Negative Regulation of Autophagy during Macrophage Infection by <i>Mycobacterium bovis</i> BCG via Protein Kinase C Activation

Mycobacterium tuberculosis (Mtb) employs various strategies to manipulate the host’s cellular machinery, overriding critical molecular mechanisms such as phagosome-lysosome fusion, which are crucial for its destruction. The Protein Kinase C (PKC) signaling pathways play a key role in regulating phagocytosis. Recent research in Interferon-activated macrophages has unveiled that PKC phosphorylates Coronin-1, leading to a shift from phagocytosis to micropinocytosis, ultimately resulting in Mtb destruction. Therefore, this study aims to identify additional PKC targets that may facilitate Mycobacterium bovis (M. bovis) infection in macrophages. Protein extracts were obtained from THP-1 cells, both unstimulated and mycobacterial-stimulated, in the presence or absence of a general PKC inhibitor. We conducted an enrichment of phosphorylated peptides, followed by their identification through mass spectrometry (LC-MS/MS). Our analysis revealed 736 phosphorylated proteins, among which 153 exhibited alterations in their phosphorylation profiles in response to infection in a PKC-dependent manner. Among these 153 proteins, 55 are involved in various cellular processes, including endocytosis, vesicular traffic, autophagy, and programmed cell death. Importantly, our findings suggest that PKC may negatively regulate autophagy by phosphorylating proteins within the mTORC1 pathway (mTOR2/PKC/Raf-1/Tsc2/Raptor/Sequestosome-1) in response to M. bovis BCG infection, thereby promoting macrophage infection

Adipose tissue IL‐18 production is independent of caspase‐1 and caspase‐11

Abstract Background Inflammation in adipose tissue, resulting from imbalanced caloric intake and energy expenditure, contributes to the metabolic dysregulation observed in obesity. The production of inflammatory cytokines, such as IL‐1β and IL‐18, plays a key role in this process. While IL‐1β promotes insulin resistance and diabetes, IL‐18 regulates energy expenditure and food intake. Previous studies have suggested that caspase‐1, activated by the Nlrp3 inflammasome in response to lipid excess, mediates IL‐1β production, whereas activated by the Nlrp1b inflammasome in response to energy excess, mediates IL‐18 production. However, this has not been formally tested. Methods Wild‐type and caspase‐1‐deficient Balb/c mice, carrying the Nlrp1b1 allele, were fed with regular chow or a high‐fat diet for twelve weeks. Food intake and mass gain were recorded weekly. At the end of the twelve weeks, glucose tolerance and insulin resistance were evaluated. Mature IL‐18 protein levels and the inflammatory process in the adipose tissue were determined. Fasting lipid and cytokine levels were quantified in the sera of the different experimental groups. Results We found that IL‐18 production in adipose tissue is independent of caspase‐1 activity, regardless of the metabolic state, while Nlrp3‐mediated IL‐1β production remains caspase‐1 dependent. Additionally, caspase‐1 null Balb/c mice did not develop metabolic abnormalities in response to energy excess from the high‐fat diet. Conclusion Our findings suggest that IL‐18 production in the adipose tissue is independent of Nlrp3 inflammasome and caspase‐1 activation, regardless of caloric food intake. In contrast, Nlrp3‐mediated IL‐1β production is caspase‐1 dependent. These results provide new insights into the mechanisms underlying cytokine production in the adipose tissue during both homeostatic conditions and metabolic stress, highlighting the distinct roles of caspase‐1 and the Nlrp inflammasomes in regulating inflammatory responses

p38 Mitogen-Activated Protein Kinase Mediates the Fas-Induced Mitochondrial Death Pathway in CD8(+) T Cells

The p38 mitogen-activated protein kinase (MAPK) signaling pathway can be activated by a variety of stress stimuli such as UV radiation and osmotic stress. The regulation and role of this pathway in death receptor-induced apoptosis remain unclear and may depend on the specific death receptor and cell type. Here we show that binding of Fas ligand to Fas activates p38 MAPK in CD8(+) T cells and that activation of this pathway is required for Fas-mediated CD8(+) T-cell death. Active p38 MAPK phosphorylates Bcl-x(L) and Bcl-2 and prevents the accumulation of these antiapoptotic molecules within the mitochondria. Consequently, a loss of mitochondrial membrane potential and the release of cytochrome c lead to the activation of caspase 9 and, subsequently, caspase 3. Therefore, the activation of p38 MAPK is a critical link between Fas and the mitochondrial death pathway and is required for the Fas-induced apoptosis of CD8(+) T cells