Injury of Macrophages Induced by Clostridium perfringens Type C Exotoxins.

Clostridium perfringens is a kind of anaerobic Gram-positive bacterium that widely exists in the intestinal tissue of humans and animals. And the main virulence factor in Clostridium perfringens is its exotoxins. Clostridium perfringens type C is the main strain of livestock disease, its exotoxins can induce necrotizing enteritis and enterotoxemia, which lead to the reduction in feed conversion, and a serious impact on breeding production performance. Our study found that treatment with exotoxins reduced cell viability and triggered intracellular reactive oxygen species (ROS) in human mononuclear leukemia cells (THP-1) cells. Through transcriptome sequencing analysis, we found that the levels of related proteins such as heme oxygenase 1 (HO-1) and ferroptosis signaling pathway increased significantly after treatment with exotoxins. To investigate whether ferroptosis occurred after exotoxin treatment in macrophages, we confirmed that the protein expression levels of antioxidant factors glutathione peroxidase 4/ferroptosis-suppressor-protein 1/the cystine/glutamate antiporter solute carrier family 7 member 11 (GPX4/FSP1/xCT), ferroptosis-related protein nuclear receptor coactivator 4/transferrin/transferrin receptor (NCOA4/TF/TFR)/ferritin and the level of lipid peroxidation were significantly changed. Based on the above results, our study suggested that Clostridium perfringens type C exotoxins can induce macrophage injury through oxidative stress and ferroptosis.

Dual-specificity phosphatase 5-mediated fatty acid oxidation promotes Mycobacterium bovis BCG -induced inflammatory responses.

Mycobacterium tuberculosis (Mtb) reprograms FAs metabolism of macrophages during infection and affects inflammatory reaction eventually, however, the mechanism remains poorly understood. Here we show that Mycobacterium bovis (BCG) induces DUSP5 expression through TLR2-MAPKs signaling pathway and promotes fatty acid oxidation (FAO). Silencing DUSP5 by adeno-associated virus vector (AAV) ameliorates lung injury and DUSP5 knockdown reduces the expression of IL-1ß, IL-6 and inactivated NF-κB signaling in BCG-infected macrophages. Of note, DUSP5 specific siRNA increases the content of free fatty acids (FFAs) and triglyceride (TG), but represses the expression of FAO associated enzymes such as CPT1A and PPARα, suggesting DUSP5 mediated FAO during BCG infection. Moreover, Inhibiting FAO by pharmacological manner suppresses IL-1ß, IL-6, TNF-α expression and relieves lung damage. Taken together, our data indicates DUSP5 mediates FAO reprogramming and promotes inflammatory response to BCG infection.

Pyroptosis of Macrophages Induced by Clostridium perfringens Beta-1 Toxin.

Clostridium perfringens beta-1 toxin (CPB1) is responsible for necrotizing enteritis and enterotoxemia. However, whether the release of host inflammatory factors caused by CPB1 is related to pyroptosis, an inflammatory form of programmed cell death, has not been reported. A construct expressing recombinant Clostridium perfringens beta-1 toxin (rCPB1) was created, and the cytotoxic activity of the purified rCPB1 toxin was assessed via CCK-8 assay. The rCPB1-induced macrophage pyroptosis by assessing changes to the expression of pyroptosis-related signal molecules and the pyroptosis pathway of macrophages using quantitative real-time PCR, immunoblotting, ELISA, immunofluorescence, and electron microscopic assays. The results showed that the intact rCPB1 protein was purified from an E. coli expression system, which exhibited moderate cytotoxicity on mouse mononuclear macrophage leukemia cells (RAW264.7), normal colon mucosal epithelial cells (NCM460), and human umbilical vein endothelial cells (HUVEC). rCPB1 could induce pyroptosis in macrophages and HUVEC cells, in part through the Caspase-1-dependent pathway. The rCPB1-induced pyroptosis of RAW264.7 cells could be blocked by inflammasome inhibitor MCC950. These results demonstrated that rCPB1 treatment of macrophages promoted the assembly of NLRP3 inflammasomes and activated Caspase 1; the activated Caspase 1 caused gasdermin D to form plasma membrane pores, leading to the release of inflammatory factors IL-18 and IL-1ß, resulting in macrophage pyroptosis. NLRP3 may be a potential therapeutic target for Clostridium perfringes disease. This study provided a novel insight into the pathogenesis of CPB1.

