Pneumolysin Is Responsible for Differential Gene Expression and Modifications in the Epigenetic Landscape of Primary Monocyte Derived Macrophages

Epigenetic modifications regulate gene expression in the host response to a diverse range of pathogens. The extent and consequences of epigenetic modification during macrophage responses to Streptococcus pneumoniae, and the role of pneumolysin, a key Streptococcus pneumoniae virulence factor, in influencing these responses, are currently unknown. To investigate this, we infected human monocyte derived macrophages (MDMs) with Streptococcus pneumoniae and addressed whether pneumolysin altered the epigenetic landscape and the associated acute macrophage transcriptional response using a combined transcriptomic and proteomic approach. Transcriptomic analysis identified 503 genes that were differentially expressed in a pneumolysin-dependent manner in these samples. Pathway analysis highlighted the involvement of transcriptional responses to core innate responses to pneumococci including modules associated with metabolic pathways activated in response to infection, oxidative stress responses and NFκB, NOD-like receptor and TNF signalling pathways. Quantitative proteomic analysis confirmed pneumolysin-regulated protein expression, early after bacterial challenge, in representative transcriptional modules associated with innate immune responses. In parallel, quantitative mass spectrometry identified global changes in the relative abundance of histone post translational modifications (PTMs) upon pneumococcal challenge. We identified an increase in the relative abundance of H3K4me1, H4K16ac and a decrease in H3K9me2 and H3K79me2 in a PLY-dependent fashion. We confirmed that pneumolysin blunted early transcriptional responses involving TNF-α and IL-6 expression. Vorinostat, a histone deacetylase inhibitor, similarly downregulated TNF-α production, reprising the pattern observed with pneumolysin. In conclusion, widespread changes in the macrophage transcriptional response are regulated by pneumolysin and are associated with global changes in histone PTMs. Modulating histone PTMs can reverse pneumolysin-associated transcriptional changes influencing innate immune responses, suggesting that epigenetic modification by pneumolysin plays a role in dampening the innate responses to pneumococci.

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Pneumolysin is responsible for differential gene expression and modifications in the epigenetic landscape of primary monocyte derived macrophages

10.1101/2020.06.08.139980 ◽

2020 ◽

Author(s):

J. Cole ◽

A. Angyal ◽

R. D. Emes ◽

T.J. Mitchell ◽

M.J. Dickman ◽

...

Keyword(s):

Gene Expression ◽

Streptococcus Pneumoniae ◽

Immune Responses ◽

Epigenetic Modification ◽

Transcriptional Response ◽

Innate Immune ◽

Global Changes ◽

Innate Immune Responses ◽

Transcriptional Responses ◽

Tnf Α

AbstractEpigenetic modifications regulate gene expression in the host response to a diverse range of pathogens. The extent and consequences of epigenetic modification during macrophage responses to Streptococcus pneumoniae, and the role of pneumolysin, a key Streptococcus pneumoniae virulence factor, in influencing these responses, are currently unknown. To investigate this, we infected human monocyte derived macrophages (MDMs) with Streptococcus pneumoniae and addressed whether pneumolysin altered the epigenetic landscape and the associated acute macrophage transcriptional response using a combined transcriptomic and proteomic approach. Transcriptomic analysis identified 503 genes that were differentially expressed in a pneumolysin-dependent manner in these samples. Pathway analysis highlighted the involvement of transcriptional responses to core innate responses to pneumococci including modules associated with metabolic pathways activated in response to infection, oxidative stress responses and NFκB, NOD-like receptor and TNF signalling pathways. Quantitative proteomic analysis confirmed pneumolysin-regulated protein expression, early after bacterial challenge, in representative transcriptional modules associated with innate immune responses. In parallel, quantitative mass spectrometry identified global changes in the relative abundance of histone post translational modifications (PTMs) upon pneumococcal challenge. We identified an increase in the relative abundance of H3K4me1, H4K16ac and a decrease in H3K9me2 and H3K79me2 in a PLY-dependent fashion. We confirmed that pneumolysin blunted early transcriptional responses involving TNF-α and IL-6 expression. Vorinostat, a histone deacetylase inhibitor, similarly downregulated TNF production, reprising the pattern observed with pneumolysin. In conclusion, widespread changes in the macrophage transcriptional response are regulated by pneumolysin and are associated with global changes in histone PTMs. Modulating histone PTMs can reverse pneumolysin-associated transcriptional changes influencing innate immune responses, suggesting that epigenetic modification by pneumolysin plays a role in dampening the innate responses to pneumococci.Author summaryPneumolysin is a toxin that contributes to how Streptococcus pneumoniae, the leading cause of pneumonia, causes disease. In this study, the toxin alters gene expression in immune cells called macrophages, one of the first lines of defence against bacteria at sites of infection. Modulation involved multiple immune responses, including generation of chemical signals coordinating responses in immune cells termed cytokines. In addition, changes were observed in histone proteins that are involved in controlling gene expression in the cell. Pneumolysin reduced early production of the cytokine TNF-α and a medicine vorinostat that modifies these ‘epigenetic’ histone modifications had a similar affect, suggesting epigenetic mechanisms contribute to the ability of pneumolysin to reduce immune responses.

