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Rationale: Chronic obstructive pulmonary disease (COPD) is a disease characterized by persistent airway inflammation and disordered macrophage function. The extent to which alterations in macrophage bioenergetics contribute to impaired antioxidant responses and disease pathogenesis has yet to be fully delineated. Objectives: Through the study of COPD alveolar macrophages (AMs) and peripheral monocyte-derived macrophages (MDMs), we sought to establish if intrinsic defects in core metabolic processes drive macrophage dysfunction and redox imbalance. Methods: AMs and MDMs from donors with COPD and healthy donors underwent functional, metabolic, and transcriptional profiling. Measurements and Main Results: We observed that AMs and MDMs from donors with COPD display a critical depletion in glycolytic- and mitochondrial respiration-derived energy reserves and an overreliance on glycolysis as a source for ATP, resulting in reduced energy status. Defects in oxidative metabolism extend to an impaired redox balance associated with defective expression of the NADPH-generating enzyme, ME1 (malic enzyme 1), a known target of the antioxidant transcription factor NRF2 (nuclear factor erythroid 2-related factor 2). Consequently, selective activation of NRF2 resets the COPD transcriptome, resulting in increased generation of TCA cycle intermediaries, improved energetic status, favorable redox balance, and recovery of macrophage function. Conclusions: In COPD, an inherent loss of metabolic plasticity leads to metabolic exhaustion and reduced redox capacity, which can be rescued by activation of the NRF2 pathway. Targeting these defects, via NRF2 augmentation, may therefore present an attractive therapeutic strategy for the treatment of the aberrant airway inflammation described in COPD. ispartof: AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE vol:207 issue:8 pages:998-1011 ispartof: location:United States status: published

Hypoxemia is a defining feature of acute respiratory distress syndrome (ARDS), an often-fatal complication of pulmonary or systemic inflammation, yet the resulting tissue hypoxia, and its impact on immune responses, is often neglected. In the present study, we have shown that ARDS patients were hypoxemic and monocytopenic within the first 48 h of ventilation. Monocytopenia was also observed in mouse models of hypoxic acute lung injury, in which hypoxemia drove the suppression of type I interferon signaling in the bone marrow. This impaired monopoiesis resulted in reduced accumulation of monocyte-derived macrophages and enhanced neutrophil-mediated inflammation in the lung. Administration of colony-stimulating factor 1 in mice with hypoxic lung injury rescued the monocytopenia, altered the phenotype of circulating monocytes, increased monocyte-derived macrophages in the lung and limited injury. Thus, tissue hypoxia altered the dynamics of the immune response to the detriment of the host and interventions to address the aberrant response offer new therapeutic strategies for ARDS.

Intratracheal instillation of apoptotic cells enhances resolution of experimental lung inflammation by incompletely understood mechanisms. We report that this intervention induces functional regulatory T lymphocytes (Tregs) in mouse lung experimentally inflamed by intratracheal administration of lipopolysaccharide. Selective depletion demonstrated that Tregs were necessary for maximal apoptotic cell–directed enhancement of resolution, and adoptive transfer of additional Tregs was sufficient to promote resolution without administering apoptotic cells. After intratracheal instillation, labeled apoptotic cells were observed in most CD11c+CD103+ myeloid dendritic cells migrating to mediastinal draining lymph nodes and bearing migratory and immunoregulatory markers, including increased CCR7 and β8 integrin (ITGB8) expression. In mice deleted for αv integrin in the myeloid line to reduce phagocytosis of dying cells by CD103+ dendritic cells, exogenous apoptotic cells failed to induce transforming growth factor-β1 expression or Treg accumulation and failed to enhance resolution of lipopolysaccharide-induced lung inflammation. We conclude that in murine lung, myeloid phagocytes encountering apoptotic cells can deploy αv integrin–mediated mechanisms to induce Tregs and enhance resolution of acute inflammation.

Acute Respiratory Distress Syndrome (ARDS), an often-fatal complication of pulmonary or systemic inflammation, has no cure. Hypoxemia is a defining feature, yet its impact on inflammation is often neglected. Patients with ARDS are monocytopenic early in the onset of the disease. Endotoxin or Streptococcus pneumoniae acute lung injury (ALI) in the context of hypoxia replicates this finding, through hypoxia-driven suppression of type I interferon signalling. This results in failed lung monocyte-derived interstitial macrophages (IM) niche expansion and unchecked neutrophilic inflammation. Administration of colony stimulating factor 1 (CSF1) rescues the monocytopenia, alters the circulating classical monocyte phenotype in hypoxic endotoxin-driven ALI and enables lung IM population expansion, thus limiting lung injury in endotoxin- and virally-induced hypoxic ALI. Hypoxia directly alters immune dynamics to the detriment of the host and manipulation of this aberrant response offers new therapeutic strategies for ARDS.

