Innate Immune Response Genes Research Articles

Possible involvement of Toll-like receptor 8-positive monocytes/macrophages in the pathogenesis of Sjögren's disease.

Sjögren's disease (SjD) is an autoimmune disease marked by lymphocytic infiltration of salivary and lacrimal glands, leading to glandular dysfunction, where CD4-positive helper T (Th) cells and their cytokines are crucial in the pathogenesis. Recent studies have demonstrated that Toll-like receptors (TLRs), particularly those recognizing immune complexes containing DNA and RNA, contribute to Th cell activation in various autoimmune diseases. This study explores the expression and function of these TLRs in SjD. DNA microarray analysis of salivary gland tissue from six SjD patients and real-time PCR (n = 32) was used to identify overexpressed TLRs. Single-cell RNA sequencing (scRNA-seq) was performed using tissue lesions and integrated with published scRNA-seq data from tissues and peripheral blood mononuclear cells to examine gene expression in macrophages and monocytes. Finally, multi-color immunofluorescence staining was conducted to confirm TLR8 expression and function in SjD lesions (n = 19). DNA microarray analysis revealed the up-regulation of TLR8, along with other TLRs and innate immune response genes in SjD. Real-time PCR showed significant up-regulation of TLR7 and TLR8. TLR8 up-regulated in both analyses. In scRNA-seq analysis, the TLR8-expressing cluster comprised macrophages and monocytes, which also produced T cell activation genes like CD86. TLR8-positive macrophages infiltrated inflammatory sites and frequently expressed CD86 in quantitative imaging approaches. These results suggest that infiltrating monocytes and macrophages may produce cytokines and chemokines through TLR8 stimulation, potentially enhancing B7 molecule expression, promoting the adaptive immune response, and contributing to SjD pathogenesis.

SARS-CoV-2 Nsp15 antagonizes the cGAS-STING-mediated antiviral innate immune responses.

Coronavirus (CoV) Nsp15 is a viral endoribonuclease (EndoU) with a preference for uridine residues. CoV Nsp15 is an innate immune antagonist which prevents dsRNA sensor recognition and stress granule formation by targeting viral and host RNAs. SARS-CoV-2 restricts and delays the host antiviral innate immune responses through multiple viral proteins, but the role of SARS-CoV-2 Nsp15 in innate immune evasion is not completely understood. Here, we generate an EndoU activity knockout rSARS-CoV-2Nsp15-H234A to elucidate the biological functions of Nsp15. Relative to wild-type rSARS-CoV-2, replication of rSARS-CoV-2Nsp15-H234A was significantly decreased in IFN-responsive A549-ACE2 cells but not in its STAT1 knockout counterpart. Transcriptomic analysis revealed upregulation of innate immune response genes in cells infected with rSARS-CoV-2Nsp15-H234A relative to wild-type virus, including cGAS-STING, cytosolic DNA sensors activated by both DNA and RNA viruses. Treatment with STING inhibitors H-151 and SN-011 rescued the attenuated phenotype of rSARS-CoV-2Nsp15-H234A. SARS-CoV-2 Nsp15 inhibited cGAS-STING-mediated IFN-β promoter and NF-κB reporter activity, as well as facilitated the replication of EV-D68 and NDV by diminishing cGAS and STING expression and downstream innate immune responses. Notably, the decline in cGAS and STING was also apparent during SARS-CoV-2 infection. The EndoU activity was essential for SARS-CoV-2 Nsp15-mediated cGAS and STING downregulation, but not all HCoV Nsp15 share the consistent substrate selectivity. In the hamster model, rSARS-CoV-2Nsp15-H234A replicated to lower titers in the nasal turbinates and lungs and induced higher innate immune responses. Collectively, our findings exhibit that SARS-CoV-2 Nsp15 serves as a host innate immune antagonist by targeting host cGAS and STING.

The Human Pathogen Mycobacterium tuberculosis and the Fish Pathogen Mycobacterium marinum Trigger a Core Set of Late Innate Immune Response Genes in Zebrafish Larvae.

