Innate Immune Response Genes Research Articles

METTL3 regulates viral m6A RNA modification and host cell innate immune responses during SARS-CoV-2 infection.

It is urgent and important to understand the relationship of the widespread severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) with host immune response and study the underlining molecular mechanism. N6-methylation of adenosine (m6A) in RNA regulates many physiological and disease processes. Here, we investigate m6A modification of the SARS-CoV-2 gene in regulating the host cell innate immune response. Our data show that the SARS-CoV-2 virus has m6A modifications that are enriched in the 3′ end of the viral genome. We find that depletion of the host cell m6A methyltransferase METTL3 decreases m6A levels in SARS-CoV-2 and host genes, and m6A reduction in viral RNA increases RIG-I binding and subsequently enhances the downstream innate immune signaling pathway and inflammatory gene expression. METTL3 expression is reduced and inflammatory genes are induced in patients with severe coronavirus disease 2019 (COVID-19). These findings will aid in the understanding of COVID-19 pathogenesis and the design of future studies regulating innate immunity for COVID-19 treatment.

ARID3a Defines a Novel Subset of Naïve B Cells in SLE Patients

Abstract Systemic Lupus Erythematosus (SLE) is a complex autoimmune disease characterized by overproduction of autoantibodies by B cells and the loss of tolerance to nucleic acids. Our previous studies show that AT-rich interacting domain 3a (ARID3a), a DNA-binding protein, is more abundant in SLE B cells than in healthy control B cells. Increased numbers of ARID3a+ B cells in SLE correlated with increased disease activity in patients. ARID3a expression in SLE patient blood occurred in naïve B cell subsets which do not express ARID3a in healthy controls. Studies by the Sanz laboratory revealed that activated naïve B cells and may be precursors for the double negative (DN) B cells linked to pathogenic responses in SLE. We hypothesize that ARID3a expression in naïve B cells also defines precursors of pathogenic B cells. In individual patients, ARID3a is bimodally expressed, such that only a fraction of the naïve B cells express ARID3a at any given time. We took advantage of this bimodal expression using single cell technology to perform RNA-seq analyses of ARID3a+ and ARID3a− SLE B cells to identify differentially expressed genes associated with ARID3a. PCA analyses of scRNA seq data show that ARID3a+ naïve B cells cluster together and away from ARID3a− cells. This allows for analyses within and among individual samples by binning cells based on ARID3a expression. Preliminary data indicate that ARID3a expression is correlated with innate immune response genes, transcription factors, and epigenetic regulators. These studies will be informative regarding how ARID3a expression contributes to disease pathogenesis in patients with SLE and will provide novel information defining a new subset of naive B cells that may be directly linked to breaches in tolerance.

Genetic variation in the immune system and malaria susceptibility in infants: a nested case\u2013control study in Nanoro, Burkina Faso

BackgroundGenetic polymorphisms in the human immune system modulate susceptibility to malaria. However, there is a paucity of data on the contribution of immunogenetic variants to malaria susceptibility in infants, who present differential biological features related to the immaturity of their adaptive immune system, the protective effect of maternal antibodies and fetal haemoglobin. This study investigated the association between genetic variation in innate immune response genes and malaria susceptibility during the first year of life in 656 infants from a birth cohort survey performed in Nanoro, Burkina Faso.MethodsSeventeen single nucleotide polymorphisms (SNPs) in 11 genes of the immune system previously associated with different malaria phenotypes were genotyped using TaqMan allelic hybridization assays in a Fluidigm platform. Plasmodium falciparum infection and clinical disease were documented by active and passive case detection. Case–control association analyses for both alleles and genotypes were carried out using univariate and multivariate logistic regression. For cytokines showing significant SNP associations in multivariate analyses, cord blood supernatant concentrations were measured by quantitative suspension array technology (Luminex).ResultsGenetic variants in IL-1β (rs1143634) and FcγRIIA/CD32 (rs1801274)—both in allelic, dominant and co-dominant models—were significantly associated with protection from both P. falciparum infection and clinical malaria. Furthermore, heterozygote individuals with rs1801274 SNP in FcγRIIA/CD32 showed higher IL-1RA levels compared to wild-type homozygotes (P = 0.024), a cytokine whose production is promoted by the binding of IgG immune complexes to Fcγ receptors on effector immune cells.ConclusionsThese findings indicate that genetic polymorphisms in genes driving innate immune responses are associated to malaria susceptibility during the first year of life, possibly by modulating production of inflammatory mediators.

Uropathogenic Escherichia coli Virulence Factor α-Hemolysin Reduces Histone Acetylation to Inhibit Expression of Proinflammatory Cytokine Genes.

