Age-mediated gut microbiota dysbiosis promotes the loss of dendritic cells tolerance.

The old age-related loss of immune tolerance inflicts a person with a wide range of autoimmune and inflammatory diseases. Dendritic cells (DCs) are the sentinels of the immune system that maintain immune tolerance through cytokines and regulatory T-cells generation. Aging disturbs the microbial composition of the gut, causing immune system dysregulation. However, the vis-à-vis role of gut dysbiosis on DCs tolerance remains highly elusive. Consequently, we studied the influence of aging on gut dysbiosis and its impact on the loss of DC tolerance. We show that DCs generated from either the aged (DCOld ) or gut-dysbiotic young (DCDysbiotic ) but not young (DCYoung ) mice exhibited loss of tolerance, as evidenced by their failure to optimally induce the generation of Tregs and control the overactivation of CD4+ T cells. The mechanism deciphered for the loss of DCOld and DCDysbiotic tolerance was chiefly through the overactivation of NF-κB, impaired frequency of Tregs, upregulation in the level of pro-inflammatory molecules (IL-6, IL-1ß, TNF-α, IL-12, IFN-γ), and decline in the anti-inflammatory moieties (IL-10, TGF-ß, IL-4, IDO, arginase, NO, IRF-4, IRF-8, PDL1, BTLA4, ALDH2). Importantly, a significant decline in the frequency of the Lactobacillus genus was noticed in the gut. Replenishing the gut of old mice with the Lactobacillus plantarum reinvigorated the tolerogenic function of DCs through the rewiring of inflammatory and metabolic pathways. Thus, for the first time, we demonstrate the impact of age-related gut dysbiosis on the loss of DC tolerance. This finding may open avenues for therapeutic intervention for treating age-associated disorders with the Lactobacillus plantarum.

Targeting dendritic cells with TLR-2 ligand-coated nanoparticles loaded with Mycobacterium tuberculosis epitope induce antituberculosis immunity.

Novel vaccination strategies are crucial to efficiently control tuberculosis, as proposed by the World Health Organization under its flagship program "End TB Strategy." However, the emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb), particularly in those coinfected with HIV-AIDS, constitutes a major impediment to achieving this goal. We report here a novel vaccination strategy that involves synthesizing a formulation of an immunodominant peptide derived from the Acr1 protein of Mtb. This nanoformulation in addition displayed on the surface a toll-like receptor-2 ligand to offer to target dendritic cells (DCs). Our results showed an efficient uptake of such a concoction by DCs in a predominantly toll-like receptor-2-dependent pathway. These DCs produced elevated levels of nitric oxide, proinflammatory cytokines interleukin-6, interleukin-12, and tumor necrosis factor-α, and upregulated the surface expression of major histocompatibility complex class II molecules as well as costimulatory molecules such as CD80 and CD86. Animals injected with such a vaccine mounted a significantly higher response of effector and memory Th1 cells and Th17 cells. Furthermore, we noticed a reduction in the bacterial load in the lungs of animals challenged with aerosolized live Mtb. Therefore, our findings indicated that the described vaccine triggered protective anti-Mtb immunity to control the tuberculosis infection.

Mycobacterium tuberculosis exploits MPT64 to generate myeloid-derived suppressor cells to evade the immune system.

Mycobacterium tuberculosis (Mtb) is a smart and successful pathogen since it can persist in the intimidating environment of the host by taming and tuning the immune system. Mtb releases MPT64 (Rv1980c) protein in high amounts in patients with active tuberculosis (TB). Consequently, we were curious to decipher the role of MPT64 on the differentiating dendritic cells (DCs) and its relation to evading the immune system. We observed that pre-exposure of differentiating DCs to MPT64 (DCMPT64) transformed them into a phenotype of myeloid-derived suppressor cells (MDSCs). DCMPT64 expressed a high level of immunosuppressive molecules PD-L1, TIM-3, nitric oxide (NO), arginase 1, IDO-1, IL-10 and TGF-ß, but inhibited the production of pro-inflammatory cytokines TNF-α, IL-6 and IL-12. DCMPT64 chemotaxis function was diminished due to the reduced expression of CCR7. DCMPT64 promoted the generation of regulatory T cells (Tregs) but inhibited the differentiation of Th1 cells and Th17 cells. Further, high lipid and methylglyoxal content, and reduced glucose consumption by DCMPT64, rendered them metabolically quiescent and consequently, reduced DCMPT64 ability to phagocytose Mtb and provided a safer shelter for the intracellular survival of the mycobacterium. The mechanism identified in impairing the function of DCMPT64 was through the increased production and accumulation of methylglyoxal. Hence, for the first time, we demonstrate the novel role of MPT64 in promoting the generation of MDSCs to favor Mtb survival and escape its destruction by the immune system.