Zebrafish is a natural host of various Mycobacterium species and a surrogate model organism for tuberculosis research. Mycobacterium marinum is evolutionarily one of the closest non-tuberculous species related to M. tuberculosis and shares the majority of virulence genes. Although zebrafish is not a natural host of the human pathogen, we have previously demonstrated successful robotic infection of zebrafish embryos with M. tuberculosis and performed drug treatment of the infected larvae. In the present study, we examined for how long M. tuberculosis can be propagated in zebrafish larvae and tested a time series of infected larvae to study the transcriptional response via Illumina RNA deep sequencing (RNAseq). Bacterial aggregates carrying fluorescently labeled M. tuberculosis could be detected up to 9 days post-infection. The infected larvae showed a clear and specific transcriptional immune response with a high similarity to the inflammatory response of zebrafish larvae infected with the surrogate species M. marinum. We conclude that M. tuberculosis can be propagated in zebrafish larvae for at least one week after infection and provide further evidence that M. marinum is a good surrogate model for M. tuberculosis. The generated extensive transcriptome data sets will be of great use to add translational value to zebrafish as a model for infection of tuberculosis using the M. marinum infection system. In addition, we identify new marker genes such as dusp8 and CD180 that are induced by M. tuberculosis infection in zebrafish and in human macrophages at later stages of infection that can be further investigated.

Impact of in ovo administration of xylo- and mannooligosaccharides on broiler chicken gut health

The intestinal mucosa creates a connection between the gut microbiota and the host. This study aimed to modify the gut microbiota of broiler chickens by in ovo stimulation with xylo-oligosaccharide (XOS) and manno-oligosaccharide (MOS) prebiotics and to determine the changes occurring in specific gut segments. Three hundred incubated eggs of Ross 308 broiler chickens on the 12th d of incubation were injected with: saline (control), xylotriose (XOS3), xylotetrose (XOS4), mannotriose (MOS3) or mannotetrose (MOS4). Tissue and digesta samples were collected post-mortem from 8 randomly selected individuals from each group, on d 42 after hatching. Gene expression analysis in the cecum and ileum was performed by RT-qPCR for a panel of genes: innate immune response genes (IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17, IL-1β, IFNγ, IFNβ), nutrient sensing and nutrient transport genes (FFAR2, FFAR4, GLUT1, GLUT2, GLUT5), host defence peptides (AvBD1, CATHL2), and barrier function genes (MUC6, CLDN1, TJAP). The relative abundance of bacteria was determined by qPCR for individual bacteria (Akkermansia muciniphilla, Bifidobacterium spp., Clostridium difficile, Escherichia coli, Faecalibacterium prausnitzii, and Lactobacillus spp.). Stimulation with prebiotics caused changes in the abundance of bacteria especially Lactobacillus spp. and Bifidobacterium spp. in the cecum. The abundance of both genera increased in each study group compared to the control group. The highest abundance of Bifidobacterium spp. in the ileum was found in the MOS3 group compared to the control group. There were changes in the XOS4 and MOS3 groups in the expression of: FFAR4, GLUT1, AvBD1, CATHL2, IL-2, IL-12, and IL-17 in the caecum. In conclusion, in ovo administration of prebiotics increased intestinal colonization by bacteria. The prebiotics influenced gene expression levels via changes in the gut microbiota.

Transcriptome analysis reveals mechanisms of metabolic detoxification and immune responses following farnesyl acetate treatment in Metisa plana

Metisa plana is a widespread insect pest infesting oil palm plantations in Malaysia. Farnesyl acetate (FA), a juvenile hormone analogue, has been reported to exert in vitro and in vivo insecticidal activity against other insect pests. However, the insecticidal mechanism of FA on M. plana remains unclear. Therefore, this study aims to elucidate responsive genes in M. plana in response to FA treatment. The RNA-sequencing reads of FA-treated M. plana were de novo-assembled with existing raw reads from non-treated third instar larvae, and 55,807 transcripts were functionally annotated to multiple protein databases. Several insecticide detoxification-related genes were differentially regulated among the 321 differentially expressed transcripts. Cytochrome P450 monooxygenase, carboxylesterase, and ATP-binding cassette protein were upregulated, while peptidoglycan recognition protein was downregulated. Innate immune response genes, such as glutathione S-transferases, acetylcholinesterase, and heat shock protein, were also identified in the transcriptome. The findings signify that changes occurred in the insect’s receptor and signaling, metabolic detoxification of insecticides, and immune responses upon FA treatment on M. plana. This valuable information on FA toxicity may be used to formulate more effective biorational insecticides for better M. plana pest management strategies in oil palm plantations.