Urinary tract infections are common and costly diseases affecting millions of people. Uropathogenic Escherichia coli (UPEC) is a primary cause of these infections and has developed multiple strategies to avoid the host immune response. Here, we dissected the molecular mechanisms underpinning UPEC inhibition of inflammatory cytokine in vitro and in vivo. We found that UPEC infection simulates nuclear factor-κB activation but does not result in transcription of cytokine genes. Instead, UPEC-mediated suppression of the metabolic enzyme ATP citrate lyase results in decreased acetyl-CoA levels, leading to reduced H3K9 histone acetylation in the promotor region of CXCL8. These effects were dependent on the UPEC virulence factor α-hemolysin and were reversed by exogenous acetate. In a murine cystitis model, prior acetate supplementation rapidly resolved UPEC-elicited immune responses and improved tissue recovery. Thus, upon infection, UPEC rearranges host cell metabolism to induce chromatin remodeling processes that subvert expression of host innate immune response genes.

Expression of innate immune response genes in upper airway samples of SARS-CoV-2 infected patients: A preliminary study.

Background & objectives:Upper respiratory mucosa is the entryway for SARS-CoV-2, and cells at this site form the first line of resistance against the pathogens. Innate immune response at this point is crucial for managing the replication and early stage symptoms of virus infection. This study was aimed to evaluate the expression of pattern recognition receptors and cytokines in upper airway cells of SARS-CoV-2 infected patients.Methods:Forty seven nasopharyngeal swab (NPS) specimens from 25 SARS-CoV-2 infected patients and 22 SARS-CoV-2 negative individuals were investigated for expression of toll-like receptors (TLRs), melanoma differentiation-associated protein 5 (MDA5), NOD-like receptors family pyrin domain containing 3 (NLRP3), angiotensin-converting enzyme 2 (ACE2), interleukin (IL) - 6, tumour necrosis factor alpha (TNFα) and type-1 interferons (IFNs) by real time reverse transcription polymerase chain reaction.Results:Increased expression of TLR2, MDA5 and ACE2 was detected in SARS-CoV-2 infected patients in comparison with controls. MDA5 expression was significantly higher in asymptomatic and mildly symptomatic SARS-CoV-2 positive patients than the patients with severe symptoms. The asymptomatic group showed significant induction of type 1 IFNs than the symptomatic group. Non-specific induction of TLR7 could be observed in nasopharyngeal (NP) cells irrespective of symptoms and SARS-CoV-2 positivity.Interpretation & conclusions:The findings suggest that increased MDA5 in NP cells of asymptomatic SARS-CoV-2 positive patients may subsequently induce type 1 IFNs to protect the individuals from further clinical severity of SARS-CoV-2 infection. A future prospective study in NPS of larger cohort needs to be performed to confirm our findings.

Survival Following Traumatic Brain Injury in Drosophila Is Increased by Heterozygosity for a Mutation of the NF-κB Innate Immune Response Transcription Factor Relish.

Traumatic brain injury (TBI) pathologies are caused by primary and secondary injuries. Primary injuries result from physical damage to the brain, and secondary injuries arise from cellular responses to primary injuries. A characteristic cellular response is sustained activation of inflammatory pathways commonly mediated by nuclear factor-κB (NF-κB) transcription factors. Using a Drosophila melanogaster TBI model, we previously found that the main proximal transcriptional response to primary injuries is triggered by activation of Toll and Imd innate immune response pathways that engage NF-κB factors Dif and Relish (Rel), respectively. Here, we found by mass spectrometry that Rel protein level increased in fly heads at 4-8 hr after TBI. To investigate the necessity of Rel for secondary injuries, we generated a null allele, Reldel , by CRISPR/Cas9 editing. When heterozygous but not homozygous, the Reldel mutation reduced mortality at 24 hr after TBI and increased the lifespan of injured flies. Additionally, the effect of heterozygosity for Reldel on mortality was modulated by genetic background and diet. To identify genes that facilitate effects of Reldel on TBI outcomes, we compared genome-wide mRNA expression profiles of uninjured and injured +/+, +/Reldel , and Reldel /Reldel flies at 4 hr following TBI. Only a few genes changed expression more than twofold in +/Reldel flies relative to +/+ and Reldel /Reldel flies, and they were not canonical innate immune response genes. Therefore, Rel is necessary for TBI-induced secondary injuries but in complex ways involving Rel gene dose, genetic background, diet, and possibly small changes in expression of innate immune response genes.

