Does neutrophil death impact Mycobacterium tuberculosis' survival?

Comment

Does neutrophil death impact Mycobacterium tuberculosis' survival?

Latest post on group Journal Club by David Stirling:

The immune mechanisms responsible for control of Mycobacterium tuberculosis (Mtb) infection and microbial countermeasures to evade this response are a subject of particular interest to our group. Our latest journal club reviewed this paper from Ulrich Schaible’s group, looking at how the mode of cell death in infected neutrophils impacts the ability of macrophages to subsequently control Mtb phagocytosed from the debris.

Unlike attenuated strains such as BCG, Mtb is capable of inducing necrosis in infected neutrophils. The researchers explored the molecular mechanisms behind this effect and found it to be an ESX-1 and reactive oxygen species (ROS)-dependent phenotype. In line with previous reports, mutant bacteria lacking RD1 did not induce substantial necrosis in infected neutrophils, with cells undergoing apoptosis instead. The ability to induce necrosis instead of apoptosis was hypothesised to be important for evasion of host immunity.

Electron microscopy revealed that bacteria from necrotic cells are loosely associated with cellular remnants, whereas those from apoptotic cells remain contained in vesicles. Immunofluorescence on macrophages which had just taken up neutrophil debris was used to investigate the fate of the bacteria upon entry into macrophages. Strikingly, endosomes containing RD1- or ESAT-6-deleted bacteria (from within apoptotic vesicles) rapidly associated with maturation markers such as LAMP1 and Rab7, whereas wild-type bacteria phagocytosed from necrotic cells did not associate with these markers and evaded degradation.

Interestingly, it was also found that inhibitors of ROS pathways could be used to block necrotic cell death in neutrophils and push the cells towards apoptosis. This allowed subsequent control of bacterial growth when the debris from these apoptotic cells was phagocytosed by macrophages.

Overall these findings highlight a fascinating new area for potential host-directed therapies in TB infection. It is possible that regulating the fate of ‘first-responder’ cells in Mtb infection, neutrophils, may in turn tune the ability of macrophages to control the infection downstream. How other components of the tissue immune response to Mtb fit into this model needs to be ascertained, but this paper certainly seems to have opened up a new avenue of investigation.

Comment

Cryptococcal IRIS: an alternatively activated immunopathology?

Comment

Cryptococcal IRIS: an alternatively activated immunopathology?

This week's blogpost comes from Lucy Bell, an Innate2Adaptive alumnus who reviews an interesting recent publication which adds insight to our understanding of immune reconstitution inflammatory syndrome.

I was interested by this recent paper by Scriven et al. in the Journal of Acquired Immunodeficiency Syndromes, entitled “The CSF Immune Response in HIV-1–Associated Cryptococcal Meningitis: Macrophage Activation, Correlates of Disease Severity, and Effect of Antiretroviral Therapy”. As the title suggests, the authors analysed the immune response within the cerebrospinal fluid (CSF) of HIV-1 positive patients with cryptococcal meningitis. They approach this by a combination of flow-cytometry based cellular assessments and multiplex cytokine measurements, and integrate this with data analysis techniques including principal component analysis (PCA) to explore the factors driving variation within their dataset.

Cryptococcal meningitis is a central nervous system infection caused by the environmentally prevalent fungus Cryptococcus neoformans, which does not cause disease in immunocompetent individuals. However, it acts as an opportunistic pathogen to cause meningitis in immunosuppressed patients, such as in advanced HIV disease (AIDS).

The part of the study that piqued my interest was their assessment of patients presenting with what the authors term ‘ART (antiretroviral therapy)-associated cryptococcal meningitis’: that is, patients who develop the infection within a short period of time after commencing ART. This likely includes patients with immune reconstitution inflammatory syndrome (IRIS), a clinical term for when patients who commence ART then either develop increased severity of an existing opportunistic infection (‘paradoxical’ IRIS), or present with a new opportunistic infection (‘unmasking’ IRIS). The pathology of IRIS is likely complex, but revolves around ART-triggered recovery of the immune response in a previously immunosuppressed tissue environment with high microbial loads, and consequent potential immunopathology.

The patient group of most interest in this paper were those who had commenced or switched to 2nd line ART in the 12 weeks prior to presentation. This definition may include individuals with unmasking IRIS (although the authors use a strict provisional case definition for cryptococcal IRIS which only one of the patient group fulfils). The authors find that this group of patients clustered separately on both PCA and non-hierarchal cluster analysis, and that the factors which distinguish the group were higher and lower CSF proportions of CD4+ T-cells & CD8+ T-cells respectively, as well as higher expression of CD206 on monocyte/macrophage cell types.