Involvement of mitochondrial fatty acid β-oxidation in the antiviral innate immune response in head kidney macrophages of large yellow croaker (Larimichthys crocea)

As a vital pathway for cellular energy production, mitochondrial fatty acid β-oxidation (FAO) is essential in regulating immune responses to bacterial pathogens and maintaining intracellular homeostasis in vertebrates. However, the specific role of FAO in antiviral innate immune response in macrophages remains insufficiently understood. In this study, virus infection simulated by poly(I:C) inhibited FAO, as indicated by the reduced expression of FAO-related genes and proteins in the head kidney of large yellow croaker, with similar results observed in poly(I:C)-stimulated macrophages. Then, inhibition of FAO by supplementary mildronate in vivo and etomoxir treatment in vitro revealed varying increases in the mRNA expression of antiviral innate immune response genes after stimulated by poly(I:C) in the head kidney and macrophages. Notably, etomoxir significantly facilitated the transcriptional up-regulation of the IFNh promoter by IRF3. Moreover, inhibiting FAO by knockdown of cpt1b promoted antiviral innate immune response triggered by poly(I:C) in macrophages. Conversely, activating FAO through overexpression of cpt1b or cpt2 significantly reduced the mRNA levels of antiviral response genes in macrophages stimulated by poly(I:C). Unlike etomoxir, cpt1b overexpression inhibited the transcriptional up-regulation of the IFNh promoter by IRF3. Furthermore, in vivo dietary palm oil feeding and in vitro exposure to palmitic acid inhibited the antiviral innate immune response triggered by poly(I:C) in the head kidney and macrophages, respectively. These effects were partly associated with FAO activation, as evidenced by etomoxir. In summary, this study elucidates FAO's critical role in regulating antiviral innate immune response in head kidney macrophages. These findings not only deepen insights into the interaction between metabolic remodeling and host immune responses, but also offer valuable guidance for developing nutritional strategies to improve antiviral immunity in aquaculture.

Sustained mucosal colonization and fecal metabolic dysfunction by Bacteroides associates with fecal microbial transplant failure in ulcerative colitis patients

Fecal microbial transplantation (FMT) offers promise for treating ulcerative colitis (UC), though the mechanisms underlying treatment failure are unknown. This study harnessed longitudinally collected colonic biopsies (n = 38) and fecal samples (n = 179) from 19 adults with mild-to-moderate UC undergoing serial FMT in which antimicrobial pre-treatment and delivery mode (capsules versus enema) were assessed for clinical response (≥ 3 points decrease from the pre-treatment Mayo score). Colonic biopsies underwent dual RNA-Seq; fecal samples underwent parallel 16S rRNA and shotgun metagenomic sequencing as well as untargeted metabolomic analyses. Pre-FMT, the colonic mucosa of non-responsive (NR) patients harbored an increased burden of bacteria, including Bacteroides, that expressed more antimicrobial resistance genes compared to responsive (R) patients. NR patients also exhibited muted mucosal expression of innate immune antimicrobial response genes. Post-FMT, NR and R fecal microbiomes and metabolomes exhibited significant divergence. NR metabolomes had elevated concentrations of immunostimulatory compounds including sphingomyelins, lysophospholipids and taurine. NR fecal microbiomes were enriched for Bacteroides fragilis and Bacteroides salyersiae strains that encoded genes capable of taurine production. These findings suggest that both effective mucosal microbial clearance and reintroduction of bacteria that reshape luminal metabolism associate with FMT success and that persistent mucosal and fecal colonization by antimicrobial-resistant Bacteroides species may contribute to FMT failure.

