Activation Of Innate Immune Pathways Research Articles

Carrageenan as a Potential Factor of Inflammatory Bowel Diseases.

Carrageenan is a widely used food additive and is seen as a potential candidate in the pharmaceutical industry. However, there are two faces to carrageenan that allows it to be used positively for therapeutic purposes. Carrageenan can be used to create edible films and for encapsulating drugs, and there is also interest in the use of carrageenan for food printing. Carrageenan is a naturally occurring polysaccharide gum. Depending on the type of carrageenan, it is used in regulating the composition of intestinal microflora, including the increase in the population of Bifidobacterium bacteria. On the other hand, the studies have demonstrated the harmfulness of carrageenan in animal and human models, indicating a direct link between diet and intestinal inflammatory states. Carrageenan changes the intestinal microflora, especially Akkermansia muciniphilia, degrades the mucous barrier and breaks down the mucous barrier, causing an inflammatory reaction. It directly affects epithelial cells by activating the pro-inflammatory nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) pathway. The mechanism is based on activation of the TLR4 receptor, alterations in macrophage activity, production of proinflammatory cytokines and activation of innate immune pathways. Carrageenan increases the content of Bacteroidetes bacteria, also causing a reduction in the number of short chain fatty acid (SCFA)-producing bacteria. The result is damage to the integrity of the intestinal membrane and reduction of the mucin layer. The group most exposed to the harmful effects of carrageenan are people suffering from intestinal inflammation, including Crohn disease (CD) and ulcerative colitis (UC).

Abstract 6406: Clinical and translational results of the academic ARIANES Phase 2 basket study: Longitudinal single-cell analysis of patient tumors identifies biological correlates of response to PARP inhibitors and anti-PD-L1 therapy

Abstract Background: Poly (ADP-ribose) polymerase inhibitors (PARPi) have immunomodulatory properties that may potentiate anti-PD-L1 therapy. To explore this, we developed the academic ARIANES phase 2 basket trial, which evaluated the PARPi rucaparib (R) and the anti-PD-L1 atezolizumab (A) in selected patients (pts). We performed single-cell RNA + T cell receptor sequencing (scRNA+TCR-seq) on 3 sequential tumor biopsies in a pts subset. Methods: ARIANES comprised 4 molecularly-selected DNA damage response deficient (DDRd), 3 platinum-sensitive, and 2 unselected cohorts of pts with non-small cell lung (NSCLC), prostate (CRPC), and other non-breast non-ovarian cancers. R was administered for 3 weeks, followed by R+A. Primary endpoint was overall response rate (ORR) at 12 weeks per cohort. scRNA+TCR-seq was performed on fresh biopsies collected at baseline, on R, and on R + A in the same lesion, with concomitant plasma collection. We tracked the evolution over time of tumor and microenvironment cells, differentially expressed genes and signatures within each cell population, and TCR clonal dynamics. Results: ARIANES enrolled 130 pts, including 27 DDRd CRPC and 3 DDRd NSCLC pts, on which we focus here. Best ORR by RECIST v1.1 was 22% (6/27) in CRPC pts, and mPFS: 7.9 months (95% CI 4 - 11.1) at data cut-off. ORR was 0% in NSCLC pts. We successfully profiled 3 sequential fresh biopsies in 2 BRCA2-mutant CRPC, 1 ATM-mutant, and 1 RAD54L-mutant NSCLC pts. Tumor, T and B lymphoid, myeloid, plasma, dendritic, NK, endothelial, and stromal cells were identified in all samples. Differential expression testing unveiled PARP1 upregulation in tumor cells on R. One CRPC pt with prolonged partial response displayed activation of innate immune sensing pathways (including cGAS-STING and IFN signaling) in tumor and immune cells, as well as JAK/STAT and CCL5, all increasing gradually on R and R + A; scTCR-seq revealed the expansion of specific T cell clonotypes on R + A. One ATM-mutant NSCLC pt with 6-month stable disease displayed increased cGAS/STING, JAK/STAT and CCL5 activation on R in NK cells only, and no TCR expansion. None of the above was observed in the 2 pts who progressed. Complete clinical and translational study results will be presented at the congress. Conclusion: Sequential scRNA+TCR-seq enables the dynamic assessment of tumor and immune cells on therapy. Our data confirms the potential of R to activate innate immune pathways in pt tumors, and R + A to induce T cell activation and clonal expansion. To our knowledge, this is the first study providing a longitudinal single-cell resolution analysis of the clonal evolution of tumor and immune cells in pts on PARPi + anti-PD-L1. We identify potential biological correlates of therapy response, including the activation of innate immune pathways and TCR expansion. Citation Format: Julien Vibert, Clémence Hénon, Lambros Tselikas, Baptiste Bonnet, Arnaud Pagès, Alice Bernard-Tessier, Léo Colmet-Daage, Roman Chabanon, Nicolas Dorvault, Clémence Astier, Anas Gazzah, Aurore Jeanson, Aurélien Parpaleix, Ronan Flippot, Natacha Naoun, Anna Patrikidou, Pernelle Lavaud, Giulia Baciarello, Emeline Colomba, Alina Fuerea, Laurence Albiges, Christophe Massard, Santiago Ponce, Yohann Loriot, Stéphane Champiat, Capucine Baldini, Fanny Bouquet, Nathalie Droin, Karim Fizazi, Damien Vasseur, Patricia Martin-Romano, Etienne Rouleau, Kaïssa Ouali, Sophie Postel-Vinay. Clinical and translational results of the academic ARIANES Phase 2 basket study: Longitudinal single-cell analysis of patient tumors identifies biological correlates of response to PARP inhibitors and anti-PD-L1 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6406.

