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Stimulation through CD40 and TLR-4 Is an Effective Host Directed Therapy against Mycobacterium tuberculosis

View Article: PubMed Central - PubMed

ABSTRACT

Tuberculosis (TB) is the leading cause of morbidity and mortality among all infectious diseases. Failure of Bacillus Calmette Guerin as a vaccine and serious side-effects and toxicity due to long-term TB drug regime are the major hurdles associated with TB control. The problem is further compounded by the emergence of drug-resistance strains of Mycobacterium tuberculosis (Mtb). Consequently, it demands a serious attempt to explore safer and superior treatment approaches. Recently, an improved understanding of host–pathogen interaction has opened up new avenues for immunotherapy for treating TB. Although, dendritic cells (DCs) show a profound role in generating immunity against Mtb, their immunotherapeutic potential needs to be precisely investigated in controlling TB. Here, we have devised an approach of bolstering DCs efficacy against Mtb by delivering signals through CD40 and TLR-4 molecules. We found that DCs triggered through CD40 and TLR-4 showed increased secretion of IL-12, IL-6, and TNF-α. It also augmented autophagy. Interestingly, CD40 and TLR-4 stimulation along with the suboptimal dose of anti-TB drugs significantly fortified their efficacy to kill Mtb. Importantly, animals treated with the agonists of CD40 and TLR-4 boosted Th1 and Th17 immunity. Furthermore, it amplified the pool of memory CD4 T cells as well as CD8 T cells. Furthermore, substantial reduction in the bacterial burden in the lungs was observed. Notably, this adjunct therapy employing immunomodulators and chemotherapy can reinvigorate host immunity suppressed due to drugs and Mtb. Moreover, it would strengthen the potency of drugs in curing TB.

No MeSH data available.


Related in: MedlinePlus

Knockdown of CD40 in the presence of TLR-4 inhibitor (TLR-4i) establishes the specificity of signaling delivered through C40.T4 in inhibiting the survival of Mtb. DCs were treated with CD40 siRNA (CD40si) for 48 h. Expression of CD40 on DCs was monitored by flowcytometry. Number in histograms indicates the percentage of CD40 negative population of DCs (A) wild-type cells; (B) CD40 knock down cells. (C) CD40 knock down and wild-type DCs were infected with Mtb followed by stimulation through C40.T4 in presence or absence of TLR-4i for 24 h. Later, cells were lysed and bacterial burden was enumerated on 21 days by CFU counting. Data shown are mean ± SD and representative of two independent experiments. CD40si: CD40 knockdown by SiRNA; TLR-4i: TLR-4 inhibitor.
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Figure 4: Knockdown of CD40 in the presence of TLR-4 inhibitor (TLR-4i) establishes the specificity of signaling delivered through C40.T4 in inhibiting the survival of Mtb. DCs were treated with CD40 siRNA (CD40si) for 48 h. Expression of CD40 on DCs was monitored by flowcytometry. Number in histograms indicates the percentage of CD40 negative population of DCs (A) wild-type cells; (B) CD40 knock down cells. (C) CD40 knock down and wild-type DCs were infected with Mtb followed by stimulation through C40.T4 in presence or absence of TLR-4i for 24 h. Later, cells were lysed and bacterial burden was enumerated on 21 days by CFU counting. Data shown are mean ± SD and representative of two independent experiments. CD40si: CD40 knockdown by SiRNA; TLR-4i: TLR-4 inhibitor.

Mentions: It was quite important for us to check the specificity of signals delivered through CD40 and TLR-4. CD40 gene in DCs was knock down by siRNA (CD40KD). Efficacy of the assay, as observed by flowcytometry, was 60% (Figures 4A,B). In addition, the specificity of TLR-4 signaling was established using TLR-4 inhibitor (TLR-4i) CLI-095. Next, CD40KD DCs infected with Mtb were stimulated with CD40A and TLR-4L in the presence or absence of TLR-4i. Interestingly, such CD40KD DCs cultured in the presence of TLR-4i followed by their stimulation through C40.T4 showed no decline in the growth of Mtb, when compared with Mtb-infected DCs triggered through C40.T4. We also observed no change in the control DCs with or without mock transfection (Figure 4C). Hence, establishing the specificity of these experiments.


