Publications by authors named "Wilhelm Bertrams"

28 Publications

  • Page 1 of 1

Tribolium castaneum defensin 1 kills Moraxella catarrhalisin an in vitro infection model but does not harm commensal bacteria.

Virulence 2021 Dec;12(1):1003-1010

Institute for Lung Research, Universities of Giessen and Marburg Lung Center, Philipps-University Marburg, Marburg, Germany.

is a bacterial pathogen that causes respiratory tract infections in humans. The increasing prevalence of antibiotic-resistant strains has created a demand for alternative treatment options. We therefore tested 23 insect antimicrobial peptides (AMPs) for their activity against in a human infection model with primary macrophages, and against commensal bacteria. Effects on bacterial growth were determined by colony counting and growth curve analysis. The inflammatory macrophage response was characterized by qPCR and multiplex ELISA. Eleven of the AMPs were active against . Defensin 1 from the red flour beetle significantly inhibited bacterial growth and reduced the number of colony forming units. This AMP also showed antibacterial activity in the infection model, reducing cytokine expression and release by macrophages. Defensin 1 had no effect on the commensal bacteria and . However, sarcotoxin 1 C from the green bottle fly was active against and , but not against . The ability of defensin 1 to inhibit but not selected commensal bacteria, and the absence of cytotoxic or inflammatory effects against human blood-derived macrophages, suggests this AMP may be suitable for development as a new therapeutic lead against antibiotic-resistant .
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http://dx.doi.org/10.1080/21505594.2021.1908741DOI Listing
December 2021

[Role of genetic factors in pneumonia and COVID-19].

Pneumologe (Berl) 2021 Mar 5:1-5. Epub 2021 Mar 5.

Institut für Lungenforschung, Universities of Gießen and Marburg Lung Center (UGMLC), Philipps-Universität Marburg, Hans-Meerwein-Str. 2, 35043 Marburg, Deutschland.

Pneumonia causes the highest mortality of all infectious diseases worldwide. The most common pathogens are bacteria but there are also epidemic or pandemic lung infections caused by influenza or coronaviruses, such as the current pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In addition to the occurrence of antibiotic resistance and immune pathologies, such as in sepsis, important challenges lie in considering the susceptibility of individual patients. Here, age, medication and comorbidities are considered; however, there is also clear evidence of genetic influences on the individual risk of developing pneumonia or developing a severe course of the disease. This article discusses the genetic influences on pneumonia and the clinical significance.
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http://dx.doi.org/10.1007/s10405-021-00385-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7934978PMC
March 2021

Pro- and Antitumorigenic Capacity of Immunoproteasomes in Shaping the Tumor Microenvironment.

Cancer Immunol Res 2021 Mar 11. Epub 2021 Mar 11.

Institute for Medical Microbiology and Hygiene, Philipps-University Marburg, Marburg, Germany.

Apart from the constitutive proteasome, the immunoproteasome that comprises the three proteolytic subunits LMP2, MECL-1, and LMP7 is expressed in most immune cells. In this study, we describe opposing roles for immunoproteasomes in regulating the tumor microenvironment (TME). During chronic inflammation, immunoproteasomes modulated the expression of protumorigenic cytokines and chemokines and enhanced infiltration of innate immune cells, thus triggering the onset of colitis-associated carcinogenesis (CAC) in wild-type mice. Consequently, immunoproteasome-deficient animals (LMP2/MECL-1/LMP7-null mice) were almost completely resistant to CAC development. In patients with ulcerative colitis with high risk for CAC, immunoproteasome-induced protumorigenic mediators were upregulated. In melanoma tumors, the role of immunoproteasomes is relatively unknown. We found that high expression of immunoproteasomes in human melanoma was associated with better prognosis. Similarly, our data revealed that the immunoproteasome has antitumorigenic activity in a mouse model of melanoma. The antitumor immunity against melanoma was compromised in immunoproteasome-deficient mice because of the impaired activity of CD8 CTLs, CD4 Th1 cells, and antigen-presenting cells. These findings show that immunoproteasomes may exert opposing roles with either pro- or antitumoral properties in a context-dependent manner.
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http://dx.doi.org/10.1158/2326-6066.CIR-20-0492DOI Listing
March 2021

Transcriptional analysis identifies potential biomarkers and molecular regulators in acute malaria infection.

Life Sci 2021 Apr 2;270:119158. Epub 2021 Feb 2.

Institute for Lung Research, Universities of Giessen and Marburg Lung Center, Philipps-Universität Marburg, Marburg, Germany; Department of Internal Medicine, Pulmonary and Critical Care Medicine, University Medical Center Giessen and Marburg, Philipps-Universität Marburg, German Center for Lung Research (DZL), Marburg, Germany; German Center for Lung Research (DZL), German Center for infectious Disease Research (DZIF), Center for Synthetic Microbiology (Synmikro), Philipps-Universität Marburg, Germany. Electronic address:

Aims: Malaria is a serious health threat in tropical countries. The causative parasite of Malaria tropica, the severe form, is the protozoan Plasmodium falciparum. In humans, it infects red blood cells, compromising blood flow and tissue perfusion. This study aims to identify potential biomarkers and RNA networks in leukocyte transcriptomes from patients suffering from Malaria tropica.

Materials And Methods: We identified differentially regulated mRNAs and microRNAs in peripheral blood leukocytes of healthy donors and Malaria patients. Genes whose expression changes were not attributable to changes in leukocyte composition were used for bioinformatics analysis and network construction. Using a previously published cohort of community-acquired pneumonia (CAP) patients, we established discriminating transcriptomic features versus Malaria. We aimed to establish differences between the patient groups by principal component (PCA) and receiving operator characteristic (ROC) analyses and in silico cell type deconvolution.