Background: Acute respiratory distress syndrome (ARDS) is a severe critical condition with a high mortality that is currently in focus given that it is associated with mortality caused by coronavirus disease 2019 (COVID-19). Neutrophils play a key role in the lung injury characteristic of non-COVID-19 ARDS and there is also accumulating evidence of neutrophil mediated lung injury in patients who succumb to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: We undertook a functional proteomic and metabolomic survey of circulating neutrophil populations, comparing patients with COVID-19 ARDS and non-COVID-19 ARDS to understand the molecular basis of neutrophil dysregulation. Results: Expansion of the circulating neutrophil compartment and the presence of activated low and normal density mature and immature neutrophil populations occurs in ARDS, irrespective of cause. Release of neutrophil granule proteins, neutrophil activation of the clotting cascade and upregulation of the Mac-1 platelet binding complex with formation of neutrophil platelet aggregates is exaggerated in COVID-19 ARDS. Importantly, activation of components of the neutrophil type I interferon responses is seen in ARDS following infection with SARS-CoV-2, with associated rewiring of neutrophil metabolism, and the upregulation of antigen processing and presentation. Whilst dexamethasone treatment constricts the immature low density neutrophil population, it does not impact upon prothrombotic hyperinflammatory neutrophil signatures. Conclusions: Given the crucial role of neutrophils in ARDS and the evidence of a disordered myeloid response observed in COVID-19 patients, this work maps the molecular basis for neutrophil reprogramming in the distinct clinical entities of COVID-19 and non-COVID-19 ARDS.

Background: Acute respiratory distress syndrome (ARDS) is a severe critical condition with a high mortality that is currently in focus given that it is associated with mortality caused by coronavirus disease 2019 (COVID-19). Neutrophils play a key role in the lung injury characteristic of non-COVID-19 ARDS and there is also accumulating evidence of neutrophil mediated lung injury in patients who succumb to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: We undertook a functional proteomic and metabolomic survey of circulating neutrophil populations, comparing patients with COVID-19 ARDS and non-COVID-19 ARDS to understand the molecular basis of neutrophil dysregulation. Results: Expansion of the circulating neutrophil compartment and the presence of activated low and normal density mature and immature neutrophil populations occurs in ARDS, irrespective of cause. Release of neutrophil granule proteins, neutrophil activation of the clotting cascade and upregulation of the Mac-1 platelet binding complex with formation of neutrophil platelet aggregates is exaggerated in COVID-19 ARDS. Importantly, activation of components of the neutrophil type I interferon responses is seen in ARDS following infection with SARS-CoV-2, with associated rewiring of neutrophil metabolism, and the upregulation of antigen processing and presentation. Whilst dexamethasone treatment constricts the immature low density neutrophil population, it does not impact upon prothrombotic hyperinflammatory neutrophil signatures. Conclusions: Given the crucial role of neutrophils in ARDS and the evidence of a disordered myeloid response observed in COVID-19 patients, this work maps the molecular basis for neutrophil reprogramming in the distinct clinical entities of COVID-19 and non-COVID-19 ARDS.

The demonstration and verification of space rapid response launch system based on data link mainly combines with the process, style and characteristics of space rapid response launch, through construction the typical mission scenarios by simulation, carries out design and verification of wide-area maneuver anti-interference networking technology system between platforms such as space and ground data link nodes, container launcher and micro-nano satellite systems, research and design of formatting message standard system, etc., to promote the integrated application of data link and space rapid response launch equipment, so as to enhance command and control, situation sharing and tactical coordination capabilities.

SummaryUnderstanding the mechanisms by which infection with SARS-CoV-2 leads to acute respiratory distress syndrome (ARDS) is of significant clinical interest given the mortality associated with severe and critical coronavirus induced disease 2019 (COVID-19). Neutrophils play a key role in the lung injury characteristic of non-COVID-19 ARDS, but a relative paucity of these cells is observed at post-mortem in lung tissue of patients who succumb to infection with SARS-CoV-2. With emerging evidence of a dysregulated innate immune response in COVID-19, we undertook a functional proteomic survey of circulating neutrophil populations, comparing patients with COVID-19 ARDS, non-COVID-19 ARDS, moderate COVID-19, and healthy controls. We observe that expansion of the circulating neutrophil compartment and the presence of activated low and normal density mature and immature neutrophil populations occurs in both COVID-19 and non-COVID-19 ARDS. In contrast, release of neutrophil granule proteins, neutrophil activation of the clotting cascade and formation of neutrophil platelet aggregates is significantly increased in COVID-19 ARDS. Importantly, activation of components of the neutrophil type I IFN responses is specific to infection with SARS-CoV-2 and linked to metabolic rewiring. Together this work highlights how differential activation of circulating neutrophil populations may contribute to the pathogenesis of ARDS, identifying processes that are specific to COVID-19 ARDS.

