Soluble immune mediators orchestrate protective in vitro granulomatous responses across Mycobacterium tuberculosis complex lineages
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eLife assessment
This study assessed the virulence and immune responses of different M. tuberculosis lineages using a 3D in vitro granuloma model. The useful findings support the functional impact of M. tuberculosis natural diversity on host-pathogen interactions but are incomplete and only partially support the claims. The study will interest researchers working on mycobacteria and understanding how genetic diversity influences virulence and immunity outcomes.
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Abstract
The members of the Mycobacterium tuberculosis complex (MTBC) causing human tuberculosis comprise ten phylogenetic lineages that differ in their geographical distribution. The human consequences of this phylogenetic diversity remain poorly understood. Here, we assessed the phenotypic properties at the host-pathogen interface of 14 clinical strains representing five major MTBC lineages. Using a human in vitro granuloma model combined with bacterial load assessment, microscopy, flow cytometry, and multiplexed-bead arrays, we observed considerable intra-lineage diversity. Yet, modern lineages were overall associated with increased growth rate and more pronounced granulomatous responses. MTBC lineages exhibited distinct propensities to accumulate triglyceride lipid droplets - a phenotype associated with dormancy - that was particularly pronounced in lineage 2 and reduced in lineage 3 strains. The most favorable granuloma responses were associated with strong CD4 and CD8 T cell activation as well as inflammatory responses mediated by CXCL9, granzyme B and TNF-alpha. Both of which showed consistent negative correlation with bacterial proliferation across genetically distant MTBC strains of different lineages. Taken together, our data indicate that different virulence strategies and protective immune traits associate with MTBC genetic diversity at lineage and strain level.
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eLife assessment
This study assessed the virulence and immune responses of different M. tuberculosis lineages using a 3D in vitro granuloma model. The useful findings support the functional impact of M. tuberculosis natural diversity on host-pathogen interactions but are incomplete and only partially support the claims. The study will interest researchers working on mycobacteria and understanding how genetic diversity influences virulence and immunity outcomes.
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Reviewer #1 (Public Review):
Summary:
This study further develops the potential of in vitro granulomas to study host-pathogen interactions in tuberculosis. It uses a human-based cellular model and a collection of M. tuberculosis isolates representative of the pathogen's diversity. It provides important methodologic information and some findings that help in defining protective responses in TB.
Strengths:
A strength of the study is the multitude of parameters addressed across different M. tuberculosis strains and donors. The inclusion of several strains of the same lineage shows that intra-lineage diversity is also relevant, illustrating how complex it is to model the immune response to M. tuberculosis.
Weaknesses:
A weakness of the study is that although several interesting findings are reported and a hypothesis proposed, the work is …
Reviewer #1 (Public Review):
Summary:
This study further develops the potential of in vitro granulomas to study host-pathogen interactions in tuberculosis. It uses a human-based cellular model and a collection of M. tuberculosis isolates representative of the pathogen's diversity. It provides important methodologic information and some findings that help in defining protective responses in TB.
Strengths:
A strength of the study is the multitude of parameters addressed across different M. tuberculosis strains and donors. The inclusion of several strains of the same lineage shows that intra-lineage diversity is also relevant, illustrating how complex it is to model the immune response to M. tuberculosis.
Weaknesses:
A weakness of the study is that although several interesting findings are reported and a hypothesis proposed, the work is mainly descriptive and correlative. Some functional data based on the current observations would strengthen the findings.
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Reviewer #2 (Public Review):
Summary:
This manuscript reports a comparison of microbial traits and host response traits in a laboratory model of infected granuloma using Mtb strains from different lineages. The authors report increased bacillary growth and granuloma formation, inversely associated with T cell activation that is characterized by CXCL9, granzyme B, and TNF expression. They therefore infer that these T cell responses are likely to be host-protective and that the greater virulence of modern Mtb lineages may be driven by their ability to avoid triggering these responses.
Strengths:
The comparison of multiple Mtb lineages in a granuloma model that enables evaluation of the potential role of multiple host cells in Mtb control offers a valuable experimental approach to studying the biological mechanisms that underpin …
Reviewer #2 (Public Review):
Summary:
This manuscript reports a comparison of microbial traits and host response traits in a laboratory model of infected granuloma using Mtb strains from different lineages. The authors report increased bacillary growth and granuloma formation, inversely associated with T cell activation that is characterized by CXCL9, granzyme B, and TNF expression. They therefore infer that these T cell responses are likely to be host-protective and that the greater virulence of modern Mtb lineages may be driven by their ability to avoid triggering these responses.
Strengths:
The comparison of multiple Mtb lineages in a granuloma model that enables evaluation of the potential role of multiple host cells in Mtb control offers a valuable experimental approach to studying the biological mechanisms that underpin differential virulence of Mtb lineages that have been previously reported in clinical and epidemiological studies.
Weaknesses:
The study is rather limited to descriptive observations and lacks experiments to test causal relationships between host and pathogen traits. Some of the presentation of the data is difficult to interpret, and some conclusions are not adequately supported by the data.
