Ovarian cancer (OVC) is one of the deadliest and most aggressive tumors in women, with an increasing incidence in recent years. Cuproptosis, a newly discovered type of programmed cell death, is caused by intracellular copper-mediated lipoylated protein aggregation and proteotoxic stress. However, the role of cuproptosis-related features in OVC remains elusive. The single-cell sequencing data from GSE154600 and bulk transcriptome data of 378 OVC patients from TCGA database. The RNA-seq and clinical data of 379 OVC patients in GSE140082 and 173 OV patients in GSE53963. The PROGENy score was calculated to assess tumor-associated pathways. Based on gene set enrichment analysis (GSEA) of the cuproptosis pathway, the single cells were divided into the cuproptosis^high and cuproptosis^low groups. The differentially expressed genes (DEGs) between the two groups were screened, and 47 prognosis-related genes were identified based on univariate cox regression analysis. Randomforest was used to construct a prognostic model. Immuno-infiltration analysis was performed using ssGSEA and xCell algorithms. In vitro and in vivo experiments were used for functional verification. Six major cell populations was identified, including fibroblast, T cell, myeloid, epithelial cell, endothelial cell, and B cell populations. The PROGENy score which revealed significant activation of the PI3K pathway in T and B cells, and activation of the TGF-β pathway in endothelial cells and fibroblasts. TIMM8B, COX8A, SSR4, HIGD2A, WASF2, PRDX5 and CLDN4 were selected to construct a prognostic model from the identified 47 prognosis-related genes. Furthermore, the cuproptosis^high and cuproptosis^low groups showed significant differences in the expression levels of the model genes, immune cell infiltration, and sensitivity to six potential drug candidates. The functional experiments showed that WASF2 is associated with cuproptotic resistance and promotes cancer cell proliferation and resistance to platinum, and its high expression is associated with poor prognosis of OVC patients. A clinically significant cuproptosis-related prognostic model was identified which can accurately predict the prognosis and immune characteristics of OVC patients. WASF2, one of the cuproptosis-related gene in the risk model, promotes the proliferation and platinum resistance of OVC cells, and leads poor prognosis.

Mycoplasma pneumoniae (M. pneumoniae, Mp) is an intracellular pathogen that causes pneumonia, tracheobronchitis, pharyngitis, and asthma in humans and can infect and survive in the host cells leading to excessive immune responses. Extracellular vesicles (EVs) from host cells carry components of pathogens to recipient cells and play a role in intercellular communication during infection. However, there is limited knowledge on whether EVs derived from M. pneumoniae-infected macrophages play as intercellular messengers and functional mechanisms. In this study, we establish a cell model of M. pneumoniae-infected macrophages that continuously secrete EVs to further asses their role as intercellular messengers and their functional mechanisms. Based on this model, we determined a method for isolating the pure EVs from M. pneumoniae-infected macrophages, which employs a sequence of operations, including differential centrifugation, filtering, and ultracentrifugation. We identified EVs and their purity using multiple methods, including electron microscopy, nanoparticle tracking analysis, Western blot, bacteria culture, and nucleic acid detection. EVs from M. pneumoniae-infected macrophages are pure, with a 30–200 nm diameter. These EVs can be taken up by uninfected macrophages and induce the production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-8 through the nuclear factor (NF)-κB, and mitogen-activated protein kinases (MAPK) signals pathway. Moreover, the expression of inflammatory cytokines induced by EVs relies on TLR2-NF-κB/JNK signal pathways. These findings will help us better understand a persistent inflammatory response and cell-to-cell immune modulation in the context of M. pneumoniae infection.

Rhodococcus equi (R. equi) is a Gram-positive coccobacillus that causes pneumonia in foals of less than 3 months, which have the ability of replication in macrophages. The ability of R. equi persist in macrophages is dependent on the virulence plasmid pVAPA. Gram-positive extracellular vesicles (EVs) carry a variety of virulence factors and play an important role in pathogenic infection. There are few studies on R. equi-derived EVs (R. equi-EVs), and little knowledge regarding the mechanisms of how R. equi-EVs communicate with the host cell. In this study, we examine the properties of EVs produced by the virulence strain R. equi 103⁺ (103⁺-EVs) and avirulenct strain R. equi 103⁻ (103⁻-EVs). We observed that 103⁺-EVs and 103⁻-EVs are similar to other Gram-positive extracellular vesicles, which range from 40 to 260 nm in diameter. The 103⁺-EVs or 103⁻-EVs could be taken up by mouse macrophage J774A.1 and cause macrophage cytotoxicity. Incubation of 103⁺-EVs or 103⁻-EVs with J774A.1 cells would result in increased expression levels of IL-1β, IL-6, and TNF-α. Moreover, the expression of TLR2, p-NF-κB, p-p38, and p-ERK were significantly increased in J774A.1 cells stimulated with R. equi-EVs. In addition, we presented that the level of inflammatory factors and expression of TLR2, p-NF-κB, p-p38, and p-ERK in J774A.1 cells showed a significant decreased when incubation with proteinase K pretreated-R. equi-EVs. Overall, our data indicate that R. equi-derived EVs are capable of mediating inflammatory responses in macrophages via TLR2-NF-κB/MAPK pathways, and R. equi-EVs proteins were responsible for TLR2-NF-κB/MAPK mediated inflammatory responses in macrophage. Our study is the first to reveal potential roles for R. equi-EVs in immune response in R. equi-host interactions and to compare the differences in macrophage inflammatory responses mediated by EVs derived from virulent strain R. equi and avirulent strain R. equi. The results of this study have improved our knowledge of the pathogenicity of R. equi.

