Cytokine Production In Response Research Articles

Phosphorylation of caspase-8 by RSKs via organ-constrained effects controls the sensitivity to TNF-induced death

Caspase-8 (Casp8) serves as an initiator of apoptosis or a suppressor of necroptosis in context-dependent manner. Members of the p90 RSK family can phosphorylate caspase-8 at threonine-265 (T265), which can inactivate caspase-8 for bypassing caspase-8-mediated blockade of necroptosis and can also decrease caspase-8 level by promoting its degradation. Mutating T265 in caspase-8 to alanine (A) in mice blocked TNF-induced necroptotic cecum damage but resulted in unexpectedly massive injury in the small intestine. Here, we show RSK1, RSK2, and RSK3 redundantly function in caspase-8 phosphorylation, and the duodenum is the most severely affected part of the small intestine when T265 phosphorylation of caspase-8 was prevented. Eliminating caspase-8 phosphorylation resulted in a duodenum-specific increase in basal caspase-8 protein level, which shall be responsible for the increased sensitivity to TNF-induced damage. Apoptosis of intestinal epithelial cells (IECs) was predominant in the duodenum of TNF-treated Rsk1−/−Rsk2−/−Rsk3−/− and Casp8T265A/T265A mice, though necroptosis was also observed. The heightened duodenal injury amplified systemic inflammatory responses, as evidenced by the contribution of hematopoietic cells to the sensitization of TNF-induced animal death. Further analysis revealed that hematopoietic and non-hematopoietic cells contributed differentially to cytokine production in response to the increased cell death. Collectively, RSKs emerges as a previously overlooked regulator that, via tissue/organ-constrained inactivating caspase-8 and/or downregulating caspase-8 protein level, controls the sensitivity to TNF-induced organ injury and animal death.

Metabolic Recovery with the Persistence of Proinflammatory Leucocyte Dysfunction After Bariatric Intervention for Obesity.

A suitable option for severe obesity treatment is a surgical approach. After surgery, metabolic markers and weight frequently return to adequate values; however, concerning systemic inflammatory mediators, the results are inconsistent. Furthermore, it has been suggested that leucocyte function may be affected even after weight normalization. This study aimed to determine if the surgical treatment of obesity influences the production of cytokines by LPS-stimulated as a function of leucocytes. We performed a cross-sectional study that investigated the production of cytokines in response to lipopolysaccharide (LPS) along a kinetic of simulation by leucocytes recovered from individuals with normal weight (NW, n = 8), persons living with obesity (Ob, n = 7), persons living with obesity and diabetes mellitus (Ob-DM, n = 17), and persons that used to live with obesity who underwent bypass surgery (fOb + bypass, n = 8) and recover normal weigh. IL-6 levels were significantly higher in the Ob and fOb + bypass groups than in NW (p = 0.043). IL-10 secretion without LPS was significantly higher in the NW group than in the other groups explored (p < 0.05). When exposed to LPS, the IL-10 levels increased in all groups except the NW group. As also observed for IL-18 and IL-33, the secretion curve of the fOb + bypass group was more similar to the Ob group, even when they had reached normal weight, as opposed to the NW group. Our results show that in patients with fOb + bypass, inflammatory and anti-inflammatory cytokine production dynamics remain disrupted even with improved metabolic control and normal weight recovery.

Immunometabolic adaptation in monocytes underpins functional changes during pregnancy

Metabolic heterogeneity is a determinant of immune cell function. The normal physiological metabolic reprogramming of pregnancy that ensures the fuel requirements of mother and baby are met, might also underpin changes in immunity that occur with pregnancy and manifest as altered responses to pathogens and changes to autoimmune disease symptoms. Using peripheral blood from pregnant women at term, we reveal that monocytes lose M2-like and gain M1-like properties accompanied by reductions in mitochondrial mass, maximal respiration, and cardiolipin content in pregnancy; glycolysis is unperturbed. We establish that muramyl dipeptide (MDP)-stimulated cytokine production relies on oxidative metabolism, then show in pregnancy reduced cytokine production in response to MDP but not LPS. Overall, mitochondrially centered metabolic capabilities of late gestation monocytes are down-regulated revealing natural plasticity in monocyte phenotype and function that could reveal targets for improving pregnancy outcomes but also yield alternative therapeutic approaches to diverse metabolic and/or immune-mediated diseases beyond pregnancy.