Fiction and Facts about BCG Imparting Trained Immunity against COVID-19.

The Bacille Calmette-Guérin or BCG vaccine, the only vaccine available against Mycobacterium tuberculosis can induce a marked Th1 polarization of T-cells, characterized by the antigen-specific secretion of IFN-γ and enhanced antiviral response. A number of studies have supported the concept of protection by non-specific boosting of immunity by BCG and other microbes. BCG is a well-known example of a trained immunity inducer since it imparts 'non-specific heterologous' immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the recent pandemic. SARS-CoV-2 continues to inflict an unabated surge in morbidity and mortality around the world. There is an urgent need to devise and develop alternate strategies to bolster host immunity against the coronavirus disease of 2019 (COVID-19) and its continuously emerging variants. Several vaccines have been developed recently against COVID-19, but the data on their protective efficacy remains doubtful. Therefore, urgent strategies are required to enhance system immunity to adequately defend against newly emerging infections. The concept of trained immunity may play a cardinal role in protection against COVID-19. The ability of trained immunity-based vaccines is to promote heterologous immune responses beyond their specific antigens, which may notably help in defending against an emergency situation such as COVID-19 when the protective ability of vaccines is suspicious. A growing body of evidence points towards the beneficial non-specific boosting of immune responses by BCG or other microbes, which may protect against COVID-19. Clinical trials are underway to consider the efficacy of BCG vaccination against SARS-CoV-2 on healthcare workers and the elderly population. In this review, we will discuss the role of BCG in eliciting trained immunity and the possible limitations and challenges in controlling COVID-19 and future pandemics.

Insight Into the Beneficial Role of Lactiplantibacillus plantarum Supernatant Against Bacterial Infections, Oxidative Stress, and Wound Healing in A549 Cells and BALB/c Mice.

Lactiplantibacillus plantarum MTCC 2621 is a well-characterized probiotic strain and is reported to possess many health benefits. However, the wound healing potential of this probiotic is yet to be explored. Here, we have assessed the antibacterial, antioxidant, and wound healing activities of cell-free supernatant of Lactiplantibacillus plantarum MTCC 2621 (Lp2621). Lp2621 exhibited excellent antibacterial activity against the indicator bacteria in the agar well diffusion assay. Lp2621 did not show any hemolytic activity. The safety of Lp2621 gel was established using the skin irritation assay in BALB/c mice, and no dermal reactions were observed. The supernatant showed 60-100% protection of A549 cells against H2O2-induced stress. In the scratch assay, Lp2621 accelerated wound healing after 24 h of treatment. The percent wound healing was significantly higher in cells treated with Lp2621 at 18-24 h posttreatment. In an excision wound healing in mice, topical application of Lp2621 gel showed faster healing than the vehicle- and betadine-treated groups. Similar wound healing activity was observed in wounds infected with Staphylococcus aureus. Histological examination revealed better wound healing in Lp2621-treated mice. Topical treatment of the wounds with Lp2621 gel resulted in the upregulation of pro-inflammatory cytokine IL-6 in the early phase of wound healing and enhanced IL-10 expression in the later phase. These findings unveil a protective role of Lp2621 against bacterial infection, oxidative stress, and wound healing.

Immunotherapeutic Role of NOD-2 and TLR-4 Signaling as an Adjunct to Antituberculosis Chemotherapy.

Tuberculosis (TB) treatment is lengthy and inflicted with severe side-effects. Here, we attempted a novel strategy to reinforce host immunity through NOD-like receptor (NOD-2) and Toll-like receptor (TLR-4) signaling in the murine model of TB. Intriguingly, we noticed that it not only bolstered the immunity but also reduced the dose and duration of rifampicin and isoniazid therapy. Further, we observed expansion in the pool of effector (CD44hi, CD62Llo, CD127hi) and central (CD44hi, CD62Lhi, CD127hi) memory CD4 T cells and CD8 T cells and increased the intracellular killing of Mycobacterium tuberculosis (Mtb) by activated dendritic cells [CD86hi, CD40hi, IL-6hi, IL-12hi, TNF-αhi, nitric oxide (NO)hi] with significant reduction in Mtb load in the lungs and spleen of infected animals. We infer that the signaling through NOD-2 and TLR-4 may be an important approach to reduce the dose and duration of the drugs to treat TB.

Deciphering the Antibacterial Role of Peptide From Bacillus subtilis subsp. spizizenii Ba49 Against Staphylococcus aureus.

