Selected β-glucans act as immune-training agents by improving anti-mycobacterial activity in human macrophages - a pilot study.

Epigenetic reprogramming of innate immune cells by β-glucan in a process called trained immunity, leads to an enhanced host response to a secondary infection. β-glucans are structural components of plants, algae, fungi and bacteria and thus recognized as non-self by human macrophages. We selected the β-glucans curdlan from Alcaligenes faecalis, WGP dispersible from Saccharomyces cerevisiae, and β-glucan-rich culture supernatant of Alternaria and investigated whether they could produce trained immunity effects leading to an increased control of virulent Mycobacterium tuberculosis. We observed a significant M. tuberculosis growth-reduction in macrophages trained with curdlan and Alternaria, which also correlated with increased IL-6 and IL-1β release. WGP dispersible-trained macrophages were stratified into ‘non responders’ and ‘responders’, according to their ability to control M. tuberculosis, with ‘responders’ producing higher IL-6 levels. The addition of neutrophils to infected macrophage cultures further enhanced macrophage control of virulent M. tuberculosis, but not in a stimuli-dependent manner. Pathway enrichment analysis of DNA methylome data also highlighted hypomethylation of genes in pathways associated with signaling and cellular reorganization and motility, and ‘responders’ to WGP-training were enriched in the interferon-gamma signaling pathway. This study adds evidence that certain β-glucans show promise as immune training agents

Innate immune responses to Mycobacterium tuberculosis infection : How extracellular traps and trained immunity can restrict bacterial growth.

Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis, and the cause of 1.5 million deaths in 2018. During a pulmonary TB infection, the bacterium reaches the lungs and is phagocytosed by cells of the innate immune system, primarily macrophages. The macrophages are either able to eradicate the bacteria or the bacteria start to replicate, and the following immune response leads to the formation of a large cluster of different cell types called a granuloma. In the granuloma the mycobacteria are contained in a latent infection, or they can start to replicate causing rupture of the granuloma and spread of the disease. Neutrophils are also innate immune cells that are rapidly recruited to the site of infection. They are phagocytes, but they also exert extracellular effector mechanisms by their release of microbicidal granule proteins, reactive oxygen species and neutrophil extracellular traps. M. tuberculosis has co-evolved and adapted to the human host making it ingenious at exploiting the human immune response, promoting its survival and replication in human host cells. The human immune system has also evolved mechanisms to limit M. tuberculosisreplication and spread. This thesis covers work on the innate immune response to TB and how neutrophils and macrophages respond to a mycobacterial infection and can control M. tuberculosis-replication. Neutrophils and macrophages can respond to M. tuberculosis by releasing extracellular traps. We demonstrated that neutrophil extracellular traps contain the danger signal heat-shock protein 72 when induced by mycobacteria, which subsequently mediate a proinflammatory activation of adjacent macrophages. Macrophages can also release extracellular traps, and we observed the release of macrophage extracellular traps in response to M. tuberculosis that grow in cord-structures. We further demonstrated that the induction of extracellular traps also required the mycobacterial virulence factor ESAT-6. Trained immunity is an epigenetically regulated memory of the innate immune system that results in a heightened response to a later encounter of the same or different pathogen. β-glucans are structural components of microbial cell walls and known inducers of trained immunity. We studied the effects of β-glucan from a bacterial source (curdlan from Alcaligenes faecalis), from yeast (WGP dispersible from Saccharomyces cerevisiae) and from the supernatant of a multicellular fungi (Alternaria) in search of functional changes in human macrophages which enhanced their anti-mycobacterial capacity. M. tuberculosis growth reduction was observed in WGP dispersible-trained macrophages when co-cultured with neutrophils. We also discovered that the interferon-gamma (IFNγ) signaling pathway, which is important for mycobacterial control, is hypomethylated in the WGP dispersible-trained macrophages. Since hypomethylation of genes typically is associated with gene activation, this suggests a more active IFNγ signaling in response to β-glucan innate immune training. Most of our studies were performed using in vitro culturing of primary human macrophages and neutrophils. However, an in vitro 3D tissue model is a valuable tool when studying complex events that occur during a TB infection that involves both multiple cell types and requires knowledge of the spatial movement of cells. In this thesis we also describe an in vitro lung tissue model, which we could use to observe the clustering of monocytes around mycobacteria and quantify the size and number of macrophage clusters. In conclusion, this thesis comprises work on innate immune functions during tuberculosis infection. We describe extracellular trap formation in macrophages and neutrophils in response to M. tuberculosis. We also explore trained immunity and how β-glucan training can enhance mycobacterial growth restriction.Tuberkulos (TB) är en infektionssjukdom som orsakade 1,5 miljoner dödsfall år 2018. Man smittas av TB via inandning av aerosoler som bildas när en sjuk person hostar eller nyser. Ett av de vanligaste symptomen vid TB är svår hosta, vilket medför att sjukdomen sprider sig vidare till andra i omgivningen. Det finns ett godkänt vaccin som ges till många spädbarn runtom i världen, men som endast ges till riskgrupper i Sverige. Detta för att det är inte ger ett bra skydd mot TB. Det finns även antibiotika som verkar mot TB, om man inte blir smittad med en antibiotika-resistent bakteriestam, men behandlingen tar upp till ett halvår och har många bieffekter. Dessutom tror man att uppemot en fjärdedel av jordens befolkning bär på latent TB, det vill säga TB som varken är smittsam eller orsakar symptom, men som kan bryta ut till aktiv sjukdom och sprida vidare infektionen till nya människor. TB orsakas av bakterien Mycobacterium tuberculosis som vid smitta transporteras ner i lungorna och träffar på celler som tillhör det medfödda immunförsvaret, som till exempel makrofager. Makrofagerna äter upp mykobakterierna och skickar ut signaler, så kallade cytokiner, till andra celler som kommer och hjälper till, däribland neutrofiler. I bästa fall lyckas makrofagen döda bakterien men ofta kan mykobakterier överleva inne i makrofagerna, för att sedan föröka sig och sprida sig vidare och på så sätt orsaka sjukdom. Mykobakterier har en unik cellvägg som fungerar som ett skydd mot yttre påverkan och gör dem svåra att avdöda. Detta skyddar dem både från att dödas av kroppens celler men skyddar även till viss del mot antibiotika. I den här avhandlingen har vi studerat funktioner hos det medfödda immunförsvaret vid infektion med M. tuberculosis. Bland annat har vi undersökt hur makrofager och neutrofiler aktiveras av mykobakterier, samspelet mellan de två celltyperna, samt hur man kan förstärka cellernas förmåga att avdöda bakterier. Om man stimulerar neutrofiler med mykobakterier kan de försvara sig genom att frisätta molekyler som är toxiska för bakterier, men som mykobakterierna inte påverkas så mycket av på grund av deras cellvägg. Som en extra försvarsmekanism kan neutrofilerna även begå självmord och i processen skicka ut strängar av DNA som kan fånga in bakterier och förhindra att de sprids i vävnaden. Dessa DNA-nät kallas NETs, och innehåller toxiska molekyler men även signalmolekyler som kan överföras till makrofager. I vårt första arbete kunde vi visa att makrofager aktiverades av signalerna som överfördes via NETs och började då utsöndra mer proinflammatoriska cytokiner. I vårt andra arbete visade vi att även makrofager kan kasta ut DNA i strängar (kallas METs) när de stimulerades av mykobakterier som växer i större repliknande strukturer. Vi visar också på en koppling mellan virulens hos bakterierna och makrofagernas frisättning av METs. I vårt tredje arbete beskriver vi en lungvävnadsmodell som kan användas för att studera tuberkulosinfektion. Vi visar att man kan använda den för att studera hur celler förflyttar sig i vävnaden och hopar sig runt mykobakterierna. I vårt fjärde arbete studerade vi ett koncept som kallas ‘tränat medfött immunförsvar’. Det medfödda immunförsvaret har ett ‘minne’ och kan tränas till att bättre försvara oss mot tuberkulos. Vi har undersökt betaglukaner som är molekyler som finns som byggstenar i olika mikroorganismer och har studerat hur cellerna som tränats med dessa blir bättre på att avdöda mykobakterier. Vi har även tittat på de tränade cellernas DNA för att hitta epigenetiska förändringar som förklarar vad som ändras i cellerna när de blir tränade. Kunskap om vad som händer när celler blir infekterade av mykobakterier, och hur de kan stimuleras eller tränas för att bättre avdöda bakterierna, är högst relevant i sökandet efter nya metoder för att förebygga och behandla TB

