Changes In Immune Gene Expression Research Articles

Biomarker Development for Cancer Immuno-oncology/Immunotherapy: Simultaneous Digital Counting of Nucleic Acids and Proteins at 800-plex

Abstract The ability to measure changes to the DNA, RNA, and protein is crucial to understanding both tumor and immune responses to therapy. NanoString® has pioneered 3D Biology™ – the ability to measure any combination of DNA, RNA, and protein simultaneously using a single detector from a small clinical sample – in order to characterize these complex interactions. Analytes of interest are labeled with fluorescent nucleic acid barcode (via direct hybridization for nucleic acids or antibody conjugated barcodes for protein) and detected via digital counting. This enables quantification of up to 800 analytes from a small sample (50,000 cells for DNA/RNA/protein, or 1–2 5μm FFPE slides for RNA alone). The cancer immunology profiling panel measures 770 mRNAs (unique signatures for 24 infiltrating immune cell types plus extensive immune-signaling pathways) plus 30 key IO proteins. We present two examples of 3D Biology in action. First, a targeted BRAFV600E inhibitor induces expression changes of immune genes which can be detected at both the RNA and protein levels. Second, in work from ongoing collaborations with the Cancer Immunotherapy Trials Network, clinical samples from patients treated with either mono- or combination immunotherapy can be stratified by gene expression. We also show that gene signatures predict presence of immune cell populations with a high degree of concordance to flow cytometry and immunohistochemistry measurements. Multiplexed RNA biomarkers have achieved success in predicting patient response to therapy, e.g. NanoString signatures predict response to pembrolizumab (anti-PD-1). Measuring DNA, RNA, and proteins in unison greatly expands the power of biomarker signatures to characterize responses to immunotherapy.

Impact of the blood meal on humoral immunity and microbiota in the gut of female Culicoides sonorensis.

Although Culicoides sonorensis is an important vector of orbiviruses causing significant disease in domestic and wild ruminants in the USA, little is known about factors contributing to midge vector competence. In other vectors such as mosquitoes, interactions among the humoral immune response, microbiota, and ingested pathogens within the vector gut directly impact pathogen survival and therefore vectoring potential. We recently described components of the humoral immune response in the reference transcriptome for adult female C. sonorensis and analysed their temporal expression profiles across several dietary states (unfed, blood, or sugar fed). Blood feeding altered the transcription of several humoral immune components of the Immune deficiency (Imd), dual‑oxidase (DUOX), and Janus Kinase and Signal Transducer and Activator of Transcription (JAK/STAT) pathways. Genes for immune effectors, such as antimicrobial peptides, were in particular highly induced. Since blood feeding also stimulated proliferation and diversification of bacterial populations colonising the gut of female midges, we infer that changes in immune gene expression were a result of fluctuations in gut microbiota. Thus, diet can indirectly (via microbiota) impact gut immune status and therefore should be carefully considered in subsequent studies assessing vector competence in biting midges.

Molecular profile of cochlear immunity in the resident cells of the organ of Corti.

BackgroundThe cochlea is the sensory organ of hearing. In the cochlea, the organ of Corti houses sensory cells that are susceptible to pathological insults. While the organ of Corti lacks immune cells, it does have the capacity for immune activity. We hypothesized that resident cells in the organ of Corti were responsible for the stress-induced immune response of the organ of Corti. This study profiled the molecular composition of the immune system in the organ of Corti and examined the immune response of non-immune epithelial cells to acoustic overstimulation.MethodsUsing high-throughput RNA-sequencing and qRT-PCR arrays, we identified immune- and inflammation-related genes in both the cochlear sensory epithelium and the organ of Corti. Using bioinformatics analyses, we cataloged the immune genes expressed. We then examined the response of these genes to acoustic overstimulation and determined how changes in immune gene expression were related to sensory cell damage.ResultsThe RNA-sequencing analysis reveals robust expression of immune-related genes in the cochlear sensory epithelium. The qRT-PCR array analysis confirms that many of these genes are constitutively expressed in the resident cells of the organ of Corti. Bioinformatics analyses reveal that the genes expressed are linked to the Toll-like receptor signaling pathway. We demonstrate that expression of Toll-like receptor signaling genes is predominantly from the supporting cells in the organ of Corti cells. Importantly, our data demonstrate that these Toll-like receptor pathway genes are able to respond to acoustic trauma and that their expression changes are associated with sensory cell damage.ConclusionThe cochlear resident cells in the organ of Corti have immune capacity and participate in the cochlear immune response to acoustic overstimulation.

