Innate Immune-related Genes Research Articles

Association of polymorphisms of innate immunity-related genes and tuberculosis susceptibility in Mongolian population

BackgroundsGenetic polymorphism of the toll-like receptor 2, 4 (TLR2, TLR4) and natural resistance-associated macrophage protein 1 (NRAMP1) genes may affect host immune response to Mycobacterium tuberculosis (Mtb) and lead to the variation of susceptibility to tuberculosis (TB) in humans. However, the association of single nucleotide polymorphisms (SNP) in these genes and the susceptibility to TB in Mongolian population has not been investigated. MethodsWe conducted a genetic association study including 197 Mongolian TB patients and 217 Mongolian healthy controls in Inner Mongolia, China. DNA of blood samples was extracted and genotyped for 5 SNPs in TLR4, 4 SNPs in TLR2 and 5 SNPs in NRAMP1 by next-generation sequencing. A logistic regression was performed and odds ratios (OR) with 95% confidence intervals (CI) were calculated to estimate the risk at TB by each SNP. ResultsThe most significant locus associated with the susceptibility to TB was TLR4 rs11536889. The frequency for allele C of TLR4 rs11536889 was 16.0% in TB patients and 23.5% in healthy controls, respectively. Rs11536889 C/C genotype of TLR4 was significantly associated with the low susceptibility against TB compared to G/G genotype in the dominant model (OR 0.62, 95% CI 0.41–0.94). ConclusionsThe TLR4 rs11536889 polymorphisms might be an indicative of the low susceptibility to TB in Mongolian population, which provides valuable information for the generation of effective strategy or measurement against TB in Mongolian population.

Reduced expression of innate immunity-related genes in lymph node metastases of luminal breast cancer patients

Immune system plays a dual role in cancer by either targeting or supporting neoplastic cells at various stages of disease, including metastasis. Yet, the exact immune-related transcriptome profiles of primary tumours (PT) and lymph node metastases (LNM) and their evolution during luminal breast cancer (BCa) dissemination remain undiscovered. In order to identify the immune-related transcriptome changes that accompany lymphatic spread, we analysed PT-LNM pairs of luminal BCa using NanoString technology. Decrease in complement C3—one of the top-downregulated genes, in LNM was validated at the protein level using immunohistochemistry. Thirty-three of 360 analysed genes were downregulated (9%), whereas only 3 (0.8%) upregulated in LNM when compared to the corresponding PT. In LNM, reduced expression was observed in genes related to innate immunity, particularly to the complement system (C1QB, C1S, C1R, C4B, CFB, C3, SERPING1 and C3AR1). In validation cohort, complement C3 protein was less frequently expressed in LNM than in PT and it was associated with worse prognosis. To conclude, local expression of the complement system components declines during lymphatic spread of non-metastatic luminal BCa, whilst further reduction of tumoral complement C3 in LNM is indicative for poor survival. This points to context-dependent role of complement C3 in BCa dissemination.

Profiles of Innate Immune Cell Infiltration and Related Core Genes in Psoriasis.

Psoriasis is an inflammatory skin disease with substantial morbidity. Numerous patients with psoriasis experience recurrence after therapy. The underlying mechanism about psoriasis is still not fully understood. Some evidences suggest that innate immunity may play an unexpected and important role in active severe psoriasis. In this work, the deconvolution algorithm CIBERSORT was conducted to identify the infiltration of innate immune cells and related core genes in psoriatic plaque. Datasets from the Gene Expression Omnibus, including skin samples from 405 psoriasis patients and 91 healthy donors, were downloaded for analysis. Considerable differences of the innate immune cell composition were uncovered between psoriatic plaque and control skin. Results revealed that γδ T cells, resting NK cells, M0 macrophages, M1 macrophages, activated dendritic cells, and neutrophils were significantly increased in psoriatic skin, while resting mast cells and active NK cells were significantly decreased. Moreover, the proportion of M0 macrophages or resting mast cells was found to be associated with disease severity. Spearman correlation analysis suggests that RORC and S100A12 genes were related to disease severity, while genes including S100A12, CLEC4C, IL-19, AIM2, IL-17F, and PPARGC1A were correlated with biologic treatment response. In conclusion, this work displays innate immune status in psoriatic skin and provides novel clues for clinical decisions and mechanism study.

The Alteration of Intestinal Microbiota Profile and Immune Response in Epinephelus coioides during Pathogen Infection.