G3BP1-dependent condensation of translationally inactive viral RNAs antagonizes infection.

G3BP1 is an RNA binding protein that condenses untranslating messenger RNAs into stress granules (SGs). G3BP1 is inactivated by multiple viruses and is thought to antagonize viral replication by SG-enhanced antiviral signaling. Here, we show that neither G3BP1 nor SGs generally alter the activation of innate immune pathways. Instead, we show that the RNAs encoded by West Nile virus, Zika virus, and severe acute respiratory syndrome coronavirus 2 are prone to G3BP1-dependent RNA condensation, which is enhanced by limiting translation initiation and correlates with the disruption of viral replication organelles and viral RNA replication. We show that these viruses counteract condensation of their RNA genomes by inhibiting the RNA condensing function of G3BP proteins, hijacking the RNA decondensing activity of eIF4A, and/or maintaining efficient translation. These findings argue that RNA condensation can function as an intrinsic antiviral mechanism, which explains why many viruses inactivate G3BP proteins and suggests that SGs may have arisen as a vestige of this antiviral mechanism.

Significant Variations in Double-Stranded RNA Levels in Cultured Skin Cells.

Endogenous double-stranded RNA has emerged as a potent stimulator of innate immunity. Under physiological conditions, endogenous dsRNA is maintained in the cell nucleus or the mitochondria; however, if protective mechanisms are breached, it leaches into the cytoplasm and triggers immune signaling pathways. Ectopic activation of innate immune pathways is associated with various diseases and senescence and can trigger apoptosis. Hereby, the level of cytoplasmic dsRNA is crucial. We have enriched dsRNA from two melanoma cell lines and primary dermal fibroblasts, including a competing probe, and analyzed the dsRNA transcriptome using RNA sequencing. There was a striking difference in read counts between the cell lines and the primary cells, and the effect was confirmed by northern blotting and immunocytochemistry. Both mitochondria (10-20%) and nuclear transcription (80-90%) contributed significantly to the dsRNA transcriptome. The mitochondrial contribution was lower in the cancer cells compared to fibroblasts. The expression of different transposable element families was comparable, suggesting a general up-regulation of transposable element expression rather than stimulation of a specific sub-family. Sequencing of the input control revealed minor differences in dsRNA processing pathways with an upregulation of oligoadenylate synthase and RNP125 that negatively regulates the dsRNA sensors RIG1 and MDA5. Moreover, RT-qPCR, Western blotting, and immunocytochemistry confirmed the relatively minor adaptations to the hugely different dsRNA levels. As a consequence, these transformed cell lines are potentially less tolerant to interventions that increase the formation of endogenous dsRNA.

Consolidative thoracic radiation therapy for extensive-stage small cell lung cancer in the era of first-line chemoimmunotherapy: preclinical data and a retrospective study in Southern Italy.

