Interrogation of the Microenvironmental Landscape in Brain Tumors Reveals Disease-Specific Alterations of Immune Cells.

Brain malignancies encompass a range of primary and metastatic cancers, including low-grade and high-grade gliomas and brain metastases (BrMs) originating from diverse extracranial tumors. Our understanding of the brain tumor microenvironment (TME) remains limited, and it is unknown whether it is sculpted differentially by primary versus metastatic disease. We therefore comprehensively analyzed the brain TME landscape via flow cytometry, RNA sequencing, protein arrays, culture assays, and spatial tissue characterization. This revealed disease-specific enrichment of immune cells with pronounced differences in proportional abundance of tissue-resident microglia, infiltrating monocyte-derived macrophages, neutrophils, and T cells. These integrated analyses also uncovered multifaceted immune cell activation within brain malignancies entailing converging transcriptional trajectories while maintaining disease- and cell-type-specific programs. Given the interest in developing TME-targeted therapies for brain malignancies, this comprehensive resource of the immune landscape offers insights into possible strategies to overcome tumor-supporting TME properties and instead harness the TME to fight cancer.

Monocyte to macrophage differentiation and changes in cellular redox homeostasis promote cell type-specific HIV latency reactivation.

HIV latency regulation in monocytes and macrophages can vary according to signals directing differentiation, polarization, and function. To investigate these processes, we generated an HIV latency model in THP-1 monocytes and showed differential levels of HIV reactivation among clonal populations. Monocyte-to-macrophage differentiation of HIV-infected primary human CD14+ and THP-1 cells induced HIV reactivation and showed that virus production increased concomitant with macrophage differentiation. We applied the HIV-infected THP-1 monocyte-to-macrophage (MLat) model to assess the biological mechanisms regulating HIV latency dynamics during monocyte-to-macrophage differentiation. We pinpointed protein kinase C signaling pathway activation and Cyclin T1 upregulation as inherent differentiation mechanisms that regulate HIV latency reactivation. Macrophage polarization regulated latency, revealing proinflammatory M1 macrophages suppressed HIV reactivation while anti-inflammatory M2 macrophages promoted HIV reactivation. Because macrophages rely on reactive-oxygen species (ROS) to exert numerous cellular functions, we disrupted redox pathways and found that inhibitors of the thioredoxin (Trx) system acted as latency-promoting agents in T-cells and monocytes, but opposingly acted as latency-reversing agents in macrophages. We explored this mechanism with Auranofin, a clinical candidate for reducing HIV reservoirs, and demonstrated Trx reductase inhibition led to ROS induced NF-κB activity, which promoted HIV reactivation in macrophages, but not in T-cells and monocytes. Collectively, cell type-specific differences in HIV latency regulation could pose a barrier to HIV eradication strategies.

Immunity in malignant brain tumors: Tumor entities, role of immunotherapy, and specific contribution of myeloid cells to the brain tumor microenvironment.

Malignant brain tumors lack effective treatment, that can improve their poor overall survival achieved with standard of care. Advancement in different cancer treatments has shifted the focus in brain tumor research and clinical trials toward immunotherapy-based approaches. The investigation of the immune cell landscape revealed a dominance of myeloid cells in the tumor microenvironment. Their exact roles and functions are the subject of ongoing research. Current evidence suggests a complex interplay of tumor cells and myeloid cells with competing functions toward support vs. control of tumor growth. Here, we provide a brief overview of the three most abundant brain tumor entities: meningioma, glioma, and brain metastases. We also describe the field of ongoing immunotherapy trials and their results, including immune checkpoint inhibitors, vaccination studies, oncolytic viral therapy, and CAR-T cells. Finally, we summarize the phenotypes of microglia, monocyte-derived macrophages, border-associated macrophages, neutrophils, and potential novel therapy targets.

SGK3 deficiency in macrophages suppresses angiotensin II-induced cardiac remodeling via regulating Ndufa13-mediated mitochondrial oxidative stress.

