Autophagy is a conserved catabolic process that degrades cytoplasmic constituents in the lysosome and thus contributes to the maintenance of intracellular homeostasis. The process of autophagy has ...been involved in many physiological and pathological processes. Therefore, there is a developing need to identify, quantify, and manipulate the autophagic process accurately in the cells. As autophagy involves dynamic and complex processes, therefore various approaches are needed to study this process precisely. In this chapter, we have tried to elaborate the approaches and methods to monitor autophagy, with a primary focus on mammalian macroautophagy. Autophagy induction can be detected using Western blotting of LC3 (marker protein for autophagosomes) in which LC3-II levels represent the quantity of autophagosomes formed on induction to a particular stimulus. This can also be confirmed by puncta formation assay using confocal microscopy. Further, the autophagic flux can be examined using bafilomycin A1 as inhibitor of autophagosome-lysosome fusion and acidification of lysosomal compartments, thereby leading to accumulation of autophagosomes which is represented by high LC3-II levels. The autophagolysosomal degradation or proteolysis which is the last step of autophagy can be analyzed by DQ-BSA assay.
Cancer associated morbidity is mostly attributed to the dissemination of tumor cells from their primary niche into the circulation known as “metastasis”. Mycobacterium indicus pranii (MIP) an ...approved immunotherapeutic agent against lung cancer (NSCLC) has shown potent anti-tumor activity in prior studies. While evaluating anti-tumor activity of MIP in mouse model, MIP treated animals typically exhibited less metastatic lesions in their pulmonary compartment. To study the role of MIP in metastasis closely, B16F10 melanoma cells were implanted subcutaneously in the mice, and the dissemination of tumor cells from the solid tumor was evaluated over a period of time. When B16F10 melanoma cells were treated with MIP in vitro, downregulation of epithelial mesenchymal transition markers was observed in these cells, which in turn suppressed the invasion, migration and adhesion of tumor cells. Notably, MIP therapy was found to be effectively reducing the metastatic burden in murine model of melanoma. Molecular characterization of MIP treated tumor cells substantiated that MIP upregulates the PPARγ expression within the tumor cells, which attenuates the NFκB/p65 levels within the nucleus, resulting in the suppression of Mmp9 expression in tumor cells. Besides that, MIP also downregulated the surface expression of chemokine receptor CXCR4 in murine melanoma cells, where chromatin immunoprecipitation confirmed the impeded recruitment of p50 and c-Rel factors to the Cxcr4 promoter, resulting in its downregulation transcriptionally. Taken together, MIP suppressed the dissemination of tumor cells in vivo, by regulating the expression of MMP9 and CXCR4 on these cells.
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MIP therapy reduced metastasis of B16F10 cells from primary tumor.Mmp9 is downregulated by MIP therapy in a PPARγ dependent manner in tumor cells.MIP therapy significantly reduced the surface expression of CXCR4 on tumor cells.c-Rel and p50 recruitment to Cxcr4 promoter is suppressed in MIP treated tumor cells.MIP and AMD3100 in combination obliterated metastasis completely in murine tumor.
The therapeutic potential of plant-based natural products is well known, yet it is actually limited by poor water solubility, low bioavailability and rapid metabolism. These issues can be solved by ...formulation with silica, gold, iron and silver nanoparticles. This manuscript discusses the synthesis and toxicity of inorganic nanoparticles used for therapeutic applications. The use of silver nanoparticles in the 0.4–30 µg dose range is optimal without any severe short-term toxicity. The bio-distribution of gold nanoparticles are controlled by their size; for instance, 5–15 nm particles show broader organ distribution, whereas 50–100 nm particles were mainly found in the liver and spleen. Curcumin-loaded silver nanoparticles decrease four times the minimum effective concentration against gram-positive and gram-negative bacteria. Mesoporous silica nanoparticles enhance the solubility of resveratrol up to about 95%. Mesoporous silica nanoparticles delivered 2.68 fold higher paclitaxel concentrations in lungs as compared to the free drug.