Zika Virus–Infected Decidual Cells Elicit a Gestational Age–Dependent Innate Immune Response and Exaggerate Trophoblast Zika Permissiveness: Implication for Vertical Transmission

Vertical transmission of the Zika virus (ZIKV) causes severe fetal defects, but the exact pathogenic mechanism is unclear. We identified up to a 10,480-fold higher expression of viral attachment factors AXL, GAS6, and PROS1 and a 3880-fold increase in ZIKV infectiousness/propagation in human term decidual stromal cells versus trophoblasts. Moreover, levels of viral attachment factors and ZIKV are significantly increased, whereas expression of innate immune response genes are significantly decreased, in human first trimester versus term decidual cells. ZIKV-infected decidual cell supernatants increased cytotrophoblasts infection up to 252-fold compared with directly infected cytotrophoblasts. Tizoxanide treatment efficiently inhibited Zika infection in both maternal and fetal cells. We conclude that ZIKV permissiveness, as well as innate immune responsiveness of human decidual cells, are gestational age dependent, and decidual cells augment ZIKV infection of primary human cytotrophoblast cultures, which are otherwise ZIKV resistant. Human decidual cells may act as reservoirs for trimester-dependent placental transmission of ZIKV, accounting for the higher Zika infection susceptibility and more severe fetal sequelae observed in early versus late pregnancy. Moreover, tizoxanide is a promising agent in preventing perinatal Zika transmission as well as other RNA viruses such as coronavirus.

Variation in intrinsic resistance of pea aphids to parasitoid wasps: A transcriptomic basis.

Evolutionary interactions between parasitoid wasps and insect hosts have been well studied at the organismal level, but little is known about the molecular mechanisms that insects use to resist wasp parasitism. Here we study the interaction between a braconid wasp (Aphidius ervi) and its pea aphid host (Acyrthosiphon pisum). We first identify variation in resistance to wasp parasitism that can be attributed to aphid genotype. We then use transcriptome sequencing to identify genes in the aphid genome that are differentially expressed at an early stage of parasitism, and we compare these patterns in highly resistant and susceptible aphid host lines. We find that resistant genotypes are upregulating genes involved in carbohydrate metabolism and several key innate immune system genes in response to parasitism, but that this response seems to be weaker in susceptible aphid genotypes. Together, our results provide a first look into the complex molecular mechanisms that underlie aphid resistance to wasp parasitism and contribute to a broader understanding of how resistance mechanisms evolve in natural populations.

Enhancing the Immune Surveillance in Multiple Myeloma Via CDK4/6 Inhibition

Deregulation of cyclin D genes is a uniform event in multiple myeloma (MM) and represent a striking addiction as observed in pan-cancer genome-wide CRISPR screening data. However, early stage Cyclin D and other cell cycle kinases inhibitors have shown a lack of single agent activity suggesting that targeting of cell cycle regulation is insufficient to produce a durable response in MM. Recent evidence recognizes the Cyclin D and CDK4 activities within the immune tumor microenvironment, supporting a previously unrecognized immunomodulatory functions of CDK4/6. This is particularly important in MM, a highly heterogeneous disease that resides in a complex ecosystem comprising of immune, endothelial, and stromal cells. We here evaluated the tumor intrinsic and extrinsic effects of CDK4/6 inhibition in MM with the goal to define rationally designed combination strategies to effectively impact MM growth. We have evaluated MM cell sensitivity to CDK4/6 inhibitors (both Palbociclib and Abemaciclib) in a panel of 32 MM cell lines and primary MM patients' samples. As expected, both inhibitors were mostly cytostatic with G0/G1 cell cycle arrest and significant impact on the pRB-E2F axis both in vitro as well as in vivo. In luminescence subcutaneous SCID models engrafted with H929 or MM1S MM cells expressing an E2F-driven luciferase reporter, treatment with low dose Palbociclib or Abemaciclib caused regression of bulky tumors, evidenced by a ~40% reduction in tumor volume at the 14-day end-point and a decreased E2F1 reporter activity. We next studied genome-wide transcriptional response to treatment using RNA-seq analysis in two MM cell lines using multiple doses and duration (24 and 72 hours) to evaluate the effect of short and long exposure to the drug. Gene set enrichment analysis (GSEA) of RNA-seq data confirmed significant downregulation of proliferation and E2F target genes at 24 hours post treatment. Interestingly, after longer exposure to both drugs we observed modulation of signatures for Ag presentation (including upregulation of HLA-A,B, and C; B2MG), innate immune response (ICAM-1 and 2; IL-8 and several CCLs) and interferon inducible genes IFN response (IRF1, IRF9, STAT1, STAT2, STAT4, OSA1, OSA2, MX1). To confirm genomic data and evaluate if CDK4/6i promotes the induction of the senescence-associated secretory phenotype (SASP) program in MM cells, we performed cytokine profile to assess the secretion of 174 soluble factors in the MM cell supernatant upon CDK4/6i. We confirmed a significant increase of chemokines involved in NK cell recruitment (CCL2, CCL4, CCL5), as well as cytokines that promote NK cell proliferation and activation. Moreover, we found that intercellular adhesion molecule-1 (ICAM-1) and the NKG2D ligands ULBP2 and MICA, required for activation of NK cell cytotoxicity and tumor cell targeting, were induced after CDK4/6i in MM cells. Although not secretory per se, these NK cell ligands are part of the transcriptional module linked to the SASP. Overall, these data suggest that, in addition to a more stable cell cycle arrest, CDK4/6i may promote MM cell immune surveillance through induction of the SASP program. To further confirm the immune effects, we tested freshly isolated NK cells from MM patients and healthy donors using in vitro MM-NK cell coculture assay and observed enhanced degranulation and cytokine production (intracellular IFN-γ and TNFα) by NK cells in response to MM cells. We finally investigated the potential of combining CDK4/6i with daratumumab in a standard 4-h ADCC assays using NK cells from MM patients as effector and MM cell lines as target. Daratumumab-mediated ADCC against MM cells was significantly augmented in the combination compared to single agent. In conclusion, we here report a novel anti-MM activity of CDK4/6i which is beyond the previously reported growth arrest. The observed ability to directly modulate the immune system along with decrease in the proliferative potential of MM cells may provide opportunities to develop unique combination approaches in MM. Disclosures Anderson: Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.. Fulciniti:NIH: Research Funding. Munshi:Takeda: Consultancy; Karyopharm: Consultancy; Amgen: Consultancy; AbbVie: Consultancy; Legend: Consultancy; Janssen: Consultancy; Adaptive: Consultancy; BMS: Consultancy; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; C4: Current equity holder in private company.

