Innate Immune Gene Expression Research Articles

METTL3 regulates viral m6A RNA modification and host cell innate immune responses during SARS-CoV-2 infection.

It is urgent and important to understand the relationship of the widespread severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) with host immune response and study the underlining molecular mechanism. N6-methylation of adenosine (m6A) in RNA regulates many physiological and disease processes. Here, we investigate m6A modification of the SARS-CoV-2 gene in regulating the host cell innate immune response. Our data show that the SARS-CoV-2 virus has m6A modifications that are enriched in the 3′ end of the viral genome. We find that depletion of the host cell m6A methyltransferase METTL3 decreases m6A levels in SARS-CoV-2 and host genes, and m6A reduction in viral RNA increases RIG-I binding and subsequently enhances the downstream innate immune signaling pathway and inflammatory gene expression. METTL3 expression is reduced and inflammatory genes are induced in patients with severe coronavirus disease 2019 (COVID-19). These findings will aid in the understanding of COVID-19 pathogenesis and the design of future studies regulating innate immunity for COVID-19 treatment.

Merkel Cell Polyomavirus Infection Induces an Antiviral Innate Immune Response in Human Dermal Fibroblasts.

Merkel cell polyomavirus (MCPyV) infects most of the human population asymptomatically, but in rare cases it leads to a highly aggressive skin cancer called Merkel cell carcinoma (MCC). MCC incidence is much higher in aging and immunocompromised populations. The epidemiology of MCC suggests that dysbiosis between the host immune response and the MCPyV infectious cycle could contribute to the development of MCPyV-associated MCC. Insufficient restriction of MCPyV by normal cellular processes, for example, could promote the incidental oncogenic MCPyV integration events and/or entry into the original cell of MCC. Progress toward understanding MCPyV biology has been hindered by its narrow cellular tropism. Our discovery that primary human dermal fibroblasts (HDFs) support MCPyV infection has made it possible to closely model cellular responses to different stages of the infectious cycle. The present study reveals that the onset of MCPyV replication and early gene expression induces an inflammatory cytokine and interferon-stimulated gene (ISG) response. The cGAS-STING pathway, in coordination with NF-κB, mediates induction of this innate immune gene expression program. Further, silencing of cGAS or NF-κB pathway factors led to elevated MCPyV replication. We also discovered that the PYHIN protein IFI16 localizes to MCPyV replication centers but does not contribute to the induction of ISGs. Instead, IFI16 upregulates inflammatory cytokines in response to MCPyV infection by an alternative mechanism. The work described herein establishes a foundation for exploring how changes to the skin microenvironment induced by aging or immunodeficiency might alter the fate of MCPyV and its host cell to encourage carcinogenesis. IMPORTANCE MCC has a high rate of mortality and an increasing incidence. Immune-checkpoint therapies have improved the prognosis of patients with metastatic MCC. Still, a significant proportion of the patients fail to respond to immune-checkpoint therapies or have a medical need for iatrogenic immune-suppression. A greater understanding of MCPyV biology could inform targeted therapies for MCPyV-associated MCC. Moreover, cellular events preceding MCC oncogenesis remain largely unknown. The present study aims to explore how MCPyV interfaces with innate immunity during its infectious cycle. We describe how MCPyV replication and/or transcription elicit an innate immune response via cGAS-STING, NF-κB, and IFI16. We also explore the effects of this response on MCPyV replication. Our findings illustrate how healthy cellular conditions may allow low-level infection that evades immune destruction until highly active replication is restricted by host responses. Conversely, pathological conditions could result in unbridled MCPyV replication that licenses MCC tumorigenesis.

