Innate Immune Pathways Research Articles

The cGAS-STING signaling pathway is modulated by urolithin A

During aging, general cellular processes, including autophagic clearance and immunological responses become compromised; therefore, identifying compounds that target these cellular processes is an important approach to improve our health span. The innate immune cGAS-STING pathway has emerged as an important signaling system in the organismal defense against viral and bacterial infections, inflammatory responses to cellular damage, regulation of autophagy, and tumor immunosurveillance. These key functions of the cGAS-STING pathway make it an attractive target for pharmacological intervention in disease treatments and in controlling inflammation and immunity. Here, we show that urolithin A (UA), an ellagic acid metabolite, exerts a profound effect on the expression of STING and enhances cGAS-STING activation and cytosolic DNA clearance in human cell lines. Animal laboratory models and limited human trials have reported no obvious adverse effects of UA administration. Thus, the use of UA alone or in combination with other pharmacological compounds may present a potential therapeutic approach in the treatment of human diseases that involves aberrant activation of the cGAS-STING pathway or accumulation of cytosolic DNA and this warrants further investigation in relevant transgenic animal models.

The DNA repair enzyme, aprataxin, plays a role in innate immune signaling.

Ataxia with oculomotor apraxia type 1 (AOA1) is a progressive neurodegenerative disorder characterized by a gradual loss of coordination of hand movements, speech, and eye movements. AOA1 is caused by an inactivation mutation in the APTX gene. APTX resolves abortive DNA ligation intermediates. APTX deficiency may lead to the accumulation of 5'-AMP termini, especially in the mitochondrial genome. The consequences of APTX deficiency includes impaired mitochondrial function, increased DNA single-strand breaks, elevated reactive oxygen species production, and altered mitochondrial morphology. All of these processes can cause misplacement of nuclear and mitochondrial DNA, which can activate innate immune sensors to elicit an inflammatory response. This study explores the impact of APTX knockout in microglial cells, the immune cells of the brain. RNA-seq analysis revealed significant differences in the transcriptomes of wild-type and APTX knockout cells, especially in response to viral infections and innate immune pathways. Specifically, genes and proteins involved in the cGAS-STING and RIG-I/MAVS pathways were downregulated in APTX knockout cells, which suggests an impaired immune response to cytosolic DNA and RNA. The clinical relevance of these findings was supported by analyzing publicly available RNA-seq data from AOA1 patient cell lines. Comparisons between APTX-deficient patient cells and healthy control cells also revealed altered immune responses and dysregulated DNA- and RNA-sensing pathways in the patient cells. Overall, this study highlights the critical role of APTX in regulating innate immunity, particularly in DNA- and RNA-sensing pathways. Our findings contribute to a better understanding of the underlying molecular mechanisms of AOA1 pathology and highlights potential therapeutic targets for this disease.

Differential expression of the genes encoding immune system components in response to Pseudomonas syringae and Pseudomonas aeruginosa in Arabidopsis thaliana.

In innate immunity, the first layer of defense against any microbial infection is triggered by the perception of pathogen-associated molecular patterns by highly specific pattern recognition receptors. The Pseudomonas syringae pv. tomato and Pseudomonas aeruginosa are plant-pathogenic bacterial species that include pathogenic strains in a wide range of different plant species. In the current study, extensive analysis including gene expression of 12 hub genes, gene ontology, protein-protein interaction, and cis-element prediction to dissect the Arabidopsis response to above-mentioned bacteria were performed. Further, we evaluated weighted co-expression network analysis (WGCNA) in the wild-type plants and coi-1 mutant line and determined changes in responsive genes at two time-points (4 and 8h) of post-treatment with P. syringae and P. aeruginosa. Compared to the wild-type plants, coi-1 mutant showed significant expression in most of the genes involved, indicating that their protein products have important role in innate immunity and RNA silencing pathways. Our findings showed that 12 hub genes were co-expressed in response to P. syringae and P. aeruginosa infections. Based on the network analysis, transcription factors, receptors, protein kinase, and pathogenesis-related protein (PR1) were involved in the immunity system. Gene ontology related to each module was involved in defense response, protein serine kinase activity, and primary miRNA processing. Based on the cis-elements prediction, MYB, MYC, WRE3, W-box, STRE, and ARE contained the most number of cis-elements in co-expressed network genes. Also, in coi-1 mutant, most responsive genes against theses pathogens were up-regulated. The knowledge gained in the gene expression analysis in response to P. syringae and P. aeruginosa in the model plant, i.e., Arabidopsis, is essential to allow us to gain more insight about the innate immunity in other crops.

