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

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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.


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C40.T4 augmented the antigen uptake capacity of DCs. (A) C40.T4 stimulated DCs were pulsed with antigen HRP for 60 min. Cells were washed and lysed to measure HRP uptake by colorimetry. Values were normalized with experimental blank and control cells kept on ice. (B) C40.T4 activated DCs were co-cultured with GFP-expressing Mtb (gMtb) for additional 4 h and were imaged through confocal microscopy. Each image represents the cells from three different fields. (C) C40.T4 activated DCs were infected with Mtb for 4 h. Later, mycobacterial uptake was enumerated by CFUs plating. Results are expressed as mean ± SD (A,C). Data are the representative of three individual experiments. “*” and “**” indicate p < 0.05 and p < 0.01, respectively.
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Figure 2: C40.T4 augmented the antigen uptake capacity of DCs. (A) C40.T4 stimulated DCs were pulsed with antigen HRP for 60 min. Cells were washed and lysed to measure HRP uptake by colorimetry. Values were normalized with experimental blank and control cells kept on ice. (B) C40.T4 activated DCs were co-cultured with GFP-expressing Mtb (gMtb) for additional 4 h and were imaged through confocal microscopy. Each image represents the cells from three different fields. (C) C40.T4 activated DCs were infected with Mtb for 4 h. Later, mycobacterial uptake was enumerated by CFUs plating. Results are expressed as mean ± SD (A,C). Data are the representative of three individual experiments. “*” and “**” indicate p < 0.05 and p < 0.01, respectively.

Mentions: Dendritic cells are highly efficient in antigen uptake (28). Therefore, we next studied whether the C40.T4 stimulation enhanced the capacity of DCs for antigen uptake. Interestingly, C40.T4 signaling showed significant (p < 0.05) augmentation in the antigen (HRP) uptake as observed by colorimetric method (Figure 2A). It is known that DCs endocytose HRP chiefly through fluid phase pinocytosis driven by constitutive membrane ruffling activity and traces of it is taken up by receptor-mediated endocytosis (29, 30). It suggests that DCs activated through C40.T4 stimulation exhibited substantial enhancement in HRP uptake through pinocytosis. Furthermore, we studied the phagocytic ability of DCs by confocal microscopy and CFU plating assay. Our results demonstrate higher uptake of GFP+Mtb by DCs upon stimulation through C40.T4 and noteworthy (p < 0.05) increase in the Mtb uptake by confocal microscopy and CFU plating, respectively (Figures 2B,C). These results suggest that the signaling of DCs through C40.T4 improves their endocytic capacity.


Stimulation through CD40 and TLR-4 Is an Effective Host Directed Therapy against Mycobacterium tuberculosis
C40.T4 augmented the antigen uptake capacity of DCs. (A) C40.T4 stimulated DCs were pulsed with antigen HRP for 60 min. Cells were washed and lysed to measure HRP uptake by colorimetry. Values were normalized with experimental blank and control cells kept on ice. (B) C40.T4 activated DCs were co-cultured with GFP-expressing Mtb (gMtb) for additional 4 h and were imaged through confocal microscopy. Each image represents the cells from three different fields. (C) C40.T4 activated DCs were infected with Mtb for 4 h. Later, mycobacterial uptake was enumerated by CFUs plating. Results are expressed as mean ± SD (A,C). Data are the representative of three individual experiments. “*” and “**” indicate p < 0.05 and p < 0.01, respectively.
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Figure 2: C40.T4 augmented the antigen uptake capacity of DCs. (A) C40.T4 stimulated DCs were pulsed with antigen HRP for 60 min. Cells were washed and lysed to measure HRP uptake by colorimetry. Values were normalized with experimental blank and control cells kept on ice. (B) C40.T4 activated DCs were co-cultured with GFP-expressing Mtb (gMtb) for additional 4 h and were imaged through confocal microscopy. Each image represents the cells from three different fields. (C) C40.T4 activated DCs were infected with Mtb for 4 h. Later, mycobacterial uptake was enumerated by CFUs plating. Results are expressed as mean ± SD (A,C). Data are the representative of three individual experiments. “*” and “**” indicate p < 0.05 and p < 0.01, respectively.
Mentions: Dendritic cells are highly efficient in antigen uptake (28). Therefore, we next studied whether the C40.T4 stimulation enhanced the capacity of DCs for antigen uptake. Interestingly, C40.T4 signaling showed significant (p < 0.05) augmentation in the antigen (HRP) uptake as observed by colorimetric method (Figure 2A). It is known that DCs endocytose HRP chiefly through fluid phase pinocytosis driven by constitutive membrane ruffling activity and traces of it is taken up by receptor-mediated endocytosis (29, 30). It suggests that DCs activated through C40.T4 stimulation exhibited substantial enhancement in HRP uptake through pinocytosis. Furthermore, we studied the phagocytic ability of DCs by confocal microscopy and CFU plating assay. Our results demonstrate higher uptake of GFP+Mtb by DCs upon stimulation through C40.T4 and noteworthy (p < 0.05) increase in the Mtb uptake by confocal microscopy and CFU plating, respectively (Figures 2B,C). These results suggest that the signaling of DCs through C40.T4 improves their endocytic capacity.

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&ndash;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-&alpha;. 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