Mycobacterium indicus pranii (MIP) known for its immunotherapeutic potential against leprosy and tuberculosis is undergoing various clinical trials and also simultaneously being studied in animal ...models to get insight into the mechanistic details contributing to its protective efficacy as a vaccine candidate. Studies have shown potential immunomodulatory properties of MIP, the most significant being the ability to induce strong Th1 type of response, enhanced expression of pro-inflammatory cytokines, activation of APCs and lymphocytes, elicitation of M.tb specific poly-functional T cells. All of these form crucial components of host-immune response during M.tb infection. Also, MIP was found to be potent inducer of autophagy in macrophages which resulted in enhanced clearance of M.tb from MIP and M.tb co-infected cells. Hence, we further examined the component/s of MIP responsible for autophagy induction. Interestingly, we found that MIP lipids and DNA were able to induce autophagy but not the protein fraction. LAM being one of the crucial components of mycobacterial cell-wall lipids and possessing the ability of immunomodulation; we isolated LAM from MIP and did a comparative study with M.tb-LAM. Stimulation with MIP-LAM resulted in significantly high secretion of pro-inflammatory cytokines and displayed high autophagy inducing potential in macrophages as compared to M.tb-LAM. Treatment with MIP-LAM enhanced the co-localization of M.tb within the phago-lysosomes and increased the clearance of M.tb from the infected macrophages. This study describes LAM to be a crucial component of MIP which has significant contribution to its immunotherapeutic efficacy against TB.
Summary
Mycobacterium indicus pranii (MIP) is an atypical mycobacterial species possessing strong immunomodulatory properties. It is a potent vaccine candidate against tuberculosis, promotes Th1 ...immune response and protects mice from tumours. In previous studies, we demonstrated higher protective efficacy of MIP against experimental tuberculosis as compared with bacillus Calmette–Guérin (BCG). Since macrophages play an important role in the pathology of mycobacterial diseases and cancer, in the present study, we evaluated the MIP in live and killed form for macrophage activation potential, compared it with BCG and investigated the underlying mechanisms. High levels of tumour necrosis factor‐α, interleukin‐12p40 (IL‐12p40), IL‐6 and nitric oxide were produced by MIP‐stimulated macrophages as compared with BCG‐stimulated macrophages. Prominent up‐regulation of co‐stimulatory molecules CD40, CD80 and CD86 was also observed in response to MIP. Loss of response in MyD88‐deficient macrophages showed that both MIP and BCG activate the macrophages in a MyD88‐dependent manner. MyD88 signalling pathway culminates in nuclear factor‐κB/activator protein‐1 (NF‐κB/AP‐1) activation and higher activation of NF‐κB/AP‐1 was observed in response to MIP. With the help of pharmacological inhibitors and Toll‐like receptor (TLR) ‐deficient macrophages, we observed the role of TLR2, TLR4 and intracellular TLRs in MIP‐mediated macrophage activation. Stimulation of HEK293 cells expressing TLR2 in homodimeric or heterodimeric form showed that MIP has a distinctly higher level of TLR2 agonist activity compared with BCG. Further experiments suggested that TLR2 ligands are well exposed in MIP whereas they are obscured in BCG. Our findings establish the higher macrophage activation potential of MIP compared with BCG and delineate the underlying mechanism.
Mycobacterium indicus pranii (MIP) is a potent vaccine candidate against tuberculosis (TB) as it has demonstrated significant protection in animal models of tuberculosis as well as in clinical ...trials. Higher protective efficacy of MIP against TB as compared to BCG provoked the efforts to gain insight into the molecular mechanisms underlying MIP mediated protection against Mycobacterium tuberculosis (M.tb). Autophagy, initially described as a cell survival mechanism during starvation, also plays a key role in host resistance to M.tb. Virulent mycobacteria like M.tb, suppresses host autophagy response to increase its survival in macrophages. Since mycobacterial species have been shown to vary widely in their autophagy-inducing properties, in the present study, we examined the autophagy inducing efficacy of MIP and its role in MIP-mediated protection against M.tb. MIP was found to be potent inducer of autophagy in macrophages. Induced autophagy was responsible for reversal of the phagosome maturation block and phagolysosome fusion inhibition in M.tb infected macrophages, which ultimately lead to significantly enhanced clearance of M.tb from the macrophages. This is an important study which further delineated the underlying mechanisms for significant immunotherapeutic activity observed in TB patients / animal models of tuberculosis, given MIP therapy along with chemotherapy.
Mycobacterium indicus pranii (MIP) is an atypical mycobacterium species with potent antitumor efficacy. Macrophages and dendritic cells (DCs) are antigen-presenting cells, playing key roles in the ...activation of antitumor immunity. We have previously shown the potent activation of macrophages and DCs by MIP, which is mediated by MyD88-TLR2 signaling axis. In the present study, we further examined the role of MyD88 and TLR2 in MIP-mediated tumor regression.
Wild-type and MyD88
mice were implanted with B16F10 tumor cells, treated with MIP or phosphate-buffered saline (PBS) and monitored for tumor growth. As expected, MIP therapy led to significant tumor regression in wild-type mice. However, antitumor efficacy of MIP was lost in MyD88
animals. Both PBS-treated (control) and MIP-treated MyD88
mice developed tumors with comparable volume. Since MyD88 relays TLR engagement signals, we analyzed the antitumor efficacy of MIP in TLR2
and TLR4
mice. It was observed that MIP therapy reduced tumor burden in wild-type and TLR4
mice but not in TLR2
mice. Tumor volume in MIP-treated TLR2
mice were comparable with those in PBS-treated wild-type animals. These results implicated the MyD88-TLR2 signaling axis in the antitumor efficacy of MIP.