Activation of mitochondrial DNA-mediated cGAS-STING pathway contributes to chronic postsurgical pain by inducing type I interferons and A1 reactive astrocytes in the spinal cord

Chronic postsurgical pain (CPSP) is increasingly recognized as a public health issue. Recent studies indicated the innate immune pathway of cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) was involved in pain regulation. However, the detailed mechanisms remain unclear. Previous studies found A1 reactive astrocytes in the spinal cord contributed to CPSP. This study aimed to investigate the roles and mechanisms of the cGAS-STING pathway in regulating the generation of A1 reactive astrocytes during CPSP. First, CPSP model was established using skin/muscle incision and retraction (SMIR) in rats. We found that cGAS-STING pathway was activated accompanied with an increase in mitochondrial DNA in the cytosol in the spinal cord following SMIR. Second, a STING inhibitor C-176 was intrathecally administrated. We found that C-176 decreased the expression of type I interferons and A1 reactive astrocytes in the spinal cord, and alleviated mechanical allodynia in SMIR rats. Third, cyclosporin A as a mitochondrial permeability transition pore blocker was intrathecally administrated. We found that cyclosporin A decreased the leakage of mitochondrial DNA and inhibited the activation of cGAS-STING pathway. Compared with C-176, cyclosporin A exhibits similar analgesic effects. The expression of type I interferons and A1 reactive astrocytes in the spinal cord were also down-regulated after intervention with cyclosporin A. Moreover, simultaneous administration of cyclosporin A and C-176 did not show synergistic effects in SMIR rats. Therefore, our study demonstrated that the cGAS-STING pathway activated by the leakage of mitochondrial DNA contributed to chronic postsurgical pain by inducing type I interferons and A1 reactive astrocytes in the spinal cord.

Whole blood transcriptomics identifies subclasses of pediatric septic shock

BackgroundSepsis is a highly heterogeneous syndrome, which has hindered the development of effective therapies. This has prompted investigators to develop a precision medicine approach aimed at identifying biologically homogenous subgroups of patients with septic shock and critical illnesses. Transcriptomic analysis can identify subclasses derived from differences in underlying pathophysiological processes that may provide the basis for new targeted therapies. The goal of this study was to elucidate pathophysiological pathways and identify pediatric septic shock subclasses based on whole blood RNA expression profiles.MethodsThe subjects were critically ill children with cardiopulmonary failure who were a part of a prospective randomized insulin titration trial to treat hyperglycemia. Genome-wide expression profiling was conducted using RNA sequencing from whole blood samples obtained from 46 children with septic shock and 52 mechanically ventilated noninfected controls without shock. Patients with septic shock were allocated to subclasses based on hierarchical clustering of gene expression profiles, and we then compared clinical characteristics, plasma inflammatory markers, cell compositions using GEDIT, and immune repertoires using Imrep between the two subclasses.ResultsPatients with septic shock depicted alterations in innate and adaptive immune pathways. Among patients with septic shock, we identified two subtypes based on gene expression patterns. Compared with Subclass 2, Subclass 1 was characterized by upregulation of innate immunity pathways and downregulation of adaptive immunity pathways. Subclass 1 had significantly worse clinical outcomes despite the two classes having similar illness severity on initial clinical presentation. Subclass 1 had elevated levels of plasma inflammatory cytokines and endothelial injury biomarkers and demonstrated decreased percentages of CD4 T cells and B cells and less diverse T cell receptor repertoires.ConclusionsTwo subclasses of pediatric septic shock patients were discovered through genome-wide expression profiling based on whole blood RNA sequencing with major biological and clinical differences.Trial Registration This is a secondary analysis of data generated as part of the observational CAF-PINT ancillary of the HALF-PINT study (NCT01565941). Registered March 29, 2012.

Inhibition of ACLY overcomes cancer immunotherapy resistance via polyunsaturated fatty acids peroxidation and cGAS-STING activation.

Adenosine 5'-triphosphate citrate lyase (ACLY) is a cytosolic enzyme that converts citrate into acetyl-coenzyme A for fatty acid and cholesterol biosynthesis. ACLY is up-regulated or activated in many cancers, and targeting ACLY by inhibitors holds promise as potential cancer therapy. However, the role of ACLY in cancer immunity regulation remains poorly understood. Here, we show that ACLY inhibition up-regulates PD-L1 immune checkpoint expression in cancer cells and induces T cell dysfunction to drive immunosuppression and compromise its antitumor effect in immunocompetent mice. Mechanistically, ACLY inhibition causes polyunsaturated fatty acid (PUFA) peroxidation and mitochondrial damage, which triggers mitochondrial DNA leakage to activate the cGAS-STING innate immune pathway. Pharmacological and genetic inhibition of ACLY overcomes cancer resistance to anti-PD-L1 therapy in a cGAS-dependent manner. Furthermore, dietary PUFA supplementation mirrors the enhanced efficacy of PD-L1 blockade by ACLY inhibition. These findings reveal an immunomodulatory role of ACLY and provide combinatorial strategies to overcome immunotherapy resistance in tumors.

The emerging roles of protein arginine methyltransferases in antiviral innate immune signaling pathways.