RNA-seq transcriptome analysis in flounder cells to compare innate immune responses to low- and high-virulence viral hemorrhagic septicemia virus.

Viral hemorrhagic septicemia virus (VHSV) is a rhabdovirus that causes high mortality in cultured flounder. Viral growth and virulence rely on the ability to inhibit the cellular innate immune response. In this study, we investigated differences in the modulation of innate immune responses of HINAE flounder cells infected with low- and high-virulence VHSV strains at a multiplicity of infection of 1 for 12 h and 24 h and performed RNA sequencing (RNA-seq)-based transcriptome analysis. A total of 193 and 170 innate immune response genes were differentially expressed by the two VHSV strains at 12 and 24 h postinfection (hpi), respectively. Of these, 73 and 77 genes showed more than a twofold change in their expression at 12 and 24 hpi, respectively. Of the genes with more than twofold changes, 22 and 11 genes showed high-virulence VHSV specificity at 12 and 24 hpi, respectively. In particular, IL-16 levels were more than two time higher and CCL20a.3, CCR6b, CCL36.1, Casp8L2, CCR7, and Trim46 levels were more than two times lower in high-virulence-VHSV-infected cells than in low-virulence-VHSV-infected cells at both 12 and 24 hpi. Quantitative PCR (qRT-PCR) confirmed the changes in expression of the ten mRNAs with the most significantly altered expression. This is the first study describing the genome-wide analysis of the innate immune response in VHSV-infected flounder cells, and we have identified innate immune response genes that are specific to a high-virulence VHSV strain. The data from this study can contribute to a greater understanding of the molecular basis of VHSV virulence in flounder.

A Comparative Study of Pathology and Host Immune Response Induced by Very Virulent Infectious Bursal Disease Virus in Experimentally Infected Chickens of Aseel and White Leghorn Breeds.

