Tyrosine kinase 2 modulates splenic B cells through type I IFN and TLR7 signaling.

Tyrosine kinase 2 (TYK2) is involved in type I interferon (IFN-I) signaling through IFN receptor 1 (IFNAR1). This signaling pathway is crucial in the early antiviral response and remains incompletely understood on B cells. Therefore, to understand the role of TYK2 in B cells, we studied these cells under homeostatic conditions and following in vitro activation using Tyk2-deficient (Tyk2-/-) mice. Splenic B cell subpopulations were altered in Tyk2-/- compared to wild type (WT) mice. Marginal zone (MZ) cells were decreased and aged B cells (ABC) were increased, whereas follicular (FO) cells remained unchanged. Likewise, there was an imbalance in transitional B cells in juvenile Tyk2-/- mice. RNA sequencing analysis of adult MZ and FO cells isolated from Tyk2-/- and WT mice in homeostasis revealed altered expression of IFN-I and Toll-like receptor 7 (TLR7) signaling pathway genes. Flow cytometry assays corroborated a lower expression of TLR7 in MZ B cells from Tyk2-/- mice. Splenic B cell cultures showed reduced proliferation and differentiation responses after activation with TLR7 ligands in Tyk2-/- compared to WT mice, with a similar response to lipopolysaccharide (LPS) or anti-CD40 + IL-4. IgM, IgG, IL-10 and IL-6 secretion was also decreased in Tyk2-/- B cell cultures. This reduced response of the TLR7 pathway in Tyk2-/- mice was partially restored by IFNα addition. In conclusion, there is a crosstalk between TYK2 and TLR7 mediated by an IFN-I feedback loop, which contributes to the establishment of MZ B cells and to B cell proliferation and differentiation.

Human IgMhiCD300a+ B Cells Are Circulating Marginal Zone Memory B Cells That Respond to Pneumococcal Polysaccharides and Their Frequency Is Decreased in People Living with HIV.

CD300a is differentially expressed among B cell subsets, although its expression in immunoglobulin (Ig)M+ B cells is not well known. We identified a B cell subset expressing CD300a and high levels of IgM (IgMhiCD300a+). The results showed that IgMhiCD300a+ B cells were CD10-CD27+CD25+IgDloCD21hiCD23-CD38loCD1chi, suggesting that they are circulating marginal zone (MZ) IgM memory B cells. Regarding the immunoglobulin repertoire, IgMhiCD300a+ B cells exhibited a higher mutation rate and usage of the IgH-VDJ genes than the IgM+CD300a- counterpart. Moreover, the shorter complementarity-determining region 3 (CDR3) amino acid (AA) length from IgMhiCD300a+ B cells together with the predicted antigen experience repertoire indicates that this B cell subset has a memory phenotype. IgM memory B cells are important in T cell-independent responses. Accordingly, we demonstrate that this particular subset secretes higher amounts of IgM after stimulation with pneumococcal polysaccharides or a toll-like receptor 9 (TLR9) agonist than IgM+CD300a- cells. Finally, the frequency of IgMhiCD300a+ B cells was lower in people living with HIV-1 (PLWH) and it was inversely correlated with the years with HIV infection. Altogether, these data help to identify a memory B cell subset that contributes to T cell-independent responses to pneumococcal infections and may explain the increase in severe pneumococcal infections and the impaired responses to pneumococcal vaccination in PLWH.

Immune stress suppresses innate immune signaling in preleukemic precursor B-cells to provoke leukemia in predisposed mice.

The initial steps of B-cell acute lymphoblastic leukemia (B-ALL) development usually pass unnoticed in children. Several preclinical studies have shown that exposure to immune stressors triggers the transformation of preleukemic B cells to full-blown B-ALL, but how this takes place is still a longstanding and unsolved challenge. Here we show that dysregulation of innate immunity plays a driving role in the clonal evolution of pre-malignant Pax5+/- B-cell precursors toward leukemia. Transcriptional profiling reveals that Myd88 is downregulated in immune-stressed pre-malignant B-cell precursors and in leukemic cells. Genetic reduction of Myd88 expression leads to a significant increase in leukemia incidence in Pax5+/-Myd88+/- mice through an inflammation-dependent mechanism. Early induction of Myd88-independent Toll-like receptor 3 signaling results in a significant delay of leukemia development in Pax5+/- mice. Altogether, these findings identify a role for innate immunity dysregulation in leukemia, with important implications for understanding and therapeutic targeting of the preleukemic state in children.