The Protein Arginine Methyltransferases (PRMTs) family is involved in various biological processes, including gene transcription, pre-mRNA splicing, mRNA translation, and protein stability. Recently, mounting evidence has shown that PRMTs also play critical roles in regulating the host antiviral immune response, either in an enzymatic activity dependent or independent manner. This review aims to provide an overview of the recent findings regarding the function and regulatory mechanisms of PRMTs in the antiviral response. These findings have the potential to aid in the discovery and design of novel therapeutic strategies for viral infections.

Proteomic Evolution from Acute to Post-COVID-19 Conditions.

Many COVID-19 survivors have post-COVID-19 conditions, and females are at a higher risk. We sought to determine (1) how protein levels change from acute to post-COVID-19 conditions, (2) whether females have a plasma protein signature different from that of males, and (3) which biological pathways are associated with COVID-19 when compared to restrictive lung disease. We measured protein levels in 74 patients on the day of admission and at 3 and 6 months after diagnosis. We determined protein concentrations by multiple reaction monitoring (MRM) using a panel of 269 heavy-labeled peptides. The predicted forced vital capacity (FVC) and diffusing capacity of the lungs for carbon monoxide (DLCO) were measured by routine pulmonary function testing. Proteins associated with six key lipid-related pathways increased from admission to 3 and 6 months; conversely, proteins related to innate immune responses and vasoconstriction-related proteins decreased. Multiple biological functions were regulated differentially between females and males. Concentrations of eight proteins were associated with FVC, %, and they together had c-statistics of 0.751 (CI:0.732-0.779); similarly, concentrations of five proteins had c-statistics of 0.707 (CI:0.676-0.737) for DLCO, %. Lipid biology may drive evolution from acute to post-COVID-19 conditions, while activation of innate immunity and vascular regulation pathways decreased over that period. (ProteomeXchange identifiers: PXD041762, PXD029437).

Effects of short-term florfenicol exposure on the gene expression pattern, midgut microbiota, and metabolome in the lepidopteran model silkworm (Bombyx mori)

Florfenicol (FF), an alternative veterinary antibiotic for chloramphenicol, has been widely utilized in livestock breeding to prevent and treat bacterial diseases. However, the toxicological effects of FF have yet to be fully disclosed. The domesticated silkworm (Bombyx mori), a lepidopteran model, was selected to assess the toxicological effects of FF dietary exposure with multi-omics. The findings showed that high-dose (250 μg/L) FF exposure increased the whole cocoon weight. High-dose FF exposure affected the species richness and community diversity of the microbiota in the silkworm midgut. Biochemical processes and innate immunity were impacted by FF exposure. The KEGG pathways impacted by the midgut microbiota and their metabolites were compared, and several pathways were found to be related to the two ecosystems. In addition, the innate immunity and lipid metabolism pathways were impacted, and some of the differentially expressed genes were enriched in these pathways. These related pathways may involve crosstalk between the midgut microbiota shift, midgut biological functions, and global gene expression. Therefore, our study also advances the application of the silkworm larval model in assessing antibiotic metabolic toxicity and provides novel insights into the potential risks of FF.

Mitochondrial nucleic acids in innate immunity and beyond.

Mitochondria participate in a wide range of cellular processes. One essential function of mitochondria is to be a platform for antiviral signaling proteins during the innate immune response to viral infection. Recently, studies have revealed that mitochondrion-derived DNAs and RNAs are recognized as non-self molecules and act as immunogenic ligands. More importantly, the cytosolic release of these mitochondrial nucleic acids (mt-NAs) is closely associated with the pathogenesis of human diseases accompanying aberrant immune activation. The release of mitochondrial DNAs (mtDNAs) via BAX/BAK activation and/or VDAC1 oligomerization activates the innate immune response and inflammasome assembly. In addition, mitochondrial double-stranded RNAs (mt-dsRNAs) are sensed by pattern recognition receptors in the cytosol to induce type I interferon expression and initiate apoptotic programs. Notably, these cytosolic mt-NAs also mediate adipocyte differentiation and contribute to mitogenesis and mitochondrial thermogenesis. In this review, we summarize recent studies of innate immune signaling pathways regulated by mt-NAs, human diseases associated with mt-NAs, and the emerging physiological roles of mt-NAs.

