Activation Of Innate Immune System Research Articles

Expansion of Fcγ Receptor IIIa-Positive Macrophages, Ficolin 1-Positive Monocyte-Derived Dendritic Cells, and Plasmacytoid Dendritic Cells Associated With Severe Skin Disease in Systemic Sclerosis.

In this study, we sought a comprehensive understanding of myeloid cell types driving fibrosis in diffuse cutaneous systemic sclerosis (dcSSc) skin. We analyzed the transcriptomes of 2,465 myeloid cells from skin biopsy specimens from 12 dcSSc patients and 10 healthy control subjects using single-cell RNA sequencing. Monocyte-derived dendritic cells (mo-DCs) were assessed using immunohistochemical staining and immunofluorescence analyses targeting ficolin-1 (FCN-1). A t-distributed stochastic neighbor embedding analysis of single-cell transcriptome data revealed 12 myeloid cell clusters, 9 of which paralleled previously described healthy control macrophage/DC clusters, and 3 of which were dcSSc-specific myeloid cell clusters. One SSc-associated macrophage cluster, highly expressing Fcγ receptor IIIA, was suggested on pseudotime analysis to be derived from normal CCR1+ and MARCO+ macrophages. A second SSc-associated myeloid population highly expressed monocyte markers FCN-1, epiregulin, S100A8, and S100A9, but was closely related to type 2 conventional DCs on pseudotime analysis and identified as mo-DCs. Mo-DCs were associated with more severe skin disease. Proliferating macrophages and plasmacytoid DCs were detected almost exclusively in dcSSc skin, the latter clustering with B cells and apparently derived from lymphoid progenitors. Transcriptional signatures in these and other myeloid populations indicate innate immune system activation, possibly through Toll-like receptors and highly up-regulated chemokines. However, the appearance and activation of myeloid cells varies between patients, indicating potential differences in the underlying pathogenesis and/or temporal disease activity in dcSSc.

DAMPs are able to skew CD4+ T cell subsets and increase the inflammatory profile in pregnant women with preeclampsia

Preeclampsia (PE) is characterized by abnormal activation of the immune system. The intense systemic inflammatory reaction, could be related to the presence of molecules released after cell stress or death, that are capable of inducing inflammation and are known as damage-associated molecular patterns (DAMP). This study evaluated the profile of T cells through the analysis of transcription factors and the cytokines produced after culture with or without DAMPs: heat shock protein 70 (Hsp70), hyaluronan (HA) and monosodium urate (MSU). Twenty pregnant women with PE, 20 normotensive (NT) pregnant women and 20 non-pregnant (NP) women were studied. The results showed polarization toward Th1/Th17 and a decrease in Th2/Treg profiles in preeclamptic women associated with elevated levels of TNF, IFN-γ, and IL-17A and diminished levels of TGF-β1 and IL-10 when compared to the normotensive group. In addition, preeclamptic women had a higher percentage of cells co-expressing T-bet/GATA-3 and T-bet/RORγt and fewer T-bet/FoxP3 cells when compared to normotensive group. MSU induced an increase in IFN-γ and IL-22 in all studied groups. MSU, HA, and Hsp70 induced significant higher production of TNF in the PE and NP groups. The PE group showed elevated levels of TGF-β1 after incubation with MSU, HA, and Hsp70, whereas HA and Hsp70 decreased TGF-β1 production in NT group. The results suggest that these alarmins may play a role in the activation of innate and adaptive immune systems by skewing CD4 + T cells and increasing the release of inflammatory cytokines, thereby contributing to the pathogenesis of this important syndrome.

NHS-IL12 and bintrafusp alfa combination therapy enhances antitumor activity in preclinical cancer models

Combinatorial immunotherapy approaches are emerging as viable cancer therapeutic strategies for improving patient responses and outcomes. This study investigated whether two such immunotherapies, with complementary mechanisms of action, could enhance antitumor activity in murine tumor models. The immunocytokine NHS-IL12, and surrogate NHS-muIL12, are designed to deliver IL-12 and muIL-12, respectively, to the tumor microenvironment (TME) to activate NK cells and CD8+ T cells and increase their cytotoxic functions. Bintrafusp alfa (BA) is a bifunctional fusion protein composed of the extracellular domains of the TGF-β receptor II to function as a TGF-β “trap” fused to a human IgG1 antibody blocking PD-L1. With this dual-targeting strategy, BA enhances efficacy over that of monotherapies in preclinical studies. In this study, NHS-muIL12 and BA combination therapy enhanced antitumor activity, prolonged survival, and induced tumor-specific antitumor immunity. This combination therapy increased tumor-specific CD8+ T cells and induced immune profiles, consistent with the activation of both adaptive and innate immune systems. In addition, BA reduced lung metastasis in the 4T1 model. Collectively, these findings could support clinical trials designed to investigate NHS-IL12 and BA combination therapy for patients with advanced solid tumors