Infiltration of monocyte-derived macrophages plays a crucial role in cardiac remodeling and dysfunction. The serum and glucocorticoid-inducible protein kinase 3 (SGK3) is a downstream factor of PI3K signaling, regulating various biological processes via an AKT-independent signaling pathway. SGK3 has been implicated in cardiac remodeling. However, the contribution of macrophagic SGK3 to hypertensive cardiac remodeling remains unclear. A cardiac remodeling model was established by angiotensin II (Ang II) infusion in SGK3-Lyz2-CRE (f/f, +) and wild-type mice to assess the function of macrophagic SGK3. Additionally, a co-culture system of SGK3-deficient or wild-type macrophages and neonatal rat cardiomyocytes (CMs) or neonatal rat fibroblasts (CFs) was established to evaluate the effects of SGK3 and the underlying mechanisms. SGK3 levels were significantly elevated in both peripheral blood mononuclear cells and serum from patients with heart failure. Macrophage SGK3 deficiency attenuated Ang II-induced macrophage infiltration, myocardial hypertrophy, myocardial fibrosis, and mitochondrial oxidative stress. RNA sequencing suggested Ndufa13 as the candidate gene in the effect of SGK3 on Ang II-induced cardiac remolding. Downregulation of Ndufa13 in CMs and CFs prevented the suppression of cardiac remodeling caused by SGK3 deficiency in macrophages. Mechanistically, the absence of SGK3 led to a reduction in IL-1ß secretion by inhibiting the NLRP3/Caspase-1/IL-1ß pathway in macrophages, consequently suppressing upregulated Ndufa13 expression and mitochondrial oxidative stress in CMs and CFs. This study provides new evidence that SGK3 is a potent contributor to the pathogenesis of hypertensive cardiac remodeling, and targeting SGK3 in macrophages may serve as a potential therapy for cardiac remodeling.

Long-term recruitment of peripheral immune cells to brain scars after a neonatal insult.

Although brain scars in adults have been extensively studied, there is less data available regarding scar formation during the neonatal period, and the involvement of peripheral immune cells in this process remains unexplored in neonates. Using a murine model of neonatal hypoxic-ischemic encephalopathy (HIE) and confocal microscopy, we characterized the scarring process and examined the recruitment of peripheral immune cells to cortical and hippocampal scars for up to 1 year post-insult. Regional differences in scar formation were observed, including the presence of reticular fibrotic networks in the cortex and perivascular fibrosis in the hippocampus. We identified chemokines with chronically elevated levels in both regions and demonstrated, through a parabiosis-based strategy, the recruitment of lymphocytes, neutrophils, and monocyte-derived macrophages to the scars several weeks after the neonatal insult. After 1 year, however, neutrophils and lymphocytes were absent from the scars. Our data indicate that peripheral immune cells are transient components of HIE-induced brain scars, opening up new possibilities for late therapeutic interventions.

Distinct spatiotemporal features of microglia and monocyte-derived macrophages in glioma.

Gliomas are the most frequent primary tumors of the brain. Glioma progression is regulated by the tumor microenvironment, which is mainly composed of tumor-associated microglia (TA-MG) and monocyte-derived macrophages (MDM). Recent studies have highlighted the distinct properties of these cells in glioma progression. However, their spatiotemporal alteration during tumor progression has not been fully explored. Using a genetic lineage tracing approach, we show that TA-MG and MDMs differ in their spatiotemporal distribution and interaction with other components of the glioma microenvironment. MDM were present only inside the tumor, whereas TA-MG accumulated both outside and inside the tumor. However, TA-MG was eliminated from the tumor mass as the tumor progressed. Depletion of MDM led to enhanced occupancy of TA-MG in the tumor core, indicating that TA-MG elimination was regulated by MDM. TA-MG and MDM are heterogeneous cell populations whose compositions and properties can change during tumor progression. Finally, MG, TA-MG and MDM were enriched in the perivascular area (PVA) compared to more distal blood vessel-associated areas. However, inside the tumor, the MDM enrichment in PVA was higher than that in TA-MG. Collectively, we established that TA-MG and MDM exhibit different spatiotemporal features in glioma, suggesting distinctive roles during tumor progression.

Pleural CD14+ monocytes/macrophages of healthy adolescents show a high expression of metallothionein family genes.