Neutrophilic inflammation is central to disease pathogenesis, for example, in chronic obstructive pulmonary disease, yet the mechanisms that retain neutrophils within tissues remain poorly understood. With emerging evidence that axon guidance factors can regulate myeloid recruitment and that neutrophils can regulate expression of a class 3 semaphorin, SEMA3F, we investigated the role of SEMA3F in inflammatory cell retention within inflamed tissues. We observed that neutrophils upregulate SEMA3F in response to proinflammatory mediators and following neutrophil recruitment to the inflamed lung. In both zebrafish tail injury and murine acute lung injury models of neutrophilic inflammation, overexpression of SEMA3F delayed inflammation resolution with slower neutrophil migratory speeds and retention of neutrophils within the tissues. Conversely, constitutive loss of sema3f accelerated egress of neutrophils from the tail injury site in fish, whereas neutrophil-specific deletion of Sema3f in mice resulted in more rapid neutrophil transit through the airways, and significantly reduced time to resolution of the neutrophilic response. Study of filamentous-actin (F-actin) subsequently showed that SEMA3F-mediated retention is associated with F-actin disassembly. In conclusion, SEMA3F signaling actively regulates neutrophil retention within the injured tissues with consequences for neutrophil clearance and inflammation resolution.

Ailiang Zhang, Kun Wang, Lianghua Ding, Xinnan Bao, Xuan Wang, Xubin Qiu, Jinbo Liu Spine Surgery, Third Affiliated Hospital of Soochow University, Changzhou, People’s Republic of China Abstract: Lumbar disc herniation (LDH) is an important cause of radiculopathy, but the underlying mechanisms are incompletely understood. Many studies suggested that local inflammation, rather than mechanical compression, results in radiculopathy induced by LDH. On the molecular and cellular level, nuclear factor-kappa B (NF-κB) and nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome have been implicated in the regulation of neuroinflammation formation and progression. In this study, the autologous nucleus pulposus (NP) was implanted in the left L5 dorsal root ganglion (DRG) to mimic LDH in rats. We investigated the expression of NF-κB and the components of NLRP3 inflammasome in the DRG neurons in rats. Western blotting and immunofluorescence for the related molecules, including NLRP3, apoptosis-associated speck-like protein containing caspase-1 activator domain (ASC), caspase-1, interleukin (IL)-1β, IL-18, IκBα, p-IκBα, p65, p-p65, and calcitonin gene-related peptide (CGRP) were examined. In the NP-treated group, the activations of NLRP3, ASC, caspase-1, IL-1β, IL-18, p-IκBα, and p-p65 in DRG neurons in rats were elevated at 1 day after surgery, and the peak occurred at 7 days. Treatment with Bay11-7082, an inhibitor of the actions of IKK-β, was able to inhibit expression and activation of the molecules (NLRP3, ASC, caspase-1, IL-1β, IL-18, p-IκBα, and p-p65) and relieve the pain in rats. Our study shows that NF-κB and NLRP3 inflammasome are involved in the maintenance of NP-induced pain, and that Bay11-7082 could alleviate mechanical allodynia and thermal hyperalgesia by inhibiting NF-κB and NLRP3 inflammasome activation. Keywords: pain, NLRP3, NF-κB, dorsal root ganglion, nucleus pulposus

Receptor tyrosine kinase like orphan receptor 2 (ROR2) regulates Wnt5a-induced cell migration by phosphorylating PI3K/Akt and activating RhoA in osteosarcoma. However, the role of Wnt5a signaling and its corresponding receptors in the regulation of osteosarcoma metastasis remains poorly understood. ROR1 monoclonal antibody (mAb) and short hairpin (sh)RNA targeting ROR2 markedly inhibited the activity of dishevelled associated activator of morphogenesis 1 (DAAM1) and RhoA and retarded cell migration in osteosarcoma. ROR1 mAb and ROR2 shRNA destroyed the microfilament formation of osteosarcoma cells. Silencing of DAAM1 (with DAAM1 shRNA) downregulated RhoA activity and inhibited cell migration. The decrease of cell migration caused by DAAM1 shRNA was rescued by wild-type DAAM1 overexpression. DAAM1 and PI3Kα/Akt were parallel signaling pathways mediating osteosarcoma cell migration in response to Wnt5a. It was concluded that Wnt5a promotes osteosarcoma cell migration via ROR1/2 receptors, and then activates DAAM1 and RhoA.

Objective: The senescence of nucleus pulposus (NP) cells induced by oxidative stress is one of the important causes of intervertebral disc degeneration (IDD). Herein, we investigated the role and action mechanism of silent information regulator 1 (SIRT1) in oxidative stress-induced senescence of rat NP cell. Methods: Premature senescence of rat NP cells was induced by sublethal concentration of hydrogen peroxide (H2O2) (100 μM). SIRT1 was activated with SRT1720 (5 μM) to explore its effect on NP cells senescence. FoxO1 and Akt were inhibited by AS1842856 (0.2 μM) and MK-2206 (5 μM), respectively, to explore the role of Akt-FoxO1-SIRT1 axis in rat NP cells. Pretreatment with the resveratrol (20 μM), a common antioxidant and indirect activator of SIRT1, was done to investigate its role in senescent rat NP cells. Results: The mRNA and protein levels of SIRT1 were decreased in H2O2-induced senescent rat NP cells, and that specific activation of SIRT1 suppresses senescence. And the Akt-FoxO1 pathway, as the upstream of SIRT1, might be involved in the regulation of H2O2-induced senescence of rat NP cells by affecting the expression of SIRT1. In addition, the resveratrol played an anti-senescence role in rat NP cells, which might affect the Akt-FoxO1-SIRT1 axis. Conclusion: SIRT1 ameliorated oxidative stress-induced senescence of rat NP cell which was regulated by Akt-FoxO1 pathway, and resveratrol exerted anti-senescence effects by affecting this signaling axis.