Indigenous breeds of young chickens in India are believed to be resistant to the classical strain of infectious bursal disease virus (IBDV). However, the mechanism underlying this resistance is obscure. Innate immunity is a key factor in defining the clinical course and pathology of microbial infections. The present study is aimed to compare the pathology of very virulent IBDV (vvIBDV) and immunological host response in experimentally infected - vaccinated and unvaccinated indigenous Aseel and commercial White Leghorn chickens. The viral loads and innate immune gene expression profiles of MDA-5, Mx, IFN-α, and IFN-β in different lymphoid organs were analyzed by quantitative PCR. The histopathological scores in Aseel birds were lower than in White Leghorns despite comparable viral loads. The degrees of histopathological lesions were fewer in vaccinated birds than in unvaccinated birds of both breeds. Analysis of innate immune response genes revealed that the cytoplasmic pattern recognition receptor MDA-5 gene was overexpressed mainly in the cecal tonsils of both vaccinated and nonvaccinated White Leghorn chickens. An increase in the expression of the IFN-α gene was seen in the cecal tonsils of Aseels, and an increase in IFN-β gene expression was seen in the thymuses of White Leghorns following vvIBDV challenge both in vaccinated and nonvaccinated birds. In addition, we observed that the Mx gene plays a minimal role, if any, in vvIBDV infection of the breeds under study. It remains interesting and important that although vvIBDV causes disease in indigenous Aseel birds, the faster clearance and reduced pathology of the virus in Aseel birds compared to White Leghorn chicken indicate some unidentified innate immune factors that are limiting IBDV in this breed. Further studies will be required to correlate kinetics of humoral and cellular immune response in relation to the virus load in different organs to illuminate the mechanism of genetic resistance in native breeds of chicken.

Impact of varied combinatorial mixture of non-fishmeal ingredients on growth, metabolism, immunity and gut microbiota of Lates calcarifer (Bloch, 1790) fry

The search for suitable fish meal replacements in aqua-diets is a salient agenda in the constant effort of making aquaculture practices more sustainable. In this study, we tested four customised diets composed by systematic inclusion of pre-selected fish meal substitutes, lupin kernel meal, BSF meal, TH and PBM on growth, metabolism, cytokine profile, gut morphology and microbiota of juvenile Lates calcarifer. Five isoproteic and isoenergetic diets were prepared viz. FM100 as a control (without fish meal substitute), while FM75, FM50, FM25 and FM0 indicates replacement of fish meal (FM) at 25%, 50%, 75%, and 100%, respectively by a mixture of four different pre-selected non-fish meal (NFM) ingredients. Fish fed FM100, FM75, FM50, FM25 exhibited consistent growth and haematological response, while the fish fed no fishmeal (FM0) showed significant decline in final body weight (FBW) and specific growth rate (SGR). The poor growth performance was correlated with a decrease in villous width, microvilli height and goblet cells density. A significant shift in abundance profile of Psychrobacter in the gut microbial profile of fish fed FM50 was noticed compared to fish fed FM100. The results of qRT-PCR showed up-regulated expression of innate immune responsive genes in the FM50 group. The adverse impacts on growth performance and gut health of fish fed FM0 suggest that the complete substitution of fishmeal is not advisable and the inclusion range of these alternatives should be decided for a species only after examining their effect on maximal physiological performance.

Transcriptome analysis based on RNA-seq of common innate immune responses of flounder cells to IHNV, VHSV, and HIRRV.

Viral hemorrhagic septicemia virus (VHSV) and hirame rhabdovirus (HIRRV) belong to the genus Novirhabdovirus and are the causative agents of a serious disease in cultured flounder. However, infectious hematopoietic necrosis virus (IHNV), a prototype of the genus Novirhabdovirus, does not cause disease in flounder. To determine whether IHNV growth is restricted in flounder cells, we compared the growth of IHNV with that of VHSV and HIRRV in hirame natural embryo (HINAE) cells infected with novirhabdoviruses at 1 multiplicity of infection. Unexpectedly, we found that IHNV grew as well as VHSV and HIRRV. For successful growth in host cells, viruses modulate innate immune responses exerted by virus-infected cells. Our results suggest that IHNV, like VHSV and HIRRV, has evolved the ability to overcome the innate immune response of flounder cells. To determine the innate immune response genes of virus-infected HINAE cells which are commonly modulated by the three novirhabdoviruses, we infected HINAE cells with novirhabdoviruses at multiplicity of infection (MOI) 1 and performed an RNA sequencing-based transcriptome analysis at 24 h post-infection. We discovered ~12,500 unigenes altered by novirhabdovirus infection and found that many of these were involved in multiple cellular pathways. After novirhabdovirus infection, 170 genes involved in the innate immune response were differentially expressed compared to uninfected cells. Among them, 9 genes changed expression by more than 2-fold and were commonly modulated by all three novirhabdoviruses. Interferon regulatory factor 8 (IRF8), C-X-C motif chemokine receptor 1 (CXCR1), Toll/interleukin-1 receptor domain-containing adapter protein (TIRAP), cholesterol 25-hydroxylase (CH25H), C-X-C motif chemokine ligand 11, duplicate 5 (CXCL11.5), and Toll-like receptor 2 (TLR2) were up-regulated, whereas C-C motif chemokine receptor 6a (CCR6a), interleukin-12a (IL12a), and Toll-like receptor 1 (TLR1) were down-regulated. These genes have been reported to be involved in antiviral responses and, thus, their modulation may be critical for the growth of novirhabdovirus in flounder cells. This is the first report to identify innate immune response genes in flounder that are commonly modulated by IHNV, VHSV, and HIRRV. These data will provide new insights into how novirhabdoviruses survive the innate immune response of flounder cells.

