The immunosuppressive drug cyclosporin A has an immunostimulatory function in CD8+ T cells.

Cyclosporin A is a well-established immunosuppressive drug used to treat or prevent graft-versus-host disease, the rejection of organ transplants, autoimmune disorders, and leukemia. It exerts its immunosuppressive effects by inhibiting calcineurin-mediated dephosphorylation of the nuclear factor of activated T cells (NFAT), thus preventing its nuclear entry and suppressing T cell activation. Here we report an unexpected immunostimulatory effect of cyclosporin A in activating the mammalian target of rapamycin complex 1 (mTORC1), a crucial metabolic hub required for T cell activation. Through screening a panel of tool compounds known to regulate mTORC1 activation, we found that cyclosporin A activated mTORC1 in CD8+ T cells in a 3-phosphoinositide-dependent protein kinase 1 (PDK1) and protein kinase B (PKB/AKT)-dependent manner. Mechanistically, cyclosporin A inhibited the calcineurin-mediated AKT dephosphorylation, thereby stabilizing mTORC1 signaling. Cyclosporin A synergized with mTORC1 pathway inhibitors, leading to potent suppression of proliferation and cytokine production in CD8+ T cells and an increase in the killing of acute T cell leukemia cells. Consequently, relying solely on CsA is insufficient to achieve optimal therapeutic outcomes. It is necessary to simultaneously target both the calcineurin-NFAT pathway and the mTORC1 pathway to maximize therapeutic efficacy.

Phagocytosis-related NADPH oxidase 2 subunit gp91phox contributes to neurodegeneration after repeated systemic challenge with lipopolysaccharides.

Repeated systemic challenge with lipopolysaccharides (LPS) can induce microglia activation and inflammatory neurodegeneration in the substantia nigra pars compacta region of mice. We now explored the role of mononuclear phagocytes associated nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX-2) in inflammatory neurodegeneration. Cybb-deficient NOX-2 knock-out (KO) and control wild type (WT) mice were treated intraperitoneally daily over four consecutive days with 1 µg/gbw/day LPS. Transcriptome analysis by RNA-seq of total brain tissue indicated increased LPS-induced upregulation of genes belonging to the reactive oxygen species and reactive nitrogen species production, complement and lysosome activation as well as apoptosis and necroptosis in WT compared to NOX-2 KO mice. Validation of up-regulated gene transcripts via qRT-PCR confirmed that LPS-challenged NOX-2 KO mice expressed lower levels of the microglial phagocytosis-related genes Nos2, Cd68, Aif1/Iba1, Cyba, Itgam, and Fcer1g compared to WT mice at Day 5 after systemic inflammatory challenge, but no significant differences in the pro-inflammatory genes Tnfα and Il1b as well as microglial IBA1 and CD68 intensities were observed between both genotypes. Furthermore, loss of tyrosine hydroxylase positive (TH+) and NeuN positive neurons in the substantia nigra pars compacta upon repeated systemic LPS application were attenuated in NOX-2 KO mice. Thus, our data demonstrate that loss of dopaminergic neurons in the substantia nigra pars compacta after repeated systemic challenge with LPS is associated with a microglial phagocytosis-related gene activation profile involving the NADPH oxidase subunit Cybb/gp91phox.

Low molecular weight polysialic acid prevents lipopolysaccharide-induced inflammatory dopaminergic neurodegeneration in humanized SIGLEC11 transgenic mice.

Parkinson's disease is one of the most common neurodegenerative diseases in the elderly population, with a pathophysiology linked to neuroinflammation, complement activation, and oxidative damage. Soluble polysialic acid with an average degree of polymerization 20 (polySia avDP20) prevents inflammation and oxidative burst in human macrophages via sialic acid-binding immunoglobulin like lectin-11 (SIGLEC11) receptor and interferes with alternative complement activation. Here, we confirmed the anti-inflammatory capacity of polySia avDP20 on cultured murine embryonic stem cell-derived microglia and analyzed the effect of polySia avDP20 in a lipopolysaccharide-triggered animal model of Parkinson's disease. We demonstrated a neuroprotective effect of intraperitoneally applied polySia avDP20 in humanized SIGLEC11 transgenic mice after repeated systemic challenge with lipopolysaccharide. Pathway enrichment analysis of the brain transcriptome on day 19 after disease initiation showed that intraperitoneal application of 10 µg/g body weight polySia avDP20 prevented excessive inflammation. In line with these data, polySia avDP20 attenuated the lipopolysaccharide-triggered increase in mRNA levels of immune-related genes (Il1b, Cd14, Myd88, Fcer1g, Itgam, C4, Cybb, Iba1 and Cd68) and cell death-related genes (Casp8, Ripk1 and Ripk3) in the brains of SIGLEC11 transgenic mice on day 19, but not on day 5. Moreover, immunohistochemistry demonstrated that polySia avDP20 reduced the lipopolysaccharide-induced increase in immunoreactivity of IBA1 and CD68 in the substantia nigra pars reticulata in SIGLEC11 transgenic and wild type mice on day 19. Furthermore, treatment with polySia avDP20 prevented the loss of dopaminergic neurons in the substantia nigra pars compacta induced by lipopolysaccharide challenge in both SIGLEC11 transgenic and wild type mice on day 19. Thus, our data demonstrate that polySia avDP20 ameliorates inflammatory dopaminergic neurodegeneration and therefore is a promising drug candidate to prevent Parkinson's disease-related inflammation and neurodegeneration.