Key Findings: We found 870 genes that were significantly differentially expressed between healthy donors and Malaria patients. E2F1, BIRC5 and CCNB1 were identified to be primarily responsible for PCA separation of these two groups. We searched for biological function and found that cell cycle processes were strongly activated. By in silico cell type deconvolution, we attribute this to an expansion of γδ T cells. Additional discrimination between CAP and Malaria yielded 445 differentially expressed genes, among which immune proteasome transcripts PSMB8, PSMB9 and PSMB10 were significantly induced in Malaria.

Significance: We identified transcripts from patient leukocytes that differentiate between healthy, Malaria and CAP, and indicate a biological context with potential pathophysiological relevance.
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http://dx.doi.org/10.1016/j.lfs.2021.119158DOI Listing
April 2021

Dietary cellulose induces anti-inflammatory immunity and transcriptional programs via maturation of the intestinal microbiota.

Gut Microbes 2020 11;12(1):1-17

Institute for Medical Microbiology and Hospital Hygiene, Philipps University , Marburg, Germany.

Although it is generally accepted that dietary fiber is health promoting, the underlying immunological and molecular mechanisms are not well defined, especially with respect to cellulose, the most ubiquitous dietary fiber. Here, the impact of dietary cellulose on intestinal microbiota, immune responses and gene expression in health and disease was examined. Lack of dietary cellulose disrupted the age-related diversification of the intestinal microbiota, which subsequently remained in an immature state. Interestingly, one of the most affected microbial genera was which is equipped with enzymes to degrade cellulose. Absence of cellulose changed the microbial metabolome, skewed intestinal immune responses toward inflammation, altered the gene expression of intestinal epithelial cells and mice showed increased sensitivity to colitis induction. In contrast, mice with a defined microbiota including showed enhanced colonic expression of intestinal IL-22 and Reg3γ restoring intestinal barrier function. This study supports the epidemiological observations and adds a causal explanation for the health promoting effects of the most common biopolymer on earth.
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http://dx.doi.org/10.1080/19490976.2020.1829962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7583510PMC
November 2020

Identification of microRNAs involved in NOD-dependent induction of pro-inflammatory genes in pulmonary endothelial cells.

PLoS One 2020 30;15(4):e0228764. Epub 2020 Apr 30.

Cardiology and Angiology, Philipps-University Marburg, Marburg, Germany.

The nucleotide-binding oligomerization domain-containing proteins (NOD) 1 and 2 are mammalian cytosolic pattern recognition receptors sensing bacterial peptidoglycan fragments in order to initiate cytokine expression and pathogen host defense. Since endothelial cells are relevant cells for pathogen recognition at the blood/tissue interface, we here analyzed the role of NOD1- and NOD2-dependently expressed microRNAs (miRNAs, miR) for cytokine regulation in murine pulmonary endothelial cells. The induction of inflammatory cytokines in response to NOD1 and NOD2 was confirmed by increased expression of tumour necrosis factor (Tnf)-α and interleukin (Il)-6. MiRNA expression profiling revealed NOD1- and NOD2-dependently regulated miRNA candidates, of which miR-147-3p, miR-200a-3p, and miR-298-5p were subsequently validated in pulmonary endothelial cells isolated from Nod1/2-deficient mice. Analysis of the two down-regulated candidates miR-147-3p and miR-298-5p revealed predicted binding sites in the 3' untranslated region (UTR) of the murine Tnf-α and Il-6 mRNA. Consequently, transfection of endothelial cells with miRNA mimics decreased Tnf-α and Il-6 mRNA levels. Finally, a novel direct interaction of miR-298-5p with the 3' UTR of the Il-6 mRNA was uncovered by luciferase reporter assays. We here identified a mechanism of miRNA-down-regulation by NOD stimulation thereby enabling the induction of inflammatory gene expression in endothelial cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0228764PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7192443PMC
July 2020

A MicroRNA Network Controls Replication in Human Macrophages via LGALS8 and MX1.

mBio 2020 03 24;11(2). Epub 2020 Mar 24.

Institute for Lung Research, Universities of Giessen and Marburg Lung Center, Philipps University Marburg, Marburg, Germany

is an important cause of pneumonia. It invades alveolar macrophages and manipulates the immune response by interfering with signaling pathways and gene transcription to support its own replication. MicroRNAs (miRNAs) are critical posttranscriptional regulators of gene expression and are involved in defense against bacterial infections. Several pathogens have been shown to exploit the host miRNA machinery to their advantage. We therefore hypothesize that macrophage miRNAs exert positive or negative control over intracellular replication. We found significant regulation of 85 miRNAs in human macrophages upon infection. Chromatin immunoprecipitation and sequencing revealed concordant changes of histone acetylation at the putative promoters. Interestingly, a trio of miRNAs (miR-125b, miR-221, and miR-579) was found to significantly affect intracellular replication in a cooperative manner. Using proteome-analysis, we pinpointed this effect to a concerted downregulation of galectin-8 (LGALS8), DExD/H-box helicase 58 (DDX58), tumor protein P53 (TP53), and then MX dynamin-like GTPase 1 (MX1) by the three miRNAs. In summary, our results demonstrate a new miRNA-controlled immune network restricting replication in human macrophages. Cases of pneumonia occur worldwide, with potentially fatal outcome. When causing human disease, injects a plethora of virulence factors to reprogram macrophages to circumvent immune defense and create a replication niche. By analyzing -induced changes in miRNA expression and genomewide chromatin modifications in primary human macrophages, we identified a cell-autonomous immune network restricting growth. This network comprises three miRNAs governing expression of the cytosolic RNA receptor DDX58/RIG-I, the tumor suppressor TP53, the antibacterial effector LGALS8, and MX1, which has been described as an antiviral factor. Our findings for the first time link TP53, LGALS8, DDX58, and MX1 in one miRNA-regulated network and integrate them into a functional node in the defense against .
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http://dx.doi.org/10.1128/mBio.03155-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7157531PMC
March 2020

Caspase-11 promotes allergic airway inflammation.

