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Isoniazid induces apoptosis of activated CD4+ T cells: implications for post-therapy tuberculosis reactivation and reinfection.

Tousif S, Singh DK, Ahmad S, Moodley P, Bhattacharyya M, Van Kaer L, Das G - J. Biol. Chem. (2014)

Bottom Line: Although this therapy is effective, it has serious disadvantages.These therapeutic agents are toxic and are associated with the development of a variety of drug-resistant TB strains.Consequently, our findings suggest that TB treatment is associated with immune impairment.

View Article: PubMed Central - PubMed

Affiliation: the School of Laboratory Medicine, College of Health Sciences, University of KwaZulu-Natal, Durban 4001, South Africa.

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Related in: MedlinePlus

INH suppresses cytokine production in splenocytes. Splenocytes were cultured and activated with 50 ng/ml phorbol 12-myristate 13-acetate and 750 ng/ml ionomycin overnight, and 10 μg/ml brefeldin A was added during the last 6 h of culture. Cells were then intracellularly stained with anti-IL4, -IFNγ, -IL9, -IL10, -IL12, or -IL22 antibodies and appropriately labeled control antibodies. Cells were acquired by a flow cytometer. Data are shown as mean ± S.D., and Student's t test was performed for estimating significance between two groups.
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Figure 3: INH suppresses cytokine production in splenocytes. Splenocytes were cultured and activated with 50 ng/ml phorbol 12-myristate 13-acetate and 750 ng/ml ionomycin overnight, and 10 μg/ml brefeldin A was added during the last 6 h of culture. Cells were then intracellularly stained with anti-IL4, -IFNγ, -IL9, -IL10, -IL12, or -IL22 antibodies and appropriately labeled control antibodies. Cells were acquired by a flow cytometer. Data are shown as mean ± S.D., and Student's t test was performed for estimating significance between two groups.

Mentions: We reasoned that INH might directly cause cell death in antigen-specific T cells. To investigate this hypothesis, we isolated spleen cells and lung T cells from infected and INH-treated animals and determined apoptosis in T cells. We found that a large number of cells stained with 7-AAD, indicating that INH induces apoptosis (data not shown). Cycling cells often have increased susceptibility to apoptotic death (27), and hence we determined whether T cell death was restricted to the activated T cell population. For this purpose, we stained the cells for expression of activation markers such as CD44 and CD69. As expected, we found that the loss of T cells was largely confined to the activated T cell population (data not shown). Apoptotic death is mediated by up-regulation of the caspase pathway, with caspase-3 playing a central role. Thus, we measured caspase-3 in apoptotic cells, and found it to be profoundly up-regulated (data not shown). To further strengthen this hypothesis, we examined the expression of FasL. We observed that FasL was dramatically up-regulated (data not shown). These observations indicated that, upon treatment with INH, activated CD4+ T cells undergo apoptosis. To find out the most probable reasons for vulnerabilities of reactivation and reinfection (Fig 2) after treatment of tuberculosis infection, we checked the status of Th1 cells in INH-treated mice. Because it is well known that Th1 cells are more susceptible to apoptosis than Th2 cells (28), we tested whether INH has a biased activity in inducing apoptosis in Th1 versus Th2 cells. For this purpose, we measured cytokines in the splenocytes. We observed that Th1 cells underwent apoptosis more rapidly than Th2 cells (Fig. 3). Th1 cells (producers of IFN-γ) play a central role in host protection against TB, and M. tuberculosis thwarts such responses by several means for its unhindered lifestyle within susceptible hosts (29). Therefore, elimination of Th1 cells by INH may be detrimental and render the host susceptible to reactivation or reinfection after treatment. In this context, the available literature indicates that patients treated with DOTS therapy are more vulnerable to disease reactivation and reinfection after completion of the therapeutic regimen (18, 19, 20). Therefore, these studies suggest that therapeutic antibiotics influence antitubercular immune responses. As susceptibility to M. tuberculosis infection is determined by Th cells (30), we reasoned that loss of CD4+ T cells during treatment may play a role in post-treatment vulnerability to TB reactivation and reinfection. To test this hypothesis, we treated animals with a course of INH and subsequently either treated the animals with dexamethasone or reinfected them with M. tuberculosis H37Rv. As shown in Fig. 2a, treatment with dexamethasone promoted M. tuberculosis reactivation in INH-treated animals, as deduced from the bacterial counts in different organs. This observation suggested that primary treatment resulted in the killing of most M. tuberculosis organisms, but did not induce complete eradication. Therefore, an alteration in the status of the immune response reactivates the latent organisms. It is interesting to note that INH treatment induces apoptosis in activated Th cells, and although INH is unable to completely eliminate the M. tuberculosis organisms, the organisms do not immediately reactivate after treatment is completed. It is highly likely that M. tuberculosis hides in secluded structures such as granulomas embedded in an immunosuppressive environment, where drug penetration is low. Previously, we have shown that mesenchymal stem cells contribute to this immunosuppressive environment (31), and other host cells may contribute as well (32).