Mycoplasma ovipneumoniae belongs to Mycoplasma, a genus containing the smallest self-replicating microorganisms, and causes infectious pleuropneumonia in goats and sheep. Nucleotide-binding oligomerization domain-containing protein (NOD2), an intracellular pattern recognition receptor, interacts with muramyl dipeptide (MDP) to recognize bacterial peptidoglycans and is involved in autophagy induction. However, there have been no reports about NOD recognition of mycoplasmas or M. ovipneumoniae-induced autophagy. In this study, we sought to determine the role of NOD2 in M. ovipneumoniae-induced autophagy using Western blotting, immunofluorescence, real-time PCR (RT-PCR), and color-changing unit (CCU) analysis. M. ovipneumoniae infection markedly increased NOD2 but did not increase NOD1 expression in RAW 264.7 cells. Treating RAW 264.7 cells with MDP significantly increased colocalization of M. ovipneumoniae and LC3, whereas treatment with NOD inhibitor, NOD-IN-1, decreased colocalization of M. ovipneumoniae and LC3. Furthermore, suppressing NOD2 expression with small interfering RNA (siRNA)-NOD2 failed to trigger M. ovipneumoniae-induced autophagy by detecting autophagy markers Atg5, beclin1, and LC3-II. In addition, M. ovipneumoniae infection significantly increased the phosphorylated c-Jun NH₂-terminal kinase (p-JNK)/JNK, p-Bcl-2/Bcl-2, beclin1, Atg5, and LC3-II ratios in RAW 264.7 cells. Treatment with JNK inhibitor, SP600126, or siRNA-NOD2 did not increase this reaction. These findings suggested that M. ovipneumoniae infection activated NOD2, and both NOD2 and JNK pathway activation promoted M. ovipneumoniae-induced autophagy. This study provides new insight into the NOD2 reorganization mechanism and the pathogenesis of M. ovipneumoniae infection. IMPORTANCEM. ovipneumoniae, which lacks a cell wall, causes infectious pleuropneumonia in goats and sheep. In the present study, we focused on the interaction between NOD and M. ovipneumoniae, as well as its association with autophagy. We showed for the first time that NOD2 was activated by M. ovipneumoniae even when peptidoglycans were not present. We also observed that both NOD2 and JNK pathway activation promoted M. ovipneumoniae-induced autophagy.

Mycoplasma pneumoniae (M. pneumoniae, Mp) is a cell wall-deficient microorganism known to cause chronic respiratory infections in both children and adults. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is an intracellular pattern recognition receptor primarily responsible for identifying muramyl dipeptide (MDP) found in bacterial cell walls. Previous experiments have demonstrated that Mycoplasma ovipneumoniae induces macrophage autophagy through NOD2. In this study, we conducted RNA-seq analysis on macrophages infected with M. pneumoniae and observed an up-regulation in the expression of genes associated with the NOD2 signaling pathway. Mechanistic investigations further revealed the involvement of the NOD2 signaling pathway in the inflammatory response of macrophages activated by M. pneumoniae. We utilized GST pull-down technology in conjunction with liquid chromatography–tandem mass spectrometry (LC–MS/MS) to pinpoint the M. pneumoniae proteins that interact with NOD2. Additionally, co-immunoprecipitation (Co-IP) and immunofluorescence co-localization techniques were used to confirm the interaction between DUF16 protein and NOD2. We found that DUF16 protein can enter macrophages and induce macrophage inflammatory response through the NOD2/RIP2/NF-κB pathway. Notably, the region spanning amino acids 13–90 was identified as a critical region necessary for DUF16-induced inflammation. This research not only broadens our comprehension of the recognition process of the intracellular receptor NOD2, but also deepens our understanding of the development of M. pneumoniae infection.