Nat Commun 2020 02 26;11(1):1055. Epub 2020 Feb 26.

School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute (TBSI)Trinity College Dublin, Dublin, Ireland.

Activated caspase-1 and caspase-11 induce inflammatory cell death in a process termed pyroptosis. Here we show that Prostaglandin E (PGE) inhibits caspase-11-dependent pyroptosis in murine and human macrophages. PGE suppreses caspase-11 expression in murine and human macrophages and in the airways of mice with allergic inflammation. Remarkably, caspase-11-deficient mice are strongly resistant to developing experimental allergic airway inflammation, where PGE is known to be protective. Expression of caspase-11 is elevated in the lung of wild type mice with allergic airway inflammation. Blocking PGE production with indomethacin enhances, whereas the prostaglandin E analog misoprostol inhibits lung caspase-11 expression. Finally, alveolar macrophages from asthma patients exhibit increased expression of caspase-4, a human homologue of caspase-11. Our findings identify PGE as a negative regulator of caspase-11-driven pyroptosis and implicate caspase-4/11 as a critical contributor to allergic airway inflammation, with implications for pathophysiology of asthma.
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http://dx.doi.org/10.1038/s41467-020-14945-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7044193PMC
February 2020

Transcriptional analysis identifies potential biomarkers and molecular regulators in pneumonia and COPD exacerbation.

Sci Rep 2020 01 14;10(1):241. Epub 2020 Jan 14.

Institute for Lung Research, Universities of Giessen and Marburg Lung Center, German Center for Lung Research (DZL), Marburg, Germany.

Lower respiratory infections, such as community-acquired pneumonia (CAP), and chronic obstructive pulmonary disease (COPD) rank among the most frequent causes of death worldwide. Improved diagnostics and profound pathophysiological insights are urgent clinical needs. In our cohort, we analysed transcriptional networks of peripheral blood mononuclear cells (PBMCs) to identify central regulators and potential biomarkers. We investigated the mRNA- and miRNA-transcriptome of PBMCs of healthy subjects and patients suffering from CAP or AECOPD by microarray and Taqman Low Density Array. Genes that correlated with PBMC composition were eliminated, and remaining differentially expressed genes were grouped into modules. One selected module (120 genes) was particularly suitable to discriminate AECOPD and CAP and most notably contained a subset of five biologically relevant mRNAs that differentiated between CAP and AECOPD with an AUC of 86.1%. Likewise, we identified several microRNAs, e.g. miR-545-3p and miR-519c-3p, which separated AECOPD and CAP. We furthermore retrieved an integrated network of differentially regulated mRNAs and microRNAs and identified HNF4A, MCC and MUC1 as central network regulators or most important discriminatory markers. In summary, transcriptional analysis retrieved potential biomarkers and central molecular features of CAP and AECOPD.
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http://dx.doi.org/10.1038/s41598-019-57108-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6959367PMC
January 2020

IL-17 CD8 T cell suppression by dimethyl fumarate associates with clinical response in multiple sclerosis.

Nat Commun 2019 12 16;10(1):5722. Epub 2019 Dec 16.

Institute for Medical Microbiology and Hospital Hygiene, University of Marburg, 35043, Marburg, Germany.

IL-17-producing CD8 (Tc17) cells are enriched in active lesions of patients with multiple sclerosis (MS), suggesting a role in the pathogenesis of autoimmunity. Here we show that amelioration of MS by dimethyl fumarate (DMF), a mechanistically elusive drug, associates with suppression of Tc17 cells. DMF treatment results in reduced frequency of Tc17, contrary to Th17 cells, and in a decreased ratio of the regulators RORC-to-TBX21, along with a shift towards cytotoxic T lymphocyte gene expression signature in CD8 T cells from MS patients. Mechanistically, DMF potentiates the PI3K-AKT-FOXO1-T-BET pathway, thereby limiting IL-17 and RORγt expression as well as STAT5-signaling in a glutathione-dependent manner. This results in chromatin remodeling at the Il17 locus. Consequently, T-BET-deficiency in mice or inhibition of PI3K-AKT, STAT5 or reactive oxygen species prevents DMF-mediated Tc17 suppression. Overall, our data disclose a DMF-AKT-T-BET driven immune modulation and suggest putative therapy targets in MS and beyond.
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http://dx.doi.org/10.1038/s41467-019-13731-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6915776PMC
December 2019

Antibacterial activity of a defensin in an infection model of .

Virulence 2019 12;10(1):902-909

Institute for Lung Research, Universities of Giessen and Marburg Lung Center, Philipps-University Marburg, Member of the German Center for Lung Research (DZL), Marburg, Germany.

