Ficolin-A induces macrophage polarization to a novel pro-inflammatory phenotype distinct from classical M1.

BACKGROUND: Macrophages are key inflammatory immune cells that orchestrate the initiation and progression of autoimmune diseases. The characters of macrophage in diseases are determined by its phenotype in response to the local microenvironment. Ficolins have been confirmed as crucial contributors to autoimmune diseases, with Ficolin-2 being particularly elevated in patients with autoimmune diseases. However, whether Ficolin-A stimulates macrophage polarization is still poorly understood. METHODS: We investigated the transcriptomic expression profile of murine bone marrow-derived macrophages (BMDMs) stimulated with Ficolin-A using RNA-sequencing. To further confirm a distinct phenotype activated by Ficolin-A, quantitative RT-PCR and Luminex assay were performed in this study. Additionally, we assessed the activation of underlying cell signaling pathways triggered by Ficolin-A. Finally, the impact of Ficolin-A on macrophages were investigated in vivo through building Collagen-induced arthritis (CIA) and Dextran Sulfate Sodium Salt (DSS)-induced colitis mouse models with Fcna-/- mice. RESULTS: Ficolin-A activated macrophages into a pro-inflammatory phenotype distinct to LPS-, IFN-γ- and IFN-γ + LPS-induced phenotypes. The transcriptomic profile induced by Ficolin-A was primarily characterized by upregulation of interleukins, chemokines, iNOS, and Arginase 1, along with downregulation of CD86 and CD206, setting it apart from the M1 and M2 phenotypes. The activation effect of Ficolin-A on macrophages deteriorated the symptoms of CIA and DSS mouse models, and the deletion of Fcna significantly alleviated the severity of diseases in mice. CONCLUSION: Our work used transcriptomic analysis by RNA-Seq to investigate the impact of Ficolin-A on macrophage polarization. Our findings demonstrate that Ficolin-A induces a novel pro-inflammatory phenotype distinct to the phenotypes activated by LPS, IFN-γ and IFN-γ + LPS on macrophages.

LLDT-8 ameliorates experimental autoimmune encephalomyelitis by mediating macrophage functions in the priming stage.

Multiple sclerosis (MS) is an inflammatory demyelinating disease in the central nervous system caused by T cell activation mediated by peripheral macrophages, resulting in severe neurological deficits and disability. Due to the currently limited and expensive treatments for MS, we here introduce an economic Chinese medicine extract, (5R)-5-Hydroxytriptolide (LLDT-8), which shows low toxicity and high immunosuppressive activity. We used the widely accepted mouse model of MS, experimental autoimmune encephalomyelitis (EAE), to examine the immunosuppressive effect of LLDT-8 in vivo. Through the RNA-sequence analysis of peripheral macrophages in EAE mice, we discovered that LLDT-8 alleviates the symptoms of EAE by inhibiting the proinflammatory effect of macrophages, thereby blocking the activation and proliferation of T cells. In all, we found that LLDT-8 could be a potential treatment for MS.

Umbilical cord blood: A promising source for allogeneic CAR-T cells.

Chimeric antigen receptor T (CAR-T) cell therapy is an effective treatment for relapsed and refractory acute lymphoblastic leukemia (R/R ALL). However, autologous CAR-T cells derived from patients with B-ALL often show poor amplification ability, exhaustion, and anergy. To overcome these limitations, allogeneic CAR-T cells may be used as effective substitutes; however, which source would be the best substitute is unclear. In this study, we compared the immunophenotype and antitumor efficacy of anti-CD19 CAR-T cells derived from healthy donor cord blood (CB), healthy donor peripheral blood (PB), and PB of B-ALL patients [PB (patient)] in vitro and NOD-Prkdcem26cd52Il2rgem26Cd22/Nju (NCG)-immunodeficient mice, respectively. The results revealed that CAR-T cells derived from healthy donor CB and PB showed a higher proportion of naive T cells and longer tumor suppression in tumor-bearing mice than those of PB (patient). PB (patient) CAR-T cells had a higher proportion of regulatory T cells (Treg cells) and released high levels of interluekin-10 (IL-10), which also suggest a poor prognosis. Thus, CAR-T cells derived from healthy donors have better antitumor efficacy than CAR-T cells derived from PB (patient), and CB may be a good source of allogeneic CAR-T cells.

[Cadmium Accumulation Characteristics of Different Heat Varieties Under Cadmium Stress].

