IL-36α inhibits melanoma by inducing pro-inflammatory polarization of macrophages.

Interleukin-36α (IL-36α) is essential for various inflammatory conditions, such as psoriasis and rheumatoid arthritis, whereas its role in tumor immunity is unclear. In this study, it was demonstrated that IL-36α could activate the NF-κB and MAPK signaling pathways in macrophages, leading to the expression of IL-1ß, IL-6, TNF-α, CXCL1, CXCL2, CXCL3, CXCL5 and iNOS. Importantly, IL-36α has significant antitumor effects, altering the tumor microenvironment and promoting the infiltration of MHC IIhigh macrophages and CD8+ T cells while decreasing the levels of monocyte myeloid-derived suppressor cells, CD4+ T cells and regulatory T cells. This ultimately results in the inhibition of tumor growth and migration. Furthermore, IL-36α synergized with the PD-L1 antibody increased the immune cells infiltration and enhanced the anti-tumor effect of the PD-L1 antibody on melanoma. Collectively, this study reveals a new role for IL-36α in promoting anti-tumor immune responses in macrophages and suggests its potential for cancer immunotherapy.

Tetraethylthiuram disulphide alleviates pulmonary fibrosis through modulating transforming growth factor-ß signalling.

Idiopathic pulmonary fibrosis (IPF) induces significant morbidity and mortality, for which there are limited therapeutic options available. Here, we found that tetraethylthiuram disulphide (disulfiram, DSF), a derivative of thiuram, used in the treatment of alcohol abuse, has an inhibitory effect on bleomycin (BLM)-induced pulmonary fibrosis via the attenuation of the fibroblast-to-myofibroblast transition, migration, and proliferation of fibroblasts. Furthermore, DSF inhibited the activation of primary pulmonary fibroblasts and fibroblast cell line under transforming growth factor-ß 1 (TGF-ß1) challenge. Mechanistically, the anti-fibrotic effect of DSF on fibroblasts depends on the inhibition of TGF-ß signalling. We further determined that DSF interrupts the interaction between SMAD3 and TGF-ß receptor Ι (TBR Ι), and identified that DSF directly binds with SMAD3, in which Trp326, Thr330, and Cys332 of SMAD3 are critical binding sites for DSF. Collectively, our results reveal a powerful anti-fibrotic function of DSF in pulmonary fibrosis through the inhibition of TGF-ß/SMAD signalling in pulmonary fibroblasts, indicating that DSF is a promising therapeutic candidate for IPF.

Design, synthesis and antitumor activities of thiazole-containing mitochondrial targeting agents.

In this study, a novel batch of thiazole-containing mitochondrial targeting agents were designed and synthesized. Four kinds of mitochondrial targeting moieties and six kinds of linkers were designed. Their structures were confirmed by NMR and HR-MS. The screening of antiproliferative activity revealed that most compounds displayed cytotoxicity on HeLa cancer cell. In particular, D1 has an IC50 value of 35.32 µmol·L-1 against HeLa cell. In addition, cellular respiratory activities were also tested on HeLa cancer cells. D1 had a basal oxygen consumption rate of 8.84 pmol·s-1·mL-1. Also, D1 inhibited the mitochondrial respiration of HeLa cell significantly at 5 µmol·L-1, as well as a complete inhibitory of oxygen consumption for cellular ATP coupling. Furthermore, the pKa, logP, and logD under different pH conditions of all the compounds were calculated by the ACD/Percepta-PhysChem Suite, and the results manifested the correlation between physicochemical properties and chemical activity of compounds. The results identify D1 as a promising mitochondria inhibitor and anticancer agent with appropriate physicochemical properties.

Natural killer group 2D receptor and its ligands in cancer immune escape.

The immune system plays important roles in tumor development. According to the immune-editing theory, immune escape is the key to tumor survival, and exploring the mechanisms of tumor immune escape can provide a new basis for the treatment of tumors. In this review, we describe the mechanisms of natural killer group 2D (NKG2D) receptor and NKG2D ligand (NKG2DL) in tumor immune responses.Natural killer (NK) cells are important cytotoxic cells in the immune system, and the activated NKG2D receptor on the NK cell surface can bind to NKG2DL expressed in tumor cells, enabling NK cells to activate and kill tumor cells. However, tumors can escape the immune clearance mediated by NKG2D receptor/NKG2DL through various mechanisms. The expression of NKG2D receptor on NK cells can be regulated by cells, molecules, and hypoxia in the tumor microenvironment. Tumor cells regulate the expression of NKG2DL at the level of transcription, translation, and post-translation and thereby escape recognition by NK cells. In particular, viruses and hormones have special mechanisms to affect the expression of NKG2D receptor and NKG2DL. Therefore, NKG2D\NKG2DL may have applications as targets for more effective antitumor therapy.

An optimized yeast display strategy for efficient scFv antibody selection using ribosomal skipping system and thermo resistant yeast.