Nowadays laparoscopic interventions enable the collection of resident macrophage populations out of the human cavities. We employed this technique to isolate pleural monocytes/macrophages from healthy young adults who underwent a correction of pectus excavatum. High quality CD14+ monocytes/macrophages (plMo/Mφ) were used for RNA-sequencing (RNA-seq) in comparison with human monocyte-derived macrophages (MDM) natural (MDM-0) or IL-4-polarized (MDM-IL4). Transcriptome analysis revealed 7166 and 7076 differentially expressed genes (DEGs) in plMo/Mφ relative to natural MDM-0 and polarized MDM-IL4, respectively. The gene set enrichment analysis, which was used to compare RNA-seq data from plMo/Mφ with single-cell (scRNA-seq) data online from human bronchial lavage macrophages, showed that plMo/Mφs are characterized by a high expression of genes belonging to the metallothionein (MT) family, and that the expression of these genes is significantly higher in plMo/Mφ than in MDM-0 or MDM-IL4. Our results provide additional insights on high MTs-expressing macrophage subsets, which seem to be present not only in bronchial lavage of healthy adults or in pleural exudates of lung cancer patients but also in pleural fluid of healthy young adults. Macrophage subsets expressing high MTs may have specific roles in lung defense, repair, and homeostasis, and require further investigations.

Macrophage niche imprinting as a determinant of macrophage identity and function.

Macrophage niches are the anatomical locations within organs or tissues consisting of various cells, intercellular and extracellular matrix, transcription factors, and signaling molecules that interact to influence macrophage self-maintenance, phenotype, and behavior. The niche, besides physically supporting macrophages, imposes a tissue- and organ-specific identity on the residing and infiltrating monocytes and macrophages. In this review, we give examples of macrophage niches and the modes of communication between macrophages and surrounding cells. We also describe how macrophages, acting against their immune defensive nature, can create a hospitable niche for pathogens and cancer cells.

The abortive SARS-CoV-2 infection of osteoclast precursors promotes their differentiation into osteoclasts.

The Coronavirus Disease 2019 (COVID-19) pandemic has resulted in the loss of millions of lives, although a majority of those infected have managed to survive. Consequently, a set of outcomes, identified as long COVID, is now emerging. While the primary target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the respiratory system, the impact of COVID-19 extends to various body parts, including the bone. This study aims to investigate the effects of acute SARS-CoV-2 infection on osteoclastogenesis, utilizing both ancestral and Omicron viral strains. Monocyte-derived macrophages, which serve as precursors to osteoclasts, were exposed to both viral variants. However, the infection proved abortive, even though ACE2 receptor expression increased postinfection, with no significant impact on cellular viability and redox balance. Both SARS-CoV-2 strains heightened osteoclast formation in a dose-dependent manner, as well as CD51/61 expression and bone resorptive ability. Notably, SARS-CoV-2 induced early pro-inflammatory M1 macrophage polarization, shifting toward an M2-like profile. Osteoclastogenesis-related genes (RANK, NFATc1, DC-STAMP, MMP9) were upregulated, and surprisingly, SARS-CoV-2 variants promoted RANKL-independent osteoclast formation. This thorough investigation illuminates the intricate interplay between SARS-CoV-2 and osteoclast precursors, suggesting potential implications for bone homeostasis and opening new avenues for therapeutic exploration in COVID-19.

Nano(bio)Materials Do Not Affect Macrophage Phenotype-A Study Conducted by the REFINE Project.

Macrophages are well known for their involvement in the biocompatibility, as well as biodistribution, of nano(bio)materials. Although there are a number of rodent cell lines, they may not fully recapitulate primary cell responses, particularly those of human cells. Isolation of tissue-resident macrophages from humans is difficult and may result in insufficient cells with which to determine the possible interaction with nano(bio)materials. Isolation of primary human monocytes and differentiation to monocyte-derived macrophages may provide a useful tool with which to further study these interactions. To that end, we developed a standard operating procedure for this differentiation, as part of the Regulatory Science Framework for Nano(bio)material-based Medical Products and Devices (REFINE) project, and used it to measure the secretion of bioactive molecules from M1 and M2 differentiated monocytes in response to model nano(bio)materials, following an initial assessment of pyrogenic contamination, which may confound potential observations. The SOP was deployed in two partner institutions with broadly similar results. The work presented here shows the utility of this assay but highlights the relevance of donor variability in responses to nano(bio)materials. Whilst donor variability can provide some logistical challenges to the application of such assays, this variability is much closer to the heterogeneous cells that are present in vivo, compared to homogeneous non-human cell lines.