In order to provide technical support for the safe utilization of heavy metal-polluted farmland, we screened wheat varieties with a low accumulation of Cd in grain via a pot experiment. For this purpose, we respectively investigated the enrichment and transport characteristics of Cd in various plant parts of 119 wheat varieties under the conditions of 1.5 mg·kg-1 (low content) and 4.0 mg·kg-1 (high content) Cd-contaminated soil and explored the correlation between the Cd content of different organs of wheat and the relationship between Cd content in soil and the uptake of Cd by wheat. The results showed that:① there were significant differences in the ability to accumulate and transport Cd in roots, stems, leaves, and grains of tested wheat varieties (P<0.05). Under the condition of low Cd content, the Cd enrichment ability of each part of the wheat plant was as follows:leaf>stem ≈root>grain; under high-content stress conditions, the Cd enrichment ability of each part of the wheat plant was:leaf>root>stem>grain; under different content conditions, the Cd transport ability of each part of the wheat plant was:leaf>stem>grain. Cd content in the wheat shoot was positively correlated with total Cd content and ion-exchangeable Cd content in soil (P<0.01) and was closely related to strong transpiration under the pot experiment. ② The correlation coefficient r of BCFCd of wheat roots, stems, leaves, and grain was -0.267 to -0.645, showing a very significant negative correlation (P<0.01), indicating that with the increase in soil Cd content, the migration and accumulation degree of Cd in wheat plant organs showed a downward trend. Moreover, the negative correlation between BCFCd and soil Cd content in stems was significantly higher than that in roots, leaves, and grains, indicating that the enrichment of Cd in wheat plants largely depended on the accumulation and transportation of stems. ③ The correlation coefficient r of Cd content between the root and stem, root and leaf, root and grain, stem and leaf, stem and grain, and leaf and grain in low-and high-content groups was 0.450-0.763, showing a very significant positive correlation (P<0.01), indicating that there was a close transport relationship among the wheat organs, and the degree of translocation from the root to stem and stem to leaf was higher than that from the stem to grain and leaf to grain. ④ Using cluster analysis and enrichment and translocation coefficient sequencing, this study screened 16 and 11 wheat varieties with low Cd accumulation under the soil cadmium content of 1.5 mg·kg-1and 4.0 mg·kg-1, respectively. Among them, Luohan 7 could be used as the optimal wheat variety with low Cd accumulation under the conditions of low-, medium-, and high-Cd content.

Enzymatic O-Glycosylation of Etoposide Aglycone by Exploration of the Substrate Promiscuity for Glycosyltransferases.

The 4-O-ß-d-glucopyranoside of DMEP ((-)-4'-desmethylepipodophyllotoxin) (GDMEP), a natural product from Podophyllum hexandrum, is the direct precursor to the topoisomerase inhibitor etoposide, used in dozens of chemotherapy regimens for various malignancies. The biosynthesis pathway for DMEP has been completed, while the enzyme for biosynthesizing GDMEP is still unclear. Here, we report the enzymatic O-glycosylation of DMEP with 53% conversion by exploring the substrate promiscuity and entrances of glycosyltransferases. Notably, we found 6 essential amino acid residues surrounding the putative substrate entrances exposed to the protein surface in UGT78D2, CsUGT78D2, and CsUGT78D2-like, and these residues may determine substrate specificity and high O-glycosylation activity toward DMEP. Our results provide an effective route for one-step synthesis of GDMEP. Identification of the key residues and entrances of glycosyltransferases will promote precise identification of glycosyltransferase biocatalysts for novel substrates and provide a rational basis for glycosyltransferase engineering.

Increasing the distance between two monomers of topoisomerase IIß under the action of antitumor agent 4ß-sulfur-(benzimidazole) 4'-demethylepipodophyllotoxin.

Topoisomerases II (Top2s) are a group of essential enzymes involved in replication, transcription, chromosome condensation, and segregation via altering DNA topology. The mechanism of the Top2s poisons such as etoposide (VP-16) was reported as stabilizing the Top2-DNA complex and engendering permanent DNA breakage. As the structurally similar compound of VP-16, a novel 4ß-sulfur-substituted 4'-demethylepipodophyllotoxin (DMEP) derivative (compound C-Bi) with superior antitumor activity was developed in our previous study. To understand the structural basis of the compound action, the crystal structure (2.54 Å) of human Top2 ß-isoform (hTop2ß) cleavage complexes stabilized by compound C-Bi was determined. However, compound C-Bi was not visible in the crystal structure. Through the comparison of the structures of hTop2ß-DNA-etoposide ternary complex and hTop2ß-DNA binary complex, it could be observed that the distance between drug-binding sites Arg503 of the two monomers was 26.62 Å in hTop2ß-DNA-etoposide ternary complex and 34.54 Å in hTop2ß-DNA binary complex, respectively. Significant twist were observed in the DNA chains of binary complex. It suggested that compound C-Bi played antitumor roles through increasing spacing of hTop2ß monomers. The changes in hTop2ß structure further caused double changes in the torsional direction and migration distance of the DNA chains, resulting in impeding religation of DNA.