Toll-like Receptor Signaling-deficient Cells Enhance Antitumor Activity of Cell-based Immunotherapy by Increasing Tumor Homing.

Cancer immunotherapy aims to activate the immune system. Some immunotherapeutic agents can be loaded in carrier cells for delivering to the tumors. However, a challenge with cell-based therapies is the selection of the appropriate cells to produce effective clinical outcomes. We hypothesize that therapies based on cells presenting a natural low proinflammatory profile ("silent cells") in the peripheral blood would result in better antitumor responses by increasing their homing to the tumor site. We studied our hypothesis in an immunotherapy model consisting of mesenchymal stromal cells (MSCs) carrying oncolytic adenoviruses for the treatment of immunocompetent mice. Toll-like receptor signaling-deficient cells (TLR4, TLR9, or MyD88 knockout) were used as "silent cells," while regular MSCs were used as control. Although in vitro migration was similar in regular and knockout carrier cells, in vivo tumor homing of silent cells was significantly higher after systemic administration. This better homing to the tumor site was highly related to the mild immune response triggered by these silent cells in peripheral blood. As a result, the use of silent cells significantly improved the antitumor efficacy of the treatment in comparison with the use of regular MSCs. While cancer immunotherapies generally aim to boost local immune responses in the tumor microenvironment, low systemic inflammation after systemic administration of the treatment may indeed enhance their tumor homing and improve the overall antitumor effect. These findings highlight the importance of selecting appropriate donor cells as therapeutic carriers in cell-based therapies for cancer treatment. Significance: Cells carrying drugs, virus, or other antitumor agents are commonly used for the treatment of cancer. This research shows that silent cells are excellent carriers for immunotherapies, improving tumor homing and enhancing the antitumor effect.

Age-dependent nasal immune responses in non-hospitalized bronchiolitis children.

Bronchiolitis in children is associated with significant rates of morbidity and mortality. Many studies have been performed using samples from hospitalized bronchiolitis patients, but little is known about the immunological responses from infants suffering from mild/moderate bronchiolitis that do not require hospitalization. We have studied a collection of nasal lavage fluid (NLF) samples from outpatient bronchiolitis children as a novel strategy to unravel local humoral and cellular responses, which are not fully characterized. The children were age-stratified in three groups, two of them (GI under 2-months, GII between 2-4 months) presenting a first episode of bronchiolitis, and GIII (between 4 months and 2 years) with recurrent respiratory infections. Here we show that elevated levels of pro-inflammatory cytokines (IL1ß, IL6, TNFα, IL18, IL23), regulatory cytokines (IL10, IL17A) and IFNγ were found in the three bronchiolitis cohorts. However, little or no change was observed for IL33 and MCP1, at difference to previous results from bronchiolitis hospitalized patients. Furthermore, our results show a tendency to IL1ß, IL6, IL18 and TNFα increased levels in children with mild pattern of symptom severity and in those in which non RSV respiratory virus were detected compared to RSV+ samples. By contrast, no such differences were found based on gender distribution. Bronchiolitis NLFs contained more IgM, IgG1, IgG3 IgG4 and IgA than NLF from their age-matched healthy controls. NLF from bronchiolitis children predominantly contained neutrophils, and also low frequency of monocytes and few CD4+ and CD8+ T cells. NLF from infants older than 4-months contained more intermediate monocytes and B cell subsets, including naïve and memory cells. BCR repertoire analysis of NLF samples showed a biased VH1 usage in IgM repertoires, with low levels of somatic hypermutation. Strikingly, algorithmic studies of the mutation profiles, denoted antigenic selection on IgA-NLF repertoires. Our results support the use of NLF samples to analyze immune responses and may have therapeutic implications.

Small Molecules as Toll-like Receptor 4 Modulators Drug and In-House Computational Repurposing.