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Reviewer #3 (Public Review):
Summary:
In "CXCL9, granzyme B, and TNF-α orchestrate protective in vitro granulomatous responses across Mycobacterium tuberculosis complex lineages", Arbués and colleagues describe the impact of mycobacterial genetic diversity on host-infection phenotypes. The authors evaluate Mtb infection and contextualize host responses, bacterial growth, and metabolic transitioning in vitro using their previously established model of blood-derived, primary human cells cultured on a collogen/fibronectin matrix. They seek to demonstrate the effectiveness of the model in determining mycobacterial strain-specific granuloma-dependent host-pathogen interactions.
Strengths and weaknesses:
Understanding the way mycobacterial genetic diversity impacts granuloma biology in tuberculosis is an important goal. One of this work's …
Reviewer #3 (Public Review):
Summary:
In "CXCL9, granzyme B, and TNF-α orchestrate protective in vitro granulomatous responses across Mycobacterium tuberculosis complex lineages", Arbués and colleagues describe the impact of mycobacterial genetic diversity on host-infection phenotypes. The authors evaluate Mtb infection and contextualize host responses, bacterial growth, and metabolic transitioning in vitro using their previously established model of blood-derived, primary human cells cultured on a collogen/fibronectin matrix. They seek to demonstrate the effectiveness of the model in determining mycobacterial strain-specific granuloma-dependent host-pathogen interactions.
Strengths and weaknesses:
Understanding the way mycobacterial genetic diversity impacts granuloma biology in tuberculosis is an important goal. One of this work's strengths is the use of primary human cells and two constituents of the pulmonary extracellular matrix to model Mtb infection. The authors and others have previously shown that Mtb-infected PBMC aggregates share important characteristics with early pulmonary TB granulomas (Arbues et al., Bio Protoc, 2020, PMID: 3659472; Guirado et al., mBio, 2015, PMID: 25691598; Kapoor et al., PloS One, 2013, PMID: 23308269). The use of multiple genetically distinct strains of Mtb defines this work and further bolsters its potential impact. However, the study is not comprehensive as lineages 6 and 7 are not tested. Experiments are primarily descriptive, and the methodologies are conventional. Correlative relationships are the manuscript's focus and functional validation is not conducted. Convoluted data presentation hampers the readers' ability to effectively evaluate many of the findings for significance. The effect sizes are generally small and most quantitative data are unitless. A further weakness of the study is a lack of any in vivo modeling.
Achievement of aims, and support for conclusions:
The main aim of this work is to extend an in vitro granuloma model to the study of a large collection of well-characterized, genetically diverse representatives of the mycobacterium tuberculosis complex (MTBC). I believe that they accomplish that aim. The work does investigate MTBC infection of aggregated PBMCs using three strains each of Mtb lineages 1-5 and H37Rv, which is not a trivial undertaking. The experimental aims are to show that MTBC genetic diversity impacts the growth and dormancy of granuloma-bound bacteria and, the host responses of granulomatous aggregation as well as macrophage apoptosis, lymphocyte activation, and soluble mediator release within granulomas. A lack of basic descriptive statistics for raw data makes it difficult to determine if benchmarks for most of the experimental aims have been reached. Although the methodologies employed should have been able to test most of these aims. The title's conclusion that CXCL9, granzyme B, and TNF orchestrate a protective granulomatous response is not tested and is not supported by the findings. Those molecules are not a focus of the work, their effects are not investigated effectively and their relationship to the granulomatous response is not determined. The authors' conclusions regarding their results are a mixed bag. Their conclusion that lineage impacts growth within granulomas is likely true and the data as presented reflect such a relationship. However, even without the basic descriptive statistics needed to evaluate the data supporting that claim, the methods employed for bacterial collection call into question whether all Mtb plated for CFU assay resided within granulomatous aggregates. Their conclusions regarding lineage's impact on dormancy are not supported, as their findings demonstrate that assays for dormancy identify replicating bacteria as being dormant. Their conclusion that strain diversity results in a spectrum of granulomatous responses in their model system is strongly supported by the results. Their conclusion that strain diversity impacts macrophage apoptosis is supported by the data but a relationship of apoptosis to the granulomatous response is not effectively evaluated. Their conclusion that lymphocyte activation is associated with reduced mycobacterial growth as an aspect of granulomas is well supported in the literature and a negative correlation between T cell activation and growth is supported by their results.
Impact on the field:
The authors contribute some valuable insights, particularly in Figure 3 and supplementary Figures 1 and 2, where data is more accessible to critique. Their identification of donor-dependent aggregation phenotypes by mycobacterial strain has the potential to enable future reverse-genetic screens for human and Mtb loci that contribute to granulomatous inflammation. Their model is a higher echelon relative to others in the field, but I don't believe that it possesses all of the necessary tissue and cellular components to effectively replicate the formation of granulomas in nature. The bulk of the data in its current form is not of high value to the community, but I think it has the potential to contribute additional novel insights if panels that display descriptive statistics are added to the figures.
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