Impacts of pineapple peel powder on growth performance, innate immunity, disease resistance, and relative immune gene expression of Nile tilapia, Oreochromis niloticus

An 8-week growth trial was conducted to examine the efficacy of pineapple peel powder (PAPP) on growth rate and immunity of Nile tilapia, O. niloticus. Three hundred Nile tilapia (20.91 ± 0.11 g) were fed five diets containing different levels of PAPP at 0, 10, 20, 30 and 40 g kg−1 PAPP, respectively. After four and eight weeks of the feeding trial, growth rates, and immune responses were tested. A challenge test using Streptococcus agalactiae and relative immune gene expression were performed after eight weeks of PAPP feeding. It was found that skin mucus and serum lysozyme, skin mucus and serum peroxidase, alternative complement, phagocytosis, and respiratory burst activities were significantly increased with the addition of PAPP. The maximum (P ≤ 0.05) innate immune values were noted in fish fed 10 g kg−1 PAPP. Similarly, the up-regulation of IL1, IL8, and LBP gene expressions were also detected in fish fed PAPP diets, with the maximum value was found in 10 g kg−1 PAPP fed fish. The relative percentage of survival (RPS) of Oreochromis niloticus after the challenge test were (56.00%, 72.00%, 60.00%, and 44.00%) for the 5, 10, 20 and 40 g kg−1 PAPP diets, respectively. Fish fed the 10 g kg−1 PAPP supplemented diet achieved the highest (P < 0.05) survival rate against S. agalactiae. Growth and feed efficiency were outstandingly (P < 0.05) enhanced in the PAPP groups. In conclusion, PAPP can be potentially used as a feed additive in Nile tilapia culture under Biofloc system.

Novel function of Nanos2 in expression of innate immunity genes and its probable roles in maintenance of pluripotency state.

Objective(s):Cell-based therapeutic approaches have witnessed significant developments during the last decade especially after approval of MSCs based treatment of graft versus host disease. Several cell-based approaches have shown immunomodulatory behavior during regeneration following the unknown cascade of events but the exact mechanisms are yet to be defined. Clinical applications of cell-based drugs are hampered all over the world because of incomplete understanding of molecular mechanisms requiring the application of mechanistic approaches to solving the mystery. Current work has given us the idea that Nanos2 enhances the cellular pluripotency characteristics while down-regulating the innate immunity genes, simultaneously.Materials and Methods:The immunomodulatory behavior of cells was studied against cells carrying the ectopic expression of Nanos2 in comparison with Stella and Oct4 individually and simultaneously using SON vector (Stella, Nanos2 and Oct4). Results:It was observed that overexpression of Nanos2 leads to down-regulation of Interferon-Stimulated Genes (ISGs)-mRNAs such as Ifitm1, lsg15, Oas2, and Oas12. Nanos2 overexpressing MEF cells have shown restrictive inflammatory effects when cells were treated with inflammatory stimuli such as LPS and Poly (I:C). Conclusion:From our recent findings in line with many others, it can be concluded that Nanos2 acts as a coin with two sides, regulating pluripotency and immunity together which enhances resistance against inflammatory stimuli. Nanos2 could be a potential candidate as a molecular drug for management of inflammation and immunomodulation but it requires a comprehensive comparative expression analysis of innate immunity genes in vitro and in vivo.

Effects of mannan oligosaccharide and synbiotic supplementation on growth performance and immune response of Gilthead Sea Bream ( Sparus aurata ) before and after thermal stress

In this study, the effects of mannan oligosaccharides (MOS) and a commercial synbiotic (Biomin) supplemented diets on growth and survival performance of gilthead sea bream after eight weeks culture at 27ºC and then ten days at 34ºC were evaluated. Before heat stress, the Biomin supplemented diet significantly increased the number of red blood cells (RBC) and eosinophils of the fish. The MOS supplemented diet increased the haematocrit, haemoglobin, lymphocytes, white blood cells (WBC) and monocytes significantly. Total immunoglobulin was significantly higher in both supplemented diets in contrast to the control group. After thermal stress, experimental diets increased the monocyte counts, lysozyme, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of the fish significantly, also the lowest concentration of cortisol was observed in the fish fed with Biomin supplemented diet. Gene expression profile showed a significantly higher expression of innate immune genes (il-1β, ghr-1, tnf-α and igf-1) in fish fed with Biomin and MOS supplemented diets. However, after thermal challenge, inflammatory response was significantly lower the fish fed with the supplemented diets. According to our results, MOS and Biomin supplemented diets enhance the immune health status of the gilthead sea bream during thermal stress.