HIV-1 Vpr combats the PU.1-driven antiviral response in primary human macrophages

HIV-1 Vpr promotes efficient spread of HIV-1 from macrophages to T cells by transcriptionally downmodulating restriction factors that target HIV-1 Envelope protein (Env). Here we find that Vpr induces broad transcriptomic changes by targeting PU.1, a transcription factor necessary for expression of host innate immune response genes, including those that target Env. Consistent with this, we find silencing PU.1 in infected macrophages lacking Vpr rescues Env. Vpr downmodulates PU.1 through a proteasomal degradation pathway that depends on physical interactions with PU.1 and DCAF1, a component of the Cul4A E3 ubiquitin ligase. The capacity for Vpr to target PU.1 is highly conserved across primate lentiviruses. In addition to impacting infected cells, we find that Vpr suppresses expression of innate immune response genes in uninfected bystander cells, and that virion-associated Vpr can degrade PU.1. Together, we demonstrate Vpr counteracts PU.1 in macrophages to blunt antiviral immune responses and promote viral spread.

MiR-33a-5p in stored red blood cells regulates genes of innate immune response and promotes inflammation.

Blood transfusion is a common therapeutic procedure in hospitalized patients. Red blood cell (RBC) units undergo various biochemical and morphological changes during storage (storage lesion). miRNAs have been studied intensively regarding cellular metabolic processes, but the effect of miRNAs on blood storage is not well defined. We performed bioinformatics analysis on the public data set of miRNA expression of RBC based on R language, and performed the Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis on the target genes of differentially expressed miRNA. The expression of miRNA differential genes in blood samples stored at different times was verified by qRT-PCR. Next, we used ELISA and qRT-PCR to verify the expression of IL-1β, IL-6, IL-12 and TNF-α in blood at day 1 and day 42. In addition, in vitro, we transfected macrophages with overexpressed miRNA, and the effects of overexpressed miRNA on macrophage polarization and the release of inflammatory factors were verified by flow cytometry and qRT-PCR and ELISA. This study combined bioinformatics analysis and experiments to discover the differentially expressed miRNAs in long-term stored blood. The results showed that compared to fresh blood samples, the inflammatory factors were significantly doubled by ELISA, as well as the higher mRNA expression at 42 day. Experimentally verified that miR-33a-5p promoted the M1 type macrophage polarization and increased the release of related inflammatory factors through PPARα/ACC2/AMPK/CPT-1a axis regulation. This study elucidates a potential mechanism of inflammatory factor accumulation in long-term stored blood, providing a theoretical basis and a potential target to prevent transfusion-related adverse reactions.

The AHR repressor limits expression of antimicrobial genes but not AHR-dependent genes in intestinal eosinophils.

Intestinal eosinophils express the aryl hydrocarbon receptor (AHR), an environmental sensor and ligand-activated transcription factor that responds to dietary or environmental ligands. AHR regulates tissue adaptation, survival, adhesion, and immune functions in intestinal eosinophils. The AHR repressor (AHRR) is itself induced by AHR and believed to limit AHR activity in a negative feedback loop. We analyzed gene expression in intestinal eosinophils from wild-type and AHRR knockout mice and found that AHRR did not suppress most AHR-dependent genes. Instead, AHRR limited the expression of a distinct small set of genes involved in the innate immune response. These included S100 proteins, antimicrobial proteins, and alpha-defensins. Using bone marrow-derived eosinophils, we found that AHRR knockout eosinophils released more reactive oxygen species upon stimulation. This work shows that the paradigm of AHRR as a repressor of AHR transcriptional activity does not apply to intestinal eosinophils. Rather, AHRR limits the expression of innate immune response and antimicrobial genes, possibly to maintain an anti-inflammatory phenotype in eosinophils when exposed to microbial signals in the intestinal environment.

Methylation patterns associated with C-reactive protein in racially and ethnically diverse populations

ABSTRACT Systemic low-grade inflammation is a feature of chronic disease. C-reactive protein (CRP) is a common biomarker of inflammation and used as an indicator of disease risk; however, the role of inflammation in disease is not completely understood. Methylation is an epigenetic modification in the DNA which plays a pivotal role in gene expression. In this study we evaluated differential DNA methylation patterns associated with blood CRP level to elucidate biological pathways and genetic regulatory mechanisms to improve the understanding of chronic inflammation. The racially and ethnically diverse participants in this study were included as 50% White, 41% Black or African American, 7% Hispanic or Latino/a, and 2% Native Hawaiian, Asian American, American Indian, or Alaska Native (total n = 13,433) individuals. We replicated 113 CpG sites from 87 unique loci, of which five were novel (CADM3, NALCN, NLRC5, ZNF792, and cg03282312), across a discovery set of 1,150 CpG sites associated with CRP level (p < 1.2E–7). The downstream pathways affected by DNA methylation included the identification of IFI16 and IRF7 CpG-gene transcript pairs which contributed to the innate immune response gene enrichment pathway along with NLRC5, NOD2, and AIM2. Gene enrichment analysis also identified the nuclear factor-kappaB transcription pathway. Using two-sample Mendelian randomization (MR) we inferred methylation at three CpG sites as causal for CRP levels using both White and Black or African American MR instrument variables. Overall, we identified novel CpG sites and gene transcripts that could be valuable in understanding the specific cellular processes and pathogenic mechanisms involved in inflammation.

