Nanoparticle Emulsions Enhance the Inhibition of NLRP3

Antibacterial delivery emulsions are potential materials for treating bacterial infections. Few studies have focused on the role and mechanism of emulsions in inflammation relief. Therefore, based on our previous analysis, in which the novel and natural Pickering emulsions stabilized by antimicrobial peptide nanoparticles were prepared, the regulation effect of emulsion on inflammasome was explored in silico, in vitro and in vivo. Firstly, the interactions between inflammasome components and parasin I or Pickering emulsion were predicted by molecular docking. Then, the inflammasome stimulation by different doses of the emulsion was tested in RAW 264.7 and THP-1 cells. Finally, in Kunming mice with peritonitis, NLRP3 and IL-1β expression in the peritoneum were evaluated. The results showed that the Pickering emulsion could combine with ALK, casp-1, NEK7, or NLRP3 to affect the assembly of the NLRP3 and further relieve inflammation. LPNE showed a dose–dependent inhibition effect on the release of IL-1β and casp-1. With the concentration of parasin I increased from 1.5 mg/mL to 3 mg/mL, the LDH activity decreased in the chitosan peptide-embedded nanoparticles emulsion (CPENE) and lipid/peptide nanoparticles emulsion (LPNE) groups. However, from 1.5 to 6 mg/mL, LPNE had a dose–dependent effect on the release of casp-1. The CPENE and parasin I-conjugated chitosan nanoparticles emulsion (PCNE) may decrease the release of potassium and chloride ions. Therefore, it can be concluded that the LPNE may inhibit the activation of the inflammasome by decreasing LDH activity, potassium and chloride ions through binding with compositions of NLRP3.     

Direct Analysis of Mitochondrial Damage Caused by Misfolded/Destabilized Proteins

Protein quality control is essential for cellular homeostasis. In this study, we examined the effect of improperly folded proteins that do not form amyloid fibrils on mitochondria, which play important roles in ATP production and cell death. First, we prepared domain 3 of the dengue envelope protein in wild type and four mutants with widely different biophysical properties in misfolded/aggregated or destabilized states. The effects of the different proteins were detected using fluorescence microscopy and Western blotting, which revealed that three of the five proteins disrupted both inner and outer membrane integrity, while the other two proteins, including the wild type, did not. Next, we examined the common characteristics of the proteins that displayed toxicity against mitochondria by measuring oligomer size, molten globule-like properties, and thermal stability. The common feature of all three toxic proteins was thermal instability. Therefore, our data strongly suggest that thermally unstable proteins generated in the cytosol can cause cellular damage by coming into direct contact with mitochondria. More importantly, we revealed that this damage is not amyloid-specific.     

IL-17A in Human Liver: Significant Source of Inflammation and Trigger of Liver Fibrosis Initiation

IL-17A is considered to guide liver inflammation and fibrosis. From twenty-two human liver samples of different fibrosis stages (F0 to F4), IL-17A, IL-22, and TGFβ1 protein expression in liver tissue lysates were analyzed. Ten paired samples of liver tissue (F0–F1 stage) and blood from the same patient were used to analyze intrahepatic and blood T-lymphoid IL-17A⁺ cells by flow cytometry. The analyses have been performed regardless of pathology, considering the stage of fibrosis. Human liver tissue was used for the primary human liver slice cultures, followed by subsequent cytokine stimulation and fibrotic markers’ analysis by ELISA. IL-17A production in human liver tissue was significantly higher in the early fibrotic stage compared with the advanced stage. Th17 T cells and, to a lesser extent, MAIT cells were the main sources of IL-17A in both compartments, the liver and the blood. Moreover, the presence of liver Th17IL-17A⁺INFγ⁺ cells was detected in the liver. IL-17A stimulation of human liver slice culture increased the expression of profibrotic and pro-inflammatory markers. IL-17A, secreted by Th17 and MAIT cells in the liver, triggered fibrosis by inducing the expression of IL-6 and profibrotic markers and could be a target for antifibrotic treatment. Further amplitude studies are needed to confirm the current results.     

Molecular Biomarkers in Peri-Implant Health and Disease: A Cross-Sectional Pilot Study

Background: The aim of this feasibility study was to investigate the concentration level of CCL-20/MIP-3α, BAFF/BLyS, IL-23, RANKL, and Osteoprotegerin in the Peri-Implant Crevicular Fluid (PICF), from patients diagnosed with peri-implant mucositis and peri-implantitis, and to compare them with PICF from patients with healthy implants. Methods: Participants with at least one dental implant with healthy peri-implant tissues, peri-implant mucositis, or peri-implantitis were included. PICF was collected using paper strips from healthy and diseased peri-implant sites (n = 19). Biomarker levels were analyzed using a custom Multiplex ELISA Assay Kit. Results: In comparison to peri-implant health, the peri-implant mucositis group showed an increased concentration of CCL-20 MIP-3α, BAFF/BLyS, IL-23, RANKL, and Osteoprotegerin. The peri-implantitis group had the lowest median concentration of Osteoprotegerin (1963 ng/mL); this group had a similar concentration of RANKL (640.84 ng/mL) when compared to the peri-implant health group. BAFF/BLyS (17.06 ng/mL) showed the highest concentration in the peri-implantitis group. Conclusions: This feasibility study suggests that IL-23 and RANKL may help to elucidate the pathogenesis during the conversion from peri-implant health to peri-implantitis. Further research is required in BAFF/BLyS for the early diagnosis of peri-implantitis.     

