Spinal cord injury (SCI) is a severe neurological complication following spinal fracture, which has long posed a challenge for clinicians. Microglia play a dual role in the pathophysiological process after SCI, both beneficial and detrimental. The underlying mechanisms of microglial actions following SCI require further exploration. The present study combined three different machine learning algorithms, namely weighted gene co-expression network analysis, random forest analysis and least absolute shrinkage and selection operator analysis, to screen for differentially expressed genes in the GSE96055 microglia dataset after SCI. It then used protein-protein interaction networks and gene set enrichment analysis with single genes to investigate the key genes and signaling pathways involved in microglial function following SCI. The results indicated that microglia not only participate in neuroinflammation but also serve a significant role in the clearance mechanism of apoptotic cells following SCI. Notably, bioinformatics analysis and lipopolysaccharide + UNC569 (a MerTK-specific inhibitor) stimulation of BV2 cell experiments showed that the expression levels of Anxa2, Myo1e and Spp1 in microglia were significantly upregulated following SCI, thus potentially involved in regulating the clearance mechanism of apoptotic cells. The present study suggested that Anxa2, Myo1e and Spp1 may serve as potential targets for the future treatment of SCI and provided a theoretical basis for the development of new methods and drugs for treating SCI.

Atherosclerosis (AS) is a chronic inflammatory vascular disease that occurs in the intima of large and medium-sized arteries with the immune system\'s involvement. It is a common pathological basis for high morbidity and mortality of cardiovascular diseases. Abnormal proliferation of apoptotic cells and necrotic cells leads to AS plaque expansion, necrotic core formation, and rupture. In the early stage of AS, macrophages exert an efferocytosis effect to engulf and degrade apoptotic, dead, damaged, or senescent cells by efferocytosis, thus enabling the regulation of the organism. In the early stage of AS, macrophages rely on this effect to slow down the process of AS. However, in the advanced stage of AS, the efferocytosis of macrophages within the plaque is impaired, which leads to the inability of macrophages to promptly remove the apoptotic cells (ACs) from the organism promptly, causing exacerbation of AS. Moreover, upregulation of CD47 expression in AS plaques also protects ACs from phagocytosis by macrophages, resulting in a large amount of residual ACs in the plaque, further expanding the necrotic core. In this review, we discussed the molecular mechanisms involved in the process of efferocytosis and how efferocytosis is impaired and regulated during AS, hoping to provide new insights for treating AS.

Synchrotron-based soft X-ray tomography (SXT), providing three-dimensional morphology and quantitative distribution of linear absorption coefficient (LAC) of the imaged objects, is widely used in many fields to obtain ultra-structure images, especially in cellular imaging. Off-line fluorescence microscopies (FMs) are combined to identify the type of organelles and status of cells. However, deformation and displacement usually occur during the transfer and loading process, which decreases the precision of two-modal images\' registration. In this paper, we report on an on-line FM, at the SXT station (BL07W) of the National Synchrotron Radiation Laboratory (NSRL), which avoids deformation and displacement. Therefore, researchers can easily find the sample and take the useful data without tedious post-processing. Combining SXT with on-line FM, we achieved the identification and high-resolution imaging of an apoptotic cell. The experiments revealed that the LAC of the nucleus of the apoptotic cell was larger than that of a normal cell, which could be explained by nucleus pyknosis of the apoptotic cell.

Allergy can cause intestinal damage, including through cell apoptosis. In this study, intestinal cell apoptosis was first observed in the β-conglycinin (β-CG) allergy model, and the effect of Lactobacillus rhamnosus GG (LGG) on reducing apoptosis of cells in the intestine and its underlying mechanisms were further investigated. Allergic mice received oral LGG daily, and intestinal tissue apoptotic cells, gut microbiota, and metabolites were evaluated six and nine days after intervention. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis revealed that LGG intervention could reduce the incidence of cell apoptosis more effectively than natural recovery (NR). The results of 16S rRNA analysis indicated that LGG intervention led to an increase in the relative abundance of Bacteroides. Metabolite analysis of intestinal contents indicated that histamine, N-acetylhistamine, N(α)-γ-glutamylhistamine, phenylalanine, tryptophan, arachidonic acid malate, and xanthine were significantly decreased, and deoxycholic acid, lithocholic acid were significantly increased after the LGG intervention on β-CG allergy; the decreases in histamine and N(α)-γ-glutamylhistamine were significant compared with those of NR. In conclusion, LGG reduces apoptosis of cells induced by β-CG allergy, which may be related to regulation of Bacteroides and the bile secretion pathway.