Select EZH2 inhibitors enhance viral mimicry effects of DNMT inhibition through a mechanism involving NFAT:AP-1 signaling.

DNA methyltransferase inhibitor (DNMTi) efficacy in solid tumors is limited. Colon cancer cells exposed to DNMTi accumulate lysine-27 trimethylation on histone H3 (H3K27me3). We propose this Enhancer of Zeste Homolog 2 (EZH2)-dependent repressive modification limits DNMTi efficacy. Here, we show that low-dose DNMTi treatment sensitizes colon cancer cells to select EZH2 inhibitors (EZH2is). Integrative epigenomic analysis reveals that DNMTi-induced H3K27me3 accumulates at genomic regions poised with EZH2. Notably, combined EZH2i and DNMTi alters the epigenomic landscape to transcriptionally up-regulate the calcium-induced nuclear factor of activated T cells (NFAT):activating protein 1 (AP-1) signaling pathway. Blocking this pathway limits transcriptional activating effects of these drugs, including transposable element and innate immune response gene expression involved in viral defense. Analysis of primary human colon cancer specimens reveals positive correlations between DNMTi-, innate immune response-, and calcium signaling-associated transcription profiles. Collectively, we show that compensatory EZH2 activity limits DNMTi efficacy in colon cancer and link NFAT:AP-1 signaling to epigenetic therapy-induced viral mimicry.

Co-inhibition of TGF-β and PD-L1 pathways in a metastatic colorectal cancer mouse model triggers interferon responses, innate cells and T cells, alongside metabolic changes and tumor resistance

ABSTRACT Colorectal cancer (CRC) is the third most prevalent cancer worldwide with a high mortality rate (20–30%), especially due to metastasis to adjacent organs. Clinical responses to chemotherapy, radiation, targeted and immunotherapies are limited to a subset of patients making metastatic CRC (mCRC) difficult to treat. To understand the therapeutic modulation of immune response in mCRC, we have used a genetically engineered mouse model (GEMM), “KPN”, which resembles the human ‘CMS4’-like subtype. We show here that transforming growth factor (TGF-β1), secreted by KPN organoids, increases cancer cell proliferation, and inhibits splenocyte activation in vitro. TGF-β1 also inhibits activation of naive but not pre-activated T cells, suggesting differential effects on specific immune cells. In vivo, the inhibition of TGF-β inflames the KPN tumors, causing infiltration of T cells, monocytes and monocytic intermediates, while reducing neutrophils and epithelial cells. Co-inhibition of TGF-β and PD-L1 signaling further enhances cytotoxic CD8+T cells and upregulates innate immune response and interferon gene signatures. However, simultaneous upregulation of cancer-related metabolic genes correlated with limited control of tumor burden and/or progression despite combination treatment. Our study illustrates the importance of using GEMMs to predict better immunotherapies for mCRC.

Abstract TP33: Knockout of Kruppel-Like Factor 4 in Brain Endothelial Cells Accelerates Vascular and Neuronal Degeneration During Aging