The innate immunity toll-like receptor 4 (TLR4) system is a receptor of paramount importance as a therapeutic target. Virtual screening following a "computer-aided drug repurposing" approach was applied to the discovery of novel TLR4 modulators with a non-lipopolysaccharide-like structure. We screened almost 29,000 approved drugs and drug-like molecules from commercial, public, and in-house academia chemical libraries and, after biological assays, identified several compounds with TLR4 antagonist activity. Our computational protocol showed to be a robust approach for the identification of hits with drug-like scaffolds as possible inhibitors of the TLR4 innate immune pathways. Our collaborative work broadens the chemical diversity for inspiration of new classes of TLR4 modulators.

Role of Toll-like receptor 4 in intravascular hemolysis-mediated injury.

Massive intravascular hemolysis is a common characteristic of several pathologies. It is associated with the release of large quantities of heme into the circulation, promoting injury in vulnerable organs, mainly kidney, liver, and spleen. Heme activates Toll-like receptor 4 (TLR4), a key regulator of the inflammatory response; however, the role of TLR4 in hemolysis and whether inhibition of this receptor may protect from heme-mediated injury are unknown. We induced intravascular hemolysis by injection of phenylhydrazine in wildtype and Tlr4-knockout mice. In this model, we analyzed physiological parameters, histological damage, inflammation and cell death in kidney, liver, and spleen. We also evaluated whether heme-mediated-inflammatory effects were prevented by TLR4 inhibition with the compound TAK-242, both in vivo and in vitro. Induction of massive hemolysis elicited acute kidney injury characterized by loss of renal function, morphological alterations of the tubular epithelium, cell death, and inflammation. These pathological effects were significantly ameliorated in the TLR4-deficient mice and in wildtype mice treated with TAK-242. In vitro studies showed that TAK-242 pretreatment reduced heme-mediated inflammation by inhibiting the TLR4/NF-κB (nuclear factor kappa B) axis. However, analysis in liver and spleen indicated that TLR4 deficiency did not protect against the toxic accumulation of heme in these organs. In conclusion, TLR4 is a key molecule involved in the renal inflammatory response triggered by massive intravascular hemolysis. TLR4 inhibition may be a potential therapeutic approach to prevent renal damage in patients suffering from hemolysis. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Senescent accelerated prone 8 (SAMP8) mice as a model of age dependent neuroinflammation.

BACKGROUND: Aging and age-related diseases are strong risk factors for the development of neurodegenerative diseases. Neuroinflammation (NIF), as the brain's immune response, plays an important role in aged associated degeneration of central nervous system (CNS). There is a need for well characterized animal models that will allow the scientific community to understand and modulate this process. METHODS: We have analyzed aging-phenotypical and inflammatory changes of brain myeloid cells (bMyC) in a senescent accelerated prone aged (SAMP8) mouse model, and compared with their senescence resistant control mice (SAMR1). We have performed morphometric methods to evaluate the architecture of cellular prolongations and determined the appearance of Iba1+ clustered cells with aging. To analyze specific constant brain areas, we have performed stereology measurements of Iba1+ cells in the hippocampal formation. We have isolated bMyC from brain parenchyma (BP) and choroid plexus plus meningeal membranes (m/Ch), and analyzed their response to systemic lipopolysaccharide (LPS)-driven inflammation. RESULTS: Aged 10 months old SAMP8 mice present many of the hallmarks of aging-dependent neuroinflammation when compared with their SAMR1 control, i.e., increase of protein aggregates, presence of Iba1+ clusters, but not an increase in the number of Iba1+ cells. We have further observed an increase of main inflammatory mediator IL-1ß, and an augment of border MHCII+Iba1+ cells. Isolated CD45+ bMyC from brain parenchyma (BP) and choroid plexus plus meningeal membranes (m/Ch) have been analyzed, showing that there is not a significant increase of CD45+ cells from the periphery. Our data support that aged-driven pro-inflammatory cytokine interleukin 1 beta (IL-1ß) transcription is enhanced in CD45+BP cells. Furthermore, LPS-driven systemic inflammation produces inflammatory cytokines mainly in border bMyC, sensed to a lesser extent by the BP bMyC, showing that IL-1ß expression is further augmented in aged SAMP8 compared to control SAMR1. CONCLUSION: Our data validate the SAMP8 model to study age-associated neuroinflammatory events, but careful controls for age and strain are required. These animals show morphological changes in their bMyC cell repertoires associated to age, corresponding to an increase in the production of pro-inflammatory cytokines such as IL-1ß, which predispose the brain to an enhanced inflammatory response after LPS-systemic challenge.