Replication and innate immune responses of two chikungunya virus genotypes in human monocyte-derived macrophages.

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne virus, which causes epidemics of fever, joint pain and rash. There are three genotypes: West African, East/Central/South/Africa (ECSA) and Asian, with the latter two predominant globally. Genotype-specific differences in clinical presentations, virulence and immunopathology have been described. Macrophages are key cells in immune responses against CHIKV. Circulating blood monocytes enter tissue to differentiate into monocyte-derived macrophages (MDMs) in response to CHIKV infection at key replication sites such as lymphoid organs and joints. This study analyses differences in replication and induced immune mediators following infection of MDMs with Asian and ECSA CHIKV genotypes. Primary human MDMs were derived from residual blood donations. Replication of Asian (MY/06/37348) or ECSA (MY/08/065) genotype strains of CHIKV in MDMs was measured by plaque assay. Nineteen immune mediators were measured in infected cell supernatants using multiplexed immunoassay or ELISA. MY/08/065 showed significantly higher viral replication at 24 h post-infection (h p.i.) but induced significantly lower expression of proinflammatory cytokines (CCL-2, CCL-3, CCL-4, RANTES and CXCL-10) and the anti-inflammatory IL-1Ra compared to MY/06/37348. No differences were seen at later time points up to 72 h p.i. During early infection, MY/08/065 induced lower proinflammatory immune responses in MDMs. In vivo, this may lead to poorer initial control of viral infection, facilitating CHIKV replication and dissemination to other sites such as joints. This may explain the consistent past findings that the ECSA genotype is associated with greater viremia and severity of symptoms than the Asian genotype. Knowledge of CHIKV genotype-specific immunopathogenic mechanisms in human MDMs is important in understanding of clinical epidemiology, biomarkers and therapeutics in areas with co-circulation of different genotypes.

Endothelial Toll-like receptor 4 is required for microglia activation in the murine retina after systemic lipopolysaccharide exposure.

BACKGROUND: Clustering of microglia around the vasculature has been reported in the retina and the brain after systemic administration of lipopolysaccharides (LPS) in mice. LPS acts via activation of Toll-like receptor 4 (TRL4), which is expressed in several cell types including microglia, monocytes and vascular endothelial cells. The purpose of this study was to investigate the effect of systemic LPS in the pigmented mouse retina and the involvement of endothelial TLR4 in LPS-induced retinal microglia activation. METHODS: C57BL/6J, conditional knockout mice that lack Tlr4 expression selectively on endothelial cells (TekCre-posTlr4loxP/loxP) and TekCre-negTlr4loxP/loxP mice were used. The mice were injected with 1 mg/kg LPS via the tail vein once per day for a total of 4 days. Prior to initiation of LPS injections and approximately 5 h after the last injection, in vivo imaging using fluorescein angiography and spectral-domain optical coherence tomography was performed. Immunohistochemistry, flow cytometry, electroretinography and transmission electron microscopy were utilized to investigate the role of endothelial TLR4 in LPS-induced microglia activation and retinal function. RESULTS: Activation of microglia, infiltration of monocyte-derived macrophages, impaired ribbon synapse organization and retinal dysfunction were observed after the LPS exposure in C57BL/6J and TekCre-negTlr4loxP/loxP mice. None of these effects were observed in the retinas of conditional Tlr4 knockout mice after the LPS challenge. CONCLUSIONS: The findings of the present study suggest that systemic LPS exposure can have detrimental effects in the healthy retina and that TLR4 expressed on endothelial cells is essential for retinal microglia activation and retinal dysfunction upon systemic LPS challenge. This important finding provides new insights into the role of microglia-endothelial cell interaction in inflammatory retinal disease.

