Activation Of Innate Immune Response Research Articles

Abstract 5254: Topical inhibition of UV-induced PD-L1 expression and inflammatory signaling by the small molecule inhibitor BMS-202

Abstract Immune evasion is a molecular hallmark of cancer, and overexpression of the immune checkpoint protein PD-L1 (CD274) has been documented in several tumors including cutaneous squamous cell carcinoma (cSCC). Notably, while baseline levels of PD-L1 in normal human epidermis are low, acute solar-simulated light (SSL) exposure increases the expression of this protein in epidermal keratinocytes, an effect which can be replicated in cultured keratinocytes and in mouse models. While current immunotherapies targeting the PD-1/PD-L1 axis using monoclonal antibodies are providing therapeutic benefits in many cancers including cSCC, small molecule inhibitors are also being developed to target PD-L1 which may be amenable for topical formulation and therefore early intervention with reduced systemic exposure. We have recently tested one such molecule, BMS-202, in vitro and in vivo. BMS-202 application to keratinocytes in culture inhibits UV-induced PD-L1 RNA and protein expression. Notably, exposure to this inhibitor also reduced UV-induced stress signaling as measured by AP-1 luciferase assay in cultured keratinocytes. Topical application of BMS-202 to immunocompetent SKH-1 mouse skin resulted in significant inhibition of SSL-induced epidermal PD-L1 as determined by immunoblot analysis. NanoString nCounter Pathway™ transcriptomic analysis of full-thickness SKH-1 mouse skin shows strong inhibition of SSL-induced inflammatory responses, chemokine activity, innate immune response and NF-κB activation in BMS-202 treated samples compared to SSL-only controls. Our results indicate that early intervention against PD-L1 expression/activity could be a viable target for skin cancer photochemoprevention. Topical application of small molecule PD-L1 inhibitors such as BMS-202 may provide novel treatment options for populations at high risk for cSCC. Citation Format: Prajakta Vaishampayan, Jana Jandova, Yuchen (Ella) Ai, Viktoria Kirschnerova, Clara Curiel-Lewandrowski, Georg T. Wondrak, Sally E. Dickinson. Topical inhibition of UV-induced PD-L1 expression and inflammatory signaling by the small molecule inhibitor BMS-202. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5254.

Magnitude and kinetics of the human immune cell response associated with severe dengue progression by single-cell proteomics.

Approximately 5 million dengue virus-infected patients progress to a potentially life-threatening severe dengue (SD) infection annually. To identify the immune features and temporal dynamics underlying SD progression, we performed deep immune profiling by mass cytometry of PBMCs collected longitudinally from SD progressors (SDp) and uncomplicated dengue (D) patients. While D is characterized by early activation of innate immune responses, in SDp there is rapid expansion and activation of IgG-secreting plasma cells and memory and regulatory T cells. Concurrently, SDp, particularly children, demonstrate increased proinflammatory NK cells, inadequate expansion of CD16+ monocytes, and high expression of the FcγR CD64 on myeloid cells, yet a signature of diminished antigen presentation. Syndrome-specific determinants include suppressed dendritic cell abundance in shock/hemorrhage versus enriched plasma cell expansion in organ impairment. This study reveals uncoordinated immune responses in SDp and provides insights into SD pathogenesis in humans with potential implications for prediction and treatment.

DNA hypomethylation mediates immune response in pan-cancer

ABSTRACT Abnormal DNA methylation is a fundamental characterization of epigenetics in cancer. Here we demonstrate that aberrant DNA methylating can modulate the tumour immune microenvironment in 16 cancer types. Differential DNA methylation in promoter region can regulate the transcriptomic pattern of immune-related genes and DNA hypomethylation mainly participated in the processes of immunity, carcinogenesis and immune infiltration. Moreover, many cancer types shared immune-related functions, like activation of innate immune response, interferon gamma response and NOD-like receptor signalling pathway. DNA methylation can further help identify molecular subtypes of kidney renal clear cell carcinoma. These subtypes are characterized by DNA methylation pattern, major histocompatibility complex, cytolytic activity and cytotoxic t lymphocyte and tumour mutation burden, and subtype with hypomethylation pattern shows unstable immune status. Then, we investigate the DNA methylation pattern of exhaustion-related marker genes and further demonstrate the role of hypomethylation in tumour immune microenvironment. In summary, our findings support the use of hypomethylation as a biomarker to understand the mechanism of tumour immune environment.