() is the most common bacterial cause of community-acquired pneumonia. Increasing rates of antibiotic-resistant strains impair therapy and necessitate alternative treatment options. In this study, we analysed insect-derived antimicrobial peptides (AMPs) for antibacterial effects on in a human infection model.AMP effects on bacterial growth were examined by colony forming unit (CFU)-assays, and growth curve measurements. Furthermore, cytotoxicity to primary human macrophages was detected by measuring lactate-dehydrogenase release to the supernatant. One AMP (Defensin 1) was tested in a model of primary human monocyte-derived macrophages infected with strain D39 and a multi-resistant clinical isolate. Inflammatory reactions were characterised by qPCR and multiplex-ELISA.In total, the antibacterial effects of 23 AMPs were characterized. Only Defensin 1 showed significant antibacterial effects against strain D39 and a multi-resistant clinical isolate. During infection of primary human macrophages with D39, Defensin 1 displayed strong antibacterial effects, and consequently reduced bacteria-induced cytokine expression and release.In summary, Defensin 1 showed profound antibacterial effectivity against D39 and a multi-resistant clinical isolate without unwanted cytotoxic or inflammatory side effects on human blood-derived macrophages.
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http://dx.doi.org/10.1080/21505594.2019.1685150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6844301PMC
December 2019

Surface Proteome of Plasma Extracellular Vesicles as Biomarkers for Pneumonia and Acute Exacerbation of Chronic Obstructive Pulmonary Disease.

J Infect Dis 2020 01;221(2):325-335

Institute for Lung Research, Universities of Giessen and Marburg Lung Center, Philipps-University Marburg, Member of the German Center for Lung Research, Marburg, Germany.

Background: Community-acquired pneumonia (CAP) and acute exacerbation of chronic obstructive pulmonary disease (AECOPD) represent a major burden of disease and death and their differential diagnosis is critical. A potential source of relevant accessible biomarkers are blood-borne small extracellular vesicles (sEVs).

Methods: We performed an extracellular vesicle array to find proteins on plasma sEVs that are differentially expressed and possibly allow the differential diagnosis between CAP and AECOPD. Plasma samples were analyzed from 21 healthy controls, 24 patients with CAP, and 10 with AECOPD . The array contained 40 antibodies to capture sEVs, which were then visualized with a cocktail of biotin-conjugated CD9, CD63, and CD81 antibodies.

Results: We detected significant differences in the protein decoration of sEVs between healthy controls and patients with CAP or AECOPD. We found CD45 and CD28 to be the best discrimination markers between CAP and AECOPD in receiver operating characteristic analyses, with an area under the curve >0.92. Additional ensemble feature selection revealed the possibility to distinguish between CAP and AECOPD even if the patient with CAP had COPD, with a panel of CD45, CD28, CTLA4 (cytotoxic T-lymphocyte-associated protein 4), tumor necrosis factor-R-II, and CD16.

Conclusion: The discrimination of sEV-associated proteins is a minimally invasive method with potential to discriminate between CAP and AECOPD.
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http://dx.doi.org/10.1093/infdis/jiz460DOI Listing
January 2020

Peptidoglycan Recognition Protein 4 Limits Bacterial Clearance and Inflammation in Lungs by Control of the Gut Microbiota.

Front Immunol 2019 20;10:2106. Epub 2019 Sep 20.

Department of Infectious Diseases and Respiratory Medicine, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin Institute of Health, Berlin, Germany.

is the most frequent cause of community-acquired pneumonia. Endogenous host defense molecules such as peptidoglycan recognition protein 4 (PGLYRP4) might influence the course of this disease. To the best of our knowledge, there are no reports on the relevance of PGLYRP4 in pneumonia. Therefore, wild type (WT) and PGLYRP4-deficient (PGLYRP4KO) mice were analyzed in an and experimental setting to examine the influence of PGLYRP4 on the course of pneumococcal pneumonia. Furthermore, caecal 16S rRNA microbiome analysis was performed, and microbiota were transferred to germfree WT mice to assess the influence of microbiotal communities on the bacterial burden. Mice lacking PGLYRP4 displayed an enhanced bacterial clearance in the lungs, and fewer mice developed bacteremia. In addition, an increased recruitment of immune cells to the site of infection, and an enhanced bacterial killing by stronger activation of phagocytes could be shown. This may depend partly on the detected higher expression of complement factors, interferon-associated genes, and the higher pro-inflammatory cytokine response in isolated primary PGLYRP4KO vs. WT cells. This phenotype is underlined by changes in the complexity and composition of the caecal microbiota of PGLYRP4KO compared to WT mice. Strikingly, we provided evidence, by cohousing and stable transfer of the respective WT or PGLYRP4KO mice microbiota into germfree WT mice, that the changes of the microbiota are responsible for the improved clearance of lung infection. In conclusion, the deficiency of PGLYRP4, a known antibacterial protein, leads to changes in the gut microbiota. Thus, alterations in the microbiota can change the susceptibility to lung infection independently of the host genotype.
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http://dx.doi.org/10.3389/fimmu.2019.02106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6763742PMC
October 2020

Intestinal development and homeostasis require activation and apoptosis of diet-reactive T cells.

J Clin Invest 2019 04 2;129(5):1972-1983. Epub 2019 Apr 2.

Institute for Medical Microbiology and Hospital Hygiene, Philipps University of Marburg, Germany.