Immunization of patients with autologous, ex vivo matured dendritic cell (DC) preparations, in order to prime antitumor T-cell responses, is the focus of intense research. Despite progress and approval of clinical approaches, significant enhancement of these personalized immunotherapies is urgently needed to improve efficacy. We show that immunotherapeutic murine and human DC, generated in the presence of the antimicrobial host defense peptide LL-37, have dramatically enhanced expansion and differentiation of cells with key features of the critical CD103+/CD141+ DC subsets, including enhanced cross-presentation and co-stimulatory capacity, and upregulation of CCR7 with improved migratory capacity. These LL-37-DC enhanced proliferation, activation and cytokine production by CD8+ (but not CD4+) T cells in vitro and in vivo. Critically, tumor antigen-presenting LL-37-DC increased migration of primed, activated CD8+ T cells into established squamous cell carcinomas in mice, and resulted in tumor regression. This advance therefore has the potential to dramatically enhance DC immunotherapy protocols.

SUMMARYAcute kidney injury (AKI) following ischemia-reperfusion injury (IRI) has a high mortality and lacks specific therapies. Here, we report that mice lacking kynurenine 3-monooxygenase (KMO) activity (Kmonull mice) are protected against AKI after renal IRI. This advances our previous work showing that KMO blockade protects against acute lung injury and AKI in experimental multiple organ failure caused by acute pancreatitis. We show that KMO is highly expressed in the kidney and exerts major metabolic control over the biologically-active kynurenine metabolites 3-hydroxykynurenine, kynurenic acid and downstream metabolites. In experimental AKI induced by unilateral kidney IRI, Kmonull mice had preserved renal function, reduced renal tubular cell injury, and fewer infiltrating neutrophils compared to wild-type (Kmowt) control mice. Together, these data confirm that flux through KMO contributes to AKI after IRI, and supports the rationale for KMO inhibition as a therapeutic strategy to protect against AKI during critical illness.

Background We have reported that the phosphatidylinositol-3 kinase (PI3K)/Akt/RhoA signaling pathway mediates Wnt5a-induced cell migration of osteosarcoma cells. However, the specific receptors responding to Wnt5a ligand remain poorly defined in osteosarcoma metastasis. Methods Wound healing assays were used to measure the migration rate of osteosarcoma cells transfected with shRNA or siRNA specific against ROR2 or indicated constructs. We evaluated the RhoA activation in osteosarcoma MG-63 and U2OS cells with RhoA activation assay. A panel of inhibitors of PI3K and Akt treated osteosarcoma cells and blocked kinase activity. Western blotting assays were employed to measure the expression and activation of Akt. Clonogenic assays were used to measure the cell proliferation of ROR2-knockdown or ROR2-overexpressed osteosarcoma cells. Results Wnt5a-induced osteosarcoma cell migration was largely abolished by shRNA or siRNA specific against ROR2. Overexpression of RhoA-CA (GFP-RhoA-V14) was able to rescue the Wnt5a-induced cell migration blocked by ROR2 knockdown. The Wnt5a-induced activation of RhoA was mostly blocked by ROR2 knockdown, and elevated by ROR2 overexpression, respectively. Furthermore, we found that Wnt5a-induced cell migration was significantly retarded by RhoA-siRNA transfection or pretreatment of HS-173 (PI3Kα inhibitor), MK-2206 (Akt inhibitor), A-674563 (Akt1 inhibitor), or CCT128930 (Akt2 inhibitor). The activation of Akt was upregulated or downregulated by transfected with ROR2-Flag or ROR2-siRNA, respectively. Lastly, Wnt5a/ROR2 signaling does not alter the cell proliferation of MG-63 osteosarcoma cells. Conclusions Taken together, we demonstrate that ROR2 receptor responding to Wnt5a ligand activates PI3K/Akt/RhoA signaling and promotes the migration of osteosarcoma cells.