Expression analysis of immune response genes in fish epithelial cells following ranavirus infection

Ranaviruses (family Iridoviridae) are a growing threat to fish and amphibian populations worldwide. The immune response to ranavirus infection has been studied in amphibians, but little is known about the responses elicited in piscine hosts. In this study, the immune response and apoptosis induced by ranaviruses were investigated in fish epithelial cells. Epithelioma papulosum cyprini (EPC) cells were infected with four different viral isolates: epizootic haematopoietic necrosis virus (EHNV), frog virus 3 (FV3), European catfish virus (ECV) and doctor fish virus (DFV). Quantitative real-time PCR (qPCR) assays were developed to measure the mRNA expression of immune response genes during ranavirus infection. The target genes included tumour necrosis factor α (TNF-α), interleukin-1β (IL-1β), β2-microglobulin (β2M), interleukin-10 (IL-10) and transforming growth factor β (TGF-β). All ranaviruses elicited changes in immune gene expression. EHNV and FV3 caused a strong pro-inflammatory response with an increase in the expression of both IL-1β and TNF-α, whereas ECV and DFV evoked transient up-regulation of regulatory cytokine TGF-β. Additionally, all viral isolates induced increased β2M expression as well as apoptosis in the EPC cells. Our results indicate that epithelial cells can serve as an in vitro model for studying the mechanisms of immune response in the piscine host in the first stages of ranavirus infection.

Modulation of rainbow trout (Oncorhynchus mykiss) intestinal immune gene expression following bacterial challenge

The mucosal immune systems of fishes are still poorly understood, and defined models for studying natural host–pathogen interactions are lacking. The objective of this study was to evaluate different challenge models and pathogens to examine the magnitude of change in intestinal immune gene expression. Rainbow trout were exposed by immersion to Yersinia ruckeri or by intraperitoneal injection with Flavobacterium psychrophilum. At 3, 9, or 10 days post-challenge, pathogen load was quantified by plate count and intestinal tissue was removed and immune gene expression measured by real-time PCR. In general, the magnitude of infection was correlated with change in immune gene transcript abundance. We found that messages for the innate immune molecules, SAA, IL-8, INF-γ and TNF-α, as well as the message for IgM, were up-regulated in intestinal tissue in both challenge paradigms. A >250-fold increase was observed in SAA and 20-fold increase of IL-8 gene transcript abundance occurred on day 10 following challenge with F. psychrophilum. Within individual fish, there was a positive correlation between bacteria load in the spleen and the increase of immune gene message between 3 and 10 days post-infection. These findings demonstrate that measurable changes in immune gene expression occur in the intestine of rainbow trout following bath challenge with Y. ruckeri or injection challenge with F. psychrophilum.

Gene expression analyses of immune responses in Atlantic salmon during early stages of infection by salmon louse (Lepeophtheirus salmonis) revealed bi-phasic responses coinciding with the copepod-chalimus transition

BackgroundThe salmon louse (Lepeophtheirus salmonis Krøyer), an ectoparasitic copepod with a complex life cycle causes significant losses in salmon aquaculture. Pesticide treatments against the parasite raise environmental concerns and their efficacy is gradually decreasing. Improvement of fish resistance to lice, through biological control methods, needs better understanding of the protective mechanisms. We used a 21 k oligonucleotide microarray and RT-qPCR to examine the time-course of immune gene expression changes in salmon skin, spleen, and head kidney during the first 15 days after challenge, which encompassed the copepod and chalimus stages of lice development.ResultsLarge scale and highly complex transcriptome responses were found already one day after infection (dpi). Many genes showed bi-phasic expression profiles with abrupt changes between 5 and 10 dpi (the copepod-chalimus transitions); the greatest fluctuations (up- and down-regulation) were seen in a large group of secretory splenic proteases with unknown roles. Rapid sensing was witnessed with induction of genes involved in innate immunity including lectins and enzymes of eicosanoid metabolism in skin and acute phase proteins in spleen. Transient (1-5 dpi) increase of T-cell receptor alpha, CD4-1, and possible regulators of lymphocyte differentiation suggested recruitment of T-cells of unidentified lineage to the skin. After 5 dpi the magnitude of transcriptomic responses decreased markedly in skin. Up-regulation of matrix metalloproteinases in all studied organs suggested establishment of a chronic inflammatory status. Up-regulation of putative lymphocyte G0/G1 switch proteins in spleen at 5 dpi, immunoglobulins at 15 dpi; and increase of IgM and IgT transcripts in skin indicated an onset of adaptive humoral immune responses, whereas MHCI appeared to be down-regulated.ConclusionsAtlantic salmon develops rapid local and systemic reactions to L. salmonis, which, however, do not result in substantial level of protection. The dramatic changes observed after 5 dpi can be associated with metamorphosis of copepod, immune modulation by the parasite, or transition from innate to adaptive immune responses.

