Aging microglia.

Microglia are the tissue-resident macrophage population of the brain, specialized in supporting the CNS environment and protecting it from endogenous and exogenous insults. Nonetheless, their function declines with age, in ways that remain to be fully elucidated. Given the critical role played by microglia in neurodegenerative diseases, a better understanding of the aging microglia phenotype is an essential prerequisite in designing better preventive and therapeutic strategies. In this review, we discuss the most recent literature on microglia in aging, comparing findings in rodent models and human subjects.

HVCN1 modulates BCR signal strength via regulation of BCR-dependent generation of reactive oxygen species.

Voltage-gated proton currents regulate generation of reactive oxygen species (ROS) in phagocytic cells. In B cells, stimulation of the B cell antigen receptor (BCR) results in the production of ROS that participate in B cell activation, but the involvement of proton channels is unknown. We report here that the voltage-gated proton channel HVCN1 associated with the BCR complex and was internalized together with the BCR after activation. BCR-induced generation of ROS was lower in HVCN1-deficient B cells, which resulted in attenuated BCR signaling via impaired BCR-dependent oxidation of the tyrosine phosphatase SHP-1. This resulted in less activation of the kinases Syk and Akt, impaired mitochondrial respiration and glycolysis and diminished antibody responses in vivo. Our findings identify unanticipated functions for proton channels in B cells and demonstrate the importance of ROS in BCR signaling and downstream metabolism.

Microglial phagocytosis in aging and Alzheimer's disease.

Microglia are the innate immune cells of the brain, which maintain homeostasis by constantly scanning and surveying the environment with their highly ramified processes. In order to exert this function, they need to phagocytose synapses as well as debris and dead cells, a process that is further amplified in pathological conditions. Importantly, it has been shown that microglia phagocytic capacity is altered in the course of neurodegenerative disease, for which aging is one of the highest risk factors. Thus, understanding how phagocytosis is impaired during aging is a priority for future research. Advances in this area are expected to significantly contribute to our understanding of normal cognition during aging, as well as changes that take place in age-associated neurodegenerative diseases. In this review, we will summarize the current knowledge on how phagocytosis is executed and affected by aging or in age-associated neurological disorders, such as Alzheimer's disease (AD). Furthermore, we will summarize both protective and deleterious consequences of altered phagocytosis in AD and where relevant in other neurodegenerative diseases.

Inhibition of oxidative metabolism leads to p53 genetic inactivation and transformation in neural stem cells.

Alterations of mitochondrial metabolism and genomic instability have been implicated in tumorigenesis in multiple tissues. High-grade glioma (HGG), one of the most lethal human neoplasms, displays genetic modifications of Krebs cycle components as well as electron transport chain (ETC) alterations. Furthermore, the p53 tumor suppressor, which has emerged as a key regulator of mitochondrial respiration at the expense of glycolysis, is genetically inactivated in a large proportion of HGG cases. Therefore, it is becoming evident that genetic modifications can affect cell metabolism in HGG; however, it is currently unclear whether mitochondrial metabolism alterations could vice versa promote genomic instability as a mechanism for neoplastic transformation. Here, we show that, in neural progenitor/stem cells (NPCs), which can act as HGG cell of origin, inhibition of mitochondrial metabolism leads to p53 genetic inactivation. Impairment of respiration via inhibition of complex I or decreased mitochondrial DNA copy number leads to p53 genetic loss and a glycolytic switch. p53 genetic inactivation in ETC-impaired neural stem cells is caused by increased reactive oxygen species and associated oxidative DNA damage. ETC-impaired cells display a marked growth advantage in the presence or absence of oncogenic RAS, and form undifferentiated tumors when transplanted into the mouse brain. Finally, p53 mutations correlated with alterations in ETC subunit composition and activity in primary glioma-initiating neural stem cells. Together, these findings provide previously unidentified insights into the relationship between mitochondria, genomic stability, and tumor suppressive control, with implications for our understanding of brain cancer pathogenesis.

Mechanisms of PD-L1/PD-1-mediated CD8 T-cell dysfunction in the context of aging-related immune defects in the Eµ-TCL1 CLL mouse model.

