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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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Cytokine induction in infected THP-1 cells.Real time PCR was carried out to estimate the mRNA expression for TNF-α, IL-1β, IL-12 and IL-10, 24 hrs after infection of THP-1 cells with M. tuberculosis H37Ra, H37Rv and three clinical isolates at MOI 10. The graph shows (A), relative mRNA expression levels presented as fold increase over specific mRNA obtained from uninfected THP-1 cells, after normalizing with housekeeping β-actin mRNA, using 2–ΔΔCt method. Three independent experiments were carried out. Data represent the means ± SD of a representative experiment. #, compared to H37Rv and $, compared to Beijing strain. (B) Shows the levels of TNF-α, IL-1β, IL-12, IL-6 and IL-10 as measured by ELISA, in the supernatant of THP-1 cells infected with different strains of MTB, 24 and 48 hrs after infection. Three such independent experiments were carried out and data represent the mean ± SD of representative experiment. #, compared to H37Rv for respective time points.
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pone-0062966-g003: Cytokine induction in infected THP-1 cells.Real time PCR was carried out to estimate the mRNA expression for TNF-α, IL-1β, IL-12 and IL-10, 24 hrs after infection of THP-1 cells with M. tuberculosis H37Ra, H37Rv and three clinical isolates at MOI 10. The graph shows (A), relative mRNA expression levels presented as fold increase over specific mRNA obtained from uninfected THP-1 cells, after normalizing with housekeeping β-actin mRNA, using 2–ΔΔCt method. Three independent experiments were carried out. Data represent the means ± SD of a representative experiment. #, compared to H37Rv and $, compared to Beijing strain. (B) Shows the levels of TNF-α, IL-1β, IL-12, IL-6 and IL-10 as measured by ELISA, in the supernatant of THP-1 cells infected with different strains of MTB, 24 and 48 hrs after infection. Three such independent experiments were carried out and data represent the mean ± SD of representative experiment. #, compared to H37Rv for respective time points.

Mentions: The mRNA expression for proinflammatory cytokines like TNF-α, IL-1β, IL-12 and anti-inflammatory cytokine IL-10, induced by different MTB strains in infected THP-1 cells, showed variable patterns (Fig. 3A). The levels of different cytokines when measured by ELISA corroborated the mRNA expression patterns (Fig. 3B). The expression of TNF-α, IL-12 and IL-1β, both at mRNA and protein level, was significantly (P<0.05) higher in EAI-5 infected THP-1 cells than those infected with H37Rv, H37Ra and Beijing strains. However, the infection with Beijing genotype induced lower levels of all cytokines mentioned above. The induction of proinflammatory response by LAM-6 was comparable with that by H37Rv. Further, there was a significant correlation among proinflammatory cytokines, (TNF-α and IL-1β, R2 = 0.879, P<0.05; TNF-α and IL-6, R2 = 0.799, P<0.05; IL-1β and IL-6, R2 = 0.927, P<0.05). There was no significant difference in expression of IL-10 mRNA among the cells infected with different strains, except for Beijing which induced significantly lower mRNA for IL-10 compared to other strains (P<0.05). However, the protein concentrations of IL-10 in the supernatants of THP-1 cells, 24 and 48 hours of post-infection, were low with no significant differences among the strains. The most interesting observation about Beijing strain was that it induced lesser amount of both pro and anti-inflammatory cytokines in THP-1 cells in contrast to their levels in supernatants of cells infected with other strains.


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)

Cytokine induction in infected THP-1 cells.Real time PCR was carried out to estimate the mRNA expression for TNF-α, IL-1β, IL-12 and IL-10, 24 hrs after infection of THP-1 cells with M. tuberculosis H37Ra, H37Rv and three clinical isolates at MOI 10. The graph shows (A), relative mRNA expression levels presented as fold increase over specific mRNA obtained from uninfected THP-1 cells, after normalizing with housekeeping β-actin mRNA, using 2–ΔΔCt method. Three independent experiments were carried out. Data represent the means ± SD of a representative experiment. #, compared to H37Rv and $, compared to Beijing strain. (B) Shows the levels of TNF-α, IL-1β, IL-12, IL-6 and IL-10 as measured by ELISA, in the supernatant of THP-1 cells infected with different strains of MTB, 24 and 48 hrs after infection. Three such independent experiments were carried out and data represent the mean ± SD of representative experiment. #, compared to H37Rv for respective time points.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646887&req=5

pone-0062966-g003: Cytokine induction in infected THP-1 cells.Real time PCR was carried out to estimate the mRNA expression for TNF-α, IL-1β, IL-12 and IL-10, 24 hrs after infection of THP-1 cells with M. tuberculosis H37Ra, H37Rv and three clinical isolates at MOI 10. The graph shows (A), relative mRNA expression levels presented as fold increase over specific mRNA obtained from uninfected THP-1 cells, after normalizing with housekeeping β-actin mRNA, using 2–ΔΔCt method. Three independent experiments were carried out. Data represent the means ± SD of a representative experiment. #, compared to H37Rv and $, compared to Beijing strain. (B) Shows the levels of TNF-α, IL-1β, IL-12, IL-6 and IL-10 as measured by ELISA, in the supernatant of THP-1 cells infected with different strains of MTB, 24 and 48 hrs after infection. Three such independent experiments were carried out and data represent the mean ± SD of representative experiment. #, compared to H37Rv for respective time points.
Mentions: The mRNA expression for proinflammatory cytokines like TNF-α, IL-1β, IL-12 and anti-inflammatory cytokine IL-10, induced by different MTB strains in infected THP-1 cells, showed variable patterns (Fig. 3A). The levels of different cytokines when measured by ELISA corroborated the mRNA expression patterns (Fig. 3B). The expression of TNF-α, IL-12 and IL-1β, both at mRNA and protein level, was significantly (P<0.05) higher in EAI-5 infected THP-1 cells than those infected with H37Rv, H37Ra and Beijing strains. However, the infection with Beijing genotype induced lower levels of all cytokines mentioned above. The induction of proinflammatory response by LAM-6 was comparable with that by H37Rv. Further, there was a significant correlation among proinflammatory cytokines, (TNF-α and IL-1β, R2 = 0.879, P<0.05; TNF-α and IL-6, R2 = 0.799, P<0.05; IL-1β and IL-6, R2 = 0.927, P<0.05). There was no significant difference in expression of IL-10 mRNA among the cells infected with different strains, except for Beijing which induced significantly lower mRNA for IL-10 compared to other strains (P<0.05). However, the protein concentrations of IL-10 in the supernatants of THP-1 cells, 24 and 48 hours of post-infection, were low with no significant differences among the strains. The most interesting observation about Beijing strain was that it induced lesser amount of both pro and anti-inflammatory cytokines in THP-1 cells in contrast to their levels in supernatants of cells infected with other strains.

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