High Dose Vardenafil Blunts the Hypertensive Effects of Toll-Like Receptor 3 Activation During Pregnancy

The maternal innate immune system plays a central role in preeclampsia (PE). Toll-like receptors (TLRs) are innate immune system receptors that recognize characteristics of extracellular endogenous ligands or pathogens, and their activation leads to a pro-inflammatory immune response. We and others have reported that excessive activation of TLRs causes pregnancy-dependent hypertension in animals and is associated with PE in women. Activation of TLR3 by poly I:C mimics the innate immune system activation by viruses that women who develop PE encounter during pregnancy. Vardenafil was approved by the FDA for erectile dysfunction but has recently been examined as a potential PE medication due to studies done with a similar drug, sildenafil. Preclinical as well as recent clinical studies demonstrate the potential effectiveness of sildenafil for PE. However, vardenafil is more potent than sildenafil and acts by increasing expression of placental growth factor in addition to increasing cGMP levels. We hypothesized that vardenafil will be more potent and effective in reducing the negative health effects in a mouse model of virus-induced PE. Pregnant mice were injected with the TLR3 agonist poly I:C (PPIC) on gestational days 13, 15, and 17. We treated PPIC mice with a high dose of vardenafil (50 mg human equivalent), a lower dose of vardenafil (20 mg human equivalent), or sildenafil (50 mg human equivalent) on gestational days 15–17 after hypertension was established. Daily i.p. injections of either high dose or low dose vardenafil significantly decreased systolic blood pressure in PPIC mice whereas sildenafil had no effect. There were no differences in body weight between the groups. The splenomegaly induced in PPIC mice was ameliorated in high dose vardenafil-treated PPIC mice, while low dose vardenafil-treated and sildenafil-treated PPIC mice still exhibited splenomegaly. High dose vardenafil-treated PPIC mice also did not exhibit any fetal demise characteristic of PPIC mice, while low dose vardenafil-treated and sildenafil-treated PPIC mice still had significantly increased incidences of fetal demise. These data support the notion that high dose vardenafil may be safe and effective at reducing blood pressure during a virus-associated hypertensive pregnancy.

Inflammation and innate immune system activation in neurodegeneration, Down syndrome, aging, and infection: Therapeutic target or partner?

AbstractBackgroundCharacteristic features/biomarkers of innate immune system activation and inflammation are associated with neurodegenerative diseases (e.g., Alzheimer’s disease [AD], Parkinson’s disease [PD]), Down syndrome [DS], normal aging, and many infectious diseases. Evidence also suggests that peripheral inflammation interacts with and mirrors inflammation in the central nervous system (CNS) and can yield useful biomarkers of the disease or disorder and potentially of the efficacy of therapies. These findings have led to the additional conclusion that developing methods to dampen inflammation would be an effective therapeutic approach. However, recent findings indicate that innate immune system activation, including inflammation, may, instead, be beneficial.MethodWe used the proinflammatory cytokine granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) to modulate the innate immune system and determine the effects on biomarker, clinical, and/or behavioral outcomes in mouse models of AD, DS, aging, and viral infection and in a clinical trial in AD.ResultRecent published and unpublished results from our laboratory and others show that treatment with GM‐CSF increases inflammatory biomarkers and also effectively reduces cognitive decline, brain pathology, and disease‐associated peripheral blood biomarkers in mouse models of AD, PD, DS, and normal aging. Interestingly, GM‐CSF treatment also reduces viral titer and mortality in mouse models of West Nile Virus and SARS‐CoV‐2 infection. Our retrospective study of patients with cognitive deficits due to chemotherapy‐induced CNS inflammation showed that recombinant human GM‐CSF (sargramostim) treatment was associated with improved cognition. A sargramostim clinical trial in PD also showed promising results. Finally, our recently‐completed clinical trial in AD showed that sargramostim treatment increased inflammatory cytokines and improved toward normal both a measure of cognition (MMSE) and blood biomarkers of amyloid (A) and Tau (T) deposition and neurodegeneration (N). Sargramostim clinical trials in COVID‐19 are underway.ConclusionTogether, these results indicate that targeted enhancement of the innate immune system and of inflammatory cytokines, specifically in response to GM‐CSF, can effectively treat many disorders that exhibit characteristic inflammation and innate immune system activation. Thus, the long‐held belief that inflammation and innate immune system activation only play a negative role in neurodegenerative diseases, DS, viral infections, and normal aging should be reassessed.