Vitamin C inhibits apoptosis in THP­1 cells in response to incubation with Mycobacterium tuberculosis.

Tuberculosis (TB) is a chronic and fatal zoonotic infectious disease caused by Mycobacterium tuberculosis (M. tb) infection. The THP-1 cell line is a cell model for studying the function, mechanism and signaling pathways of macrophages; macrophages are the primary host cells of M. tb. Macrophages are important for the progression of tuberculosis, as they affect the release of various inflammatory cytokines, including IL-1ß, IL-6 and TNF-α. Vitamin C is a trace element for the human body. Its biological efficacy depends on its redox abilities and its role as a cofactor in several enzymatic reactions. However, whether vitamin C can protect THP-1 cells from M. tb infection has not yet been reported. The present study aimed to further investigate the effects of vitamin C on M. tb infection-induced THP-1 cell injury and its mechanism. In the present study, MTT assay, reverse transcription-quantitative PCR, EdU cell proliferation assay, western blotting, immunohistochemistry, flow cytometry and TUNEL staining assays were used to assess the cell viability, inflammation and apoptotic levels of THP-1 cells induced by M. tb following vitamin C treatment. The effect of vitamin C on M. tb infection was also assessed using Balb/c mice; pulmonary injury was assessed by H&E staining of the lung tissue. The results demonstrated that vitamin C markedly attenuated cellular damage caused by M. tb infection. The results demonstrated that vitamin C reduced the expression of M. tb-induced apoptosis-related proteins (Cleaved-caspase-9, Cleaved-caspase-3, Bcl-2, Cyt-c) and inflammatory factors (IL-1ß, IL-6, NLRP3, TNF-α, IL-8, NF-κB) in THP-1 cells and reduced apoptosis. Overall, these results suggested that vitamin C may reduce lung damage caused by M. tb infection.

Lycium barbarum polysaccharide attenuates Pseudomonas-aeruginosa pyocyanin-induced cellular injury in mice airway epithelial cells.

Background: Lycium barbarum berries have been utilized in Asia for many years. However, the mechanisms of its lung-defensive properties are indeterminate. Objective: We investigate whether L. barbarum polysaccharide (LBP) could weaken Pseudomonas aeruginosa infection-induced lung injury. Design: Mice primary air-liquid interface epithelial cultures were pretreated with LBP and subsequently treated with pyocyanin (PCN). Lung injury, including apoptosis, inflammation, and oxidative stress, was estimated by western blot, enzyme-linked immunosorbent assay, and real-time quantitative polymerase chain reaction, Real-time qPCR (Q-PCR). Flow cytometry was used to test cell apoptosis. Moreover, Balb/c mice were used to evaluate the tissue injury. We used hematoxylin-eosin staining and immunofluorescence to detect the expression of related proteins and tissue damage in mouse lungs and spleen. Results: The flow cytometric analysis shows the potential of LBP to reduce time-dependent cell death by PCN. Mechanistically, LBP reduces PCN-induced expression of proapoptotic proteins and caspase3 and induces the activation of Bcl-2 in mice bronchial epithelial cells. Similarly, LBP reduces PCN-induced intracellular reactive oxygen species (ROS) production. Moreover, LBP inhibits the production of inflammatory cytokines, Interleukin (IL-1ß), Tumor Necrosis Factor (TNF), IL-6, and IL-8. Our study confirms the ability of LBP to retard PCN-induced injury in mice lung and spleen. Conclusions: The inhibition of PCN-induced lung injury by LBP is capable of protecting mice cells from injury.

Vitamin D3 promotes autophagy in THP-1 cells infected with Mycobacterium tuberculosis.