Impaired Integrated Stress Response and Mitochondrial Integrity Modulate Genotoxic Stress Impact and Lower the Threshold for Immune Signalling.

Mitochondria-nucleus communication during stress dictates cellular fate with consequences on the etiopathology of multiple age-related diseases. Impaired mitochondrial quality control through loss of function of the mitochondrial protease HtrA2 associates with accumulation of damaged mitochondria and triggers the integrated stress response, implicating the transcription factor CHOP. Here we have employed a combined model of impaired mitochondria quality control, namely HtrA2 loss of function, and/or integrated stress response, namely CHOP loss of function, and genotoxicity to address the distinctive roles of these cellular components in modulating intracellular and intercellular responses. The genotoxic agents employed were cancer therapeutic agents such as irradiation with X-ray and protons or treatment with the radiomimetic bleomycin. The irradiation had an enhanced effect in inducing DNA damage in cells with CHOP loss of function, while the bleomycin treatment induced more DNA damage in all the transgenic cells as compared to the control. The genetic modifications impaired the transmission of DNA damage signalling intercellularly. Furthermore, we have dissected the signalling pathways modulated by irradiation in selected genotypes with RNA sequencing analysis. We identified that loss of HtrA2 and CHOP function, respectively, lowers the threshold where irradiation may induce the activation of innate immune responses via cGAS-STING; this may have a significant impact on decisions for combined therapeutic approaches for various diseases.

Mitochondrion-targeted antioxidant SkQ1 prevents rapid animal death caused by highly diverse shocks

The response to stress involves the activation of pathways leading either to protection from the stress origin, eventually resulting in development of stress resistance, or activation of the rapid death of the organism. Here we hypothesize that mitochondrial reactive oxygen species (mtROS) play a key role in stress-induced programmed death of the organism, which we called “phenoptosis” in 1997. We demonstrate that the synthetic mitochondria-targeted antioxidant SkQ1 (which specifically abolishes mtROS) prevents rapid death of mice caused by four mechanistically very different shocks: (a) bacterial lipopolysaccharide (LPS) shock, (b) shock in response to intravenous mitochondrial injection, (c) cold shock, and (d) toxic shock caused by the penetrating cation C12TPP. Importantly, under all these stresses mortality was associated with a strong elevation of the levels of pro-inflammatory cytokines and administration of SkQ1 was able to switch off the cytokine storms. Since the main effect of SkQ1 is the neutralization of mtROS, this study provides evidence for the role of mtROS in the activation of innate immune responses mediating stress-induced death of the organism. We propose that SkQ1 may be used clinically to support patients in critical conditions, such as septic shock, extensive trauma, cooling, and severe infection by bacteria or viruses.

Spatial characterization and quantification of CD40 expression across cancer types

BackgroundCD40, a TNF receptor family member, is expressed by a variety of immune cells and is involved in the activation of both adaptive and innate immune responses. Here, we used quantitative immunofluorescence (QIF) to evaluate CD40 expression on the tumor epithelium of solid tumors in large patient cohorts of lung, ovarian, and pancreatic cancers.MethodsTissue samples from nine different solid tumors (bladder, breast, colon, gastric, head and neck, non-small cell lung cancer (NSCLC), ovarian, pancreatic and renal cell carcinoma), constructed in tissue microarray format, were initially assessed for CD40 expression by QIF. CD40 expression was then evaluated on the large available patient cohorts for three of the tumor types demonstrating high CD40 positivity rate; NSCLC, ovarian and pancreatic cancer. The prognostic impact of CD40 expression on tumor cells was also investigated.ResultsCD40 expression on tumor cells was found to be common, with 80% of the NSCLC population, 40% of the ovarian cancer population, and 68% of the pancreatic adenocarcinoma population displaying some degree of CD40 expression on cancer cells. All of three of these cancer types displayed considerable intra-tumoral heterogeneity of CD40 expression, as well as partial correlation between expression of CD40 on tumor cells and on surrounding stromal cells. CD40 was not found to be prognostic for overall survival in NSCLC, ovarian cancer, or pancreatic adenocarcinoma.ConclusionsThe high percentage of tumor cells expressing CD40 in each of these solid tumors should be considered in the development of therapeutic agents designed to target CD40.