Epinephelus coioides, or grouper, is a high economic value fish species that plays an important role in the aquaculture industry in Asia. However, both viral and bacterial diseases have threatened grouper for many years, especially nervous necrosis virus, grouper iridovirus and Vibrio harveyi, which have caused a bottleneck in the grouper industry. Currently, intestinal microbiota can provide novel insights into the pathogenesis-related factors involved in pathogen infection. Hence, we investigated the comparison of intestinal microbiota communities in control group and pathogen-infected grouper through high-throughput sequencing of the 16S rRNA gene. Our results showed that microbial diversity was decreased, whereas microbial richness was increased during pathogen infection. The individuals in each group were distributed distinctly on the PLSDA diagram, especially the GIV group. Proteobacteria and Firmicutes were the most abundant bacterial phyla in all groups. Interestingly, beneficial genera, Faecalibacterium and Bifidobacterium, predominated in the intestines of the control group. In contrast, the intestines of pathogen-infected grouper had higher levels of harmful genera such as Sphingomonas, Atopostipes, Staphylococcus and Acinetobacter. Additionally, we investigated the expression levels of innate and adaptive immune-related genes after viral and bacterial infection. The results revealed that immunoglobulin T and proinflammatory cytokine levels in the intestine increased after pathogen infection. Through these unique bacterial compositions in diseased and uninfected fish, we could establish a novel therapeutic approach and bacterial marker for preventing and controlling these diseases.

Targeting XPO1 enhances innate immune response and inhibits KSHV lytic replication during primary infection by nuclear stabilization of the p62 autophagy adaptor protein

Nucleocytoplasmic transport of signaling modulators is essential for regulating cellular responses to extracellular stimulation and stress, as well as pathogen infection. Exportin 1 (XPO1), also known as chromosomal maintenance 1 (CRM1), mediates nuclear export of proteins, rRNAs, snRNAs, and some mRNAs. In this study, we have identified an essential role of XPO1 in regulating Kaposi’s sarcoma-associated herpesvirus (KSHV) lytic replication during primary infection of primary human umbilical vein endothelial cells. Treatment with an XPO1 inhibitor KPT-8602 and short hairpin RNA (shRNA)-mediated knockdown of XPO1 reduced KSHV lytic replication but had no effect on KSHV entry and trafficking. XPO1 inhibition induced retention of autophagy adaptor protein p62 (SQSTM1) in the nucleus, which enhanced activation of TBK1 and IRF3. As a result, nuclear accumulation of p62 increased expression of innate immune-related genes including IRF7, ISG15, IFIT1, IFIT2, and IFIT3, leading to a reduction of KSHV lytic replication. These results illustrate a novel mechanism by which XPO1 mediates innate immune response and KSHV replication, and identify XPO1 as a potential therapeutic target and KPT-8602 as a promising therapeutic agent for KSHV infection.

Transcriptome analysis of Mycoplasma bovis stimulated bovine peripheral blood mononuclear cells

Mycoplasma bovis is a pathogenic bacterium in bovines that causes huge global economic losses. Numerous factors play important roles in M. bovis pathogenesis; however, the host immune response involved in M. bovis infection has not been fully elucidated. We aimed to determine the characteristics of the host immune response to Mycoplasma infection. We evaluated the responsiveness of bovine peripheral blood mononuclear cells (PBMCs) stimulated with M. bovis via microarray analysis. The transcriptional abundance of innate immune-related genes IL-36A, IL-27, IFN-γ, and IL-17 in PBMCs increased after M. bovis exposure. Upon M. bovis infection, there was increased expression of the lymphocyte activated genes basic leucine zipper transcription factor (BATF) and signaling lymphocytic activation molecule family members 1 and 7 (SLAMF 1 and SLAMF 7) in PBMCs compared with that in unstimulated cells. The study revealed that the transcriptional abundance of innate immunity genes in PBMCs increased during M. bovis infection. This induced the activation of PBMCs, giving rise to an immune response, which is followed by the development of the inflammatory response. The results from this study could be used as the basis for the development of novel vaccine candidates against M. bovis.

Investigation of herpesviruses in dementia patients and associated inflammatory gene modulation