Consolidative thoracic radiotherapy (TRT) has been commonly used in the management of extensive-stage small cell lung cancer (ES-SCLC). Nevertheless, phase III trials exploring first-line chemoimmunotherapy have excluded this treatment approach. However, there is a strong biological rationale to support the use of radiotherapy (RT) as a boost to sustain anti-tumor immune responses. Currently, the benefit of TRT after chemoimmunotherapy remains unclear. The present report describes the real-world experiences of 120 patients with ES-SCLC treated with different chemoimmunotherapy combinations. Preclinical data supporting the hypothesis of anti-tumor immune responses induced by RT are also presented. A total of 120 ES-SCLC patients treated with chemoimmunotherapy since 2019 in the South of Italy were retrospectively analyzed. None of the patients included in the analysis experienced disease progression after undergoing first-line chemoimmunotherapy. Of these, 59 patients underwent TRT after a multidisciplinary decision by the treatment team. Patient characteristics, chemoimmunotherapy schedule, and timing of TRT onset were assessed. Safety served as the primary endpoint, while efficacy measured in terms of overall survival (OS) and progression-free survival (PFS) was used as the secondary endpoint. Immune pathway activation induced by RT in SCLC cells was explored to investigate the biological rationale for combining RT and immunotherapy. Preclinical data supported the activation of innate immune pathways, including the STimulator of INterferon pathway (STING), gamma-interferon-inducible protein (IFI-16), and mitochondrial antiviral-signaling protein (MAVS) related to DNA and RNA release. Clinical data showed that TRT was associated with a good safety profile. Of the 59 patients treated with TRT, only 10% experienced radiation toxicity, while no ≥ G3 radiation-induced adverse events occurred. The median time for TRT onset after cycles of chemoimmunotherapy was 62 days. Total radiation dose and fraction dose of TRT include from 30 Gy in 10 fractions, up to definitive dose in selected patients. Consolidative TRT was associated with a significantly longer PFS than systemic therapy alone (one-year PFS of 61% vs. 31%, p<0.001), with a trend toward improved OS (one-year OS of 80% vs. 61%, p=0.027). Multi-center data from establishments in the South of Italy provide a general confidence in using TRT as a consolidative strategy after chemoimmunotherapy. Considering the limits of a restrospective analysis, these preliminary results support the feasibility of the approach and encourage a prospective evaluation.

RNA m5C methylation: a potential modulator of innate immune pathways in hepatocellular carcinoma.

RNA 5-methylcytosine (m5C) methylation plays a crucial role in hepatocellular carcinoma (HCC). As reported, aberrant m5C methylation is closely associated with the progression, therapeutic efficacy, and prognosis of HCC. The innate immune system functions as the primary defense mechanism in the body against pathogenic infections and tumors since it can activate innate immune pathways through pattern recognition receptors to exert anti-infection and anti-tumor effects. Recently, m5C methylation has been demonstrated to affect the activation of innate immune pathways including TLR, cGAS-STING, and RIG-I pathways by modulating RNA function, unveiling new mechanisms underlying the regulation of innate immune responses by tumor cells. However, research on m5C methylation and its interplay with innate immune pathways is still in its infancy. Therefore, this review details the biological significance of RNA m5C methylation in HCC and discusses its potential regulatory relationship with TLR, cGAS-STING, and RIG-I pathways, thereby providing fresh insights into the role of RNA methylation in the innate immune mechanisms and treatment of HCC.

Abstract C077: STC-15, a small molecule inhibitor of the RNA methyltransferase METTL3, activates anti-tumor immunity and reshapes the tumor microenvironment

Abstract Background METTL3 is the RNA methyltransferase responsible for the deposition of N-6-methyladenosine modification (m6A) on mRNA, to regulate its stability, splicing and protein translation. Small molecule inhibitors of METTL3 catalytic activity have previously demonstrated anti-tumor efficacy in pre-clinical models of acute myeloid leukemia (AML) and solid tumors. Inhibition of METTL3 was shown to induce a cell-intrinsic innate immune response through activation of the double strand RNA (dsRNA) sensing machinery and interferon (IFN) signalling. Here we present new pre-clinical data, investigating the combination of METTL3 inhibitor (METTL3i) with DNA damaging therapies. We show that METTL3 inhibition profoundly changes the tumor microenvironment (TME), and we further demonstrate the generation of durable immune memory in mice treated with METTL3i, with or without anti-PD1 therapy. Materials &amp; Methods The orally bioavailable small molecule clinical-stage METTL3i STC-15, or the equipotent tool compound STM3675 were used in these studies. To characterise transcriptomic changes following METTL3 inhibition, RNA sequencing was performed. Induction of specific genes was validated by Western Blot, and cytokine secretion assessed by ELISA. In-vitro cell viability was assessed by Cell TiterGlo (Promega). In vivo efficacy studies used subcutaneous MC38 and GL261 syngeneic tumor models, and characterisation of the TME was performed by flow cytometry. Results Treatment with METTL3i sensitized cells to DNA damaging agents such as doxorubicin. On the molecular level, further enhancement of the IFN pathway by the combination treatment was observed. Moreover, combination of METTL3i with radiation treatment (RT) in vivo in the MC38 colorectal syngeneic mouse model demonstrated a clear benefit of the combination over each of the single treatments. METTL3i alone or in combination with RT led to a major TME remodulation, reducing the number of Tregs, M2 macrophages and neutrophils, and increasing the number of M1 macrophages and CD8+ T-cells in tumors. The GL261 syngeneic glioma model is exquisitely sensitive to anti-PD1 treatment. Similarly, METTL3i treatment led to multiple complete regressions, and the combination of METTL3i and anti-PD1 further accelerated the occurrence of regressions. Following a 3-weeks’ observation period, tumor free mice were re-inoculated with GL261 cells. All tumor-free mice remained tumor free after the rechallenge. Conclusions In pre-clinical cancer models, treatment with METTL3i such as STC-15 resulted in activation of innate immune pathways and subsequent enhancement of cytotoxic T-cell activity. In-vivo, STC-15 had a profound effect on the TME, shifting it from an immunosuppressive, pro-tumorigenic state to an immunostimulatory, anti-tumor state. In combination with anti-PD1, METTL3 inhibitors generate a durable anti-tumor response and lasting immune memory. A First-in-Human dose escalating study evaluating STC-15 monotherapy in patients with solid tumors is ongoing (NCT05584111). Citation Format: Yaara Ofir-Rosenfeld, Lina Vasiliauskaitė, Joanna Obacz, Georgia Tsagkogeorga, Josefin-Beate Holz, Jerry McMahon, Oliver Rausch. STC-15, a small molecule inhibitor of the RNA methyltransferase METTL3, activates anti-tumor immunity and reshapes the tumor microenvironment [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C077.

