Targeting of Mycobacterium tuberculosis (MTB) PE/PPE antigens that induce type 1 helper T cell (Th1) and Th17 responses represents a crucial strategy for the development of tuberculosis (TB) vaccines. However, only a few PE/PPE antigens induce these responses. Here, we sought to determine how the cell wall–associated antigen PPE60 (Rv3478) activates dendritic cell (DC) maturation and T-cell differentiation. We observed that PPE60 induces DC maturation by augmenting the protein expression of cluster of differentiation 80 (CD80) and CD86 and major histocompatibility complex (MHC) class I and MHC class II on the cell surface. PPE60 also stimulated the production of tumor necrosis factor-α (TNFα), interleukin (IL)-1β, IL-6, IL-12p70, and IL-23p19 but not IL-10. This induction was mediated by Toll-like receptor 2 (TLR2) and followed by activation of p38, c-Jun N-terminal kinase (JNK), and NF-κB signaling. PPE60 enhanced MHC-II expression and promoted antigen processing by DCs in a TLR2-dependent manner. Moreover, PPE60-stimulated DCs directed naïve CD4⁺ T cells to produce IFN-γ, IL-2, and IL-17A, expanding the Th1 and Th17 responses, along with activation of T-bet and RAR-related orphan receptor C (RORγt) but not GATA-3. Moreover, PPE60 activated the NLRP3 inflammasome followed by caspase-1–dependent IL-1β and IL-18 synthesis in DCs. Of note, pharmacological inhibition of NLRP3 activation specifically attenuated IFN-γ and IL-17A secretion into the supernatant from CD4⁺ T cells cocultured with PPE60-activated DCs. These findings indicate that PPE60 induces Th1 and Th17 immune responses by activating DCs in a TLR2-dependent manner, suggesting PPE60's potential for use in MTB vaccine development.

Trained monocytes and macrophages produce reactive oxygen species (ROS), which trigger antioxidative glutathione (GSH) response to buffer the rising ROS. However, whether and how the trained immunity is shaped by GSH synthesis remains unknown. Here, we report that β-glucan-trained macrophages from mice harboring a myeloid-specific deletion of the catalytic subunit of glutamate-cysteine ligase (Gclc) showed impaired GSH synthesis and decreased proinflammatory cytokine production in response to lipopolysaccharide challenge. Gclc deficiency compromised the activation of mammalian target of rapamycin-1 (mTOR) and expression of c-Myc transcription factors, abrogating the energy utilization and the metabolic reprogramming that allows β-glucan-trained macrophages to switch to glycolysis and glutaminolysis. Furthermore, Gclc deletion repressed effective H3K27me3 demethylation in the promoters of immunometabolic genes, such as Gls, Hk2, and Glut1, in β-glucan-trained macrophages by promoting the methyltransferase enhancer of zeste homolog 2 (EZH2). In vivo, myeloid-specific ablation of Gclc decreased the secretion of proinflammatory cytokines upon rechallenge with Candida albicans and these animals were less protected against the infection, compared with control littermates. Moreover, pharmacological inhibition of EZH2 enhanced the trained immunity response against Candida infection in Gclc-deficient mouse and human peripheral blood mononuclear cells treated with GCLC inhibitor buthionine sulfoximine (BSO). Thus, antioxidative GSH synthesis supports an environment conducive to β-glucan-induced metabolic and epigenetic reprogramming in trained immunity, allowing exploration of its functional consequences in autoimmune or inflammatory disease.

TLR2-dependent cellular signaling in Mycobacterium tuberculosis-infected macrophages causes apoptosis and inhibits class II major histocompatibility complex (MHC-II) molecules antigen processing, leading to evasion of surveillance. Mycobacterium tuberculosis (Mtb) lipoproteins are an important class of Toll-like receptor (TLR) ligand, and identified as specific components that mediate these effects. In this study, we identified and characterized Mtb lipoprotein Rv1016c (lpqT) as a cell wall associated-protein that was exposed on the cell surface and enhanced the survival of recombinants M. smegmatis_Rv1016c under stress conditions. We found that Rv1016c lipoprotein was a novel TLR2 ligand and able to induce macrophage apoptosis in a both dose- and time-dependent manner. Additionally, apoptosis induced by Rv1016c was reserved in THP-1 cells blocked with anti-TLR-2 Abs or in TLR2-/- mouse macrophages, indicating that Rv1016c-induced apoptosis is dependent on TLR2. Moreover, we demonstrated that Rv1016c lipoprotein inhibited IFN-γ-induced MHC-II expression and processing of soluble antigens in a TLR2 dependent manner. Class II transactivator (CIITA) regulates MHC II expression. In this context, Rv1016c lipoprotein diminished IFN-γ-induced expression of CIITA IV through TLR2 and MAPK Signaling. TLR2-dependent apoptosis and inhibition of MHC-II Ag processing induced by Rv1016c during mycobacteria infection may promote the release of residual bacilli from apoptotic cells and decrease recognition by CD4+ T cells. These mechanisms may allow intracellular Mtb to evade immune surveillance and maintain chronic infection.