In systemic autoimmune diseases such as systemic lupus erythematosus (SLE), B cell tolerance is lost and there is a production of autoantibodies that drive pathology. The specificities of these antibodies are towards a wide range of autoantigens including proteins such as serum factors including cytokines as well as towards nucleic acids and modified glycolipids. It is known that endosomal pattern recognition receptors are involved in specific responses but if they drive specificity towards a specific group of autoantigens is not known. Here, we used syngeneic apoptotic cells alone to break B cell tolerance and investigated the antibody response in Unc93b1 mutant mice that lack signalling from the TLR3, TLR7 and TLR9 receptors. We found that specific B cell responses known from patients with SLE including antibodies towards Ro-52/60, La, cardiolipin as well as DNA were all significantly lower in the knockout mice. Thus, we found that endosomal TLR receptors were involved in break of tolerance and drive B cell responses for protein, nucleic acid and modified lipid antigens. This pinpoints these receptors as key drivers for the full range of antibody driven pathology in SLE and suggests that targeting of endosomal TLR driven responses will quench all B cell driven autoreactivity.

We have recently reported that capping protein regulator and myosin 1 linker 3 (CARMIL3), first identified as an oncofetal-like gene, is required for metastasis of breast and prostate cancer cells via regulating the actin cytoskeletal dynamics near the plasma membrane. Here, we demonstrate a novel function of CARMIL3 as an essential regulator of the transcription of several key proinflammatory cytokines in macrophages engulfing apoptotic cells and/or exposed to lipopolysaccharides (LPS). CARMIL3-deficient macrophages expressed strongly abrogated levels of interleukin (IL)-6, TNF-α, IL-1β and IL-23 in response to LPS, whereas IL-10 expression was enhanced. An RNA-seq analysis of CARMIL3-deficient and wild-type (WT) RAW264.7 cells stimulated with LPS revealed many differentially expressed genes, impacting several important inflammatory pathways. At the molecular level, CARMIL3 deficiency caused a strong impairment in LPS-activated nuclear factor-κB (NF-κB) signaling with decreased IKKα/β and IκBα phosphorylation and severely reduced p65 protein levels. This study uncovers a crucial role of CARMIL3 in impacting the balance between inflammation and tissue homeostasis via regulating major cytokines production in phagocytic cells.

Skin avulsion is commonly seen in individuals exposed to heavy shearing forces. Subcutaneous tissue detachment and bone fractures usually accompany skin avulsion. Thus, the estimation of the extent of damaged tissue is very important. Currently, the viability of skin and subcutaneous tissue is determined by clinical observations, and these observations always underestimate the true extent of the avulsed skin. Herein, we synthesized an innovative probe, CH1055-GRRRDEVDK (CH1055-GK), which can specifically bind to caspase-3 so as to image skin avulsion and define necrotic regions. Our uptake and binding affinity tests in apoptotic cells and evaluation of the probe ex vivo and in vivo showed that the probe has a strong ability to bind caspase-3 in skin avulsion models and that it vividly detected the necrotic area in avulsed skins. Furthermore, the increased fluorescence intensity of the probe in the avulsed skin showed a larger affected area than that determined by clinical observations in live mice. Consequently, our results indicated that observation of the caspase-3-targeted probe CH1055-GK via NIR-II imaging allowed the clear detection of skin avulsion in subjects, indicating its potential as an imaging tool for the early diagnosis of skin avulsion and the determination of necrotic margins.