On the use of a coupled mathematical model for understanding the dynamics of multiple sclerosis

Multiple sclerosis (MS) is characterised by the attack on the myelin sheath, oligodendrocytes, and neurons by immune system cells. For this reason, MS is classified as an autoimmune and demyelinating disease. This work aims to study the influence of both the innate and adaptive immune response activation at the onset and development of MS through the coupling of two mathematical models in different compartments, both based on differential equations. The proposed model is, to the best of our knowledge, the first mathematical model based on differential equations that represent the spatio-temporal dynamics of the innate immune response activation in the brain tissue and the posterior activation of the adaptive cellular and humoral immune system responses in the lymph node through antigen presentation, culminating on the attack to the oligodendrocytes and plaque formation.

Biomarker Profiles Associated with COVID-19 Severity and Mortality.

The aim of this study was to analyze biomarkers that might predict the severity and progression of the SARS-CoV-2 infection, both in the acute phase and after recovery. Unvaccinated patients infected with the original strain of COVID-19 requiring ward (Group 1, n = 48) or ICU (Group 2, n = 41) admission were included. At the time of admission (visit 1), a clinical history was acquired, and blood samples were obtained. One and six months after discharge from the hospital (visits 2 and 3, respectively), a clinical history, lung function tests, and blood samples were carried out. At visit 2, patients also underwent a chest CT scan. Different cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-17A, G-CSF, GM-CSF, IFN-ɣ, MCP-1, MIP-1β, and TNF-α) and lung fibrosis biomarkers (YKL-40 and KL-6) were measured in blood samples obtained at visits 1, 2, and 3. At visit 1, IL-4, IL-5, and IL-6 levels were higher in Group 2 (p = 0.039, 0.011, and 0.045, respectively), and IL-17 and IL-8 levels were higher in Group 1 (p = 0.026 and 0.001, respectively). The number of patients in Groups 1 and 2 who died during hospitalization was 8 and 11, respectively. YKL-40 and KL-6 levels were higher in patients who died. Serum YKL-40 and KL-6 levels determined at visit 2 correlated negatively with FVC (p = 0.022 and p = 0.024, respectively) and FEV1 (p = 0.012 and p = 0.032, respectively) measured at visit 3. KL-6 levels also correlated negatively with the diffusing capacity of the lungs for carbon monoxide (DLCO, p = 0.001). Patients who required ICU admission had higher levels of Th2 cytokines, while patients admitted to the ward showed an innate immune response activation, with IL-8 release and Th1/Th17 lymphocyte contribution. Increased levels of YKL-40 and KL-6 were associated with mortality in COVID-19 patients.

Cardiomyocyte infection by Trypanosoma cruzi promotes innate immune response and glycolysis activation.

