Activation Of Innate Immune Response Research Articles

Identification of a Multi-Messenger RNA Signature as Type 2 Diabetes Mellitus Candidate Genes Involved in Crosstalk between Inflammation and Insulin Resistance.

Type 2 Diabetes Mellitus (T2DM) is a metabolic disease associated with inflammation widening the scope of immune-metabolism, linking the inflammation to insulin resistance and beta cell dysfunction. New potential and prognostic biomarkers are urgently required to identify individuals at high risk of β-cell dysfunction and pre-DM. The DNA-sensing stimulator of interferon genes (STING) is an important component of innate immune signaling that governs inflammation-mediated T2DM. NOD-like receptor (NLR) reduces STING-dependent innate immune activation in response to cyclic di-GMP and DNA viruses by impeding STING-TBK1 interaction. We proposed exploring novel blood-based mRNA signatures that are selective for components related to inflammatory, immune, and metabolic stress which may reveal the landscape of T2DM progression for diagnosing or treating patients in the pre-DM state. In this study, we used microarray data set to identify a group of differentially expressed mRNAs related to the cGAS/STING, NODlike receptor pathways (NLR) and T2DM. Then, we comparatively analyzed six mRNAs expression levels in healthy individuals, prediabetes (pre-DM) and T2DM patients by real-time PCR. The expressions of ZBP1, DDX58, NFKB1 and CHUK were significantly higher in the pre-DM group compared to either healthy control or T2DM patients. The expression of ZBP1 and NFKB1 mRNA could discriminate between good versus poor glycemic control groups. HSPA1B mRNA showed a significant difference in its expression regarding the insulin resistance. Linear regression analysis revealed that LDLc, HSPA1B and NFKB1 were significant variables for the prediction of pre-DM from the healthy control. Our study shed light on a new finding that addresses the role of ZBP1 and HSPA1B in the early prediction and progression of T2DM.

The role of bacterial cyclic di-adenosine monophosphate in the host immune response.

Cyclic di-adenosine monophosphate (c-di-AMP) is a second messenger which is widely used in signal transduction in bacteria and archaea. c-di-AMP plays an important role in the regulation of bacterial physiological activities, such as the cell cycle, cell wall stability, environmental stress response, and biofilm formation. Moreover, c-di-AMP produced by pathogens can be recognized by host cells for the activation of innate immune responses. It can induce type I interferon (IFN) response in a stimulator of interferon genes (STING)-dependent manner, activate the nuclear factor kappa B (NF-κB) pathway, inflammasome, and host autophagy, and promote the production and secretion of cytokines. In addition, c-di-AMP is capable of triggering a host mucosal immune response as a mucosal adjuvant. Therefore, c-di-AMP is now considered to be a new pathogen-associated molecular pattern in host immunity and has become a promising target in bacterial/viral vaccine and drug research. In this review, we discussed the crosstalk between bacteria and host immunity mediated by c-di-AMP and addressed the role of c-di-AMP as a mucosal adjuvant in boosting evoked immune responses of subunit vaccines. The potential application of c-di-AMP in immunomodulation and immunotherapy was also discussed in this review.

Transcription-independent regulation of STING activation and innate immune responses by IRF8 in monocytes

Sensing of cytosolic DNA of microbial or cellular/mitochondrial origin by cGAS initiates innate immune responses via the adaptor protein STING. It remains unresolved how the activity of STING is balanced between a productive innate immune response and induction of autoimmunity. Here we show that interferon regulatory factor 8 (IRF8) is essential for efficient activation of STING-mediated innate immune responses in monocytes. This function of IRF8 is independent of its transcriptional role in monocyte differentiation. In uninfected cells, IRF8 remains inactive via sequestration of its IRF-associated domain by its N- and C-terminal tails, which reduces its association with STING. Upon triggering the DNA sensing pathway, IRF8 is phosphorylated at Serine 151 to allow its association with STING via the IRF-associated domain. This is essential for STING polymerization and TBK1-mediated STING and IRF3 phosphorylation. Consistently, IRF8-deficiency impairs host defense against the DNA virus HSV-1, and blocks DNA damage-induced cellular senescence. Bone marrow-derived mononuclear cells which have an autoimmune phenotype due to deficiency of Trex1, respond to IRF-8 deletion with reduced pro-inflammatory cytokine production. Peripheral blood mononuclear cells from systemic lupus erythematosus patients are characterized by elevated phosphorylation of IRF8 at the same Serine residue we find to be important in STING activation, and in these cells STING is hyper-active. Taken together, the transcription-independent function of IRF8 we describe here appears to mediate STING activation and represents an important regulatory step in the cGAS/STING innate immune pathway in monocytes.