AbstractBackgroundAlzheimer’s disease (AD) and vascular contributions to cognitive impairment and dementia (VCID) are the leading causes of dementia worldwide, with the two frequently found co‐morbid in dementia patients at autopsy. Studies have connected multiple herpesviruses to the development and pathogenesis of AD. The goal of this study was to determine the prevalence of four common herpesviruses in a cohort of AD and VCID patients, and to identify differential gene expression as a function of viral presence. We hypothesize AD and VCID cases will possess more latent virus than control cases, and cases with latent virus present will show a down‐regulation of innate immune related genes, and an upregulation of cell survival genes and transcriptional suppressors.MethodUsing droplet‐based PCR, we quantified the prevalence of four herpesviruses (herpes simplex virus 1, herpes simplex virus 2, cytomegalovirus, and human herpes virus 6) in a subset of brain autopsy tissue obtained from the Sanders‐Brown Center on Aging Alzheimer’s Disease Center (UK‐ADC). RNA and genomic DNA was extracted from the superior medial temporal gyrus and cerebellum of 49 cases (n=18 control, n=10 vascular pathology, n=20 AD, n=1 mixed AD+VCI). Genomic DNA was used to determine the presence of latent virus and cDNA to determine actively replicating virus. The RNA from these regions was run on the NanoString neuroinflammatory panel which quantifies a number of genes, ranging from neuroinflammatory markers to cell specific functional genes.ResultWe identified an increased amount of latent viral populations in the AD patients relative to control patients. NanoString analysis supported our initial hypothesis regarding gene regulation relating to innate immunity and cell survival, but we also observed downregulation of insulin‐like growth factor, synapse‐associated protein 97, and cell death‐inducing DFFA‐like effector b in the presence of certain latent viral populations.ConclusionOur results demonstrate the prevalence of herpesviruses within an AD cohort, supporting research suggesting a link between the two. Further, it shows alterations in the expression of neuroinflammatory genes as a function of latent virus presence.

Experimental and population-genetic evidence for inflammation control functions of long noncoding RNAs and a novel tRNA-like transcript arising from the human NEAT1-MALAT1 genomic region

Abstract Background Uncontrolled inflammation is a key driver of atherosclerosis, myocardial infarction (MI), and multiple other diseases. Beyond proteins and microRNAs, long noncoding RNAs (lncRNAs) are implicated in inflammation control. We previously reported suppression of lncRNA NEAT1 in circulating immune cells of post-MI patients. In mice lacking lncRNAs NEAT1 or MALAT1 we observed major immune disturbances affecting monocyte-macrophage and T cell differentiation and rendering the immune system unstable and highly vulnerable to immune stress. Here, we report functions of a novel tRNA-type transcript arising from the NEAT1-MALAT1 gene cluster, and on genetic heterogeneity of this region in the human population. Methods and results While previously investigated mice were deficient in the entire NEAT1 or MALAT1 locus, we here aimed to selectively disrupted only the novel 59-nt tRNA-like transcript “menRNA” with hitherto unknown functions. Through CRISPR/Cas9 editing we developed 4 human THP-1 monocyte-macrophage cell line clones with deletions of different extension all of which prevented, however, normal transcript folding and formation of “menRNA”. Transcriptome mapping of all clones by RNA-sequencing identified dysregulation of innate immunity-related genes (IFI16, IFITM3, IRAK3, IRF2BP2, IRF3), chemokine and interleukin receptors (CCR10, IL11RA, IL12RB2, IL23A), cell surface receptors (CD37, CD40LG, CD72, FOCAD, ITGA6, MAEA, THY1), macrophage function-associated genes (ELANE, GRN, MIF, MMP25, MST1P2, PRTN3), tRNA-processing transcripts (GARS, QRSL1P3, QTRT1P1, THG1L, VARS), and small nucleolar RNAs (SNORA26.62.64, SNORD65.112). These data and functional assays indicate functions of NEAT1-derived “menRNA” distinct from those previously described for MALAT1-derived mascRNA. As multiple data suggest inflammation control functions of the NEAT1-MALAT1 region, we investigated the extent of genetic variability of this region in humans. In cohorts from the SHIP study coordinated by the Institute for Community Medicine Greifswald, screening of this region for sequence variants and possible phenotype associations was conducted the results of which are given in Figure 1. Consistent with prior findings, a MALAT1 SNP with very low minor allele frequency (MAF=0.01) was associated (p=0.0062) with systemic low level inflammation (CRP >3.0 mg/L). Unexpected was the association (p<0.01) of eight SNPs (low MAF=0.09 for all) with BMI >35 kg/m2 and LDL >164 mg/dl. Conclusions First, selective disruption of menRNA formation in human monocyte-macrophages provides evidence that this novel type of noncoding RNA has immunoregulatory functions. Second, the phenotype associations of SNPs within the NEAT1-MALAT1 gene cluster warrant further in-depth investigation of the molecular basis of these associations, and of their allele frequencies in cardiovascular disease patient cohorts. The first three and the last authors contributed equally to this work. Figure 1 Funding Acknowledgement Type of funding source: Other. Main funding source(s): “Transcriptome analysis of circulating immune cells to improve the assessment of prognosis and the response to novel anti-inflammatory treatments after myocardial infarction”; DZHK Shared Expertise project B19-006_SE FKZ 81X2100257

Development and validation of a fourteen- innate immunity-related gene pairs signature for predicting prognosis head and neck squamous cell carcinoma