The formin protein dishevelled-associated activator of morphogenesis 1 (DAAM1) polymerizes straight actin filaments and mediates migration of cancer cells. However, how DAAM1 governs cell haptotaxis in response to collagen remains unexplored in breast cancer cells. We hypothesized that DAAM1 mediates invadopodia extension and cell haptotaxis in response to type IV collagen in association with integrin receptors. Using Boyden chamber membranes coated with type IV collagen, we show here that type IV collagen activates both DAAM1 and Ras homolog family member A (RHOA) and promotes haptotaxis of MDA-MB-231 and MDA-MB-453 breast cancer cells, a process abolished by treatment with the integrin αvβ3 inhibitor cyclo(-RGDfK). shRNA-mediated knockdown of DAAM1 or a dominant-negative DAAM1 mutation (N-DAAM1) significantly decreased collagen-induced RHOA activity and the assembly of stress fibers, invadopodia extension, and cell haptotaxis. Immunoprecipitation and pulldown assays revealed that integrin αvβ3 is associated with, but only indirectly binds to, the C-terminal DAD domain of DAAM1 in mammalian cells. Blockade of RHOA activation with a specific inhibitor (CCG-1423) or via a dominant-negative RHOA mutation (RHOA-N19) suppressed collagen-induced invadopodia extension and haptotaxis of the MDA-MB-231 and MDA-MB-453 cells. Immunoblotting and immunofluorescence assays indicated high DAAM1 and RHOA expression in invadopodia, which was abolished by cyclo(-RGDfK) treatment or DAAM1 knockdown. These findings have uncovered an integrin αvβ3/DAAM1/RHOA signaling pathway for type IV collagen–induced invadopodia extension and haptotaxis in breast cancer cells. Targeting this pathway may be a means for reducing invasiveness and metastasis of breast cancer.

The purpose of this study was to investigate the diagnostic value of the apparent diffusion coefficient (ADC), measured by diffusion-weighted magnetic resonance imaging (MRI), for the diagnosis of breast lesions presenting as mass and non-mass-like enhancement (NMLE). The breast MRI studies of 174 patients were reviewed retrospectively. A total of 188 histologically confirmed lesions were analyzed and classified into 127 mass enhancement (86 malignant and 41 benign) and 61 NMLE (42 malignant and 19 benign). The ADC values were measured using a spin-echo echo-planner-imaging (SE-EPI) sequence with b=1,000 s/mm(2). Diagnostic performance was evaluated using receiver operating characteristic (ROC) analysis. The mean ADC was 0.99 ± 0.22 × 10(-3)mm(2)/s for invasive cancer, 1.23 ± 0.33 × 10(-3)mm(2)/s for ductal carcinoma in situ (DCIS), and 1.52 ± 0.35 × 10(-3)mm(2)/s for benign adenosis. The mean ADC of all NMLE lesions was 1.44 ± 0.41 × 10(-3)mm(2)/s, which is higher than the mean ADC of all mass lesions, 1.12 ± 0.33 × 10(-3)mm(2)/s. In the ROC analysis, the optimal cutoff ADC value for differentiating benign from malignant lesions was 1.05 × 10(-3)mm(2)/s for mass lesions and 1.35 × 10(-3)mm(2)/s for NMLE. In conclusion, ADC values can be used for the diagnosis of invasive and DCIS as well as benign tumors. The NMLE lesions tend to have higher ADC values than mass lesions; therefore, the morphological appearance of a lesion needs to be considered when using the ADC value for diagnosis.

Background We have reported that the phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway mediated Wnt5a-induced osteosarcoma cell migration. However, the signaling pathways regulating Wnt5a/PI3K/Akt-mediated cell migration remains poorly defined in osteosarcoma cells. Methods We evaluated the activations of RhoA, Rac1 and Cdc42 in osteosarcoma MG-63 and U2OS cells with small G-protein activation assay. Boyden chamber assays were used to confirm the migration of cells transfected indicated constructs or siRNA specific against RhoA. A panel of inhibitors of PI3K and Akt treated osteosarcoma cells and blocked kinase activity. Western blotting and RhoA activation assay were employed to measure the effect of kinase inhibitors and activations of RhoA and Akt. Results We found that Wnt5a had a potent stimulatory effect on RhoA activation, but not on Rac1 and Cdc42 activations. Wnt5a-induced cell migration was largely abolished by siRNA specific against RhoA. DN-RhoA (GFP-RhoA-N19) was also capable of retarding Wnt5a-induced cell migration, but the overexpression of CA-RhoA (GFP-RhoA-V14) was not able to accelerate cell migration. The Wnt5a-induced activation of RhoA was mostly blocked by pretreatment of LY294002 (PI3K inhibitor) and MK-2206 (Akt inhibitor). Furthermore, we found that the Wnt5a-induced activation of RhoA was mostly blocked by pretreatment of HS-173 (PI3Kα inhibitor). Lastly, the phosphorylation of Akt (p-Ser473) was not altered by transfection with siRNA specific against RhoA or DN-RhoA (GFP-RhoA-N19). Conclusions Taken together, we demonstrate that RhoA acts as the downstream of PI3K/Akt signaling (specific PI3Kα, Akt1 and Akt2 isoforms) and mediated Wnt5a-induced the migration of osteosarcoma cells. Electronic supplementary material The online version of this article (doi:10.1186/s12935-017-0396-8) contains supplementary material, which is available to authorized users.