An Attenuated Strain of Human Cytomegalovirus for the Establishment of a Subviral Particle Vaccine.

Human cytomegalovirus (HCMV) infection is associated with severe disease conditions either following congenital transmission of the virus or viral reactivation in immunosuppressed individuals. Consequently, the establishment of a protective vaccine is of high medical need. Several candidates have been tested in preclinical and clinical studies, yet no vaccine has been licensed. Subviral dense bodies (DB) are a promising vaccine candidate. We have recently provided a GMP-compliant protocol for the production of DB, based on a genetically modified version of the HCMV laboratory strain Towne, expressing the pentameric complex of envelope protein gH-gL-pUL128-131 (Towne-UL130rep). In this work, we genetically attenuated Towne-UL130rep by abrogating the expression of the tegument protein pUL25 and by fusing the destabilizing domain ddFKBP to the N-terminus of the IE1- and IE2-proteins of HCMV. The resulting strain, termed TR-VAC, produced high amounts of DB under IE1/IE2 repressive conditions and concomitant supplementation of the viral terminase inhibitor letermovir to the producer cell culture. TR-VAC DB retained the capacity to induce neutralizing antibodies. A complex pattern of host protein induction was observed by mass spectrometry following exposure of primary human monocytes with TR-VAC DB. Human monocyte-derived dendritic cells (DC) moderately increased the expression of activation markers and MHC molecules upon stimulation with TR-VAC DB. In a co-culture with autologous T cells, the TR-VAC DB-stimulated DC induced a robust HCMV-specific T cell-activation and –proliferation. Exposure of donor-derived monocytic cells to DB led to the activation of a rapid innate immune response. This comprehensive data set thus shows that TR-VAC is an optimal attenuated seed virus strain for the production of a DB vaccine to be tested in clinical studies.

Innate Immune Response and Inflammasome Activation During SARS-CoV-2 Infection.

The novel coronavirus SARS-CoV-2, responsible for the COVID-19 outbreak, has become a pandemic threatening millions of lives worldwide. Recently, several vaccine candidates and drugs have shown promising effects in preventing or treating COVID-19, but due to the development of mutant strains through rapid viral evolution, urgent investigations are warranted in order to develop preventive measures and further improve current vaccine candidates. Positive-sense-single-stranded RNA viruses comprise many (re)emerging human pathogens that pose a public health problem. Our innate immune system and, in particular, the interferon response form an important first line of defense against these viruses. Flexibility in the genome aids the virus to develop multiple strategies to evade the innate immune response and efficiently promotes their replication and infective capacity. This review will focus on the innate immune response to SARS-CoV-2 infection and the virus’ evasion of the innate immune system by escaping recognition or inhibiting the production of an antiviral state. Since interferons have been implicated in inflammatory diseases and immunopathology along with their protective role in infection, antagonizing the immune response may have an ambiguous effect on the clinical outcome of the viral disease. This pathology is characterized by intense, rapid stimulation of the innate immune response that triggers activation of the Nod-like receptor family, pyrin-domain-containing 3 (NLRP3) inflammasome pathway, and release of its products including the pro-inflammatory cytokines IL-6, IL-18, and IL-1β. This predictive view may aid in designing an immune intervention or preventive vaccine for COVID-19 in the near future.