Chagas cardiomyopathy, a disease caused by Trypanosoma cruzi (T. cruzi) infection, is a major contributor to heart failure in Latin America. There are significant gaps in our understanding of the mechanism for infection of human cardiomyocytes, the pathways activated during the acute phase of the disease, and the molecular changes that lead to the progression of cardiomyopathy. To investigate the effects of T. cruzi on human cardiomyocytes during infection, we infected induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) with the parasite and analyzed cellular, molecular, and metabolic responses at 3 hours, 24 hours, and 48 hours post infection (hpi) using transcriptomics (RNAseq), proteomics (LC-MS), and metabolomics (GC-MS and Seahorse) analyses. Analyses of multiomic data revealed that cardiomyocyte infection caused a rapid increase in genes and proteins related to activation innate and adaptive immune systems and pathways, including alpha and gamma interferons, HIF-1α signaling, and glycolysis. These responses resemble prototypic responses observed in pathogen-activated immune cells. Infection also caused an activation of glycolysis that was dependent on HIF-1α signaling. Using gene editing and pharmacological inhibitors, we found that T. cruzi uptake was mediated in part by the glucose-facilitated transporter GLUT4 and that the attenuation of glycolysis, HIF-1α activation, or GLUT4 expression decreased T. cruzi infection. In contrast, pre-activation of pro-inflammatory immune responses with LPS resulted in increased infection rates. These findings suggest that T. cruzi exploits a HIF-1α-dependent, cardiomyocyte-intrinsic stress-response activation of glycolysis to promote intracellular infection and replication. These chronic immuno-metabolic responses by cardiomyocytes promote dysfunction, cell death, and the emergence of cardiomyopathy.

Impact of Toll-Like Receptor 2 and 9 Gene Polymorphisms on COVID-19: Susceptibility, Severity, and Thrombosis.

Toll-like receptors (TLRs) play an important role in activation of innate and adaptive immune responses. We aimed to detect the association between TLR2 rs5743708 G>A and TLR9 rs5743836 C>T variants and COVID-19 disease susceptibility, severity, and thrombosis by using neutrophil extracellular traps (NETs). We included 100 adult COVID-19 patients as well as 100 age- and gender-matched normal controls. Participants were genotyped for TLR2 rs5743708 and TLR9 rs5743836. Citrullinated Histone (H3) was detected as an indicator of NETs. The mutant (G/A and C/C) genotypes and (A and C) alleles of TLR2 rs5743708 and TLR9 rs5743836, respectively, have been significantly related to a higher risk of COVID-19 infection, representing a significant risk factor for the severity of COVID-19. There was no significant association between the two variants and citrullinated histone (H3). TLR2 rs5743708 and TLR9 rs5743836 variants have been significantly related to a higher risk and severity of COVID-19 infection but had no effect on thrombus formation.

The Potential of Dendritic-Cell-Based Vaccines to Modulate Type 3 Innate Lymphoid Cell Populations.

Dendritic cell (DC) vaccines are a type of immunotherapy that relies on the communication of DCs with other aspects of the immune system. DCs are potent antigen-presenting cells involved in the activation of innate immune responses and education of adaptive immunity, making them ideal targets for immunotherapies. Innate lymphoid cells (ILCs) are relatively newly identified in the field of immunology and have important roles in health and disease. The studies described here explored the communications between type 3 ILCs (ILC3s) and DCs using a murine model of DC-based vaccination. Local and systemic changes in ILC3 populations following the administration of a DC vaccine were observed, and upon challenge with B16F10 melanoma cells, changes in ILC3 populations in the lungs were observed. The interactions between DCs and ILC3s should be further explored to determine the potential that their communications could have in health, disease, and the development of immunotherapies.

Spermatozoa-induced seminal vesiculitis in mice.

Seminal vesiculitis is a common inflammation in the male genital tract. Etiologically, microbial infection and non-infectious factors can be responsible for seminal vesiculitis. The pathogenic triggers and mechanisms underlying non-infectious seminal vesiculitis remain unclear. To demonstrate that spermatozoa can induce seminal vesiculitis in mice, which could be attributable to spermatozoa-induced innate immune responses in seminal vesicular epithelial cells. Spermatozoa from epididymis were injected into seminal vesicles at the tail of the gland. Histopathology of seminal vesicles were examined by hematoxylin-eosin staining. Infiltration of leukocytes were identified by immunohistochemistry. Seminal vesicular epithelial cells were isolated from 5-week-old mice and cell types were detected by immunofluoresence. Western blot and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to detect protein and gene expression levels. In vivo, local injection of epididymal spermatozoa into seminal vesicles resulted in seminal vesiculitis characterized by tissue swelling and leukocyte infiltration. In vitro, spermatozoa induced the expression of pro-inflammatory cytokines and chemokines, including TNF-α, IL-6, CXCL10, and MCP1, and the activation of NF-κB in seminal vesicular epithelial cells. Spermatozoa may induce seminal vesiculitis through the activation of innate immune responses in seminal vesicular epithelial cells, which provide novel insights into the mechanisms underlying non-infectious seminal vesiculitis.