T-cell defects, immune suppression, and poor antitumor immune responses are hallmarks of chronic lymphocytic leukemia (CLL), and PD-1/PD-L1 inhibitory signaling has emerged as a major immunosuppressive mechanism. However, the effect of different microenvironments and the confounding influence of aging are poorly understood. The current study uses the Eµ-TCL1 mouse model, which replicates human T-cell defects, as a preclinical platform to longitudinally examine patterns of T-cell dysfunction alongside developing CLL and in different microenvironments, with a focus on PD-1/PD-L1 interactions. The development of CLL was significantly associated with changes in T-cell phenotype across all organs and function. Although partly mirrored in aging wild-type mice, CLL-specific T-cell changes were identified. Murine CLL cells highly expressed PD-L1 and PD-L2 in all organs, with high PD-L1 expression in the spleen. CD3(+)CD8(+) T cells from leukemic and aging healthy mice highly expressed PD-1, identifying aging as a confounder, but adoptive transfer experiments demonstrated CLL-specific PD-1 induction. Direct comparisons of PD-1 expression and function between aging CLL mice and controls identified PD-1(+) T cells in CLL as a heterogeneous population with variable effector function. This is highly relevant for therapeutic targeting of CD8(+) T cells, showing the potential of reprogramming and selective subset expansion to restore antitumor immunity.

Expression of polymeric immunoglobulin receptor and stromal activity in pancreatic ductal adenocarcinoma.

BACKGROUND/OBJECTIVES: Polymeric immunoglobulin receptor (pIgR) traffics Immunoglobulins (IgA and IgM) through epithelial cells in normal mucosae but neither are expressed in the normal pancreas. Recent work from our laboratory suggested pIgR may be upregulated in pancreatic ductal adenocarcinoma (PDAC). Our aim was to assess the role of pIgR in human PDAC. METHODS: pIgR expression was manipulated (siRNA and shRNA) in cell lines to evaluate its subsequent effect on cell behaviour in 2D assays as well as 3D organotypics models. Tissue Microarrays of 88 patients with PDAC were analysed after pIgR, αSMA, E-Cadherin and Picrosirius Red staining to assess their role as a combined bio-marker panel. RESULTS: Cytokines such as interleukin 4 (IL4) and Tumour Necrosis Factor (TNFα) could not modulate pIgR expression in PDAC cell lines despite this effect being seen in other studies. Down-regulation in pIgR expression in Capan1 cancer cell line resulted in reduction of cellular proliferation, adhesion and migration in 2D assays. In 3D physiomimetic organotypic models, pIgR downregulation resulted in reduced cancer cell invasion, alteration of apico-basal polarity and diminished stromal activity. In human PDAC, decreased E-cadherin expression correlates with increased pIgR expression through pancreatic intra-epithelial neoplasia (PanIN) progression. In combination with enhanced stromal indices (α-smooth muscle action (SMA) and Picrosirius red), low pIgR scores had a trend towards better survival. CONCLUSION: pIgR may be involved in PDAC progression and may be linked stromal activity. Further work on its precise role is mandated in in vivo models, to understand its influence on cancer progression.

Enhanced activation of an amino-terminally truncated isoform of the voltage-gated proton channel HVCN1 enriched in malignant B cells.

HVCN1 (Hydrogen voltage-gated channel 1) is the only mammalian voltage-gated proton channel. In human B lymphocytes, HVCN1 associates with the B-cell receptor (BCR) and is required for optimal BCR signaling and redox control. HVCN1 is expressed in malignant B cells that rely on BCR signaling, such as chronic lymphocytic leukemia (CLL) cells. However, little is known about its regulation in these cells. We found that HVCN1 was expressed in B cells as two protein isoforms. The shorter isoform (HVCN1S) was enriched in B cells from a cohort of 76 CLL patients. When overexpressed in a B-cell lymphoma line, HVCN1S responded more profoundly to protein kinase C-dependent phosphorylation. This more potent enhanced gating response was mediated by increased phosphorylation of the same residue responsible for enhanced gating in HVCN1L, Thr(29). Furthermore, the association of HVCN1S with the BCR was weaker, which resulted in its diminished internalization upon BCR stimulation. Finally, HVCN1S conferred a proliferative and migratory advantage as well as enhanced BCR-dependent signaling. Overall, our data show for the first time, to our knowledge, the existence of a shorter isoform of HVCN1 with enhanced gating that is specifically enriched in malignant B cells. The properties of HVCN1S suggest that it may contribute to the pathogenesis of BCR-dependent B-cell malignancies.

