Tuberculosis - Clinical Value of Initial Host Molecular Signalling and Molecular Fingerprinting

Mycobacterium tuberculosis (Mtb) is principally a pulmonary pathogen infecting one-third of the world's population and causing two million deaths annually. The only approved tuberculosis vaccine today is the Mycobacterium bovis bacilli Calmette-Guérin (BCG). BCG vaccine is used as a benchmark to compare the immunogenicity of new vaccines, but not much is known about its mechanisms to induce protection. We investigated the initial events of mycobacterial activation of airway epithelial cells (AECs) through the signalling pathways of toll like receptors (TLRs) and the G- protein coupled receptors (GPCR; CXCR1, CXCR2). Our data indicate that mycobacteria attenuate epithelial pro-inflammatory production by supressing NF-B activation, thereby supporting the production of the anti-inflammatory cytokines IL-22 and IL-10. BCG infection of AECs also resulted in epithelial actin redistribution that involved the MAPK signalling pathway. This study demonstrated that BCG infection of AECs manipulated the GPCRs to suppress epithelial signalling pathways. Future vaccine strategies could thus be improved by targeting GPCRs.In a second part of the thesis, we investigated the expression and function of GPCRs in a simple whole blood model from patients with pulmonary TB and in subjects with latent TB infection (LTBI). We found variations in GPCRs as pulmonary TB patients had significantly increased CXCR1 expression on blood cells compared to LTBI subjects and controls. These variations in receptor expression were linked to disease progression and affected the immune response against Mycobacterium tuberculosis (Mtb). As an airborne infection, tuberculosis (TB) has no boundaries and easily spreads by migration from one region to another. In this study, 93 patient Mtb-isolates, previously genotypically analysed by standard techniques, were re-analysed by whole genome sequencing (WGS). Compared to the standard genotyping, WGS had an overall high match in identifying cluster transmissions in this patient population. When comparing the different techniques individually, WGS and epidemiological data had the highest cluster similarity, while MIRU-VNTR had less cluster resolution. We can conclude that WGS is well suited for identifying transmission clusters in settings with low TB incidence

supplementary file 3 (data set).xlsx

this is a raw data for a project titled ''Knowledge and information sources of Helicobacter pylori among Jordanian population: a cross-sectional study.''</p

Anti-SARS-Cov-2 S-RBD IgG Formed after BNT162b2 Vaccination Can Bind C1q and Activate Complement

Background. The ability of vaccine-induced antibodies to bind C1q could affect pathogen neutralization. In this study, we investigated C1q binding and subsequent complement activation by anti-spike (S) protein receptor-binding domain (RBD) specific antibodies produced following vaccination with either the mRNA vaccine BNT162b2 or the inactivated vaccine BBIBP-CorV. Methods. Serum samples were collected in the period of July 2021-March 2022. Participants’ demographic data, type of vaccine, date of vaccination, as well as adverse effects of the vaccine were recorded. The serum samples were incubated with S protein RBD-coated plates. Levels of human IgG, IgA, IgM, C1q, and mannose-binding lectin (MBL) that were bound to the plate, as well as formed C3d, and C5b-9 were compared between different groups of participants. Results. A total of 151 samples were collected from vaccinated (n=116) and nonvaccinated (n=35) participants. Participants who received either one or two doses of BNT162b2 formed higher levels of anti-RBD IgG and IgA than participants who received BBIBP-CorV. The anti-RBD IgG formed following either vaccine bound C1q, but significantly more C1q binding was observed in participants who received BNT162b2. Subsequently, C5b-9 formation was significantly higher in participants who received BNT162b2, while no significant difference in C5b-9 formation was found between the nonvaccinated and BBIBP-CorV groups. The formation of C5b-9 was strongly correlated to C1q binding and not to MBL binding, additionally, the ratio of formed C5b-9/bound C1q was significantly higher in the BNT162b2 group. Conclusion. Anti-RBD IgG formed following vaccination can bind C1q with subsequent complement activation, and the degree of terminal complement pathway activation differed between vaccines, which could play a role in the protection offered by COVID-19 vaccines. Further investigation into the correlation between vaccine protection and vaccine-induced antibodies’ ability to activate complement is required

Mycobacteria Manipulate G-Protein-Coupled Receptors to Increase Mucosal Rac1 Expression in the Lungs

Mycobacterium bovis bacille Calmette-Guérin (BCG) is currently the only approved vaccine against tuberculosis (TB). BCG mimics M. tuberculosis (Mtb) in its persistence in the body and is used as a benchmark to compare new vaccine candidates. BCG was originally designed for mucosal vaccination, but comprehensive knowledge about its interaction with epithelium is currently lacking. We used primary airway epithelial cells (AECs) and a murine model to investigate the initial events of mucosal BCG interactions. Furthermore, we analysed the impact of the G-protein-coupled receptors (GPCRs), CXCR1 and CXCR2, in this process, as these receptors were previously shown to be important during TB infection. BCG infection of AECs induced GPCR-dependent Rac1 up-regulation, resulting in actin redistribution. The altered distribution of the actin cytoskeleton involved the MAPK signalling pathway. Blocking of the CXCR1 or CXCR2 prior to infection decreased Rac1 expression, and increased epithelial transcriptional activity and epithelial cytokine production. BCG infection did not result in epithelial cell death as measured by p53 phosphorylation and annexin. This study demonstrated that BCG infection of AECs manipulated the GPCRs to suppress epithelial signalling pathways. Future vaccine strategies could thus be improved by targeting GPCRs

Mycobacteria control epithelial TLR responses.