Abstract A172: TP-317, a first-in-class resolvin E1 small molecule, drives adjuvant efficacy in solid tumors by engaging innate and adaptive anti-tumor immunity in the tumor microenvironment (TME) and has neoadjuvant potential

Abstract Background: TP-317 is a novel salt of resolvin E1 (RvE1), an endogenous lipid agonist of ChemR23 that stimulates tumor debris phagocytosis/clearance by myeloid cells (Sulciner et al., 2018, J.Exp.Med). We recently reported TP-317 anti-tumor efficacy in murine solid tumors as monotherapy and in combination with immune checkpoint inhibitors (ICI) and chemotherapy (Kipper, 2023, Can. Res, 83 (7_Supp: 1135). Here we elaborate on the mechanism of action (MOA) of TP-317 in the adjuvant setting and report TP-317 efficacy in a neoadjuvant regimen Methods: 125-240 mm3 established subcutaneous murine melanoma (B16F10) and pancreatic (Panc02-H7; KPC) tumors were treated with placebo, TP-317 or ICI and tumors excised at 12-14 days for bulk RNAseq or flow cytometry. Placebo, TP-317 (300 ug/kg s.c. 2 hr prior to and Q6D after resection), cisplatin (5 mg/kg i.p. 24 hrs prior to resection) or anti-PD1 (8 mg/kg i.p. 24 hr prior to resection only) or TP-317 + anti-PD1 were evaluated in the LLC lung tumor model with tumors resected when reaching ~ 1500 mm3. Endpoints were lung/lymph node metastases or survival. Results: RNAseq analysis showed that adjuvant TP-317 broadly engages innate (macrophage, dendritic, NK cells) and adaptive (CD8, CD4, B cells) immune pathways in the TME of PANC2-H7, KPC and B16F10 models. Depletion studies suggest TP-317 efficacy is CD8- and NK cell-dependent, consistent with the RNAseq data and flow cytometry, which confirmed expression of ChemR23 (RvE1 receptor) on M1/M2 macrophages, conventional cDC1/cDC2, NK and lymphoid subsets. In the TME of PANC2-H7, KPC and B16-F10 models, TP-317 induced a gene signature with upregulated IFNg responses, effector CD8/NK receptors, antigen-presenting pathways, cDC1/NK cell cross-talk, and chemokines/cytokines involved in anti-tumor immunity. In the B16F10 model TP-317 + dual ICI combined therapy synergistically upregulated an IFNg response signature relative to dual ICI therapy. In the LLC resection model, TP-317 monotherapy reduced lung (p<0.05) and lymph node metastases (p<0.01) 19 days post-resection, but cisplatin was not effective. Neoadjuvant cisplatin or anti-PD1 monotherapy did not improve survival relative to placebo, but TP-317 combined with neoadjuvant anti-PD1 induced long term survival (>100 days) in 3 of 7 treated mice (p <0.05 vs placebo or anti-PD1). Conclusions: In adjuvant therapy of murine tumors, TP-317 broadly modulates innate/adaptive immune cell compartments in the TME, consistent with resetting the immunogenic tone and favoring anti-tumor immunity. Mechanistic synergy was observed for TP-317 combined with dual ICI therapy. TP-317 monotherapy shows metastasis control when initated prior to surgical resection and long term survival when combined with neoadjuvant anti-PD1 in the LLC model of NSCLC. Citation Format: John F Parkinson, Eva Rothenberger, Abigail Kelly, Diane Bielenberg, Ahmed Attaya, Sui Huang, Lance Pflieger, Wayne Klohs, Gary Mathias, Dipak Panigrahy. TP-317, a first-in-class resolvin E1 small molecule, drives adjuvant efficacy in solid tumors by engaging innate and adaptive anti-tumor immunity in the tumor microenvironment (TME) and has neoadjuvant potential [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A172.

The role of immunological signaling in disrupted proteostasis in the gut‐brain axis

AbstractBackgroundThe pathophysiological mechanism underlying the progressive neurodegenerative process in Alzheimer’s disease (AD) is not well understood. Hence, there is no disease‐modifying treatment. It is then imperative to identify the triggering molecular factors of the disease and the mechanisms supporting molecular spreading of toxic assemblies. Our search for these key clues in the periphery is founded on the fact that peripheral dysfunction, particularly in the gastrointestinal (GI) system, can arise decades before the onset of the classic symptoms in neurodegenerative disorders. In fact, the risk of AD increases significantly in patients with inflammatory bowel disease (IBD) when compared with the non‐IBD population. Furthermore, Tau accumulates in Crohn’s Disease guts and in sigmoid colon of AD and FTD patients. Thus, we are investigating the physiological and molecular mechanisms that support Tau spreading in the prodromal stages, particularly, along the gut‐brain axis.MethodTo test whether corrupted proteins expressed in the gut will spread their pathology to the brain, we employed the Gal4/UAS system and expressed wild‐type or mutant human Tau in selected populations of gut epithelium cells. We then monitor behavior on aging flies expressing Tau in the gut epithelium using Drosophila activity monitors and analyzed their circadian patterns of activity along with several sleep parameters.ResultWe observed that flies selectively expressing full‐length‐2N4R human Tau in enteroendocrine cells of the gut exhibited a significant impairment in their daily activity pattern that specifically affected their behavior during the light phase (day) but not during the dark period (night). Interestingly, these flies also showed an increased sleepiness during the day without affecting their nocturnal sleep pattern.ConclusionOur results support the presence of spreading mechanisms for Tau from gut epithelial cells to the brain; hence, affecting the neuronal physiology that sustain daily activity patterns. We are now investigating the pathways and mechanisms that pathological Tau in the gut employs to target these brain regions responsible for the behavioral phenotypes: transsynaptic spreading or humoral signaling via the innate immune system. We are dissecting the 3 major pathways of innate immunity in Drosophila (Imd‐Toll‐TNF) for their role in disease progression and the involvement of their target antimicrobial peptides.