Novel phenotypical and functional sub-classification of liver macrophages highlights changes in population dynamics in experimental mouse models.

Liver macrophages are critical components of systemic immune system defense mechanisms. F4/80high Kupffer cells (KCs) are the predominant liver-resident macrophages and the first immune cells to contact pathogens entering the liver. F4/80low monocyte-derived macrophages (MoMφs) are essential macrophages that modulate liver immune functions. Here we report a novel method of identifying subpopulations of these two populations using traditional flow cytometry and examine each subpopulation for its putative roles in the pathogenesis of an experimental non-alcoholic steatohepatitis model. Using male C57BL/6 mice, we isolated and analyzed liver non-parenchymal cells by flow cytometry. We identified F4/80high and F4/80low macrophage populations and characterized subpopulations using uniform manifold approximation and projection. We identified three subpopulations in F4/80high macrophages: CD163(+) KCs, CD163(-) KCs, and liver capsular macrophages. CD163(+) KCs had higher phagocytic and bactericidal activities and more complex cellular structures than CD163(-) KCs. We also identified four subpopulations of F4/80low MoMφs based on Ly6C and MHC class II expression: infiltrating monocytes, pro-inflammatory MoMφs, Ly6C(-) monocytes, and conventional dendritic cells. CCR2 knock-out mice expressed lower levels of these monocyte-derived cells, and the count varied by subpopulation. In high-fat- and cholesterol-diet-fed mice, only one subpopulation, pro-inflammatory MoMφs, significantly increased in count. This indicates that changes to this subpopulation is the first step in the progression to non-alcoholic steatohepatitis. The community can use our novel subpopulation and gating strategy to better understand complex immunological mechanisms in various liver disorders through detailed analysis of these subpopulations.

Cardiac macrophages undergo dynamic changes after coxsackievirus B3 infection and promote the progression of myocarditis.

Although most patients with acute viral myocarditis recover spontaneously, some patients progress to heart failure. Perturbations in innate immunity may partially explain the heterogeneity of clinical outcomes. As the most abundant immune cells in the heart, cardiac macrophages have heterogeneous origins, including embryonic-derived resident macrophages (ResMϕs) and monocyte-derived macrophages (MoMFs). However, the time course change and role of cardiac macrophage subsets has not been fully explored. In the present study, we found that BALB/c mice had prolonged MoMF accumulation and low proportions of ResMϕs that could not be restored to normal levels. MoMFs of BALB/c mice generally exhibit an M1-dominant functional phenotype. Moreover, the preferential depletion of MoMF by a C-C chemokine receptor type 2 (CCR2) inhibitor resulted in improved acute myocarditis and chronic fibrosis, as well as the recovery of ResMϕs number and reduced CD4+ T cell expansion. Hence, immunomodulatory therapy that targets the balance among cardiac macrophages and modulates their function is expected to prevent the progression of cardiac injury to overt heart failure and improve adverse outcomes.

Immunological Features of Non-neuronal Brain Cells: Implications for Alzheimer's Disease Immunotherapy.

An interaction network exists among cells within the brain, maintaining brain homeostasis and ensuring its functional plasticity. In addition to neurons, participating cells include astrocytes, oligodendrocytes, and microglia. Peripheral immune cells, such as monocytes and lymphocytes, have also been found to play an important role in supporting the brain in health and assisting in its repair. Here, we describe the multiple immune-specific modes of cellular dialogue among cells within the mammalian brain and their crosstalk with the periphery in both health and disease. We further suggest that interventions directed at boosting the peripheral immune response can restore the balance between the brain and the immune system and can rewire their communication to modify chronic neurodegenerative diseases.

Acute and Chronic Macrophage Differentiation Modulates TREM2 in a Personalized Alzheimer's Patient-Derived Assay.