An elevated level of interleukin-17A in a Senegalese malaria cohort is associated with rs8193038 IL-17A genetic variant

Malaria infection is a multifactorial disease partly modulated by host immuno-genetic factors. Recent evidence has demonstrated the importance of Interleukin-17 family proinflammatory cytokines and their genetic variants in host immunity. However, limited knowledge exists about their role in parasitic infections such as malaria. We aimed to investigate IL-17A serum levels in patients with severe and uncomplicated malaria and gene polymorphism’s influence on the IL-17A serum levels. In this research, 125 severe (SM) and uncomplicated (UM) malaria patients and 48 free malaria controls were enrolled. IL-17A serum levels were measured with ELISA. PCR and DNA sequencing were used to assess host genetic polymorphisms in IL-17A. We performed a multivariate regression to estimate the impact of human IL-17A variants on IL-17A serum levels and malaria outcomes. Elevated serum IL-17A levels accompanied by increased parasitemia were found in SM patients compared to UM and controls (P < 0.0001). Also, the IL-17A levels were lower in SM patients who were deceased than in those who survived. In addition, the minor allele frequencies (MAF) of two IL-17A polymorphisms (rs3819024 and rs3748067) were more prevalent in SM patients than UM patients, indicating an essential role in SM. Interestingly, the heterozygous rs8193038 AG genotype was significantly associated with higher levels of IL-17A than the homozygous wild type (AA). According to our results, it can be concluded that the IL-17A gene rs8193038 polymorphism significantly affects IL-17A gene expression. Our results fill a gap in the implication of IL-17A gene polymorphisms on the cytokine level in a malaria cohort. IL-17A gene polymorphisms also may influence cytokine production in response to Plasmodium infections and may contribute to the hyperinflammatory responses during severe malaria outcomes.

TRPV4 Regulates the Macrophage Metabolic Response to Limit Sepsis-induced Lung Injury.

Sepsis is a systemic inflammatory response that requires effective macrophage metabolic functions to resolve ongoing inflammation. Previous work showed that the mechanosensitive cation channel, transient receptor potential vanilloid 4 (TRPV4), mediates macrophage phagocytosis and cytokine production in response to lung infection. Here, we show that TRPV4 regulates glycolysis in a stiffness-dependent manner by augmenting macrophage glucose uptake by GLUT1. In addition, TRPV4 is required for LPS-induced phagolysosome maturation in a GLUT1-dependent manner. In a cecal slurry mouse model of sepsis, TRPV4 regulates sepsis-induced glycolysis as measured by BAL fluid (BALF) lactate and sepsis-induced lung injury as measured by BALF total protein and lung compliance. TRPV4 is necessary for bacterial clearance in the peritoneum to limit sepsis-induced lung injury. It is interesting that BALF lactate is increased in patients with sepsis compared with healthy control participants, supporting the relevance of lung cell glycolysis to human sepsis. These data show that macrophage TRPV4 is required for glucose uptake through GLUT1 for effective phagolysosome maturation to limit sepsis-induced lung injury. Our work presents TRPV4 as a potential target to protect the lung from injury in sepsis.

Abstract WP162: Myelin Suppresses Macrophage Responses to Diverse Stimuli

Introduction: In intracerebral hemorrhage (ICH), blood-derived macrophages initially contribute to a neurotoxic inflammatory response. Understanding the endogenous signals that control this response could help limit these effects. Uptake of myelin debris limits macrophage inflammatory cytokine production in response to LPS, possibly due to the cholesterol present in myelin. However, the pathways by which myelin suppresses macrophage responses and the broader impacts of myelin on macrophage phenotype are poorly understood. Methods and Results: RNA sequencing was used to examine impacts of myelin in diverse conditions. BMDMs were pretreated with myelin, cholesterol or T0901317, an agonist of the cholesterol-response transcription factor LXR. The cells were stimulated with Type I cytokines, Type II cytokines, and different TLR agonists to mimic the diverse stimuli encountered in the inflamed brain (Fig 1A). Myelin suppressed responses to all stimulations, as did T0901317. Cholesterol suppressed the response to LPS and had little effect on other stimulations, including the ICH-relevant alarmin S100A9 (Fig 2). Gene Ontology Enrichment showed that many of the inflammatory gene pathways suppressed by myelin were unique for each stimulation although lipid metabolism pathways were shared across stimulations. Conclusions: Myelin broadly limits macrophage responses to diverse stimulations, partially though not completely through cholesterol and activation of LXR. Cholesterol does not suppress the response to S100A9, suggesting that other components of myelin or activation of phagocytic receptors contribute to suppression of the macrophage response during ICH.

Dual CAR-T Cells Targeting CD19 and CD37 Are Effective in Target Antigen Loss B-cell Tumor Models.