Regulation of Metabolic Homeostasis By TRAF6 Contributes to the Leukemia Progression

TNF receptor associated factor 6 (TRAF6) is an E3 ubiquitin ligase that mediates innate immune signaling that converges on key mediators of Toll-like receptor and interleukin-1 signaling. Dysregulated TRAF6 expression and innate immune pathway activation is implicated in the pathogenesis of various myeloid malignancies. For instance, increased TRAF6-mediated signaling in hematopoietic stem and progenitor cells (HSPCs) results in myelodysplastic syndrome-like cellular features, such as the functional impairment of HSPCs. On the other hand, our previous study indicated that loss of TRAF6 induces the leukemic transformation of pre-leukemic cells ( Muto et al. Cell Stem Cell). These paradoxical observations suggests that TRAF6 acts as both an oncogene and tumor suppressor gene in hematopoietic cells in a disease type- and context-dependent manner. However, the direct consequence of TRAF6 loss in AML cells, rather than in pre-leukemic cells, has not yet been explored. Therefore, the role of TRAF6 in the pathogenesis of AML remains obscure. In this study, we found elevated level of TRAF6 expression in AML cells, and determined the effect of TRAF6 loss on the cellular and molecular features of AML cells. We proposed a novel role of TRAF6 as a regulator of metabolic homeostasis, which is important for the progression of AML. To determine the requirement of TRAF6 in AML, we first evaluated cell the effect of TRAF6 loss in AML cell lines on proliferative capacities. Multiple types of human AML cell lines showed significant growth impairment upon TRAF6-knockdown. Furthermore, transplantation of TRAF6-deficient HSPCs expressing the leukemic oncogene MLL-AF9 to the recipient mice resulted in suppression of overt leukemia. These findings confirm that loss of TRAF6 suppresses leukemic cell function in vitro and in vivo. To assess the molecular mechanism of inhibitory effect of TRAF6 loss in AML, we next performed gene expression analysis using human AML database and TRAF6-knockdown AML cell lines. Interestingly, we found that reduced levels of TRAF6 in AML is associated with changes in the expression of genes involved in mitochondria-related processes. Our extensive in vitro validation experiments including extracellular flux assay and metabolome analysis confirmed that TRAF6 loss in AML cells induces dynamic changes in metabolites and the impairment of mitochondrial function. To identify the mechanism of metabolic alterations by TRAF6 loss in leukemia, we compared the upregulated genes in TRAF6-knockdown AML cells with the 130 previously identified essential genes for AML cell survival in vitro and in vivo ( Yamauchi et al. Cancer Cell). We focused on O-GlcNAc transferase ( OGT) as a gene associated in mitochondrial function and AML cell survival. Importantly, TRAF6 gene expression in human AML patient samples was positively correlated with the expression of OGT. To confirm the importance of OGT in the regulation of mitochondrial function mediated by TRAF6 in leukemia, we utilized the inhibitor of O-GlcNAcase (OGA), a remover of O-GlcNAc from its substrates. The treatment with MK8719 (OGA inhibitor) mildly, but significantly, restored the number of TRAF6 knockdown AML cells, which was correlated with the changes in mitochondrial function, indicating that O-GlcNAc modification regulated by TRAF6 is important for AML progression. In summary, We provide evidence for the oncogenic function of TRAF6 in leukemia, and shed light on the novel TRAF6/OGT/O-GlcNAc axis that regulates the metabolic reprogramming required for leukemogenesis.

