Summary Microglia play critical roles in brain development, homeostasis, and neurological disorders. Here, we report that human microglial-like cells (iMGL) can be differentiated from iPSCs to study their function in neurological diseases, like Alzheimer’s disease (AD). We find that iMGLs develop in vitro similarly to microglia in vivo and whole transcriptome analysis demonstrates that they are highly similar to cultured adult and fetal human microglia. Functional assessment of iMGLs reveals that they secrete cytokines in response to inflammatory stimuli, migrate and undergo calcium transients, and robustly phagocytose CNS substrates. iMGLs were used to examine the effects of Aβ fibrils and brain-derived tau oligomers on AD-related gene expression and to interrogate mechanisms involved in synaptic pruning. Furthermore, iMGLs transplanted into transgenic mice and human brain organoids resemble microglia in vivo. Together, these findings demonstrate that iMGLs can be used to study microglial function, providing important new insight into human neurological disease.

The innate immune system is strongly implicated in the pathogenesis of Alzheimer's disease (AD). In contrast, the role of adaptive immunity in AD remains largely unknown. However, numerous clinical trials are testing vaccination strategies for AD, suggesting that T and B cells play a pivotal role in this disease. To test the hypothesis that adaptive immunity influences AD pathogenesis, we generated an immune-deficient AD mouse model that lacks T, B, and natural killer (NK) cells. The resulting "Rag-5xfAD" mice exhibit a greater than twofold increase in β-amyloid (Aβ) pathology. Gene expression analysis of the brain implicates altered innate and adaptive immune pathways, including changes in cytokine/chemokine signaling and decreased Ig-mediated processes. Neuroinflammation is also greatly exacerbated in Rag-5xfAD mice as indicated by a shift in microglial phenotype, increased cytokine production, and reduced phagocytic capacity. In contrast, immune-intact 5xfAD mice exhibit elevated levels of nonamyloid reactive IgGs in association with microglia, and treatment of Rag-5xfAD mice or microglial cells with preimmune IgG enhances Aβ clearance. Last, we performed bone marrow transplantation studies in Rag-5xfAD mice, revealing that replacement of these missing adaptive immune populations can dramatically reduce AD pathology. Taken together, these data strongly suggest that adaptive immune cell populations play an important role in restraining AD pathology. In contrast, depletion of B cells and their appropriate activation by T cells leads to a loss of adaptiveinnate immunity cross talk and accelerated disease progression.is the leading cause of age-related neurodegeneration, affecting over 5.2 million people in the United States alone (1). Pathologically, AD is characterized by two hallmark protein aggregates, amyloid-β (Aβ) plaques and neurofibrillary tangles, that are accompanied by neuroinflammation, including microgliosis, elevated cytokine production, and activation of complement pathways (2-5). Initially, microglia respond to and surround plaques, degrading Aβ by phagocytosis (for review, see refs. 6-8). However, chronic activation of these cells shift microglia to a more proinflammatory and less phagocytic state (9, 10). Although much of the data implicating microglia in AD has come from neuropathological investigation, recent genome-wide association studies have provided the first genetic evidence (to our knowledge) linking microglia dysfunction to AD, with the discovery of risk polymorphisms in several immune system genes: CR1, TREM2, CD33, HLA-DRB5, MS4A6A, and ABCA7 (8,(11)(12)(13)(14)(15).In contrast to the field's increasing understanding of the role of innate immunity in AD, comparatively little is known about whether the adaptive immune system might also influence AD. Those studies that have examined these peripheral populations have largely focused on questions about their potential as biomarkers or their role in active Aβ immunization (3, 16). However, the adaptive and innate immune systems rarely fu...

Genome-wide association studies (GWAS) have identified more than 40 loci associated with Alzheimer’s disease (AD), but the causal variants, regulatory elements, genes and pathways remain largely unknown, impeding a mechanistic understanding of AD pathogenesis. Previously, we showed that AD risk alleles are enriched in myeloid-specific epigenomic annotations. Here, we show that they are specifically enriched in active enhancers of monocytes, macrophages and microglia. We integrated AD GWAS with myeloid epigenomic and transcriptomic datasets using analytical approaches to link myeloid enhancer activity to target gene expression regulation and AD risk modification. We identify AD risk enhancers and nominate candidate causal genes among their likely targets (including AP4E1, AP4M1, APBB3, BIN1, MS4A4A, MS4A6A, PILRA, RABEP1, SPI1, TP53INP1, and ZYX) in twenty loci. Fine-mapping of these enhancers nominates candidate functional variants that likely modify AD risk by regulating gene expression in myeloid cells. In the MS4A locus we identified a single candidate functional variant and validated it in human induced pluripotent stem cell (hiPSC)-derived microglia and brain. Taken together, this study integrates AD GWAS with multiple myeloid genomic datasets to investigate the mechanisms of AD risk alleles and nominates candidate functional variants, regulatory elements and genes that likely modulate disease susceptibility.