B regulatory cells in cancer.

B regulatory cells are a newly described subpopulation of B cells that appear to play important roles in autoimmunity and more recently, in cancer. In this review we summarize our current knowledge of B regulatory cells, as well as the body of evidence pointing towards a role for B cells in general, and B regulatory cells in particular, in promoting tumor growth.

Regulation of immune responses by proton channels.

The identification of the HVCN1 gene, encoding the only mammalian voltage-gated proton channel, prompted a number of studies on how proton channels affect cellular functions. As their expression is mainly restricted to immune cells, it is not surprising that proton channels regulate different aspects of immune responses. In this review, I will examine the current knowledge of voltage-gated proton channels in both innate and adaptive responses and assess the remaining outstanding questions.

Assessing mRNA translation in mouse adult microglia and bone-marrow-derived macrophages.

Protein synthesis, or mRNA translation, is the biological process through which genetic information stored in messenger RNAs is encoded into proteins. Here, we present an optimized protocol for assessing the translation rate in mouse adult microglia and cultured bone-marrow-derived macrophages. We describe steps for isolating cells, treating them with a puromycin-analog probe, and fluorescently labeling the puromycylated-polypeptide chains. We then detail their quantification by flow cytometry or with a fluorescent plate reader. For complete details on the use and execution of this protocol, please refer to Keane et al. (2021).1.

Systemic alterations in neutrophils and their precursors in early-stage chronic obstructive pulmonary disease.

Systemic inflammation is established as part of late-stage severe lung disease, but molecular, functional, and phenotypic changes in peripheral immune cells in early disease stages remain ill defined. Chronic obstructive pulmonary disease (COPD) is a major respiratory disease characterized by small-airway inflammation, emphysema, and severe breathing difficulties. Using single-cell analyses we demonstrate that blood neutrophils are already increased in early-stage COPD, and changes in molecular and functional neutrophil states correlate with lung function decline. Assessing neutrophils and their bone marrow precursors in a murine cigarette smoke exposure model identified similar molecular changes in blood neutrophils and precursor populations that also occur in the blood and lung. Our study shows that systemic molecular alterations in neutrophils and their precursors are part of early-stage COPD, a finding to be further explored for potential therapeutic targets and biomarkers for early diagnosis and patient stratification.

Immunoglobulin heavy chain locus chromosomal translocations in B-cell precursor acute lymphoblastic leukemia: rare clinical curios or potent genetic drivers?

Chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus define common subgroups of B-cell lymphoma but are rare in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Recent fluorescent in situ hybridization and molecular cloning studies have identified several novel IGH translocations involving genes that play important roles in normal hemopoiesis, including the cytokine receptor genes CRLF2 and EPOR, all members of the CCAAT enhancer-binding protein gene family, as well as genes not normally expressed in hemopoietic cells including inhibitor of DNA binding 4. IGH translocation results in deregulated target gene expression because of juxtaposition with IGH transcriptional enhancers. However, many genes targeted by IGH translocations are also more commonly deregulated in BCP-ALL as a consequence of other genetic or epigenetic mechanisms. For example, interstitial genomic deletions also result in deregulated CRLF2 expression, whereas EPOR expression is deregulated as a consequence of the ETV6-RUNX1 fusion. The possible clinical importance of many of the various IGH translocations in BCP-ALL remains to be determined from prospective studies, but CRLF2 expression is associated with a poor prognosis. Despite their rarity, IGH chromosomal translocations in BCP-ALL therefore define not only new mechanisms of B-cell transformation but also clinically important subgroups of disease and suggest new targeted therapeutic approaches.

Voltage-gated proton channels maintain pH in human neutrophils during phagocytosis.