Chimeric antigen receptor T (CAR-T) cells targeting multiple antigens (Ag), may reduce the risk of immune escape following the loss of the target Ag and further increase the efficacy of treatment. We developed dual-targeting CAR-T cells that target CD19 and CD37 Ags and evaluated their antitumor effects. CD19/CD37 dual CAR-T cells were generated using cotransduction and simultaneous gene transfer of two types of lentiviral vectors transferring CD19CAR or CD37CAR genes, including the intracellular domains of CD28 and CD3ζ signaling domains. These dual CAR-T cells contained three fractions: CD19/CD37 bispecific CAR-T cells, single CD19CAR-T cells, and single CD37CAR-T cells. In the functional evaluation of CAR-T cells in vitro, CD19/CD37 dual CAR-T cells showed adequate proliferation and cytokine production in response to CD19 and CD37 antigen stimulation alone or in combination. Evaluation of intracellular signaling revealed that dual CAR-T cell-mediated signals were comparable with single CAR-T cells in response to CD19- and CD37-positive B-cell tumors. Although the cytotoxicity of CD19/CD37 dual CAR-T cells in both CD19- and CD37-positive B-cell tumors was similar to that of single CD19 and CD37CAR-T cells, against CD19 and CD37 Ag-heterogeneous tumor, dual CAR-T cells demonstrated significantly superior tumor lysis compared with single CAR-T cells. Furthermore, CD19/CD37 dual CAR-T cells effectively suppressed Ag-heterogeneous Raji cells in a xenograft mouse model. Collectively, these results suggest that CD19/CD37 dual CAR-T cells may be effective target-Ag-loss B-cell tumor models in vitro and in vivo, which represents a promising treatment for patients with relapsed/refractory B-cell malignancies.

Consequences of mitochondrial DNA depletion in microglial cells

AbstractBackgroundMitochondrial dysfunction is an early and prominent feature of Alzheimer’s disease (AD). We have recently published that lower brain mitochondrial DNA copy number (mtDNAcn) is associated with a greater odds of AD neuropathological change and worse cognitive performance. As it is known that microglial cells play an important role in AD, we want to address how a reduction in mtDNAcn may affect microglial function.Methodsmicroglial BV2 cells were treated with ethidium bromide (etbr) (1ug/ml) for 5 days to achieve a 95% reduction of mitochondrial‐encoded genes expression. Cells were characterized by RNAseq and Seahorse experiments. BV2 cells were challenged with latex beads and 10uM zymosan‐pHrodo for 24 hours and phagocytosis was analyzed by flow cytometry. BV2 cells were stimulated with LPS (100ng/ml) for 3 hours to measure cytokine release by qPCR and ELISA. After BV2 stimulation, fresh media (without LPS or etbr) was conditioned for 6 hours and collected to treat neurons (SH‐SY5Y cells) and human primary astrocytes for 24 hours. RNA and protein samples were collected.ResultsmtDNAcn reduction led to a dramatic reduction in oxidative phosphorylation and a mild reduction in proliferation. RNAseq data showed an increase in the hypoxia signature and cellular senescence, coupled with a reduction in biosynthetic process and response to interferon. No changes were observed in phagocytic capacity. However, after LPS stimulation, etbr‐treated cells showed a reduction in the production of IL1B and IL6 cytokines, suggesting an impairment in the response to challenge. Neurons exposed to media from etbr‐treated BV2 cells showed an increase in MAPT and APOE expression. Samples from astrocytes are currently being analyzed.ConclusionsmtDNAcn reduction impairs mitochondrial respiration, which affects the production of cytokines in response to stimulus and leads to cell senescence in microglial cells. Cytokines released by microglia affected neuronal expression of AD‐related genes.

Multiplex Prime Editing and PASSIGE TM for Non-Viral Generation of an Allogeneic CAR-T Cell Product