Background: Blockage of autophagic pathways had been reported in IPF. Exosomes are microvesicles that mediate a variety of biological functions. Compromised autophagy can modify cellular exosomes release. Aims: We aimed to study alveolar macrophage (AMo) autophagy and exosomes in the BALf of patients with fibrotic interstitial lung disease (ILD). We hypothesized that impaired autophagy …

Activation of TGF-β by dendritic cells (DCs) expressing αvβ8 integrin is essential for the generation of intestinal regulatory T cells (Tregs) that in turn promote tolerance to intestinal Ags. We have recently shown that αvβ8 integrin is preferentially expressed by CD103+ DCs and confers their ability to activate TGF-β and generate Tregs. However, how these DCs become specialized for this vital function is unknown. In this study, we show that β8 expression is controlled by a combination of factors that include DC lineage and signals derived from the tissue microenvironment and microbiota. Specifically, our data demonstrate that TGF-β itself, along with retinoic acid and TLR signaling, drives expression of αvβ8 in DCs. However, these signals only result in high levels of β8 expression in cells of the cDC1 lineage, CD8α+, or CD103+CD11b− DCs, and this is associated with epigenetic changes in the Itgb8 locus. Together, these data provide a key illustrative example of how microenvironmental factors and cell lineage drive the generation of regulatory αvβ8-expressing DCs specialized for activation of TGF-β to facilitate Treg generation.

There is increasing evidence that topical application of mitomycin-C can be beneficial in reducing epidural scar adhesion. However, the ideal concentration of mitomycin-C is unknown. The purpose of this study was to verify its efficacy for preventing epidural adhesion and the immediate electrophysiological responses caused by it in a laminectomy model. Seventy rats underwent laminectomy at L-1 and L-2. Cotton pads soaked with saline and various concentrations of mitomycin-C (0.1 mg/ml, 0.3 mg/ml, 0.5 mg/ml and 0.7 mg/ml) were applied to the exposed dura for 5 min. Spine somatosensory evoked potentials (SSEP) were monitored in preoperative and the immediate electrophysiological responses of mitomycin-C used. Four weeks postlaminectomy the rats were killed. The area of epidural scar tissue and degree of epidural adhesion were determined by 7.0 T Micro MR imaging. Macroscopic evaluations were performed according to the Rydell standard. The results showed that severe epidural adhesion was formed in the saline group and no dural adherence or incomplete adhesions were found in the mitomycin-C group. The Rydell classification and the degree of epidural adhesion and the area of the scar in 0.5 mg/ml group and 0.7 mg/ml mitomycin-C group revealed a significant decrease compared with the control group and 0.1 mg/ml group and 0.3 mg/ml mitomycin-C group. The spine sensory evoked potentials did not alter obviously in both preoperative and the immediate electrophysiological responses of mitomycin-C used. In conclusion, locally applied mitomycin-C in a concentration of 0.5 mg/ml and 0.7 mg/ml mitomycin-C may be the optimal concentration in preventing postlaminectomy epidural adhesion.

Astrocytes play an important role in protecting neurons during ischemia and reperfusion in the central nervous system. Although many studies have shown that oxygen-glucose deprivation (OGD) can induce astrocyte apoptosis, the role of PERK/eIF2 alpha/ATF4 integrated stress response (ISR) in astrocyte apoptosis mediated by oxygen-glucose-serum deprivation (OGSD)/restoration remains uncertain. Astrocytes were subjected to a combination of oxygen, glucose, and serum deprivation for 8h followed by restoration. Hoechst 33342 staining was performed to quantify apoptotic astrocytes and cell viability was assessed with Cell Counting Kit-8 (CCK8). Immunocytochemical analysis and Western blotting for some related molecules, including pancreatic ER stress kinase (PERK), p-PERK, eukaryotic initiation factor 2 alpha (eIF2 alpha), p-eIF2 alpha, activating transcription factor 4 (ATF4), caspase-12, were examined. Caspase activation and apoptosis were detected in neonatal rat astrocytes from spinal cord subjected to OGSD/restoration. We also observed an increase in cytoplasmic staining of p-eIF2 alpha in astrocytes (8h OGSD/15 min restoration) compared with that of non-treated cells. In addition, we found the sequential activation of PERK, eIF2 alpha, and ATF4 during OGSD/restoration by Western blotting. These results indicate that both the PERK/eIF2 alpha/ATF4 ISR and activation of caspase-12 may be involved in apoptosis of spinal cord astrocytes induced by OGSD/restoration.

As a basic testing element, the thermocouples are widely used in the safety and energy efficiency testing of the electronic and electrical products, new energy products and so on. The accuracy of all thermocouples can't be assured for they are mostly calibrated by sampling. In view of this situation, an efficient and convenient method for the intermediate check of the thermocouple is proposed in this paper. Taking 0 degree and 100 degree which can be easily obtained in the nature as the reference standard temperature, the deviation of the testing thermocouples and the reference standard temperature can be determined and the qualification rate of the thermocouples can be obtained. This method can promote the quality control of the laboratory.