Molecular characterization, expression and response to immune challenges of 3 members of the toll-like receptor superfamily 11 in the golden pompano (Trachinotus ovatus)

Toll-like receptors (TLRs) are one of most important innate immune receptors, and play critical roles in pathogen recognition and activation of both innate and adaptive immune responses. In this study, we identified 2 tlr13 and 1 tlr23 genes in golden pompano (Trachinotus ovatus) by whole-genome screening, and their transcription patterns in response to various immune challenges were investigated. The open reading frames (ORFs) of, Totlr13–1, Totlr13–2 and Totlr23 are 2868, 2844 and 2802 bp in length, encoding 955, 947 and 933 amino acids, respectively. Multiple sequences alignment and protein structure analysis showed that these TLR receptors share high sequence homology. Phylogenetic analysis showed that Tlr13 comprises 2 subclades including Tlr13–1 and Tlr13–2, implying two Tlr13 paralogs may be widely existed in teleost. These three tlrs are extensively distributed and show a consistent pattern, with high expression levels in brain, kidney and spleen. In brain, intraperitoneal (IP) injection of both LPS and poly I:C induced Totlr23 expression at 24 h, while promoted Totlr13–1 and Totlr13–2 expressions at 12 h. In kidney and spleen, Totlr23 expression was dramatically increased at 3 h after LPS and poly I:C administration whereas Totlr13–1 and Totlr13–2 expressions slightly increased at 6 h and 24 h after LPS stimulation. Results revealed that both Tlr13s and Tlr23 may be involved in immune response in various tissues. These findings will expand our understanding about the evolutionary history and functional characteristics of the TLR11 superfamily in vertebrate.

827 Psoriasis exacerbation by obesity reflects reduced adiponectin regulation of PPAR-γ/Th17 pathway activation and is reversal by adiponectin receptor agonism

Although obesity-related deficiency of adipokines is known to contribute to increased psoriasis severity and decreased clinical response to interventions, the mechanism by which it exacerbates psoriasis is poorly defined. We hypothesized that adipokines suppress the activation of innate immune responses in keratinocytes and skin-resident Th17/γδ T cells. In preliminary studies, we confirmed that diet-induced obese (DIO) mice had significantly lower serum adiponectin levels than lean mice when subjected to topical imiquimod (IMQ) treatments to induce the psoriasis-like disease, supporting the use of this model for studies.

Proteomic and Metabolomic Characterization of SARS-CoV-2-Infected Cynomolgus Macaque at Early Stage.

Although tremendous effort has been exerted to elucidate the pathogenesis of severe COVID-19 cases, the detailed mechanism of moderate cases, which accounts for 90% of all patients, remains unclear yet, partly limited by lacking the biopsy tissues. Here, we established the COVID-19 infection model in cynomolgus macaques (CMs), monitored the clinical and pathological features, and analyzed underlying pathogenic mechanisms at early infection stage by performing proteomic and metabolomic profiling of lung tissues and sera samples from COVID-19 CMs models. Our data demonstrated that innate immune response, neutrophile and platelet activation were mainly dysregulated in COVID-19 CMs. The symptom of neutrophilia, lymphopenia and massive “cytokines storm”, main features of severe COVID-19 patients, were greatly weakened in most of the challenged CMs, which are more semblable as moderate patients. Thus, COVID-19 model in CMs is rational to understand the pathogenesis of moderate COVID-19 and may be a candidate model to assess the safety and efficacy of therapeutics and vaccines against SARS-CoV-2 infection.

Prediction of Deleterious Non-Synonymous Single Nucleotide Polymorphisms (Nssnps) of Human TLR7 Gene

Toll-like receptors (TLRs) play a key role in innate immune response activation against viruses. TLR7, one of the TLRs family, is potentially important in controlling viral infection and the production of vaccines against the virus. The wide spectrum of discrepancy in response to antiviral drugs among different populations which is emerged by some pandemics like COVID-19 might be due to their different TLR7 single nucleotide polymorphisms (SNPs). The present study aimed to investigate the consequences of 401 non-synonymous missense SNPs (nsSNPs) within TLR7 on its protein structure, stability, and function by using specific bioinformatics tools. Seven bioinformatics tools were used to investigate 401 TLR7 nsSNPs from the dbSNP database. The results showed that the six variations, rs1171508003 (R262H), rs35160120 (F580S), rs968155471 (H587Q), rs202028806 (Y871D), rs1331496205 (W933S), and rs181600414 (R1004W), were found to be extremely deleterious by all of the employed bioinformatics tools. All six variations showed an impact on the protein’s structure, function, and stability. Among them, Y871D (rs202028806) and R1004W (rs181600414) were revealed as the most damaging nsSNPs. This study suggested that the predicted six damaging variants of TLR7 could indirectly or directly destabilize the structure of protein and deviate its function to some extent.