Discovery of RSV-Induced BRD4 Protein Interactions Using Native Immunoprecipitation and Parallel Accumulation-Serial Fragmentation (PASEF) Mass Spectrometry.

Respiratory Syncytial Virus (RSV) causes severe inflammation and airway pathology in children and the elderly by infecting the epithelial cells of the upper and lower respiratory tract. RSV replication is sensed by intracellular pattern recognition receptors upstream of the IRF and NF-κB transcription factors. These proteins coordinate an innate inflammatory response via Bromodomain-containing protein 4 (BRD4), a protein that functions as a scaffold for unknown transcriptional regulators. To better understand the pleiotropic regulatory function of BRD4, we examine the BRD4 interactome and identify how RSV infection dynamically alters it. To accomplish these goals, we leverage native immunoprecipitation and Parallel Accumulation—Serial Fragmentation (PASEF) mass spectrometry to examine BRD4 complexes isolated from human alveolar epithelial cells in the absence or presence of RSV infection. In addition, we explore the role of BRD4’s acetyl-lysine binding bromodomains in mediating these interactions by using a highly selective competitive bromodomain inhibitor. We identify 101 proteins that are significantly enriched in the BRD4 complex and are responsive to both RSV-infection and BRD4 inhibition. These proteins are highly enriched in transcription factors and transcriptional coactivators. Among them, we identify members of the AP1 transcription factor complex, a complex important in innate signaling and cell stress responses. We independently confirm the BRD4/AP1 interaction in primary human small airway epithelial cells. We conclude that BRD4 recruits multiple transcription factors during RSV infection in a manner dependent on acetyl-lysine binding domain interactions. This data suggests that BRD4 recruits transcription factors to target its RNA processing complex to regulate gene expression in innate immunity and inflammation.

Abstract PO048: Loss of intra-tumoral RIG-I immune signaling is a potential microbiome-mediated mechanism underlying poor anti-tumor immunity and immunotherapy resistance in mucosal melanoma

Abstract Cutaneous melanoma (CM) has one of the highest response rates across cancers to immune checkpoint blockade (ICB) therapies, but mucosal melanoma (MM) responds poorly to ICB. MM patients have very few effective treatments options, making it critical to understand and improve anti-tumor immunity in these patients. MM is a rare subtype of melanoma, accounting for approximately 1% of all malignant melanoma diagnoses. MM occurs on mucosal surfaces such as nasal, vaginal, and anorectal, which provide a highly unique tumor microenvironment compared to CM arising in the skin. We compared the tumor immune microenvironment between CM and MM by multiplex immunofluorescence and discovered MM has decreased frequency of infiltrating immune cells. Using RNA sequencing, we investigated intra-tumoral differences in global gene expression between CM and MM that might explain the lack of immune cell infiltration and ICB response in MM. Compared to CM, MM had decreased expression of numerous innate immune genes in the RIG-I pathogen-sensing pathway. The RIG-I pathway produces an inflammatory response to destroy foreign pathogens, but is also important for ICB sensitivity and anti-tumor immunity in CM. Studies in CM demonstrate the RIG-I pathway enhances antigen presentation and promotes a favorable immune cell profile in the tumor microenvironment. Interestingly, the tumor microbiome, which is vastly different between CM and MM, has been shown to regulate the RIG-I pathway. We measured total fungal and bacterial load in MM and CM primary tumors and found increased levels of both bacteria and fungi in MM compared to CM. This raises the possibility that microbiome-mediated suppression of the RIG-I pathway underlies poor anti-tumor immunity and ICB response in MM, and that re-activation of this pathway may be a novel therapeutic strategy for overcoming ICB resistance in MM patients. Direct RIG-I agonists are still in clinical development, but FDA-approved hypomethylating agents, particularly 5’ aza-deoxycytidine (decitabine), can activate the RIG-I pathway and improve anti-tumor immunity by increasing the expression of RIG-I pathway activating genes, endogenous RNA retroviruses, tumor antigens, and natural killer (NK) cell ligands. We treated MM cell lines with decitabine and observed strong induction of RIG-I pathway genes, NK ligands, and tumor antigens. In conclusion, we have identified the loss of intra-tumoral RIG-I signaling as a potential microbiome-mediated mechanism underlying the poor tumor immune microenvironment and ICB response in MM. Combining decitabine, which is FDA-approved and now available orally, with ICB represents a novel treatment strategy for these difficult to treat MM patients that can be rapidly translated into the clinic. Citation Format: Morgan MacBeth, Richard Tobin, Robert Van Gulick, Martin D. McCarter, William A. Robinson, Kasey L. Couts. Loss of intra-tumoral RIG-I immune signaling is a potential microbiome-mediated mechanism underlying poor anti-tumor immunity and immunotherapy resistance in mucosal melanoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO048.

