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Drug resistant clinical isolates of Mycobacterium tuberculosis from different genotypes exhibit differential host responses in THP-1 cells.

Chakraborty P, Kulkarni S, Rajan R, Sainis K - PLoS ONE (2013)

Bottom Line: EAI-5 strain from ancient lineage 1, induced higher proinflammatory responses, higher apoptosis and moderate intracellular growth compared to other strains, in contrast, for Beijing strain of modern lineage 2, all three parameters were lowest among the clinical isolates.Thus, these profiles were specific to their respective lineages and/or genotypes and independent of their drug resistance status.Further, a positive correlation, among TNF-α, IL-1β, IL-6 and IL-12 induced in infected THP-1 cells was demonstrated.

View Article: PubMed Central - PubMed

Affiliation: Radiation Medicine Centre, Bio-Medical Group, Bhabha Atomic Research Centre, Mumbai, India.

ABSTRACT
Mycobacterium tuberculosis (MTB) persistently infects and survives within the host macrophages. Substantial genotypic variation exists among MTB strains which correlate with their interactions with the host. The present study was designed to establish a correlation, if any, between infection and induction of innate immune response by genetically diverse drug resistant MTB isolates from India. For this purpose, three clinical isolates from ancient and modern lineages, along with H37Ra and H37Rv were evaluated for intracellular growth, phagocytic index, induction of proinflammatory cytokines and apoptosis following infection in THP-1 cell line. A wide variation in the induction of cytokines was revealed subsequent to infection with different strains. EAI-5 strain from ancient lineage 1, induced higher proinflammatory responses, higher apoptosis and moderate intracellular growth compared to other strains, in contrast, for Beijing strain of modern lineage 2, all three parameters were lowest among the clinical isolates. Further, the responses induced by LAM-6 from modern lineage 4 were at a moderate level, similar to the laboratory strain H37Rv which also belongs to lineage 4. Thus, these profiles were specific to their respective lineages and/or genotypes and independent of their drug resistance status. Further, a positive correlation, among TNF-α, IL-1β, IL-6 and IL-12 induced in infected THP-1 cells was demonstrated. In addition, induction of all pro-inflammatory cytokines correlated well with the host cell apoptosis. A positive correlation was observed between phagocytic index in the category of '>10 bacilli/cell' and induction of apoptosis, only for virulent strains, indicating that initial accumulation of MTB strains inside the host cell may be an important determining factor for different innate responses.

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Intracellular growth of different MTB strains.THP-1 cells were infected with H37Ra, H37Rv, EAI, LAM and Beijing strains of MTB at MOI of 10 for 4 hrs and after removing extracellular bacteria, the infected cells were further incubated with medium for 1–5 days. After each incubation time, the infected cells were lysed. The lysates of infected cells were either inoculated in radiorespirometry vial containing LJ medium with 14C acetate or serially diluted and plated for CFU assay. For radiorespirometry (A), the counts were taken in a Liquid Scintillation counter (LSC) five days after vial preparation and for CFU assay (B) colonies were counted after 30 days of plating. Three such independent experiments were carried out and the data points represent mean ± SEM from all three experiments.
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pone-0062966-g002: Intracellular growth of different MTB strains.THP-1 cells were infected with H37Ra, H37Rv, EAI, LAM and Beijing strains of MTB at MOI of 10 for 4 hrs and after removing extracellular bacteria, the infected cells were further incubated with medium for 1–5 days. After each incubation time, the infected cells were lysed. The lysates of infected cells were either inoculated in radiorespirometry vial containing LJ medium with 14C acetate or serially diluted and plated for CFU assay. For radiorespirometry (A), the counts were taken in a Liquid Scintillation counter (LSC) five days after vial preparation and for CFU assay (B) colonies were counted after 30 days of plating. Three such independent experiments were carried out and the data points represent mean ± SEM from all three experiments.

Mentions: The intracellular growth was monitored by radiorespirometry and as CFU. Figure 2A shows the cumulative response (CPM obtained for 14CO2 released by viable bacilli), observed by radiorespirometry technique for intracellular bacterial load of different MTB strains at different time points. Fig. 2B shows CFU counts obtained for corresponding time points after infection. The radiorespirometry data correlated well with CFU counts (R2 = 0.98, P<0.05), confirming that the signal observed in radiorespirometry was from intracellular bacteria. A gradual increase in intracellular bacilli was observed for all the strains, with H37Rv and LAM-6 showing significantly higher intracellular bacillary growth compared to H37Ra. Though, THP-1 cells infected by different strains showed different percentages of cells in three different categories mentioned above, the total number of intracellular bacteria on day zero were in the range of 6.6 × 104 to 7.1 × 104 (as per CFU assay), which were not significantly different to give differences in cpm values in the radiorespirometry assay.