While CAR-T cell therapy represents a major advance in personalized immunotherapy, the autologous nature of commercially available CAR-T products have delayed its broad application beyond a subset of hematological malignancies. In particular, manufacturing autologous CAR-T therapies is costly and time-consuming, and success relies on the fitness of a patient's cells. An allogeneic off-the-shelf CAR-T product could overcome these cell quality and quantity issues and could be accessed on-demand. However, a successful allogeneic CAR-T product will require multiplex gene knockout in addition to stable CAR integration to prevent graft-versus-host disease (GvHD) and graft rejection by the patient's immune system. Strategies for the delivery and expression of CAR transgenes include semi-random integration using lentivirus or transposons, which risk unintended gene disruption or activation. Targeted integration can be achieved using nucleases in combination with a template for homology-directed repair (HDR), but efficiency of this approach is generally low and there are risks associated with the induction of double-strand breaks (DSBs), including p53 activation and chromothripsis. This is further complicated in a situation where multiplex editing is required, as multiple DSBs can lead to gross chromosomal rearrangements. Alternatively, base editors have been used for targeted gene disruption without the induction of DSBs via single nucleotide conversion to disrupt a splice site or introduce a single premature stop (pmSTOP) codon. However, this approach is limited to transition mutations and cannot be used to integrate large genetic cargo, which is required to generate CAR-T cells. Prime Editing (PE) can be used for precise and programmable gene disruption via multiple strategies, including introduction of multiple pmSTOP codons, splice site disruption, frameshift mutations, or deletion of large segments of regulatory or coding sequence. Further, Prime-Assisted Site-Specific Integrase Gene Editing (PASSIGE) can be used to precisely and flexibly integrate large genetic cargo at a specific locus. Together, PE-mediated gene knock out and PASSIGE can be tailored to generate a more broadly applicable, potentially safer, and more effective CAR-T cell product. To evaluate the efficacy of an all-PE non-viral approach to allogeneic CAR-T cell generation, PASSIGE and PE mediated gene knockout were used to generate CD19-CAR-T cells. Multiplex PE precisely disrupted expression of the endogenous T cell receptor alpha constant ( TRAC) and beta-2-microglobulin ( B2M) loci in over 90% of human T cells. Co-delivery of a non-viral DNA donor template with PASSIGE editing components resulted in targeted integration of a 3.5 kb CD19-CAR transgene expression cassette at the TRAC locus in over 60% of the T cells, with no observed impact on T cell viability, phenotype, or functionality. CD19 CAR-T cells generated using PASSIGE show potent anti-tumor activity and cytokine production in response to CD19 + tumor cell lines in vitro and in vivo. The PASSIGE-generated CD19 CAR-T cells reduce tumor burden and prolong survival of mice bearing CD19 + tumors. These results show that a PE platform can be used to generate multiplex edited CAR-T cells without the need for viral vectors and without causing DSBs. This modular, one-step, non-viral delivery Prime Editing platform expands the applicability of T cell therapies for the treatment of tumors and immune diseases.

Immunological Phenotyping of Mice with a Point Mutation in Cdk4.

Cyclin-dependent kinases (CDKs) play a crucial role in regulation of the mammalian cell cycle. CDK4 and CDK6 control the G1/S restriction checkpoint through their ability to associate with cyclin D proteins in response to growth factor signals. CDK4 deficiency in mice gives rise to a range of endocrine-specific phenotypes including diabetes, infertility, dwarfism, and atrophy of the anterior pituitary. Although CDK6 deficiency can cause thymic atrophy due to a block in the double-negative (DN) to double-positive (DP) stage of T cell development, there are no overt defects in immune cell development reported for CDK4-deficient mice. Here, we examined the impact of a novel N-ethyl-N-nitrosourea-induced point mutation in the gene encoding CDK4 on immune cell development. Mutant mice (Cdk4wnch/wnch) showed normal development and differentiation of major immune cell subsets in the thymus and spleen. Moreover, T cells from Cdk4wnch/wnch mice exhibited normal cytokine production in response to in vitro stimulation. However, analysis of the mixed bone marrow chimeras revealed that Cdk4wnch/wnch-derived T cell subsets and NK cells are at a competitive disadvantage compared to Cdk4+/+-derived cells in the thymus and periphery of recipients. These results suggest a possible role for the CDK4wnch mutation in the development of some immune cells, which only becomes apparent when the Cdk4wnch/wnch mutant cells are in direct competition with wild-type immune cells in the mixed bone marrow chimera.

Feeding Saccharomyces cerevisiae fermentation postbiotic products alters immune function and the lung transcriptome of preweaning calves with an experimental viral-bacterial coinfection

Bovine respiratory disease causes morbidity and mortality in cattle of all ages. Supplementing with postbiotic products from Saccharomyces cerevisiae fermentation (SCFP) has been reported to improve growth and provide metabolic support required for immune activation in calves. The objective of this study was to determine effects of SCFP supplementation on the transcriptional response to coinfection with bovine respiratory syncytial virus (BRSV) and Pasteurella multocida in the lung using RNA sequencing. Twenty-three calves were enrolled and assigned to 2 treatment groups: control (n = 12) or SCFP-treated (n = 11, fed 1 g/d SmartCare in milk and 5 g/d NutriTek on starter grain; both from Diamond V Mills Inc.). Calves were infected with ∼104 median tissue culture infectious dose per milliliter of BRSV, followed 6 d later by intratracheal inoculation with ∼1010 cfu of Pasteurella multocida (strain P1062). Calves were euthanized on d 10 after viral infection. Blood cells were collected and assayed on d 0 and 10 after viral infection. Bronchoalveolar lavage (BAL) cells were collected and assayed on d 14 of the feeding period (preinfection) and d 10 after viral infection. Blood and BAL cells were assayed for proinflammatory cytokine production in response to stimulation with lipopolysaccharide (LPS) or a combination of polyinosinic:polycytidylic acid and imiquimod, and BAL cells were evaluated for phagocytic and reactive oxygen species production capacity. Antemortem and postmortem BAL and lesioned and nonlesioned lung tissue samples collected at necropsy were subjected to RNA extraction and sequencing. Sequencing reads were aligned to the bovine reference genome (UMD3.1) and edgeR version 3.32.1 used for differential gene expression analysis. Supplementation with SCFP did not affect the respiratory burst activity or phagocytic activity of either lung or blood immune cells. Immune cells from the peripheral blood of SCFP-supplemented calves produced increased quantities of IL-6 in response to toll-like receptor stimulation, whereas cells from the BAL of SCFP-treated calves secreted fewer proinflammatory cytokines and less tumor necrosis factor-α (TNF-α) and IL-6 in response to the same stimuli. Transcriptional responses in lung tissues and BAL samples from SCFP-fed calves differed from the control group. The top enriched pathways in SCFP-treated lungs were associated with decreased expression of inflammatory responses and increased expression of plasminogen and genes involved in glutathione metabolism, supporting effective lung repair. Our results indicate that supplementing with SCFP postbiotics modulates both systemic and mucosal immune responses, leading to increased resistance to bovine respiratory disease.