Innate immune-gene expression during experimental amyloodiniosis in European seabass (Dicentrarchus labrax)

The ectoparasite protozoan Amyloodinium ocellatum (AO) is the causative agent of amyloodiniosis in European seabass (ESB, Dicentrarchus labrax). There is a lack of information about basic molecular immune response mechanisms of ESB during AO infestation. Therefore, to compare gene expression between experimental AO-infested ESB tissues and uninfested ESB tissues (gills and head kidney) RNA-seq was adopted. The RNA-seq revealed multiple differentially expressed genes (DEG), namely 679 upregulated genes and 360 downregulated genes in the gills, and 206 upregulated genes and 170 downregulated genes in head kidney. In gills, genes related to the immune system (perforin, CC1) and protein binding were upregulated. Several genes involved in IFN related pathways were upregulated in the head kidney. Subsequently, to validate the DEG from amyloodiniosis, 26 ESB (mean weight 14 g) per tank in triplicate were bath challenged for 2 h with AO (3.5 × 106/tank; 70 dinospores/mL) under controlled conditions (26−28 °C and 34‰ salinity). As a control group (non-infested), 26 ESB per tank in triplicate were also used. Changes in the expression of innate immune genes in gills and head kidney at 2, 3, 5, 7 and 23 dpi were analysed using real-time PCR. The results indicated that the expression of cytokines (CC1, IL-8) and antimicrobial peptide (Hep) were strongly stimulated and reached a peak at 5 dpi in the early infestation stage, followed by a gradual reduction in the recovery stage (23 dpi). Noticeably, the immunoglobulin (IgM) expression was higher at 23 dpi compared to 7 dpi. Furthermore, in-situ hybridization showed positive signals of CC1 mRNA in AO infested gills compared to the control group. Altogether, chemokines were involved in the immune process under AO infestation and this evidence allows a better understanding of the immune response in European seabass during amyloodiniosis.

Modulation of growth performance, gut microflora, non-specific immunity and gene expression of proinflammatory cytokines in shabout (Tor grypus) upon dietary prebiotic supplementation

In this study, the effect of dietary supplementation of mannan oligosaccharide (MOS) + β-glucan (Immunogen®) was investigated on growth performance, body composition, gut microflora, innate immune responses and gene expression of some proinflammatory cytokines in shabout (Tor grypus). Shabout fingerlings (35 ± 1.2 g) were fed with basal diet (control) or basal diet supplemented with Immunogen® at 0.5, 1 and 1.5% of feed for 90 days. According to the results, growth parameters were significantly improved in fish fed with prebiotic (1 and 1.5%) for 90 days (p < 0.05). The carcass protein content was significantly higher in fish nourished by prebiotic at 1.5% of feed for 90 days compared to fish received the basal diet (p < 0.05). Feeding with various levels of Immunogen® resulted in the significant promotion of the population of intestinal Lactobacillus spp. in the prebiotic-treated groups relative to the control group (p < 0.05). Serum total globulin was significantly higher in all prebiotic groups relative to the control group at day 60. Serum bactericidal and lysozyme activities were significantly (p < 0.05) elevated after feeding with dietary prebiotic at all intervals (days 30, 60 and 90). However, the highest serum bactericidal activities were recorded in fish fed with Immunogen® at 1.5% of diet (p < 0.05). The transcription levels of interleukin 1 beta (IL-1β), interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-α) were significantly increased in the head kidney of fish treated with dietary prebiotic at all intervals. The results show that dietary supplementation with Immunogen®, particularly at the level of 1.5%, can positively alter growth parameters, carcass protein, intestinal microflora and immune responses of shabout.