The impact of food antigens on intestinal homeostasis and immune function is poorly understood. Here, we explored the impact of dietary antigens on the phenotype and fate of intestinal T cells. Physiological uptake of dietary proteins generated a highly activated CD44+Helios+CD4+ T cell population predominantly in Peyer patches. These cells are distinct from regulatory T cells and develop independently of the microbiota. Alimentation with a protein-free, elemental diet led to an atrophic small intestine with low numbers of activated T cells, including Tfh cells and decreased amounts of intestinal IgA and IL-10. Food-activated CD44+Helios+CD4+ T cells in the Peyer patches are controlled by the immune checkpoint molecule PD-1. Blocking the PD-1 pathway rescued these T cells from apoptosis and triggered proinflammatory cytokine production, which in IL-10-deficient mice was associated with intestinal inflammation. In support of these findings, our study of patients with Crohn's disease revealed significantly reduced frequencies of apoptotic CD4+ T cells in Peyer patches as compared with healthy controls. These results suggest that apoptosis of diet-activated T cells is a hallmark of the healthy intestine.
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http://dx.doi.org/10.1172/JCI98929DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6486345PMC
April 2019

Intracellular bacteria engage a STING-TBK1-MVB12b pathway to enable paracrine cGAS-STING signalling.

Nat Microbiol 2019 04 25;4(4):701-713. Epub 2019 Feb 25.

Department of Biomedicine, Aarhus University, Aarhus, Denmark.

The innate immune system is crucial for eventual control of infections, but may also contribute to pathology. Listeria monocytogenes is an intracellular Gram-positive bacteria and a major cause of food-borne disease. However, important knowledge on the interactions between L. monocytogenes and the immune system is still missing. Here, we report that Listeria DNA is sorted into extracellular vesicles (EVs) in infected cells and delivered to bystander cells to stimulate the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) pathway. This was also observed during infections with Francisella tularensis and Legionella pneumophila. We identify the multivesicular body protein MVB12b as a target for TANK-binding kinase 1 phosphorylation, which is essential for the sorting of DNA into EVs and stimulation of bystander cells. EVs from Listeria-infected cells inhibited T-cell proliferation, and primed T cells for apoptosis. Collectively, we describe a pathway for EV-mediated delivery of foreign DNA to bystander cells, and suggest that intracellular bacteria exploit this pathway to impair antibacterial defence.
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http://dx.doi.org/10.1038/s41564-019-0367-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433288PMC
April 2019

ncRNAs in Inflammatory and Infectious Diseases.

Methods Mol Biol 2019 ;1912:3-32

Institute for Lung Research, Universities of Giessen and Marburg Lung Center, Member of the German Center for Lung Research (DZL), Philipps-University Marburg, Marburg, Germany.

Inflammatory and infectious diseases are among the main causes of morbidity and mortality worldwide. Inflammation is central to maintenance of organismal homeostasis upon infection, tissue damage, and malignancy. It occurs transiently in response to diverse stimuli (e.g., physical, radioactive, infective, pro-allergenic, or toxic), and in some cases may manifest itself in chronic diseases. To limit the potentially deleterious effects of acute or chronic inflammatory responses, complex transcriptional and posttranscriptional regulatory networks have evolved, often involving nonprotein-coding RNAs (ncRNA). MicroRNAs (miRNAs) are a class of posttranscriptional regulators that control mRNA translation and stability. Long ncRNAs (lncRNAs) are a very diverse group of transcripts >200 nt, functioning among others as scaffolds or decoys both in the nucleus and the cytoplasm. By now, it is well established that miRNAs and lncRNAs are implicated in all major cellular processes including control of cell death, proliferation, or metabolism. Extensive research over the last years furthermore revealed a fundamental role of ncRNAs in pathogen recognition and inflammatory responses. This chapter reviews and summarizes the current knowledge on regulatory ncRNA networks in infection and inflammation.
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http://dx.doi.org/10.1007/978-1-4939-8982-9_1DOI Listing
June 2019

Proviral MicroRNAs Detected in Extracellular Vesicles From Bronchoalveolar Lavage Fluid of Patients With Influenza Virus-Induced Acute Respiratory Distress Syndrome.

J Infect Dis 2019 01;219(4):540-543

Institute for Lung Research, Universities of Giessen and Marburg Lung Center.

Influenza A virus (IAV) causes severe respiratory infections and alveolar epithelial damage resulting in acute respiratory distress syndrome (ARDS). Extracellular vesicles (EVs) have been shown to mediate cellular crosstalk in inflammation by transfer of microRNAs (miRNAs). In this study, we found significant changes in the miRNA composition of EVs in the bronchoalveolar lavage fluid from patients with IAV-induced ARDS. Among the 9 significantly deregulated microRNAs, miR-17-5p was upregulated in patients' BALF and in EVs of IAV-infected lung epithelial cells (A549). In these cells, transfer of miR-17-5p strongly downregulated expression of the antiviral factor Mx1 and significantly enhanced IAV replication.
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http://dx.doi.org/10.1093/infdis/jiy554DOI Listing
January 2019

Sphingosine Kinase 1 Regulates Inflammation and Contributes to Acute Lung Injury in Pneumococcal Pneumonia via the Sphingosine-1-Phosphate Receptor 2.

Crit Care Med 2018 03;46(3):e258-e267

Department of Infectious Diseases and Pulmonary Medicine, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.

Objectives: Severe pneumonia may evoke acute lung injury, and sphingosine-1-phosphate is involved in the regulation of vascular permeability and immune responses. However, the role of sphingosine-1-phosphate and the sphingosine-1-phosphate producing sphingosine kinase 1 in pneumonia remains elusive. We examined the role of the sphingosine-1-phosphate system in regulating pulmonary vascular barrier function in bacterial pneumonia.