Inhibition of autophagy in microglia and macrophages exacerbates innate immune responses and worsens brain injury outcomes

ABSTRACT Excessive and prolonged neuroinflammation following traumatic brain injury (TBI) contributes to long-term tissue damage and poor functional outcomes. However, the mechanisms contributing to exacerbated inflammatory responses after brain injury remain poorly understood. Our previous work showed that macroautophagy/autophagy flux is inhibited in neurons following TBI in mice and contributes to neuronal cell death. In the present study, we demonstrate that autophagy is also inhibited in activated microglia and infiltrating macrophages, and that this potentiates injury-induced neuroinflammatory responses. Macrophage/microglia-specific knockout of the essential autophagy gene Becn1 led to overall increase in neuroinflammation after TBI. In particular, we observed excessive activation of the innate immune responses, including both the type-I interferon and inflammasome pathways. Defects in microglial and macrophage autophagy following injury were associated with decreased phagocytic clearance of danger/damage-associated molecular patterns (DAMP) responsible for activation of the cellular innate immune responses. Our data also demonstrated a role for precision autophagy in targeting and degradation of innate immune pathways components, such as the NLRP3 inflammasome. Finally, inhibition of microglial/macrophage autophagy led to increased neurodegeneration and worse long-term cognitive outcomes after TBI. Conversely, increasing autophagy by treatment with rapamycin decreased inflammation and improved outcomes in wild-type mice after TBI. Overall, our work demonstrates that inhibition of autophagy in microglia and infiltrating macrophages contributes to excessive neuroinflammation following brain injury and in the long term may prevent resolution of inflammation and tissue regeneration. Abbreviations: Becn1/BECN1, beclin 1, autophagy related; CCI, controlled cortical impact; Cybb/CYBB/NOX2: cytochrome b-245, beta polypeptide; DAMP, danger/damage-associated molecular patterns; Il1b/IL1B/Il-1β, interleukin 1 beta; LAP, LC3-associated phagocytosis; Map1lc3b/MAP1LC3/LC3, microtubule-associated protein 1 light chain 3 beta; Mefv/MEFV/TRIM20: Mediterranean fever; Nos2/NOS2/iNOS: nitric oxide synthase 2, inducible; Nlrp3/NLRP3, NLR family, pyrin domain containing 3; Sqstm1/SQSTM1/p62, sequestosome 1; TBI, traumatic brain injury; Tnf/TNF/TNF-α, tumor necrosis factor; Ulk1/ULK1, unc-51 like kinase 1.

Low expression of NR1H3 correlates with macrophage infiltration and indicates worse survival in breast cancer.

Background: Nuclear receptor NR1H3 is a key regulator of macrophage function and lipid homeostasis. Here, we aimed to visualize the prognostic value and immunological characterization of NR1H3 in breast cancer. Methods: The expression pattern and prognostic value of NR1H3 were analyzed via multiple databases, including TIMER2, GEPIA2 and Kaplan-Meier Plotter. TISIDB, TIMER2 and immunohistochemical analysis were used to investigate the correlation between NR1H3 expression and immune infiltration. GO enrichment analysis, KEGG analysis, Reactome analysis, ConsensusPathDB and GeneMANIA were used to visualize the functional enrichment of NR1H3 and signaling pathways related to NR1H3. Results: We demonstrated that the expression of NR1H3 was significantly lower in breast cancer compared with adjacent normal tissues. Kaplan-Meier survival curves showed shorter overall survival in basal breast cancer patients with low NR1H3 expression, and poorer prognosis of relapse-free survival in breast cancer patients with low NR1H3 expression. NR1H3 was mainly expressed in immune cells, and its expression was closely related with infiltrating levels of tumor-infiltrating immune cells in breast cancer. Additionally, univariate and multivariate analysis indicated that the expression of NR1H3 and the level of macrophage infiltration were independent prognostic factors for breast cancer. Gene interaction network analysis showed the function of NR1H3 involved in regulating of innate immune response and macrophage activation. Moreover, NR1H3 may function as a predictor of chemoresponsiveness in breast cancer. Conclusion: These findings suggest that NR1H3 serves as a prognostic biomarker and contributes to the regulation of macrophage activation in breast cancer.