Histone deacetylase HDA-1 modulates mitochondrial stress response and longevity

The ability to detect, respond and adapt to mitochondrial stress ensures the development and survival of organisms. Caenorhabditis elegans responds to mitochondrial stress by activating the mitochondrial unfolded protein response (UPRmt) to buffer the mitochondrial folding environment, rewire the metabolic state, and promote innate immunity and lifespan extension. Here we show that HDA-1, the C. elegans ortholog of mammalian histone deacetylase (HDAC) is required for mitochondrial stress-mediated activation of UPRmt. HDA-1 interacts and coordinates with the genome organizer DVE-1 to induce the transcription of a broad spectrum of UPRmt, innate immune response and metabolic reprogramming genes. In rhesus monkey and human tissues, HDAC1/2 transcript levels correlate with the expression of UPRmt genes. Knocking down or pharmacological inhibition of HDAC1/2 disrupts the activation of the UPRmt and the mitochondrial network in mammalian cells. Our results underscore an evolutionarily conserved mechanism of HDAC1/2 in modulating mitochondrial homeostasis and regulating longevity.

Identification of Hub genes associated with infection of three lung cell lines by SARS-CoV-2 with integrated bioinformatics analysis.

Identification of Hub genes associated with infection of three lung cell lines by SARS-CoV-2 with integrated bioinformatics analysis.

Abstract 962: The dipeptidyl peptidase inhibitor BXCL701 activates innate immunity followed by adaptive immunity on a molecular and cellular level in a mouse model of pancreatic cancer

Abstract The oral DPP8/9 inhibitor BXCL701 in combination with an anti-PD-1 antibody (aPD-1) [AACR 2017] or in triple combination with aPD-1 and pegylated IL-2 (NKTR-214) [ASCO 2018] has demonstrated inhibition of tumor growth or complete regression respectively in animal models of pancreatic cancer. In the present study, the mechanism of action of BXCL701 has been elucidated in a sub-chronic pharmacokinetic / pharmacodynamic study at molecular and cellular level, by administering BXCL701 as a single agent. BXCL701 (20 μg) was administered orally every day for 14 days in tumor (Pan02) bearing mice. Tumor and serum samples were harvested after 0, 1, 8 and 16 hrs after BXCL701 administration on day 1, 7 and 14. Serum samples were analysed for cytokines and tumor tissues were analysed for infiltrating immune cells and gene expression. BXCL701 is known to inhibit regulatory proteases DPP8/9, consequently activating Nlrp1b inflammasome, which in turn activates pro-caspase-1 to mediate pyroptosis in mouse macrophages [Okondo et al, 2018]. Caspase-1 is involved in cleavage of pro-IL-1β and pro-IL-18 to their active forms, IL-1β and IL-18 respectively [Walle et al, 2016,]. IL-18 was observed to be significantly (p<0.05) upregulated (>50 fold) at 8 hrs on day 1 and achieved steady state levels by day 7 in BXCL701-treated animals. Other cytokines like IL-1β, IFN-γ, G-CSF, IL-5, IL-6, CXCL9, MCP-1, KC and Eotaxin were also observed to be upregulated in BXCL701-treated animals at the 8 hr timepoint on day 1, 7 and 14 in comparison to day 1, 0 hr. BXCL701 significantly upregulates T cells (total T cells, CD4+ T helper cells and CD8+ T cells) infiltration along with NK cells within the tumor microenvironment. It also appears to enhance antigen presentation by upregulating MHC class I genes and MHC class I expressing cells within the tumor. The mechanism of action of BXCL701 was evaluated at the molecular level as well. The comparison of genes in tumor tissues from BXCL701-treated animals vs respective vehicle-treated animals on Day 7 and 14 demonstrated that the upregulated gene clusters were innate and adaptive immune response genes, T-cell receptor genes and MHC genes. Also, genes associated with T cell and NK cell mediated apoptosis and cytolysis e.g. FasL, GzmA, were upregulated and indicates enhanced cell death (e.g. upregulation of pdcd1) within tumor. On the other hand, downregulated gene clusters belonged to functional categories like cell cycle, DNA repair, several genes associated with cancer progression (GPCRs and Olfactory receptors) and extra cellular matrix (ECM) modification (collagen and metalloproteases). In conclusion, BXCL701-treatment induces innate and adaptive immune responses that leads to tumor growth inhibition probably via inducing cell death and reducing ECM modification. Citation Format: Veena Agarwal, John MacDougall, shubhendu Trivedi, Dimple Bhatia, Zeenia Jagga, Hemant Banga, Diane Healy, Sreenivas Adurthi, Vince O'Neill. The dipeptidyl peptidase inhibitor BXCL701 activates innate immunity followed by adaptive immunity on a molecular and cellular level in a mouse model of pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 962.