Abstract C077: STC-15, a small molecule inhibitor of the RNA methyltransferase METTL3, activates anti-tumor immunity and reshapes the tumor microenvironment

Abstract Background METTL3 is the RNA methyltransferase responsible for the deposition of N-6-methyladenosine modification (m6A) on mRNA, to regulate its stability, splicing and protein translation. Small molecule inhibitors of METTL3 catalytic activity have previously demonstrated anti-tumor efficacy in pre-clinical models of acute myeloid leukemia (AML) and solid tumors. Inhibition of METTL3 was shown to induce a cell-intrinsic innate immune response through activation of the double strand RNA (dsRNA) sensing machinery and interferon (IFN) signalling. Here we present new pre-clinical data, investigating the combination of METTL3 inhibitor (METTL3i) with DNA damaging therapies. We show that METTL3 inhibition profoundly changes the tumor microenvironment (TME), and we further demonstrate the generation of durable immune memory in mice treated with METTL3i, with or without anti-PD1 therapy. Materials & Methods The orally bioavailable small molecule clinical-stage METTL3i STC-15, or the equipotent tool compound STM3675 were used in these studies. To characterise transcriptomic changes following METTL3 inhibition, RNA sequencing was performed. Induction of specific genes was validated by Western Blot, and cytokine secretion assessed by ELISA. In-vitro cell viability was assessed by Cell TiterGlo (Promega). In vivo efficacy studies used subcutaneous MC38 and GL261 syngeneic tumor models, and characterisation of the TME was performed by flow cytometry. Results Treatment with METTL3i sensitized cells to DNA damaging agents such as doxorubicin. On the molecular level, further enhancement of the IFN pathway by the combination treatment was observed. Moreover, combination of METTL3i with radiation treatment (RT) in vivo in the MC38 colorectal syngeneic mouse model demonstrated a clear benefit of the combination over each of the single treatments. METTL3i alone or in combination with RT led to a major TME remodulation, reducing the number of Tregs, M2 macrophages and neutrophils, and increasing the number of M1 macrophages and CD8+ T-cells in tumors. The GL261 syngeneic glioma model is exquisitely sensitive to anti-PD1 treatment. Similarly, METTL3i treatment led to multiple complete regressions, and the combination of METTL3i and anti-PD1 further accelerated the occurrence of regressions. Following a 3-weeks’ observation period, tumor free mice were re-inoculated with GL261 cells. All tumor-free mice remained tumor free after the rechallenge. Conclusions In pre-clinical cancer models, treatment with METTL3i such as STC-15 resulted in activation of innate immune pathways and subsequent enhancement of cytotoxic T-cell activity. In-vivo, STC-15 had a profound effect on the TME, shifting it from an immunosuppressive, pro-tumorigenic state to an immunostimulatory, anti-tumor state. In combination with anti-PD1, METTL3 inhibitors generate a durable anti-tumor response and lasting immune memory. A First-in-Human dose escalating study evaluating STC-15 monotherapy in patients with solid tumors is ongoing (NCT05584111). Citation Format: Yaara Ofir-Rosenfeld, Lina Vasiliauskaitė, Joanna Obacz, Georgia Tsagkogeorga, Josefin-Beate Holz, Jerry McMahon, Oliver Rausch. STC-15, a small molecule inhibitor of the RNA methyltransferase METTL3, activates anti-tumor immunity and reshapes the tumor microenvironment [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C077.

At the Crossroads of the cGAS-cGAMP-STING Pathway and the DNA Damage Response: Implications for Cancer Progression and Treatment.

The evolutionary conserved DNA-sensing cGAS-STING innate immunity pathway represents one of the most important cytosolic DNA-sensing systems that is activated in response to viral invasion and/or damage to the integrity of the nuclear envelope. The key outcome of this pathway is the production of interferon, which subsequently stimulates the transcription of hundreds of genes. In oncology, the situation is complex because this pathway may serve either anti- or pro-oncogenic roles, depending on context. The prevailing understanding is that when the innate immune response is activated by sensing cytosolic DNA, such as DNA released from ruptured micronuclei, it results in the production of interferon, which attracts cytotoxic cells to destroy tumors. However, in tumor cells that have adjusted to significant chromosomal instability, particularly in relapsed, treatment-resistant cancers, the cGAS-STING pathway often supports cancer progression, fostering the epithelial-to-mesenchymal transition (EMT). Here, we review this intricate pathway in terms of its association with cancer progression, giving special attention to pancreatic ductal adenocarcinoma and gliomas. As the development of new cGAS-STING-modulating small molecules and immunotherapies such as oncolytic viruses involves serious challenges, we highlight several recent fundamental discoveries, such as the proton-channeling function of STING. These discoveries may serve as guiding lights for potential pharmacological advancements.