BackgroundImmune-related genes is closely related to the occurrence and prognosis of head and neck squamous cell carcinoma (HNSCC). At the same time, immune-related genes have great potential as prognostic markers in many types of cancer. The prognosis of HNSCC is still poor currently, and it may be effective to predict the clinical outcome of HNSCC by immunogenic analysis.MethodsRNASeq and clinical follow-up information were downloaded from The Cancer Genome Atlas (TCGA), the MINiML format GSE65858 chip expression data was downloaded from NCBI, and immune-related genes was downloaded from the InnateDB database. Immune-related genes in 519 HNSC patients were integrated from TCGA dataset. By using multivariate COX analysis and Lasso regression, robust immune-related gene pairs (IRGPs) that predict clinical outcomes of HNSCC were identified. Finally, a risk prognostic model related to immune gene pair was established and verified by clinical features, test sets and GEO external validation set.ResultsA total of 699 IRGPs were significantly correlated with the prognosis of HNSCC patients. Fourteen robust IRGPs were finally obtained by Lasso regression and a prognostic risk prediction model was constructed. Risk score of each sample were calculated based on Risk models and divided into the high-risk group (Risk-H) and low Risk group (Risk-L). Risk models were able to stratify the risk in patients with TNM Stage, Age, gender, and smoking history, and the AUC > 0.65 in training set and test set, shows that 14-IRGPs signature in patients with HNSCC has excellent classification performance. In addition, 14-IRGPs had the highest average C index compared with the prognostic characteristics and T, N, and Age of the 3 previously reported HNSCC.ConclusionThis study constructed 14-IRGPs as a novel prognostic marker for predicting survival in HNSCC patients.

CpG‐ODN 2007 protects zebrafish ( Danio rerio ) against Vibrio vulnificus infection

Vibrio vulnificus is an aquatic pathogen that can cause primary sepsis and soft tissue infection. CpG oligodeoxynucleotides (CpG-ODN) are a type of essential immunomodulators, which can trigger or enhance immune responses in mammals, fish and humans. In this study, we evaluated the effect of CpG-ODN 2007 as a potential immunostimulant for zebrafish (Danio rerio) infected with V. vulnificus strain FJ03-X2. Fish injected with the CpG-ODN 2007 showed a lower mortality rate compared with the fish that did not receive treatment; the survival rates of the CpG-ODN 2007-treated group and PBS-treated group were 85% and 57.9% respectively. Also, our in vitro results demonstrated that CpG-ODN 2007 could effectively reduce the toxicity of V. vulnificus strain FJ03-X2 to zebrafish embryonic fibroblast (ZF4) cells. Furthermore, we assessed innate immune-related genes (TNF, IL-1β, IFNg1-2, MyD88, lysozyme, IL-10, IL-22, IL-26 and TLRs) in expression patterns of V. vulnificus strain FJ03-X2 infected zebrafish or ZF4 cells with and without CpG-ODN 2007 treatment. We found that CpG-ODN 2007 could reduce the toxicity of V. vulnificus to ZF4 cells and regulate the immune system to control the overexpression of specific genes. To sum up, our data indicated that CpG-ODN 2007 protects zebrafish against Vibrio vulnificus infection.

Regulation of gene expression by miRNA-455-3p, upregulated in the conjunctival epithelium of patients with Stevens\u2013Johnson syndrome in the chronic stage

To investigate the role of miRNA in the pathogenesis underlying ocular surface complications in patients with Stevens–Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN) in the chronic stage. Using oligonucleotide microarrays, we performed comprehensive miRNA analysis of the conjunctival epithelium of SJS/TEN patients with severe ocular complications (SOC) in the chronic stage (n = 3). Conjunctival epithelium of patients with conjunctival chalasis (n = 3) served as the control. We confirmed the down- and up-regulation of miRNA of interest by quantitative real-time polymerase chain reaction (RT-PCR) assays using the conjunctival epithelium from 6 SJS/TEN with SOC patients and 7 controls. We focused on miRNA-455-3p, which is significantly upregulated in the conjunctival epithelium of the SJS/TEN patients, and investigated its function by inhibiting miR-455-3p in primary human conjunctival epithelial cells (PHCjEs). Comprehensive miRNA expression analysis showed that the expression of 5 kinds of miRNA was up-regulated more than fivefold, and that the expression of another 5 kinds of miRNA was down-regulated by less than one-fifth. There was a significant difference between the SJS/TEN patients and the controls [analysis of variance (ANOVA) p < 0.05]. Quantitative miRNA PCR assay showed that hsa-miR-31* and hsa-miR-455-3p were significantly up-regulated in the conjunctival epithelium of the SJS/TEN patients. Comprehensive gene expression analysis of PHCjEs transfected with the hsa-miR-455-3p inhibitor and quantitative RT PCR assay showed that ANKRD1, CXCL8, CXCL2, GEM, PTGS2, RNASE8, IL6, and CXCL1 were down-regulated by the hsa-miR-455-3p inhibitor. Quantitative RT-PCR, focused on the genes that tended to be up-regulated in SJS/TEN with SOC, revealed that the expression of IL1A, KPRP, IL36G, PPP1R3C, and ADM was significantly down-regulated in PHCjEs transfected with the hsa-miR-455-3p inhibitor. Our results suggest that miRNA-455-3p could regulate many genes including innate immune related genes in human conjunctival epithelium, and that its up-regulation contributes to the pathogenesis on the ocular surface in SJS/TEN patients with the SOC in the chronic stage. Our findings may lead to the development of new treatments using the miRNA-455-3p inhibitor.