Abstract 462: The mutated chromatin modifier gene, H3F3AK27M, is a druggable target of DNA hypomethylating agents in pediatric high-grade glioma

Abstract Purpose: With the current standard of care for Pediatric high-grade glioma (pHGG) patients the 5-year survival rate remain low at 5-35%. Thus, there is an urgent need to develop improved therapies for pHGG patients. A specific point mutation of histone H3 (H3F3A) has been identified at amino acid 27 (lysine to methionine (K27M)), resulting in inhibition of EZH2 (histone H3K27 tri-methylase), and reduced global levels of H3K27 tri-methylation (H3K27me3). The H3K27M alteration can be found in the majority (~80%) of pHGGs and represents an attractive target for diagnostic and treatment. Since the H3K27me3 mark correlate with gene silencing, mutations resulting in global decrease in H3K27me3 may result in aberrant gene regulation. Majority of demethylated promoters remain silenced after treatment with hypomethylating agent (HMA) due to the presence of repressive H3K27me3. HMAs treatment is also rendered ineffective in case of repression of endogenous retroviruses (ERVs) due to the presence of H3K27me3. Hence, pHGGs harboring the H3K27M mutation with low levels of H3K27me3 represent an attractive therapeutic target for treatment with HMAs. HMAs are approved by FDA for treatment of several forms of human cancers. Methods: We had introduced mutant H3K27M in human pediatric glioma cell (SF188). Pediatric glioma cell lines (wild type and endogenous or endogenous expressed with H3K27M) were treated with DNA hypomethylating agents (HMAs). Cell doubling time was calculated. DNA methylation and gene expression were monitored at different time points. Additional RNA-seq data were obtained from pediatric glioma patient and patient derived cell lines. Results: We demonstrate that pHGG cells carrying H3K27M mutation are more aggressive and showed down-regulation of innate immune response genes (immune deficient or immune cold) and up-regulation of cancer stem like genes as compared to the wild-type pHGG cells. These findings are further confirmed with published data from patient-derived pHGG cells. The prolonged antitumor effects in pHGG cells with H3K27M mutation observed after HMA treatment may be due to the failure of DNA remethylation and upregulated innate immune response pathway genes due to ERV reactivation. Conclusion: Our findings suggest that HMA treatment may be effective against pHGGs with the H3K27M mutation and critical for converting immune deficient cells into immune reactive cells (immune hot). The success of this project will lead to clinical trials for pHGG patients to evaluate HMAs with immune-based therapies in pHGG patients, for whom there are currently no effective treatments. Citation Format: Hemant Gujar, Lian Zhang, Hong-Tao Li, Hitoshi Otani, Daniel J. Weisenberger, Anat Erdreich-Epstein, Gangning Liang. The mutated chromatin modifier gene, H3F3AK27M, is a druggable target of DNA hypomethylating agents in pediatric high-grade glioma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 462.

Fetal Lymphoid Organ Immune Responses to Transient and Persistent Infection with Bovine Viral Diarrhea Virus.

Bovine Viral Diarrhea Virus (BVDV) fetal infections occur in two forms; persistent infection (PI) or transient infection (TI), depending on what stage of gestation the fetus is infected. Examination of lymphoid organs from both PI and TI fetuses reveals drastically different fetal responses, dependent upon the developmental stage of the fetal immune system. Total RNA was extracted from the thymuses and spleens of uninfected control, PI, and TI fetuses collected on day 190 of gestation to test the hypothesis that BVDV infection impairs the innate and adaptive immune response in the fetal thymus and spleen of both infection types. Transcripts of genes representing the innate immune response and adaptive immune response genes were assayed by Reverse Transcription quatitative PCR (RT-qPCR) (2−ΔΔCq; fold change). Genes of the innate immune response, interferon (IFN) inducible genes, antigen presentation to lymphocytes, and activation of B cells were downregulated in day 190 fetal PI thymuses compared to controls. In contrast, innate immune response genes were upregulated in TI fetal thymuses compared to controls and tended to be upregulated in TI fetal spleens. Genes associated with the innate immune system were not different in PI fetal spleens; however, adaptive immune system genes were downregulated, indicating that PI fetal BVDV infection has profound inhibitory effects on the expression of genes involved in the innate and adaptive immune response. The downregulation of these genes in lymphocytes and antigen-presenting cells in the developing thymus and spleen may explain the incomplete clearance of BVDV and the persistence of the virus in PI animals while the upregulation of the TI innate immune response indicates a more mature immune system, able to clear the virus.