Toll-Like Receptors in Acute Kidney Injury.

Acute kidney injury (AKI) is an important health problem, affecting 13.3 million individuals/year. It is associated with increased mortality, mainly in low- and middle-income countries, where renal replacement therapy is limited. Moreover, survivors show adverse long-term outcomes, including increased risk of developing recurrent AKI bouts, cardiovascular events, and chronic kidney disease. However, there are no specific treatments to decrease the adverse consequences of AKI. Epidemiological and preclinical studies show the pathological role of inflammation in AKI, not only at the acute phase but also in the progression to chronic kidney disease. Toll-like receptors (TLRs) are key regulators of the inflammatory response and have been associated to many cellular processes activated during AKI. For that reason, a number of anti-inflammatory agents targeting TLRs have been analyzed in preclinical studies to decrease renal damage during AKI. In this review, we updated recent knowledge about the role of TLRs, mainly TLR4, in the initiation and development of AKI as well as novel compounds targeting these molecules to diminish kidney injury associated to this pathological condition.

The TLR4-MyD88 Signaling Axis Regulates Lung Monocyte Differentiation Pathways in Response to Streptococcus pneumoniae.

Streptococcus pneumoniae is the main cause of bacterial pneumonia, a condition that currently produces significant global morbidity and mortality. The initial immune response to this bacterium occurs when the innate system recognizes common motifs expressed by many pathogens, events driven by pattern recognition receptors like the Toll-like family receptors (TLRs). In this study, lung myeloid-cell populations responsible for the innate immune response (IIR) against S. pneumoniae, and their dependence on the TLR4-signaling axis, were analyzed in TLR4-/- and Myeloid-Differentiation factor-88 deficient (MyD88-/-) mice. Neutrophils and monocyte-derived cells were recruited in infected mice 3-days post-infection. Compared to wild-type mice, there was an increased bacterial load in both these deficient mouse strains and an altered IIR, although TLR4-/- mice were more susceptible to bacterial infection. These mice also developed fewer alveolar macrophages, weaker neutrophil infiltration, less Ly6Chigh monocyte differentiation and a disrupted classical and non-classical monocyte profile. The pro-inflammatory cytokine profile (CXCL1, TNF-α, IL-6, and IL-1ß) was also severely affected by the lack of TLR4 and no induction of Th1 was observed in these mice. The respiratory burst (ROS production) after infection was profoundly dampened in TLR4-/- and MyD88-/- mice. These data demonstrate the complex dynamics of myeloid populations and a key role of the TLR4-signaling axis in the IIR to S. pneumoniae, which involves both the MyD88 and TRIF (Toll/IL-1R domain-containing adaptor-inducing IFN-ß) dependent pathways.

ICOS deficiency hampers the homeostasis, development and function of NK cells.

Signaling through the inducible costimulator ICOS is required for the homeostasis and function of various immune cell populations, with an outstanding role in the generation and maintenance of germinal centers. Very recently, it has been suggested that the clinical phenotype of ICOS-deficient patients is much broader than initially anticipated and the innate immune response might be also affected. However, the role of the ICOS/ICOS-Ligand axis in the homeostasis and development of innate NK cells is not known, and reports on its participation in NK cell activation are scarce. NK cells may express low levels of ICOS that are markedly enhanced upon activation. We show here that ICOS-deficient (ICOS-KO) mice present low NK cell numbers and defects in the homeostasis of these cells, with delayed maturation and altered expression of the developmental NK cell markers CD122, NK1.1, CD11b or CD27. Our experiments in mixed bone marrow chimera mice indicate that, both, cell-intrinsic defects of ICOS-KO NK and deficiencies in the milieu of these mice contribute to the altered phenotype. ICOS-deficient NK cells show impaired production of IFN-γ and cytotoxicity, and a final outcome of defects in NK cell-mediated effector function during the response to poly(I:C) or vaccinia virus infection in vivo. Interestingly, we show that murine innate cells like IL-2-cultured NK and bone marrow-derived dendritic cells can simultaneously express ICOS and ICOS-Ligand; both molecules are functional in NK intracellular signaling, enhancing early phosphorylation of Akt and Erk, or IFN-γ secretion in IL-2-activated NK cells. Our study shows the functional importance of the ICOS/ICOS-L pair in NK cell homeostasis, differentiation and activity and suggests novel therapeutic targets for NK manipulation.