Dimethyl Fumarate Protects against Lipopolysaccharide- (LPS-) Induced Sepsis through Inhibition of NF-κB Pathway in Mice.

Sepsis is one of the most severe complications and causes of mortality in the clinic. It remains a great challenge with no effective treatment for clinicians worldwide. Inhibiting the release of proinflammatory cytokines during sepsis is considered as an important strategy for treating sepsis and improving survival. In the present study, we have observed the effect of dimethyl fumarate (DMF) on lipopolysaccharide- (LPS-) induced sepsis and investigated the possible mechanism. By screening a subset of the Johns Hopkins Drug Library, we identified DMF as a novel inhibitor of nitric oxide synthesis in LPS-stimulated RAW264.7 cells, suggesting that DMF could be a potential drug to treat sepsis. To further characterize the effect of DMF on LPS signaling, TNF-α, MCP-1, G-CMF, and IL-6 expression levels were determined by using cytokine array panels. In addition, an endotoxemia model with C57BL/6 mice was used to assess the in vivo efficacy of DMF on sepsis. The survival rate was assessed, and HE staining was performed to investigate histopathological damage to the organs. DMF was found to increase the survival of septic mice by 50% and attenuate organ damage, consistent with the reduction in IL-10, IL-6, and TNF-α (inflammatory cytokines) in serum. In vitro experiments revealed DMF's inhibitory effect on the phosphorylation of p65, IκB, and IKK, suggesting that the primary inhibitory effects of DMF can be attributed, at least in part, to the inhibition of phosphorylation of IκBα, IKK as well as nuclear factor-κB (NF-κB) upon LPS stimulation. The findings demonstrate that DMF dramatically inhibits NO and proinflammatory cytokine production in response to LPS and improves survival in septic mice, raising the possibility that DMF has the potential to be repurposed as a new treatment of sepsis.

Subcutaneous Bacillus Calmette-Guérin Administration Induces Innate Training in Monocytes in Preweaned Holstein Calves.

The bacillus Calmette-Guérin (BCG) vaccine, administered to prevent tuberculosis, is a well-studied inducer of trained immunity in human and mouse monocytes. We have previously demonstrated that aerosol BCG administration induces innate training in calves. The current study aimed to determine whether s.c. BCG administration could induce innate training, identify the cell type involved, and determine whether innate training promoted resistance to bovine respiratory syncytial virus (BRSV) infection, a major cause of bovine respiratory disease in preweaned calves. A total of 24 calves were enrolled at 1-3 d of age and blocked by age into two treatment groups (BCG, n = 12; control, n = 12). BCG was given s.c. to preweaned calves. The control calves received PBS. We observed a trained phenotype, demonstrated by enhanced cytokine production in response to invitro stimulation with LPS (TLR-4 agonist) in PBMCs and CD14+ monocytes from the BCG group 2 wk (IL-1β, p = 0.002) and 4 wk (IL-1β, p = 0.005; IL-6, p = 0.013) after BCG administration, respectively. Calves were experimentally infected via aerosol inoculation with BRSV strain 375 at 5 wk after BCG administration and necropsied on day 8 postinfection. There were no differences in disease manifestation between the treatment groups. Restimulation of bronchoalveolar lavage fluid cells isolated on day 8 after BRSV infection revealed enhanced IL-1β (p = 0.014) and IL-6 (p = 0.010) production by the BCG group compared with controls. In conclusion, results from our study show that s.c. administration of the BCG vaccine can induce trained immunity in bovine monocytes and influence cytokine production in the lung environment after BRSV infection.