Transglutaminase 2 (TG2), a protein cross-linking enzyme with many additional biological functions, acts as coreceptor for integrin β3. We have previously shown that TG2−/− mice develop an age-dependent autoimmunity due to defective in vivo clearance of apoptotic cells. Here we report that TG2 on the cell surface and in guanine nucleotide-bound form promotes phagocytosis. Besides being a binding partner for integrin β3, a receptor known to mediate the uptake of apoptotic cells via activating Rac1, we also show that TG2 binds MFG-E8 (milk fat globulin EGF factor 8), a protein known to bridge integrin β3 to apoptotic cells. Finally, we report that in wild-type macrophages one or two engulfing portals are formed during phagocytosis of apoptotic cells that are characterized by accumulation of integrin β3 and Rac1. In the absence of TG2, integrin β3 cannot properly recognize the apoptotic cells, is not accumulated in the phagocytic cup, and its signaling is impaired. As a result, the formation of the engulfing portals, as well as the portals formed, is much less efficient. We propose that TG2 has a novel function to stabilize efficient phagocytic portals.

Spinal cord injury (SCI) occurs frequently and is a leading cause of permanent disability in young adults. Many immune inhibitors including tacrolimus (FK506) are shown to be helpful in the regeneration of neural tissue following spinal cord injury. FTY720 belongs to a new class of immunosuppressants. The combination of FTY720 and tacrolimus has been reported to elicit synergistic immunosuppresive effects in rat allograft models without causing critical adverse effects. This study was to determine whether the combination of FTY720 and tacrolimus is superior to FTY720 or tacrolimus alone in the treatment of SCI. Forty-eight rats were subjected to a weight-drop contusion at the tenth thoracic level (a 10-g rod dropped from a height of 25 mm). At 30 min after the operation, they were randomly divided into four groups and received treatment with either FTY720 (0.5 mg/kg), tacrolimus (0.5 mg/kg), FTY720 + tacrolimus (0.5 mg/kg and 0.5 mg/kg respectively) or saline via gavage. Functional recovery was evaluated during 42 days after SCI via open-field test, inclined plane test, footprint analysis, somatosensory evoked potentials (SSEPs), and electron microscopic analysis. Rats from three treatment groups showed significantly better locomotor functional outcomes, higher SSEP amplitude, shorter SSEP latency, and milder pathological changes compared with those of control group. Moreover, rats treated with a combination of FTY720 and tacrolimus demonstrated significantly greater functional recovery by day 14 after SCI than those treated with either FTY720 or tacrolimus alone. These results suggest that the combination of FTY720 and tacrolimus could be a potentially effective therapeutic strategy to treat SCI.

The pattern recognition receptor CD36 initiates a signaling cascade that promotes microglial activation and recruitment to beta-amyloid deposits in the brain. In the present study we identify the focal adhesion-associated proteins p130Cas, Pyk2, and paxillin as novel members of the tyrosine kinase signaling pathway downstream of CD36 and show that assembly of this complex is essential for microglial migration. In primary microglia and macrophages exposed to beta-amyloid, the scaffolding protein p130Cas is rapidly tyrosine-phosphorylated and co-localizes with CD36 to membrane ruffles contemporaneous with F-actin polymerization. These beta-amyloid-stimulated events are not detected in CD36 null cells and are dependent on CD36 activation of Src family tyrosine kinases. Fyn, a Src kinase known to interact with CD36, co-precipitates with p130Cas and is an essential upstream intermediate in the signaling pathways leading to phosphorylation of the p130Cas substrate domain. Furthermore, the p130Cas-interacting kinase Pyk2 and the cytoskeletal adapter protein paxillin also demonstrate CD36-dependent phosphorylation, identifying these focal adhesion molecules as additional members of this beta-amyloid signaling cascade. Disruption of this p130Cas complex by small interfering RNA silencing inhibits p44/42 mitogen-activated protein kinase phosphorylation and microglial migration, illustrating the importance of this pathway in microglial activation and recruitment. Together, these data are the first to identify the signaling cascade that directly links CD36 to the actin cytoskeleton and, thus, implicates it in diverse processes such as cellular migration, adhesion, and phagocytosis.

It has previously been demonstrated that a dose-dependent enhancement of immune response is derived from immunization with several copies of the CpG motif. Following that lead, we sought to incorporate a higher copy number of CpG motifs into an expression construct to evaluate the augmentation of immune responses. By multiple insertions, 30 copies of the CpG motif were cloned into the backbone of an expression construct encoding the foot-and-mouth disease virus (FMDV) capsid protein VP1. After intramuscular immunization, an augmented immune response with significantly increased levels of the specific antibody, T-cell proliferation, and IFN-gamma in Balb/c mice was observed. Compared to chemically synthesized CpG ODN, application of such a multicopy of CpG sequences within the expression backbone for DNA vaccination strategy is feasible and warranted.

We previously found that oxygen-glucose-serum deprivation/restoration (OGSD/R) induces apoptosis of spinal cord astrocytes, possibly via caspase-12 and the integrated stress response, which involves protein kinase R-like endoplasmic reticulum kinase (PERK), eukaryotic initiation factor 2-alpha (eIF2α) and activating transcription factor 4 (ATF4). We hypothesized that edaravone, a low molecular weight, lipophilic free radical scavenger, would reduce OGSD/R-induced apoptosis of spinal cord astrocytes. To test this, we established primary cultures of rat astrocytes, and exposed them to 8 hours/6 hours of OGSD/R with or without edaravone (0.1, 1, 10, 100 μM) treatment. We found that 100 μM of edaravone significantly suppressed astrocyte apoptosis and inhibited the release of reactive oxygen species. It also inhibited the activation of caspase-12 and caspase-3, and reduced the expression of homologous CCAAT/enhancer binding protein, phosphorylated (p)-PERK, p-eIF2α, and ATF4. These results point to a new use of an established drug in the prevention of OGSD/R-mediated spinal cord astrocyte apoptosis via the integrated stress response.