Hypercapnia selectively modulates LPS-induced changes in innate immune and DNA replication-related gene transcription in the macrophage.

Hypercapnia, the elevation of CO2 in blood and tissues, commonly occurs in severe acute and chronic respiratory diseases and is associated with increased risk of death. Recent studies have shown that hypercapnia inhibits expression of select innate immune genes and suppresses host defence against bacterial and viral pneumonia in mice. In the current study, we evaluated the effect of culture under conditions of hypercapnia (20% CO2) versus normocapnia (5% CO2), both with normoxia, on global gene transcription in human THP-1 and mouse RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS). We found that hypercapnia selectively downregulated transcription of LPS-induced genes associated with innate immunity, antiviral response, type I interferon signalling, cytokine signalling and other inflammatory pathways in both human and mouse macrophages. Simultaneously, hypercapnia increased expression of LPS-downregulated genes associated with mitosis, DNA replication and DNA repair. These CO2-induced changes in macrophage gene expression help explain hypercapnic suppression of antibacterial and antiviral host defence in mice and reveal a mechanism that may underlie, at least in part, the high mortality of patients with severe lung disease and hypercapnia.

Integral assessment of markers of the local infectious and inflammatory process in women with preterm birth in multiple pregnancies

Hypothesis/Aims of study. Premature birth in multiple pregnancies remains an important object of research, since it is the main factor in poor perinatal outcomes, and their heterogeneous mechanisms determine the ineffectiveness of prediction and prevention methods. In the pathogenesis of premature birth, as is known, one of the leading links is inflammation caused by infections of the lower genital tract (40%). In multiple pregnancies, which in most cases occur as a result of assisted reproductive technology treatment (70%) and are mainly accompanied by complications, pregravid preparation and antenatal observation include more careful control and correction of local infectious and inflammatory processes. In this regard, the persisting high rate of premature birth in multiple pregnancies (about 54%) demonstrates the ambiguity of the opinion about the suppressive role of the infectious factor in the induction of premature birth and determines the need for studying its contribution to multifactorial genesis. The aim of this study was to conduct an integral assessment of markers of the local infectious and inflammatory process in women with PB in multiple pregnancies.&#x0D; Study design, materials and methods. We performed a comprehensive study of the bacteriological composition of the lower genital tract discharge using microscopic, bacteriological, and molecular biological methods (Femoflor 16 test) and assessed the local inflammatory status (ImmunoQuantex test) in 30 pregnant women with dichorionic diamniotic twins. The main group consisted of women with premature birth (n = 13), the control group comprising those with term birth (n = 16), while patients with induced premature birth (n = 2) were not included in the comparative analysis.&#x0D; Results. This study was the first to determine the features of vaginal microbiocenosis and the local immune status in women with premature birth in multiple pregnancies. In general, the study cohort had a low inflammatory status and normal or intermediate types of vaginal biotope. The most common disruptions (24.1%) were vaginal dysbiosis, expressed in a small amount of Lactobacillius spp., and non-specific vaginitis associated with Mycoplasma hominis. The local immune status of women with premature birth was characterized by a relative decrease in the mRNA expression of such innate immunity genes as IL1B, TNF, TLR4, and GATA3. An integrated assessment of the studied parameters based on the data obtained allowed us to build a mathematical model for predicting premature birth with the probability of 87.6%.&#x0D; Conclusion. The integral assessment of infectious and inflammatory markers is important from the standpoint of not only their possible identification as predictors, but also a general understanding of the genesis of premature birth.

CF monocyte-derived macrophages have an attenuated response to extracellular vesicles secreted by airway epithelial cells.