Tuberculosis (TB) is a major disease that causes mortality worldwide. The lethality of this disease is a result of the contagious bacteria Mycobacterium tuberculosis (M.tb). Infection can inhibit phagosomal maturation, with M.tb mainly attacking macrophages and inhibiting autophagy and apoptosis. Vitamin D has been used to treat tuberculosis, whereby the active metabolite, 1,25-dihydroxyvitamin D, may enhance the immune response to M.tb. Moreover, macrophages infected with M.tb have a high demand for Ca2+. However, the mechanisms by which vitamin D3 protects against and treats TB remain unclear. In the present study, MTT assay showed that vitamin D3 decreased the viability of THP-1 cells in a dose- and time-dependent manner. Autophagy-related factors in THP-1 cells infected with M.tb were analyzed by western blotting and RT-qPCR and the results demonstrated that vitamin D3 significantly increased the expression level of p62, LC3Ⅱ/LC3Ⅰ, Beclin-1, ATG-5 and AMPK in THP-1 cells following M.tb infection. The Ca2+ concentration assay demonstrated that vitamin D3 may promoted cellular autophagy by inhibiting the concentration of Ca2+. Furthermore, the effect of vitamin D3 on M.tb infection was also assessed using Balb/c mice; pulmonary injury was assessed by H&E staining of the lungs tissue. The results demonstrated that vitamin D3 markedly attenuated cellular damage caused by M.tb infection. In conclusion, the present study indicated that vitamin D3 may activate cell autophagy signals by inhibiting the concentration of Ca2+. These data may improve understanding of the effect of vitamin D3 on M.tb infection and help determine the underlying mechanism of vitamin D3 to alleviate and treat the inflammatory response caused by TB.

PD-1-siRNA Delivered by Attenuated Salmonella Enhances the Antitumor Effect of Chloroquine in Colon Cancer.

The widespread appearance of drug tolerance and the low efficiency of single treatment have severely affected the survival time of the patients with colorectal cancer. Exploring new treatment options and combined treatment strategies have become the key to improving the prognosis. The combination of immunotherapy and chemotherapy have shown good clinical expectations. Here, we studied the cooperative effects of chloroquine, an anti-malarial drug that is now widely used in anti-tumor research, and RNA interference (RNAi) targeting the immune checkpoint molecule Programmed Death-1 (PD-1) delivered with attenuated Salmonella. Our results show that chloroquine can not only significantly inhibit the survival of colon cancer cells and induce apoptosis, but also effectively inhibit cell invasion and migration. The results of in vivo experiments show that chloroquine can increase the expression of PD-1 in tumor tissues. Combining chloroquine and PD-1 siRNA can further inhibit the growth and metastases of colon cancer and induce apoptosis. The mechanism underlying this phenomenon is the occurrence of chloroquine-induced apoptosis and the effective immune response caused by the attenuated Salmonella carrying PD-1 siRNA. This study suggests that the combined application of PD-1-based immunotherapy and anti-cancer drugs has become a new expectation for clinical treatment of colorectal cancer.

Transcriptomic analyses reveal dynamic changes of defense response in Glycyrrhiza uralensis leaves under enhanced ultraviolet-B radiation.

The amount of solar ultraviolet-B (UV-B) radiation reaching the Earth's surface is increasing due to stratospheric ozone dynamics and global climate change. Increased UV-B radiation poses a major threat to ecosystems. Although many studies have focused on the potential effects of enhanced UV-B radiation on plants, the dynamic changes of defense response in plants under continuous UV-B radiation remains enigmatic. In this study, we investigated the effect of UV-B radiation at 0.024 W/m2 on the UVR8-and reactive oxygen species (ROS-) signaling pathways, antioxidant system, and wax synthesis of G. uralensis. These parameters were investigated at different UV-B radiation stages (2 h, 6 h, 12 h, 24 h, 48 h, and 96 h). The results revealed that the uvr8 expression level was significantly repressed after 2 h of UV-B radiation, partly because G. uralensis rapidly acclimated to UV-B. Significant H2O2 accumulation occurred after 12 h UV-B radiation, resulting in activation of the ROS signaling pathway and the antioxidant system. After 24 h of UV-B radiation, wax synthesis was enhanced alongside a decrease in the capacity of the main antioxidant system. The dynamic and ordered changes in these pathways reveal how different strategies function in G. uralensis at different times during adaption to enhanced UV-B radiation. This study will help us better understand dynamic changes of defense response in plant under enhanced UV-B radiation, further providing fundamental knowledge to develop plant resistance gene resources.