As we get older, various aspects of our bodies start to decline, including brain function. The mechanisms behind the decline in brain function due to aging are still not well understood. The blood-brain barrier (BBB) is crucial for maintaining brain health and homeostasis by controlling both passive and active exchange of materials between the blood and brain. This barrier is predominantly composed of endothelial cells, which deteriorate in both aging and neurodegenerative disorders. Endothelial cells are also highly enriched in the Kruppel-like factor 4 (KLF4) transcription factor, which is reduced in expression as we age. In this study, we report that endothelial-specific KLF4 KO mice demonstrate accelerated brain aging, including accelerated BBB deterioration, neurodegeneration, and cognitive decline. Single-cell RNA sequencing from brain endothelial cells of endothelial-specific KLF4 KO mice demonstrate aberrantly high expression of adaptive and innate immune response genes, and live animal brain imaging across the lifespan reveals accelerated aging-related BBB deterioration, decreased blood flow, and impaired neurovascular coupling. Endothelial-specific KLF4 KO mice show increased neuroinflammation and neutrophil infiltration. Our data suggest that diminished endothelial cell KLF signaling with age could be a novel therapeutic target to preserve health and function of the aging brain.

Host and Microbe Blood Metagenomics Reveals Key Pathways Characterizing Critical Illness Phenotypes.

Rationale: Two molecular phenotypes of sepsis and acute respiratory distress syndrome, termed hyperinflammatory and hypoinflammatory, have been consistently identified by latent class analysis in numerous cohorts, with widely divergent clinical outcomes and differential responses to some treatments; however, the key biological differences between these phenotypes remain poorly understood.Objectives: We used host and microbe metagenomic sequencing data from blood to deepen our understanding of biological differences between latent class analysis-derived phenotypes and to assess concordance between the latent class analysis-derived phenotypes and phenotypes reported by other investigative groups (e.g., Sepsis Response Signature [SRS1-2], molecular diagnosis and risk stratification of sepsis [MARS1-4], reactive and uninflamed).Methods: We analyzed data from 113 patients with hypoinflammatory sepsis and 76 patients with hyperinflammatory sepsis enrolled in a two-hospital prospective cohort study. Molecular phenotypes had been previously assigned using latent class analysis.Measurements and Main Results: The hyperinflammatory and hypoinflammatory phenotypes of sepsis had distinct gene expression signatures, with 5,755 genes (31%) differentially expressed. The hyperinflammatory phenotype was associated with elevated expression of innate immune response genes, whereas the hypoinflammatory phenotype was associated with elevated expression of adaptive immune response genes and, notably, T cell response genes. Plasma metagenomic analysis identified differences in prevalence of bacteremia, bacterial DNA abundance, and composition between the phenotypes, with an increased presence and abundance of Enterobacteriaceae in the hyperinflammatory phenotype. Significant overlap was observed between these phenotypes and previously identified transcriptional subtypes of acute respiratory distress syndrome (reactive and uninflamed) and sepsis (SRS1-2). Analysis of data from the VANISH trial indicated that corticosteroids might have a detrimental effect in patients with the hypoinflammatory phenotype.Conclusions: The hyperinflammatory and hypoinflammatory phenotypes have distinct transcriptional and metagenomic features that could be leveraged for precision treatment strategies.

Markers and mechanisms of death in Drosophila.

Parameters correlated with age and mortality in Drosophila melanogaster include decreased negative geotaxis and centrophobism behaviors, decreased climbing and walking speed, and darkened pigments in oenocytes and eye. Cessation of egg laying predicts death within approximately 5days. Endogenous green fluorescence in eye and body increases hours prior to death. Many flies exhibit erratic movement hours before death, often leading to falls. Loss of intestinal barrier integrity (IBI) is assayed by feeding blue dye ("Smurf" phenotype), and Smurf flies typically die within 0-48h. Some studies report most flies exhibit Smurf, whereas multiple groups report most flies die without exhibiting Smurf. Transgenic reporters containing heat shock gene promoters and innate immune response gene promoters progressively increase expression with age, and partly predict remaining life span. Innate immune reporters increase with age in every fly, prior to any Smurf phenotype, in presence or absence of antibiotics. Many flies die on their side or supine (on their back) position. The data suggest three mechanisms for death of Drosophila. One is loss of IBI, as revealed by Smurf assay. The second is nervous system malfunction, leading to erratic behavior, locomotor malfunction, and falls. The aged fly is often unable to right itself after a fall to a side-ways or supine position, leading to inability to access the food and subsequent dehydration/starvation. Finally, some flies die upright without Smurf phenotype, suggesting a possible third mechanism. The frequency of these mechanisms varies between strains and culture conditions, which may affect efficacy of life span interventions.