Nrf2 Plays a Protective Role Against Intravascular Hemolysis-Mediated Acute Kidney Injury.

Massive intravascular hemolysis is associated with acute kidney injury (AKI). Nuclear factor erythroid-2-related factor 2 (Nrf2) plays a central role in the defense against oxidative stress by activating the expression of antioxidant proteins. We investigated the role of Nrf2 in intravascular hemolysis and whether Nrf2 activation protected against hemoglobin (Hb)/heme-mediated renal damage in vivo and in vitro. We observed renal Nrf2 activation in human hemolysis and in an experimental model of intravascular hemolysis promoted by phenylhydrazine intraperitoneal injection. In wild-type mice, Hb/heme released from intravascular hemolysis promoted AKI, resulting in decreased renal function, enhanced expression of tubular injury markers (KIM-1 and NGAL), oxidative and endoplasmic reticulum stress (ER), and cell death. These features were more severe in Nrf2-deficient mice, which showed decreased expression of Nrf2-related antioxidant enzymes, including heme oxygenase 1 (HO-1) and ferritin. Nrf2 activation with sulforaphane protected against Hb toxicity in mice and cultured tubular epithelial cells, ameliorating renal function and kidney injury and reducing cell stress and death. Nrf2 genotype or sulforaphane treatment did not influence the severity of hemolysis. In conclusion, our study identifies Nrf2 as a key molecule involved in protection against renal damage associated with hemolysis and opens novel therapeutic approaches to prevent renal damage in patients with severe hemolytic crisis. These findings provide new insights into novel aspects of Hb-mediated renal toxicity and may have important therapeutic implications for intravascular hemolysis-related diseases.

NFATc3 controls tumour growth by regulating proliferation and migration of human astroglioma cells.

Calcium/Calcineurin/Nuclear Factor of Activated T cells (Ca/CN/NFAT) signalling pathway is the main calcium (Ca2+) dependent signalling pathway involved in the homeostasis of brain tissue. Here, we study the presence of NFATc members in human glioma by using U251 cells and a collection of primary human glioblastoma (hGB) cell lines. We show that NFATc3 member is the predominant member. Furthermore, by using constitutive active NFATc3 mutant and shRNA lentiviral vectors to achieve specific silencing of this NFATc member, we describe cytokines and molecules regulated by this pathway which are required for the normal biology of cancer cells. Implanting U251 in an orthotopic intracranial assay, we show that specific NFATc3 silencing has a role in tumour growth. In addition NFATc3 knock-down affects both the proliferation and migration capacities of glioma cells in vitro. Our data open the possibility of NFATc3 as a target for the treatment of glioma.

CD45 expression discriminates waves of embryonic megakaryocytes in the mouse.

Embryonic megakaryopoiesis starts in the yolk sac on gestational day 7.5 as part of the primitive wave of hematopoiesis, and it continues in the fetal liver when this organ is colonized by hematopoietic progenitors between day 9.5 and 10.5, as the definitive hematopoiesis wave. We characterized the precise phenotype of embryo megakaryocytes in the liver at gestational day 11.5, identifying them as CD41++CD45-CD9++CD61+MPL+CD42c+ tetraploid cells that express megakaryocyte-specific transcripts and display differential traits when compared to those present in the yolk sac at the same age. In contrast to megakaryocytes from adult bone marrow, embryo megakaryocytes are CD45- until day 13.5 of gestation, as are both the megakaryocyte progenitors and megakaryocyte/erythroid-committed progenitors. At gestational day 11.5, liver and yolk sac also contain CD41+CD45+ and CD41+CD45- cells. These populations, and that of CD41++CD45-CD42c+ cells, isolated from liver, differentiate in culture into CD41++CD45-CD42c+ proplatelet-bearing megakaryocytes. Also present at this time are CD41-CD45++CD11b+ cells, which produce low numbers of CD41++CD45-CD42c+ megakaryocytes in vitro, as do fetal liver cells expressing the macrophage-specific Csf receptor-1 (Csf1r/CD115) from MaFIA transgenic mice, which give rise poorly to CD41++CD45-CD42c+ embryo megakaryocytes both in vivo and in vitro In contrast, around 30% of adult megakaryocytes (CD41++CD45++CD9++CD42c+) from C57BL/6 and MaFIA mice express CD115. We propose that differential pathways operating in the mouse embryo liver at gestational day 11.5 beget CD41++CD45-CD42c+ embryo megakaryocytes that can be produced from CD41+CD45- or from CD41+CD45+ cells, at difference from those from bone marrow.