Bacillus Calmette-Guérin (BCG) vaccination induces a type of immune memory known as "trained immunity", characterized by the immunometabolic and epigenetic changes in innate immune cells. However, the molecular mechanism underlying the strategies for inducing and/or boosting trained immunity in alveolar macrophages remains unknown. Here, we found that mucosal vaccination with the recombinant strain rBCG^PPE27 significantly augmented the trained immune response in mice, facilitating a superior protective response against Mycobacterium tuberculosis and non-related bacterial reinfection in mice when compared to BCG. Mucosal immunization with rBCG^PPE27 enhanced innate cytokine production by alveolar macrophages associated with promoted glycolytic metabolism, typical of trained immunity. Deficiency of the mammalian target of rapamycin complex 2 and hexokinase 1 abolished the immunometabolic and epigenetic rewiring in mouse alveolar macrophages after mucosal rBCG^PPE27 vaccination. Most noteworthy, utilizing rBCG^PPE27's higher-up trained effects: The single mucosal immunization with rBCG^PPE27-adjuvanted coronavirus disease (CoV-2) vaccine raised the rapid development of virus-specific immunoglobulin G antibodies, boosted pseudovirus neutralizing antibodies, and augmented T helper type 1-biased cytokine release by vaccine-specific T cells, compared to BCG/CoV-2 vaccine. These findings revealed that mucosal recombinant BCG vaccine induces lung-resident memory macrophages and enhances trained immunity via reprogramming mTORC2- and HK-1-mediated aerobic glycolysis, providing new vaccine strategies for improving tuberculosis (TB) or coronavirus variant vaccinations, and targeting innate immunity via mucosal surfaces.

Trained immunity induced by β‐glucan insult drives the functional reprogramming of macrophages to the hyperinflammatory status, contributing to developing or maintaining inflammatory diseases. Inflammatory arthritis is characterized by an idiopathically hyperinflammatory response, a phenotype similar to that of trained immunity, and its etiology involves environmental factors such as β‐glucan exposure. However, whether trained immunity contributes to inflammatory arthritis progression, as well as the reciprocal interactions, remains elusive. The study shows that β‐glucan‐induced experimental trained immunity heighten inflammation and arthritis severity in collagen‐induced arthritis (CIA) rat model. Trained macrophages by β‐glucan, upon adoptive transfer, further intensify symptoms. In arthritis progression, trained macrophages reduce fibroblast‐like synoviocytes’ (FLS) lipid peroxidation, lessening sensitivity to iFSP1‐induced ferroptosis through interleukin‐1 beta (IL‐1β)/N‐acetyltransferase 10 (NAT10)/ferroptosis suppressor protein 1 (FSP1) mRNA ac4C modification. A therapeutic approach targeting trained immunity, combining low‐dose iFSP1 and Remodelin, mitigates arthritis severity and restores ferroptosis sensitivity. Additionally, this interplay between CIA induction and β‐glucan training creates a feedback loop reinforcing trained immune memory, accelerating disease deterioration. The findings highlight trained immunity induced by endogenous or exogenous insult, such as β‐glucan, as an unexplored mechanism of inflammation dysregulation in the pathogenesis of inflammatory arthritis, opening avenues for the therapeutic approaches by targeting trained immunity.

To successfully metastasize, cancer cells must evade detachment induced cell death, known as anoikis. Unraveling the mechanisms that gastric cancer (GC) circumvent anoikis and achieve peritoneal metastasis especially during unanchored growth, could significantly improve patient outcomes. Our study reveals that GC cells exhibit increased lipid peroxidation, MDA production, and cell death during suspension culture, which can be mitigated by the intervention with liproxstatin-1 and ferrostatin-1. We discovered that oleic acid (OA) or adipocytes stimulate lipid accumulation in GC cells, thereby inhibiting lipid peroxidation and cell death. Lipid mass spectrometry confirmed an upregulation of triglyceride synthesis, indicating that the accumulation of lipid droplet may confer resistance to ferroptosis during suspension growth. In vitro assays demonstrated that OA not only induces lipid droplet accumulation but also upregulates the expression of ferroptosis suppressor protein 1 (FSP1), a process that can be abrogated by the double knockout of GPD1/1L genes. Additionally, we have demonstrated that a decrease in the ubiquitination of FSP1 in GC cells upon lipid droplet accumulation, as well as silencing or pharmacological targeting FSP1, promotes ferroptosis and disrupts the peritoneal metastatic potential of GC cells. Collectively, our findings highlight the potential of FSP1 as a promising therapeutic target for metastatic gastric cancer.