An increase in antibiotic resistance has led to escalating the need for the development of alternate therapy. Antimicrobial peptides (AMPs) are at the forefront of replacing conventional antibiotics, showing slower development of drug resistance, antibiofilm activity, and the ability to modulate the host immune response. The ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens that jeopardize most conventional antibiotics are known to be involved in severe respiratory tract, bloodstream, urinary tract, soft tissue, and skin infections. Among them, S. aureus is an insidious microbe and developed resistance against conventional antibiotics. In the present study, an AMP (named as peptide-Ba49) isolated from Bacillus subtilis subsp. spizizenii strain from Allium cepa (the common onion) exhibited strong antibacterial efficacy against S. aureus ATCC 25923. The mode of action of this peptide-Ba49 on S. aureus was deciphered through various sensitive probes, i.e., DiSC3 (5) and H2DCFDA, suggesting the peptide-Ba49 to be acting upon through change in membrane potential and by triggering the production of reactive oxygen species (ROS). This induced disruption of the cell membrane was further supported by morphological studies using scanning electron microscopy (SEM). Investigations on a possible post-antibiotic effect (PAE) of peptide-Ba49 showed prolonged PAE against S. aureus. Furthermore, the peptide-Ba49 prevented the formation of S. aureus biofilm at low concentration and showed its potential to degrade the mature biofilm of S. aureus. The peptide-Ba49 also exhibited intracellular killing potential against S. aureus ATCC 25923 in the macrophage cells, and moreover, peptide-Ba49 was found to bolster the fibroblast cell migration in the scratch assay at low concentration, exhibiting a wound healing efficacy of this peptide. These studies demonstrated that peptide-Ba49 isolated from the strain B. subtilis subsp. spizizenii could be a therapeutic candidate to combat the pathogenic S. aureus infections.

Cumulative Signaling Through NOD-2 and TLR-4 Eliminates the Mycobacterium Tuberculosis Concealed Inside the Mesenchymal Stem Cells.

For a long time, tuberculosis (TB) has been inflicting mankind with the highest morbidity and mortality. Although the current treatment is extremely potent, a few bacilli can still hide inside the host mesenchymal stem cells (MSC). The functional capabilities of MSCs are known to be modulated by TLRs, NOD-2, and RIG-1 signaling. Therefore, we hypothesize that modulating the MSC activity through TLR-4 and NOD-2 can be an attractive immunotherapeutic strategy to eliminate the Mtb hiding inside these cells. In our current study, we observed that MSC stimulated through TLR-4 and NOD-2 (N2.T4) i) activated MSC and augmented the secretion of pro-inflammatory cytokines; ii) co-localized Mtb in the lysosomes; iii) induced autophagy; iv) enhanced NF-κB activity via p38 MAPK signaling pathway; and v) significantly reduced the intracellular survival of Mtb in the MSC. Overall, the results suggest that the triggering through N2.T4 can be a future method of immunotherapy to eliminate the Mtb concealed inside the MSC.

A multiple T cell epitope comprising DNA vaccine boosts the protective efficacy of Bacillus Calmette-Guérin (BCG) against Mycobacterium tuberculosis.

BACKGROUND: Approximately 80% - 90% of individuals infected with latent Mycobacterium tuberculosis (Mtb) remain protected throughout their life-span. The release of unique, latent-phase antigens are known to have a protective role in the immune response against Mtb. Although the BCG vaccine has been administered for nine decades to provide immunity against Mtb, the number of TB cases continues to rise, thereby raising doubts on BCG vaccine efficacy. The shortcomings of BCG have been associated with inadequate processing and presentation of its antigens, an inability to optimally activate T cells against Mtb, and generation of regulatory T cells. Furthermore, BCG vaccination lacks the ability to eliminate latent Mtb infection. With these facts in mind, we selected six immunodominant CD4 and CD8 T cell epitopes of Mtb expressed during latent, acute, and chronic stages of infection and engineered a multi-epitope-based DNA vaccine (C6). RESULT: BALB/c mice vaccinated with the C6 construct along with a BCG vaccine exhibited an expansion of both CD4 and CD8 T cell memory populations and augmented IFN-γ and TNF-α cytokine release. Furthermore, enhancement of dendritic cell and macrophage activation was noted. Consequently, illustrating the elicitation of immunity that helps in the protection against Mtb infection; which was evident by a significant reduction in the Mtb burden in the lungs and spleen of C6 + BCG administered animals. CONCLUSION: Overall, the results suggest that a C6 + BCG vaccination approach may serve as an effective vaccination strategy in future attempts to control TB.

Predominance of M2 macrophages in gliomas leads to the suppression of local and systemic immunity.