Effect of mevalonate, zoledronate and BCG on monocyte/macrophage phenotype

Cells of innate immunity, mainly monocytes/macrophages, form a long-term nonspecific immunological memory during the initial encounter with the pathogen, the so-called trained immunity. Mevalonate pathway metabolites play an important role in the formation of trained immunity. The aim of this investigation was to study the effect of modulators of mevalonate pathway, mevalonate and zoledronate, on the formation of trained immunity in human and animal monocytes/ macrophages.Material and methods. Human monocyte-like cell lines THP-1 and U-937, peritoneal macrophages of BALB/c mice were used. Trained immunity was induced in vitro by incubation of THP-1 and U-937 monocyte-like cell lines for 24 and 72 hours with inactivated Mycobacteria of BCG vaccine strain, and in vivo by intraperitoneal administration of BCG to BALB/c mice with isolation of peritoneal macrophages on day 7 after infection (lag phase). Cell hyperreactivity was assessed by response to a second stimulus with bacterial lipopolysaccharide (LPS) and mevalonate, zoledranate in the presence or absence of LPS. Lactate, cytokine (IL-1β, TNF-α, IL-10), nitric oxide and glucose level was measured in conditioned media from cells.Results and discussion. The study showed that monocyte-like cell lines THP-1 and U-937 responded differently by cytokine production, lactate, and glucose consumption to BCG stimulus in the presence or absence of lag phase. Mevalonate and zoledronate alone or in combination with LPS also stimulated cytokine production in different ways. The presence of lag phase for human monocyte-like cells is essential for the level of cytokine production and glucose consumption. Peritoneal macrophages have been shown to enhance pro-inflammatory cytokine production in response to LPS, mevalonate, and zoledronate.Conclusions. Mevalonate and zoledronate induce trained immunity in monocytes/macrophages.

Defects in immune response to Toxoplasma gondii are associated with enhanced HIV-1-related neurocognitive impairment in co-infected patients.

Human immunodeficiency virus-1 (HIV-1) and Toxoplasma gondii can invade the central nervous system and affect its functionality. Advanced HIV-1 infection has been associated with defects in immune response to T. gondii, leading to reactivation of latent infections and development of toxoplasmic encephalitis. This study evaluates relationship between changes in immune response to T. gondii and neurocognitive impairment in HIV-1/T. gondii co-infected patients, across different stages of HIV-1 infection. The study assessed the immune response to T. gondii by measuring cytokine production in response to parasite antigens, and also neurocognitive functions by performing auditory and visual P300 cognitive evoked potentials, short term memory (Sternberg) and executive function tasks (Wisconsin Card Sorting Test-WCST) in 4 groups of individuals: HIV-1/T. gondii co-infected (P2), HIV-1-infected/T. gondii-non-infected (P1), HIV-1-non-infected/T. gondii-infected (C2) and HIV-1-non-infected/T. gondii-non-infected (C1). Patients (P1 and P2) were grouped in early/asymptomatic (P1A and P2A) or late/symptomatic (P1B/C and P2B/C) according to peripheral blood CD4+ T lymphocyte counts (>350 or <350/μL, respectively). Groups were compared using T-student or U-Mann-Whitney tests as appropriate, p<0.05 was considered as significantly. For P300 waves, HIV-1-infected patients (P1) had significantly longer latencies and significantly smaller amplitudes than uninfected controls, but HIV-1/T. gondii co-infected patients (P2) had significantly longer latencies and smaller amplitude than P1. P1 patients had significantly poorer results than uninfected controls in Sternberg and WCST, but P2 had significantly worse results than P1. HIV-1 infection was associated with significantly lower production of IL-2, TNF-α and IFN-γ in response to T. gondii from early/asymptomatic stages, when comparing P2 patients to C2 controls. These findings may indicate impairment in anti-parasitic response in co-infected patients, facilitating early limited reactivation of the parasitic latent infection, therefore creating cumulative damage in the brain and affecting neurocognitive functions from asymptomatic stages of HIV-1 infection, as suggested by defects in co-infected patients in this study.

Modeling the innate inflammatory cGAS/STING pathway: sexually dimorphic effects on microglia and cognition in obesity and prediabetes.