Data-driven discovery of innate immunomodulators via machine learning-guided high throughput screening.

The innate immune response is vital for the success of prophylactic vaccines and immunotherapies. Control of signaling in innate immune pathways can improve prophylactic vaccines by inhibiting unfavorable systemic inflammation and immunotherapies by enhancing immune stimulation. In this work, we developed a machine learning-enabled active learning pipeline to guide in vitro experimental screening and discovery of small molecule immunomodulators that improve immune responses by altering the signaling activity of innate immune responses stimulated by traditional pattern recognition receptor agonists. Molecules were tested by in vitro high throughput screening (HTS) where we measured modulation of the nuclear factor κ-light-chain-enhancer of activated B-cells (NF-κB) and the interferon regulatory factors (IRF) pathways. These data were used to train data-driven predictive models linking molecular structure to modulation of the NF-κB and IRF responses using deep representational learning, Gaussian process regression, and Bayesian optimization. By interleaving successive rounds of model training and in vitro HTS, we performed an active learning-guided traversal of a 139 998 molecule library. After sampling only ∼2% of the library, we discovered viable molecules with unprecedented immunomodulatory capacity, including those capable of suppressing NF-κB activity by up to 15-fold, elevating NF-κB activity by up to 5-fold, and elevating IRF activity by up to 6-fold. We extracted chemical design rules identifying particular chemical fragments as principal drivers of specific immunomodulation behaviors. We validated the immunomodulatory effect of a subset of our top candidates by measuring cytokine release profiles. Of these, one molecule induced a 3-fold enhancement in IFN-β production when delivered with a cyclic di-nucleotide stimulator of interferon genes (STING) agonist. In sum, our machine learning-enabled screening approach presents an efficient immunomodulator discovery pipeline that has furnished a library of novel small molecules with a strong capacity to enhance or suppress innate immune signaling pathways to shape and improve prophylactic vaccination and immunotherapies.

Перспективные иммуномодулирующие эффекты бактериальных лизатов в профилактике и лечении острых и реккурентных респираторных инфекций у детей

The incidence of influenza and acute respiratory infections (ARI) continues to grow, including due to limited opportunities for immunoprophylaxis of the most common respiratory infections, in particular, acute respiratory viral infections (ARVI). Attempts are being made to reduce the children susceptibility to respiratory pathogens through the rational use of nonspecific immunoprophylactic agents, among which bacterial lysates (BL) occupy a special place. BL key patterns, contributing to the prevention of viral infections, are innate immune response activation, excessive inflammatory response prevention, adaptive immune response stimulation. BL contain fragments of inactivated various pathogenic bacteria strains that are important in the ARI etiology. Obtaining lysates of bacterial strains is possible by mechanical or chemical lysis. The article discusses the results of preclinical and clinical studies on BL, as well as suggests the expediency of further study concerning the BL pharmacodynamic effects within the modern concept of trained immunity. The authors conclude that BL as a trained immunity inducer according to the rationally selected regimen can contribute to the respiratory infections' prevention both during COVID-19 pandemic and post-pandemic period. KEYWORDS: acute respiratory infections, acute respiratory viral infections, influenza, trained immunity, immunoprophylaxis, bacterial lysate. FOR CITATION: Zaplatnikov A.L., Kanner E.V., Kanner I.D., Girina A.A., Farber I.M., Maximov M.L. Promising immunomodulatory effects of bacterial lysates in the prevention and treatment of acute and recurrent respiratory infections in children. Russian Journal of Woman and Child Health. 2023;6(3):290–297 (in Russ.). DOI: 10.32364/2618-8430-2023-6-3-12.