TILRR Promotes Migration of Immune Cells Through Induction of Soluble Inflammatory Mediators.

TILRR has been identified as an important modulator of inflammatory responses. It is associated with NF-κB activation, and inflammation. Our previous study showed that TILRR significantly increased the expression of many innate immune responsive genes and increased the production of several pro-inflammatory cytokines/chemokines by cervical epithelial cells. In this study, we evaluated the effect of TILRR-induced pro-inflammatory cytokines/chemokines on the migration of immune cells. The effect of culture supernatants of TILRR-overexpressed cervical epithelial cells on the migration of THP-1 monocytes and MOLT-4 T-lymphocytes was evaluated using Transwell assay and a novel microfluidic device. We showed that the culture supernatants of TILRR-overexpressed HeLa cells attracted significantly more THP-1 cells (11–40%, p = 0.0004–0.0373) and MOLT-4 cells (14–17%, p = 0.0010–0.0225) than that of controls. The microfluidic device-recorded image analysis showed that significantly higher amount with longer mean cell migration distance of THP-1 (p < 0.0001–0.0180) and MOLT-4 (p < 0.0001–0.0025) cells was observed toward the supernatants of TILRR-overexpressed cervical epithelial cells compared to that of the controls. Thus, the cytokines/chemokines secreted by the TILRR-overexpressed cervical epithelial cells attracted immune cells, such as monocytes and T cells, and may potentially influence immune cell infiltration in tissues.

Abstract A31: The two most potent PARP inhibitors increase expression levels of innate immune response genes in BRCA wild-type HGSC tumors

Abstract Background: Poly (ADP-ribose) polymerase (PARP) inhibitors have transformed treatment of ovarian cancer in the past decade. Most of the currently active PARP inhibitors have similar ability to inhibit PARP’s enzymatic activity. However, it has been previously reported that velipairb, rucaparib, olaparib, niraparib, and talazoparib vary in their PARP trapping potency levels. Veliparib was also characterized to be mainly a catalytic inhibitor while the other four drugs were determined to be both PARP trappers and catalytic inhibitors, with talazoparib being the most potent PARP trapper. Responses in many patients with HGSC tumors are limited, particularly those women without known mutations in BRCA1 or BRCA2. Recently, PARP inhibitors have been reported to influence innate immune responses. For these reasons, there are ongoing clinical trials testing the safety and efficacy of PARP inhibitors in combination with various immune checkpoint inhibitors. The objective of this study is to compare the effect of the five most clinically active PARP inhibitors on type 1 interferon regulated genes and the PD-L1 gene. Methods: Two high-grade serous ovarian cancer cell lines were used in this study: CAOV3 (TP53 mutant) and OVCAR3 (TP53 mutant, CCNE1-amplified, and EMSY-amplified). The cell lines were treated for 48 hours with the PARP inhibitors listed above as well as a vehicle control (DMSO). Gene expression was measured using TaqMan assays. CXCL10 (IP-10) protein expression levels were determined via an ELISA assay. Summary of the Data: Talazoparib upregulated CXCL10 gene expression in both OVCAR3 and CAOV3 cell lines. These results were translated to the protein level, as we observed an increase in IP-10 protein expression levels after talazoparib treatment in the OVCAR3 cell line. IP-10 protein levels were also significantly increased when treated with the second most potent PARP inhibitor, niraparib. A statistically significant effect was not observed after treatment with the three other PARPis. Futhermore, CD274 (PD-L1) gene expression levels were significantly increased after talazoparib and not veliparib treatment in the CAOV3 cell line. Conclusions: Our data suggest that there are distinct effects exhibited among PARP inhibitors on innate immune pathways. The two most potent PARP inhibitors, niraparib and talazoparib, had the greatest effect on these pathways, specifically in BRCA wild-type HGSCs, compared to the three other drugs. These finding support the idea that we need to define the best PARP inhibitor to use in future clinical trials when combining this group of anticancer agents with immune checkpoint inhibitors. Citation Format: Monica Wielgos-Bonvallet, Danhui Weng, Petar Jelinic, Douglas A. Levine. The two most potent PARP inhibitors increase expression levels of innate immune response genes in BRCA wild-type HGSC tumors [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A31.