Intracellular triggering of inflammation by the extracellular bacterium Pseudomonas aeruginosa

Abstract P.aeruginosa is an extracellular, Gram-negative opportunistic pathogen. One of the most important virulence factors during infection is the type III secretion system (T3SS). This system is found exclusively in Gram-negative bacteria and it forms a conduit between the bacteria and the host cell through which effector molecules can be translocated. These effectors alter the function of the host cell to promote survival of the bacterium. Infections are detected initially by the innate immune system via germ-line encoded receptors, pathogen recognition receptors (PRRs). These receptors recognise conserved microbial patterns, known as pathogen-associated molecular patterns and molecules which signal danger, danger-associated molecular patterns. PRRs are both membrane bound, such as Toll-like receptors (TLRs), and cytosolic, such as Nod-like receptors (NLRs). Some NLRs are involved in the formation of multimeric protein complexes, the Nod-signalosome and inflammasomes. These lead to the activation of NF-κB and the activation of caspase-1 and subsequent proteolytic processing of interleukin-1β (IL-1β) into its mature form. Both processes contribute to the inflammatory response following infection. In this study we sought to elucidate whether P.aeruginosa is able to trigger cytosolic PRRs and the mechanism of this activation. Initially we studied inflammasome activation by P.aeruginosa. We demonstrated that P.aeruginosa is able to activate the NLRC4/ASC-inflammasome complex. This was found to be dependent on a functional T3SS, but independent of any effectors passing through the system. The activation was discovered by detection of processed, and thus active caspase-1 fragments, as well as by secretion of mature IL-1β. The mechanism of the inflammasome activation was then investigated. We found that the NLRC4-dependent inflammasome activation is also dependent on extracellular potassium. An increase of extracellular potassium leads to a complete abrogation of inflammasome activation by P.aeruginosa and Salmonella. To further elucidate this finding, we investigated the leakiness of the pore formed by the T3SS in the host cell membrane. No flux of ions or small molecules could be detected in the host cell membrane following infection. However, host-membrane repair mechanisms were triggered, which could be detected by lysosomal-associated membrane protein (LAMP)-1-specific staining of the plasma membrane. We hypothesize a role for membrane potential in triggering of inflammasome activation by bacteria possessing a secretion system. Potassium-efflux has previously been identified as a activator of the NLRP3 inflammasome, but no changes in intracellular potassium could be found during this study. The activation of the NLRC4 inflammasome by the Pseudomonal strain PA103 was shown, in this study, to be independent of flagellin. Instead, the bacterial molecule responsible for inflammasome activation was shown to be pilin. Pilin is important for attachment to the host cell and the function of the T3SS. We showed that a strain lacking pilin were still able to translocate effectors through its T3SS. However, it was unable to activate the inflammasome complex. Transfection of purified pilin into cells was shown to trigger inflammasome activation. This was found to be dependent on caspase-1 but independent of NLRC4 and ASC, which is not in agreement with the results found for live bacteria. We hypothesised that the reason for this is the delivery method used, since a T3SS and infection delivers proteins and molecules differently compared to a transfection reagent. Finally, the role for Nod1 in infection by P.aeruginosa was explored. We could not identify Nod1-dependent NF-κB-activation using luciferase reporter gene assays. We therefore hypothesise that Nod1 does not have a role in the innate immune response to P.aeruginosa. In conclusion, we have identified NLRC4- and ASC-dependent inflammasome activation by P.aeruginosa. This activation was shown to be dependent on a functional T3SS and the surface protein pilin, as well as extracellular potassium. This describes a novel NLRC4-activation mechanism dependent on potassium and identifies pilin as a PRR-trigger for the first time

Pseudomonas aeruginosa pilin activates the inflammasome

IL-1 beta is produced from inactive pro-IL-1 beta by activation of caspase-1 brought about by a multi-subunit protein platform called the inflammasome. Many bacteria can trigger inflammasome activity through flagellin activation of the host protein NLRC4. However, strains of the common human pathogen Pseudomonas aeruginosa lacking flagellin can still activate the inflammasome. We set out to identify what non-flagellin components could produce this activation. Using mass spectroscopy, we identified an inflammasome-activating factor from P. aeruginosa as pilin, the major component of the type IV bacterial pilus. Purified pilin introduced into mouse macrophages by liposomal delivery activated caspase-1 and led to secretion of mature IL-1 beta, as did recombinant pilin purified from Escherichia coli. This was dependent on caspase-1 but not on the host inflammasome proteins NLRC4, NLRP3 or ASC. Mutants of P. aeruginosa strain PA103 lacking pilin did not activate the inflammasome following infection of macrophages with live bacteria. Type III secretion remained intact in the absence of pili, showing this was not due to a lack of effector delivery. Our observations show pilin is a novel activator of the inflammasome in addition to flagellin and the recently described PrgJ protein family, the basal body rod component of the type III apparatu

100 years of the Bacillus Calmette-Guerin vaccine

Immunogenetics and cellular immunology of bacterial infectious disease

Editorial: Exploring Immune Variability in Susceptibility to Tuberculosis Infection in Humans.