Mutations in CFTR alter macrophage responses, for example, by reducing their ability to phagocytose and kill bacteria. Altered macrophage responses may facilitate bacterial infection and inflammation in the lungs, contributing to morbidity and mortality in cystic fibrosis (CF). Extracellular vesicles (EVs) are secreted by multiple cell types in the lungs and participate in the host immune response to bacterial infection, but the effect of EVs secreted by CF airway epithelial cells (AEC) on CF macrophages is unknown. This report examines the effect of EVs secreted by primary AEC on monocyte-derived macrophages (MDM) and contrasts responses of CF and wild type (WT) MDM. We found that EVs generally increase pro-inflammatory cytokine secretion and expression of innate immune genes in MDM, especially when EVs are derived from AEC exposed to Pseudomonas aeruginosa and that this effect is attenuated in CF MDM. Specifically, EVs secreted by P. aeruginosa exposed AEC (EV-PA) induced immune response genes and increased secretion of proinflammatory cytokines, chemoattractants, and chemokines involved in tissue repair by WT MDM, but these effects were less robust in CF MDM. We attribute attenuated responses by CF MDM to differences between CF and WT macrophages because EVs secreted by CF AEC or WT AEC elicited similar responses in CF MDM. Our findings demonstrate the importance of AEC EVs in macrophage responses and show that the Phe508del mutation in CFTR attenuates the innate immune response of MDM to EVs.

Uropathogenic Escherichia coli Virulence Factor α-Hemolysin Reduces Histone Acetylation to Inhibit Expression of Proinflammatory Cytokine Genes.

Urinary tract infections are common and costly diseases affecting millions of people. Uropathogenic Escherichia coli (UPEC) is a primary cause of these infections and has developed multiple strategies to avoid the host immune response. Here, we dissected the molecular mechanisms underpinning UPEC inhibition of inflammatory cytokine in vitro and in vivo. We found that UPEC infection simulates nuclear factor-κB activation but does not result in transcription of cytokine genes. Instead, UPEC-mediated suppression of the metabolic enzyme ATP citrate lyase results in decreased acetyl-CoA levels, leading to reduced H3K9 histone acetylation in the promotor region of CXCL8. These effects were dependent on the UPEC virulence factor α-hemolysin and were reversed by exogenous acetate. In a murine cystitis model, prior acetate supplementation rapidly resolved UPEC-elicited immune responses and improved tissue recovery. Thus, upon infection, UPEC rearranges host cell metabolism to induce chromatin remodeling processes that subvert expression of host innate immune response genes.

Evaluation of indirect diagnostic tests and PBMC expression of innate immune genes in subclinical mastitis in dairy cows

The aim of this study was to estimate the prevalence of subclinical mastitis (SCM), its innate immunity and to compare the efficacy of indirect diagnostic tests [California mastitis test (CMT), differential electrical conductivity (DEC), absolute EC, enzyme based color detection method (Tanucheck kit) and brothymol blue strip (BTB) tests] to milk somatic cell count (SCC) method using 200×103 cells/ml as a cut off value in Deoni (N=27) and HF crossbred (N=32) cows. Cumulative prevalence of SCM was 38% and 63% in Deoni and HF crossbred cows, respectively on milk SCC basis (N=215). Breed, udder health status and its interaction had significant effect on SCC level. The overall sensitivity and specificity of above indirect tests were 38 and 99%; 52 and 72%; 52 and 69%; 76 and 31%, and 1 and 100%, respectively. mRNA expression of serum amyloid A (SAA), IL-1β and TNF- α genes were significantly up-regulated while, TNF-α gene was significantly down-regulated in PBMC of SCM affected HF crossbred and Deoni cows, respectively. It is concluded that, DEC is more suitable indirect method to detect SCM and mRNA expression of SAA and TNF-α was strongly related to SCM in HF crossbred and Deoni cows, respectively.

G-quadruplex-forming nucleic acids interact with splicing factor 3B subunit 2 and suppress innate immune gene expression.

