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Background: Giardia duodenalis is an extracellular protozoan parasite that causes giardiasis in mammals. The presentation of giardiasis ranges from asymptomatic to severe diarrhea, and the World Health Organization lists it in the Neglected Diseases Initiative. Extracellular vesicles (EVs) are a key mediator of intracellular communication. Although previous studies have shown that G. intestinalis can regulate a host's innate immune response, the role of G. intestinalis EVs (GEVs) in triggering a G. intestinalis-induced innate immune response remains to be further explored. Methods: In this study, GEVs, G. intestinalis and GEVs + G. intestinalis were inoculated into macrophages, respectively. The transcription and secretion levels of proinflammatory cytokines, including interleukin (IL)-1β, IL-6 and tumor necrosis factor alpha (TNF-α), were measured using real-time quantitative PCR (qPCR) and enzyme-linked immunosorbent assays (ELISAs). The phosphorylation levels of the MAPK, AKT and NF-κB signaling pathways in GEV-stimulated mouse macrophages were examined using western blotting and immunofluorescence methods. The roles of activated pathways in the GEV-triggered inflammatory response were determined using inhibition assays, western blotting and ELISAs. Results: The results showed that pretreatment with GEVs enhanced with G. intestinalis (GEVs + G. intestinalis) induced IL-1β, IL-6 and TNF-α transcription and secretion from mouse macrophages compared to stimulation with either GEVs or G. intestinalis alone. Inoculation of mouse macrophages with GEVs upregulated the phosphorylation levels of the p38 MAPK, p44/42 MAPK (Erk1/2), AKT and NF-κB signaling pathways and led to the nuclear translocation of NF-κB p65. Blocking the activated p38, Erk and NF-κB signaling pathways significantly downregulated the secretion of proinflammatory cytokines, and blocking the activated AKT signaling pathway demonstrated reverse effects. Conclusions: The results of this study reveal that GEVs can enhance G. intestinalis-induced inflammatory response levels in mouse macrophages through activation of the p38, ERK and NF-κB signaling pathways. The role of GEVs in regulating host cell immune responses may provide insights into exploring the underlying mechanisms in G. intestinalis–host interactions. [ABSTRACT FROM AUTHOR]

Polyporaceae is an important fungal family that has been a source of natural products with a range of pharmaceutical activities in China. In our previous study, two polysaccharides, PCWPW and PCWPS, with significant antioxidant and antidepressant activity were obtained from Poria cocos. In this study, we evaluated their potential molecular mechanisms in the immunomodulation of macrophages. PCWPW and PCWPS were characterized by GC–MS analysis to contain 1,3-linked Glcp. ELISA assays results demonstrated that the secretion of TNF-α was significantly enhanced by PCWPW/PCWPS. RNA-seq data demonstrated that PCWPS treatment modulated the expression of immune-related genes in macrophages, which was further confirmed by RT-qPCR assays. The activation of TNF-α secretion was found to be mannose receptor (MR) dependent and suppressed by MR inhibitor pretreatment. Moreover, the amount of TNF-α cytokine secretion in PCWPW/PCWPS-induced RAW264.7 cells was decreased when pretreated with NF-κB or MAPK signaling pathway inhibitors. Collectively, our results suggested that PCWPW and PCWPS possessed immunomodulatory activity that regulates TNF-α expression through the NF-κB/MAPK signaling pathway by binding to mannose receptors. Therefore, PCWPW and PCWPS isolated from Poria cocos have potential as drug candidates for immune-related disease treatment. [ABSTRACT FROM AUTHOR]