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Effect of aerobic exercise on innate immune responses and inflammatory mediators in the spinal cord of diabetic rats

Comparative Exercise Physiology ◽

10.3920/cep190050 ◽

2020 ◽

Vol 16 (4) ◽

pp. 293-301

Author(s):

A. Kaki ◽

M. Nikbakht ◽

A.H. Habibi ◽

H.F. Moghadam

Keyword(s):

Spinal Cord ◽

Neuropathic Pain ◽

Immune Responses ◽

Diabetic Neuropathy ◽

Aerobic Exercise ◽

Inflammatory Mediators ◽

Innate Immune ◽

Moderate Intensity ◽

Innate Immune Responses ◽

Tnf Α

Neuronal inflammation is one of the pathophysiological causes of diabetes neuropathic pain. The purpose of this research was to determine the effect of aerobic exercise on innate immune responses and inflammatory mediators in the spinal dorsal horn in rats with diabetic neuropathic pain. 40 eight-week-old male Wistar rats (weight range 220±10.2 g) were randomly divided into four groups of (1) sedentary diabetic neuropathy (SDN), (2) training diabetic neuropathy (TDN), (3) training control (TC), and (4) sedentary control (SC). Diabetes was induced by injection of streptozocin (50 mg/kg). Following confirmation of behavioural tests for diabetes neuropathy, the training groups performed 6 weeks of moderate-intensity aerobic exercise on the treadmill. The expression of Toll like receptor (TLR)4, TLR2, tumour necrosis factor (TNF)-α, interleukin (IL)-1β and IL-10 genes in L4-L6 spinal cord sensory neurons was measured by Real Time PCR. Two-way ANOVA and Bonferroni’s post hoc tests were used for statistical analysis. After performing aerobic exercise protocol, the TDN compared to the SDN showed a significant decrease in the mean score of pain in the formalin test and a significant increase in the latency in Tail-Flick test was observed. The expression of TLR4, TLR2, TNF-α and IL-1β genes was significantly higher in the SDN than in the SC group (P<0.05). The expression of the above genes in the TDN was significantly lower than the SDN group (P<0.05). Also, the expression level of IL-10 gene was significantly higher in the TDN than the SDN group (P<0.05). Aerobic exercise improved sensitivity of nociceptors to pain-inducing agents in diabetic neuropathy due to inhibition of inflammatory receptors and increased levels of anti-inflammatory agents in the nervous system. Thus, aerobic exercise should be used as a non-pharmacological intervention for diabetic patients to reduce neuropathic pain.