Design: Controlled, in vitro, ex vivo, and in vivo laboratory study.

Subjects: Female wild-type and SphK1-deficient mice, 8-10 weeks old. Human postmortem lung tissue, human blood-derived macrophages, and pulmonary microvascular endothelial cells.

Interventions: Wild-type and SphK1-deficient mice were infected with Streptococcus pneumoniae. Pulmonary sphingosine-1-phosphate levels, messenger RNA expression, and permeability as well as lung morphology were analyzed. Human blood-derived macrophages and human pulmonary microvascular endothelial cells were infected with S. pneumoniae. Transcellular electrical resistance of human pulmonary microvascular endothelial cell monolayers was examined. Further, permeability of murine isolated perfused lungs was determined following exposition to sphingosine-1-phosphate and pneumolysin.

Measurements And Main Results: Following S. pneumoniae infection, murine pulmonary sphingosine-1-phosphate levels and sphingosine kinase 1 and sphingosine-1-phosphate receptor 2 expression were increased. Pneumonia-induced lung hyperpermeability was reduced in SphK1 mice compared with wild-type mice. Expression of sphingosine kinase 1 in macrophages recruited to inflamed lung areas in pneumonia was observed in murine and human lungs. S. pneumoniae induced the sphingosine kinase 1/sphingosine-1-phosphate system in blood-derived macrophages and enhanced sphingosine-1-phosphate receptor 2 expression in human pulmonary microvascular endothelial cell in vitro. In isolated mouse lungs, pneumolysin-induced hyperpermeability was dose dependently and synergistically increased by sphingosine-1-phosphate. This sphingosine-1-phosphate-induced increase was reduced by inhibition of sphingosine-1-phosphate receptor 2 or its downstream effector Rho-kinase.

Conclusions: Our data suggest that targeting the sphingosine kinase 1-/sphingosine-1-phosphate-/sphingosine-1-phosphate receptor 2-signaling pathway in the lung may provide a novel therapeutic perspective in pneumococcal pneumonia for prevention of acute lung injury.
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http://dx.doi.org/10.1097/CCM.0000000000002916DOI Listing
March 2018

THP-1-derived macrophages render lung epithelial cells hypo-responsive to Legionella pneumophila - a systems biology study.

Sci Rep 2017 09 20;7(1):11988. Epub 2017 Sep 20.

Institute for Lung Research, Universities of Giessen and Marburg Lung Center, Philipps-University Marburg, Member of the German Center for Lung Research (DZL), Marburg, Germany.

Immune response in the lung has to protect the huge alveolar surface against pathogens while securing the delicate lung structure. Macrophages and alveolar epithelial cells constitute the first line of defense and together orchestrate the initial steps of host defense. In this study, we analysed the influence of macrophages on type II alveolar epithelial cells during Legionella pneumophila-infection by a systems biology approach combining experimental work and mathematical modelling. We found that L. pneumophila-infected THP-1-derived macrophages provoke a pro-inflammatory activation of neighboring lung epithelial cells, but in addition render them hypo-responsive to direct infection with the same pathogen. We generated a kinetic mathematical model of macrophage activation and identified a paracrine mechanism of macrophage-secreted IL-1β inducing a prolonged IRAK-1 degradation in lung epithelial cells. This intercellular crosstalk may help to avoid an overwhelming inflammatory response by preventing excessive local secretion of pro-inflammatory cytokines and thereby negatively regulating the recruitment of immune cells to the site of infection. This suggests an important but ambivalent immunomodulatory role of macrophages in lung infection.
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http://dx.doi.org/10.1038/s41598-017-12154-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5607273PMC
September 2017

Legionella pneumophila infection activates bystander cells differentially by bacterial and host cell vesicles.

Sci Rep 2017 07 24;7(1):6301. Epub 2017 Jul 24.

Institute for Lung Research, German Center for Lung Research, Universities of Giessen and Marburg Lung Centre, Philipps-University Marburg, 35043, Marburg, Germany.

Extracellular vesicles from eukaryotic cells and outer membrane vesicles (OMVs) released from gram-negative bacteria have been described as mediators of pathogen-host interaction and intercellular communication. Legionella pneumophila (L. pneumophila) is a causative agent of severe pneumonia. The differential effect of bacterial and host cell vesicles in L. pneumophila infection is unknown so far. We infected THP-1-derived or primary human macrophages with L. pneumophila and isolated supernatant vesicles by differential centrifugation. We observed an increase of exosomes in the 100 k pellet by nanoparticle tracking analysis, electron microscopy, and protein markers. This fraction additionally contained Legionella LPS, indicating also the presence of OMVs. In contrast, vesicles in the 16 k pellet, representing microparticles, decreased during infection. The 100 k vesicle fraction activated uninfected primary human alveolar epithelial cells, A549 cells, and THP-1 cells. Epithelial cell activation was reduced by exosome depletion (anti-CD63, or GW4869), or blocking of IL-1β in the supernatant. In contrast, the response of THP-1 cells to vesicles was reduced by a TLR2-neutralizing antibody, UV-inactivation of bacteria, or - partially - RNase-treatment of vesicles. Taken together, we found that during L. pneumophila infection, neighbouring epithelial cells were predominantly activated by exosomes and cytokines, whereas myeloid cells were activated by bacterial OMVs.
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http://dx.doi.org/10.1038/s41598-017-06443-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5524687PMC
July 2017

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

PLoS One 2017 26;12(4):e0176204. Epub 2017 Apr 26.