Pineal Gland Hormone Melatonin Inhibits Migration of Hematopoietic Stem/Progenitor Cells (HSPCs) by Downregulating Nlrp3 Inflammasome and Upregulating Heme Oxygenase-1 (HO-1) Activity.

Hematopoietic stem progenitor cells (HSPCs) follow the diurnal circulation rhythm in peripheral blood (PB) with nadir during late night and peak at early morning hours. The level of these cells in PB correlates with activation of innate immunity pathways, including complement cascade (ComC) that drives activation of Nlrp3 inflammasome. To support this, mice both in defective ComC activation as well as Nlrp3 inflammasome do not show typical changes in the diurnal level of circulating HSPCs. Migration of HSPCs is also impaired at the intracellular level by the anti-inflammatory enzyme heme oxygenase-1 (HO-1) which is an inhibitor of Nlrp3 inflammasome. It is also well known that circadian rhythm mediates PB level of melatonin released from the pineal gland. Since trafficking of HSPCs is driven by innate immunity-induced sterile inflammation and melatonin has an anti-inflammatory effect, we hypothesized that melatonin could negatively impact the release of HSPCs from BM into PB by inhibiting Nlrp3 inflammasome activation. We provide anevidence that melatonin being a ''sleep regulating pineal hormone'' directly inhibits migration of HSPCs both in vitro migration assays and in vivo during pharmacological mobilization. This correlated with inhibition of cholesterol synthesis required for a proper membrane lipid raft (MLRs) formation and an increase in expression of HO-1-an inhibitor of Nlrp3 inflammasome. Since melatonin is a commonly used drug, this should be considered while preparing a patient for the procedure of HSPCs mobilization. More importantly, our studies shed more mechanistic light on a role of melatonin in the diurnal circulation of HSPCs.

Synergistic immunostimulation for tumor sensitization with a biomineralized DNA sponge

The activation of stimulator of interferon genes (STING) plays a key role for provoking effective innate and adaptive immunity, yet is constrained by the limited clinical translation of STING agonists. Herein, we construct a smart DNA sponge to deliver STING agonists for robust cancer immunotherapy via the synergistic activation of innate immune pathways. The compact DNA sponge (Mn-CpG), assembling through a simple rolling circle amplification reaction, is encoded with numerous STING agonist (Mn2+) and toll-like receptor 9 (TLR9) agonist (CpG oligodeoxynucleotide) and allows their on-demand cellular delivery We show that Mn-CpG sponge can trigger the synergistic type I interferon response and robust T cell priming through a convenient coordination of different signaling pathways. In a poorly immunogenic breast cancer model, the method achieves the boosted immune responses with significantly inhibited tumor growth and metastasis.

Multi-omics Approach in Kidney Transplant: Lessons Learned from COVID-19 Pandemic.

Multi-omics approach has advanced our knowledge on transplantation-associated clinical outcomes, such as acute rejection and infection, and emerging omics data are becoming available in kidney transplant and COVID-19. Herein, we discuss updated findings of multi-omics data on kidney transplant outcomes, as well as COVID-19 and kidney transplant. Transcriptomics, proteomics, and metabolomics revealed various inflammation pathways associated with kidney transplantation-related outcomes and COVID-19. Although multi-omics data on kidney transplant and COVID-19 is limited, activation of innate immune pathways and suppression of adaptive immune pathways were observed in the active phase of COVID-19 in kidney transplant recipients. Multi-omics analysis has led us to a deeper exploration and a more comprehensive understanding of key biological pathways in complex clinical settings, such as kidney transplantation and COVID-19. Future multi-omics analysis leveraging multi-center biobank collaborative will further advance our knowledge on the precise immunological responses to allograft and emerging pathogens.

Gut Microbiota and the Microvasculature.

The gut microbiota is increasingly recognized as an actuating variable shaping vascular development and endothelial cell function in the intestinal mucosa but also affecting the microvasculature of remote organs. In the small intestine, colonization with gut microbiota and subsequent activation of innate immune pathways promotes the development of intricate capillary networks and lacteals, influencing the integrity of the gut-vascular barrier as well as nutrient uptake. Since the liver yields most of its blood supply via the portal circulation, the hepatic microcirculation steadily encounters microbiota-derived patterns and active signaling metabolites that induce changes in the organization of the liver sinusoidal endothelium, influencing immune zonation of sinusoids and impacting on metabolic processes. In addition, microbiota-derived signals may affect the vasculature of distant organ systems such as the brain and the eye microvasculature. In recent years, this gut-resident microbial ecosystem was revealed to contribute to the development of several vascular disease phenotypes.