Isoniazid induces apoptosis of activated CD4+ T cells: implications for post-therapy tuberculosis reactivation and reinfection.

Tousif S, Singh DK, Ahmad S, Moodley P, Bhattacharyya M, Van Kaer L, Das G - J. Biol. Chem. (2014)

INH suppresses cytokine production in splenocytes. Splenocytes were cultured and activated with 50 ng/ml phorbol 12-myristate 13-acetate and 750 ng/ml ionomycin overnight, and 10 μg/ml brefeldin A was added during the last 6 h of culture. Cells were then intracellularly stained with anti-IL4, -IFNγ, -IL9, -IL10, -IL12, or -IL22 antibodies and appropriately labeled control antibodies. Cells were acquired by a flow cytometer. Data are shown as mean ± S.D., and Student's t test was performed for estimating significance between two groups.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215201&req=5

Figure 3: INH suppresses cytokine production in splenocytes. Splenocytes were cultured and activated with 50 ng/ml phorbol 12-myristate 13-acetate and 750 ng/ml ionomycin overnight, and 10 μg/ml brefeldin A was added during the last 6 h of culture. Cells were then intracellularly stained with anti-IL4, -IFNγ, -IL9, -IL10, -IL12, or -IL22 antibodies and appropriately labeled control antibodies. Cells were acquired by a flow cytometer. Data are shown as mean ± S.D., and Student's t test was performed for estimating significance between two groups.
Mentions: We reasoned that INH might directly cause cell death in antigen-specific T cells. To investigate this hypothesis, we isolated spleen cells and lung T cells from infected and INH-treated animals and determined apoptosis in T cells. We found that a large number of cells stained with 7-AAD, indicating that INH induces apoptosis (data not shown). Cycling cells often have increased susceptibility to apoptotic death (27), and hence we determined whether T cell death was restricted to the activated T cell population. For this purpose, we stained the cells for expression of activation markers such as CD44 and CD69. As expected, we found that the loss of T cells was largely confined to the activated T cell population (data not shown). Apoptotic death is mediated by up-regulation of the caspase pathway, with caspase-3 playing a central role. Thus, we measured caspase-3 in apoptotic cells, and found it to be profoundly up-regulated (data not shown). To further strengthen this hypothesis, we examined the expression of FasL. We observed that FasL was dramatically up-regulated (data not shown). These observations indicated that, upon treatment with INH, activated CD4+ T cells undergo apoptosis. To find out the most probable reasons for vulnerabilities of reactivation and reinfection (Fig 2) after treatment of tuberculosis infection, we checked the status of Th1 cells in INH-treated mice. Because it is well known that Th1 cells are more susceptible to apoptosis than Th2 cells (28), we tested whether INH has a biased activity in inducing apoptosis in Th1 versus Th2 cells. For this purpose, we measured cytokines in the splenocytes. We observed that Th1 cells underwent apoptosis more rapidly than Th2 cells (Fig. 3). Th1 cells (producers of IFN-γ) play a central role in host protection against TB, and M. tuberculosis thwarts such responses by several means for its unhindered lifestyle within susceptible hosts (29). Therefore, elimination of Th1 cells by INH may be detrimental and render the host susceptible to reactivation or reinfection after treatment. In this context, the available literature indicates that patients treated with DOTS therapy are more vulnerable to disease reactivation and reinfection after completion of the therapeutic regimen (18, 19, 20). Therefore, these studies suggest that therapeutic antibiotics influence antitubercular immune responses. As susceptibility to M. tuberculosis infection is determined by Th cells (30), we reasoned that loss of CD4+ T cells during treatment may play a role in post-treatment vulnerability to TB reactivation and reinfection. To test this hypothesis, we treated animals with a course of INH and subsequently either treated the animals with dexamethasone or reinfected them with M. tuberculosis H37Rv. As shown in Fig. 2a, treatment with dexamethasone promoted M. tuberculosis reactivation in INH-treated animals, as deduced from the bacterial counts in different organs. This observation suggested that primary treatment resulted in the killing of most M. tuberculosis organisms, but did not induce complete eradication. Therefore, an alteration in the status of the immune response reactivates the latent organisms. It is interesting to note that INH treatment induces apoptosis in activated Th cells, and although INH is unable to completely eliminate the M. tuberculosis organisms, the organisms do not immediately reactivate after treatment is completed. It is highly likely that M. tuberculosis hides in secluded structures such as granulomas embedded in an immunosuppressive environment, where drug penetration is low. Previously, we have shown that mesenchymal stem cells contribute to this immunosuppressive environment (31), and other host cells may contribute as well (32).

Bottom Line: Although this therapy is effective, it has serious disadvantages.These therapeutic agents are toxic and are associated with the development of a variety of drug-resistant TB strains.Consequently, our findings suggest that TB treatment is associated with immune impairment.

View Article: PubMed Central - PubMed

Affiliation: the School of Laboratory Medicine, College of Health Sciences, University of KwaZulu-Natal, Durban 4001, South Africa.

Show MeSH
Related in: MedlinePlus