Organic cranberry pomace and its ethanolic extractives as feed supplement in broiler: impacts on serum Ig titers, liver and bursal immunity

With the pressure to reduce antibiotics use in poultry production, cost-effective alternative products need to be developed to enhance the bird's immunity. The present study evaluated the efficacy of cranberry fruit by-products to modulate immunity in broiler chickens. Broiler Cobb 500 chicks were fed a control basal diet, basal diet supplemented with bacitracin (BACI, 55 ppm), cranberry pomace at 1% and 2% (CP2), or cranberry pomace ethanolic extract at 150 and 300 ppm (COH300) for 30 d. Blood sera were analyzed at days 21 and 28 of age for Ig levels by ELISA. The innate and adaptive immune-related gene expression levels in the liver and bursa of Fabricius were investigated at 21 d of age by quantitative polymerase chain reaction arrays. At day 21, the highest IgY level was found in the blood serum of the CP2-fed birds. In the liver, 13 of the 22 differentially expressed genes were downregulated across all treatments compared with the control. Expression of genes belonging to innate immunity such as caspase 1 apoptosis–related cysteine peptidase, chemokine receptor 5, interferon gamma, myeloid differentiation primary response gene 88, and Toll-like receptor 3 were significantly downregulated mainly in BACI- and COH300-fed birds. In the bursa, 5 of 9 genes associated with the innate immunity were differentially expressed. The expression of anti-inflammatory IL-10 gene was upregulated in all treatment groups in bursa compared with the control. The expression of transferrin gene was significantly upregulated in livers of birds fed COH300 and in bursa of birds fed BACI, indicating feeding practices and organ-dependant modulation of this gene in broiler. Overall results of this study showed that cranberry product feed supplementation modulated the innate immune and suppressed proinflammatory cytokines in broilers, providing a platform for future investigations to develop berry products in poultry feeding.

Abstract 4443: Inhibition of PGE2/EP4 pathway by ONO-4578/BMS-986310, a novel EP4 antagonist, promotes T cell activation and myeloid cell differentiation to dendritic cells

Abstract Prostaglandin E2 (PGE2) contributes to immunosuppression in the tumor microenvironment through one or more of its receptors (EP1-EP4), especially EP4 which is expressed at high levels on myeloid cells. ONO-4578/BMS-986310 is a novel EP4-selective antagonist that potently inhibits the PGE2-induced cAMP production in CHO cells expressing mouse and human EP4 receptors with single-digit nM IC50. Furthermore, ONO-4578/BMS-986310 shows excellent pharmacokinetic properties after oral dosing in mouse. ONO-4578/ BMS-986310 reverses PGE2-mediated suppression of T cell activation and promotes DC differentiation in vitro. Moreover, treatment with ONO-4578/BMS-986310 inhibits growth of CT26 and MC38 tumors in syngeneic mouse. The anti-tumor effect of ONO-4578/BMS-986310 is abrogated in immuno-deficient mice bearing CT26 tumors indicating that such effect is immune-mediated. ONO-4578/BMS-986310 treatment significantly prolongs survival of mice with highly aggressive SB28 gliomas in the brain. Flow cytometric analysis reveals that ONO-4578/BMS-986310 reduces infiltration of M2 macrophages in MC38 tumors compared with control mice. Microarray analysis of MC38 tumor tissues demonstrates up-regulation of cytotoxic T lymphocytes, natural killer cells and innate immunity-related genes after treatment with ONO-4578/BMS-986310. Finally, a combination of ONO-4578/BMS-986310 and anti-PD-1 antibody results in enhanced anti-tumor effects in GL261 and MC38 tumor-bearing mice compared with anti-PD-1 monotherapy.Our data demonstrate that ONO-4578/BMS-986310 promotes anti-tumor immunity and may be useful in the treatment of cancer patients. ONO-4578/BMS-986310 is being evaluated as monotherapy and in combination with nivolumab in an ongoing Ph1/1b clinical trials in patients with advanced solid tumors (NCT03155061, NCT03661632). Citation Format: Tohru Kotani, Hirotsugu Takano, Tomoko Yoshida, Ryota Hamasaki, Gary Kohanbash, Kazuhiko Takeda, Hideho Okada. Inhibition of PGE2/EP4 pathway by ONO-4578/BMS-986310, a novel EP4 antagonist, promotes T cell activation and myeloid cell differentiation to dendritic cells [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 4443.