A multi-center, prospective cohort study of whole blood gene expression in the tuberculosis-diabetes interaction

Diabetes mellitus (DM) increases tuberculosis (TB) severity. We compared blood gene expression in adults with pulmonary TB, with or without diabetes mellitus (DM) from sites in Brazil and India. RNA sequencing (RNAseq) performed at baseline and during TB treatment. Publicly available baseline RNAseq data from South Africa and Romania reported by the TANDEM Consortium were also analyzed. Across the sites, differentially expressed genes varied for each condition (DM, TB, and TBDM) and no pattern classified any one group across all sites. A concise signature of TB disease was identified but this was expressed equally in TB and TBDM. Pathway enrichment analysis failed to distinguish TB from TBDM, although there was a trend for greater neutrophil and innate immune pathway activation in TBDM participants. Pathways associated with insulin resistance, metabolic dysfunction, diabetic complications, and chromosomal instability were positively correlated with glycohemoglobin. The immune response to pulmonary TB as reflected by whole blood gene expression is substantially similar with or without comorbid DM. Gene expression pathways associated with the microvascular and macrovascular complications of DM are upregulated during TB, supporting a syndemic interaction between these coprevalent diseases.

A Chemical Proteomics Approach to Discover Regulators of Innate Immune Signaling.

Innate immune pathways are tightly regulated to balance an appropriate response to infectious agents and tolerable levels of inflammation. Dysregulation of innate immune pathways can lead to severe autoinflammatory disorders or susceptibility to infections. Here, we aimed to identify kinases in common cellular pathways that regulate innate immune pathways by combining small-scale kinase inhibitor screening with quantitative proteomics. We found that inhibitors of kinases ATM, ATR, AMPK, and PLK1 reduced the induction of interferon-stimulated gene expression in response to innate immune pathway activation by poly(I:C) transfection. However, siRNA depletion of these kinases did not validate findings with kinase inhibitors, suggesting that off-target effects may explain their activities. We mapped the effects of kinase inhibitors to various stages in innate immune pathways. Determining the mechanisms by which kinase inhibitors antagonize these pathways may illuminate novel mechanisms of innate immune pathway control.

Inactivation of p53 provides a competitive advantage to del(5q) myelodysplastic syndrome hematopoietic stem cells during inflammation.

Inflammation is associated with the pathogenesis of myelodysplastic syndromes (MDS) and emerging evidence suggests that MDS hematopoietic stem and progenitor cells (HSPC) exhibit an altered response to inflammation. Deletion of chromosome 5 (del(5q)) is the most common chromosomal abnormality in MDS. Although this MDS subtype contains several haploinsufficient genes that impact innate immune signaling, the effects of inflammation on del(5q) MDS HSPC remains undefined. Utilizing a model of del(5q)-like MDS, inhibiting the IRAK1/4-TRAF6 axis improved cytopenias, suggesting that activation of innate immune pathways contributes to certain clinical features underlying the pathogenesis of low-risk MDS. However, low-grade inflammation in the del(5q)-like MDS model did not contribute to more severe disease but instead impaired the del(5q)-like HSPC as indicated by their diminished numbers, premature attrition and increased p53 expression. Del(5q)-like HSPC exposed to inflammation became less quiescent, but without affecting cell viability. Unexpectedly, the reduced cellular quiescence of del(5q) HSPC exposed to inflammation was restored by p53 deletion. These findings uncovered that inflammation confers a competitive advantage of functionally defective del(5q) HSPC upon loss of p53. Since TP53 mutations are enriched in del(5q) AML following an MDS diagnosis, increased p53 activation in del(5q) MDS HSPC due to inflammation may create a selective pressure for genetic inactivation of p53 or expansion of a pre-existing TP53-mutant clone.

Chronic activation of Toll-like receptor 2 induces an ichthyotic skin phenotype.

