsible for the release of "self" antigens, such as hypoxia, cell cytotoxicity, and DNA damage. In this paper, we confirm and extend our initial observation on a different cohort of individuals, and we show that also the plasma of these individuals is enriched in IgG antibodies with specificities for adipocyte-derived antigens. Adipocyte-specific IgG secreted in the obese AT are significantly correlated with those present in plasma. Using immunoprecipitation and mass spectrometry, we have identified these antigenic specificities. The antigens are almost exclusively intracellular or cell-associated, usually not recognized as "self" antigens, but they are released by cells dying in the AT. We also show for the first time that the adipocytes in the obese AT contribute to the secretion of IgG autoimmune antibodies and this seems to be due to their expression of the antigen-presenting molecules CD1d and, to a much lesser extent, MHC class II, as our mechanistic experiments performed in mice have shown. These results may lead to the development of novel therapeutic strategies to control autoimmunity.

Staphylococcus epidermidis Boosts Innate Immune Response by Activation of Gamma Delta T Cells and Induction of Perforin-2 in Human Skin.

Perforin-2 (P-2) is an antimicrobial protein with unique properties to kill intracellular bacteria. Gamma delta (GD) T cells, as the major T cell population in epithelial tissues, play a central role in protective and pathogenic immune responses in the skin. However, the tissue-specific mechanisms that control the innate immune response and the effector functions of GD T cells, especially the cross-talk with commensal organisms, are not very well understood. We hypothesized that the most prevalent skin commensal microorganism, Staphylococcus epidermidis, may play a role in regulating GD T cell-mediated cutaneous responses. We analyzed antimicrobial protein P-2 expression in human skin at a single cell resolution using an amplified fluorescence in situ hybridization approach to detect P-2 mRNA in combination with immunophenotyping. We show that S. epidermidis activates GD T cells and upregulates P-2 in human skin ex vivo in a cell-specific manner. Furthermore, P-2 upregulation following S. epidermidis stimulation correlates with increased ability of skin cells to kill intracellular Staphylococcus aureus. Our findings are the first to reveal that skin commensal bacteria induce P-2 expression, which may be utilized beneficially to modulate host innate immune responses and protect from skin infections.

Mesoporous Silicon Particles Favor the Induction of Long-Lived Humoral Responses in Mice to a Peptide-Based Vaccine.

Vaccinology faces the challenge of developing improved immunization approaches that are able to induce long-term immunity with the desired Th profile according to the pathology. In this context, new vehicles for efficient antigen delivery that exert adjuvant effects play a critical role in addressing this goal. Herein, mesoporous silicon particles (PSiP) were assessed as carriers for a peptide-based vaccine targeting the receptor for advanced glycation end products (RAGE), which is a relevant receptor in Alzheimer´s disease and other diseases. A RAGE peptide was adsorbed onto PSiP (PSiP vaccine) and administered to BALB/c mice, leading to immune responses that were similar in magnitude to those induced by the soluble peptide. However, the response induced by PSiP lasted for a significantly longer period when compared with the behavior of the group immunized with the peptide alone. Therefore, PSiP are proposed as carriers to enhance immune memory, which is critical in vaccination. This study opens interesting perspectives related to the application of PSiP in vaccinology.

Cicatrización de tejido óseo y gingival en cirugías de terceros molares inferiores. Estudio comparativo entre el uso de fibrina rica en plaquetas versus cicatrización fisiológica / Gingival and bone tissue healing in lower third molar surgeries. Comparative study between use of platelet rich fibrin versus physiological healing

Los terceros molares retenidos son dientes que se encuentran ligados a una serie de patologías en la cavidad bucal, por lo que se requiere su extracción quirúrgica en la mayoría de los casos. Los procedimientos quirúrgicos para extraer terceros molares retenidos, traen consigo efectos propios de la cirugía. El objetivo de este estudio fue determinar la efectividad cicatrizante en tejido óseo y gingival con el uso de la fibrina rica en plaquetas en la cirugía de terceros molares inferiores en el Centro Quirúrgico de la Facultad de Odontología de la Universidad Central del Ecuador en el periodo de mayoseptiembre del 2015, mediante un estudio comparativo realizado en 30 pacientes que cumplieron los criterios de inclusión. Se controló a los pacientes a los ocho días mediante observación directa de las heridas, y a los 60 días posteriores a la intervención quirúrgica una toma radiográfica panorámica digital de maxilares, analizada en el software RadiAnt DICOM Viewer. Los resultados obtenidos en cicatrización de tejido blando fueron mediante la prueba de χ2 p < 0.001 y para tejido óseo mediante la prueba t de Student p = 0.015.

Retained third molars are teeth linked to several conditions of the mouth, therefore, in most cases, surgical extraction is required. Surgical procedures undertaken to extract retained third molars bring about surgical procedures effects. The aim of the present study was to determine healing effectiveness in bone and gingival tissue with use of platelet rich fibrin in surgical procedures involving lower third molar extraction performed at the Surgical Center of the School of Dentistry, Central University of Ecuador in the period comprised May-September 2015. A comparative study was performed of 30 patients meeting inclusion criteria. Eight days after extraction, patients were controlled by means of direct observation of surgical site; 60 days after extraction, a digital panoramic X-ray of the jaws was taken and analyzed with software RadiAnt DICOM Viewer. For soft tissue, healing results were obtained with χ2 test p < 0.001, and for bone tissue results were obtained with t-Student test p = 0.015.