CD206 (also known as mannose receptor) is a marker of ‘alternative activation’ of macrophages,  driven by stimulation with interleukin-4 and interleukin-13 produced by T-helper 2 (Th2) CD4+ T-cells, as opposed to classical activation driven by interferon gamma produced by T helper 1 (Th1) cells. This marries somewhat with previous work from the same group, which found high levels of activated macrophages and Th2 cytokines in the CSF of patients with possible paradoxical cryptococcal IRIS. The authors hypothesise that the shift in macrophage phenotype could be driven directly by reduction in the HIV viral load, which has been shown in other models to be inversely correlated with CD206 expression. However, they do not speculate further on the precise host:pathogen interactions which might drive this.

We have also noted evidence of Th2 immune responses in patients with IRIS (unmasking TB-IRIS in our case) which poses the question of whether dysregulated Th2 responses might be a common process driving immune reconstitution-associated immunopathology across different opportunistic infections, and if so, how does commencing ART trigger this? Further studies such as the one in this paper utilising specimens from the site of disease, as well as the opportunity to assess immune responses in vivo with increasing levels of dimensionality via -omics technologies, should allow us to further explore these and related questions.

Comment

Does Mycobacterium tuberculosis thrive in a lactate-rich environment?

Comment

Does Mycobacterium tuberculosis thrive in a lactate-rich environment?

Last week’s journal club paper was presented by Lavanya Mane, a member of Prof Rob Heyderman’s and Prof Robert Wilkinson’s groups. She presented the paper “Lactate oxidation facilitates growth of Mycobacterium tuberculosis in human macrophages”. Below, she provides her commentary and opinion on the paper.

The nutritional flexibility of M. tuberculosis (Mtb) along with its ability to co-metabolise multiple substrates is well-known, and lends the pathogen immense adaptability. In response to an infection, immune cells (including macrophages) switch from oxidative phosphorylation to aerobic glycolysis in order to meet increased energy requirements, generating lactate in the process - a process often referred to as the Warburg effect.

Researchers from Hannover Medical School demonstrate for the first time that Mtb can utilise L-lactate as a sole carbon source for growth in vitro, and that this is contingent on the presence of a mycobacterial quinone-dependent lactate dehydrogenase, LldD2. Carbon-13 labelling reveals lactate is metabolised predominantly via gluconeogenesis to generate biomass. As gluconeogenic enzymes have previously been shown to be crucial for intracellular survival of M. tuberculosis, this highlights the benefit of lactate utilisation.

Removal of LldD2 in the knockout strain ∆lldd2 renders the organisms unable to proliferate in human monocyte-derived macrophages. The authors interpret this to mean lactate oxidation is essential for intracellular growth of Mtb in macrophages, although the study itself shows that LldD2 has a role in metabolising other compounds. In addition, the authors demonstrate that excessive lactate considerably slows the growth of Mtb in culture, speculating this may be related to lactate production correlating with increases in oxidative stress.

Therefore, this paper seems to demonstrate that through expression of the lactate dehydrogenase LldD2 within a macrophage that has been activated and undergoing a Warburg switch in its metabolism, Mtb can not only remove accumulated lactate to prevent ROS build-up but also consume lactate as a source of energy to facilitate its survival and growth. Looks like Mtb is once again making the most of its environment to its own advantage and it also renders LldD2 an attractive drug target for researchers with the skills to make things like that!

Warburg effect (Vander Heiden et al 2009)

Warburg effect (Vander Heiden et al 2009)

 

 

Comment

Are mycobacteria calling the shots in human infections?

Comment

Are mycobacteria calling the shots in human infections?

M tuberculosis phagocytosis by a macrophage (source)

M tuberculosis phagocytosis by a macrophage (source)

Our eye has recently been drawn to 2 papers from Lalita Ramakrishnan's laboratory, both using the zebrafish larvae in vivo model to focus on the role of mycobacterial phenolic glycolipids (PGLs) in orchestrating the host immune response following infection with Mycobacterium marinum or Mycobacterium leprae.

The first paper was presented by Carolin Turner in our group's journal club last week, and demonstrated that PGLs expressed on M marinum directs infected resident macrophages to secrete CCL2, recruiting CCR2-expressing monocytes to the site of infection. Amazingly, these same cells that are recruited are more permissive to bacterial replication, thus seemingly favouring bacterial survival in the host.

Concurrently, the same group published a paper in Cell demonstrating that PGL expression by M leprae was responsible for macrophage activation, reactive nitrogen species generation and consequent nerve damage, a feature characteristic of most clinical presentations of leprosy.