Abstract 774: Microdissected gene expression profiling of recurrent nasopharyngeal carcinoma

Abstract Nasopharyngeal cancer (NPC) is an EBV-driven epithelial carcinoma with a recurrence rate of 30%. Biological pathways and predictive markers for recurrence remain understudied. Further, NPC tumors are associated with an intense inflammatory infiltrate, masking gene expression signals from the tumor epithelial compartment. We perform laser-capture microdissection on a preliminary cohort of 12 tumors, including primary tumors from recurrent and non-recurrent cases matched for stage of disease. Smart-3SEQ, a novel 3’ end RNA-Seq technique which allows for the accurate quantification of transcript abundance in FFPE samples, was performed to obtain gene expression libraries. Tumor epithelial and microenvironment regions from were separately microdissected, giving a total of 42 histologically-pure gene expression libraries. Raw reads were mapped to the human hg19 and EBV genomes and gene expression counts were obtained with Subread. Differential gene expression and gene set enrichment was performed in R with DESeq2 and fgsea respectively. We observed that primary NPC tumors that did not recur were significantly enriched for immune processes, including leukocyte mediated immunity, innate immune response and lymphocyte activation (padj < 1E-9 for all). The top differentially expressed genes in this group included mediators of inflammation such as STAT1 (padj = 0.0204). Critically, these gene expression changes were observed within the tumor epithelial compartment. In contrast, primary NPC tumors that eventually recurred were enriched for known oncogenes including BCL2 (padj = 0.000420). These findings suggest that heterogeneity in gene expression between NPC tumors play an important role in treatment response. Further work to define gene expression subtypes of NPC tumors will be helpful for patient stratification and personalized care. Citation Format: Joshua Tay, Wei Keat Teo, Chuan Keng Goh, Bing Cheng Wu, Kwok Seng Loh. Microdissected gene expression profiling of recurrent nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 774.

Extracellular Hsp90α stimulates a unique innate gene profile in microglial cells with simultaneous activation of Nrf2 and protection from oxidative stress.

Delivery of exogenous heat shock protein 90α (Hsp90α) and/or its induced expression in neural tissues has been suggested as a potential strategy to combat neurodegenerative disease. However, within a neurodegenerative context, a pro-inflammatory response to extracellular Hsp90α (eHsp90α) could undermine strategies to use it for therapeutic intervention. The aim of this study was to investigate the biological effects of eHsp90α on microglial cells, the primary mediators of inflammatory responses in the brain. Transcriptomic profiling by RNA-seq of primary microglia and the cultured EOC2 microglial cell line treated with eHsp90α showed the chaperone to stimulate activation of innate immune responses in microglia that were characterized by an increase in NF-kB-regulated genes. Further characterization showed this response to be substantially lower in amplitude than the effects of other inflammatory stimuli such as fibrillar amyloid-β (fAβ) or lipopolysaccharide (LPS). Additionally, the toxicity of conditioned media obtained from microglia treated with fAβ was attenuated by addition of eHsp90α. Using a co-culture system of microglia and hippocampal neuronal cell line HT22 cells separated by a chamber insert, the neurotoxicity of medium conditioned by microglia treated with fAβ was reduced when eHsp90α was also added. Mechanistically, eHsp90α was shown to activate Nrf2, a response which attenuated fAβ-induced nitric oxide production. The data thus suggested that eHsp90α protects against fAβ-induced oxidative stress. We also report eHsp90α to induce expression of macrophage receptor with collagenous structure (Marco), which would permit receptor-mediated endocytosis of fAβ.