Parallel declines in cognition, motivation, and locomotion in aging mice: Association with immune gene upregulation in the medial prefrontal cortex

Aging in humans is associated with parallel changes in cognition, motivation, and motoric performance. Based on the human aging literature, we hypothesized that this constellation of age-related changes is mediated by the medial prefrontal cortex and that it would be observed in aging mice. Toward this end, we performed detailed assessments of cognition, motivation, and motoric behavior in aging mice. We assessed behavioral and cognitive performance in C57Bl/6 mice aged 6, 18, and 24 months, and followed this with microarray analysis of tissue from the medial prefrontal cortex and analysis of serum cytokine levels. Multivariate modeling of these data suggested that the age-related changes in cognition, motivation, motor performance, and prefrontal immune gene expression were highly correlated. Peripheral cytokine levels were also correlated with these variables, but less strongly than measures of prefrontal immune gene upregulation. To determine whether the observed immune gene expression changes were due to prefrontal microglial cells, we isolated CD11b-positive cells from the prefrontal cortex and subject them to next-generation RNA sequencing. Many of the immune changes present in whole medial prefrontal cortex were enriched in this cell population. These data suggest that, as in humans, cognition, motivation, and motoric performance in the mouse change together with age and are strongly associated with CNS immune gene upregulation.

Gene expression responses to restricted feeding and extracted soybean meal in Atlantic salmon (Salmo salar L.)

We assessed effects of feed restriction and inclusion of 200 g kg−1 extracted soybean meal in the diet on gene expression in Atlantic salmon using a cDNA microarray (SFA2.0) and real-time qPCR. The trial lasted for 54 days. Restricted feeding and soybean inclusion reduced the thermal growth coefficient by respectively 51% and 22% compared with fish fed with the fishmeal-based control diet to satiation. Soybean decreased distal intestinal expression of lysosomal (cathepsins C, D, L, Y and Z) and extracellular proteases while genes involved in responses to cellular stress were up-regulated. Expression changes of immune genes suggested both pro- and anti-inflammatory regulation. The hepatic responses to soybean and restricted feeding were highly similar, which could be because of negative effects of soybean meal on digestion and nutrient absorption. We observed up-regulation of ribosomal proteins and down-regulation of genes involved in lipid and steroid metabolism. Of note, growth reduction in both study groups was associated with coordinated down-regulation of genes involved in oxidative and cellular stress responses, metabolism of xenobiotics and protein degradation. High expression of stress-related genes in salmon fed with the control diet suggests that maximum growth rates can be associated with health problems.

Expression of immune-related genes in rohu Labeo rohita (Hamilton) by experimental freshwater lice Argulus siamensis (Wilson) infection

The crustacean ectoparasite, Argulus poses one of the major threats to carp culture due to absence of any suitable control measure. The study was undertaken to determine the expression of immune-related genes in three major immunocompetent organs viz., kidney, skin and liver of rohu (Labeo rohita) during experimental freshwater lice Argulus siamensis infection. Results showed that the expression of TLR 22-like, lysozyme G and β2-microglobulin genes in kidney was significantly (P≤0.05) down-regulated in lice-infected fish. On the other hand, no significant difference (P>0.05) in CXCa, lysozyme C, TNFα and complement component 3 (C3) expression was found between uninfected control and different degrees of lice infected fish. In the skin, the expression of TLR 22-like and TNFα genes were significantly up-regulated whereas that of C3 was significantly (P≤0.05) down-regulated in lice-infected fish with respect to control fish. The expression of CXCa, lysozyme C and transferrin was not detected in the skin samples of fish. In the liver, the expression of CXCa, lysozyme G, β2-microglobulin and transferrin was significantly (P≤0.05) up-regulated in lice-infected fish with respect to control fish whereas expression of C3 was significantly (P≤0.05) down-regulated in lice-infected fish. The expression of TLR 22-like, lysozyme C, TNFα genes was not detected in the liver samples of fish. This study indicates that majority of the genes showed down-regulation in kidney tissue whereas up-regulation in liver and skin tissues except C3 in Argulus-infected fish. We show that infection with this parasite irrespective of intensity can also result in immune gene expression changes in tissues situated away from the site of parasite attachment and feeding. The information obtained here could be useful towards understanding the susceptibility of rohu to argulosis and mechanisms involved in protection of rohu to ectoparasitic infections, which is causing immense economic losses to freshwater aquaculture sector.

