Objective: To elucidate the correlation of expression of CC chemokine receptor 5 (CCR5) with degrees of inflammatory cells infiltration and expression of β-arrestin2 in biopsic intestinal mucosa of the patients with inflammatory bowel disease (IBD). Methods: Paraffin sections were derived from 53 patients with active IBD, 26 patients with remissive IBD and 30 healthy people. Immunohistochemical envision two-step method was used to test the expression of CCR5 and β-arrestin2 in biopsic intestinal mucosa. HE and toluidine blue staining were used to detect the pathological cytological analysis and classification in lamina propria of colonic mucosa. Results: The positive rate, strong positive rate and immunohistochemical score of CCR5 expression in active IBD were significantly higher than that in normal controls and remissive IBD (p < .05). CCR5 expression had no obvious correlation with clinical severity, lesion distribution and endoscopic classification of active IBD. Neutrophils, eosinophils and lymphocytes in active IBD were significantly higher than that in normal controls and remissive IBD (p < .05), while the lymphocyte grade had a positive correlation with CCR5 expression (p = .042, r = .286). Mastocytes in active IBD, remissive IBD and normal controls had no obvious difference (p > .05). β-arrestin2 expression was significantly lower in active IBD than that in remissive IBD and normal controls, and it had a negative correlation with CCR5 expression (p = .01, r = −.247). Conclusions: CCR5 is highly expressed in active IBD, and it has positive correlation with lymphocyte grade and negative correlation with expression of β-arrestin2.

Transcription of the subgenomic mRNA of alphaviruses initiates at an internal site, called the promoter, which is highly conserved. To determine the functional significance of this conservation, we used an approach that randomizes positions -13 to -9 of the promoter to generate a library containing all possible sequences within this region, including the wild-type sequence. Viruses in the mixed population with more-efficient promoters were selected for during passaging in mammalian (BHK-21) cells. Results from early passage populations indicate that a large number of different promoters are functionally active. Analysis of eight individual viruses found that although each contained a promoter with different degrees of sequence identity to the wild-type sequence, all eight viruses produced progeny. This suggests that the mechanism for transcription allows for a diversity of sequences to serve as promoters. Further passaging of the viral library led to a population consensus sequence that increasingly resembled the wild-type sequence, despite the fact that these promoters are not constrained by the need to encode the carboxyl terminus of the nsP4 protein. Thus, conservation of the region of the promoter from -13 to -9 is in large part due to selection for promoter function, and the wild-type sequence and sequences closely similar to it seem to be optimal for promoter function in BHK-21 cells.

Mesenchymal stem cell (MSC) transplantation has been demonstrated to be promising in the treatment of inflammatory bowel disease (IBD). Azathioprine (AZA) is widely used in IBD patients. Infliximab, as a representative of biological therapy for IBD, is important in the treatment regimen. In the present study we investigated the effects of AZA and infliximab on the cell proliferation, cell cycle and apoptosis of the MSCs derived from the bone marrow of Sprague‑Dawley (SD) rats in vitro in order to provide preliminary data for optimizing the treatment of IBD. MSCs derived from the bone marrow of rats were either cultured in various concentrations of AZA‑ or infliximab‑supplemented medium for 24, 48 and 72 h, respectively. The growth curves of MSCs were obtained. The apoptosis and the cell cycle of the MSCs were analyzed by flow cytometry. AZA decreased the proliferation of MSCs by 66% and increased apoptosis at 0.20 mg/ml for 72 h (P<0.05). The percentage of necrotic cells increased markedly in MSCs treated with 0.30 mg/ml AZA for 72 h (P<0.05). As the exposure time increased, the percentage of MSCs in phase G0‑G1 increased and that in phase S decreased in AZA groups exceeding 0.20 mg/ml (P<0.05). However, infliximab had a minimal impact on the cell proliferation, apoptosis and cell cycle of the MSCs. AZA was able to inhibit cell proliferation and induce apoptosis of the MSCs in vitro. Infliximab did not affect the cell proliferation, apoptosis and cell cycle of the MSCs derived from rats.

