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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.


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Triggering through C40.T4 showed augmentation in autophagy. DCs infected with Mtb were stimulated through C40.T4, CD40, TLR-4 for 24 h and assessed for (A) NO in SNs by Greiss assay. (B) Expression of LC3 was detected in the whole cells lysates of DCs stimulated for 2 h through C40.T4 by immunoblotting. Actin was used as a loading control. Densitometric data show the LC3I/actin and LC3II/actin ratio. (C) LC3 puncta formation was demonstrated by immunofluorescence staining. Starved DCs were used as a positive control; (D,E) Bar graphs depict the percentage of LC3 puncta positive cells and LC3 puncta per cells, respectively. (F) DCs were stimulated through C40.T4 for 5 h were later incubated with acridine orange for 15 min to visualize autophagosomes by fluorescence microscopy (40×). Orange dots indicate the acidic vacuoles. (G) BeclinKD DCs were infected with Mtb followed by stimulation with C40.T4. Later, cells were lysed and bacterial burden was quantified by CFU assay. Data represented as the mean ± SD (A,D,E,G); immunoblotting (B); fluorescent microscopy (C,F); are of 2–3 independent experiments. “*” and “**” indicate p < 0.05 and p < 0.01, respectively.
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Figure 6: Triggering through C40.T4 showed augmentation in autophagy. DCs infected with Mtb were stimulated through C40.T4, CD40, TLR-4 for 24 h and assessed for (A) NO in SNs by Greiss assay. (B) Expression of LC3 was detected in the whole cells lysates of DCs stimulated for 2 h through C40.T4 by immunoblotting. Actin was used as a loading control. Densitometric data show the LC3I/actin and LC3II/actin ratio. (C) LC3 puncta formation was demonstrated by immunofluorescence staining. Starved DCs were used as a positive control; (D,E) Bar graphs depict the percentage of LC3 puncta positive cells and LC3 puncta per cells, respectively. (F) DCs were stimulated through C40.T4 for 5 h were later incubated with acridine orange for 15 min to visualize autophagosomes by fluorescence microscopy (40×). Orange dots indicate the acidic vacuoles. (G) BeclinKD DCs were infected with Mtb followed by stimulation with C40.T4. Later, cells were lysed and bacterial burden was quantified by CFU assay. Data represented as the mean ± SD (A,D,E,G); immunoblotting (B); fluorescent microscopy (C,F); are of 2–3 independent experiments. “*” and “**” indicate p < 0.05 and p < 0.01, respectively.

Mentions: Involvement of NO is a classical mechanism related with the killing of intracellular pathogens. Hence, we thought to monitor the change in the NO secretion. Importantly, magnitude of NO secretion was significantly (p < 0.01) enhanced in Mtb-infected DCs triggered through C40.T4, as compared to untreated or CD40 or TLR-4 triggered DCs (Figure 6A). Furthermore, our Western blotting experiments also showed considerable enhancement in the expression of iNOS (Figure S3A in Supplementary Material). We confirmed the specificity of NO on the survival of Mtb by culturing C40.T4 activated DCs with iNOS inhibitor (N-monomethyl l-arginine). We observed the recovery of viable bacteria in the presence of iNOS inhibitor (Figure S3B in Supplementary Material). However, only marginal restoration in the survival of Mtb was achieved, indicating the involvement of an additional mechanism. Hence, we thought to monitor autophagy, which is considered to be quite crucial in inhibiting the intracellular survival of Mtb (31, 32). Intriguingly, C40.T4 triggered DCs exhibited significantly higher conversion of LC3I to LC3II, a hallmark phenomenon of autophagy (Figure 6B). This information was corroborated with intracellular LC3 staining for puncta formation (Figures 6C–E). Furthermore, acidic vacuoles were stained with acridine orange dye to show the induction of autophagy (Figure 6F). In addition, we have knocked down the expression of beclin in DCs through siRNA prior to Mtb infection (Figure S3C in Supplementary Material). We observed that beclin knock down DCs if triggered through C40.T4 fails to restrict the Mtb growth (Figure 6G). These results suggest that C40.T4-induced autophagy significantly contributes in constraining the Mtb growth.