IL-15-dependent immune crosstalk between natural killer cells and dendritic cells in HIV-1 elite controllers

As the principal effector cell population of the innate immune system, natural killer (NK) cells may make critical contributions to natural, immune-mediated control of HIV-1 replication. Using genome-wide assessments of activating and inhibitory chromatin features, we demonstrate here that cytotoxic NK (cNK) cells from elite controllers (ECs) display elevated activating histone modifications at the interleukin 2 (IL-2)/IL-15 receptor β chain and the BCL2 gene loci. These histone changes translate into increased responsiveness of cNK cells to paracrine IL-15 secretion, which coincides with higher levels of IL-15 transcription by myeloid dendritic cells in ECs. The distinct immune crosstalk between these innate immune cell populations results in improved IL-15-dependent cNK cell survival and cytotoxicity, paired with a metabolic profile biased toward IL-15-mediated glycolytic activities. Together, these results suggest that cNK cells from ECs display a programmed IL-15 response signature and support the emerging role of innate immune pathways in natural, drug-free control of HIV-1.

Abstract A163: Exoribonuclease XRN1 is a therapeutic vulnerability in tumors with intrinsically elevated type I interferon signaling

Abstract Background: 5′→3′ exoribonuclease 1 (XRN1) degrades single stranded mRNA from the 5′→3′ direction and plays a key role in endogenous cellular mRNA turnover. In addition, XRN1 can degrade double-stranded RNA (dsRNA), and as such plays a role in innate immunity by prevention of dsRNA accumulation and pPKR induction in virus-infected cells. We identified XRN1 as a putative selective vulnerability in tumor cells with intrinsic elevation of a Type I Interferon Stimulated Gene (TISG) signature through analysis of publicly available CRISPR screen data across 483 tumor cell lines. We further explored the role of XRN1 in a set of lung cancer cell lines with differential TISG expression in order to validate XRN1 as a compelling target for oncology drug development. Materials and Methods: NCI-H1650, NCI-H1703, NCI-H1944, and NCI-H838 lung adenocarcinoma cell lines were infected with lentiviruses containing Cas9 and sgRNAs targeting XRN1. Non-targeting sgRNAs were used as negative controls. XRN1 knockout (KO) cells and controls were evaluated in proliferation, apoptosis, qPCR and western blot assays. Results: Consistent with a selective dependency on XRN1 in cells with high intrinsic interferon signaling, XRN1 KO via CRISPR leads to robust anti-proliferative effects in TISG high NCI-H1650 and NCI-H1703 cells, but not in TISG low NCI-H838 and NCI-H1944 cells. Annexin V staining of NCI-H1650 cells upon XRN1 KO demonstrates that apoptosis is induced in TISG high cells. XRN1 KO in the TISG high setting leads to robust upregulation of Interferon-β mRNA consistent with activation of the MDA5 pathway by elevation of cytoplasmic dsRNA. Similarly, XRN1 loss induces pPKR activation in TISG high, but not TISG low cells. As pPKR activation triggers translational shutdown, TISG high selective pPKR elevation is consistent with the cell death observed. Exogenous treatment with Interferon-β or dsRNA mimetic Poly I:C to induce elevated Type I Interferon expression sensitizes NCI-H838 cells to XRN1 KO, with effects on cell proliferation. Conclusions: Taken together, these results demonstrate a critical role of XRN1 in cancer cells with intrinsically elevated Type I Interferon signaling. Cell death is triggered via accumulation of dsRNA and downstream activation of the MDA5 and PKR innate immune pathways. These data suggest that XRN1, through its regulation of dsRNA burden, is a compelling oncology target for TISG high tumors. Conflict of Interests: All authors are current or former employees and shareholders of Accent Therapeutics, Inc. Citation Format: Maureen M Lynes, Sophie A Shen, Sunaina P Nayak, Brian A Sparling, Scott Ribich, Stephen J Blakemore, Serena J Silver. Exoribonuclease XRN1 is a therapeutic vulnerability in tumors with intrinsically elevated type I interferon signaling [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A163.

Transcriptomic and Translatomic Analyses Reveal Insights into the Signaling Pathways of the Innate Immune Response in the Spleens of SPF Chickens Infected with Avian Reovirus.