A sciatic nerve transection and repair model was established in Sprague-Dawley rats by transecting the tendon of obturator internus muscle in the greater sciatic foramen and suturing with nylon sutures. The models were treated with tacrolimus gavage (4 mg/kg per day) for 0, 2, 4 and 6 weeks. Specimens were harvested at 6 weeks of intragastric administration. Masson staining revealed that the collagen fiber content and scar area in the nerve anastomosis of the sciatic nerve injury rats were significantly reduced after tacrolimus administration. Hematoxylin-eosin staining showed that tacrolimus significantly increased myelinated nerve fiber density, average axon diameter and myelin sheath thickness. Intragastric administration of tacrolimus also led to a significant increase in the recovery rate of gastrocnemius muscle wet weight and the sciatic functional index after sciatic nerve injury. The above indices were most significantly improved at 6 weeks after of tacrolimus gavage. The myelinated nerve fiber density in the nerve anastomosis and the sciatic nerve functions had a significant negative correlation with the scar area, as detected by Spearman’s rank correlation analysis. These findings indicate that tacrolimus can promote peripheral nerve regeneration and accelerate the recovery of neurological function through the reduction of scar formation.

Background & Aims Immune responses in the intestine are controlled by regulatory T cells (Tregs), which prevent inflammation in response to commensal bacteria. A specific population of intestinal dendritic cells (DCs), marked by expression of CD103, generate Tregs more efficiently than other DC populations through mechanisms that involve retinoic acid and transforming growth factor (TGF)-β. However, it is not clear how CD103+ DCs are specialized for this function. We investigated the ability of CD103+ DCs to promote Treg generation through activation of TGF-β and the role of integrins with the αv subunit in this process. Methods Naïve T cells were cultured with purified DCs from mesenteric lymph nodes (MLNs) or intestines of wild-type and αv conditional knockout mice to assess generation of Tregs. Antigens were administered orally to mice, and antigen-specific generation of Tregs was measured in intestinal tissues. Expression of the integrin αv subunit was measured in purified subpopulations of DCs by quantitative polymerase chain reaction and immunoblot analyses. Results In vitro, CD103+ DCs generated more Tregs in the presence of latent TGF-β than other MLN DCs. Efficient generation of Tregs required expression of the integrin αv subunit by DCs; mice that lacked αv in immune cells did not convert naïve T cells to intestinal Tregs in response to oral antigen. CD103+ DCs derived from the MLNs selectively expressed high levels of integrin αvβ8 compared with other populations of DCs. Conclusions Expression of αvβ8 is required for CD103+ DCs to become specialized and activate latent TGF-β and generate Tregs during the induction of tolerance to intestinal antigens in mice.

Transglutaminase 2 (TG2) is a protein crosslinking enzyme with many additional biological functions. We have previously shown that in TG2(-/-) mice the in vivo clearance of apoptotic cells is defective leading to autoimmunity. TG2 contributes to the formation of phagocytic portals by binding to both integrin beta(3), a known phagocytic receptor, and its bridging molecule, MFG-E8. In TG2 null macrophages integrin beta(3) cannot accumulate around the apoptotic cells and its signaling is impaired. In the present study we describe a subline of TG2 null mice, in which a compensatory increase in integrin beta(3) expression, which resulted alone in a high receptor concentration around the apoptotic cells without the requirement for accumulation, partially corrected the defect in integrin beta(3) signaling. Our data provide a proof for the concept that the function of TG2 is to stabilize accumulated integrin beta(3) concentration in the phagocytic cup.

Wnt5a is classified as a non-transforming Wnt family member and plays complicated roles in oncogenesis and cancer metastasis. However, Wnt5a signaling in osteosarcoma progression remains poorly defined. In this study, we found that Wnt5a stimulated the migration of human osteosarcoma cells (MG-63), with the maximal effect at 100 ng/ml, via enhancing phosphorylation of phosphatidylinositol-3 kinase (PI3K)/Akt. PI3K and Akt showed visible signs of basal phosphorylation and elevated phosphorylation at 15 min after stimulation with Wnt5a. Pharmaceutical inhibition of PI3K with LY294002 significantly blocked the Wnt5a-induced activation of Akt (p-Ser473) and decreased Wnt5a-induced cell migration. Akt siRNA remarkably inhibited Wnt5a-induced cell migration. Additionally, Wnt5a does not alter the total expression and phosphorylation of β-catenin in MG-63 cells. Taken together, we demonstrated for the first time that Wnt5a promoted osteosarcoma cell migration via the PI3K/Akt signaling pathway. These findings could provide a rationale for designing new therapy targeting osteosarcoma metastasis.