Influence of chronic microglial activation and aging on the medial septal cholinergic cell number and morphology

Pathological ageing, involving chronic oxidative and inflammatory stress increases the risk of cognitive degenerative diseases such as dementia, including Alzheimer's disease (AD). Inflammatory stress contributes to unregulated aberrant activation of innate immune system leading to "neuroinflammation". Microglia, the resident immune cells of the CNS, can either trigger chronic neuroinflammatory pathways resulting in gradual neurodegeneration or promote neuroprotection and repair via down regulation of neuroinflammation. Augmented glial activation, accompanied by increased levels of pro-inflammatory cytokines such as IL-6 and TNF-α are biomarkers of ageing and age-related diseases, and shown to contribute to neurodegeneration. AD is characterized by a progressive downregulation of markers within cholinergic basal forebrain (BF) neurons, cholinergic cell loss and diminished cortical choline acetyltransferase (ChAT) activity associated with cognitive decline. While the underlying mechanism leading to cholinergic loss is still unknown, several studies suggest chronic inflammatory processes may contribute to the pathology. Within the BF, the medial septum (MS) provides most of the cholinergic innervations into the hippocampus and the cortex, hence this study investigated the effects of aging and chronic neuroinflammation on the septal cholinergic cell numbers and morphology. 12 and 24 months old GFAP-IL6 (presenting continuous IL6 expression under the GFAP promoter, contributing to chronic microglia activation) and ChATBAC -eGFP (cholinergic neurones tagged by GFP to represent the effect of healthy ageing) mice were used. Immunohistochemistry combined with unbiased stereology were used to estimate the number of Iba1+ microglia and ChAT+ cholinergic cells in the MS. The number of Iba1+ microglia in the MS showed a significant 1.2 and 1.4-fold increase in the GFAP-IL6 mice at 12 and 24 months respectively, compared to WT cohorts. Meanwhile, the number of ChAT+ cells in the MS are significantly reduced by 1.4 and 1.2-folds in the GFAP-IL6 mice at 12 and 24 months respectively, compared to WT cohorts. Differences in microglial and cholinergic cell morphology between GFAP-IL6 and WT was also investigated. These findings demonstrate a potential direct effect of chronic microglia activation on cholinergic cell number in the MS which can exacerbate throughout ageing leading to neurodegenerative impairments.

Assessment of Markers of Gut Integrity and Inflammation in Non-Celiac Gluten Sensitivity After a Gluten Free-Diet.

PurposeMarkers for gut integrity and inflammation have received increasing interest as intestinal permeability and innate immune system activation are suggested as possible pathophysiological mechanisms in non-celiac gluten sensitivity (NCGS). We aimed to assess relevant biomarkers in NCGS by analyzing serum levels of gut integrity and permeability markers, pro-inflammatory cytokines and antigliadin IgG in patients with suspected NCGS on a gluten-free diet (GFD), and compare them to serum levels in patients with irritable bowel syndrome (IBS) and healthy controls (HC).Patients and MethodsSerum samples collected from patients with suspected NCGS on a GFD (n=20, 14 women, 21–62 years), IBS (n=20, 16 women, 24–67 years) and HC (n=20, 14 women, 21–54 years) were analyzed. IBS severity scoring system (IBS-SSS) was applied to evaluate gastrointestinal symptoms.ResultsThe IBS-SSS score was higher in subjects with suspected NCGS and IBS patients compared to HC (p<0.0001). No significant differences were found in the serum levels of any of the gut integrity and permeability markers, cytokines or antigliadin IgG antibodies between the three groups. However, positive correlations were observed between claudin-1 and i-FABP, and between claudin-1 and antigliadin IgG antibodies.ConclusionNo differences in serum levels of gut integrity and permeability markers, pro-inflammatory cytokines or antigliadin IgG antibodies were found among patients with suspected NCGS on a GFD, IBS and HC.

In vitro functional genetic modification of canine adenovirus type 2 genome by CRISPR/Cas9