<p>The impact of TLRs on mycobacterial modulated epithelial signalling was studied by Western blotting prior to infection and three days after infection. (a) Blocking of TLR2 (p = 0.0063) or TLR4 (p = 0.0047) prior to infection or stimulation with 19 kDa significantly increased epithelial pCREB production (p = 0.0163). (b) Blocking of TLRs or 19 kDa stimulation of epithelial cells had an non-significant impact on pGSK3βα expression. (c) Blocking of TLR2 or TLR4 before mycobacterial infection of primary epithelial cells non-significantly restored the NF-κB values to background levels. Data are presented as mean ± SEM of three separate experiments; *p<0.05 and **p<0.01.</p

Mycobacteria bypass epithelial NF-κB signalling.

<p>(a) Infection of primary epithelial cells did not induce NF-κB activation quantified by ELISA, but an early activation of c-Jun proteins in epithelial cells was observed. (b) Epithelial GSK3αβ-pathway was analysed by Phospho-kinase array upon mycobacteria infection. In the beginning of infection, live mycobacteria, the virulence factors LAM and 19 kDa, and the TLR4 agonist LPS, induced comparable induction of p38, pAkt and pGSK3αβ. During the first 24 h, LPS induced higher increase of pCREB protein levels than mycobacteria (p = 0.0017). Third day of infection, mycobacteria significantly increased epithelial pCREB compared to medium control (p = 0.0357) or LPS (p = 0.0089). Epithelial stimulation with LAM induced an increase in pGSK3αβ and pAkt phosphorylation (p = 0.001 respectively p = 0.0196) during the later stages of infection compared to the early time-point. Generally, mycobacteria induced a more persistent increase of the investigated transcription factors three days after infection in primary epithelial cell than the controls LPS, 19 kDa and LAM. Data are presented as mean ± SEM of three separate experiments; **p<0.01 and ***p<0.001.</p

Mycobacteria modulate epithelial signalling pathways.

<p>Several molecules in the TLR-signalling pathway were analysed by Western blotting upon mycobacterial infection. (a–b) We could confirm that mycobacterial infection did not induce NF-κB- or IκB-activation. Mycobacterial suppression of primary epithelial (b) (p = 0.002) IκB and (d) (p = 0.0148) pGSK3αβ proteins were mostly pronounced at 24 hours of infection. The phosphorylated forms of (c) (p = 0.0163) CREB and (d) (p = 0.0248) GSK3αβ proteins reached highest levels 72 hours after infection. (e) Mycobacterial infection increased the Fos family of AP-1 proteins, as c-Fos protein levels significantly increased 72 hours after infection (p = 0.0038). (f) Mycobacteria induced two peaks of pERK1/2 protein levels, after 24 hours (p<0.001) and after 72 hours (p = 0.0034) of infection. (g) Epithelial cells express PPARγ protein, but mycobacterial infection did not significantly increase epithelial PPARγ amount. Data are presented as mean ± SEM of three experiments; *p<0.05, **p<0.01 and ***p<0.001.</p

Controlled epithelial cytokine secretion.

<p>Mycobacterial control of induced transcriptional factors was analysed as epithelial cytokine secretion from six hours up to three days after infection. Infection induced a significant (a) IL-6 and (b) IL-10 secretion that peaked at 72 hours (p = 0.0425 and p = 0.0186 compared to LPS). (c) Mycobacterial infection of primary epithelial cells induced an early significant IL-22 secretion (p = 0.0463 compared to LPS) that ended 24 hours after infection. Data are presented as mean ± SEM of four separate experiments; *p<0.05 and **p<0.01.</p

Innate Immune Responses after Airway Epithelial Stimulation with <i>Mycobacterium bovis</i> Bacille-Calmette Guérin

<div><p><i>Mycobacterium bovis</i> bacilli Calmette-Guerin (BCG) is used as a benchmark to compare the immunogenicity of new vaccines against tuberculosis. This live vaccine is administered intradermal, but several new studies show that changing the route to mucosal immunisation represents an improved strategy. We analysed the immunomodulatory functions of BCG on human neutrophils and primary airway epithelial cells (AECs), as the early events of mucosal immune activation are unclear. Neutrophils and the primary epithelial cells were found to express the IL-17A receptor subunit IL-17RA, while the expression of IL-17RE was only observed on epithelial cells. BCG stimulation specifically reduced neutrophil IL-17RA and epithelial IL-17RE expression. BCG induced neutrophil extracellular traps (NETs), but did not have an effect on apoptosis as measured by transcription factor forkhead box O3 (FOXO3). BCG stimulation of AECs induced CXCL8 secretion and neutrophil endothelial passage towards infected epithelia. Infected epithelial cells and neutrophils were not found to be a source of IL-17 cytokines or the interstitial collagenase MMP-1. However, the addition of IFNγ or IL-17A to BCG stimulated primary epithelial cells increased epithelial IL-6 secretion, while the presence of IFNγ reduced neutrophil recruitment. Using our model of mucosal infection we revealed that BCG induces selective mucosal innate immune responses that could lead to induction of vaccine-mediated protection of the lung.</p></div