Podocytes are new cellular targets of haemoglobin-mediated renal damage.

Recurrent and massive intravascular haemolysis induces proteinuria, glomerulosclerosis, and progressive impairment of renal function, suggesting podocyte injury. However, the effects of haemoglobin (Hb) on podocytes remain unexplored. Our results show that cultured human podocytes or podocytes isolated from murine glomeruli bound and endocytosed Hb through the megalin-cubilin receptor system, thus resulting in increased intracellular Hb catabolism, oxidative stress, activation of the intrinsic apoptosis pathway, and altered podocyte morphology, with decreased expression of the slit diaphragm proteins nephrin and synaptopodin. Hb uptake activated nuclear factor erythroid-2-related factor 2 (Nrf2) and induced expression of the Nrf2-related antioxidant proteins haem oxygenase-1 (HO-1) and ferritin. Nrf2 activation and Hb staining was observed in podocytes of mice with intravascular haemolysis. These mice developed proteinuria and showed podocyte injury, characterized by foot process effacement, decreased synaptopodin and nephrin expression, and podocyte apoptosis. These pathological effects were enhanced in Nrf2-deficient mice, whereas Nrf2 activation with sulphoraphane protected podocytes against Hb toxicity both in vivo and in vitro. Supporting the translational significance of our findings, we observed podocyte damage and podocytes stained for Hb, HO-1, ferritin and phosphorylated Nrf2 in renal sections and urinary sediments of patients with massive intravascular haemolysis, such as atypical haemolytic uraemic syndrome and paroxysmal nocturnal haemoglobinuria. In conclusion, podocytes take up Hb both in vitro and during intravascular haemolysis, promoting oxidative stress, podocyte dysfunction, and apoptosis. Nrf2 may be a potential therapeutic target to prevent loss of renal function in patients with intravascular haemolysis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Spatially restricted JAG1-Notch signaling in human thymus provides suitable DC developmental niches.

A key unsolved question regarding the developmental origin of conventional and plasmacytoid dendritic cells (cDCs and pDCs, respectively) resident in the steady-state thymus is whether early thymic progenitors (ETPs) could escape T cell fate constraints imposed normally by a Notch-inductive microenvironment and undergo DC development. By modeling DC generation in bulk and clonal cultures, we show here that Jagged1 (JAG1)-mediated Notch signaling allows human ETPs to undertake a myeloid transcriptional program, resulting in GATA2-dependent generation of CD34+ CD123+ progenitors with restricted pDC, cDC, and monocyte potential, whereas Delta-like1 signaling down-regulates GATA2 and impairs myeloid development. Progressive commitment to the DC lineage also occurs intrathymically, as myeloid-primed CD123+ monocyte/DC and common DC progenitors, equivalent to those previously identified in the bone marrow, are resident in the normal human thymus. The identification of a discrete JAG1+ thymic medullary niche enriched for DC-lineage cells expressing Notch receptors further validates the human thymus as a DC-poietic organ, which provides selective microenvironments permissive for DC development.

Altered marginal zone and innate-like B cells in aged senescence-accelerated SAMP8 mice with defective IgG1 responses.