Neuroinflammation plays a pivotal role in the pathogenesis of Alzheimer`s disease (AD). Brain macrophage populations differentially modulate the immune response to AD pathology according to the disease stage. Triggering receptor expressed on myeloid cells 2 (TREM2) is known to play a protective role in AD and has been postulated as a putative therapeutic target. Whether, and to which extent TREM2 expression can be modulated in the aged macrophage population of the brain is unknown, emphasizing the need for a human, patient-specific model. Using cells from AD patients and matched controls (CO) we designed an assay based on monocyte-derived macrophages to mimic brain-infiltrating macrophages and to assess the individualized TREM2 synthesis in vitro. We systematically assessed the effects of short-term (acute-2 days) and long-term (chronic-10 days) M1- (LPS), M2- (IL-10, IL-4, TGF-ß), and M0- (vehicle) macrophage differentiation on TREM2 synthesis. Moreover, the effects of retinoic acid (RA), a putative TREM2 modulator, on individualized TREM2 synthesis were assessed. We report increased TREM2 synthesis after acute M2- compared to M1-differentiation in CO- but not AD-derived cells. Chronic M2- and M0-differentiation however resulted in an increase of TREM2 synthesis in both AD- and CO-derived cells while chronic M1-differentiation increased TREM2 in AD-derived cells only. Moreover, chronic M2- and M0-differentiation improved the amyloid-ß (Aß) uptake of the CO-derived whereas M1-differentiation of the AD-derived cells. Interestingly, RA-treatment did not modulate TREM2. In the age of personalized medicine, our individualized model could be used to screen for potential drug-mediated treatment responses in vitro. Triggering receptor expressed on myeloid cells 2 (TREM2) has been postulated as a putative therapeutic target in Alzheimer's disease (AD). Using cells from AD patients and matched controls (CO), we designed a monocyte-derived macrophages (Mo-MФs) assay to assess the individualized TREM2 synthesis in vitro. We report increased TREM2 synthesis after acute M2- compared to M1- macrophage differentiation in CO- but not AD-derived cells. Chronic M2- and M0- differentiation however resulted in an increase of TREM2 synthesis in both AD- and CO-derived cells while chronic M1-differentiation increased TREM2 in AD-cells only.

TNF-α-dependent lung inflammation upregulates PD-L1 in monocyte-derived macrophages to contribute to lung tumorigenesis.

Chronic inflammation, which is dominated by macrophage-involved inflammatory responses, is an instigator of cancer initiation. Macrophages are the most abundant immune cells in healthy lungs, and associated with lung tumor development and promotion. PD-L1 is a negative molecule in macrophages and correlated with an immunosuppressive function in tumor environment. Macrophages expressing PD-L1, rather than tumor cells, exhibits a critical role in tumor growth and progression. However, whether and how PD-L1 in macrophages contributes to inflammation-induced lung tumorigenesis requires further elucidation. Here, we found that higher expression of PD-L1 in CD11b+ CD206+ macrophages was positively correlated with tumor progression and PD-1+ CD8+ T cells population in human adenocarcinoma patients. In the urethane-induced inflammation-driven lung adenocarcinoma (IDLA) mouse model, the infiltration of circulating CD11bhigh F4/80+ monocyte-derived macrophages (MoMs) was increased in pro-tumor inflamed lung tissues and lung adenocarcinoma. PD-L1 was mainly upregulated in MoMs associated with enhanced T cells exhaustion in lung tissues. Anti-PD-L1 treatment can reduce T cells exhaustion at pro-tumor inflammatory stage, and then inhibit tumorigenesis in IDLA. The pro-tumor lung inflammation depended on TNF-α to upregulate PD-L1 and CSN6 expression in MoMs, and induced cytokines production by alveolar type-II cells (AT-II). Furthermore, inflammatory AT-II cells could secret TNF-α to upregulate PD-L1 expression in bone-marrow driven macrophages (BM-M0). Inhibition of CSN6 decreased PD-L1 expression in TNF-α-activated macrophage in vitro, suggesting a critical role of CSN6 in PD-L1 upregulation. Thus, pro-tumor inflammation can depend on TNF-α to upregulate PD-L1 in recruited MoMs, which may be essential for lung tumorigenesis.

The Role of Macrophages in Atherosclerosis: Participants and Therapists.