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Commensal and Opportunistic Bacteria Present in the Microbiota in Atlantic Cod (Gadus morhua) Larvae Differentially Alter the Hosts’ Innate Immune Responses

Microorganisms ◽

10.3390/microorganisms10010024 ◽

2021 ◽

Vol 10 (1) ◽

pp. 24

Author(s):

Ragnhild Inderberg Vestrum ◽

Torunn Forberg ◽

Birgit Luef ◽

Ingrid Bakke ◽

Per Winge ◽

...

Keyword(s):

Immune System ◽

Immune Responses ◽

Atlantic Cod ◽

Innate Immune ◽

Rrna Gene ◽

Innate Immune Responses ◽

Transcriptional Responses ◽

Gut Morphology ◽

Germ Free ◽

Opportunistic Bacteria

The roles of host-associated bacteria have gained attention lately, and we now recognise that the microbiota is essential in processes such as digestion, development of the immune system and gut function. In this study, Atlantic cod larvae were reared under germ-free, gnotobiotic and conventional conditions. Water and fish microbiota were characterised by 16S rRNA gene analyses. The cod larvae’s transcriptional responses to the different microbial conditions were analysed by a custom Agilent 44 k oligo microarray. Gut development was assessed by transmission electron microscopy (TEM). Water and fish microbiota differed significantly in the conventional treatment and were dominated by different fast-growing bacteria. Our study indicates that components of the innate immune system of cod larvae are downregulated by the presence of non-pathogenic bacteria, and thus may be turned on by default in the early larval stages. We see indications of decreased nutrient uptake in the absence of bacteria. The bacteria also influence the gut morphology, reflected in shorter microvilli with higher density in the conventional larvae than in the germ-free larvae. The fact that the microbiota alters innate immune responses and gut morphology demonstrates its important role in marine larval development.

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Screening for Toxoplasma gondii-Regulated Transcriptional Responses in Lipopolysaccharide-Activated Macrophages

Infection and Immunity ◽

10.1128/iai.74.3.1916-1923.2006 ◽

2006 ◽

Vol 74 (3) ◽

pp. 1916-1923 ◽

Cited By ~ 17

Author(s):

Chiang W. Lee ◽

Soumaya Bennouna ◽

Eric Y. Denkers

Keyword(s):

Toxoplasma Gondii ◽

Defense Mechanisms ◽

Transcriptional Response ◽

Interleukin 12 ◽

Enzyme Linked Immunosorbent Assay ◽

Cell Mediated Immunity ◽

Transcriptional Responses ◽

Necrosis Factor Alpha ◽

Reverse Transcription Pcr ◽

Tnf Α

ABSTRACT Toxoplasma gondii-infected macrophages are blocked in production of the proinflammatory cytokines interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-α) upon activation with lipopolysaccharide (LPS). Here, we used pathway-focused cDNA arrays to identify additional T. gondii-regulated transcriptional responses. Parasite infection decreased 57 (inclusive of IL-12 and TNF-α) and increased expression of 7 of 77 LPS-activated cytokine and cytokine-related genes. Interestingly, we found that the LPS-induced transcriptional response of the anti-inflammatory cytokine IL-10 was synergistically increased by T. gondii, results that we validated by conventional reverse transcription-PCR and enzyme-linked immunosorbent assay. Importantly, although the parasite exerted disparate effects in LPS-signaling leading to TNF-α versus IL-10 production, both responses required functional Toll-like receptor 4. We suggest that these effects represent parasite defense mechanisms to avoid or delay induction of antimicrobial activity and/or T-cell-mediated immunity during Toxoplasma infection.

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Toll-Like Receptor-Initiated Testicular Innate Immune Responses in Mouse Leydig Cells

Endocrinology ◽

10.1210/en.2011-0031 ◽

2011 ◽

Vol 152 (7) ◽

pp. 2827-2836 ◽

Cited By ~ 50

Author(s):

Tao Shang ◽

Xiaoyan Zhang ◽

Tao Wang ◽

Bing Sun ◽

Tingting Deng ◽

...