G‐quadruplex (G4), a non‐canonical higher‐order structure formed by guanine‐rich nucleic acid sequences, affects various genetic events in cis, including replication, transcription and translation. Whereas up‐regulation of innate immune/interferon‐stimulated genes (ISGs) is implicated in cancer progression, G4‐forming oligonucleotides that mimic telomeric repeat‐containing RNA suppress ISG induction in three‐dimensional (3D) culture of cancer cells. However, it is unclear how G4 suppresses ISG expression in trans. In this study, we found that G4 binding to splicing factor 3B subunit 2 (SF3B2) down‐regulated STAT1 phosphorylation and ISG expression in 3D‐cultured cancer cells. Liquid chromatography‐tandem mass spectrometry analysis identified SF3B2 as a G4‐binding protein. Either G4‐forming oligonucleotides or SF3B2 knockdown suppressed ISG induction, whereas Phen‐DC3, a G4‐stabilizing compound, reversed the inhibitory effect of G4‐forming oligonucleotides on ISG induction. Phen‐DC3 inhibited SF3B2 binding to G4 in vitro. SF3B2‐mediated ISG induction appeared to occur independently of RNA splicing because SF3B2 knockdown did not affect pre‐mRNA splicing under the experimental conditions, and pharmacological inhibition of splicing by pladienolide B did not repress ISG induction. These observations suggest that G4 disrupts the ability of SF3B2 to induce ISGs in cancer. We propose a new mode for gene regulation, which employs G4 as an inhibitory trans‐element.

Why is it worth remembering the lung microbiome in ICU patients?

In recent years commensal microorganisms are not just "passive occupants", but important element of homeostasis. There are numerous reports documenting the composition and role of the gut, skin or vagina microbiome but the role of commensal orga-nisms living in the lungs is relatively unknown. Pulmonary microbiome impact on the immune response of the host organism and may indicate new therapeutic directions. Lung microbiome, by modulating the expression of innate immunity genes, causes an increase in the concentration of interleukin (IL)-5, IL-10, interferon γ and C-C motif chemokine ligand 11, affects the toll-like receptor-4-dependent response of pulmonary macrophages and modulate the production of antibacterial peptides contained in the mucus. It is documented that disorders of the lung microbiome contribute to asthma or chronic obstructive pulmonary disease. However it is known that pulmonary dysbiosis also occurs in critically ill patients. It is possible, therefore, that microbiota-targeted therapy may constitute the future therapeutic direction in ICU.

The Dbl-1/TGF-β Signaling Pathway Regulates Pathogen-Specific Innate Immune Responses in &lt;i&gt;Caenorhabditis Elegans&lt;/i&gt;

Generating specific, robust innate immune responses to different bacteria is vital for animal survival. Here, we address the role of transforming growth factor β (TGF- β) DBL-1 in regulating signature host defense responses in Caenorhabditis elegans to human opportunistic Gram-negative and Gram-positive pathogens. Canonical DBL-1 signaling is required to suppress avoidance behavior in response to Gram-negative, but not Gram-positive bacteria. SMA-4/co-Smad acts independently of other DBL-1 core signaling components to suppress avoidance to Gram-positive bacteria, and sma-4 expression is specifically induced in response to Gram-positive bacteria. DBL-1 signaling differentially regulates expression of target innate immunity genes depending on the pathogen. Animals activate DBL-1 pathway activity in response to Gram-negative bacteria and repress it in response to Gram-positive bacteria, demonstrating bacteria-responsive regulation of DBL-1 signaling. These findings highlight a central role for TGF-β and identify a novel role for a co-Smad in tailoring a suite of bacteria-specific host defenses.

Expression of innate immune response genes in upper airway samples of SARS-CoV-2 infected patients: A preliminary study.

Background & objectives:Upper respiratory mucosa is the entryway for SARS-CoV-2, and cells at this site form the first line of resistance against the pathogens. Innate immune response at this point is crucial for managing the replication and early stage symptoms of virus infection. This study was aimed to evaluate the expression of pattern recognition receptors and cytokines in upper airway cells of SARS-CoV-2 infected patients.Methods:Forty seven nasopharyngeal swab (NPS) specimens from 25 SARS-CoV-2 infected patients and 22 SARS-CoV-2 negative individuals were investigated for expression of toll-like receptors (TLRs), melanoma differentiation-associated protein 5 (MDA5), NOD-like receptors family pyrin domain containing 3 (NLRP3), angiotensin-converting enzyme 2 (ACE2), interleukin (IL) - 6, tumour necrosis factor alpha (TNFα) and type-1 interferons (IFNs) by real time reverse transcription polymerase chain reaction.Results:Increased expression of TLR2, MDA5 and ACE2 was detected in SARS-CoV-2 infected patients in comparison with controls. MDA5 expression was significantly higher in asymptomatic and mildly symptomatic SARS-CoV-2 positive patients than the patients with severe symptoms. The asymptomatic group showed significant induction of type 1 IFNs than the symptomatic group. Non-specific induction of TLR7 could be observed in nasopharyngeal (NP) cells irrespective of symptoms and SARS-CoV-2 positivity.Interpretation & conclusions:The findings suggest that increased MDA5 in NP cells of asymptomatic SARS-CoV-2 positive patients may subsequently induce type 1 IFNs to protect the individuals from further clinical severity of SARS-CoV-2 infection. A future prospective study in NPS of larger cohort needs to be performed to confirm our findings.

