Effects of Cytokines IFN-γ and TGF-β on the Functional Activity of Blood Mononuclear Cells against Giardia lamblia

Background: This study aimed to analyze cultures of mononuclear (MN) cells with Giardia lamblia to determine the levels of the cytokines IFN-γ and TGF-β and the functional activity of MN cells after incubation with cytokines. Methods: This study was conducted in 2018 in Barra do Garças, Mato Grosso State, Brazil. Blood samples were collected from 60 healthy volunteer donors to obtain leukocytes. The levels of IFN-γ and TGF-β were quantified in trophozoite cell culture supernatants. Superoxide release, phagocytosis, microbicidal activity, apoptosis and intracellular calcium release were analyzed. Results: The cytokines evaluated were detected in the culture supernatant of MN cells and G. lamblia. Regardless of the type of cytokine, MN cells increased superoxide release in the presence of G. lamblia. Phagocytosis, microbicidal activity and apoptosis were higher when MN phagocytes were treated with cytokines. The highest microbicidal activity and apoptosis rates were observed in MN cells cultured with TGF-β. IFN-γ increased the release of intracellular calcium by MN phagocytes. Conclusion: Cytokines play a beneficial role in the host by activating MN cells against G. lamblia. In addition, phagocytosis causes G. lamblia death and that the modulation of the functional activity of blood MN phagocytes by cytokines is an alternative mechanism for eliminating G. lamblia.

Manuka Honey Modulates the Inflammatory Behavior of a dHL-60 Neutrophil Model under the Cytotoxic Limit

Recent work has shown that Manuka honey, an increasingly popular wound additive with potent antibacterial properties, also has anti-inflammatory properties. However, little research has been done examining its effect on neutrophils. This study investigates the hypothesis that Manuka honey reduces neutrophil superoxide release and chemotaxis and reduces the activation of the inflammatory nuclear factor-κB (NF-κB) signaling pathway under honey’s cytotoxic limit. A differentiated HL-60 cell line was used as a neutrophil model and cultured in various concentrations of Manuka honey for 3 and 24 hours to measure cytotoxicity via mitochondrial activity and visual trypan-exclusion count. Cytochrome C and Boyden chamber assays were used to measure the effect of Manuka honey on superoxide release and chemotaxis toward fMLP, respectively. Additionally, a Western blot for NF-κB inhibitorα(IκBα) was performed to measure Manuka honey’s effect on the NF-κB pathway via IκBαphosphorylation. The results indicate a cytotoxic limit of 3-5% v/v. The presence of 1% honey decreased superoxide release at 24 hours. The 0.5, 1, and 3% honey concentrations reduced chemotaxis and IκBαphosphorylation in a dose-dependent fashion. These results suggest that Manuka honey significantly reduces neutrophil recruitment and inflammatory behavior in the wound site in a dose-dependent fashion under the cytotoxic limit.

Peptoanaerobacter stomatis Primes Human Neutrophils and Induces Granule Exocytosis

ABSTRACT Peptoanaerobacter stomatis is a newly appreciated taxon associated with periodontal diseases; however, little is known about the organism's pathogenic potential or its interaction with the host immune response. Neutrophils are the most abundant innate immune cell present in the gingival tissue and function to constrain the oral microbial challenge. However, some periodontal pathogens have developed strategies to evade phagocytosis and killing by neutrophils. Therefore, to begin to understand the role of P. stomatis in periodontitis, we studied its interactions with human neutrophils. Our data showed that after 30 min of incubation, neutrophils failed to engulf P. stomatis efficiently; however, when P. stomatis was internalized, it was promptly eradicated. P. stomatis challenge induced a robust intracellular respiratory burst; however, this response did not contribute to bacterial killing. Minimal superoxide release was observed by direct bacterial challenge; however, P. stomatis significantly increased N-formyl-methionyl-leucyl phenylalanine (fMLF)-stimulated superoxide release to an extent similar to that of cells primed with tumor necrosis factor alpha (TNF-α). When neutrophils were challenged with P. stomatis, 52% of the bacterium-containing phagosomes were enriched for the specific granule marker lactoferrin and 82% with the azurophil granule marker elastase. P. stomatis challenge stimulated exocytosis of the four neutrophil granule subtypes. Moreover, P. stomatis susceptibility to extracellular killing could be attributed to the exocytosis of antimicrobial components present in neutrophil granules. Priming neutrophils for an enhanced respiratory burst together with promoting granule content release could contribute to the chronic inflammation and tissue destruction that characterize periodontal diseases.

Berbamine Exerts Anti-Inflammatory Effects via Inhibition of NF-κB and MAPK Signaling Pathways

Background/Aims: This study aimed to investigate the anti-inflammatory activity of Berbamine (BER), a bisbenzylisoquinoline alkaloid extracted from Berberis amurensis (Xiao Bo An), and the underlying mechanisms. Methods: Macrophages and neutrophils were treated with BER in vitro and stimulated with LPS and fMLP. The effects of BER on the expression of pro-inflammatory mediators in macrophages were evaluated with quantitative RT-PCR and ELISA. The effects of BER on the activation and superoxide release of neutrophils were determined with flow cytometry and WST-1 reduction test. The inhibitory effects of BER on the activation of signaling pathways related to inflammatory response in macrophages were evaluated by western blot analysis. In addition, a mouse peritonitis model was made by peritoneal injection of thioglycollate medium and anti-inflammatory effects of BER were investigated in vivo by quantitative analysis of pro-inflammatory factor production and leukocyte exudation. Results: BER significantly inhibited inflammatory factor expression by LPS-stimulated macrophages and suppressed activation and superoxide release of fMLP-stimulated neutrophils. In the mouse peritonitis model, BER significantly inhibited the activation of macrophages and exudation of neutrophils. According to analysis, BER significantly suppressed phosphorylation of NF-κB and MAPK (JNK and ERK1/2) signaling pathways in LPS-stimulated macrophages. Conclusions: Collectively, data from this study suggest that BER has anti-inflammatory potential, which is effected via inhibition of NF-κB and MAPK signaling pathways, and thus holds promise for treatment of inflammatory disease.