Keyword(s):

Immune Responses ◽

Tyrosine Kinases ◽

Leydig Cells ◽

Innate Immune ◽

Inflammatory Factors ◽

Innate Immune Responses ◽

Polyinosinic:Polycytidylic Acid ◽

Tnf Α ◽

Microbial Infections ◽

Local Cell

The testis is an immunoprivileged site, where the local cell-initiated testicular innate immune responses play a crucial role in defense against microbial infections. Mechanisms modulating the testicular cell-built defense system remain to be clarified. In this article, we demonstrate that Leydig cells, a major cell population in the testicular interstitium, initiate innate immunity through the activation of Toll-like receptors (TLRs). Several TLRs are expressed in mouse Leydig cells; among these, TLR3 and TLR4 are expressed at relatively high levels compared with other TLR members. Both TLR3 and TLR4 can be activated by their agonists (polyinosinic:polycytidylic acid and lipopolysaccharide) in Leydig cells and subsequently induce the production of inflammatory factors, such as IL-1β, IL-6, TNF-α, and type 1 interferons (IFN) (IFN-α and IFN-β). Notably, the activation of TLR3 and TLR4 suppresses steroidogenesis by Leydig cells. Further, we provide evidence that Axl and Mer receptor tyrosine kinases are expressed in Leydig cells and regulate TLR-mediated innate immune responses negatively. Data presented here describe a novel function of Leydig cells in eliciting testicular innate immune responses that should contribute to the protection of the testis from microbial infections.

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Brassica rapa L. activates macrophages via Toll-like receptors

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbaa075 ◽

2021 ◽

Vol 85 (3) ◽

pp. 656-665

Author(s):

Fuka Takahashi ◽

Katsunori Endo ◽

Rina Matsui ◽

Kana Yamamoto ◽

Sachi Tanaka

Keyword(s):

Brassica Rapa ◽

Polymyxin B ◽

Peritoneal Macrophages ◽

Interferon Γ ◽

Insoluble Fraction ◽

Innate Immune ◽

Innate Immune Responses ◽

Toll Like Receptor ◽

Tnf Α ◽

Necrosis Factor

ABSTRACT Macrophages can initiate innate immune responses against microbes and cancer. The aim of this study was to elucidate the effects of Brassica rapa L. on macrophages. The production of interleukin (IL)-6, tumor necrosis factor (TNF)-α, and interferon-γ induced by the insoluble fraction of B. rapa L. was decreased in macrophage-depleted spleen cells compared with controls. The insoluble fraction of B. rapa L. induced expression of H-2Kb, I-Ab, CD40, and CD86, production of cytokines and nitric oxide, and phagocytic activity in RAW264 cells. After treatment with the insoluble fraction, IL-6 and TNF-α production was significantly decreased by anti-Toll-like receptor (TLR)2 mAb or polymyxin B compared with the control. Furthermore, insoluble fraction-mediated cytokine production was significantly lower in peritoneal macrophages from TLR2−/− and TLR4−/− mice compared with wild-type mice. These results suggest that B. rapa L. is a potentially effective immunomodulator for activating macrophages to prevent infections.

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Innate immune responses at the asymptomatic stage of influenza A viral infections of Streptococcus pneumoniae colonized and non-colonized mice

Scientific Reports ◽

10.1038/s41598-021-00211-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Fabian Cuypers ◽

Alexander Schäfer ◽

Sebastian B. Skorka ◽

Surabhi Surabhi ◽

Lea A. Tölken ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Immune Responses ◽