Single-cell analysis of arthritogenic alphavirus-infected human synovial fibroblasts links low abundance of viral RNA to induction of innate immunity and arthralgia-associated gene expression

ABSTRACT Infection by (re-)emerging RNA arboviruses including Chikungunya virus (CHIKV) and Mayaro virus primarily cause acute febrile disease and transient polyarthralgia. However, in a significant subset of infected individuals, debilitating arthralgia persists for weeks over months up to years. The underlying immunopathogenesis of chronification of arthralgia upon primary RNA-viral infection remains unclear. Here, we analysed cell-intrinsic responses to ex vivo arthritogenic alphaviral infection of primary human synovial fibroblasts isolated from knee joints, one the most affected joint types during acute and chronic CHIKV disease. Synovial fibroblasts were susceptible and permissive to alphaviral infection. Base-line and exogenously added type I interferon (IFN) partially and potently restricted infection, respectively. RNA-seq revealed a CHIKV infection-induced transcriptional profile that comprised upregulation of expression of several hundred IFN-stimulated and arthralgia-mediating genes. Single-cell virus-inclusive RNA-seq uncovered a fine-tuned switch from induction to repression of cell-intrinsic immune responses depending on the abundance of viral RNA in an individual cell. Specifically, responses were most pronounced in cells displaying low-to-intermediate amounts of viral RNA and absence of virus-encoded, fluorescent reporter protein expression, arguing for efficient counteraction of innate immunity in cells expressing viral antagonists at sufficient quantities. In summary, cell-intrinsic sensing of viral RNA that potentially persists or replicates at low levels in synovial fibroblasts and other target cell types in vivo may contribute to the chronic arthralgia induced by alphaviral infections. Our findings might advance our understanding of the immunopathophysiology of long-term pathogenesis of RNA-viral infections.

Survival Following Traumatic Brain Injury in Drosophila Is Increased by Heterozygosity for a Mutation of the NF-κB Innate Immune Response Transcription Factor Relish.

Traumatic brain injury (TBI) pathologies are caused by primary and secondary injuries. Primary injuries result from physical damage to the brain, and secondary injuries arise from cellular responses to primary injuries. A characteristic cellular response is sustained activation of inflammatory pathways commonly mediated by nuclear factor-κB (NF-κB) transcription factors. Using a Drosophila melanogaster TBI model, we previously found that the main proximal transcriptional response to primary injuries is triggered by activation of Toll and Imd innate immune response pathways that engage NF-κB factors Dif and Relish (Rel), respectively. Here, we found by mass spectrometry that Rel protein level increased in fly heads at 4-8 hr after TBI. To investigate the necessity of Rel for secondary injuries, we generated a null allele, Reldel , by CRISPR/Cas9 editing. When heterozygous but not homozygous, the Reldel mutation reduced mortality at 24 hr after TBI and increased the lifespan of injured flies. Additionally, the effect of heterozygosity for Reldel on mortality was modulated by genetic background and diet. To identify genes that facilitate effects of Reldel on TBI outcomes, we compared genome-wide mRNA expression profiles of uninjured and injured +/+, +/Reldel , and Reldel /Reldel flies at 4 hr following TBI. Only a few genes changed expression more than twofold in +/Reldel flies relative to +/+ and Reldel /Reldel flies, and they were not canonical innate immune response genes. Therefore, Rel is necessary for TBI-induced secondary injuries but in complex ways involving Rel gene dose, genetic background, diet, and possibly small changes in expression of innate immune response genes.