Institute for Lung Research/iLung, German Center for Lung Research, Universities of Giessen and Marburg Lung Center, Philipps-University Marburg, Marburg, Germany.

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0176204PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5406027PMC
September 2017

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages.

J Vis Exp 2017 02 22(120). Epub 2017 Feb 22.

Institute for Lung Research, Universities of Giessen and Marburg Lung Center, Philipps-University Marburg; German Center for Lung Research; Department of Medicine, Pulmonary and Critical Care Medicine, University Medical Center Giessen and Marburg;

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.
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http://dx.doi.org/10.3791/55146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409326PMC
February 2017

An Interactive Macrophage Signal Transduction Map Facilitates Comparative Analyses of High-Throughput Data.

J Immunol 2017 03 30;198(5):2191-2201. Epub 2017 Jan 30.

Labor für Systemtumorimmunologie, Hautklinik, Friedrich-Alexander-Universität Erlangen-Nürnberg und Universitätsklinikum Erlangen, 91054 Erlangen, Germany;

Macrophages (Mϕs) are key players in the coordination of the lifesaving or detrimental immune response against infections. The mechanistic understanding of the functional modulation of Mϕs by pathogens and pharmaceutical interventions at the signal transduction level is still far from complete. The complexity of pathways and their cross-talk benefits from holistic computational approaches. In the present study, we reconstructed a comprehensive, validated, and annotated map of signal transduction pathways in inflammatory Mϕs based on the current literature. In a second step, we selectively expanded this curated map with database knowledge. We provide both versions to the scientific community via a Web platform that is designed to facilitate exploration and analysis of high-throughput data. The platform comes preloaded with logarithmic fold changes from 44 data sets on Mϕ stimulation. We exploited three of these data sets-human primary Mϕs infected with the common lung pathogens , , or -in a case study to show how our map can be customized with expression data to pinpoint regulated subnetworks and druggable molecules. From the three infection scenarios, we extracted a regulatory core of 41 factors, including TNF, CCL5, CXCL10, IL-18, and IL-12 p40, and identified 140 drugs targeting 16 of them. Our approach promotes a comprehensive systems biology strategy for the exploitation of high-throughput data in the context of Mϕ signal transduction. In conclusion, we provide a set of tools to help scientists unravel details of Mϕ signaling. The interactive version of our Mϕ signal transduction map is accessible online at https://vcells.net/macrophage.
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http://dx.doi.org/10.4049/jimmunol.1502513DOI Listing
March 2017

Genome-wide Chromatin Profiling of Legionella pneumophila-Infected Human Macrophages Reveals Activation of the Probacterial Host Factor TNFAIP2.

J Infect Dis 2016 08 29;214(3):454-63. Epub 2016 Apr 29.

Institute for Lung Research/iLung Department of Medicine, Pulmonary, and Critical Care Medicine, University Medical Center Marburg, Philipps-University Universities of Giessen and arburg Lung Centre, German Center for Lung Research.

Background: Legionella pneumophila is a causative agent of severe pneumonia. Infection leads to a broad host cell response, as evident, for example, on the transcriptional level. Chromatin modifications, which control gene expression, play a central role in the transcriptional response to L. pneumophila

Methods: We infected human-blood-derived macrophages (BDMs) with L. pneumophila and used chromatin immunoprecipitation followed by sequencing to screen for gene promoters with the activating histone 4 acetylation mark.

Results: We found the promoter of tumor necrosis factor α-induced protein 2 (TNFAIP2) to be acetylated at histone H4. This factor has not been characterized in the pathology of L. pneumophila TNFAIP2 messenger RNA and protein were upregulated in response to L. pneumophila infection of human-BDMs and human alveolar epithelial (A549) cells. We showed that L. pneumophila-induced TNFAIP2 expression is dependent on the NF-κB transcription factor. Importantly, knock down of TNFAIP2 led to reduced intracellular replication of L. pneumophila Corby in A549 cells.

Conclusions: Taken together, genome-wide chromatin analysis of L. pneumophila-infected macrophages demonstrated induction of TNFAIP2, a NF-κB-dependent factor relevant for bacterial replication.
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http://dx.doi.org/10.1093/infdis/jiw171DOI Listing
August 2016

Legionella pneumophila-Derived Outer Membrane Vesicles Promote Bacterial Replication in Macrophages.

PLoS Pathog 2016 04 22;12(4):e1005592. Epub 2016 Apr 22.

Institute for Lung Research, Universities of Giessen and Marburg Lung Center, Philipps-University Marburg, Member of the German Center for Lung Research (DZL), Marburg, Germany.