DUSP5 (dual-specificity protein phosphatase 5) suppresses BCG-induced autophagy via ERK 1/2 signaling pathway.

Autophagy is considered as an effective strategy for host cells to eliminate intracellular Mycobacterium tuberculosis (Mtb). Dual-specificity phosphatase 5 (DUSP5) is an endogenous phosphatase of ERK1/2, and plays an important role in host innate immune responses, its function in autophagy regulation however remains unexplored. In the present study, the function of DUSP5 in autophagy in Mycobacterium bovis Bacillus Calmette-Guerin (BCG)-infected RAW264.7 cells, a murine macrophage-like cell line, was examined by assessing the alteration of the cell morphology, expression of autophagy markers, and ERK1/2 signaling activation. The results demonstrated that the BCG infection could induce DUSP5 expression and activate ERK1/2 signaling in RAW264.7 cells; an activation of ERK1/2 signaling contributed to autophagic process in RAW264.7 cells. Moreover, DUSP5 knockdown increased the expression of autophagy-related proteins (Atgs), including LC3-II, Beclin1, Atg5 and Atg7. However, an overexpression of DUSP5 exhibited an opposite effect. Mechanistically, DUSP5 could inhibit the formation of autophagosome by suppressing the phosphorylation of signaling molecules in ERK1/2 signaling cascade. This study thus demonstrated a novel role of DUSP5 in modulating autophagy inRAW264.7 cells in response to BCG infection in particular, and autophagy macrophage to Mtb in general.

Modulation of Wnt/ß-catenin signaling in IL-17A-mediated macrophage polarization of RAW264.7 cells.

Macrophages play pivotal roles in host defense and immune homeostasis, which have two major functional polarization states, the classically activated M1 and the alternatively activated M2. Interleukin (IL)-17A is an immune modulator able to shape macrophage phenotypes. Wnt/ß-catenin is a developmental signaling pathway that plays crucial roles in morphogenesis and tissue homeostasis, which has also been recently demonstrated playing roles in immune regulation. A growing amount of evidence suggests that both Wnt and IL-17A signaling are involved in macrophage polarization. However, their interaction in macrophage polarization remains elusive. The aim of present study was to explore impacts of Wnt/ß-catenin on IL-17A-mediated macrophage M1/M2 polarization in murine monocyte/macrophage-like cell line RAW264.7. Results revealed that IL-17A activated Wnt/ß-catenin signaling and induced macrophage M1 polarization, but inhibited M2 polarization. In contrast, the activation of Wnt/ß-catenin signaling led to the inhibition of M1 macrophage polarization but the promotion of M2 polarization. Importantly, the activation of Wnt/ß-catenin also showed abilities to inhibit the IL-17A-induced M1 macrophage polarization while diminishing the IL-17A-inhibited M2 polarization. Molecular analysis further uncovered that the JAK/STAT signaling pathway was involved in the interaction of Wnt/ß-catenin and IL-17A in the modulation of macrophage polarization. These results suggested that the Wnt/ß-catenin signaling modulated IL-17A-altered macrophage polarization in part by regulating the JAK/STAT signaling pathway. This study thus revealed a novel function of Wnt/ß-catenin signaling in regulating IL-17A-altered macrophage polarization.

N-acetylcysteine (NAC) Attenuating Apoptosis and Autophagy in RAW264.7 Cells in Response to Incubation with Mycolic Acid from Bovine Mycobacterium tuberculosis Complex.