Phagocytosis of microbial invaders represents a fundamental defense mechanism of the innate immune system. The subsequent killing of microbes is initiated by the respiratory burst, in which nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generates vast amounts of superoxide anion, precursor to bactericidal reactive oxygen species. Cytoplasmic pH regulation is crucial because NADPH oxidase functions optimally at neutral pH, yet produces enormous quantities of protons. We monitored pH(i) in individual human neutrophils during phagocytosis of opsonized zymosan, using confocal imaging of the pH sensing dye SNARF-1, enhanced by shifted excitation and emission ratioing, or SEER. Despite long-standing dogma that Na(+)/H(+) antiport regulates pH during the phagocyte respiratory burst, we show here that voltage-gated proton channels are the first transporter to respond. During the initial phagocytotic event, pH(i) decreased sharply, and recovery required both Na(+)/H(+) antiport and proton current. Inhibiting myeloperoxidase attenuated the acidification, suggesting that diffusion of HOCl into the cytosol comprises a substantial acid load. Inhibiting proton channels with Zn(2+) resulted in profound acidification to levels that inhibit NADPH oxidase. The pH changes accompanying phagocytosis in bone marrow phagocytes from HVCN1-deficient mice mirrored those in control mouse cells treated with Zn(2+). Both the rate and extent of acidification in HVCN1-deficient cells were twice larger than in control cells. In summary, acid extrusion by proton channels is essential to the production of reactive oxygen species during phagocytosis.

Loss of mTORC2-induced metabolic reprogramming in monocytes uncouples migration and maturation from production of proinflammatory mediators.

Monocyte migration to the sites of inflammation and maturation into macrophages are key steps for their immune effector function. Here, we show that mechanistic target of rapamycin complex 2 (mTORC2)-dependent Akt activation is instrumental for metabolic reprogramming at the early stages of macrophage-mediated immunity. Despite an increased production of proinflammatory mediators, monocytes lacking expression of the mTORC2 component Rictor fail to efficiently migrate to inflammatory sites and fully mature into macrophages, resulting in reduced inflammatory responses in vivo. The mTORC2-dependent phosphorylation of Akt is instrumental for the enhancement of glycolysis and mitochondrial respiration, required to sustain monocyte maturation and motility. These observations are discussed in the context of therapeutic strategies aimed at selective inhibition of mTORC2 activity.

Loss of voltage-gated hydrogen channel 1 expression reveals heterogeneous metabolic adaptation to intracellular acidification by T cells.

Voltage-gated hydrogen channel 1 (Hvcn1) is a voltage-gated proton channel, which reduces cytosol acidification and facilitates the production of ROS. The increased expression of this channel in some cancers has led to proposing Hvcn1 antagonists as potential therapeutics. While its role in most leukocytes has been studied in depth, the function of Hvcn1 in T cells remains poorly defined. We show that Hvcn1 plays a nonredundant role in protecting naive T cells from intracellular acidification during priming. Despite sharing overall functional impairment in vivo and in vitro, Hvcn1-deficient CD4+ and CD8+ T cells display profound differences during the transition from naive to primed T cells, including in the preservation of T cell receptor (TCR) signaling, cellular division, and death. These selective features result, at least in part, from a substantially different metabolic response to intracellular acidification associated with priming. While Hvcn1-deficient naive CD4+ T cells reprogram to rescue the glycolytic pathway, naive CD8+ T cells, which express high levels of this channel in the mitochondria, respond by metabolically compensating mitochondrial dysfunction, at least in part via AMPK activation. These observations imply heterogeneity between adaptation of naive CD4+ and CD8+ T cells to intracellular acidification during activation.

Identification of Thr29 as a critical phosphorylation site that activates the human proton channel Hvcn1 in leukocytes.

Voltage-gated proton channels and NADPH oxidase function cooperatively in phagocytes during the respiratory burst, when reactive oxygen species are produced to kill microbial invaders. Agents that activate NADPH oxidase also enhance proton channel gating profoundly, facilitating its roles in charge compensation and pH(i) regulation. The "enhanced gating mode" appears to reflect protein kinase C (PKC) phosphorylation. Here we examine two candidates for PKC-delta phosphorylation sites in the human voltage-gated proton channel, H(V)1 (Hvcn1), Thr(29) and Ser(97), both in the intracellular N terminus. Channel phosphorylation was reduced in single mutants S97A or T29A, and further in the double mutant T29A/S97A, by an in vitro kinase assay with PKC-delta. Enhanced gating was evaluated by expressing wild-type (WT) or mutant H(V)1 channels in LK35.2 cells, a B cell hybridoma. Stimulation by phorbol myristate acetate enhanced WT channel gating, and this effect was reversed by treatment with the PKC inhibitor GF109203X. The single mutant T29A or double mutant T29A/S97A failed to respond to phorbol myristate acetate or GF109203X. In contrast, the S97A mutant responded like cells transfected with WT H(V)1. We conclude that under these conditions, direct phosphorylation of the proton channel molecule at Thr(29) is primarily responsible for the enhancement of proton channel gating. This phosphorylation is crucial to activation of the proton conductance during the respiratory burst in phagocytes.