Bacillus licheniformis BCR 4-3 increases immune response and survival of Litopenaeus vannamei challenged with Vibrio parahaemolyticus IPNGS16

Immunoprophylactic alternatives are needed to fight against acute hepatopancreatic necrosis disease (AHPND). This study addressed the effects of different routes of Bacillus administration (via diet or culture water) on growth performance, survival, and immune parameters of Litopenaeus vannamei challenged with Vibrio parahaemolyticus-AHPND. Bacillus mixture and Bacillus licheniformis BCR 4-3 were added to feed (1 × 106 CFU g−1) or rearing water (1 × 106, 2 × 106, 3 × 106 CFU L−1). Animals were challenged with a determined median lethal V. parahaemolyticus concentration. Survival, growth performance, and immune response were determined. The results showed that B. licheniformis BCR 4-3 (3 × 106 CFU L−1) administration via culture water significantly (P < 0.05) increased survival (53.4%) in shrimp challenged with V. parahaemolyticus; growth performance was also significantly (P < 0.05) higher compared to the control group. Moreover, the relative expression of innate immune-related genes, such as lysozyme, penaeidin4, crustin, and superoxide dismutase, significantly (P < 0.05) upregulated in shrimp fed B. licheniformis BCR4-3 compared with the control group. In conclusion, the administration of probiotic B. licheniformis BCR4-3 in feed and culture water increased resistance in L. vannamei against V. parahaemolyticus-AHPND and was found associated with an upregulation of key antimicrobial innate immune-related genes.

Abstract 969: Pharmacologic induction of innate immune signaling via STING directly drives homologous recombination deficiency

Abstract Poly (ADP-ribose) polymerase inhibitors (PARPi) are FDA approved in a subset of patients with ovarian cancer or metastatic breast cancers who harbor BRCA gene mutations. Although these mutations, which generate homologous recombination deficiencies (HRD), have been the main predictor to PARPi sensitivity, responses to therapy have not been durable and have failed for the majority of sporadic triple negative breast cancers (TNBC). We previously reported that DNA methyltransferase inhibitor (DNMTi) azacytidine (Aza) improves the efficacy of a new generation of PARPi, Talazoparib (Tal), through increased trapping of cytotoxic PARP-DNA complexes in both BRCA-mutant and -proficient TNBC. These trapped complexes lead to increased and persistent levels of lethal double strand breaks (DSBs), suggesting that DSB repair may also be impaired with this treatment. Indeed, Aza/Tal treatment in BRCA-proficient TNBC cell lines significantly downregulates expression of HR and Fanconi Anemia (FA) genes, notably FANCD2, and decreases HR activity, thus generating HRD. DNMTi have also been established to induce a viral mimicry response which upregulate type I interferon (IFN) signaling and production of inflammatory cytokines. In the present study, we elucidate the link between Aza/Tal facilitated HRD and induction of innate immune and inflammatory related genes, mediated through a STING dependent mechanism, which we term as a pathogen mimicry response (PMR). Gene set enrichment analysis of RNA-Seq data derived from mono- and combination-treated TNBC cell line MDA-MB-231, reveals enrichment of innate immune and cytosolic DNA sensing pathways with significant increases of TNFα/NF-κB and IFNαβ gene sets. Overlap between HRD and immune related signaling was evaluated using the STRING database, which reveals a significant interaction specifically between FA and TNFα/NF-κB and IFNαβ pathway genes. Tal driven cytosolic DNA as well as an Aza augmentation in STING protein expression, emerges as the key node in Aza/Tal induced innate immune signaling. Transient knockdown of STING is able to significantly rescue Aza- and combination-induced repression of FANCD2. Critically, in the presence of FANCD2 rescue facilitated by STING-specific inhibition, Aza-induced HRD was also reversed. These STING perturbation data suggest that Aza-induced HRD is dependent on presence of competent STING signaling. This inverse relationship was validated in both METABRIC TNBC dataset and TCGA data sets including lung adenocarcinoma and lung squamous cell carcinoma, colon adenocarcinoma, and AML, thus suggesting broad applicability of this observed transcriptional program independent of pharmacologic intervention. Induction of PMR to drive HRD suggests that DNMTi-PARPi therapy strategies can expand the therapeutic scope of PARPi to encompass treatment of BRCA-proficient cancers. Citation Format: Lena J. McLaughlin, Aksinija A. Kogan, Stephen B. Baylin, Michael J. Topper, Feyruz V. Rassool. Pharmacologic induction of innate immune signaling via STING directly drives homologous recombination deficiency [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 969.