Bovine tuberculosis is an airborne infectious disease caused by organisms of the Mycobacterium tuberculosis (MTB) complex. Mycolic acid (MA) is the main lipid component of the cell membrane of MTB. It is non-enzymatically reduced by NAD(P)H and further produces reactive oxygen species (ROS), which can cause oxidative stress in human cells. N-acetylcysteine (NAC) is a synthetic precursor of glutathione (GSH) and exhibits anti-ROS activity. However, the underlying mechanisms of its protective properties remain uncertain. Herein, after pre-incubation of RAW264.7 cells with NAC, the factors associated with apoptosis and autophagy were measured. Mechanistically, NAC could reduce MA-induced expression of pro-apoptotic and pro-autophagy proteins. At the mRNA level, NAC can inhibit AMPK and activate mTOR expression. The results indicate that NAC might regulate autophagy in RAW264.7 cells through the AMPK/mTOR pathway. To further prove the effect of NAC on MA, ICR mice were used to evaluate the lung injury. Hematoxylin-eosin (HE) staining was performed on the lung. The results show that NAC could reduce cell injury induced by MA. In conclusion, our research showed that NAC attenuates apoptosis and autophagy in response to incubation with mycolic acid.Bovine tuberculosis is an airborne infectious disease caused by organisms of the Mycobacterium tuberculosis (MTB) complex. Mycolic acid (MA) is the main lipid component of the cell membrane of MTB. It is non-enzymatically reduced by NAD(P)H and further produces reactive oxygen species (ROS), which can cause oxidative stress in human cells. N-acetylcysteine (NAC) is a synthetic precursor of glutathione (GSH) and exhibits anti-ROS activity. However, the underlying mechanisms of its protective properties remain uncertain. Herein, after pre-incubation of RAW264.7 cells with NAC, the factors associated with apoptosis and autophagy were measured. Mechanistically, NAC could reduce MA-induced expression of pro-apoptotic and pro-autophagy proteins. At the mRNA level, NAC can inhibit AMPK and activate mTOR expression. The results indicate that NAC might regulate autophagy in RAW264.7 cells through the AMPK/mTOR pathway. To further prove the effect of NAC on MA, ICR mice were used to evaluate the lung injury. Hematoxylin-eosin (HE) staining was performed on the lung. The results show that NAC could reduce cell injury induced by MA. In conclusion, our research showed that NAC attenuates apoptosis and autophagy in response to incubation with mycolic acid.

Modulation of Wnt/ß-catenin signaling in IL-17A-mediated macrophage polarization of RAW264. 7 cells

Macrophages play pivotal roles in host defense and immune homeostasis, which have two major functional polarization states, the classically activated M1 and the alternatively activated M2. Interleukin (IL)-17A is an immune modulator able to shape macrophage phenotypes. Wnt/β-catenin is a developmental signaling pathway that plays crucial roles in morphogenesis and tissue homeostasis, which has also been recently demonstrated playing roles in immune regulation. A growing amount of evidence suggests that both Wnt and IL-17A signaling are involved in macrophage polarization. However, their interaction in macrophage polarization remains elusive. The aim of present study was to explore impacts of Wnt/β-catenin on IL-17A-mediated macrophage M1/M2 polarization in murine monocyte/macrophage-like cell line RAW264.7. Results revealed that IL-17A activated Wnt/β-catenin signaling and induced macrophage M1 polarization, but inhibited M2 polarization. In contrast, the activation of Wnt/β-catenin signaling led to the inhibition of M1 macrophage polarization but the promotion of M2 polarization. Importantly, the activation of Wnt/β-catenin also showed abilities to inhibit the IL-17A-induced M1 macrophage polarization while diminishing the IL-17A-inhibited M2 polarization. Molecular analysis further uncovered that the JAK/STAT signaling pathway was involved in the interaction of Wnt/β-catenin and IL-17A in the modulation of macrophage polarization. These results suggested that the Wnt/β-catenin signaling modulated IL-17A-altered macrophage polarization in part by regulating the JAK/STAT signaling pathway. This study thus revealed a novel function of Wnt/β-catenin signaling in regulating IL-17A-altered macrophage polarization.

Capsular Polysaccharide is a Main Component of Mycoplasma ovipneumoniae in the Pathogen-Induced Toll-Like Receptor-Mediated Inflammatory Responses in Sheep Airway Epithelial Cells.