Glioblastoma is a highly prevalent and aggressive form of primary brain tumor. It represents approximately 56% of all the newly diagnosed gliomas. Macrophages are one of the major constituents of tumor-infiltrating immune cells in the human gliomas. The role of immunosuppressive macrophages is very well documented in correlation with the poor prognosis of patients suffering from breast, prostate, bladder and cervical cancers. The current study highlights the correlation between the tumor-associated macrophage phenotypes and glioma progression. We observed an increase in the pool of M2 macrophages in high-grade gliomas, as confirmed by their CD68 and CD163 double-positive phenotype. In contrast, less M1 macrophages were noticed in high-grade gliomas, as evidenced by the down-regulation in the expression of CCL3 marker. In addition, we observed that higher gene expression ratio of CD163/CCL3 is associated with glioma progression. The Kaplan-Meier survival plots indicate that glioma patients with lower expression of M2c marker (CD163), and higher expression of M1 marker (CCL3) had better survival. Furthermore, we examined the systemic immune response in the peripheral blood and noted a predominance of M2 macrophages, myeloid-derived suppressor cells and PD-1+ CD4 T cells in glioma patients. Thus, the study indicates a high gene expression ratio of CD163/CCL3 in high-grade gliomas as compared to low-grade gliomas and significantly elevated frequency of M2 macrophages and PD-1+ CD4 T cells in the blood of tumor patients. These parameters could be used as an indicator of the early diagnosis and prognosis of the disease.

A Facile Approach for Synthesis and Intracellular Delivery of Size Tunable Cationic Peptide Functionalized Gold Nanohybrids in Cancer Cells.

Peptide-based drug delivery systems have become a mainstay in the contemporary medicinal field, resulting in the design and development of better pharmaceutical formulations. However, most of the available reports employ tedious multiple reaction steps for the conjugation of bioactive cationic peptides with drug delivery vehicles. To overcome these limitations, the present work describes a one-step approach for facile and time efficient synthesis of highly cationic cell penetrating peptide functionalized gold nanoparticles and their intracellular delivery. The nanoconstruct was synthesized by the reduction of gold metal ions utilizing cell penetrating peptide (CPP), which facilitated the simultaneous synthesis of metal nanoparticles and the capping of the peptide over the nanoparticle surface. The developed nanoconstruct was thoroughly characterized and tested for intracellular delivery into HeLa cells. Intriguingly, a high payload of cationic peptide over gold particles was achieved, in comparison to conventional conjugation methods. Moreover, this method also provides the ability to control the size and peptide payload of nanoparticles. The nanoconstructs produced showed enhanced cancer cell penetration (µM) and significant cytotoxic effect compared to unlabeled gold nanoparticles. Therefore, this novel approach may also have significant future potential to kill intracellular hidden dreaded pathogens like the human immunodeficiency virus, Mycobacterium tuberculosis, and so forth.

Reinforcing the Functionality of Mononuclear Phagocyte System to Control Tuberculosis.

The mononuclear phagocyte system (MPS) constitutes dendritic cells, monocytes, and macrophages. This system contributes to various functions that are essential for maintaining homeostasis, activation of innate immunity, and bridging it with the adaptive immunity. Consequently, MPS is highly important in bolstering immunity against the pathogens. However, MPS is the frontline cells in destroying Mycobacterium tuberculosis (Mtb), yet the bacterium prefers to reside in the hostile environment of macrophages. Therefore, it may be very interesting to study the struggle between Mtb and MPS to understand the outcome of the disease. In an event when MPS predominates Mtb, the host remains protected. By contrast, the situation becomes devastating when the pathogen tames and tunes the host MPS, which ultimately culminates into tuberculosis (TB). Hence, it becomes extremely crucial to reinvigorate MPS functionality to overwhelm Mtb and eliminate it. In this article, we discuss the strategies to bolster the function of MPS by exploiting the molecules associated with the innate immunity and highlight the mechanisms involved to overcome the Mtb-induced suppression of host immunity. In future, such approaches may provide an insight to develop immunotherapeutics to treat TB.

Bolstering Immunity through Pattern Recognition Receptors: A Unique Approach to Control Tuberculosis.

The global control of tuberculosis (TB) presents a continuous health challenge to mankind. Despite having effective drugs, TB still has a devastating impact on human health. Contributing reasons include the emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb), the AIDS-pandemic, and the absence of effective vaccines against the disease. Indeed, alternative and effective methods of TB treatment and control are urgently needed. One such approach may be to more effectively engage the immune system; particularly the frontline pattern recognition receptor (PRR) systems of the host, which sense pathogen-associated molecular patterns (PAMPs) of Mtb. It is well known that 95% of individuals infected with Mtb in latent form remain healthy throughout their life. Therefore, we propose that clues can be found to control the remainder by successfully manipulating the innate immune mechanisms, particularly of nasal and mucosal cavities. This article highlights the importance of signaling through PRRs in restricting Mtb entry and subsequently preventing its infection. Furthermore, we discuss whether this unique therapy employing PRRs in combination with drugs can help in reducing the dose and duration of current TB regimen.