Comparative innate immune gene expression in chicken intestine after in vivo challenge with commensal (Campylobacter) and pathogenic (Salmonella) bacteria

Campylobacter jejuni is a commensal bacterium in chickens and a major cause of food poisoning in humans. It is unknown whether the intestinal immune response has a role in establishing the commensal state. The innate immune responses of 4 week‐old broiler chickens to C. jejuni challenge (108 bacteria) were studied and compared with an avian pathogen, Salmonella enterica. Challenged and control birds were sacrificed 6 hours post‐challenge, RNA was extracted and cDNA synthesised. Selected genes involved in pathogen detection and innate immune response to bacterial infection were profiled in the caecum by quantitative real‐time PCR.A significant increase in the expression of TLR21 was observed after challenge with both types of bacteria (P < 0.05) compared to control birds. TLR4 expression was increased 4‐fold after C. jejuni challenge only (P = 0.02). IL6 was increased in response to both commensal and pathogenic challenges (P < 0.02). In contrast, IL8 mRNA expression was decreased 4‐fold in response to C. jejuni but increased 5‐fold to S. enterica challenge (P = 0). Antimicrobial peptides were differentially expressed in both models. Specifically, AvBD6 was increased by 24‐fold in response to S. enterica infection (P = 0).Results indicate that C. jejuni induces early innate immune gene expression changes, some similar to and others distinct from pathogenic challengeFunding: FIRM, Ireland

Changes in immune gene expression and resistance to bacterial infection in lobster ( Homarus gammarus) post-larval stage VI following acute or chronic exposure to immune stimulating compounds

Real-time PCR was used to measure changes in transcript abundance of genes encoding important immune proteins, namely prophenoloxidase ( proPO gene), β-1,3-glucan binding protein ( βGBP gene) and a 12.2 kDa antimicrobial peptide ( amp gene) in post-larval stage VI (PLVI) juveniles of the European lobster, Homarus gammarus. Gene expression was studied in both healthy PLVI and following single or repeat exposure to a range of compounds claimed to induce immune reactivity. A single acute (3-h) exposure to any of the tested stimulants did not produce a significant increase in expression of either the proPO or βGBP genes, measured 6 h after stimulation. However, there were a small sub-group of positive responders, identified mainly from βGBP expression, within the experimental groups stimulated with either a β-1,3-glucan or an alginate. There was also no significant increase in the expression of any of the three genes tested 24 h after repeated weekly (3-h) exposures to a either the β-1,3-glucan or the alginate over the longer (36-day) period. The results do show that amp is expressed at an extremely high level compared to proPO or βGBP in healthy animals and a significant correlation was found between the expression of proPO and both βGBP and amp, irrespective of whether or not the larvae were stimulated. None of the immune stimulated compounds improved survival of PLVI challenged with the opportunistic pathogen, Listonella anguillarum, or the lobster pathogen, Aerococcus viridans var. homari. Thus, we found no evidence to support recent claims that immunity and disease resistance can be primed or promoted within a given population of crustaceans or that these animals exhibit functional immune memory to some soluble immune elicitors.

Increasing Burn Size Dramatically Influences the Immune Response to Burn Injury

Increasing total body surface area percentage (%TBSA) following burn injury leads to an increased risk of immune dysfunction, infection, and sepsis, yet the cellular mechanism responsible for this relationship is poorly understood. In this study, we hypothesize that increasing %TBSA burn area correlates with predictable changes in immune gene expression. To test this hypothesis, we analyzed the expression of several gene families important in inflammation and innate immunity in human peripheral blood monocytes following burn injury. We enrolled patients with 9% to 59% TBSA burn injury. We drew an initial peripheral blood sample with 24 hours of burn injury and a follow up blood sample within 10 days of injury. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll extraction, cells lysed and mRNA extracted. Total RNA was reverse transcribed and subsequent cDNA used for real-time quantitative PCR analysis of NF-kappaB signaling genes as well as newly described and novel intracellular pathogen receptors genes (CLR). Gene expression was determined by log fold-increase or decrease over baseline. Data compared using chi-square, with significance defined as p<0.05.