Aging has a strong impact on the activity of the immune system, enhancing susceptibility to pathogens and provoking a predominant pre-inflammatory status, whereas dampening responses to vaccines in humans and mice. Here, we demonstrate a loss of marginal zone B lymphocytes (MZ, CD19+CD45R+CD21++CD23lo) and a decrease of naive B cells (CD19+IgD+), whereas there is an enhancement of a CD19+CD45Rlo innate-like B cell population (B1REL) and the so-called aged B cell compartment (ABC, CD45R+CD21loCD23loCD5-CD11b-) in aged senescence-accelerated (SAMP8) mice but not in aged senescence-resistant (SAMR1) mice. These changes in aged SAMP8 mice were associated with lower IgG isotype levels, displaying low variable gene usage repertoires of the immunoglobulin heavy chain (VH) diversity, with a diminution on IgG1-memory B cells (CD11b-Gr1-CD138-IgM-IgD-CD19+CD38+IgG1+), an increase in T follicular helper (TFH, CD4+CXCR5+PD1+) cell numbers, and an altered MOMA-1 (metallophilic macrophages) band in primary follicles. LPS-mediated IgG1 responses were impaired in the B1REL and ABC cell compartments, both in vitro and in vivo. These data demonstrate the prominent changes to different B cell populations and in structural follicle organization that occur upon aging in SAMP8 mice. These novel results raise new questions regarding the importance of the cellular distribution in the B cell layers, and their effector functions needed to mount a coordinated and effective humoral response.

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry.

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

PSGL-1 on Leukocytes is a Critical Component of the Host Immune Response against Invasive Pneumococcal Disease.

Bacterial uptake by phagocytic cells is a vital event in the clearance of invading pathogens such as Streptococcus pneumoniae. A major role of the P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes against invasive pneumococcal disease is described in this study. Phagocytosis experiments using different serotypes demonstrated that PSGL-1 is involved in the recognition, uptake and killing of S. pneumoniae. Co-localization of several clinical isolates of S. pneumoniae with PSGL-1 was demonstrated, observing a rapid and active phagocytosis in the presence of PSGL-1. Furthermore, the pneumococcal capsular polysaccharide and the main autolysin of the bacterium--the amidase LytA--were identified as bacterial ligands for PSGL-1. Experimental models of pneumococcal disease including invasive pneumonia and systemic infection showed that bacterial levels were markedly increased in the blood of PSGL-1-/- mice. During pneumonia, PSGL-1 controls the severity of pneumococcal dissemination from the lung to the bloodstream. In systemic infection, a major role of PSGL-1 in host defense is to clear the bacteria in the systemic circulation controlling bacterial replication. These results confirmed the importance of this receptor in the recognition and clearance of S. pneumoniae during invasive pneumococcal disease. Histological and cellular analysis demonstrated that PSGL-1-/- mice have increased levels of T cells migrating to the lung than the corresponding wild-type mice. In contrast, during systemic infection, PSGL-1-/- mice had increased numbers of neutrophils and macrophages in blood, but were less effective controlling the infection process due to the lack of this functional receptor. Overall, this study demonstrates that PSGL-1 is a novel receptor for S. pneumoniae that contributes to protection against invasive pneumococcal disease.

Aldosterone Induces Renal Fibrosis and Inflammatory M1-Macrophage Subtype via Mineralocorticoid Receptor in Rats.

We aimed to evaluate macrophages heterogeneity and structural, functional and inflammatory alterations in rat kidney by aldosterone + salt administration. The effects of treatment with spironolactone on above parameters were also analyzed. Male Wistar rats received aldosterone (1 mgkg-1d-1) + 1% NaCl for 3 weeks. Half of the animals were treated with spironolactone (200 mg kg-1d-1). Systolic and diastolic blood pressures were elevated (p<0.05) in aldosterone + salt-treated rats. Relative kidney weight, collagen content, fibronectin, macrophage infiltrate, CTGF, Col I, MMP2, TNF-α, CD68, Arg2, and SGK-1 were increased (p<0.05) in aldosterone + salt-treated rats, being reduced by spironolactone (p<0.05). Increased iNOS and IFN-γ mRNA gene expression (M1 macrophage markers) was observed in aldosterone + salt rats, whereas no significant differences were observed in IL-10 and gene ArgI mRNA expression or ED2 protein content (M2 macrophage markers). All the observed changes were blocked with spironolactone treatment. Macrophage depletion with liposomal clodronate reduced macrophage influx and inflammatory M1 markers (INF-γ or iNOS), whereas interstitial fibrosis was only partially reduced after this intervention, in aldosterone plus salt-treated rats. In conclusion, aldosterone + salt administration mediates inflammatory M1 macrophage phenotype and increased fibrosis throughout mineralocorticoid receptors activation.