Differences in gene expression profiles in early and late stage rhodesiense HAT individuals in Malawi.

T. b. rhodesiense is the causative agent of Rhodesian human African trypanosomiasis (r-HAT) in Malawi. Clinical presentation of r-HAT in Malawi varies between foci and differs from East African HAT clinical phenotypes. The purpose of this study was to gain more insights into the transcriptomic profiles of patients with early stage 1 and late stage 2 HAT disease in Malawi. Whole blood from individuals infected with T. b. rhodesiense was used for RNA-Seq. Control samples were from healthy trypanosome negative individuals matched on sex, age range, and disease foci. Illumina sequence FASTQ reads were aligned to the GRCh38 release 84 human genome sequence using HiSat2 and differential analysis was done in R Studio using the DESeq2 package. XGR, ExpressAnalyst and InnateDB algorithms were used for functional annotation and gene enrichment analysis of significant differentially expressed genes. RNA-seq was done on 23 r-HAT case samples and 28 healthy controls with 7 controls excluded for downstream analysis as outliers. A total of 4519 genes were significant differentially expressed (p adjusted <0.05) in individuals with early stage 1 r-HAT disease (n = 12) and 1824 genes in individuals with late stage 2 r-HAT disease (n = 11) compared to controls. Enrichment of innate immune response genes through neutrophil activation was identified in individuals with both early and late stages of the disease. Additionally, lipid metabolism genes were enriched in late stage 2 disease. We further identified uniquely upregulated genes (log2 Fold Change 1.4-2.0) in stage 1 (ZNF354C) and stage 2 (TCN1 and MAGI3) blood. Our data add to the current understanding of the human transcriptome profiles during T. b. rhodesiense infection. We further identified biological pathways and transcripts enriched than were enriched during stage 1 and stage 2 r-HAT. Lastly, we have identified transcripts which should be explored in future research whether they have potential of being used in combination with other markers for staging or r-HAT.

Differential Isoforms Signature of Splicing Factor Mutant Myeloid Neoplasia

Exclusivity of splicing factor (SF) mutations is a near-dogma in myeloid neoplasia (MN). Such exclusivity is often reflected in morphopathologic features as in the case of SF3B1 mutations associated with ringed sideroblasts. Consequences of SF mutations are hard to reconcile given the central role of RNA splicing in normal physiology and the multitude of alternative splicing changes occurring upon mutations. Historically, distinct gene isoforms play key roles in hematopoiesis and might be tumor associated. We performed global transcript level expression analysis to identify SF specific differentially expressed isoforms associated with isoform disequilibrium. Such analysis prompted to the distinction between common and exclusive isoforms possibly discerning novel mechanistic insights in the pathogenesis of SF mutant MN. Of particular importance is the discovery of novel isoforms not expressed in normal condition. Total RNA isolated from 100 whole bone marrows of MN patients with six SF mutations ( DDX41, PRPF8, SF3B1, SRSF2, U2AF1, ZRSR2) and healthy subjects was subjected to RNASeq library prep (Kapa RNAHyperPrep Kit with rRNA depletion). Transcript abundance was generated using spliced alignment to the genome by STAR followed by Salmon-basedquantification. We then applied bootstrapping to capture variation in abundance estimates since transcript-level quantification is amenable to variation, in contrast with gene level, due to the difficulty of unique mapping of short read sequences. Differential isoform testing was done using edgeR accounting for overdispersion due to isoform uncertainty. Mutant samples were selected with VAF &amp;gt;15% for better estimation of isoforms vs clonality. Differential SF isoform usage was defined in comparison to healthy controls. In total, 1681 unique transcripts in 1418 unique genes (positively/negatively dysregulated) at a threshold of .05 P-value and absolute logfold change of 2.0 were selected. Among patients with SF mutations, patients carrying sole U2AF1 had the highest number of distinct isoforms (n=189), which accounted for about 20% of isoforms in the entire cohort of U2AF1 mutant patients. DDX41 mutations had the second highest (n=161), while ZRSR2 had the lowest number of distinct isoforms(n=48). We categorized the features in groups I1-I6 according to the number of SF sharing isoforms. I1 indicates the group of patients carrying isoforms which where distinct per each SF ( Fig1). U2AF1I1 included genes previously reported to be associated with U2AF1 ( e.g., GNAS) and genes restricted to neutrophil degranulation. DDX41I1 was enriched in innate immune response gene isoforms including HLA-DQA1 and SAMHD1 and known genes predisposing to myeloid and blood disorders ( e.g. CSF1R, RPL15). SRSF2 isoform signature was mainly characterized by upregulation of INF-gamma and beta, IFN-induced OAS1 and TLR response pathways ( e.g., IRAK2). Specific isoforms were shared between SFs. SF3B1 shared 35 isoforms with PRPF8 and 8 with SRSF2, including Stathmin1, a gene expressed in high proliferative MN phenotypes. Different isoforms in the same gene were also associated with different groups. For instance, DDX5 isoform-219 producing a 65-residues transcript was shared between PRPF8 and SF3B1 (I2) while isoform-234 producing 614-residues transcript was shared between PRPF8, DDX41, and ZRSR2 mutants (I3). Beside the unique isoform signature per each SF, we imputed any differential isoform changes in myeloid genes often associated with SF mutations. Isoforms for the TET triad were analyzed and resulted in a 110 residues transcript (TET2-208) significantly downregulated (FDR &amp;lt; .05) in all SF, except SRSF2. We then focused on the discovery of exclusive isoforms. In total, 115 unique transcripts (lncRNAs, 20; protein coding, 95) were expressed in all SF mutant patients but not expressed in healthy controls. GDF15 isoform-201 encoding a 308-residues transcript was detected in SF3B1, DDX41, and ZRSR2 mutants. LncRNAs have gained attention in regulating gene expression in MN. We found 9 novel lncRNA transcripts in DDX41, SF3B1, PRPF8, U2AF1 and ZRSR2 mutants, with one being common in all of them (ENSG00000273445 chromosome 2p11.2). In sum, our study focusing solely on transcript level analysis describes the first isoform landscape of SF mutant MN and launches the idea that isoform analysis might shed light on the diversity and uniqueness of SF mutations.