OBJECTIVES: Establish a method to restrict unexpected fragments including stop codons in scFv library and generate a thermo resistant strain for screening of thermal stable scFv sequences. RESULTS: Here, we have constructed a T2A-Leu2 system for selection of yeast surface display libraries that blocks amplification of "stop codon" plasmids within the library, thereby increasing the quality of the library and efficiency of the selection screen. Also, we generated a temperature-resistant yeast strain, TR1, and validated its combined use with T2A-Leu2 for efficient screening. Thus, we developed a general approach for a fast and efficient screening of scFv libraries using a ribosomal skipping system and thermo-resistant yeast. CONCLUSIONS: The method highlights the utility of the T2A-Leu2-based ribosomal skipping strategy for increasing the quality of the input library for selection, along with an optimized selection protocol based on thermo-resistant yeast cells.

The chemical constituents from Urtica fissa leaves.

A new ceramide urticamide (1), two new secolignans urticalactones I (2) and Ⅱ (3), and a new flavonoid glycoside urticaside (4), together with 15 known compounds (4-19), were isolated from the leaves of Urtica fissa, a folk medicine for rheumatism arthritis in China. The active evaluation results showed that 1, 2, 3, 8, and 13 possessed the potent anti-inflammatory. They could inhibit the release of NO and TNF-α in lipopolysaccharide (LPS) stimulated RAW 264.7 cells, with IC50 values less than 4.0 µM.

A novel anti-IL-33 antibody recognizes an epitope FVLHN of IL-33 and has a therapeutic effect on inflammatory diseases.

As a crucial member of the Interleukin-1 (IL-1) family, IL-33 plays an indispensable role in modulating inflammatory responses. Here, we developed an effective anti-human IL-33 monoclonal antibody (mAb) named 5H8. Importantly, we have identified an epitope (FVLHN) of IL-33 protein as a recognition sequence for 5H8, which plays an important role in mediating the biological activity of IL-33. We observed that 5H8 significantly suppressed IL-33-induced IL-6 expression in bone marrow cells and mast cells in a dose-dependent manner in vitro. Furthermore, 5H8 effectively relievedHDM-induced asthma and PR8-induced acute lung injury in vivo. These findings indicate that targeting the FVLHN epitope is critical for inhibiting IL-33 function. In addition, wedetected that the Tm value of 5H8 was 66.47℃ and the KD value was 173.0 pM, which reflected that 5H8 had good thermal stability and high affinity. Taken together, our data suggest that our newly developed 5H8 antibody has potential as a therapeutic antibody for treating inflammatory diseases.

Macrophage LMO7 deficiency facilitates inflammatory injury via metabolic-epigenetic reprogramming.

Inflammatory bowel disease (IBD) is a formidable disease due to its complex pathogenesis. Macrophages, as a major immune cell population in IBD, are crucial for gut homeostasis. However, it is still unveiled how macrophages modulate IBD. Here, we found that LIM domain only 7 (LMO7) was downregulated in pro-inflammatory macrophages, and that LMO7 directly degraded 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) through K48-mediated ubiquitination in macrophages. As an enzyme that regulates glycolysis, PFKFB3 degradation led to the glycolytic process inhibition in macrophages, which in turn inhibited macrophage activation and ultimately attenuated murine colitis. Moreover, we demonstrated that PFKFB3 was required for histone demethylase Jumonji domain-containing protein 3 (JMJD3) expression, thereby inhibiting the protein level of trimethylation of histone H3 on lysine 27 (H3K27me3). Overall, our results indicated the LMO7/PFKFB3/JMJD3 axis is essential for modulating macrophage function and IBD pathogenesis. Targeting LMO7 or macrophage metabolism could potentially be an effective strategy for treating inflammatory diseases.

Neuroimmune Interaction: A Widespread Mutual Regulation and the Weapons for Barrier Organs.

Since the embryo, the nervous system and immune system have been interacting to regulate each other's development and working together to resist harmful stimuli. However, oversensitive neural response and uncontrolled immune attack are major causes of various diseases, especially in barrier organs, while neural-immune interaction makes it worse. As the first defense line, the barrier organs give a guarantee to maintain homeostasis in external environment. And the dense nerve innervation and abundant immune cell population in barrier organs facilitate the neuroimmune interaction, which is the physiological basis of multiple neuroimmune-related diseases. Neuroimmune-related diseases often have complex mechanisms and require a combination of drugs, posing challenges in finding etiology and treatment. Therefore, it is of great significance to illustrate the specific mechanism and exact way of neuro-immune interaction. In this review, we first described the mutual regulation of the two principal systems and then focused on neuro-immune interaction in the barrier organs, including intestinal tract, lungs and skin, to clarify the mechanisms and provide ideas for clinical etiology exploration and treatment.

SARS-CoV-2 vaccination in androgen sensitive phenotypes - A study on associated factors for SARS-CoV-2 vaccination and its adverse effects among androgenetic alopecia and benign prostate hyperplasia patients.

