DAMPs and Innate Immune Training

The ability to remember a previous encounter with pathogens was long thought to be a key feature of the adaptive immune system enabling the host to mount a faster, more specific and more effective immune response upon the reencounter, reducing the severity of infectious diseases. Over the last 15 years, an increasing amount of evidence has accumulated showing that the innate immune system also has features of a memory. In contrast to the memory of adaptive immunity, innate immune memory is mediated by restructuration of the active chromatin landscape and imprinted by persisting adaptations of myelopoiesis. While originally described to occur in response to pathogen-associated molecular patterns, recent data indicate that host-derived damage-associated molecular patterns, i.e. alarmins, can also induce an innate immune memory. Potentially this is mediated by the same pattern recognition receptors and downstream signaling transduction pathways responsible for pathogen-associated innate immune training. Here, we summarize the available experimental data underlying innate immune memory in response to damage-associated molecular patterns. Further, we expound that trained immunity is a general component of innate immunity and outline several open questions for the rising field of pathogen-independent trained immunity

Trained innate immunity, long-lasting epigenetic modulation, and skewed myelopoiesis by heme

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Hormesis and Defense of Infectious Disease

Infectious diseases are a global health burden and remain associated with high social and economic impact. Treatment of affected patients largely relies on antimicrobial agents that act by directly targeting microbial replication. Despite the utility of host specific therapies having been assessed in previous clinical trials, such as targeting the immune response via modulating the cytokine release in sepsis, results have largely been frustrating and did not lead to the introduction of new therapeutic tools. In this article, we will discuss current evidence arguing that, by applying the concept of hormesis, already approved pharmacological agents could be used therapeutically to increase survival of patients with infectious disease via improving disease tolerance, a defense mechanism that decreases the extent of infection-associated tissue damage without directly targeting pathogenic microorganisms

Hormesis and Defense of Infectious Disease

Infectious diseases are a global health burden and remain associated with high social and economic impact. Treatment of affected patients largely relies on antimicrobial agents that act by directly targeting microbial replication. Despite the utility of host specific therapies having been assessed in previous clinical trials, such as targeting the immune response via modulating the cytokine release in sepsis, results have largely been frustrating and did not lead to the introduction of new therapeutic tools. In this article, we will discuss current evidence arguing that, by applying the concept of hormesis, already approved pharmacological agents could be used therapeutically to increase survival of patients with infectious disease via improving disease tolerance, a defense mechanism that decreases the extent of infection-associated tissue damage without directly targeting pathogenic microorganisms

microRNA-125a-3p is regulated by MyD88 in Legionella pneumophila infection and targets NTAN1.

Legionella pneumophila (L. pneumophila) is a causative agent of severe pneumonia. It is highly adapted to intracellular replication and manipulates host cell functions like vesicle trafficking and mRNA translation to its own advantage. However, it is still unknown to what extent microRNAs (miRNAs) are involved in the Legionella-host cell interaction.WT and MyD88-/- murine bone marrow-derived macrophages (BMM) were infected with L. pneumophila, the transcriptome was analyzed by high throughput qPCR array (microRNAs) and conventional qPCR (mRNAs), and mRNA-miRNA interaction was validated by luciferase assays with 3´-UTR mutations and western blot.L. pneumophila infection caused a pro-inflammatory reaction and significant miRNA changes in murine macrophages. In MyD88-/- cells, induction of inflammatory markers, such as Ccxl1/Kc, Il6 and miR-146a-5p was reduced. Induction of miR-125a-3p was completely abrogated in MyD88-/- cells. Target prediction analyses revealed N-terminal asparagine amidase 1 (NTAN1), a factor from the n-end rule pathway, to be a putative target of miR-125a-3p. This interaction could be confirmed by luciferase assay and western blot.Taken together, we characterized the miRNA regulation in L. pneumophila infection with regard to MyD88 signaling and identified NTAN1 as a target of miR-125a-3p. This finding unravels a yet unknown feature of Legionella-host cell interaction, potentially relevant for new treatment options

Azithromycin does not improve disease severity in acute experimental pancreatitis.

Acute pancreatitis is a severe systemic disease triggered by a sterile inflammation and initial local tissue damage of the pancreas. Immune cells infiltrating into the pancreas are main mediators of acute pancreatitis pathogenesis. In addition to their antimicrobial potency, macrolides possess anti-inflammatory and immunomodulatory properties which are routinely used in patients with chronic airway infections and might also beneficial in the treatment of acute lung injury. We here tested the hypothesis that the macrolide antibiotic azithromycin can improve the course of acute experimental pancreatitis via ameliorating the damage imposed by sterile inflammation, and could be used as a disease specific therapy. However, our data show that azithromycin does not have influence on caerulein induced acute pancreatitis in terms of reduction of organ damage, and disease severity. Furthermore Infiltration of immune cells into the pancreas or the lungs was not attenuated by azithromycin as compared to controls or ampicillin treated animals with acute experimental pancreatitis. We conclude that in the chosen model, azithromycin does not have any beneficial effects and that its immunomodulatory properties cannot be used to decrease disease severity in the model of caerulein-induced pancreatitis in mice

KC, IL-6 and microRNAs are differentially regulated in wild type and <i>MyD88</i>^<i>-/-</i> BMMs upon infection with <i>Legionella pneumophila</i>.