Inhibition of Tissue Matrix Metalloproteinases Interferes with Mycobacterium tuberculosis-Induced Granuloma Formation and Reduces Bacterial Load in a Human Lung Tissue Model

Granulomas are hallmarks of pulmonary tuberculosis (TB) and traditionally viewed as host-protective structures. However, recent evidence suggest that Mycobacterium tuberculosis (Mtb) uses its virulence factors to stimulate the formation of granuloma. In the present study, we investigated the contribution of matrix metalloproteinases (MMPs), host enzymes that cause degradation of the extracellular matrix, to granuloma formation and bacterial load in Mtb-infected tissue. To this end, we used our lung tissue model for TB, which is based on human lung-derived cells and primary human monocyte-derived macrophages. Global inhibition of MMPs in the Mtb-infected tissue model reduced both granuloma formation and bacterial load. The infection caused upregulation of a set of MMPs (MMP1, 3, 9, and 12), and this finding could be validated in lung biopsies from patients with non-cavitary TB. Data from this study indicate that MMP activation contributes to early TB granuloma formation, suggesting that host-directed, MMP-targeted intervention could be considered as adjunct therapy to TB treatment.Funding Agencies|Swedish Research Council [2014-02289, 2015-02593]; Swedish Heart. Lung Foundation [20130685, 20150709]</p

Corticosteroids protect infected cells against mycobacterial killing in vitro

The effect of corticosteroids on human physiology is complex and their use in tuberculosis patients remains controversial. In a high-throughput screening approach designed to discover virulence inhibitors, several corticosteroids were found to prevent cytolysis of fibroblasts infected with mycobacteria. Further experiments with Mycobacterium tuberculosis showed anti-cytolytic activity in the 10 nM range, but no effect on bacterial growth or survival in the absence of host cells at 20 mu M. The results from a panel of corticosteroids with various affinities to the glucocorticoid- and mineralocorticoid receptors indicate that the inhibition of cytolysis most likely is mediated through the glucocorticoid receptor. Using live-imaging of M. tuberculosis-infected human monocyte-derived macrophages, we also show that corticosteroids to some extent control intracellular bacteria. In vitro systems with reduced complexity are to further study and understand the interactions between bacterial infection, immune defense and cell signaling. (C) 2019 The Authors. Published by Elsevier Inc

The Cording Phenotype of Mycobacterium tuberculosis Induces the Formation of Extracellular Traps in Human Macrophages

The causative agent of tuberculosis, Mycobacterium tuberculosis, shares several characteristics with organisms that produce biofilms during infections. One of these is the ability to form tight bundles also known as cords. However, little is known of the physiological relevance of the cording phenotype. In this study, we investigated whether cord-forming M. tuberculosis induce the formation of macrophage extracellular traps (METs) in human monocyte-derived macrophages. Macrophages have previously been shown to produce extracellular traps in response to various stimuli. We optimized bacterial culturing conditions that favored the formation of the cord-forming phenotype as verified by scanning electron microscopy. Microscopy analysis of METs formation during experimental infection of macrophages with M. tuberculosis revealed that cord-forming M. tuberculosis induced significantly more METs compared to the non-cording phenotype. Deletion of early secreted antigenic target-6 which is an important virulence factor of M. tuberculosis, abrogated the ability of the bacteria to induce METs. The release of extracellular DNA from host cells during infection may represent a defense mechanism against pathogens that are difficult to internalize, including cord-forming M. tuberculosis.Funding Agencies|Swedish Research Council [2012-3349, 2015-02593]; Swedish Heart Lung Foundation [20130685, 20150709]</p