Background: Androgen sensitivity, which was established as the leading etiology of androgenetic alopecia (AGA) and benign prostatic hyperplasia (BPH), plays an important role in SARS-CoV-2 infection. Vaccination is essential for AGA and BPH patients in view of the high risk from SARS-CoV-2 infection. Purpose: We aimed to investigate the associated factors for SARS-CoV-2 vaccination and its side effects in populations with AGA and BPH. Method: We collected the data on SARS-CoV-2 vaccination and adverse reactions of male AGA and BPH patients visited the outpatient of Xiangya hospital by telephone and web-based questionnaires. Vaccination rate and adverse reactions were compared by different vaccine types and use of anti-androgen therapy. Result: A total of 457 AGA patients and 397 BPH patients were recruited in this study. Among which, 92.8% AGA patients and 61.0% BPH patients had at least the first dose of SARS-CoV-2 vaccination (p < 0.001). Having comorbidities and use of anti-androgen therapy increased the risk of un-vaccination among AGA by 2.875 and 3.729 times, respectively (p < 0.001). Around 31.1% AGA patients and 9.5% BPH patients presented adverse reactions, which were mostly mild. Anti-androgen therapy increased the inclination of injection site pain after vaccination (18.7% vs 11.9%; OR: 1.708, 95% CI: 1.088-2.683, p = 0.019). Conclusion: Co-existence of other systemic diseases and anti-androgen therapy were the limiting factors for SARS-CoV-2 unvaccination, especially in AGA patients. The importance of SARS-CoV-2 vaccines should be strengthened and popularized in androgen sensitive phenotypes.

Protein Kinase C δ (PKCδ) Attenuates Bleomycin Induced Pulmonary Fibrosis via Inhibiting NF-κB Signaling Pathway.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and lethal interstitial lung disease characterized by consistent pulmonary inflammation. Although protein kinase C delta (PKCδ) is involved in broad scope cellular response, the role of PKCδ in IPF is complicated and has not been fully defined yet. Here, we reported that PKCδ deficiency (PKCδ-/-) aggravated bleomycin (BLM)-induced pulmonary fibrosis and inflammation. Upon challenge with BLM, the pulmonary capillary permeability, immune cell infiltration, inflammatory cytokine production, and collagen deposition were enhanced in PKCδ-/- mice compared to that in PKCδ+/+ mice. In response to poly(I:C) stimulation, PKCδ deficient macrophages displayed an increased production of IL-1ß, IL-6, TNF-α, and IL-33, which were associated with an enhanced NF-κB activation. Furthermore, we found that PKCδ could directly bind to and phosphorylate A20, an inhibitory protein of NF-κB signal. These results suggested that PKCδ may inhibit the NF-κB signaling pathway via enhancing the stability and activity of A20, which in turn attenuates pulmonary fibrosis, suggesting that PKCδ is a promising target for treating pulmonary fibrosis.

MK2 Is Required for Neutrophil-Derived ROS Production and Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a chronic disease that is commonly accompanied by increased inflammatory responses and elevated reactive oxygen species (ROS) of the gastrointestinal tract. Here, we found that MAPK-activated protein kinase 2 (MK2) modulates ROS production and is required for dextran sulfate sodium (DSS)-induced IBD in the mouse model. Genetic ablation of MK2 in the myeloid lineage cells (MK2Lyz2-KO) protected against DSS-induced colitis injury. In response to DSS challenge, compared to MK2lyz2-WT mice, MK2Lyz2-KO mice exhibited less damage of epithelial and goblet cells, decreased generation of interleukin (IL)-6, tumor necrosis factor (TNF)-α, and ROS, as well as reduced Ki67-positive cells and concentrations of myeloperoxidase (MPO) in the intestinal epithelium. Furthermore, upon treatment with formylated peptide N-formyl-methionyl-leucyl-phenylalanine (fMLF), the generation of ROS was attenuated in MK2-deficient neutrophils, in which the phosphorylation of Akt and p38 MAPK was also reduced. Collectively, these findings indicated that MK2 is required for neutrophil-derived ROS production and IBD, and MK2 and ROS are promising therapeutic targets for IBD.

Efficient strategy for introducing large and multiple changes in plasmid DNA.

While the QuikChange site-directed mutagenesis method and its later modifications are extremely useful and simple, they suffer from several drawbacks. Here, we propose a new method, named LFEAP mutagenesis (Ligation of Fragment Ends After PCR) for creating various mutations in plasmid by leveraging three existing concepts: inverse PCR, single primer PCR, and sticky-end assembly. The first inverse PCR on the target plasmid yielded linearized DNA fragments with mutagenic ends, and a second single primer PCR resulted in complementary single-stranded DNA fragments with the addition of overhangs at the 5' end of each strand. The resulting single strands were then annealed to produce double-stranded DNA with free 5' single-stranded DNA tails. These products with compatible sticky ends were efficiently assembled into a circular, mutagenized plasmid. With this strategy, multiple simultaneous changes (up to 15) and mutations in large plasmids (up to 50 kb) were achieved with high efficiency and fidelity. LFEAP mutagenesis is a versatile method that offers significant advantages for introducing large and multiple changes in plasmid DNA.