AbstractGenetically modified oncolytic adenoviruses have been proposed as a vehicle for cancer therapy. However, several concerns, such as toxicity to normal cells and organs, lack of suitable cell surface receptors to allow viral entry to the desired cell type(s), and activation of both innate and adaptive immune systems in patients, restrict the successful clinical application of adenoviral-mediated cancer gene therapy. Successful virotherapy will require efficient transductional and transcriptional targeting to enhance therapeutic efficacy by ensuring targeted adenoviral infection, replication, and/or therapeutic transgene expression. Targeted modification of viral components, such as viral capsid, fiber knob, and the insertion of transgenes for expression, are prerequisites for the necessary transductional and transcriptional targeting of adenovirus. However, the conventional approach to modify the adenoviral genome is complex, time consuming, and expensive. It is dependent on the presence of unique restriction enzyme sites that may or may not be present in the target location. Clustered regularly interspaced short palindromic repeat (CRISPR) along with the RNA-guided nuclease Cas9 (CRISPR/Cas9) is one of the most powerful tools that has been adopted for precise genome editing in a variety of cells and organisms. However, the ability of the CRISPR/Cas9 system to precisely and efficiently make genetic modification, as well as introduce gene replacements, in adenoviral genomes, remains essentially unknown. Herein the ability of in vitro CRISPR/CAS9-mediated editing of the canine adenovirus type 2 (CAV2) genome to promote targeted modification of the viral genome was assessed. To demonstrate the feasibility of this goal, CRISPR/Cas9 has been used to successfully insert the RFP (red fluorescent protein) reporter construct into the CAV2 genome. Initial results demonstrated high efficiency and accuracy for in vitro CRISPR-mediated editing of the large CAV2 genome. Furthermore, this application was expanded, using multiple guide RNAs, to conduct gene replacement in the CAV2 genome by substituting a portion of the E3 gene with a construct designed to express a single chain antibody to canine PD-1. Thus, this work provides a significantly improved and efficient method for targeted editing of adenoviruses to generate altered and potentially therapeutic viral genomes in the shortest possible time.

A novel gut microbiome therapeutic derived from dietary polyphenols attenuates neuroinflammation in vivo in a model of C9orf72‐mediated frontotemporal dementia

AbstractBackgroundFrontotemporal dementia (FTD) is a neurodegenerative disorder with various genetic etiologies, the most prevalent of which include G4C2 expansions in the c9orf72 gene. Expansions produce dipeptide repeat proteins (DPR), forming aggregates with deleterious impacts on cellular processes and correlating with neuroinflammation. Glycine‐Arginine (GR) aggregates are the only significantly correlated DPR found in clinically relevant neuroanatomical regions of the brain. Our current study investigates the contribution of innate immunity to FTD pathogenesis and the attenuation of neuropathologies with the brain bioavailable microbiome‐derived metabolite, 3‐hydroxybenzoic acid (3‐HBA).MethodMice were transfected via intracerebroventricular injection with AAV9‐GFP(GR)100 or AAV9‐GFP and treated starting on Day 28 with either PBS vehicle or 50 mg kg‐1 of 3‐HBA. At 14 weeks mice were sacrificed and prefrontal cortex (PFC), somatosensory cortex (CTX), and hippocampal (HIPP) tissue was dissected and prepared for RNA sequencing. Differentially expressed genes (DEGs) were identified using DESeq2 and pathway alterations were detected using Ingenuity Pathway Analysis. ELISA for IL‐1β was conducted on PFC, CTX, and HIPP tissue. To determine the ability of 3‐HBA to reduce secretion of IL‐1β, primary microglia pretreated with 3‐HBA were stimulated with LPS+ATP and IL‐1β concentration was analyzed by ELISA.ResultDEGs associated with FTD pathology, protein expression levels of the pro‐inflammatory cytokine IL‐1β, and subsequent amelioration via treatment with 3‐HBA were demonstrated to be a result of contributions from the innate immune system and inflammasome activation in microglia. Inflammatory pathways differentially enriched among treatment groups include TREM1 Signaling, the complement system, and NFκB signaling. GR‐100 Vehicle treated animals exhibited elevated IL‐β in cortical tissue. 3‐HBA was efficacious in vivo in rescuing IL‐1β levels in GR‐100 treated mice to levels of GFP animals. Primary microglia pretreated with 3‐HBA and stimulated with LPS+ATP significantly reduced IL‐1β release, signifying the efficacy of 3‐HBA in attenuating innate immune reactivity.ConclusionPathways and genes associated with the innate immune system show enrichment in a model of FTD and are rescued by administration of 3‐HBA. Overexpression of IL‐1β in the cortex of GR‐100 Vehicle animals is attenuated by the administration of 3‐HBA. Our data implicates a gut‐brain axis mechanism of treatment for key FTD associated neuropathologies.

