There was a strong correlation between BCG and M. Thus, greater differential mycobacteria growth occurred in the modified BCG MGIT and was dependent upon the infection status of the subject. Ex vivo differential mycobacterial control is associated with disease state in pre-treatment whole blood. Samples were collected from volunteers before starting anti-TB treatment. Points represent the mean of duplicates; bars represent mean values with SD. Significant increases in the growth of both BCG and M.
Similar changes in the growth of M. Enhanced mycobacterial control decreases following TB treatment. A paired t- test was performed to assess net growth before and after treatment.
Given that whole blood samples contain several cellular constituents, which may have anti-mycobacterial functions, we examined which factors correlated with the mycobacterial growth rate. For M. In a reversal of the pre-treatment relationships, significant positive correlations between M. However, significant increases in the proportion of intermediate monocytes in the M. There were no changes in classical monocytes in any group data not shown. Thus mycobacterial growth inhibition was associated with more intermediate and fewer non-classical monocytes and altered subset frequencies typically normalised following successful treatment.
Altered proportions of intermediate and non-classical monocyte subsets are associated with differential mycobacterial control.
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The proportions of classical A , intermediate B , and non-classical C monocytes were calculated from the total monocyte population. A negative correlation between intermediate monocytes and net growth D , and a positive correlation between non-classical monocytes and net growth E of BCG were seen.
The latter was also seen with M. The proportions of intermediate F and non-classical G monocytes are shown. Points are single values and bars represent the mean with SD. Classical memory BC mBC were significantly reduced in the active group compared with healthy controls Fig. Increases in activated and atypical memory B cells occur in M.
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Points are single values and bars represent the median with interquartile range. Serodiagnostics are as yet poorly characterised in the context of M. There was a weaker association between anti-culture filtrate IgG1 and M. IgG1 responses to RD1 restricted M. Antigens used were M. Optical densities ODs are reported following subtraction of the background and plotted on a Log 10 scale. Points represent the mean of duplicates and bars are the median values with the interquartile range.
Twenty-three analytes were detected. Clustering analysis identified four homogeneous clusters defining two broad groups of individuals: the red and blue clusters comprising active and some LTBI patients, and the grey and the black clusters of LTBI and control patients Fig.
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Spearman correlation coefficients showed significant negative associations between M. Serum cytokine analysis reveals discrete clusters, which delineate the spectrum of M. Samples were clustered according to Spearman correlation distance and the four most distinct clusters were coloured in black, grey, blue and red.
Identifying individuals with LTBI at risk of developing active disease as candidates for preventative treatment will be crucial to control TB, and fundamental to achieving this is to understand the full array of infection.
We present evidence supporting the concept of a heterogenous spectrum of M. The simple ordinal readout of anti-mycobacterial immunity reflects the summative combination of multiple individual cellular and non-cellular constituents. We hypothesise that the improved control seen in this model may reflect a general state of immune activation, generated in response to high in vivo bacillary loads. This may also account for the wide spectrum of mycobacterial growth control seen among participants with LTBI, with many overlapping with the active and control groups.
We suggest that some individuals with a high bacillary burden but subclinical disease demonstrate enhanced immune activation and thus bacillary control similar to the active group, whilst those with quiescent infection cluster with the healthy controls. This is supported by human intradermal BCG vaccination studies, which have shown induction of immune activation and improved growth control in MGIAs 10 , Such a principle is well established in animal models of infection, where prior activation macrophages has been shown to enhance control of Salmonella typhimurium The significant increases in mycobacterial net growth observed after treatment, which were of greatest magnitude among the active disease patients, are consistent with our hypothesis.
Individuals with the highest bacillary burden, and the greatest immune activation, demonstrated larger changes in growth control after therapy, supported by resolution of pre-treatment hematological abnormalities. While significant differences in growth control occurred between all groups when using BCG, the M.
We have hypothesised that differential mycobacterial control results from immune activation and suggest that the increased virulence of M. This observation has important implications for further work to assess infection stratification using this MGIA. The heterogeneous responses in the M. While this heterogeneity did not appear to be associated with different disease states e. The increased ML ratio, neutrophil and platelet counts, and reduced hemoglobin concentrations seen here before treatment among individuals with active disease, and some with LTBI, are consistent with other reports 13 , 14 , 15 , 16 , Such profiles may result in altered immune-modulatory and anti-mycobacterial functions, resulting in enhanced ex vivo control.