Previous studies have reported the pathogenic role of C-reactive protein (CRP) during diabetic kidney disease (DKD) in human CRP transgenic and Crp-/- mice. However, because humans and mice have inverse acute phase expression patterns of CRP and serum amyloid P component, this could lead to the inaccurate evaluation of CRP function with the above-mentioned CRP transgenic mouse. But different from mice, rats have the same acute phase protein expression pattern as human, which might avoid this problem and be a better choice for CRP function studies. To dispel this doubt and accurately define the role of CRP during diabetic nephropathy, we created the first Crp-/- rat model, which we treated with streptozocin to induce DKD for in vivo studies. Moreover, an established cell line (human kidney 2) was used to further investigate the pathologic mechanisms of CRP. We found that CRP promotes epithelial-mesenchymal transition (EMT) through Wnt/β-catenin and ERK1/2 signaling, which are dependent on CRP binding to FcγRII on apoptotic cells. By promoting EMT, CRP was demonstrated to accelerate the development of DKD. We thus present convincing evidence demonstrating CRP as a therapeutic target for DKD treatment.-Zhang, L., Shen, Z.-Y., Wang, K., Li, W., Shi, J.-M., Osoro, E. K., Ullah, N., Zhou, Y., Ji, S.-R. C-reactive protein exacerbates epithelial-mesenchymal transition through Wnt/β-catenin and ERK signaling in streptozocin-induced diabetic nephropathy.

The accumulation of airway apoptotic cells may be an important factor causing airway hyper-responsiveness (AHR). Whether the apoptotic cells can be promptly removed is related to the occurrence and course of asthma. In recent years, studies have shown that Rac1 is involved in many cellular biological activities including the formation and elimination of apoptotic cells. In this study, based on the analysis of airway local cells and related factors in asthmatic mice, we evaluated the expression of Rac1 in airway epithelial cells or phagocytes and analysed its relationship with the incidence of apoptosis or scavenging of apoptotic cells. Our data showed that the expression level of Rac1 in asthmatic mice decreased significantly, while the expression of IL-33 increased obviously. The airway epithelial cell line was stimulated by curcumin at 50 μmol/L for 24-48 hours; more than 50% of the cells were apoptotic, and of which, about 20% were late apoptosis. Rac1 inhibitor (NSC23766) can enhance the apoptosis effect. In addition, the ability of phagocytosis and migration in the epithelial cells or macrophages was increased following the application of Rac1 inhibitors or specific siRNA in a dose-dependent manner, and the expression level of IL-33 was simultaneously increased after blocking Rac1. It is suggested that the down regulation of Rac1 in asthma may contribute to the apoptosis of airway epithelial cells and affect the clearance of apoptotic cells, which will lead to the aggregation of the apoptotic cells in the respiratory tract and participate in AHR.

Apoptotic cells, by externalizing phosphatidylserine (PS) as a hallmark feature, are procoagulant. However, the mechanism by which apoptotic cells activate coagulation system remains unknown. Intrinsic coagulation pathway is initiated by coagulation factor XII (FXII) of contact activation system. The purpose of this study was to determine whether FXII is involved in procoagulant activity of apoptotic cells. Using western blotting and chromogenic substrate assay, we found that incubation with apoptotic cells, but not with viable cells, resulted in rapid cleavage and activation of FXII in the presence of prekallikrein and high molecular weight kininogen (HK), other two components of contact activation system. As detected by flow cytometry, FXII bound to apoptotic cells in a concentration-dependent manner, which was inhibited by annexin V and PS liposome. Direct association of FXII with PS was confirmed in a surface plasmon resonance assay. Clotting time of FXII-deficient plasma induced by apoptotic cells was significantly prolonged, which was fully reversed by replenishment with FXII. Corn trypsin inhibitor, a FXII inhibitor, completely prevented apoptotic cells-induced intrinsic tenase complex formation. Consistently, apoptotic cells significantly increased thrombin production in normal plasma, which was not affected by an inhibitory anti-tissue factor antibody. However, blocking of PS by annexin V, inhibition of FXII, or the deficiency of FXII suppressed apoptotic cells-induced thrombin generation. Addition of purified FXII to FXII-deficient plasma recovered thrombin generation to the normal plasma level. In conclusion, FXII binds to apoptotic cells via PS and becomes activated, thereby constituting a novel mechanism mediating the procoagulant activity of apoptotic cells.