Mycoplasma ovipneumoniae (M. ovipneumoniae) is characterized as an etiological agent of primary atypical pneumonia that specifically infects sheep and goat. In an attempt to better understand the pathogen-host interaction between the invading M. ovipneumoniae and airway epithelial cells, we investigated the host inflammatory responses against capsular polysaccharide (designated as CPS) of M. ovipneumoniae using sheep bronchial epithelial cells cultured in an air-liquid interface (ALI) model. Results showed that CPS derived from M. ovipneumoniae could activate toll-like receptor- (TLR-) mediated inflammatory responses, along with an elevated expression of nuclear factor kappa B (NF-κB), activator protein-1 (AP-1), and interferon regulatory factor 3 (IRF3) as well as various inflammatory-associated mediators, representatively including proinflammatory cytokines, such as IL1ß, TNFα, and IL8, and anti-inflammatory cytokines such as IL10 and TGFß of TLR signaling cascade. Mechanistically, the CPS-induced inflammation was TLR initiated and was mediated by activations of both MyD88-dependent and MyD88-independent signaling pathways. Of importance, a blockage of CPS with specific antibody led a significant reduction of M. ovipneumoniae-induced inflammatory responses in sheep bronchial epithelial cells. These results suggested that CPS is a key virulent component of M. ovipneumoniae, which may play a crucial role in the inflammatory response induced by M. ovipneumoniae infections.

Capsular Polysaccharide of Mycoplasma ovipneumoniae Induces Sheep Airway Epithelial Cell Apoptosis via ROS-Dependent JNK/P38 MAPK Pathways.

In an attempt to better understand the pathogen-host interaction between invading Mycoplasma ovipneumoniae (M. ovipneumoniae) and sheep airway epithelial cells, biological effects and possible molecular mechanism of capsular polysaccharide of M. ovipneumoniae (CPS) in the induction of cell apoptosis were explored using sheep bronchial epithelial cells cultured in air-liquid interface (ALI). The CPS of M. ovipneumoniae was first isolated and purified. Results showed that CPS had a cytotoxic effect by disrupting the integrity of mitochondrial membrane, accompanied with an increase of reactive oxygen species and decrease of mitochondrial membrane potential (ΔΨm). Of importance, the CPS exhibited an ability to induce caspase-dependent cell apoptosis via both intrinsic and extrinsic apoptotic pathways. Mechanistically, the CPS induced extrinsic cell apoptosis by upregulating FAS/FASL signaling proteins and cleaved-caspase-8 and promoted a ROS-dependent intrinsic cell apoptosis by activating a JNK and p38 signaling but not ERK1/2 signaling of mitogen-activated protein kinases (MAPK) pathways. These findings provide the first evidence that CPS of M. ovipneumoniae induces a caspase-dependent apoptosis via both intrinsic and extrinsic apoptotic pathways in sheep bronchial epithelial cells, which may be mainly attributed by a ROS-dependent JNK and p38 MAPK signaling pathways.

The Generation and Characterization of Recombinant Protein and Antibodies of Clostridium perfringens Beta2 Toxin.

Introduction. Clostridium perfringens (C. perfringens) beta2 toxin (CPB2) is an important virulent factor of necrotic enteritis in both animals and humans. However, studies of its pathogenic roles and functional mechanisms have been hampered due to the difficulty of purification and lack of specific antibodies against this toxin. Methods. A recombinant His-tagged C. perfringens beta2 (rCPB2) toxin and monoclonal antibodies (McAbs) against CPB2 were generated and characterized by assays of cytotoxicity, immunoblotting, ELISA, neutralization, and immunofluorescence. Results. A His-tagged rCPB2 with integrity and cytotoxicity of native CPB2 was purified from E. coli expressing system, which exhibited a moderate cytotoxicity on NCM460 human intestinal epithelial cells. The rCPB2 could induce apoptotic cell death rather than necrotic death in part through a pathway involved in caspase-3 signaling. Mechanistically, rCPB2 was able to first bind to cell membrane and dynamically translocate into cytoplasm for its cytotoxic activity. Three McAbs 1E23, 2G7 and 2H7 were characterized to be able to immunologically react with CPB2 and neutralize rCPB2 cytotoxicity on NCM460 cells. Conclusion. These results indicated the rCPB2 and antibodies generated in this study are useful tools for studies of biological functions and pathogenic mechanisms of CPB2 in future, which warrants for further investigations.