Th17, Th22, and Myeloid-Derived Suppressor Cell Population Dynamics and Response to IL-6 in 4T1 Mammary Carcinoma

Immunotherapies relying on type 1 immunity have shown robust clinical responses in some cancers yet remain relatively ineffective in solid breast tumors. Polarization toward type 2 immunity and expansion of myeloid-derived suppressor cells (MDSC) confer resistance to therapy, though it remains unclear whether polarization toward type 3 immunity occurs or has a similar effect. Therefore, we investigated the involvement of type 3 T_h17 and T_h22 cells and their association with expanding MDSC populations in the 4T1 mouse mammary carcinoma model. T_h17 and T_h22 were detected in the earliest measurable mass at d 14 and remained present until the final sampling on d 28. In peripheral organs, T_h17 populations were significantly higher than the non-tumor bearing control and peaked early at d 7, before a palpable tumor had formed. Peripheral T_h22 proportions were also significantly increased, though at later times when tumors were established. To further address the mechanism underlying type 3 immune cell and MDSC recruitment, we used CRISPR-Cas9 to knock out 4T1 tumor production of interleukin-6 (4T1-IL-6-KO), which functions in myelopoiesis, MDSC recruitment, and T_h maturation. While 4T1-IL-6-KO tumor growth was similar to the control, the reduced IL-6 significantly expanded the total CD4⁺ T_h population and T_h17 in tumors, while T_h22 and MDSC were reduced in all tissues; this suggests that clinical IL-6 depletion combined with immunotherapy could improve outcomes. In sum, 4T1 mammary carcinomas secrete IL-6 and other factors, to polarize and reshape T_h populations and expand distinct T_h17 and T_h22 populations, which may facilitate tumor growth and confer immunotherapy resistance.     

Effects of Sulforaphane-Induced Cell Death upon Repeated Passage of Either P-Glycoprotein-Negative or P-Glycoprotein-Positive L1210 Cell Variants

The expression of the membrane ABCB1 transporter in neoplastic cells is one of the most common causes of reduced sensitivity to chemotherapy. In our previous study, we investigated the effect of a single culture of ABCB1-negative (S) and ABCB1-positive variants of L1210 cells (R and T) in the presence of sulforaphane (SFN). We demonstrated that SFN induces the onset of autophagy more markedly in S cells than in R or T cells. In the current study, we focused on the effect of the repeated culture of S, R and T cells in SFN-containing media. The repeated cultures increased the onset of autophagy compared to the simple culture, mainly in S cells and to a lesser extent in R and T cells, as indicated by changes in the cellular content of 16 and 18 kDa fragments of LC3B protein or changes in the specific staining of cells with monodansylcadaverine. We conclude that SFN affects ABCB1-negative S cells more than ABCB1-positive R and T cells during repeated culturing. Changes in cell sensitivity to SFN appear to be related to the expression of genes for cell-cycle checkpoints, such as cyclins and cyclin-dependent kinases.     

Unhealthy Diets Induce Distinct and Regional Effects on Intestinal Inflammatory Signalling Pathways and Long-Lasting Metabolic Dysfunction in Rats

The intestinal epithelium is a principal site for environmental agents’ detection. Several inflammation- and stress-related signalling pathways have been identified as key players in these processes. However, it is still unclear how the chronic intake of inadequate nutrients triggers inflammatory signalling pathways in different intestinal regions. We aimed to evaluate the impact of unhealthy dietary patterns, starting at a younger age, and the association with metabolic dysfunction, intestinal inflammatory response, and obesity in adulthood. A rat model was used to evaluate the effects of the consumption of sugary beverages (HSD) and a Western diet (WD), composed of ultra-processed foods. Both diets showed a positive correlation with adiposity index, but a positive correlation was found between the HSD diet and the levels of blood glucose and triglycerides, whereas the WD diet correlated positively with triglyceride levels. Moreover, a distinct inflammatory response was associated with either the WD or HSD diets. The WD induced an increase in TLR2, TLR4, and nuclear factor-kappa B (NF-κB) intestinal gene expression, with higher levels in the colon and overexpression of the inducible nitric oxide synthase. In turn, the HSD diet induced activation of the TLR2-mediated NF-κB signalling pathway in the small intestine. Altogether, these findings support the concept that early intake of unhealthy foods and nutrients are a main exogenous signal for disturbances of intestinal immune mechanisms and in a region-specific manner, ultimately leading to obesity-related disorders in later life.     

Selective Fluorescent Probes for High-Throughput Functional Diagnostics of the Human Multidrug Transporter P-Glycoprotein (ABCB1)

The multidrug transporter ABCB1 (MDR1, Pgp) plays an important role in the absorption, distribution, metabolism, and elimination of a wide range of pharmaceutical compounds. Functional investigation of the ABCB1 expression is also essential in many diseases, including drug-resistant cancer, inflammatory conditions, or Alzheimer disease. In this study, we examined the potential interaction of the ABCB1 multidrug transporter with a group of commercially available viability dyes that are generally considered not to penetrate into intact cells. Here, we demonstrate that the slow cellular accumulation of TO-PRO™-1 (TP1) or TO-PRO™-3 (TP3) was strongly inhibited by ABCB1-dependent dye extrusion. TP1/3 dye accumulation was not affected by the presence of ABCC1 or ABCG2, while this uptake was increased to the level in the ABCB1-negative cells by a specific P-glycoprotein inhibitor, Tariquidar. We suggest that TP compounds can be used as highly sensitive, selective, non-toxic, and stable dyes to examine the functional expression and properties of the ABCB1 multidrug transporter, especially in microplate-based high-throughput flow cytometry assays. In addition, we demonstrate the applicability of the TP dyes to efficiently select and separate even a very low number of Pgp-expressing intact cells.