Prediabetes uncovers differential gene expression at fasting and in response to oral glucose load in immune cells

Metabolic disorders including diabetes are associated with immune cell dysfunction. However, the effect of normal glucose metabolism or impairment thereof on immune cell gene expression is not well known. Hence, in this cross-sectional pilot study, we sought to determine the differences in gene expression in the peripheral blood mono-nuclear cells (PBMCs) of normal glucose tolerant (NGT) and prediabetic (PD) Asian Indian men, at fasting and in response to 75g oral glucose load. Illumina HT12 bead chip-based microarray was performed on PBMCs at fasting and 2-h post load conditions for NGT (N=6) and PD (N=9) subjects. Following normalization and due quality control of the raw data, differentially expressed genes (DEGs) under different conditions within and across the two groups were identified using GeneSpring GX V12.0 software. Paired and unpaired Student's t-tests were applied along with fold change cut-offs for appropriate comparisons. Validation of the microarray data was carried out through real-time qPCR analysis. Significantly regulated biological pathways were analyzed by employing DEGs and DAVID resource. Deconvolution of the DEGs between NGT and PD subjects at fasting was performed using CIBERSORT and genes involved in regulatory T-cell (Treg) function were further analyzed for biological significance. Glucose load specifically altered the expression of 112 genes in NGT and 356 genes in PD subjects. Biological significance analysis revealed transient up-regulation of innate and adaptive immune response related genes following oral glucose load in NGT individuals, which was not observed in PD subjects. Instead, in the PD group, glucose load led to an increase in the expression of pro-atherogenic and anti-angiogenic genes. Comparison of gene expression at fasting state in PD versus NGT revealed 21,707 differentially expressed genes. Biological significance analysis of the immune function related genes between these two groups (at fasting) revealed higher gene expression of members of the TLR signaling, MHC class II molecules, and T-cell receptor, chemotaxis and adhesion pathways in PD subjects. Expression of interferon-γ (IFN-γ) and TNFα was higher and that of type-1 interferons and TGF-β was lower at fasting state in PD subjects compared to NGT. Additionally, expression of multiple proteasome subunits and protein arginine methyl transferase genes (PRMTs) were higher and that of Treg specific genes was significantly distinct at fasting in PD subjects compared to NGT. Prediabetes uncovers constitutive TLR activation, enhanced IFN-γ signaling, and Treg dysfunction at fasting along with altered gene expression response to oral glucose load.

Association of TNF rs668920841 and INRA111 polymorphisms with caprine brucellosis: A case-control study of candidate genes involved in innate immunity

Caprine brucellosis is an infectious, contagious zoonotic disease caused by Brucella melitensis. Multiple factors, including host genetics, can influence the outcome of the exposure to Brucella; and it is expected that genetic variants that affect the host innate immune response could have a key role in Brucella infection and pathogenesis. In this study, we evaluated if polymorphisms in innate immunity-related genes are associated with results of Brucella infection in goats. Nine polymorphisms within interferon gamma (IFNG), tumor necrosis factor (TNF), MyD88 innate immune signal transduction adaptor (MYD88), interleukin 10 (IL10) and IL-10 receptor subunit alpha (IL10RA) genes and two molecular markers (BMS2753 and INRA111) were resolved by PCR-capillary electrophoresis in samples from 81 seronegative and 61 seropositive goats for brucellosis. A heterozygous genotype at INRA111, a microsatellite near the VRK serine/threonine kinase 2 (VRK2) gene, was associated with absence of Brucella-specific antibodies in goats naturally exposed to the pathogen (P = .004). Conversely, variants in the TNF gene (rs668920841) and near the IFN gamma receptor 1 (IFNGR1) gene (microsatellite BMS2753) were significantly associated with presence of Brucella-specific antibodies at allelic (P = .042 and P = .046) and genotypic level (P = .012 and P = .041, respectively). Moreover, an in silico analysis predicted a functional role of the insertion-deletion polymorphism rs668920841 on the transcriptional regulation of the caprine TNF gene. Altogether, these results contribute to the identification of genetic factors that have a putative effect on the resistance / susceptibility phenotype of goats to Brucella infection.