Editorial on the Research Topic - Exploring Immune Variability in Susceptibility to Tuberculosis Infection in Humans. No abstract available

T Cell Responses to Neural Autoantigens Are Similar in Alzheimer’s Disease Patients and Age-Matched Healthy Controls

Alzheimer’s disease (AD), a chronic multifactorial and complex neurodegenerative disorder is a leading cause of dementia. Recently, neuroinflammation has been hypothesized as a contributing factor to AD pathogenesis. The role of adaptive immune responses against neuronal antigens, which can either confer protection or induce damage in AD, has not been fully characterized. Here, we measured T cell responses to several potential antigens of neural origin including amyloid precursor protein (APP), amyloid beta (Aβ), tau, α-synuclein, and transactive response DNA binding protein (TDP-43) in patients with AD and age-matched healthy controls (HC). Antigen-specific T cell reactivity was detected for all tested antigens, and response to tau-derived epitopes was particularly strong, but no significant differences between individuals with AD and age-matched HC were identified. We also did not observe any correlation between the antigen-specific T cell responses and clinical variables including age, gender, years since diagnosis and cognitive score. Additionally, further characterization did not reveal any differences in the relative frequency of major Peripheral Blood Mononuclear Cells (PBMC) subsets, or in the expression of genes between AD patients and HC. These observations have not identified a key role of neuronal antigen-specific T cell responses in AD

A quantitative analysis of complexity of human pathogen-specific CD4 T cell responses in healthy M. tuberculosis infected South Africans

Author Summary: Human pathogen-specific immune responses are tremendously complex and the techniques to study them ever expanding. There is an urgent need for a quantitative analysis and better understanding of pathogen-specific immune responses. Mycobacterium tuberculosis (Mtb) is one of the leading causes of mortality due to an infectious agent worldwide. Here, we were able to quantify the Mtb-specific response in healthy individuals with Mtb infection from South Africa. The response is highly diverse and 66 epitopes are required to capture 80% of the total reactivity. Our study also show that the majority of the identified epitopes are restricted by multiple HLA alleles. Thus, technical advances are required to capture and characterize the complete pathogen-specific response. This study demonstrates further that the approach combining identified epitopes into "megapools" allows capturing a large fraction of the total reactivity. This suggests that this technique is generally applicable to the characterization of immunity to other complex pathogens. Together, our data provide for the first time a quantitative analysis of the complex pathogen-specific T cell response and provide a new understanding of human infections in a natural infection setting

α-Synuclein-specific T cell reactivity is associated with preclinical and early Parkinson’s disease

A diagnosis of motor Parkinson’s disease (PD) is preceded by a prolonged premotor phase with accumulating neuronal damage. Here we examined the temporal relation between α-synuclein (α-syn) T cell reactivity and PD. A longitudinal case study revealed that elevated α-syn-specific T cell responses were detected prior to the diagnosis of motor PD, and declined after. The relationship between T cell reactivity and early PD in two independent cohorts showed that α-syn-specific T cell responses were highest shortly after diagnosis of motor PD and then decreased. Additional analysis revealed significant association of α-syn-specific T cell responses with age and lower levodopa equivalent dose. These results confirm the presence of α-syn-reactive T cells in PD and show that they are most abundant immediately after diagnosis of motor PD. These cells may be present years before the diagnosis of motor PD, suggesting avenues of investigation into PD pathogenesis and potential early diagnosis

Analysis of host responses to Mycobacterium tuberculosis antigens in a multi-site study of subjects with different TB and HIV infection states in sub-Saharan Africa.

BACKGROUND: Tuberculosis (TB) remains a global health threat with 9 million new cases and 1.4 million deaths per year. In order to develop a protective vaccine, we need to define the antigens expressed by Mycobacterium tuberculosis (Mtb), which are relevant to protective immunity in high-endemic areas. METHODS: We analysed responses to 23 Mtb antigens in a total of 1247 subjects with different HIV and TB status across 5 geographically diverse sites in Africa (South Africa, The Gambia, Ethiopia, Malawi and Uganda). We used a 7-day whole blood assay followed by IFN-γ ELISA on the supernatants. Antigens included PPD, ESAT-6 and Ag85B (dominant antigens) together with novel resuscitation-promoting factors (rpf), reactivation proteins, latency (Mtb DosR regulon-encoded) antigens, starvation-induced antigens and secreted antigens. RESULTS: There was variation between sites in responses to the antigens, presumably due to underlying genetic and environmental differences. When results from all sites were combined, HIV- subjects with active TB showed significantly lower responses compared to both TST(-) and TST(+) contacts to latency antigens (Rv0569, Rv1733, Rv1735, Rv1737) and the rpf Rv0867; whilst responses to ESAT-6/CFP-10 fusion protein (EC), PPD, Rv2029, TB10.3, and TB10.4 were significantly higher in TST(+) contacts (LTBI) compared to TB and TST(-) contacts fewer differences were seen in subjects with HIV co-infection, with responses to the mitogen PHA significantly lower in subjects with active TB compared to those with LTBI and no difference with any antigen. CONCLUSIONS: Our multi-site study design for testing novel Mtb antigens revealed promising antigens for future vaccine development. The IFN-γ ELISA is a cheap and useful tool for screening potential antigenicity in subjects with different ethnic backgrounds and across a spectrum of TB and HIV infection states. Analysis of cytokines other than IFN-γ is currently on-going to determine correlates of protection, which may be useful for vaccine efficacy trials