A combination of burn wound injury and Pseudomonas infection elicits unique gene expression that enhances bacterial pathogenicity.

The interaction between an underlying disease process and a specific pathogen may lead to the unique expression of genes that affect bacterial pathogenesis. These genes may not be observed during infection in the absence of, or with a different underlying process or infection during the underlying process with a different pathogen. To test this hypothesis, we used Nanostring technology to compare gene transcription in a murine-burned wound infected with P. aeruginosa. The Nanostring probeset allowed the simultaneous direct comparison of immune response gene expression in both multiple host tissues and P. aeruginosa in conditions of burn alone, infection alone, and burn with infection. While RNA-Seq is used to discover novel transcripts, NanoString could be a technique to monitor specific changes in transcriptomes between samples and bypass the additional adjustments for multispecies sample processing or the need for the additional steps of alignment and assembly required for RNASeq. Using Nanostring, we identified arginine and IL-10 as important contributors to the lethal outcome of burned mice infected with P. aeruginosa. While other examples of altered gene transcription are in the literature, our study suggests that a more systematic comparison of gene expression in various underlying diseases during infection with specific bacterial pathogens may lead to the identification of unique host-pathogen interactions and result in more precise therapeutic interventions.

Innate immune responses reverse HIV cognitive disease in mice: Profile by RNAseq in the brain

Antiretroviral therapy controls immunodeficiency in people with HIV but many develop mild neurocognitive disorder. Here we investigated HIV brain disease by infecting mice with the chimeric HIV, EcoHIV, and probing changes in brain gene expression during infection and reversal with polyinosinic-polycytidylic acid (poly I:C). EcoHIV-infected C57BL/6 mice were treated with poly I:C and monitored by assay of learning in radial arm water maze, RNAseq of striatum, and QPCR of virus burden and brain transcripts. Poly I:C reversed EcoHIV-associated cognitive impairment and reduced virus burden. Major pathways downregulated by infection involved neuronal function, these transcriptional changes were normalized by poly I:C treatment. Innate immune responses were the major pathways induced in EcoHIV-infected, poly I:C treated mice. Our findings provide a framework to identify brain cell genes dysregulated by HIV infection and identify a set of innate immune response genes that can block systemic infection and its associated dysfunction in the brain.