Deregulated expression of cytokine receptor gene, CRLF2, is involved in lymphoid transformation in B-cell precursor acute lymphoblastic leukemia.

We report 2 novel, cryptic chromosomal abnormalities in precursor B-cell acute lymphoblastic leukemia (BCP-ALL): a translocation, either t(X;14)(p22;q32) or t(Y;14)(p11;q32), in 33 patients and an interstitial deletion, either del(X)(p22.33p22.33) or del(Y)(p11.32p11.32), in 64 patients, involving the pseudoautosomal region (PAR1) of the sex chromosomes. The incidence of these abnormalities was 5% in childhood ALL (0.8% with the translocation, 4.2% with the deletion). Patients with the translocation were older (median age, 16 years), whereas the patients with the deletion were younger (median age, 4 years). The 2 abnormalities result in deregulated expression of the cytokine receptor, cytokine receptor-like factor 2, CRLF2 (also known as thymic stromal-derived lymphopoietin receptor, TSLPR). Overexpression of CRLF2 was associated with activation of the JAK-STAT pathway in cell lines and transduced primary B-cell progenitors, sustaining their proliferation and indicating a causal role of CRLF2 overexpression in lymphoid transformation. In Down syndrome (DS) ALL and 2 non-DS BCP-ALL cell lines, CRLF2 deregulation was associated with mutations of the JAK2 pseudokinase domain, suggesting oncogenic cooperation as well as highlighting a link between non-DS ALL and JAK2 mutations.

Pancreatic Cancer Chemotherapy Is Potentiated by Induction of Tertiary Lymphoid Structures in Mice.

BACKGROUND AND AIMS: The presence of tertiary lymphoid structures (TLSs) may confer survival benefit to patients with pancreatic ductal adenocarcinoma (PDAC), in an otherwise immunologically inert malignancy. Yet, the precise role in PDAC has not been elucidated. Here, we aim to investigate the structure and role of TLSs in human and murine pancreatic cancer. METHODS: Multicolor immunofluorescence and immunohistochemistry were used to fully characterize TLSs in human and murine (transgenic [KPC (KrasG12D, p53R172H, Pdx-1-Cre)] and orthotopic) pancreatic cancer. An orthotopic murine model was developed to study the development of TLSs and the effect of the combined chemotherapy and immunotherapy on tumor growth. RESULTS: Mature, functional TLSs are not ubiquitous in human PDAC and KPC murine cancers and are absent in the orthotopic murine model. TLS formation can be induced in the orthotopic model of PDAC after intratumoral injection of lymphoid chemokines (CXCL13/CCL21). Coadministration of systemic chemotherapy (gemcitabine) and intratumoral lymphoid chemokines into orthotopic tumors altered immune cell infiltration ,facilitating TLS induction and potentiating antitumor activity of chemotherapy. This resulted in significant tumor reduction, an effect not achieved by either treatment alone. Antitumor activity seen after TLS induction is associated with B cell-mediated dendritic cell activation. CONCLUSIONS: This study provides supportive evidence that TLS induction may potentiate the antitumor activity of chemotherapy in a murine model of PDAC. A detailed understanding of TLS kinetics and their induction, owing to multiple host and tumor factors, may help design personalized therapies harnessing the potential of immune-oncology.

mTOR-dependent translation amplifies microglia priming in aging mice.

Microglia maintain homeostasis in the brain. However, with age, they become primed and respond more strongly to inflammatory stimuli. We show here that microglia from aged mice had upregulated mTOR complex 1 signaling controlling translation, as well as protein levels of inflammatory mediators. Genetic ablation of mTOR signaling showed a dual yet contrasting effect on microglia priming: it caused an NF-κB-dependent upregulation of priming genes at the mRNA level; however, mice displayed reduced cytokine protein levels, diminished microglia activation, and milder sickness behavior. The effect on translation was dependent on reduced phosphorylation of 4EBP1, resulting in decreased binding of eIF4E to eIF4G. Similar changes were present in aged human microglia and in damage-associated microglia, indicating that upregulation of mTOR-dependent translation is an essential aspect of microglia priming in aging and neurodegeneration.