The influence of anesthesia and surgery on fear extinction

Accumulating evidence has demonstrated significant clinical post-traumatic stress disorder (PTSD) symptoms after anesthesia or surgery. Fear extinction dysfunction is a notable feature of PTSD. Although anesthetics and surgery profoundly affect memory processes, their designated effects on fear extinction have not been dissertated. Previous studies have suggested that innate immune system activation disrupts fear extinction, and surgery has been shown to increase the inflammatory response. Thus, in the current study, we examined the effects of propofol, sevoflurane, dexmedetomidine and surgery on fear extinction in adolescent mice, and further tested whether dexmedetomidine could reverse the injury effect of surgery on fear extinction through its anti-inflammatory effects. Our results showed that propofol (200 mg/kg) impaired the acquisition and recall of cued fear extinction, and surgery disrupted cued fear extinction acquisition/recall and consolidation. In contrast to cued fear extinction, contextual fear extinction was not affected by propofol or surgery. Moreover, dexmedetomidine prevented surgery-induced impairment of cued extinction acquisition and recall but not consolidation. Finally, TNF-α and IL-6 levels in the ventromedial prefrontal cortex were not necessary for the dexmedetomidine treatment effect of surgery-induced fear extinction dysfunction. The study results showed that propofol and surgery selective impaired the cued fear extinction stage in adolescent mice, and dexmedetomidine may unleash a protective effect in preventing postoperative PTSD.

Abstract 10926: Importance of Mitochondrial DNA Damage in Cigarette Smoke Extract-Induced Activation of Innate Immune System

Objective: Smoking is a well-known risk factor for atherosclerosis, yet the mechanism by which smoking causes atherosclerosis remains unclear. It has been reported that inflammation and DNA damage are involved in the formation and development of atherosclerosis. We have previously reported that nuclear DNA damage was increased in peripheral mononuclear cells of smokers compared to those of non-smokers, and that DNA damage was accumulated in atherosclerotic lesions. In this study, to clarify the mechanism by which smoking induces inflammation in vascular endothelial cells, we investigated the effect of cigarette smoke extract (CSE) on both nuclear and mitochondrial DNA damage and the subsequent cellular response in human umbilical vein endothelial cells (HUVEC). Methods and Results: CSE increased double-strand breaks in HUVEC. Notably, this increase was detected relatively late (after 3 days of CSE exposure) compared to other genotoxic agents. CSE also increased oxidative DNA damage both in the nucleus and mitochondria and induced mitochondrial dysfunction. Mitochondrial dysfunction decreased cytosolic protein levels of ICAD, an inhibitor of caspase-activated DNase (CAD), which causes nuclear DNA fragmentation, and increased CAD in the nucleus, suggesting that this is one of the mechanisms of nuclear DNA double-strand breaks. In addition, continuous stimulation by CSE induced the accumulation of cytosolic DNA. Interestingly, real-time PCR analysis revealed that the accumulated DNA in the cytosol was derived not only from the nucleus but also from mitochondria. We further examined whether DNA accumulated in the cytosol activated cytosolic DNA-sensing pathways. The production of cGAMP, a second messenger in cGAS (a DNA-sensing receptor) signaling, was increased by CSE. We also found that the mRNA expression of IL-6 was increased by CSE, and that the increase was suppressed by si-cGAS. Conclusions: This study showed that continuous exposure to CSE induces not only nuclear but also mitochondrial DNA damage, which leads to cytosolic DNA accumulation, and evokes chronic inflammation via the cGAS-STING pathway.

Overexpression of Human TLR8 Causes Fatal Anemia in SLE-Prone Mice By Altering the Bone Marrow Erythropoietic Niche