Stimulation through CD40 and TLR-4 Is an Effective Host Directed Therapy against Mycobacterium tuberculosis
Triggering through C40.T4 showed augmentation in autophagy. DCs infected with Mtb were stimulated through C40.T4, CD40, TLR-4 for 24 h and assessed for (A) NO in SNs by Greiss assay. (B) Expression of LC3 was detected in the whole cells lysates of DCs stimulated for 2 h through C40.T4 by immunoblotting. Actin was used as a loading control. Densitometric data show the LC3I/actin and LC3II/actin ratio. (C) LC3 puncta formation was demonstrated by immunofluorescence staining. Starved DCs were used as a positive control; (D,E) Bar graphs depict the percentage of LC3 puncta positive cells and LC3 puncta per cells, respectively. (F) DCs were stimulated through C40.T4 for 5 h were later incubated with acridine orange for 15 min to visualize autophagosomes by fluorescence microscopy (40×). Orange dots indicate the acidic vacuoles. (G) BeclinKD DCs were infected with Mtb followed by stimulation with C40.T4. Later, cells were lysed and bacterial burden was quantified by CFU assay. Data represented as the mean ± SD (A,D,E,G); immunoblotting (B); fluorescent microscopy (C,F); are of 2–3 independent experiments. “*” and “**” indicate p < 0.05 and p < 0.01, respectively.
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Figure 6: Triggering through C40.T4 showed augmentation in autophagy. DCs infected with Mtb were stimulated through C40.T4, CD40, TLR-4 for 24 h and assessed for (A) NO in SNs by Greiss assay. (B) Expression of LC3 was detected in the whole cells lysates of DCs stimulated for 2 h through C40.T4 by immunoblotting. Actin was used as a loading control. Densitometric data show the LC3I/actin and LC3II/actin ratio. (C) LC3 puncta formation was demonstrated by immunofluorescence staining. Starved DCs were used as a positive control; (D,E) Bar graphs depict the percentage of LC3 puncta positive cells and LC3 puncta per cells, respectively. (F) DCs were stimulated through C40.T4 for 5 h were later incubated with acridine orange for 15 min to visualize autophagosomes by fluorescence microscopy (40×). Orange dots indicate the acidic vacuoles. (G) BeclinKD DCs were infected with Mtb followed by stimulation with C40.T4. Later, cells were lysed and bacterial burden was quantified by CFU assay. Data represented as the mean ± SD (A,D,E,G); immunoblotting (B); fluorescent microscopy (C,F); are of 2–3 independent experiments. “*” and “**” indicate p < 0.05 and p < 0.01, respectively.
Mentions: Involvement of NO is a classical mechanism related with the killing of intracellular pathogens. Hence, we thought to monitor the change in the NO secretion. Importantly, magnitude of NO secretion was significantly (p < 0.01) enhanced in Mtb-infected DCs triggered through C40.T4, as compared to untreated or CD40 or TLR-4 triggered DCs (Figure 6A). Furthermore, our Western blotting experiments also showed considerable enhancement in the expression of iNOS (Figure S3A in Supplementary Material). We confirmed the specificity of NO on the survival of Mtb by culturing C40.T4 activated DCs with iNOS inhibitor (N-monomethyl l-arginine). We observed the recovery of viable bacteria in the presence of iNOS inhibitor (Figure S3B in Supplementary Material). However, only marginal restoration in the survival of Mtb was achieved, indicating the involvement of an additional mechanism. Hence, we thought to monitor autophagy, which is considered to be quite crucial in inhibiting the intracellular survival of Mtb (31, 32). Intriguingly, C40.T4 triggered DCs exhibited significantly higher conversion of LC3I to LC3II, a hallmark phenomenon of autophagy (Figure 6B). This information was corroborated with intracellular LC3 staining for puncta formation (Figures 6C–E). Furthermore, acidic vacuoles were stained with acridine orange dye to show the induction of autophagy (Figure 6F). In addition, we have knocked down the expression of beclin in DCs through siRNA prior to Mtb infection (Figure S3C in Supplementary Material). We observed that beclin knock down DCs if triggered through C40.T4 fails to restrict the Mtb growth (Figure 6G). These results suggest that C40.T4-induced autophagy significantly contributes in constraining the Mtb growth.

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