Combined inhibition of Wee1 and Chk1 as a therapeutic strategy in multiple myeloma.

Multiple myeloma (MM) is a hematological malignancy characterized by an abnormal clonal proliferation of malignant plasma cells. Despite the introduction of novel agents that have significantly improved clinical outcome, most patients relapse and develop drug resistance. MM is characterized by genomic instability and a high level of replicative stress. In response to replicative and DNA damage stress, MM cells activate various DNA damage signaling pathways. In this study, we reported that high CHK1 and WEE1 expression is associated with poor outcome in independent cohorts of MM patients treated with high dose melphalan chemotherapy or anti-CD38 immunotherapy. Combined targeting of Chk1 and Wee1 demonstrates synergistic toxicities on MM cells and was associated with higher DNA double-strand break induction, as evidenced by an increased percentage of γH2AX positive cells subsequently leading to apoptosis. The therapeutic interest of Chk1/Wee1 inhibitors' combination was validated on primary MM cells of patients. The toxicity was specific of MM cells since normal bone marrow cells were not significantly affected. Using deconvolution approach, MM patients with high CHK1 expression exhibited a significant lower percentage of NK cells whereas patients with high WEE1 expression displayed a significant higher percentage of regulatory T cells in the bone marrow. These data emphasize that MM cell adaptation to replicative stress through Wee1 and Chk1 upregulation may decrease the activation of the cell-intrinsic innate immune response. Our study suggests that association of Chk1 and Wee1 inhibitors may represent a promising therapeutic approach in high-risk MM patients characterized by high CHK1 and WEE1 expression.

Identifying an Optimal Neuroinflammation Treatment Using a Nanoligomer Discovery Engine.

Acute activation of innate immune response in the brain, or neuroinflammation, protects this vital organ from a range of external pathogens and promotes healing after traumatic brain injury. However, chronic neuroinflammation leading to the activation of immune cells like microglia and astrocytes causes damage to the nervous tissue, and it is causally linked to a range of neurodegenerative diseases such as Alzheimer's diseases (AD), Multiple Sclerosis (MS), Parkinson's disease (PD), and many others. While neuroinflammation is a key target for a range of neuropathological diseases, there is a lack of effective countermeasures to tackle it, and existing experimental therapies require fairly invasive intracerebral and intrathecal delivery due to difficulty associated with the therapeutic crossover between the blood-brain barrier, making such treatments impractical to treat neuroinflammation long-term. Here, we present the development of an optimal neurotherapeutic using our Nanoligomer Discovery Engine, by screening downregulation of several proinflammatory cytokines (e.g., Interleukin-1β or IL-1β, tumor necrosis factor-alpha or TNF-α, TNF receptor 1 or TNFR1, Interleukin 6 or IL-6), inflammasomes (e.g., NLRP1), key transcription factors (e.g., nuclear factor kappa-B or NF-κβ) and their combinations, as upstream regulators and canonical pathway targets, to identify and validate the best-in-class treatment. Using our high-throughput drug discovery, target validation, and lead molecule identification via a bioinformatics and artificial intelligence-based ranking method to design sequence-specific peptide molecules to up- or downregulate gene expression of the targeted gene at will, we used our discovery engine to perturb and identify most effective upstream regulators and canonical pathways for therapeutic intervention to reverse neuroinflammation. The lead neurotherapeutic was a combination of Nanoligomers targeted to NF-κβ (SB.201.17D.8_NF-κβ1) and TNFR1 (SB.201.18D.6_TNFR1), which were identified using in vitro cell-based screening in donor-derived human astrocytes and further validated in vivo using a mouse model of lipopolysaccharide (LPS)-induced neuroinflammation. The combination treatment SB_NI_111 was delivered without any special formulation using a simple intraperitoneal injection of low dose (5 mg/kg) and was found to significantly suppress the expression of LPS-induced neuroinflammation in mouse hippocampus. These results point to the broader applicability of this approach towards the development of therapies for chronic neuroinflammation-linked neurodegenerative diseases, sleep countermeasures, and others, and the potential for further investigation of the lead neurotherapeutic molecule as reversible gene therapy.