Anemia is a common hematologic abnormality in patients with active systemic lupus erythematosus (SLE), a disease characterized by innate immune system activation by nucleic acid containing immune complexes and cell debris. Work in mouse models has shown that overactivation of several cytoplasmic innate immune sensors by either self-DNA or self-RNA specifically leads to erythroid cell death in the fetal liver while sparing other hematopoietic cell lineages.The endosomal receptors TLR7 and TLR8 both recognize ssRNA in humans. TLR7 overexpression in mice causes a lupus syndrome that includes the development of mild to moderate anemia (Hb >10) and thrombocytopenia and is associated with spleen histiocytosis, autoimmune hemolysis, erythrophagocytosis and compensatory stress erythropoiesis in the spleen. A recent study demonstrated that patients with TLR8 gain of function mutations present with immunodeficiency, inflammation and bone marrow failure. However the role of TLR8 in SLE has been difficult to study in mice because it has a 5 amino acid deletion that attenuates its RNA binding capacity.To address the role of TLR8 in SLE, we overexpressed human TLR8 in a lupus mouse model (huTLR8tg.Sle1.Yaa) using a BAC transgene. 50% of homozygous huTLR8tg.Sle1.Yaa mice developed severe anemia (Hb<9) resulting in early mortality starting at 3-4.5 months of age. This phenotype required both the Sle1 and Yaa loci that promote the formation of high titer anti-chromatin and anti-RNA antibodies and onset of nephritis at >6 months of age. There was no difference in autoantibody titers between Sle1.Yaa wt and huTLR8tg mice and early death was not due to premature onset of renal disease. All mice had normal RBC indices prior to 10 weeks of age. Anemia was associated with an increase in bone marrow (BM) trabecular bone and a decrease in erythroblastic islands (EBI) in the BM with compensatory stress erythropoiesis leading to reticulocytosis and vast splenomegaly. RBC half-life was normal even after the development of reticulocytosis, but decreased in severely anemic mice as a pre-terminal event. Using CFSE labeling of RBCs we showed that hemophagocytosis occurred in vivo as a result of expansion of phagocytic red pulp macrophages.Flow cytometry of BMs from transgenic mice showed normal erythroid progenitors but a block at the late CFU-E/early proerythroblast stage leading to overall decreased terminal erythroid differentiation. Single cell RNAseq of EBIs isolated from transgenic BMs confirmed the block in erythropoiesis. The erythroblast cluster proximal to the block had a signature of mitochondrial stress and decreased proliferation. Spleen EBIs from transgenic mice were characterized by a low frequency of early erythroblasts compared with BM EBIs. We found 6 related clusters of EBI central macrophages in the BMs of both wt and transgenic mice. In transgenic mice, most of these displayed an inflammatory and Type 1 IFN signature. One cluster (M2) expressed all the classical central macrophage phenotypic markers in wt mice but was characterized in transgenic mice by downregulation of multiple phagocytic receptors and a 5-fold decrease of VCAM1 expression. Loss of VCAM1 and downregulation of CD169 in central macrophages of transgenic BMs was confirmed by flow cytometry. By contrast spleen central macrophages from transgenic mice retained VCAM and CD169 expression.Together, these results suggest that failure of BM erythropoiesis in huTLR8 transgenic SLE.Yaa lupus-prone mice, in which the acquisition of anti-nucleic acid autoantibodies drives excess innate stimulus through TLR7 and huTLR8, is due to a block in differentiation from CFU-E to the early proerythroblast stage; this is associated with an inflammatory phenotype specifically in BM erythroblastic island central macrophages and down regulation of adhesion and phagocytic receptors. Stress erythropoiesis in the spleens is associated with vast expansion of red pulp macrophages with phagocytic properties and fatal anemia is associated with a decrease in red blood cell half-life, suggesting that excessive RBC phagocytosis, coupled with insufficient erythroblast progenitors, eventually exceeds the capability of stress erythropoiesis to replace the RBC mass. DisclosuresKalfa: Agios Pharmaceuticals, Inc.: Other: Steering Committee, Research Funding; FORMA Therapeutics, Inc: Research Funding. Paulson: Forma Therapeutics: Consultancy. Blanc: Keros Therapeutics, Inc.: Consultancy.

237 Impact of innate immunity on pemphigus vulgaris disease manifestation

Pemphigus vulgaris is a chronic autoimmune blistering disease characterized by antibody production against Desmogleins 3 (Dsg3) and 1 (Dsg1) causing acantholysis. Increased levels of innate cytokines detected in the blister fluid suggest a role of innate immune system activity. To elucidate the discrepancy between binding of pathogenic antibodies and a lack of blister formation, we focused on the role of innate cofactors supporting acantholysis. Here, we used a dispase-based keratinocyte-dissociation assay with a human immortalized keratinocyte cell line (HaCaT) and human primary keratinocytes.

IL-1\u03b2 and HMGB1 are anti-neurogenic to endogenous neural stem cells in the sclerotic epileptic human hippocampus

BackgroundThe dentate gyrus exhibits life-long neurogenesis of granule-cell neurons, supporting hippocampal dependent learning and memory. Both temporal lobe epilepsy patients and animal models frequently have hippocampal-dependent learning and memory difficulties and show evidence of reduced neurogenesis. Animal and human temporal lobe epilepsy studies have also shown strong innate immune system activation, which in animal models reduces hippocampal neurogenesis. We sought to determine if and how neuroinflammation signals reduced neurogenesis in the epileptic human hippocampus and its potential reversibility.MethodsWe isolated endogenous neural stem cells from surgically resected hippocampal tissue in 15 patients with unilateral hippocampal sclerosis. We examined resultant neurogenesis after growing them either as neurospheres in an ideal environment, in 3D cultures which preserved the inflammatory microenvironment and/or in 2D cultures which mimicked it.Results3D human hippocampal cultures largely replicated the cellular composition and inflammatory environment of the epileptic hippocampus. The microenvironment of sclerotic human epileptic hippocampal tissue is strongly anti-neurogenic, with sustained release of the proinflammatory proteins HMGB1 and IL-1β. IL-1β and HMGB1 significantly reduce human hippocampal neurogenesis and blockade of their IL-1R and TLR 2/4 receptors by IL1Ra and Box-A respectively, significantly restores neurogenesis in 2D and 3D culture.ConclusionOur results demonstrate a HMGB1 and IL-1β-mediated environmental anti-neurogenic effect in human TLE, identifying both the IL-1R and TLR 2/4 receptors as potential drug targets for restoring human hippocampal neurogenesis in temporal lobe epilepsy.

