METHODS: Immunohistochemistry was performed on GCA temporal artery biopsy specimens (n = 12) and aortas (n = 10) for detection of YKL-40, its receptor interleukin-13 receptor α2 (IL-13Rα2), macrophage markers PU.1 and CD206, and the tissue-destructive protein matrix metalloproteinase 9 (MMP-9). Ten noninflamed temporal artery biopsy specimens served as controls. In vitro experiments with granulocyte-macrophage colony-stimulating factor (GM-CSF)- or macrophage colony-stimulating factor (M-CSF)-skewed monocyte-derived macrophages were conducted to study the dynamics of YKL-40 production. Next, small interfering RNA-mediated knockdown of YKL-40 in GM-CSF-skewed macrophages was performed to study its effect on MMP-9 production. Finally, the angiogenic potential of YKL-40 was investigated by tube formation experiments using human microvascular endothelial cells (HMVECs).
RESULTS: YKL-40 was abundantly expressed by a CD206+MMP-9+ macrophage subset in inflamed temporal arteries and aortas. GM-CSF-skewed macrophages from GCA patients, but not healthy controls, released significantly higher levels of YKL-40 compared to M-CSF-skewed macrophages (P = 0.039). In inflamed temporal arteries, IL-13Rα2 was expressed by macrophages and endothelial cells. Functionally, knockdown of YKL-40 led to a 10-50% reduction in MMP-9 production by macrophages, whereas exposure of HMVECS to YKL-40 led to significantly increased tube formation.
CONCLUSION: In GCA, a GM-CSF-skewed, CD206+MMP-9+ macrophage subset expresses high levels of YKL-40 which may stimulate tissue destruction and angiogenesis through IL-13Rα2 signaling. Targeting YKL-40 or GM-CSF may inhibit macrophages that are currently insufficiently suppressed by glucocorticoids.
MATERIALS AND METHODS: An in vitro monocyte recruitment model utilizing THP-1 and HUVECs was developed to evaluate TNF-α-induced monocyte adhesion and trans-endothelial migration. To study the role of Nrf2 for MA-mediated anti-inflammatory effects, Nrf2 inhibitor ML385 was used as the pharmacological inhibitor. The expression of Nrf2, monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule 1 (VCAM-1), cluster of differentiation 36 (CD36), and scavenger receptor type A (SR-A) in HUVECs and THP-1 macrophages were investigated using RT-qPCR and Western blotting. The NF-κB activity was determined using NF-κB (p65) Transcription Factor Assay Kit.
KEY FINDINGS: The results showed opposing effects of MA on Nrf2 expression in HUVECs and THP-1 macrophages. MA suppressed TNF-α-induced Nrf2 expression in HUVECs, but enhanced its expression in THP-1 macrophages. Combined effects of MA and ML385 suppressed MCP-1, VCAM-1, and SR-A expressions. Intriguingly, at the protein level, ML385 selectively inhibited SR-A but enhanced CD36 expression. Meanwhile, ML385 further enhanced MA-mediated inhibition of NF-κB activity in HUVECs. This effect, however, was not observed in THP-1 macrophages.
SIGNIFICANCE: MA attenuated foam cell formation by suppressing VCAM-1, MCP-1, and SR-A expression, as well as NF-κB activity, possibly through Nrf2 inhibition. The involvement of Nrf2 for MA-mediated anti-inflammatory effects however differs between HUVECs and macrophages. Future investigations are warranted for a detailed evaluation of the contributing roles of Nrf2 in foam cells formation.
AIM OF THE STUDY: To evaluate the anti-inflammatory activity as well as the preliminary mechanism of S. ferruginea parasitizing on Tecoma stans.
MATERIALS AND METHODS: The anti-inflammatory capability of freeze-dried stem aqueous extract was assessed via inhibition of inflammatory cytokines interleukin- (IL-) 1β, IL-6, IL-10, and tumor necrosis factor-alpha (TNF-α) production in lipopolysaccharide (LPS) and interferon-γ (IFN-γ) stimulated RAW 264.7 macrophages. The underlying anti-inflammatory mechanism was deciphered through reverse transcriptase and real time quantitative polymerase chain reactions (RT-PCR and qPCR) for inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), IL-1β, and TNF-α mRNA expression.
RESULTS: The results exhibited that aqueous extract of freeze-dried S. ferruginea stem sample concentration-dependently inhibited IL-1β protein production along with the down regulation of iNOS and IL-1β mRNA expression. Moreover, it significantly suppressed the protein release of IL-6 and IL-10 in a concentration-dependent manner. However, it slightly reduced TNF-α at higher sample concentration (250 μg/mL) without affecting the mRNA expression levels of COX-2 and TNF-α.