Currently, atherosclerosis, characterized by the dysfunction of lipid metabolism and chronic inflammation in the intimal space of the vessel, is considered to be a metabolic disease. As the most abundant innate immune cells in the body, macrophages play a key role in the onset, progression, or regression of atherosclerosis. For example, macrophages exhibit several polarization states in response to microenvironmental stimuli; an increasing proportion of macrophages, polarized toward M2, can suppress inflammation, scavenge cell debris and apoptotic cells, and contribute to tissue repair and fibrosis. Additionally, specific exosomes, generated by macrophages containing certain miRNAs and effective efferocytosis of macrophages, are crucial for atherosclerosis. Therefore, macrophages have emerged as a novel potential target for anti-atherosclerosis therapy. This article reviews the role of macrophages in atherosclerosis from different aspects: origin, phenotype, exosomes, and efferocytosis, and discusses new approaches for the treatment of atherosclerosis.

Effects of autologous serum on TREM2 and APOE in a personalized monocyte-derived macrophage assay of late-onset Alzheimer's patients.

BACKGROUND: Age-associated deterioration of the immune system contributes to a chronic low-grade inflammatory state known as "inflammaging" and is implicated in the pathogenesis of late-onset Alzheimer's disease (LOAD). Whether changes in the tissue environment caused by circulatory factors associated with aging may alter the innate immune response is unknown. Monocyte-derived macrophages (Mo-MФs) infiltrating the brain alongside microglia are postulated to play a modulatory role in LOAD and both express triggering receptor expressed on myeloid cells 2 (TREM2). Apolipoprotein E (APOE) acts as a ligand for TREM2, and their role in amyloid beta (Aß) clearance highlights their importance in LOAD. However, the influence of the patient's own milieu (autologous serum) on the synthesis of TREM2 and APOE in infiltrating macrophages remains unknown. OBJECTIVES: To functionally assess patient-specific TREM2 and APOE synthesis, we designed a personalized assay based on Mo-MФs using monocytes from LOAD patients and matched controls (CO). We assessed the influence of each participant's own milieu, by examining the effect of short- (1 day) and long- (10 days) term differentiation of the cells in the presence of the donor´s autologous serum (AS) into M1-, M2- or M0-macrophages. Additionally, sex differences and Aß-uptake ability in short- and long-term differentiated Mo-MФs were assessed. RESULTS: We showed a time-dependent increase in TREM2 and APOE protein levels in LOAD- and CO-derived cells. While AS did not differentially modulate TREM2 compared to standard fetal calf serum (FCS), AS decreased APOE levels in M2 macrophages but increased levels in M1 macrophages. Interestingly, higher levels of TREM2 and lower levels of APOE were detected in female- than in male- LOAD patients. Finally, we report decreased Aß-uptake in long-term differentiated CO- and LOAD-derived cells, particularly in APOEε4(+) carriers. CONCLUSIONS: We demonstrate for the first time the suitability of a personalized Mo-MФ cell culture-based assay for studying functional TREM2 and APOE synthesis in a patient's own aged milieu. Our strategy may thus provide a useful tool for future research on diagnostic and therapeutic aspects of personalized medicine.

Cytocidal macrophages in symbiosis with CD4 and CD8 T cells cause acute diabetes following checkpoint blockade of PD-1 in NOD mice.

Autoimmune diabetes is one of the complications resulting from checkpoint blockade immunotherapy in cancer patients, yet the underlying mechanisms for such an adverse effect are not well understood. Leveraging the diabetes-susceptible nonobese diabetic (NOD) mouse model, we phenocopy the diabetes progression induced by programmed death 1 (PD-1)/PD-L1 blockade and identify a cascade of highly interdependent cellular interactions involving diabetogenic CD4 and CD8 T cells and macrophages. We demonstrate that exhausted CD8 T cells are the major cells that respond to PD-1 blockade producing high levels of IFN-γ. Most importantly, the activated T cells lead to the recruitment of monocyte-derived macrophages that become highly activated when responding to IFN-γ. These macrophages acquire cytocidal activity against ß-cells via nitric oxide and induce autoimmune diabetes. Collectively, the data in this study reveal a critical role of macrophages in the PD-1 blockade-induced diabetogenesis, providing new insights for the understanding of checkpoint blockade immunotherapy in cancer and infectious diseases.