Innate Immune System Activation and Neuroinflammation in Down Syndrome and Neurodegeneration: Therapeutic Targets or Partners?

Innate immune system activation and inflammation are associated with and may contribute to clinical outcomes in people with Down syndrome (DS), neurodegenerative diseases such as Alzheimer’s disease (AD), and normal aging. In addition to serving as potential diagnostic biomarkers, innate immune system activation and inflammation may play a contributing or causal role in these conditions, leading to the hypothesis that effective therapies should seek to dampen their effects. However, recent intervention studies with the innate immune system activator granulocyte-macrophage colony-stimulating factor (GM-CSF) in animal models of DS, AD, and normal aging, and in an AD clinical trial suggest that activating the innate immune system and inflammation may instead be therapeutic. We consider evidence that DS, AD, and normal aging are accompanied by innate immune system activation and inflammation and discuss whether and when during the disease process it may be therapeutically beneficial to suppress or promote such activation.

Hepatic Macrophages Express Melanoma Differentiation-Associated Gene 5 in Nonalcoholic Steatohepatitis.

The activation of innate immune system is essential for the pathogenesis of nonalcoholic steatohepatitis (NASH). Among pattern recognition receptors, it is well-characterized that toll-like receptors (TLRs) are deeply involved in the development of NASH to reflect exposure of the liver to gut-driven endotoxins. In contrast, it has not been elucidated whether retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) are similarly implicated in the disease progression. In the present study, we examined the expression of melanoma differentiation-associated antigen 5 (MDA5), known to be a member of RLRs, in a diet-induced murine model of NASH. The liver tissues were collected from C57BL/6J mice at 1, 3, and 6weeks after choline-deficient L-amino acid-defined high-fat diet (CDAHFD), and the expression of MDA5 was analyzed by western blotting, immunofluorescence (IF), and real-time quantitative PCR (qPCR). The results of western blotting showed that hepatic expression of MDA5 was increased at 3 and 6weeks. In IF, MDA5-positive cells co-expressed F4/80 and CD11b, indicating they were activated macrophages, and these cells began to appear at 1week after CDAHFD. The mRNA expression of MDA5 was significantly upregulated at 1week. Additionally, we performed IF using liver biopsy specimens collected from 11 patients with nonalcoholic fatty liver diseases (NAFLD), and found that MDA5-positive macrophages were detected in eight out of eleven patients. In an in vitro study, MDA5 was induced upon stimulation with lipopolysaccharide in murine bone marrow-derived macrophages and THP-1 cells. Our findings suggest that MDA5 may be involved in the inflammation of NASH.

Histotripsy Ablation Alters the Tumor Microenvironment and Promotes Immune System Activation in a Subcutaneous Model of Pancreatic Cancer.

Pancreatic cancer is a significant cause of cancer-related deaths in the United States with an abysmal five-year overall survival rate that is under 9%. Reasons for this mortality include the lack of late-stage treatment options and the immunosuppressive tumor microenvironment. Histotripsy is an ultrasound-guided, noninvasive, nonthermal tumor ablation therapy that mechanically lyses targeted cells. To study the effects of histotripsy on pancreatic cancer, we utilized an in vitro model of pancreatic adenocarcinoma and compared the release of potential antigens following histotripsy treatment to other ablation modalities. Histotripsy was found to release immune-stimulating molecules at magnitudes similar to other nonthermal ablation modalities and superior to thermal ablation modalities, which corresponded to increased innate immune system activation in vivo. In subsequent in vivo studies, murine Pan02 tumors were grown in mice and treated with histotripsy. Flow cytometry and rtPCR were used to determine changes in the tumor microenvironment over time compared to untreated animals. In mice with pancreatic tumors, we observed significantly increased tumor-progression-freeand general survival, with increased activation of the innate immune system 24 h posttreatment and decreased tumor-associated immune cell populations within 14 days of treatment. This study demonstrates the feasibility of using histotripsy for pancreatic cancer ablation and provides mechanistic insight into the initial innate immune system activation following treatment. Further work is needed to establish the mechanisms behind the immunomodulation of the tumor microenvironment and immune effects.

HIV Viremia Is Associated With APOL1 Variants and Reduced JC-Viruria.