Deletion of Alzheimer's disease-associated CD33 results in an inflammatory human microglia phenotype.

Genome-wide association studies demonstrated that polymorphisms in the CD33/sialic acid-binding immunoglobulin-like lectin 3 gene are associated with late-onset Alzheimer's disease (AD). CD33 is expressed on myeloid immune cells and mediates inhibitory signaling through protein tyrosine phosphatases, but the exact function of CD33 in microglia is still unknown. Here, we analyzed CD33 knockout human THP1 macrophages and human induced pluripotent stem cell-derived microglia for immunoreceptor tyrosine-based activation motif pathway activation, cytokine transcription, phagocytosis, and phagocytosis-associated oxidative burst. Transcriptome analysis of the macrophage lines showed that knockout of CD33 as well as knockdown of the CD33 signaling-associated protein tyrosine phosphatase, nonreceptor type 6 (PTPN6) led to constitutive activation of inflammation-related pathways. Moreover, deletion of CD33 or expression of Exon 2-deleted CD33 (CD33ΔE2 /CD33m) led to increased phosphorylation of the kinases spleen tyrosine kinase (SYK) and extracellular signal-regulated kinase 1 and 2 (ERK1 and 2). Transcript analysis by quantitative real-time polymerase chain reaction confirmed increased levels of interleukin (IL) 1B, IL8, and IL10 after knockout of CD33 in macrophages and microglia. In addition, upregulation of the gene transcripts of the AD-associated phosphatase INPP5D was observed after knockout of CD33. Functional analysis of macrophages and microglia showed that phagocytosis of aggregated amyloid-ß1-42 and bacterial particles were increased after knockout of CD33 or CD33ΔE2 expression and knockdown of PTPN6. Furthermore, the phagocytic oxidative burst during uptake of amyloid-ß1-42 or bacterial particles was increased after CD33 knockout but not in CD33ΔE2 -expressing microglia. In summary, deletion of CD33 or expression of CD33ΔE2 in human macrophages and microglia resulted in putative beneficial phagocytosis of amyloid ß1-42 , but potentially detrimental oxidative burst and inflammation, which was absent in CD33ΔE2 -expressing microglia.

Therapeutic potential to target sialylation and SIGLECs in neurodegenerative and psychiatric diseases.

Sialic acids, commonly found as the terminal carbohydrate on the glycocalyx of mammalian cells, are pivotal checkpoint inhibitors of the innate immune system, particularly within the central nervous system (CNS). Sialic acid-binding immunoglobulin-like lectins (SIGLECs) expressed on microglia are key players in maintaining microglial homeostasis by recognizing intact sialylation. The finely balanced sialic acid-SIGLEC system ensures the prevention of excessive and detrimental immune responses in the CNS. However, loss of sialylation and SIGLEC receptor dysfunctions contribute to several chronic CNS diseases. Genetic variants of SIGLEC3/CD33, SIGLEC11, and SIGLEC14 have been associated with neurodegenerative diseases such as Alzheimer's disease, while sialyltransferase ST8SIA2 and SIGLEC4/MAG have been linked to psychiatric diseases such as schizophrenia, bipolar disorders, and autism spectrum disorders. Consequently, immune-modulatory functions of polysialic acids and SIGLEC binding antibodies have been exploited experimentally in animal models of Alzheimer's disease and inflammation-induced CNS tissue damage, including retinal damage. While the potential of these therapeutic approaches is evident, only a few therapies to target either sialylation or SIGLEC receptors have been tested in patient clinical trials. Here, we provide an overview of the critical role played by the sialic acid-SIGLEC axis in shaping microglial activation and function within the context of neurodegeneration and synaptopathies and discuss the current landscape of therapies that target sialylation or SIGLECs.

Reenacting Neuroectodermal Exposure of Hematopoietic Progenitors Enables Scalable Production of Cryopreservable iPSC-Derived Human Microglia.