The formation and release of outer membrane vesicles (OMVs) is a phenomenon of Gram-negative bacteria. This includes Legionella pneumophila (L. pneumophila), a causative agent of severe pneumonia. Upon its transmission into the lung, L. pneumophila primarily infects and replicates within macrophages. Here, we analyzed the influence of L. pneumophila OMVs on macrophages. To this end, differentiated THP-1 cells were incubated with increasing doses of Legionella OMVs, leading to a TLR2-dependent classical activation of macrophages with the release of pro-inflammatory cytokines. Inhibition of TLR2 and NF-κB signaling reduced the induction of pro-inflammatory cytokines. Furthermore, treatment of THP-1 cells with OMVs prior to infection reduced replication of L. pneumophila in THP-1 cells. Blocking of TLR2 activation or heat denaturation of OMVs restored bacterial replication in the first 24 h of infection. With prolonged infection-time, OMV pre-treated macrophages became more permissive for bacterial replication than untreated cells and showed increased numbers of Legionella-containing vacuoles and reduced pro-inflammatory cytokine induction. Additionally, miRNA-146a was found to be transcriptionally induced by OMVs and to facilitate bacterial replication. Accordingly, IRAK-1, one of miRNA-146a's targets, showed prolonged activation-dependent degradation, which rendered THP-1 cells more permissive for Legionella replication. In conclusion, L. pneumophila OMVs are initially potent pro-inflammatory stimulators of macrophages, acting via TLR2, IRAK-1, and NF-κB, while at later time points, OMVs facilitate L. pneumophila replication by miR-146a-dependent IRAK-1 suppression. OMVs might thereby promote spreading of L. pneumophila in the host.
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http://dx.doi.org/10.1371/journal.ppat.1005592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4841580PMC
April 2016

MicroRNAs Constitute a Negative Feedback Loop in Streptococcus pneumoniae-Induced Macrophage Activation.

J Infect Dis 2016 07 16;214(2):288-99. Epub 2016 Mar 16.

Institute for Lung Research, German Center for Lung Research, Universities of Giessen and Marburg Lung Center Department of Medicine, Pulmonary and Critical Care Medicine, University Medical Center Giessen and Marburg, Philipps-University Marburg.

Streptococcus pneumoniae causes high mortality as a major pneumonia-inducing pathogen. In pneumonia, control of innate immunity is necessary to prevent organ damage. We assessed the role of microRNAs (miRNAs) as regulators in pneumococcal infection of human macrophages. Exposure of primary blood-derived human macrophages with pneumococci resulted in transcriptional changes in several gene clusters and a significant deregulation of 10 microRNAs. Computational network analysis retrieved miRNA-146a as one putatively important regulator of pneumococci-induced host cell activation. Its induction depended on bacterial structural integrity and was completely inhibited by blocking Toll-like receptor 2 (TLR-2) or depleting its mediator MyD88. Furthermore, induction of miRNA-146a release did not require the autocrine feedback of interleukin 1β and tumor necrosis factor α released from infected macrophages, and it repressed the TLR-2 downstream mediators IRAK-1 and TRAF-6, as well as the inflammatory factors cyclooxygenase 2 and interleukin 1β. In summary, pneumococci recognition induces a negative feedback loop, preventing excessive inflammation via miR-146a and potentially other miRNAs.
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http://dx.doi.org/10.1093/infdis/jiw109DOI Listing
July 2016

Systems Medicine for Lung Diseases: Phenotypes and Precision Medicine in Cancer, Infection, and Allergy.

Methods Mol Biol 2016 ;1386:119-33

Laboratory of Systems Tumor Immunology, Department of Dermatology, Faculty of Medicine, University of Erlangen-Nurnberg, Erlangen, Germany.

Lung diseases cause an enormous socioeconomic burden. Four of them are among the ten most important causes of deaths worldwide: Pneumonia has the highest death toll of all infectious diseases, lung cancer kills the most people of all malignant proliferative disorders, chronic obstructive pulmonary disease (COPD) ranks third in mortality among the chronic noncommunicable diseases, and tuberculosis is still one of the most important chronic infectious diseases. Despite all efforts, for example, by the World Health Organization and clinical and experimental researchers, these diseases are still highly prevalent and harmful. This is in part due to the specific organization of tissue homeostasis, architecture, and immunity of the lung. Recently, several consortia have formed and aim to bring together clinical and molecular data from big cohorts of patients with lung diseases with novel experimental setups, biostatistics, bioinformatics, and mathematical modeling. This "systems medicine" concept will help to match the different disease modalities with adequate therapeutic and possibly preventive strategies for individual patients in the sense of precision medicine.
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http://dx.doi.org/10.1007/978-1-4939-3283-2_8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7153428PMC
October 2016

TLR9- and Src-dependent expression of Krueppel-like factor 4 controls interleukin-10 expression in pneumonia.

Eur Respir J 2013 Feb 31;41(2):384-91. Epub 2012 May 31.

Dept of Internal Medicine/Infectious Diseases and Respiratory Medicine, Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, Berlin, Germany.

The release of potent pro-inflammatory mediators is crucial to mounting an efficient host response during infection. However, excessive inflammation may lead to deleterious tissue damage. This is highlighted in severe pneumococcal pneumonia, in which the delicate balance between a robust inflammatory response necessary to kill pneumococci and the loss of organ function determines the outcome of the disease. We assessed the regulation of the potent anti-inflammatory cytokine interleukin (IL)-10 in pneumococcal infection via Western blot, ELISA and chromatin immunoprecipitation analysis. Streptococcus pneumoniae induced IL-10 expression in mouse lungs and human lung epithelial cells. Pneumococcal infection resulted in a strong induction of Krueppel-like factor (KLF)4 expression in vivo and in vitro. The induction of both IL-10 and KLF4 is mediated by a pathway involving bacterial DNA, Toll-like receptor (TLR)9, MyD88 and Src kinase. KLF4 is recruited to the il10 promoter, and small-interfering RNA-mediated knockdown of KLF4 expression blocked IL-10 expression during pneumococcal infection. In conclusion, KLF4 is induced in a bacterial DNA-TLR9-Src-dependent manner and regulates IL-10 expression, linking the detection of bacterial DNA by TLR9 to the control of an inflammatory response.
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http://dx.doi.org/10.1183/09031936.00196311DOI Listing
February 2013