Dissecting transcriptional heterogeneity in multiple primary lung cancer by single cell RNA sequencing.

e20516 Background: The progression of multiple primary lung cancer has been involved in complex cell changes in composition and metabolic function. However, the role of immune related cells proliferation and epithelial cells differentiation during the synchronous multiple primary lung cancer development is still understudied. Methods: In this study, we performed scRNA sequencing of 167,397 cells from six patients with multiple primary lung cancer, combined with bulk RNAseq and whole exome sequencing. Results: We firstly revealed that both naïve and memory T cells participated in the differentiation of CD8+ T cells. One end state of CD8+ T cells is exhausted T cells and they response to stimulus and positive regulated the cell death. The Other end state is cytotoxic T cells which mainly implicated in regulation of cytokine production included positive regulation of myeloid leukocyte cytokine production involved in immune response, immune effector process, macrophage cytokine production and positive regulation of cytokine production involved in immune response. Multiple functional Treg cells and naïve T cells contributed to the CD4+ T cells origin. In addition, B cells with two main kinds of function also plays an important role in immune response. One is that the regulation and activation of innate immune response have been highly activated. The other were enriched in pathways that antigen binding, processing and presentation including MHC class II Antigen Presentation. We also revealed the cellular metabolic functions during the tumor invasion. The positive regulation of blood vessel diameter has been observed in endothelial cells, while angiogenesis has not been found in early glandular neoplasia of the lung. The development of epithelial cells mainly consists of two function states. One is that some cells response to the stress from immune, and the other is that some will die programmatically and begin to cell circle due to selective pressure with the development of tumor in multiple primary lung cancer. Conclusions: Our study showed a complete landscape of different dynamic cellular changes which might pinpoint the key cellular mechanisms of synchronous multiple primary lung cancer and therefore provided new clues for pathogenesis of tumors.

A phase 1/2 study of RTX-224, an engineered red blood cell expressing 4-1BB ligand and membrane-bound IL-12, for the treatment of patients with select advanced solid tumors.

TPS2680 Background: RTX-224 is a genetically engineered off-the-shelf, allogeneic red blood cell that expresses the costimulatory molecule 4-1BB ligand (4-1BBL) and cytokine interleukin-12 (IL-12) on the cell surface. The use of 4-1BB and IL-12 agonists for cancer immunotherapy has been limited due to systemic toxicities. Unlike agonist antibodies and recombinant cytokines, which are distributed systemically, Red Cell Therapeutics, such as RTX-224, are restricted to the vasculature and spleen, which may limit toxicities previously observed with agonist 4-1BB monoclonal antibodies and recombinant IL-12. RTX-224 is designed to be a broad immune agonist of both adaptive and innate responses that activates and expands effector and memory CD8+ and CD4+ T cells, cytotoxic natural killer (NK) cells and produces inflammatory cytokines and chemokines, leading to enhanced antigen presentation. In preclinical studies, the combined activation of both adaptive and innate immune responses by the murine surrogate of RTX-224 led to antitumor activity while the restricted biodistribution improved the safety profile. Methods: The RTX-224-01 study is a Phase 1/2, first-in-human, multi-center, dose-escalation and expansion study of RTX-224 in patients with relapsed or refractory urothelial cancer, squamous cell carcinoma of the head and neck, non-small cell lung cancer, triple negative breast cancer and cutaneous melanoma. Safety, tolerability, pharmacokinetics and pharmacodynamics and anti-tumor activity of RTX-224 will be assessed. Approximately 28 patients will be enrolled across dose level cohorts to identify the recommended Phase 2 dose (RP2D). The starting dose is 100 million (1x108) cells administered intravenously every 3 weeks (Q3W) and the dose will be escalated by half-log increments following a Bayesian logarithmic regression model (BLRM) with overdose control. Following RP2D selection, each of the 5 expansion cohorts will enroll approximately 20 patients. Pharmacodynamic and exploratory biomarker studies correlative to clinical response will be evaluated on peripheral blood and paired tumor biopsies. Multiple technologies will be employed to profile the innate and adaptive responses following RTX-224 treatment. The study is open and enrolling patients in Phase 1. Clinical trial information: NCT05219578.