Respiratory Tract ◽

Influenza A ◽

Innate Immune ◽

Upper Respiratory Tract ◽

Innate Immune Responses ◽

Local Inflammatory Response ◽

Inflammatory Monocytes ◽

Asymptomatic Stage

AbstractSeasonal Influenza A virus (IAV) infections can promote dissemination of upper respiratory tract commensals such as Streptococcus pneumoniae to the lower respiratory tract resulting in severe life-threatening pneumonia. Here, we aimed to compare innate immune responses in the lungs of healthy colonized and non-colonized mice after IAV challenge at the initial asymptomatic stage of infection. Responses during a severe bacterial pneumonia were profiled for comparison. Cytokine and innate immune cell imprints of the lungs were analyzed. Irrespective of the colonization status, mild H1N1 IAV infection was characterized by a bi-phasic disease progression resulting in full recovery of the animals. Already at the asymptomatic stage of viral infection, the pro-inflammatory cytokine response was as high as in pneumococcal pneumonia. Flow cytometry analyses revealed an early influx of inflammatory monocytes into the lungs. Neutrophil influx was mostly limited to bacterial infections. The majority of cells, except monocytes, displayed an activated phenotype characterized by elevated CCR2 and MHCII expression. In conclusion, we show that IAV challenge of colonized healthy mice does not automatically result in severe co-infection. However, a general local inflammatory response was noted at the asymptomatic stage of infection irrespective of the infection type.

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Crosstalk Between NDP52 and LUBAC in Innate Immune Responses, Cell Death, and Xenophagy

Frontiers in Immunology ◽

10.3389/fimmu.2021.635475 ◽

2021 ◽

Vol 12 ◽

Author(s):

Hirohisa Miyashita ◽

Daisuke Oikawa ◽

Seigo Terawaki ◽

Daijiro Kabata ◽

Ayumi Shintani ◽

...

Keyword(s):

Immune Responses ◽

Hela Cells ◽

Nuclear Factor Κb ◽

Suppressive Effect ◽

Innate Immune ◽

Bacterial Invasion ◽

Innate Immune Responses ◽

Apoptotic Pathway ◽

Ubiquitin Chain ◽

Tnf Α

Nuclear dot protein 52 kDa (NDP52, also known as CALCOCO2) functions as a selective autophagy receptor. The linear ubiquitin chain assembly complex (LUBAC) specifically generates the N-terminal Met1-linked linear ubiquitin chain, and regulates innate immune responses, such as nuclear factor-κB (NF-κB), interferon (IFN) antiviral, and apoptotic pathways. Although NDP52 and LUBAC cooperatively regulate bacterial invasion-induced xenophagy, their functional crosstalk remains enigmatic. Here we show that NDP52 suppresses canonical NF-κB signaling through the broad specificity of ubiquitin-binding at the C-terminal UBZ domain. Upon TNF-α-stimulation, NDP52 associates with LUBAC through the HOIP subunit, but does not disturb its ubiquitin ligase activity, and has a modest suppressive effect on NF-κB activation by functioning as a component of TNF-α receptor signaling complex I. NDP52 also regulates the TNF-α-induced apoptotic pathway, but not doxorubicin-induced intrinsic apoptosis. A chemical inhibitor of LUBAC (HOIPIN-8) cancelled the increased activation of the NF-κB and IFN antiviral pathways, and enhanced apoptosis in NDP52-knockout and -knockdown HeLa cells. Upon Salmonella-infection, colocalization of Salmonella, LC3, and linear ubiquitin was detected in parental HeLa cells to induce xenophagy. Treatment with HOIPIN-8 disturbed the colocalization and facilitated Salmonella expansion. In contrast, HOIPIN-8 showed little effect on the colocalization of LC3 and Salmonella in NDP52-knockout cells, suggesting that NDP52 is a weak regulator in LUBAC-mediated xenophagy. These results indicate that the crosstalk between NDP52 and LUBAC regulates innate immune responses, apoptosis, and xenophagy.

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Glucose and glutamine availability regulate HepG2 transcriptional responses to low oxygen

Wellcome Open Research ◽

10.12688/wellcomeopenres.14839.1 ◽

2018 ◽

Vol 3 ◽

pp. 126 ◽

Cited By ~ 4

Author(s):

Alvina G. Lai ◽

Donall Forde ◽

Wai Hoong Chang ◽

Fang Yuan ◽

Xiaodong Zhuang ◽

...