CONCLUSIONS: This study suggests that S. ferruginea parasitizing on Tecoma stans exerted anti-inflammatory capability attributed to inhibition of iNOS and IL-1β mRNA expression, NO creation, IL-1β, IL-6, IL-10, and TNF-α protein production, indicating this plant might be a useful plant-derived candidate against inflammation.
METHODS: Herein, we have engineered antibiotic-loaded (doxycycline or vancomycin) LPHNPs with cationic and zwitterionic lipids and examined the effects on their physicochemical characteristics (size and charge), antibiotic entrapment efficiency, and the in vitro intracellular bacterial killing efficiency against Mycobacterium smegmatis or Staphylococcus aureus infected macrophages.
RESULTS: The incorporation of cationic or zwitterionic lipids in the LPHNP formulation resulted in a size reduction in LPHNPs formulations and shifted the surface charge of bare NPs towards positive or neutral values. Also observed were influences on the drug incorporation efficiency and modulation of the drug release from the biodegradable polymeric core. The therapeutic efficacy of LPHNPs loaded with vancomycin was improved as its minimum inhibitory concentration (MIC) (2 µg/mL) versus free vancomycin (4 µg/mL). Importantly, our results show a direct relationship between the cationic surface nature of LPHNPs and its intracellular bacterial killing efficiency as the cationic doxycycline or vancomycin loaded LPHNPs reduced 4 or 3 log CFU respectively versus the untreated controls.
CONCLUSION: In our study, modulation of surface charge in the nanomaterial formulation increased macrophage uptake and intracellular bacterial killing efficiency of LPHNPs loaded with antibiotics, suggesting alternate way for optimizing their use in biomedical applications.
MAIN BODY: In this review, we summarized the evidence and unique properties of TME in pancreatic cancer that may contribute to its resistance towards immunotherapies as well as strategies to overcome those barriers. We reviewed the current strategies and future perspectives of combination therapies that (1) promote T cell priming through tumor associated antigen presentation; (2) inhibit tumor immunosuppressive environment; and (3) break-down the desmoplastic barrier which improves tumor infiltrating lymphocytes entry into the TME.
CONCLUSIONS: It is imperative for clinicians and scientists to understand tumor immunology, identify novel biomarkers, and optimize the position of immunotherapy in therapeutic sequence, in order to improve pancreatic cancer clinical trial outcomes. Our collaborative efforts in targeting pancreatic TME will be the mainstay of achieving better clinical prognosis among pancreatic cancer patients. Ultimately, pancreatic cancer will be a treatable medical condition instead of a death sentence for a patient.
METHODS: The release of prostaglandin E2 (PGE2) and pro-inflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-1β in a culture supernatant was determined by ELISA. Determination of cyclooxygenase-2 (COX-2) protein and the activation of MAPKs molecules (JNK, ERK and p38 MAPK), NF-κB and Akt in LPS-induced U937 human macrophages were investigated by immunoblot technique. The relative gene expression levels of COX-2 and pro-inflammatory cytokines were measured by using qRT-PCR. The major metabolites of P. amarus were qualitatively and quantitatively analyzed in the extract by using validated reversed-phase high performance liquid chromatography (HPLC) methods.
RESULTS: P. amarus extract significantly inhibited the production of pro-inflammatory mediators (TNF-α, IL-1β, PGE2) and COX-2 protein expression in LPS-induced U937 human macrophages. P. amarus-pretreatment also significantly downregulated the increased mRNA transcription of pro-inflammatory markers (TNF-α, IL-1β, and COX-2) in respective LPS-induced U937 macrophages. It downregulated the phosphorylation of NF-κB (p65), IκBα, and IKKα/β and restored the degradation of IκBα, and attenuated the expression of Akt, JNK, ERK, and p38 MAPKs phosphorylation in a dose-dependent manner. P. amarus extract also downregulated the expression of upstream signaling molecules, TLR4 and MyD88, which play major role in activation of NF-κB, MAPK and PI3K-Akt signaling pathways. The quantitative amounts of lignans, phyllanthin, hypophyllahtin and niranthin, and polyphenols, gallic acid, geraniin, corilagin, and ellagic acid in the extract were determined by HPLC analysis.
CONCLUSION: The study revealed that P. amarus targeted the NF-κB, MAPK and PI3K-Akt signaling pathways to exert its anti- inflammatory effects by downregulating the prospective inflammatory signaling mediators.