Ichthyosis defines a group of chronic conditions that manifest phenotypically as a thick layer of scales, often affecting the entire skin. While the gene mutations that lead to ichthyosis are well documented, the actual signalling mechanisms that lead to scaling are poorly characterized; however, recent publications suggest that common mechanisms are active in ichthyotic tissue and in analogous models of ichthyosis. To determine common mechanisms of hyperkeratosis that may be easily targeted with small-molecule inhibitors. We combined gene expression analysis of gene-specific short hairpin RNA (shRNA) knockdowns in rat epidermal keratinocytes (REKs) of two genes mutated in autosomal recessive congenital ichthyosis (ARCI), Tgm1 and Alox12b, and proteomic analysis of skin scale from patients with ARCI, as well as RNA sequencing data from rat epidermal keratinocytes treated with the Toll-like receptor 2 (TLR2) agonist Pam3CSK4. We identified common activation of the TLR2 pathway. Exogenous TLR2 activation led to increased expression of important cornified envelope genes and, in organotypic culture, caused hyperkeratosis. Conversely, blockade of TLR2 signalling in keratinocytes from patients with ichthyosis and our shRNA models reduced the expression of keratin 1, a structural protein overexpressed in ichthyosis scale. A time course of TLR2 activation in REKs revealed that although there was rapid initial activation of innate immune pathways, this was rapidly superseded by widespread upregulation of epidermal differentiation-related proteins. Both nuclear factor kappa B phosphorylation and GATA3 upregulation was associated with this switch, and GATA3 overexpression was sufficient to increase keratin 1 expression. Taken together, these data define a dual role for TLR2 activation during epidermal barrier repair that may be a useful therapeutic modality in treating diseases of epidermal barrier dysfunction.

Role of pyroptosis in hemostasis activation in sepsis.

Sepsis is frequently associated with hemostasis activation and thrombus formation, and systematic hemostatic changes are associated with a higher risk of mortality. The key events underlying hemostasis activation during sepsis are the strong activation of innate immune pathways and the excessive inflammatory response triggered by invading pathogens. Pyroptosis is a proinflammatory form of programmed cell death, that defends against pathogens during sepsis. However, excessive pyroptosis can lead to a dysregulation of host immune responses and organ dysfunction. Recently, pyroptosis has been demonstrated to play a prominent role in hemostasis activation in sepsis. Several studies have demonstrated that pyroptosis participates in the release and coagulation activity of tissue factors. In addition, pyroptosis activates leukocytes, endothelial cells, platelets, which cooperate with the coagulation cascade, leading to hemostasis activation in sepsis. This review article attempts to interpret the molecular and cellular mechanisms of the hemostatic imbalance induced by pyroptosis during sepsis and discusses potential therapeutic strategies.

An AGS-associated mutation in ADAR1 catalytic domain results in early-onset and MDA5-dependent encephalopathy with IFN pathway activation in the brain

BackgroundAicardi–Goutières syndrome (AGS) is a severe neurodegenerative disease with clinical features of early-onset encephalopathy and progressive loss of intellectual abilities and motor control. Gene mutations in seven protein-coding genes have been found to be associated with AGS. However, the causative role of these mutations in the early-onset neuropathogenesis has not been demonstrated in animal models, and the mechanism of neurodegeneration of AGS remains ambiguous.MethodsVia CRISPR/Cas-9 technology, we established a mutant mouse model in which a genetic mutation found in AGS patients at the ADAR1 coding gene (Adar) loci was introduced into the mouse genome. A mouse model carrying double gene mutations encoding ADAR1 and MDA-5 was prepared using a breeding strategy. Phenotype, gene expression, RNA sequencing, innate immune pathway activation, and pathologic studies including RNA in situ hybridization (ISH) and immunohistochemistry were used for characterization of the mouse models to determine potential disease mechanisms.ResultsWe established a mouse model bearing a mutation in the catalytic domain of ADAR1, the D1113H mutation found in AGS patients. With this mouse model, we demonstrated a causative role of this mutation for the early-onset brain injuries in AGS and determined the signaling pathway underlying the neuropathogenesis. First, this mutation altered the RNA editing profile in neural transcripts and led to robust IFN-stimulated gene (ISG) expression in the brain. By ISH, the brains of mutant mice showed an unusual, multifocal increased expression of ISGs that was cell-type dependent. Early-onset astrocytosis and microgliosis and later stage calcification in the deep white matter areas were observed in the mutant mice. Brain ISG activation and neuroglial reaction were completely prevented in the Adar D1113H mutant mice by blocking RNA sensing through deletion of the cytosolic RNA receptor MDA-5.ConclusionsThe Adar D1113H mutation in the ADAR1 catalytic domain results in early-onset and MDA5-dependent encephalopathy with IFN pathway activation in the mouse brain.