Asthma is a heterogeneous chronic inflammatory disorder of the airways with a complex etiology, which involves a variety of cells and cellular components. Therefore, the aim of the study was to investigate the effects and mechanisms of antagonistic peptides that specifically bind to the first and second extracellular loops of CCR5 (GH and HY peptides, respectively) and anti-interleukin-23 subunit p19 (anti-IL-23p19) in the airway and thereby mediate inflammation and the IL-23/T helper 17 (Th17) cell pathway in asthmatic mice. An experimental asthma model using BALB/c mice was induced by ovalbumin (OVA) and treated with peptides that are antagonistic to CCR5 or with anti-IL-23p19. The extents of the asthmatic inflammation and mucus production were assessed. In addition, bronchoalveolar lavage fluid (BALF) was collected, the cells were counted, and the IL-4 level was detected by ELISA. The IL-23/Th17 pathway-related protein and mRNA levels in the lung tissues were measured, and the positive production rates of Th17 cells in the thymus, spleen, and peripheral blood were detected. The groups treated with one of the two peptides and/or anti-IL-23p19 showed significant reductions in allergic inflammation and mucus secretion; decreased expression levels of IL-23p19, IL-23R, IL-17A and lactoferrin (LTF); and reduced proportions of Th17 cells in the thymus, spleen, and peripheral blood. Specifically, among the four treatment groups, the anti-IL-23p19 with HY peptide group exhibited the lowest positive production rate of Th17 cells. Our data also showed a significant and positive correlation between CCR5 and IL-23p19 protein expression. These findings suggest that the administration of peptides antagonistic to CCR5 and/or anti-IL-23p19 can reduce airway inflammation in asthmatic mice, most likely through inhibition of the IL-23/Th17 signaling pathway, and the HY peptide can alleviate inflammation not only through the IL-23/Th17 pathway but also through other mechanisms that result in the regulation of inflammation.

Liu J, et al. Allergy Asthma Immunol Res. 2021 Jan;13(1):106-121. https://doi.org/10.4168/aair.2021.13.1.106

Objective: To investigate the effects of interferon-γ (IFN-γ) on the proliferation and viability of human embryonic lung Mrc-5 fibroblasts in vitro and the expression of the A Disintegrin and Metalloprotease 33 (ADAM33) gene and to explore the mechanism of airway remodeling. Methods: Mrc-5 fibroblasts were sensitized with Dermatophagoides farinae 1 (Derf1) in vitro to mimic in vivo conditions observed in bronchial asthma. An inverted fluorescence microscope was used to observe changes in cell morphology before and after treatment. The viability of Mrc-5 cells was tested using the Cell Counting kit-8 (CCK8). Expression of the ADAM33 gene and protein in Mrc-5 cells was assessed using qPCR and Western blotting, respectively. Results: Different concentrations of Derf1 increased cell growth and the expression of the ADAM33 gene in Mrc-5 cells, and these changes were most obvious in the 10 µg/ml group. In contrast, IFN-γ decreased cell growth and the expression of the ADAM33 gene in both Mrc-5 cells and Derf1-induced Mrc-5 cells, and these changes were most obvious in the 10 ng/ml group. The negative effects of 10 ng/ml IFN-γ were the most significant at 32 hours. Conclusions: Derf1-induced Mrc-5 cells successfully imitated the in vivo conditions observed in patients with asthma. IFN-γ inhibited the proliferation and viability of Mrc-5 cells, and Derf1-induced Mrc-5 cells were more sensitive to IFN-γ treatment. IFN-γ treatment significantly downregulated the expression of the ADAM33 gene in a concentration- and time-dependent manner. IFN-γ may participate in airway remodeling in asthma by regulating the expression of the ADAM33 gene.