Human microglia, as innate immune cells of the central nervous system (CNS), play a central role in the pathogenesis of a large number of neurological and psychiatric disorders. However, experimental access to primary human microglia for biomedical applications such as disease modeling is extremely limited. While induced pluripotent stem cells (iPSCs) could provide an alternative source of microglia, the reenactment of their complex ontogenesis with a yolk sac origin and subsequent priming upon CNS invasion has remained a challenge. Here, we report a developmentally informed in vitro differentiation method for large-scale production and cryopreservation of iPSC-derived microglia (iPSdMiG). Specifically, iPSCs were propagated in conditions yielding both yolk sac hematopoietic derivatives and early neuroepithelial cells. To enable large-scale production, we implemented 3D bioreactor-based dynamic culture conditions and the use of novel mesh macrocarriers. Under these conditions, microglia could be harvested across a time period of at least 6 weeks, with 1 × 106 iPSCs giving rise to up to 45 × 106 iPSdMiG. The transcriptomic profile of iPSdMiG showed high similarity to adult human microglia, and harvested cells were immunopositive for typical microglial markers. In addition, iPSdMiG were able to secrete pro-inflammatory cytokines, engaged in phagocytotic activity, produced reactive oxygen species and lent themselves to co-culture studies in neural 2D and 3D systems. Importantly, iPSdMiG were efficiently cryopreserved, enabling the establishment of donor-specific microglia cell banks for disease modeling, drug discovery and eventually cell therapy. Main points. Scalable generation of iPSC-derived multi-lineage embryoid bodies on macrocarriers, reproducibly releasing microglia exhibiting characteristic markers and function. Cells are transcriptomically similar to primary human microglia and cryopreservable.

Metabolic regulation of immune responses to cancer.

The tumor microenvironment is an ecosystem composed of multiple types of cells, such as tumor cells, immune cells, and cancer-associated fibroblasts. Cancer cells grow faster than non-cancerous cells and consume larger amounts of nutrients. The rapid growth characteristic of cancer cells fundamentally alters nutrient availability in the tumor microenvironment and results in reprogramming of immune cell metabolic pathways. Accumulating evidence suggests that cellular metabolism of nutrients, such as lipids and amino acids, beyond being essential to meet the bioenergetic and biosynthetic demands of immune cells, also regulates a broad spectrum of cellular signal transduction, and influences immune cell survival, differentiation, and anti-tumor effector function. The cancer immunometabolism research field is rapidly evolving, and exciting new discoveries are reported in high-profile journals nearly weekly. Therefore, all new findings in this field cannot be summarized within this short review. Instead, this review is intended to provide a brief introduction to this rapidly developing research field, with a focus on the metabolism of two classes of important nutrients-lipids and amino acids-in immune cells. We highlight recent research on the roles of lipids and amino acids in regulating the metabolic fitness and immunological functions of T cells, macrophages, and natural killer cells in the tumor microenvironment. Furthermore, we discuss the possibility of "editing" metabolic pathways in immune cells to act synergistically with currently available immunotherapies in enhancing anti-tumor immune responses.

Reporter cell assay for human CD33 validated by specific antibodies and human iPSC-derived microglia.

CD33/Sialic acid-binding Ig-like lectin 3 (SIGLEC3) is an innate immune receptor expressed on myeloid cells and mediates inhibitory signaling via tyrosine phosphatases. Variants of CD33 are associated with Alzheimer's disease (AD) suggesting that modulation of CD33 signaling might be beneficial in AD. Hence, there is an urgent need for reliable cellular CD33 reporter systems. Therefore, we generated a CD33 reporter cell line expressing a fusion protein consisting of the extracellular domain of either human full-length CD33 (CD33M) or the AD-protective variant CD33ΔE2 (D2-CD33/CD33m) linked to TYRO protein tyrosine kinase binding protein (TYROBP/DAP12) to investigate possible ligands and antibodies for modulation of CD33 signaling. Application of the CD33-specific antibodies P67.6 and 1c7/1 to the CD33M-DAP12 reporter cells resulted in increased phosphorylation of the kinase SYK, which is downstream of DAP12. CD33M-DAP12 but not CD33ΔE2-DAP12 expressing reporter cells showed increased intracellular calcium levels upon treatment with CD33 antibody P67.6 and partially for 1c7/1. Furthermore, stimulation of human induced pluripotent stem cell-derived microglia with the CD33 antibodies P67.6 or 1c7/1 directly counteracted the triggering receptor expressed on myeloid cells 2 (TREM2)-induced phosphorylation of SYK and decreased the phagocytic uptake of bacterial particles. Thus, the developed reporter system confirmed CD33 pathway activation by CD33 antibody clones P67.6 and 1c7/1. In addition, data showed that phosphorylation of SYK by TREM2 activation and phagocytosis of bacterial particles can be directly antagonized by CD33 signaling.