Variants in the Apolipoprotein L1 (APOL1) gene (G1-rs60910145, rs73885319, G2-rs71785313) are common in Africans and in individuals of recent African ancestry and are associated with an increased risk of non-diabetic chronic kidney disease (CKD) and in particular of HIV associated nephropathy (HIVAN). In light of the significantly increased risk of HIVAN in carriers of two APOL1 risk alleles, a role in HIV infectivity has been postulated in the mechanism of APOL1 associated kidney disease. Herein, we aim to explore the association between HIV viremia and APOL1 genotype. In addition, we investigated interaction between BK and JC viruria, CKD and HIV viremia. A total of 199 persons living with HIV/AIDS (comprising 82 CKD cases and 117 controls) from among the participants in the ongoing Human Heredity and Health in Africa (H3Africa) Kidney Disease Research Network case control study have been recruited. The two APOL1 renal risk alleles (RRA) genotypes were associated with a higher risk of CKD (OR 12.6, 95% CI 3.89–40.8, p < 0.0001). Even a single APOL1 RRA was associated with CKD risk (OR 4.42, 95% CI 1.49–13.15, p = 0.007). The 2 APOL1 RRA genotypes were associated with an increased probability of having HIV viremia (OR 2.37 95% CI 1.0–5.63, p = 0.05). HIV viremia was associated with increased CKD risk (OR 7.45, 95% CI 1.66–33.35, P = 0.009) and with a significant reduction of JC virus urine shedding (OR 0.35, 95% CI 0.12–0.98, p = 0.046). In contrast to prior studies, JC viruria was not associated with CKD but was restricted in patients with HIV viremia, regardless of CKD status. These findings suggest a role of APOL1 variants in HIV infectivity and emphasize that JC viruria can serve as biomarker for innate immune system activation.

CSF Proteomic Alzheimer's Disease-Predictive Subtypes in Cognitively Intact Amyloid Negative Individuals.

We recently discovered three distinct pathophysiological subtypes in Alzheimer’s disease (AD) using cerebrospinal fluid (CSF) proteomics: one with neuronal hyperplasticity, a second with innate immune system activation, and a third subtype with blood–brain barrier dysfunction. It remains unclear whether AD proteomic subtype profiles are a consequence of amyloid aggregation, or might exist upstream from aggregated amyloid. We studied this question in 127 older individuals with intact cognition and normal AD biomarkers in two independent cohorts (EMIF-AD MBD and ADNI). We clustered 705 proteins measured in CSF that were previously related to AD. We identified in these cognitively intact individuals without AD pathology three subtypes: two subtypes were seen in both cohorts (n = 49 with neuronal hyperplasticity and n = 44 with blood–brain barrier dysfunction), and one only in ADNI (n = 12 with innate immune activation). The proteins specific for these subtypes strongly overlapped with AD subtype protein profiles (overlap coefficients 92%–71%). Longitudinal p181-tau and amyloid β 1–42 (Aβ42) CSF analysis showed that in the hyperplasticity subtype p181-tau increased (β = 2.6 pg/mL per year, p = 0.01) and Aβ42 decreased over time (β = −4.4 pg/mL per year, p = 0.03), in the innate immune activation subtype p181-tau increased (β = 3.1 pg/mL per year, p = 0.01) while in the blood–brain barrier dysfunction subtype Aβ42 decreased (β = −3.7 pg/mL per year, p = 0.009). These findings suggest that AD proteomic subtypes might already manifest in cognitively normal individuals and may predispose for AD before amyloid has reached abnormal levels.

Transcriptional landscape of PTEN loss in primary prostate cancer

BackgroundPTEN is the most frequently lost tumor suppressor in primary prostate cancer (PCa) and its loss is associated with aggressive disease. However, the transcriptional changes associated with PTEN loss in PCa have not been described in detail. In this study, we highlight the transcriptional changes associated with PTEN loss in PCa.MethodsUsing a meta-analysis approach, we leveraged two large PCa cohorts with experimentally validated PTEN and ERG status by Immunohistochemistry (IHC), to derive a transcriptomic signature of PTEN loss, while also accounting for potential confounders due to ERG rearrangements. This signature was expanded to lncRNAs using the TCGA quantifications from the FC-R2 expression atlas.ResultsThe signatures indicate a strong activation of both innate and adaptive immune systems upon PTEN loss, as well as an expected activation of cell-cycle genes. Moreover, we made use of our recently developed FC-R2 expression atlas to expand this signature to include many non-coding RNAs recently annotated by the FANTOM consortium. Highlighting potential novel lncRNAs associated with PTEN loss and PCa progression.ConclusionWe created a PCa specific signature of the transcriptional landscape of PTEN loss that comprises both the coding and an extensive non-coding counterpart, highlighting potential new players in PCa progression. We also show that contrary to what is observed in other cancers, PTEN loss in PCa leads to increased activation of the immune system. These findings can help the development of new biomarkers and help guide therapy choices.