Drug resistant clinical isolates of Mycobacterium tuberculosis from different genotypes exhibit differential host responses in THP-1 cells.

Chakraborty P, Kulkarni S, Rajan R, Sainis K - PLoS ONE (2013)

Intracellular growth of different MTB strains.THP-1 cells were infected with H37Ra, H37Rv, EAI, LAM and Beijing strains of MTB at MOI of 10 for 4 hrs and after removing extracellular bacteria, the infected cells were further incubated with medium for 1–5 days. After each incubation time, the infected cells were lysed. The lysates of infected cells were either inoculated in radiorespirometry vial containing LJ medium with 14C acetate or serially diluted and plated for CFU assay. For radiorespirometry (A), the counts were taken in a Liquid Scintillation counter (LSC) five days after vial preparation and for CFU assay (B) colonies were counted after 30 days of plating. Three such independent experiments were carried out and the data points represent mean ± SEM from all three experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646887&req=5

pone-0062966-g002: Intracellular growth of different MTB strains.THP-1 cells were infected with H37Ra, H37Rv, EAI, LAM and Beijing strains of MTB at MOI of 10 for 4 hrs and after removing extracellular bacteria, the infected cells were further incubated with medium for 1–5 days. After each incubation time, the infected cells were lysed. The lysates of infected cells were either inoculated in radiorespirometry vial containing LJ medium with 14C acetate or serially diluted and plated for CFU assay. For radiorespirometry (A), the counts were taken in a Liquid Scintillation counter (LSC) five days after vial preparation and for CFU assay (B) colonies were counted after 30 days of plating. Three such independent experiments were carried out and the data points represent mean ± SEM from all three experiments.
Mentions: The intracellular growth was monitored by radiorespirometry and as CFU. Figure 2A shows the cumulative response (CPM obtained for 14CO2 released by viable bacilli), observed by radiorespirometry technique for intracellular bacterial load of different MTB strains at different time points. Fig. 2B shows CFU counts obtained for corresponding time points after infection. The radiorespirometry data correlated well with CFU counts (R2 = 0.98, P<0.05), confirming that the signal observed in radiorespirometry was from intracellular bacteria. A gradual increase in intracellular bacilli was observed for all the strains, with H37Rv and LAM-6 showing significantly higher intracellular bacillary growth compared to H37Ra. Though, THP-1 cells infected by different strains showed different percentages of cells in three different categories mentioned above, the total number of intracellular bacteria on day zero were in the range of 6.6 × 104 to 7.1 × 104 (as per CFU assay), which were not significantly different to give differences in cpm values in the radiorespirometry assay.

Bottom Line: EAI-5 strain from ancient lineage 1, induced higher proinflammatory responses, higher apoptosis and moderate intracellular growth compared to other strains, in contrast, for Beijing strain of modern lineage 2, all three parameters were lowest among the clinical isolates.Thus, these profiles were specific to their respective lineages and/or genotypes and independent of their drug resistance status.Further, a positive correlation, among TNF-α, IL-1β, IL-6 and IL-12 induced in infected THP-1 cells was demonstrated.

View Article: PubMed Central - PubMed

Affiliation: Radiation Medicine Centre, Bio-Medical Group, Bhabha Atomic Research Centre, Mumbai, India.

ABSTRACT
Mycobacterium tuberculosis (MTB) persistently infects and survives within the host macrophages. Substantial genotypic variation exists among MTB strains which correlate with their interactions with the host. The present study was designed to establish a correlation, if any, between infection and induction of innate immune response by genetically diverse drug resistant MTB isolates from India. For this purpose, three clinical isolates from ancient and modern lineages, along with H37Ra and H37Rv were evaluated for intracellular growth, phagocytic index, induction of proinflammatory cytokines and apoptosis following infection in THP-1 cell line. A wide variation in the induction of cytokines was revealed subsequent to infection with different strains. EAI-5 strain from ancient lineage 1, induced higher proinflammatory responses, higher apoptosis and moderate intracellular growth compared to other strains, in contrast, for Beijing strain of modern lineage 2, all three parameters were lowest among the clinical isolates. Further, the responses induced by LAM-6 from modern lineage 4 were at a moderate level, similar to the laboratory strain H37Rv which also belongs to lineage 4. Thus, these profiles were specific to their respective lineages and/or genotypes and independent of their drug resistance status. Further, a positive correlation, among TNF-α, IL-1β, IL-6 and IL-12 induced in infected THP-1 cells was demonstrated. In addition, induction of all pro-inflammatory cytokines correlated well with the host cell apoptosis. A positive correlation was observed between phagocytic index in the category of '>10 bacilli/cell' and induction of apoptosis, only for virulent strains, indicating that initial accumulation of MTB strains inside the host cell may be an important determining factor for different innate responses.

Show MeSH
Related in: MedlinePlus