The TLR7/8 agonist R848 optimizes host and tumor immunity to improve therapeutic efficacy in murine lung cancer.

Treatment with the Toll-like receptor 7 (TLR7) agonist, resiquimod (R848), is effective in various types of cancer, such as breast, pancreatic and colorectal cancer. The reported antitumor effect of R848 in lung cancer is considered to be achieved by targeting macrophages. In the present study, it was demonstrated that TLR7 expression on various immune cell types initially rises, then declines in the late stage of lung cancer. Intraperitoneal injection of R848 resulted in a reduction in tumor burden and prolonged survival in both subcutaneous and metastatic lung cancer models in C57BL/6 mice. Initial treatment with R848 at an early stage was found to be the optimal choice. Systemic injection of R848 promoted the activation of innate and adaptive immune responses. Systemic administration of R848 upregulated TLR7 expression in dendritic cells (DCs) and enhanced the activation of DCs and natural killer (NK) cells. Moreover, this treatment also resulted in increased production of T helper cell-associated cytokines in serum, including IFN-γ, TNF-α and IL-2. In addition, continuous treatment with R848 increased the proportion of DCs, NK and CD8+ T cells, and reduced that of Foxp3+ regulatory T cells in the tumor microenvironment. These findings supported the use of R848 treatment for lung cancer via TLR7 targeting and provided insight into the underlying therapeutic mechanism.

Potential role of green tea amino acid l-theanine in the activation of innate immune response by enhancing expression of cytochrome b558 responsible for the reactive oxygen species-generating ability of leukocytes.

l-Theanine (N-ethyl- l-glutamine) is an analog of l-glutamine and l-glutamic acid, accounts for up to 50% of all free amino acids in green tea, and elicits an umami taste. As l-theanine also shows various physiological activities including immune response-modifying activities, it is expected to be an excellent health-promoting phytochemical agent. To know the influences of l-theanine on the human innate immune response, we investigated the effect of l-theanine on the superoxide anion (O2 - )-generating system of leukocytes using U937 cells. The O2 - -generating system in leukocytes consists of membrane cytochrome b558 protein (a complex of p22-phox and gp91-phox proteins) and cytosolic p40-phox, p47-phox, and p67-phox proteins. Addition of 500 μM l-theanine caused remarkable enhancement of the all-trans retinoic acid (ATRA)-induced O2 - -generating activity (to ~470% of ATRA-treated cells), but not l-glutamine and l-glutamic acid. Semiquantitative RT-PCR showed that the transcription level of gp91-phox is significantly increased in ATRA and l-theanine-co-treated cells. Chromatin immunoprecipitation revealed that l-theanine enhances acetylations of Lys-9 and Lys-14 residues of histone H3 within the chromatin surrounding the promoter region of the gp91-phox gene. Immunoblotting demonstrated that membrane cytochrome b558 proteins remarkably accumulate in ATRA + l-theanine-treated cells. These results suggested that l-theanine brings about a remarkable accumulation of cytochrome b558 protein via upregulating the transcription of the gp91-phox gene during leukocyte differentiation, resulting in marked augmentation of the O2 - -generating ability, which is one of the most important functions of leukocytes responsible for the innate immune system.

Mobilization of innate and adaptive antitumor immune responses by the RNP-targeting antibody ATRC-101