Bacillary growth correlated with hemoglobin concentration and iron is known to augment bacillary replication in MGIA models 18 , The negative association between ML ratio and mycobacterial growth may reflect the balance between innate and adaptive responses in the assay and could prove a useful marker of mycobacterial disease risk 14 , Interestingly, after treatment the correlation between mycobacterial growth and ML ratio reversed, resulting in profiles very similar to those reported among healthy individuals, suggesting that treatment alters not only the absolute number of myeloid and lymphoid cells, but also their function We used an exploratory multi-platform approach to further characterise the spectrum of mycobacterial control profiles defined by the MGIA.
We observed increased frequencies of intermediate monocytes and decreases in non-classical monocytes among individuals with active disease and LTBI, which changed significantly in the active group following anti-TB treatment, findings consistent with other reports 22 , There is biological plausibility for the significant correlations between pre-treatment intermediate negative and non-classical positive monocytes and MGIA bacillary growth. The sequestration of non-classical monocytes in active disease would account for the reduction in peripheral frequencies, while enhanced numbers of inflammatory intermediate monocytes in vivo may result in the improved ex vivo mycobacterial control.
While there is increasing evidence of B cell responses to intracellular pathogens, little is known about B cell subsets in TB 26 , We found that individuals with active TB had significant increases in activated and atypical mBCs and significantly fewer classical mBC. We observed strong negative correlations between the proportion of atypical or activated mBCs and ex vivo mycobacterial growth.
Atypical mBCs are hypo-responsive cells which are thought to be generated from dysfunctional germinal centers or pre-existing classical mBCs 28 , The resulting diminished classical mBC frequencies are in agreement with our observations. Increased proportions of atypical mBCs occur in active TB and LTBI, and these cells have impaired capacity for proliferation, immunoglobulin and cytokine production, which resolves following treatment In TB, expanded atypical mBCs and failure of B cell responses could contribute to reactivation of LTBI, akin to the reduced malaria immunity observed following repeated Plasmodium falciparum infections, which result in expanded atypical mBC populations The ability to discriminate between active TB and LTBI or controls by IgG1 or IgG2 responses is consistent with evidence that antibody titers are related to mycobacterial load and disease severity 32 , One of the difficulties with TB serodiagnostics is the large overlap in responses, particularly between uninfected healthy controls and LTBI Prior environmental mycobacteria exposure and BCG vaccination could account for high IgG background levels.
The strong negative association of these responses with ex vivo mycobacterial growth suggests that this M. Hierarchical cluster analysis highlighted several correlations between high analyte levels and enhanced mycobacterial control. The chemokine IP CXCL10 is involved in the regulation of innate and adaptive immune responses through the recruitment of monocytes and activated T-cells to sites of inflammation High levels of IP have been reported in active TB disease, possibly as a non-specific indication of inflammation and immune activation, thus reflecting disease activity 38 , PDGF-BB, an angiogenic growth factor, was also negatively associated with ex vivo mycobacterial growth.
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Recent studies have shown increased serum levels of PDGF-BB in patients with pulmonary TB, which was associated with pulmonary fibrosis and reduced following treatment 40 , There were some limitations to this study. Peripheral blood samples are unlikely to simulate the complexity of the pulmonary granuloma in TB, however they are easily accessible and are frequently used in diagnostics, allowing comparison between studies.
In addition, while we consider our findings biologically plausible, they are correlative observations. We did not have the opportunity to investigate underlying mechanisms, which will be an area for future studies. Finally, we acknowledge that this exploratory work was not a biomarker study and suggest that further studies in independent cohorts are required to validate the MGIA findings and associated immune responses presented here.
Using a novel approach we have described the spectrum of TB using a functional MGIA and further characterised immune profiles associated with mycobacterial control responses Fig. Our findings may have value in identifying subclinical active TB disease and possibly in determining reactivation risk. A spectrum of M. We propose that the delineation of the spectrum of M.
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Studies using samples collected from human volunteers were conducted in accordance with the principles of the Declaration of Helsinki, Good Clinical Practice, and local ethical and regulatory requirements. All volunteers provided informed consent for study participation. In addition, samples were also collected from healthy uninfected volunteers with no past medical history of TB, exposure to M.
All experimental work using M. In addition, the number of colony forming units cfu for each dilution was determined by solid culture on 7H10 agar plates. A standard curve of time-to-positivity TTP against cfu was derived and linear regression analysis generated an equation to convert experimental TTP to cfu. This assay was performed as previously described A smaller net growth value indicates less bacillary replication and therefore represents greater mycobacterial control. Peripheral blood mononuclear cells PBMC were separated from fresh heparinized whole blood obtained from study participants by density gradient centrifugation.