The prevalence of obesity, prediabetes, and diabetes continues to grow worldwide. These metabolic dysfunctions predispose individuals to neurodegenerative diseases and cognitive impairment, including dementias such as Alzheimer's disease and Alzheimer's disease related dementias (AD/ADRD). The innate inflammatory cGAS/STING pathway plays a pivotal role in metabolic dysfunction and is an emerging target of interest in multiple neurodegenerative diseases, including AD/ADRD. Therefore, our goal was to establish a murine model to specifically target the cGAS/STING pathway to study obesity- and prediabetes-induced cognitive impairment. We performed two pilot studies in cGAS knockout (cGAS-/-) male and female mice designed to characterize basic metabolic and inflammatory phenotypes and examine the impact of high-fat diet (HFD) on metabolic, inflammatory, and cognitive parameters. cGAS-/- mice displayed normal metabolic profiles and retained the ability to respond to inflammatory stimuli, as indicated by an increase in plasma inflammatory cytokine production in response to lipopolysaccharide injection. HFD feeding caused expected increases in body weight and decreases in glucose tolerance, although onset was accelerated in females versus males. While HFD did not increase plasma or hippocampal inflammatory cytokine production, it did alter microglial morphology to a state indicative of activation, particularly in female cGAS-/- mice. However, HFD negatively impacted cognitive outcomes in male, but not female animals. Collectively, these results suggest that cGAS-/- mice display sexually dimorphic responses to HFD, possibly based on differences in microglial morphology and cognition.

QRICH1 is a negative regulator of T cell activation via the CARD11 signalosome

Abstract Controlled lymphocyte activation is crucial for defending against pathogens while preventing diseases such as autoimmunity and lymphoma. In T lymphocytes, the scaffold protein and signaling hub CARD11 mediates NF-κB, JNK, and mTOR pathway activation downstream of T cell receptor (TCR) engagement. We recently identified QRICH1 as a previously unrecognized CARD11 interactor recruited to the CARD11 complex downstream of TCR engagement and discovered that QRICH1 protein levels are upregulated in wild-type activated mouse primary CD8+ T cells. Overexpression of QRICH1 in 293T and Jurkat T cells dose-dependently inhibited NF-κB activation by wild-type CARD11 as well as by constitutively active and oncogenic CARD11 mutants. QRICH1-deficient Jurkat T cells had increased NF-κB activation and IL-2 secretion downstream of TCR stimulation. To study the in vivo role of QRICH1 in immunity, we generated a novel QRICH1 conditional KO mouse model. Compared to control mice, QRICH1 T cell KO mice had decreased splenic CD4+ and CD8+ T cells, despite no differences in thymic CD4+, CD8+, or CD4+CD8+ T cell populations. The CD8+ T cell population in QRICH1 T cell KO mice was skewed towards an effector memory phenotype. QRICH1-deficient primary CD8+ T cells had increased proliferation and production of pro-inflammatory cytokines IFN-γ and TNF-α in response to ex vivo stimulation with anti-CD3/CD28, and QRICH1-deficient OT-1 CD8+ T cells also had increased cytokine production in response to antigen-specific stimulation with Ova peptide. Our findings reveal that QRICH1 negatively regulates T cell activation by modulating CARD11’s signaling output. This work was supported by NIH grants R01AI148143, F31CA254167, and T32GM007445.

CD38: an ecto-enzyme with functional diversity in T cells.

CD38, a nicotinamide adenine dinucleotide (NAD)+ glycohydrolase, is considered an activation marker of T lymphocytes in humans that is highly expressed during certain chronic viral infections. T cells constitute a heterogeneous population; however, the expression and function of CD38 has been poorly defined in distinct T cell compartments. We investigated the expression and function of CD38 in naïve and effector T cell subsets in the peripheral blood mononuclear cells (PBMCs) from healthy donors and people with HIV (PWH) using flow cytometry. Further, we examined the impact of CD38 expression on intracellular NAD+ levels, mitochondrial function, and intracellular cytokine production in response to virus-specific peptide stimulation (HIV Group specific antigen; Gag). Naïve T cells from healthy donors showed remarkably higher levels of CD38 expression than those of effector cells with concomitant reduced intracellular NAD+ levels, decreased mitochondrial membrane potential and lower metabolic activity. Blockade of CD38 by a small molecule inhibitor, 78c, increased metabolic function, mitochondrial mass and mitochondrial membrane potential in the naïve T lymphocytes. PWH exhibited similar frequencies of CD38+ cells in the T cell subsets. However, CD38 expression increased on Gag-specific IFN-γ and TNF-α producing cell compartments among effector T cells. 78c treatment resulted in reduced cytokine production, indicating its distinct expression and functional profile in different T cell subsets. In summary, in naïve cells high CD38 expression reflects lower metabolic activity, while in effector cells it preferentially contributes to immunopathogenesis by increasing inflammatory cytokine production. Thus, CD38 may be considered as a therapeutic target in chronic viral infections to reduce ongoing immune activation.