Macrophages and dendritic cells (DCs) play key role in the recognition of mycobacterial infection and mounting of antimycobacterial immunity. In case of macrophages, recognition of BCG and other ...mycobacteria has been attributed predominantly to MyD88-dependent singling. Interestingly, in previous study with bone marrow-derived DCs, we have shown that BCG promotes the survival of wild-type and MyD88
cells to the comparable levels. In the present study, we further examined MyD88
DC's response to BCG.
Bone marrow-derived DCs from wild-type and MyD88
mice were stimulated with BCG and analyzed for cytokine secretion. As expected, BCG-stimulated wild-type DCs produced significant amount of TNF-α and IL-12p40 in response to BCG. Interestingly, BCG-stimulated MyD88
DCs were also found to secret significantly higher levels of TNF-α and IL-12p40, compared with unstimulated DCs. We further observed that wild-type DCs produced significant level of immunosuppressive cytokine IL-10 in response to BCG, whereas MyD88
DCs secreted very low amount of IL-10 when stimulated with BCG. These findings demonstrated that MyD88
DCs exhibit a skewed cytokine response to BCG.
The evolving tumor secretes various immunosuppressive factors that reprogram the tumor microenvironment (TME) to become immunologically cold. Consequently, various immunosuppressive cells like Tregs ...are recruited into the TME which in turn subverts the anti-tumor response of dendritic cells and T cells.Tumor immunotherapy is a popular means to rejuvenate the immunologically cold TME into hot.
(MIP) has shown strong immunomodulatory activity in different animal and human tumor models and has been approved for treatment of lung cancer (NSCLC) patients as an adjunct therapy. Previously, MIP has shown TLR2/9 mediated activation of antigen presenting cells/Th1 cells and their enhanced infiltration in mouse melanoma but the underlying mechanism by which it is modulating these immune cells is not yet known.
This study reports for the first time that MIP immunotherapy involves type 1 interferon (IFN) signaling as one of the major signaling pathways to mediate the antitumor responses. Further, it was observed that MIP therapy significantly influenced frequency and activation of different subsets of T cells like regulatory T cells (Tregs) and CD8+ T cells in the TME. It reduces the migration of Tregs into the TME by suppressing the expression of CCL22, a Treg recruiting chemokine on DCs and this process is dependent on type 1 IFN. Simultaneously, in a type 1 IFN dependent pathway, it enhances the activation and effector function of the immunosuppressive tumor resident DCs which in turn effectively induce the proliferation and effector function of the CD8
T cells.
This study also provides evidence that MIP induced pro-inflammatory responses including induction of effector function of conventional dendritic cells and CD8
T cells along with reduction of intratumoral Treg frequency are essentially mediated in a type 1 IFN-dependent pathway.
TB-IRIS is an abnormal inflammatory response in a subset of HIV-TB co-infected patients shortly after initiation of anti-retroviral therapy (ART). Therapy in these patients could have greatly ...improved the life expectancy as ART reconstitutes the function and number of CD4+ T cells and many patients see improvement in symptoms but paradoxically up to 54% of co-infected patients develop TB-IRIS. Different studies have indicated that both innate and adaptive immunity are involved in the pathology of IRIS but the role of macrophages in abnormal activation of CD4+ T cells is poorly understood. Since macrophages are one of the major antigen-presenting cells and are infected by M.tb at a high frequency, they are very much likely to be involved in the development of TB-IRIS. In this study, we have developed a mouse model of experimental IRIS, in which M.tb-infected T-cell knockout mice undergo a fatal inflammatory disease after CD4+ T cell reconstitution. Lung macrophages and blood monocytes from M.tb-infected TCRβ
mice showed upregulated expression of cell surface activation markers and also showed higher mRNA expression of inflammation-associated chemokines and matrix metalloproteases responsible for tissue damage. Furthermore, cytokine and TLR signaling feedback mechanism to control excessive inflammation was also found to be dysregulated in these macrophages under lymphopenic conditions. Previous studies have shown that hyperactive CD4+ T cells are responsible for disease induction and our study shows that somehow macrophages are in a higher activated state when infected with M.tb in an immune-deficient condition, which results in excessive activation of the adoptively transferred CD4+ T cells. Understanding of the mechanisms underlying the pathophysiology of TB-IRIS would facilitate identification of prospective biomarkers for disease development in HIV-TB co-infected patients before starting antiretroviral therapy.
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