As ever, even elegant studies such as these, when performed in animal models raise questions about how the findings translate to a natural infection in humans. It is notable that the clarity (literally) offered by transparent zebrafish larvae is offset by the lack of any adaptive immune response at the time the experiments were performed. Thus, we do not know how a T cell response, ubiquitous in mycobacterial infections, would tip the balance in the host-pathogen interactions described.

Nevertheless, it is striking how mycobacterial surface glycoproteins can apparently drive the pathogenesis of infection with seeming impunity. SImilar to immune evasive actions of PGLs described here, another virulence factor expressed by M tuberculosis, ESAT-6, can provide the bacteria a mechanism to escape the hostile phagolysosome. Overall, these findings would suggest that studying the biology of different clinical strains of pathogenic mycobacteria may yield significant information on the determinants of disease in humans, and thus complement well the work of those of us focusing predominantly on the host's attempt to contain the infection. In some instances, it may be that from the moment the bacteria arrive, the host's fate is already out of their control.

Comment

Comment

Meeting report: 10th International Conference on the Pathogenesis of Mycobacterial Infections

WP_20170824_08_35_42_Rich_LI.jpg

Between 23rd and 25th August 2017, we attended the 10th International Conference on the Pathogenesis of Mycobacterial Infections meeting in Stockholm. It was a small gathering (100 or so attendants) but over 2 days a large number of topics were covered. Below are a few of our (probably biased!) highlights:

Day 1 kicked off with a keynote address from Joel Ernst, who underlined the importance of focusing on diversity in host response to Mycobacterium tuberculosis (Mtb) infection (clinical & immunological in order to determine the critical determinants of clinical outcome in tuberculosis infection. Gunilla Kallenius followed this up with a diagnostic orientated keynote address, readdressing diagnostic urinary lipoarabinomanna (LAM) detection in TB disease. She described how microbead concentration of LAM in urine may significantly increase the sensitivity of a diagnostic test who's use to date has been limited to confirmation of TB in patients coinfected with advanced HIV disease.

WP_20170824_09_05_41_Panorama.jpg

The morning session on day 2 focused on mycobacterial biology, with our highlight being Roland Brosch who proposed a mechanism of action for a putative novel TB vaccine. He demonstrated that by lacking ESX-1 secretion system, BCG is unable to escape the phagolysosome and access the cytosol, in contrast to the ESX-1 expressing Mtb. Whilst this approach allows the bacteria to escape the phagolysosome environment, it results in triggering of cGAS and an ensuing type I IFN and inflammasome driven response. He demonstrated that transfecting the Mycobacterium marinum ESX-1 machinery into BCG generated a bacterial species of low virulence (like BCG) but with the ability to access the macrophage cytosol and trigger inflammation. Consequently he demonstrated that such a recombinant BCG was superior in protecting mice in a Mtb challenge model. We felt this was an innovative and exciting combination of bacterial biology and host response to guide both understanding of the host-pathogen interaction and also provide direct insight into putative new vaccine development.

WP_20170824_20_51_05_Pro_LI.jpg

Another highlight of day 2 was Simone Joosten who described the phenotype of HLA-E restricted T cells present in active TB diseae, which carry an unusual phenotype in that they have cytolytic ability but secrete Th2 cytokines. This perked our interest as Th2 responses in the context of Tb are rare. However, in our own work looking at inflammation at the site of TST challenge, we were also surprised to observe enrichment of Th2 responses in the context of unmasking TB-IRIS. In this setting, we surmised that these responses may be responsible for driving the IRIS pathology, and it would be of interest to see if this was coupled by the presence of the same HLA-E restricted T cells described by Simone. 

Obviously our day 3 highlight was our own Gabriele Pollara presenting work on IL-17-driven pathogenic immune responses in TB disease (manuscript on its way, we promise!), but in fact the morning session was filled with other interesting talks. Adrian Martineau provided a highly humorous and informative run through his extensive and thorough efforts to explore a role for Vitamin D in tuberculosis infection. He reminded us that Vitamin D attenuates inflammatory responses to TB in vitro, but also that this has yet to translate to clinical benefit in active TB or prevention of disease in those with latentinfection.

WP_20170824_18_33_51_Rich_LI.jpg

Finally Deepak Kashaul showed some new and exciting data that blockade of the hose IDO enzyme in the macaque model of TB infection could remove an immunosuppressive block on granuloma T cells to access the bacteria-rich core of the granuloma. He acknowledged limitations that this was a low dose challenge experiment (i.e. not full blown active disease) and that this may have prevented significant levels of immunopathology using this approach. Nevertheless, this was an elegant demonstration of the power of performing host-directed intervention studies in the context of challenge experiments in an animal model most aligned to immunopathology in humans. In turn, the findings from this study may directly inform clinical therapeutic options in man more readily.

Comment