Rational design of a vaccine for Alzheimer’s disease using computationally-derived conformational B cell epitopes to selectively target toxic amyloid-beta oligomers (S26.004)

<h3>Objective:</h3> Design an optimal amyloid-beta (Abeta) vaccine to elicit a robust and durable antibody response against toxic Abeta oligomers (ABO) without inducing potentially detrimental B or T cell responses against plaque or normal Abeta. <h3>Background:</h3> Abeta vaccines have the potential to protect against disease but also carry the risk of eliciting proinflammatory T cell responses causing meningoencephalitis, and plaque-reactive antibodies that can increase the risk of brain edema (ARIA-E). To circumvent these issues and induce an antibody response that selectively targets soluble toxic ABO, we designed a vaccine consisting of a computationally-derived conformational B cell epitope of ABO, coupled to KLH as a carrier protein to provide T cell help. <h3>Design/Methods:</h3> Mice received 3 immunizations, 4 weeks apart, with vaccine conjugate in alum or QS-21 as adjuvants. Serum titers and IgG subtypes of antibodies to the peptide epitope were measured by ELISA. The selectivity of serum antibodies for toxic ABO versus monomers or plaque was assessed by SPR and immunohistochemistry, respectively. T helper responses to the peptide and to KLH were evaluated by ELISPOT analysis of splenic lymphocytes. <h3>Results:</h3> A robust antibody response against the ABO epitope was observed with both adjuvants and was remarkably maintained unabated out to 6 months after the last immunization. The serum antibodies reacted with ABO only, not monomers or plaque. ELISPOT analysis showed T helper cytokine production in response to stimulation with KLH but not the ABO epitope thereby confirming that the peptide only contains a B cell epitope. <h3>Conclusions:</h3> A vaccine consisting of an ABO-restricted conformational B cell epitope conjugated to KLH produced a strong Abeta antibody response with no measurable pro-inflammatory T cell response against Abeta. In addition, the oligomer selectivity of the antibodies focused the response on pathogenic ABO, potentially reducing the risk of ARIA-E associated with binding to plaque and vascular deposits of Abeta. <b>Disclosure:</b> Dr. Kaplan has received personal compensation for serving as an employee of ProMIS Neurosciences. Dr. Kaplan has stock in ProMIS Neurosciences. Dr. Kaplan has received intellectual property interests from a discovery or technology relating to health care. The institution of Dr. Napper has received research support from Weston Family Foundation. Ms. Scruten has nothing to disclose. Dr. Gibbs has received personal compensation in the range of $50,000-$99,999 for serving as a Consultant for Promis Neuroscinces. Ms. Coutts has nothing to disclose. Dr. Cashman has received personal compensation for serving as an employee of ProMIS Neurosciences. Dr. Cashman has received personal compensation in the range of $5,000-$9,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Mitsubishi-Tanabe. Dr. Cashman has stock in ProMIS Neurosciences. The institution of Dr. Cashman has received research support from ProMIS Neurosciences. Dr. Cashman has received intellectual property interests from a discovery or technology relating to health care. Dr. Cashman has a non-compensated relationship as a BoD memeber with ALS Society of BC that is relevant to AAN interests or activities.

191 Treatment with GM-CSF Reverses TBI-Induced Immune Suppression in a Combined Injury Model

INTRODUCTION: Traumatic brain injury (TBI) is a potentially devastating injury for which there are no pharmacotherapies. Frequently, TBI is accompanied by injuries outside the CNS, and we have previously shown that TBI with concurrent extra-cranial injury reliably leads to post-injury immunosuppression, which puts patients at risk for infection and ultimately a poor outcome. To reverse post-injury immune suppression, we have proposed the use of the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF). METHODS: To induce post-injury immunosuppression, we used a previously described model. 28 day old rats were injured via controlled cortical impact and then had 25% blood volume removed. Following injury, animals were treated with vehicle, 5 μg/kg, or 50 μg/kg GM-CSF daily for 7 days. Blood was obtained pre-injury, and on 1, 3, and 7 days post-injury. Brain and spleen were harvested at 7 days post-injury. Adaptive and innate immune function (measured by ex vivo cytokine production in response to an immunostimulant) were assessed in blood and spleen. Flow cytometry was performed on blood and spleen samples. Cytokine levels at the lesion and penumbra were measured by ELISA. RESULTS: As shown previously, injury induced reduction of both innate and adaptive immune function (approximately 50% decrease), which was reversed with GM-CSF. Treatment with GM-CSF significantly expanded the percentage of monocytes. In spleen, injury was associated with an increase in percentage of CD4 cells expressing programmed death-1 (PD-1), and this was also reversed with GM-CSF. ELISA for cytokines in brain samples from lesion and penumbra from GM-CSF treated animals were not significantly different from vehicle. CONCLUSIONS: These data add to a body of literature suggesting that severe trauma leads to immune suppression. Our findings suggest that treatment with GM-CSF may normalize post-injury immune response.