Immunotherapy approaches focusing on T cells have provided breakthroughs in treating solid tumors. However, there remains an opportunity to drive anticancer immune responses via other cell types, particularly myeloid cells. ATRC-101 was identified via a target-agnostic process evaluating antibodies produced by the plasmablast population of B cells in a patient with non-small cell lung cancer experiencing an antitumor immune response during treatment with checkpoint inhibitor therapy. Here, we describe the target, antitumor activity in preclinical models, and data supporting a mechanism of action of ATRC-101. Immunohistochemistry studies demonstrated tumor-selective binding of ATRC-101 to multiple nonautologous tumor tissues. In biochemical analyses, ATRC-101 appears to target an extracellular, tumor-specific ribonucleoprotein (RNP) complex. In syngeneic murine models, ATRC-101 demonstrated robust antitumor activity and evidence of immune memory following rechallenge of cured mice with fresh tumor cells. ATRC-101 increased the relative abundance of conventional dendritic cell (cDC) type 1 cells in the blood within 24 h of dosing, increased CD8+ T cells and natural killer cells in blood and tumor over time, decreased cDC type 2 cells in the blood, and decreased monocytic myeloid-derived suppressor cells in the tumor. Cellular stress, including that induced by chemotherapy, increased the amount of ATRC-101 target in tumor cells, and ATRC-101 combined with doxorubicin enhanced efficacy compared with either agent alone. Taken together, these data demonstrate that ATRC-101 drives tumor destruction in preclinical models by targeting a tumor-specific RNP complex leading to activation of innate and adaptive immune responses.

Abstract 465: Epitranscriptomic Modification Of Mir-483 Impairs Endothelial Function

Emerging evidence indicates that oxidative stress causes the hydroxylation of guanine (G) to generate 8-oxo-7,8-dihydro guanosine ( 8 OH-G) in microRNAs (miRs), which induces the guanine-to-uracil (G-to-U) transversion and changes the miR targetomes. However, whether and how the 8 OH-G-modified miRs are involved in vascular endothelial dysfunction has never been explored. Using 8 OH-G miR crosslinking immunoprecipitation sequencing ( 8 OH-G miR-CLIP-seq), we sorted 8 OH-G miRs enriched in ECs underwent ox-LDL treatment. CLIP-seq analysis revealed that 8 OH-G-miR-483 was among the most abundant 8 OH-G miR species in ECs induced by ox-LDL. Likewise, H 2 O 2 and disturbed flow induced the miR-483 to 8 OH-G miR-483 transversion in cultured ECs. In C57 mice, the level of 8 OH-G-miR-483 was ~22-fold higher in the aortic arch (AA, under atheroprone flow) than the thoracic aorta (TA, under atheroprotective flow). Transcriptomic profiling by RNA-seq followed by principal component analysis (PCA) revealed a transcriptomic segregation between ECs overexpressing miR-483 and 8 OH-G miR-483, indicating that 8 OH-G miR-483 altered the transcriptomes. Gene Ontology (GO) analysis of the 8745 differentially expressed genes (DEGs, fold change >1.5, P<0.05) indicated that miR-483 suppressed genes involved in the TGF-β signaling, inflammatory response, cellular senescence, positive regulation of cell adhesion, and activation of innate immune response. In contrast, 8 OH-G miR-483 downregulated genes were largely correlated with angiogenesis, NO biosynthetic process, EC development, EC barrier function, and cell junction maintenance. Thus, the miR-483 to 8 OH-G miR-483 transversion would impair EC function. Functionally, the monocyte adhesion was significantly increased, and nitric oxide bioavailability decreased in ECs overexpressing 8 OH-G miR-483. Collectively, this study demonstrates that redox burden incurred by cardiovascular risk factors is a culprit of the miR-483 to 8 OH-G miR-483 transversion. Such epitranscriptomic modification of miR-483 causes endothelial dysfunction via its targetome shifting.

Defining complex mechanistic interactions and responses by macrophages during Mycobacterium abscessus infection