Transcriptome analysis reveals molecular strategies in gills and heart of large yellow croaker (Larimichthys crocea) under hypoxia stress

The gills and heart are two major targets of hypoxia in fish. However, the molecular responses in fish gills and heart to hypoxia challenge remain unclear. Here, RNA-Seq technology was used to study the gene expression profiles in gills and heart of large yellow croaker (Larimichthys crocea) at 6, 24, and 48 h after hypoxia stress. A total of 1,546 and 2,746 differentially expressed genes (DEGs) were identified in gills and heart, respectively. Expression changes of nine genes in each tissue were further validated by the qPCR. Based on KEGG and Gene ontology enrichments, we found that various innate immunity-related genes, such as complement components (C1qs, C2, C3, C6, and C7), chemokines (CCL3, CCL17, CCL19, CCL25, and CXCL8_L3), chemokine receptors (CCR9, CXCR1, and CXCR3), and nitric oxide synthase (NOS), were significantly down-regulated in gills and/or heart, suggesting that innate immune processes mediated by these genes may be inhibited by hypoxia. The genes involved in both glycolysis pathway (LDHA) and tricarboxylic acid cycle (IDH2 and OGDH) were up-regulated in gills and heart of hypoxic large yellow croakers, possibly because gill and heart tissues need enough energy to accelerate gas exchange and blood circulation. Hypoxia also affected the ion transport in gills of large yellow croaker, through down-regulating the expression levels of numerous classical ion transporters, including HVCN1, SLC20A2, SLC4A4, RHBG, RHCG, and SCN4A, suggesting an energy conservation strategy to hypoxia stress. All these results indicate that the immune processes, glycolytic pathways, and ion transport were significantly altered in gills and/or heart of large yellow croaker under hypoxia, possibly contributing to maintain cellular energy balance during hypoxia. Our data, therefore, afford new information to understand the tissue-specific molecular responses of bony fish to hypoxia stress.

Identification of Reference Genes in Chicken Intraepithelial Lymphocyte Natural Killer Cells Infected with Very-virulent Infectious Bursal Disease Virus

Due to the limitations in the range of antibodies recognising avian viruses, quantitative real-time PCR (RT-qPCR) is still the most widely used method to evaluate the expression of immunologically related genes in avian viruses. The objective of this study was to identify suitable reference genes for mRNA expression analysis in chicken intraepithelial lymphocyte natural killer (IEL-NK) cells after infection with very-virulent infectious bursal disease virus (vvIBDV). Fifteen potential reference genes were selected based on the references available. The coefficient of variation percentage (CV%) and average count of these 15 genes were determined by NanoString technology for control and infected samples. The M and V values for shortlisted reference genes (ACTB, GAPDH, HMBS, HPRT1, SDHA, TUBB1 and YWHAZ) were calculated using geNorm and NormFinder. GAPDH, YWHAZ and HMBS were the most stably expressed genes. The expression levels of three innate immune response related target genes, CASP8, IL22 and TLR3, agreed in the NanoString and RNA sequencing (RNA-Seq) results using one or two reference genes for normalisation (not HMBS). In conclusion, GAPDH and YWHAZ could be used as reference genes for the normalisation of chicken IEL-NK cell gene responses to infection with vvIBDV.

Hepcidin-Expressing Fish Eggs as A Novel Food Supplement to Modulate Immunity against Pathogenic Infection in Zebrafish (Danio rerio)

Hepcidin antimicrobial peptides are difficult to produce in prokaryotic expression systems due to their complex structure and antimicrobial activity. Although synthetic hepcidin provides an alternative to solve this issue, its high cost limits its practical application in various industries. The present study used zebrafish eggs as bioreactors to produce convict cichlid (Amatitlania nigrofasciata) hepcidin (AN-hepc) using the oocyte-specific zona pellucida (zp3) promoter. The expression plasmid pT2-ZP3-AN-hepc-ZP3-EGFP, using EGFP as a reporter of AN-hepc expression, was designed to establish the transgenic line Tg(ZP3:AN-hepc:ZP3:EGFP) for the expression of AN-hepc. The AN-hepc peptide was produced successfully in fertilized eggs, as evidenced by RT-PCR and Western blotting. The AN-hepc-expressing eggs exhibited antimicrobial activity against a variety of aquatic pathogens and antibiotic-resistant pathogens, suggesting that the AN-hepc expressed in fish eggs was bioactive. The immunomodulatory effects of AN-hepc-expressing fertilized eggs on zebrafish innate immunity were evaluated by determining the expression of indicator genes after feeding with AN-hepc-expressing fertilized eggs for two months. Zebrafish supplementation with AN-hepc-expressing fertilized eggs significantly increased the expression of innate immunity-related genes, including IL-1β, IL-6, IL-15, TNF-α, NF-κb, complement C3b, lysozyme and TLR-4a. The zebrafish administered AN-hepc-expressing eggs exhibited higher cumulative survival than fish supplemented with wild-type and control eggs after infection with Aeromonas hydrophila and Streptococcus iniae. In conclusion, the present results showed that supplementation with AN-hepc-expressing fish eggs enhanced zebrafish innate immunity against pathogenic infections, suggesting that fertilized eggs containing AN-hepc have the potential to be developed as a food supplement for improving health status in aquaculture.