Avian reovirus (ARV) infection is prevalent in farmed poultry and causes viral arthritis and severe immunosuppression. The spleen plays a very important part in protecting hosts against infectious pathogens. In this research, transcriptome and translatome sequencing technology were combined to investigate the mechanisms of transcriptional and translational regulation in the spleen after ARV infection. On a genome-wide scale, ARV infection can significantly reduce the translation efficiency (TE) of splenic genes. Differentially expressed translational efficiency genes (DTEGs) were identified, including 15 upregulated DTEGs and 396 downregulated DTEGs. These DTEGs were mainly enriched in immune regulation signaling pathways, which indicates that ARV infection reduces the innate immune response in the spleen. In addition, combined analyses revealed that the innate immune response involves the effects of transcriptional and translational regulation. Moreover, we discovered the key gene IL4I1, the most significantly upregulated gene at both the transcriptional and translational levels. Further studies in DF1 cells showed that overexpression of IL4I1 could inhibit the replication of ARV, while inhibiting the expression of endogenous IL4I1 with siRNA promoted the replication of ARV. Overexpression of IL4I1 significantly downregulated the mRNA expression of IFN-β, LGP2, TBK1 and NF-κB; however, the expression of these genes was significantly upregulated after inhibition of IL4I1, suggesting that IL4I1 may be a negative feedback effect of innate immune signaling pathways. In addition, there may be an interaction between IL4I1 and ARV σA protein, and we speculate that the IL4I1 protein plays a regulatory role by interacting with the σA protein. This study not only provides a new perspective on the regulatory mechanisms of the innate immune response after ARV infection but also enriches the knowledge of the host defense mechanisms against ARV invasion and the outcome of ARV evasion of the host's innate immune response.

M 6a-Son-CCL5 Axis Controls HSC Fate and Inflammation

Blood stem cells intricately regulate their fate, balancing self-renewal and differentiation through symmetric and asymmetric divisions. When exposed to various stresses they can rapidly undergo symmetric commitment divisions to generate differentiated progenitors for immune regeneration or driving inflammation. In previous studies, we and others found that m 6A RNA methylation controls symmetric commitment and inflammation in hematopoietic stem cells (HSCs) (Cheng and Luo et al., Cell Reports 2019). However, the precise molecular mechanisms underlying the role of m 6A and its influence on the inflammatory program in stem cells remain elusive. To uncover the dynamic RNA methylation during HSC commitment, we employed a recently developed tool called DART-seq, that fuses the YTH domain of the m 6A reader protein YTHDF2 with the RNA editing protein APOBEC1. We identified 300 m 6A sites and approximately 250-300 gene targets within HSCs and MPPs (padj beta binomial < 0.05). We found m 6A sites in HSCs were enriched for: innate immune response pathways including NFκB and Toll-like receptor cascades, cellular signaling processes encompassing RhoGTPase and ERK/MAPK signaling targets, and metabolic pathways associated with amino acid transport and fatty acid oxidation. In contrast, the m 6A targets enriched in MPPs were associated with lineage differentiation and myeloid programs such as MLL, GATA1, PU.1, and CEBPD. By integrating our findings with two additional m 6A mapping datasets, Son was a shared m 6A target with increased m 6A modification during the transition from HSCs to MPPs. SON, an RNA binding protein residing in nuclear speckles, has been implicated in a myriad of cellular processes including splicing control and transcriptional repression. Notably, de novo heterozygous loss-of-function variants in SON (ZTTK Syndrome) results in hematological symptoms, neurological impairments, and developmental delays. The genetic deletion of METTL3 ( Mettl3 conditional knockout mice; Mettl3 cKO) or METTL3 inhibitor STM2457 treatment, resulted in a twofold increase in Son transcripts in both HSCs and MPPs, but reduced SON protein abundance by 50%. Notably, SON exhibited asymmetric segregation during HSC division, with its abundance displaying a strong correlation with the commitment marker NUMB. Collectively, these findings suggest that m 6A modification regulates the abundance of SON protein and implicates SON in HSC commitment fate. Reintroduction of SON in m 6A-deficient HSCs effectively rescued commitment defects (cKO EV 9.74% vs. cKO+SON 21.43%). Furthermore, SON overexpression partially restored in vivo engraftment defects in Mettl3 cKO LSKs (cKO EV 1.5% vs. cKO+SON 4.5%). Intriguingly, the functional rescue was accomplished using a fragment containing the RNA-binding domain of SON (cKO EV 1.2% vs. cKO+SON-RB 12.3%), highlighting the significance of SON's RNA-binding ability. Additionally, deletion of SON in RosaCas9 WT LSKs using sgRNAs resulted in a substantial reduction in engraftment (WT EV 35.4% vs. WT+sgSON 11%), whereas SON overexpression in WT LSKs enhanced engraftment (WT EV 6% vs. WT+SON 39%). These results demonstrate that SON is a positive regulator of stem cell engraftment and function. Moreover, SON overexpression effectively rescued specific clusters identified through scRNA-seq analysis in Mettl3 KO (KOsp1-1 and CCL5+) by 50%. In bulk RNA-seq analysis, we identified 257 upregulated genes and 227 downregulated genes between the Mettl3 cKO EV and Mettl3 cKO SON groups (padj<0.05). Remarkably, SON overexpression significantly downregulated inflammatory pathways that were aberrantly upregulated upon m 6A loss. Furthermore, SON overexpression indirectly rescued dsRNA formation in Mettl3 cKO LSKs, aligning with the role of m 6A in suppressing dsRNA formation in HSPCs. SON overexpression reduced Ccl5 through binding to its transcript and decreasing nascent transcription. Strikingly, depletion of CCL5 significantly rescued Mettl3 cKO LSK engraftment (cKO 1.6% vs. cKO+shCCL5 5.3%), indicating that CCL5 is sufficient to mediate the effects downstream of SON. Moreover, co-culturing WT HSCs with CCL5 resulted in a symmetric commitment defect similar to that observed with m 6A loss. In summary, our study delineates the critical m 6A-SON-CCL5 axis governing HSC symmetric commitment fate and inflammation control.