Brain aging and neurodegeneration are associated with prominent microglial reactivity and activation of innate immune response pathways, commonly referred to as neuroinflammation. One such pathway, the type I interferon response, recognizes viral or mitochondrial DNA in the cytoplasm via activation of the recently discovered cyclic dinucleotide synthetase cGAS and the cyclic dinucleotide receptor STING. Here we show that the FDA-approved antiviral drug ganciclovir (GCV) induces a type I interferon response independent of its canonical thymidine kinase target. Inhibition of components of the STING pathway, including STING, IRF3, Tbk1, extracellular IFNβ, and the Jak-Stat pathway resulted in reduced activity of GCV and its derivatives. Importantly, functional STING was necessary for GCV to inhibit inflammation in cultured myeloid cells and in a mouse model of multiple sclerosis. Collectively, our findings uncover an unexpected new activity of GCV and identify the STING pathway as a regulator of microglial reactivity and neuroinflammation.

Chronic hypoxia is an inciting factor for the development of pulmonary arterial hypertension. The mechanisms involved in the development of hypoxic pulmonary hypertension (HPH) include hypoxia-inducible factor 1 (HIF-1)-dependent transactivation of genes controlling pulmonary arterial smooth muscle cell (PASMC) intracellular calcium concentration ([Ca 2+ ] i ) and pH. Recently, digoxin was shown to inhibit HIF-1 transcriptional activity.In this study, we tested the hypothesis that digoxin could prevent and reverse the development of HPH. Mice were injected daily with saline or digoxin and exposed to room air or ambient hypoxia for 3 wk. Treatment with digoxin attenuated the development of right ventricle (RV) hypertrophy and prevented the pulmonary vascular remodeling and increases in PASMC [Ca 2+ ] i , pH, and RV pressure that occur in mice exposed to chronic hypoxia. When started after pulmonary hypertension was established, digoxin attenuated the hypoxia-induced increases in RV pressure and PASMC pH and [Ca 2+ ] i . These preclinical data support a role for HIF-1 inhibitors in the treatment of HPH.acriflavine | cardiac glycosides | pulmonary circulation P rolonged exposure to hypoxia occurs in physiological and pathological contexts, such as during a high-altitude sojourn or as a result of chronic obstructive pulmonary disease, respectively. Chronic hypoxia (CH) triggers maladaptive responses in the lung vasculature, leading to the development of hypoxic pulmonary hypertension (HPH) (1). Typically, HPH results from arteriolar constriction followed by vascular wall remodeling, which includes both thickening of the wall due to smooth muscle cell (SMC) and fibroblast proliferation, as well as extension of SMCs into previously nonmuscular precapillary arterioles. Over time, HPH causes right ventricle hypertrophy (RVH), which can lead to right ventricular (RV) failure and death.Although incompletely understood, some of the alterations in pulmonary arterial SMCs (PASMCs) that underlie the development of HPH have been delineated (2, 3). For example, in HPH, alterations in Ca 2+ and pH homeostasis contribute to growth and contraction of PASMCs. Our previous work demonstrated that HPH is characterized by increased PASMC intracellular pH (pH i ) due to increased activity and expression of Na + /H + exchanger isoform 1 (NHE1) (4, 5), and elevated intracellular Ca 2+ concentration ([Ca 2+ ] i ) (6) due to increased expression of canonical transient receptor potential (TRPC) proteins and enhanced Ca 2+ entry through nonselective cation channels (7). Critical aspects of the pathogenesis of HPH, including both the increase in basal [Ca 2+ ] i and alkalinization of PASMCs, are mediated by hypoxia-inducible factor 1 (HIF-1) (5, 7-9).HIF-1 is a heterodimeric transcription factor composed of HIF-1α and HIF-1β subunits that regulates the expression of hundreds of genes in response to hypoxia, including many genes associated with HPH (10). HIF-1β is ubiquitously expressed, whereas HIF-1α expression is O 2 -r...