Abstract Phagocytosis by macrophages is critical to eradicate invading pathogens. Phagocytosis is regulated by parallel networks that control the binding and uptake of particles, subsequent lysosome delivery, and activation of downstream innate immune responses. However, whether distinct host pathways drive unique responses to targeted substrates within the lung environment remains unknown. Mycobacterium abscessus (MAB) is a rapidly growing non-tuberculous mycobacterium that infects patients with chronic lung diseases, like cystic fibrosis. While interactions between MAB and macrophages contribute to its pathogenesis, how macrophages bind, phagocytose, and control MAB remains entirely unclear. We hypothesize that understanding key MAB-macrophage interactions will identify critical host targets that can be inhibited to prevent disease. To test this hypothesis, using a genome-wide knockout library of immortalized bone marrow derived macrophages, we completed a forward genetic screen to identify host pathways that contribute to fluorescent MAB uptake. Our results show glycosaminoglycan synthesis (sGAGs) in macrophages is required to efficiently take up MAB. Both enzymatic and genetic approaches found key sGAG biosynthesis regulators, UGDH, B3GLCT, B4GALT7 and B3GAT3, influence macrophage interactions with MAB and other pathogens like Mycobacterium tuberculosis. Using a recently developed alveolar macrophage model, we are now dissecting the role of sGAGs in lung specific macrophages. Future work will determine how sGAG pathways modulate bacterial replication, cytokine responses and disease progression following MAB infection with the goal of defining key mechanisms driving host-pathogen interactions in the lungs.

PD-L1 back signaling manipulates IL-6 signaling to coordinate innate immune responses

Abstract Programmed death ligand-1 (PD-L1) interaction with PD-1 on T cells limits immune activation to prevent immunopathology in healthy tissues and the development of autoimmunity. While ligation of PD-1 by PD-L1 leads to T cell-intrinsic PD-1 signaling, PD-L1 back signaling into the ligand-bearing cell remains poorly defined. We recently outlined a major role for PD-L1 back signaling in regulating dermal dendritic cell (DC) migration to the lymph node following a type I interferon inducing stimulus or infection. Similar to prior studies in IFNβ treated cancer cells, we noted increased pSTAT3 post DC activation that was unchanged with addition of CCL21. Increased pSTAT3 occurred in Pdl1−/− DCs and in DCs from mice in which 3 cytoplasmic residues were mutated to alanine (Pdl1CM). Since STAT3 propagates signalling through the IL-6 receptor, we hypothesized that increased IL-6 signaling may explain elevated pSTAT3 in Pdl1−/− and Pdl1CM DCs. To test whether PDL1 back signaling contributed to STAT3-associated cytokine responses, we generated bone marrow derived DCs from Pdl1−/− and Pdl1CM mice and observed significantly higher levels of IL-6 compared to WT controls following activation. We next asked if PD-L1 back signaling is important for proper innate activation as PD-L1 is upregulated on many innate immune populations early after systemic L. monocytogenes (LM) infection. We found loss of PD-L1 back signaling following infection led to differences in circulating levels of many innate immune cells and reduced IL-6 in the serum. These findings demonstrate that PD-L1 back signaling in innate immune cells may assist in coordinating IL-6 signaling and proper activation of early innate immune responses during systemic LM infection. Supported by grants from NIH (R01 AI155474, T32 AI074491 - "Molecular Mechanisms of Immune Tolerance")

Biomimetic Nanomedicine-Triggered in Situ Vaccination for Innate and Adaptive Immunity Activations for Bacterial Osteomyelitis Treatment.

The development of bacterial vaccines for inducing an immunoresponse against infectious diseases such as osteomyelitis is of great significance and importance. However, the responsiveness of bacterial immunotherapy remains far from being satisfactory, largely due to the erratic antigen epitopes of bacteria. Herein, we report an in situ vaccination strategy for the immunotherapy of bacterial infection based on an osteomyelitis model using a biomimetic nanomedicine named as HMMP, which was constructed by engineering PpIX-encapsulated hollow MnOx with a hybrid membrane exfoliated from both macrophage and tumor cell lines. The as-established HMMP features a burst bacterial antigen release as the in situ vaccine by the augmented sonodynamic treatment and the resultant priming of antigen-presenting cells for the following activations of both cellular and humoral adaptive immunities against bacterial infections. This treatment regimen not only triggers initial bacterial regression in the established osteomyelitis model but also simultaneously generates robust systemic antibacterial immunity against poorly immunogenic secondary osteomyelitis in the contralateral knee and additionally confers long-lasting bacteria-specific immune memory responses to prevent infection relapse. Thus, our study provides a proof of concept of in situ vaccination for the activation of both innate and adaptive antibacterial immune responses, providing an individual-independent bacterial immunotherapy.