<p>WT and <i>MyD88</i>^<i>-/-</i> macrophages were infected with <i>L</i>. <i>pneumophila</i> for 24 h at a MOI of 0.25, and KC release was determined by ELISA (A). The expression patterns of selected mRNAs were analysed by qPCR (B). microRNAs were investigated by Taqman Low Density Array. The top 4 differentially regulated microRNAs as ranked by p-value are shown. The relative log₂ fold induction in infected <i>MyD88</i>^<i>-/-</i> vs. infected WT cells (left column), in infected <i>MyD88</i>^<i>-/-</i> vs. uninfected <i>MyD88</i>^<i>-/-</i> <i>cells</i> (middle column) or in infected WT vs. uninfected WT cells (right column) is depicted (C). Selected microRNAs were validated by qPCR (D). mRNA samples were normalized against GAPDH, while microRNA samples were normalized against snRNA U6 (C) or snoRNA202 (D). Data are shown as mean & SD of at least three independent experiments. Statistical tests were one-way ANOVA with post-hoc intergroup comparison (A) and Student´s T-Test with Bonferroni-Holm adjustment for multiple testing (B) and (D). *p<0.05 WT uninfected vs. WT MOI 0.25, ^§§p<0.05 <i>MyD88</i>^<i>-/-</i> MOI 0.25 vs. WT MOI 0.25 (A), *p_adj<0.05 <i>MyD88</i>^<i>-/-</i> vs. WT (B) and (D).</p

Single-cell RNA sequencing and analysis of rodent blood stage Plasmodium

Summary: Bulk RNA sequencing of Plasmodium spp., the causative parasite of malaria, fails to discriminate developmental-stage-specific gene regulation. Here, we provide a protocol that uses single-cell RNA sequencing of FACS-sorted Plasmodium-chabaudi-chabaudi-AS-infected red blood cells (iRBCs) to characterize developmental-stage-specific modulation of gene expression during malaria blood stage. We describe steps for infecting mice, monitoring disease progression, preparing iRBCs, and single-cell sequencing iRBCs. We then detail procedures for analyzing scRNA-seq data.For complete details on the use and execution of this protocol, please refer to Ramos et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

microRNA-125a-3p is regulated by MyD88 in <i>Legionella pneumophila</i> infection and targets NTAN1

<div><p>Background</p><p><i>Legionella pneumophila</i> (<i>L</i>. <i>pneumophila</i>) is a causative agent of severe pneumonia. It is highly adapted to intracellular replication and manipulates host cell functions like vesicle trafficking and mRNA translation to its own advantage. However, it is still unknown to what extent microRNAs (miRNAs) are involved in the <i>Legionella</i>-host cell interaction.</p><p>Methods</p><p>WT and <i>MyD88</i>^-/- murine bone marrow-derived macrophages (BMM) were infected with <i>L</i>. <i>pneumophila</i>, the transcriptome was analyzed by high throughput qPCR array (microRNAs) and conventional qPCR (mRNAs), and mRNA-miRNA interaction was validated by luciferase assays with 3´-UTR mutations and western blot.</p><p>Results</p><p><i>L</i>. <i>pneumophila</i> infection caused a pro-inflammatory reaction and significant miRNA changes in murine macrophages. In <i>MyD88</i>^-/- cells, induction of inflammatory markers, such as <i>Ccxl1/Kc</i>, <i>Il6</i> and miR-146a-5p was reduced. Induction of miR-125a-3p was completely abrogated in <i>MyD88</i>^-/- cells. Target prediction analyses revealed N-terminal asparagine amidase 1 (NTAN1), a factor from the n-end rule pathway, to be a putative target of miR-125a-3p. This interaction could be confirmed by luciferase assay and western blot.</p><p>Conclusion</p><p>Taken together, we characterized the miRNA regulation in <i>L</i>. <i>pneumophila</i> infection with regard to MyD88 signaling and identified NTAN1 as a target of miR-125a-3p. This finding unravels a yet unknown feature of <i>Legionella</i>-host cell interaction, potentially relevant for new treatment options.</p></div

microRNA-125a-3p targets NTAN1 and regulates it on protein level.

<p>microRNA-125a-3p shows partial homology to the 3´UTR of <i>Ntan1</i> mRNA. To destroy the sequence compatibility in the seed region, 3 bases were exchanged (bold, underlined). MEF cells were co-transfected with the microRNA-125a-3p or a scramble sequence and the psiCheck2 vector carrying either the wild type <i>Ntan1</i> 3´UTR or the mutated sequence. Relative luminescence units (RLU) were determined after 72 h. Renilla luciferase signal was normalized against firefly luciferase. Relative luminescence units were calculated as a percentage of signal in scramble transfected cells (A). Raw264.7 cells were transfected with microRNA-125a-3p or a scramble sequence (5 nM), and NTAN1 protein levels were determined by western blot after 72 h. Densitometric analysis was performed with normalization against Tubulin. One representative blot is shown (B). In an interaction network, which was built from the STRING database, NTAN1 is shown to be associated with the N-end rule pathway. The observed interactions are based on experimental data (purple edges), co-expression (black edges) and textmining (green edges). Only direct interactions with an interaction score of minimum 0.4 (i.e. medium confidence) are shown. Interaction partners include arginyltransferase 1 (Ate1) and members of the ubiquitin protein ligase E3 component n-recognin (Ubr) family (C). Data are shown as mean & SD of at least four independent experiments. Statistical tests were one-way ANOVA with post-hoc intergroup comparison (A) and Mann Whitney U test (B). **p<0.01 Vector WT + miR-125a-3p vs. Vector Mutant + miR-125a-3p (A), *p<0.05 miR-125a-3p vs. Scramble (B).</p