Stimulation through CD40 and TLR-4 Is an Effective Host Directed Therapy against Mycobacterium tuberculosis
Knockdown of CD40 in the presence of TLR-4 inhibitor (TLR-4i) establishes the specificity of signaling delivered through C40.T4 in inhibiting the survival of Mtb. DCs were treated with CD40 siRNA (CD40si) for 48 h. Expression of CD40 on DCs was monitored by flowcytometry. Number in histograms indicates the percentage of CD40 negative population of DCs (A) wild-type cells; (B) CD40 knock down cells. (C) CD40 knock down and wild-type DCs were infected with Mtb followed by stimulation through C40.T4 in presence or absence of TLR-4i for 24 h. Later, cells were lysed and bacterial burden was enumerated on 21 days by CFU counting. Data shown are mean ± SD and representative of two independent experiments. CD40si: CD40 knockdown by SiRNA; TLR-4i: TLR-4 inhibitor.
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Related In: Results  -  Collection

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Figure 4: Knockdown of CD40 in the presence of TLR-4 inhibitor (TLR-4i) establishes the specificity of signaling delivered through C40.T4 in inhibiting the survival of Mtb. DCs were treated with CD40 siRNA (CD40si) for 48 h. Expression of CD40 on DCs was monitored by flowcytometry. Number in histograms indicates the percentage of CD40 negative population of DCs (A) wild-type cells; (B) CD40 knock down cells. (C) CD40 knock down and wild-type DCs were infected with Mtb followed by stimulation through C40.T4 in presence or absence of TLR-4i for 24 h. Later, cells were lysed and bacterial burden was enumerated on 21 days by CFU counting. Data shown are mean ± SD and representative of two independent experiments. CD40si: CD40 knockdown by SiRNA; TLR-4i: TLR-4 inhibitor.
Mentions: It was quite important for us to check the specificity of signals delivered through CD40 and TLR-4. CD40 gene in DCs was knock down by siRNA (CD40KD). Efficacy of the assay, as observed by flowcytometry, was 60% (Figures 4A,B). In addition, the specificity of TLR-4 signaling was established using TLR-4 inhibitor (TLR-4i) CLI-095. Next, CD40KD DCs infected with Mtb were stimulated with CD40A and TLR-4L in the presence or absence of TLR-4i. Interestingly, such CD40KD DCs cultured in the presence of TLR-4i followed by their stimulation through C40.T4 showed no decline in the growth of Mtb, when compared with Mtb-infected DCs triggered through C40.T4. We also observed no change in the control DCs with or without mock transfection (Figure 4C). Hence, establishing the specificity of these experiments.

View Article: PubMed Central - PubMed

ABSTRACT

Tuberculosis (TB) is the leading cause of morbidity and mortality among all infectious diseases. Failure of Bacillus Calmette Guerin as a vaccine and serious side-effects and toxicity due to long-term TB drug regime are the major hurdles associated with TB control. The problem is further compounded by the emergence of drug-resistance strains of Mycobacterium tuberculosis (Mtb). Consequently, it demands a serious attempt to explore safer and superior treatment approaches. Recently, an improved understanding of host–pathogen interaction has opened up new avenues for immunotherapy for treating TB. Although, dendritic cells (DCs) show a profound role in generating immunity against Mtb, their immunotherapeutic potential needs to be precisely investigated in controlling TB. Here, we have devised an approach of bolstering DCs efficacy against Mtb by delivering signals through CD40 and TLR-4 molecules. We found that DCs triggered through CD40 and TLR-4 showed increased secretion of IL-12, IL-6, and TNF-α. It also augmented autophagy. Interestingly, CD40 and TLR-4 stimulation along with the suboptimal dose of anti-TB drugs significantly fortified their efficacy to kill Mtb. Importantly, animals treated with the agonists of CD40 and TLR-4 boosted Th1 and Th17 immunity. Furthermore, it amplified the pool of memory CD4 T cells as well as CD8 T cells. Furthermore, substantial reduction in the bacterial burden in the lungs was observed. Notably, this adjunct therapy employing immunomodulators and chemotherapy can reinvigorate host immunity suppressed due to drugs and Mtb. Moreover, it would strengthen the potency of drugs in curing TB.

No MeSH data available.


Related in: MedlinePlus