Abstract 12645: Role of Viral Rna-Triggered Innate Immune Activation on Cardiac Arrhythmias Associated With Covid-19

Background: The COVID-19 pandemic has highlighted how little is known about how double stranded RNA (dsRNA) viruses like the SARS-CoV-2 impact cardiac physiology acutely and long-term. While it is unclear if the cardiac effects are direct or indirect, adverse effects in patients are common, including cardiac inflammation, electrophysiological changes in QT interval, atrial fibrillation, bradyarrhythmia, and ventricular tachycardia. Mechanisms underlying these COVID-19-associated arrhythmias are unknown. Methods: We assess the effects of SARS-CoV-2 infection on cardiac electrophysiology and inflammatory pathway activation in a hamster model over a 4-week timespan. We further test if activation of antiviral innate immune responses with a dsRNA mimetic, Poly I:C (PI:C) induces similar effects in uninfected hearts and in human iPSC-derived cardiomyocytes (hiPSC-CMs), examining the determinants of RNAseL activation, interferon (IFN) signaling, and cytokine release in vivo and in vitro. Results: Intranasal SARS-CoV-2 USA-WA1 infection of hamsters caused ECG abnormalities including bradycardia and cardiac conduction defects including sinus pauses, 2 nd and 3 rd degree Atrio-Ventricular block. Bradycardia peaked at 4-5 days after infection. Mean RR interval increased from 182±4ms pre- to 233±6ms post-infection, without significant increases in mock-infected hamsters. In the absence of viral infection, ventricular injection of PI:C induced similar arrhythmias and induction of IFN-stimulated genes. PI:C treatment of hiPSC-CMs induced robust increases in type-I IFN pathway proteins STAT1/pSTAT1, OAS2 (48% increase) and OAS3 (32% increase) and a robust cytokine response. Negative effects on excitation-contraction coupling after 72hr of PI:C treatment, manifested as a 25% reduction in hiPSC-CM Ca 2+ transient peaks. In vitro studies showed increased RNA degradation after PI:C, consistent with activation of the OAS/RNAseL dsRNA sensor pathway. Conclusions: COVID-19 in the hamster model recapitulates a subset of arrhythmias observed in COVID-19 patients. Activation of the viral dsRNA innate immune sensor pathway, even in the absence of active cardiac viral replication, may contribute to SARS-CoV-2-associated cardiac dysfunction.

Proteomic profiling of extracellular vesicles derived from ARPE-19 cells challenged with Aspergillus flavus and Candida albicans: application in fungal endophthalmitis.

Extracellular vesicles (EVs) are nano-sized-particles that play an important role in cellular cross-talk. The aim of this study was to understand the proteomic cargo of EVs, released by Retinal Pigment Epithelial (RPE) cells challenged with Candida albicans (C-CA) and Aspergillus flavus (C-AF). EVs were isolated from culture supernatant of retinal cells infected with fungal pathogens and characterized by dynamic light scattering, SEM, and western blot. EV proteome was then evaluated by mass spectrometry (LC-MS/MS). Isolated EVs were approximately 120-150nm and higher in number in infected group compared to control. Proteomic profiling of EVs from infected cells, showed a total of 419 and 254 differentially expressed proteins, of which 218 were upregulated in C-CA group and 81 proteins were upregulated in C-AF group. Gene ontology revealed majority of proteins associated with transport, cell migration, and in activation of innate immune response. Proteins identified were annexins, calpain, and Sorcin proteins. Additionally, KEGG analysis unveiled involvement of MAPK, HIF-1, and PI3K-AKT signalling pathways. Proteomic results indicate that EVs cargo derived from fungal-infected retinal cells can activate immune signalling pathways and might contribute to the pathogenesis of endophthalmitis, indicating the potential use of EVs as theranostic marker for management of fungal infections.