Keyword(s):

Gene Expression ◽

Mass Spectrometry ◽

Transcriptional Response ◽

Global Changes ◽

Low Oxygen ◽

Transcriptional Responses ◽

Dna Hydroxymethylation ◽

Modified Dna ◽

The Impact

Background: Little is known about the impact of nutrients on cellular transcriptional responses, especially in face of environmental stressors such as oxygen deprivation. Hypoxia-inducible factors (HIF) coordinate the expression of genes essential for adaptation to oxygen-deprived environments. A second family of oxygen-sensing genes known as the alpha-ketoglutarate-dependent dioxygenases are also implicated in oxygen homeostasis and epigenetic regulation. The relationship between nutritional status and cellular response to hypoxia is understudied. In vitro cell culture systems frequently propagate cells in media that contains excess nutrients, and this may directly influence transcriptional response in hypoxia. Methods: We studied the effect of glucose and glutamine concentration on HepG2 hepatoma transcriptional response to low oxygen and expression of hypoxia inducible factor-1α (HIF-1α). Mass spectrometry confirmed low oxygen perturbation of dioxygenase transcripts resulted in changes in DNA methylation. Results: Under normoxic conditions, we observed a significant upregulation of both HIF-target genes and oxygen-dependent dioxygenases in HepG2 cells cultured with physiological levels of glucose or glutamine relative to regular DMEM media, demonstrating that excess glutamine/glucose can mask changes in gene expression. Under hypoxic conditions, CA9 was the most upregulated gene in physiological glutamine media while TETs and FTO dioxygenases were downregulated in physiological glucose. Hypoxic regulation of these transcripts did not associate with changes in HIF-1α protein expression. Downregulation of TETs suggests a potential for epigenetic modulation. Mass-spectrometry quantification of modified DNA bases confirmed our transcript data. Hypoxia resulted in decreased DNA hydroxymethylation, which correlated with TETs downregulation. Additionally, we observed that TET2 expression was significantly downregulated in patients with hepatocellular carcinoma, suggesting that tumour hypoxia may deregulate TET2 expression resulting in global changes in DNA hydroxymethylation. Conclusion: Given the dramatic effects of nutrient availability on gene expression, future in vitro experiments should be aware of how excess levels of glutamine and glucose may perturb transcriptional responses.

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Age-dependent impairment of disease tolerance is associated with a robust transcriptional response following RNA virus infection in Drosophila

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkab116 ◽

2021 ◽

Author(s):

Lakbira Sheffield ◽

Noah Sciambra ◽

Alysa Evans ◽

Eli Hagedorn ◽

Casey Goltz ◽

...

Keyword(s):

Rna Virus ◽

Model Organism ◽

Transcriptional Response ◽

Innate Immune ◽

Full Potential ◽

Flock House Virus ◽

Virus Infections ◽

Transcriptional Responses ◽

Disease Tolerance ◽

Age Dependent

Abstract Advanced age in humans is associated with greater susceptibility to and higher mortality rates from infections, including infections with some RNA viruses. The underlying innate immune mechanisms, which represent the first line of defense against pathogens, remain incompletely understood. Drosophila melanogaster is able to mount potent and evolutionarily conserved innate immune defenses against a variety of microorganisms including viruses and serves as an excellent model organism for studying host-pathogen interactions. With its relatively short lifespan, Drosophila also is an organism of choice for aging studies. Despite numerous advantages that this model offers, Drosophila has not been used to its full potential to investigate the response of the aged host to viral infection. Here we show that, in comparison to younger flies, aged Drosophila succumb more rapidly to infection with the RNA-containing Flock House Virus (FHV) due to an age-dependent defect in disease tolerance. Relative to younger individuals, we find that older Drosophila mount transcriptional responses characterized by differential regulation of more genes and genes regulated to a greater extent. We show that loss of disease tolerance to FHV with age associates with a stronger regulation of genes involved in apoptosis, some genes of the Drosophila Immune deficiency (IMD) NF-kB pathway and genes whose products function in mitochondria and mitochondrial respiration. Our work shows that Drosophila can serve as a model to investigate host-virus interactions during aging and furthermore sets the stage for future analysis of the age-dependent mechanisms that govern survival and control of virus infections at older age.

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