Molecular characterization and functional analysis of a beta-1,3-glucan recognition protein from oriental fruit moth Grapholita molesta (Lepidoptera: Tortricidae).

The beta-1,3-glucan recognition protein (BGRP) is an important pattern recognition protein (PRP), which plays an important role in immune recognition and signaling pathway of insect innate immunity. Herein, a BGRP gene was obtained from the transcriptome of Grapholita molesta and its expression was verified by PCR. The full cDNA of the GmBGRP gene was 1691 bp encoding 486 amino acid residues. The calculated molecular mass of the mature protein was 54.83 kDa with an estimated pI of 6.14. The amino acid sequence of GmBGRP was highly homologous to BGRPs of other lepidopterans including Leguminivora glycinivorella BGRP-3. Expression profile of GmBGRP at different developmental stages and different tissues of 5th instar larvae showed that the expression level of this gene tends to slightly increase and then decrease at the adult stage, with the highest at the pupa stage; and mainly expressed in the epidermis, fat body and hemocytes compared with other tissues. In addition, we investigated the transcription levels of other immune-related genes, such as Serine-1, Serine-2, Serine-3, Serpin, SRCB (scavenger receptor gene), Toll, PPO (prophenoloxidase) upon GmBGRP gene silencing, indicating that GmBGRP expression is associated with immune responses of G. molesta. This was further supported by the upregulation of the mRNA level of GmBGRP following fungal infection. Taken together, these results provide experimental evidence for the role of GmBGRP gene in immune defense in G. molesta larvae.

Salmonella enterica serovar Choleraesuis vector outperforms alum as an adjuvant, increasing a cross-protective immune response against Glaesserella parasuis

The adjuvant and/or vector significantly affect a vaccine's efficacy. Although traditional adjuvants such as alum have contributed to vaccine development, deficiencies in the induction of cellular and mucosal immunity have limited their further promotion. Salmonella vectors have unique advantages for establishing cellular and mucosal immunity due to mucosal pathways of invasion and intracellular parasitism. In addition, Salmonella vectors can activate multiple innate immune pathways, thereby promoting adaptive immune responses. In this work, the attenuated Salmonella enterica serovar Choleraesuis (S. Choleraesuis) vector rSC0016 was used to deliver the conserved protective antigen HPS_06257 of Glaesserella parasuis (G. parasuis), generating a novel recombinant strain rSC0016(pS-HPS_06257). The rSC0016(pS-HPS_06257) can express and deliver the HPS_06257 protein to the lymphatic system of the host. In comparison to HPS_06257 adjuvanted with alum, rSC0016(pS-HPS_06257) significantly increased TLR4 and TLR5 activation in mice as well as the levels of proinflammatory cytokines. In addition, rSC0016 promoted a greater degree of maturation in bone marrow-derived dendritic cells (BMDCs) than alum. The specific humoral, mucosal, and cellular immune responses against HPS_06257 in mice immunized with rSC0016(pS-HPS_06257) were significantly higher than those of HPS_06257 adjuvanted with alum. HPS_06257 delivered by the S. Choleraesuis vector induces a Th1-biased Th1/Th2 mixed immune response, while HPS adjuvanted with alum can only induce a Th2-biased immune response. HPS_06257 adjuvanted with alum only causes opsonophagocytic activity (OPA) responses against a homologous strain (G. parasuis serotype 5, GPS5), whereas rSC0016(pS-HPS_06257) could generate cross-OPA responses against a homologous strain and a heterologous strain (G. parasuis serotype 12, GPS12). Ultimately, HPS_06257 adjuvanted with alum protected mice against lethal doses of GPS5 challenge by 60 % but failed to protect mice against lethal doses of GPS12. In contrast, mice immunized with rSC0016(pS-HPS_06257) had 100 % or 80 % survival when challenged with lethal doses of GPS5 or GPS12, respectively. Altogether, the S. Choleraesuis vector rSC0016 could potentially generate an improved innate immune response and an improved adaptive immunological response compared to the traditional alum adjuvant, offering a novel concept for the development of a universal G. parasuis vaccine.