Platelet TLR9 Promotes Pulmonary Thrombo-Inflammation in Sickle Cell Disease

Sickle cell disease (SCD) is a monogenic disorder, which affects millions of people worldwide. Erythrocyte sickling, chronic hemolysis and ischemia-reperfusion injury promote sterile inflammation in SCD.Recent evidence suggests that activation of innate immune pathways in platelets contribute to development of acute chest syndrome (ACS), a type of acute lung injury in SCD by promoting occlusion of lung arterioles by neutrophil-platelet-erythrocyte aggregates (pulmonary thrombo-inflammation). Although, TLR9 is functionally expressed on the surface of activated platelets and may bind to circulating double stranded DNA, the role of platelet TLR-9 in promoting pulmonary thrombo-inflammation in SCD remains elusive. Our initial findings using intravenous lipopolysaccharide (0.1 μg/kg LPS) or oxy-hemoglobin (10 μmole/kg oxy-Hb) triggered model of vaso-occlusive crisis in transgenic-humanized SCD mice reveal that pulmonary vaso-occlusion is associated with elevated surface-expression of platelet TLR-9 in SCD mice. Unlike SCD mice, surface-expression of TLR9 was undetectable in the platelets of control mice challenged with either IV LPS or oxy-Hb. These findings suggest a potential role for platelet TLR9 in promoting lung vaso-occlusion and injury in SCD. Currently, studies are in progress to identify the innate immune signaling downstream to platelet TLR9, contributing to pulmonary thrombo-inflammation in SCD.

Novel Evidence That Pineal Gland Hormone Melatonin Inhibits Migration of Hematopoietic Stem/Progenitor Cells (HSPCs) By Inhibiting Nlrp3 Inflammasome and Upregulating Heme Oxygenase-1 (HO-1) - Implications to Understand Better Diurnal Trafficking of HSPCs and Anti-Inflammatory Effects of Melatonin

Background. Hematopoietic stem progenitor cells (HSPCs) follow the diurnal circulation rhythm in peripheral blood (PB) with nadir during late night and pick at early morning hours. We have reported that the level of these cells in PB correlates with activation of innate immunity pathways, including complement cascade (ComC) (Stem Cell Rev Rep. 2018;14(5):677-685) that drives activation of Nlrp3 inflammasome (Stem Cell Rev Rep. 2020;16(2):335-343). Therefore, mice both in defective ComC activation as well as Nlrp3 inflammasome do not show typical changes in the diurnal level of circulating HSPCs. Cell migration is also inhibited by the anti-inflammatory enzyme heme oxygenase-1 (HO-1), which inhibits Nlrp3 inflammasome (Leukemia. 2022;36(1):23-32). It is well known that circadian rhythm is regulated by the release of melatonin from the pineal gland, which also has some anti-inflammatory effects (J. Pineal Res. 2018;65:e12525). Aim of the study.Since stem cell trafficking is driven by innate immunity-induced sterile inflammation and melatonin has an anti-inflammatory effect, we hypothesized that melatonin could negatively impact the release of HSPCs from bone marrow (BM) into PB by inhibiting Nlrp3 inflammasome activation. Materials and Methods. To test this hypothesis, first, we tested the expression of melatonin receptors in murine and human HSPCs. We employed Transwell system to see the effect of melatonin on the migration of human and murine HSPCs to the SDF-1 gradient. Next, we performed pharmacological mobilization of normal mice with G-CSF or AMD3100 in the presence of melatonin. Finally, employing Glow assay, we evaluated the effect of melatonin on activation of Nlrp3 inflammasome in HSPCs stimulated by BM chemotactic factors and the effect of melatonin on expression of migration inhibitory HO-1. Since membrane lipid rafts (MLRs) are crucial for the proper response of cells to chemoattractants, we also evaluated the effect of melatonin on the expression of enzymes (SREBP2, HMGCs, and HMGCR) involved in the synthesis of cholesterol and sphingolipids that are major constituents of MLRs. Results. We noticed that HSPCs express melatonin receptors, and melatonin inhibits in vitro Transwell assays migration of HSPCs and negatively affects mobilization of these cells into PB in response to G-CSF and AMD3100 administration. We also observed that melatonin inhibits Nlrp3 inflammasome activation and the formation of MLRs. This is explained by a decrease in the expression of enzymes such as SREBP2, HMGCs, and HMGCR involved in cholesterol synthesis leading to impaired formation of MLRs. Moreover, melatonin increases in HSPCs expression of heme oxygenase-1 (HO-1), that is a known inhibitor of Nlrp3 inflammasome and a negative regulator of cell migration. Conclusions. We provide for the first-time evidence that melatonin being a ''sleep regulating pineal hormone'' directly inhibits migration of HSPCs in vitro assays and in vivo their release from BM in response to G-CSF and AMD3100. This is correlated with inhibition of cholesterol synthesis, which results in defective assembly of MLRs, as well as a decrease in Nlrp3 inflammasome activity and an increase in expression of HO-1. Since melatonin is a commonly used shelf drug, this should be considered while preparing a patient for the procedure of HSPCs mobilization. More importantly, our studies shed more light on molecular mechanisms of melatonin involvement in the diurnal circulation of HSPCs.