Vibrio harveyi is a zoonotic pathogen that can infect humans through wounds and cause severe inflammatory responses. Previous studies have reported that the Toll like receptors (TLR) mediated MAPK, AKT and NF-κB signaling pathways are involved in innate immune system resistance to pathogen invasion. However, the molecular mechanism of these pathways, as well as their involvement in V. harveyi infection remains elusive. This study established a V. harveyi infection model using murine peritoneal macrophages (PMs). Various techniques, including western blotting, ELISA, RT-qPCR, immunofluorescence, inhibition assays, were used to explore the roles of TLRs, MAPK, AKT and NF-κB signaling pathways in V. harveyi -induced inflammatory responses. ELISA assays showed that V. harveyi infection triggered proinflammatory cytokines secretion in PMs. RT-qPCR and inhibition assays showed that TLR2 participated in V. harveyi infection and up-regulated the proinflammatory cytokines secretion in murine PMs. Western blotting data showed that the phosphorylation of p38, JNK, AKT, and NF-κB p65 were significantly increased partly mediated by TLR2. In addition, immunofluorescence assays revealed that the NF-κB p65 translocated into nucleus in response to V. harveyi infection. The secretion of IL-1β, IL-6, IL-12, and TNF-α were considerably reduced when the p38 MAPK and NF-κB signaling pathways were blocked, whereas blocking of AKT significantly increased the expression of IL-1β, IL-6, IL-12, and TNF-α. These findings indicate that V. harveyi infection induces inflammatory responses in murine PMs via activation of p38 MAPK and NF-κB pathways, which are partly mediated by TLR2, but are inhibited by PI3K/AKT pathways. • V. harveyi induces inflammatory responses in murine PMs. • The MAPK, AKT and NF-κB pathways were activated against V. harveyi infection in PMs. • V. harveyi induce inflammation in PMs via p38 MAPK and NF-κB pathways. • Activation of AKT pathway relieve V. harveyi induced inflammatory responses in PMs. [ABSTRACT FROM AUTHOR]

Vibrio alginolyticus is an important zoonotic marine pathogenic bacterium. Previous studies on the mechanism of innate immune against V. alginolyticus infection have been limited to aquatic animals, however, how V. alginolyticus activates mammalian immune cells has not been fully clarified. Here, ELISA combined RT-qPCR assays were used to detect the secretion and transcription level of pro-inflammatory cytokines and TLRs during V. alginolyticus infection of mice peritoneal macrophages (PMϕs). Western blotting was used to explore the phosphorylation levels of p38, JNK, ERK, AKT and NF-κB protein. Immunofluorescence assay was used to determine the location of NF-κB protein. Inhibition assay was used to study the role of up-regulated TLR in activated signaling pathways and the role of these pathways in the release of pro-inflammatory cytokines. Our data showed that V. alginolyticus can up-regulate the expression levels of IL-1β, IL-6, IL-12 and TNF-α in PMϕs. In addition, V. alginolyticus stimulation activated the phosphorylation of p38, JNK and ERK were TLR2 heterodimers-dependent, whereas inhibitors of SB203580 (p38), SCH772984 (ERK) and SP600125 (JNK) significantly reduced IL-1β, IL-6, IL-12 and TNF-α production. We further revealed that V. alginolyticus activated the signaling pathways of AKT via TLR2 heterodimers. The inhibitor of MK-2206 2HCl (AKT) negatively regulated the IL-1β, IL-6 and TNF-α release levels. Moreover, V. alginolyticus infection of PMϕs resulted in TLR2 heterodimers-mediated activation of NF-κB and induced translocation of phosphorylated NF-κB protein from the cytoplasm into the nucleus via IκBα degradation. V. alginolyticus induced IL-1β, IL-6, IL-12 and TNF-α release were blocked by the specific NF-κB inhibitor, BAY 11–7082. Taken together, our results suggested that activation of the TLR2 heterodimers-mediated downstream signaling pathways NF-κB, MAPK and AKT is responsible for inflammatory response during Vibrio alginolyticus infection in vitro. • V. alginolyticus activate TLR2-mediated MAPK, AKT and NF-κB pathways in mouse PMϕs. • V. alginolyticus activate MAPK and NF-κB pathways to enhance inflammatory response. • Cytokines levels are negatively regulated by AKT pathway in infected PMϕs. [ABSTRACT FROM AUTHOR]