Promote Leukocyte Infiltration Research Articles

Targeting TNF-α: The therapeutic potential of certolizumab pegol in the early period of cerebral ischemia reperfusion injury in mice

The neuroinflammatory response triggered by cerebral ischemia–reperfusion injury (CIRI) is characterized by the upsurge of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, which promote leukocyte infiltration and subsequent accumulation in the ischemic zone. This accumulation further intensifies inflammation and aggravates ischemic damage. Certolizumab pegol (CZP), a monoclonal antibody targeting TNF-α, is widely used in treating various inflammatory diseases. This study explored the therapeutic potential of CZP in a mouse model of CIRI, induced by middle cerebral artery occlusion (MCAO), focusing on its influence on the microglial inflammatory response. In vitro analyses revealed that CZP markedly inhibits TNF-α-stimulated inflammation in primary microglia with an EC50 of 1.743 ng/mL. In vivo, MCAO mice treated with CZP (10 μg/mouse, i.p.) for 3 days showed reduced infarct volume, partially improved neurological function, and diminished blood–brain barrierdisruption. Additionally, CZP treatment curtailed microglial activation and the release of pro-inflammatory mediators in the early stages of stroke. It also favorably modulated microglial M1/M2 polarization, rebalanced Th17/Treg cells dynamics, and inhibited Caspase-8-mediated GSDMD cleavage, preventing microglial pyroptosis. Collectively, this study described that the treatment with CZP reversed damaging process caused by CIRI, offering a promising therapeutic strategy for the treatment of ischemic stroke.

Substance P Promotes Leukocyte Infiltration in the Liver and Lungs of Mice with Sepsis: A Key Role for Adhesion Molecules on Vascular Endothelial Cells.

Substance P (SP), encoded by the Tac1 gene, has been shown to promote leukocyte infiltration and organ impairment in mice with sepsis. Neurokinin-1 receptor (NK1R) is the major receptor that mediates the detrimental impact of SP on sepsis. This investigation studied whether SP affects the expression of adhesion molecules, including intercellular cell adhesion molecule-1 (ICAM1) and vascular cell adhesion molecule-1 (VCAM1) on vascular endothelial cells in the liver and lungs, contributing to leukocyte infiltration in these tissues of mice with sepsis. Sepsis was induced by caecal ligation and puncture (CLP) surgery in mice. The actions of SP were inhibited by deleting the Tac1 gene, blocking NK1R, or combining these two methods. The activity of myeloperoxidase and the concentrations of ICAM1 and VCAM1 in the liver and lungs, as well as the expression of ICAM1 and VCAM1 on vascular endothelial cells in these tissues, were measured. The activity of myeloperoxidase and the concentration of ICAM1 and VCAM1 in the liver and lungs, as well as the expression of ICAM1 and VCAM1 on vascular endothelial cells in these tissues, increased in mice with CLP surgery-induced sepsis. Suppressing the biosynthesis of SP and its interactions with NK1R attenuated CLP surgery-induced alterations in the liver and lungs of mice. Our findings indicate that SP upregulates the expression of ICAM1 and VCAM1 on vascular endothelial cells in the liver and lungs, thereby increasing leukocyte infiltration in these tissues of mice with CLP surgery-induced sepsis by activating NK1R.

Organ-on-a-chip for studying immune cell adhesion to liver sinusoidal endothelial cells: the potential for testing immunotherapies and cell therapy trafficking.

Immunotherapy has changed the landscape of treatment options for patients with hepatocellular cancer. Checkpoint inhibitors are now standard of care for patients with advanced tumours, yet the majority remain resistant to this therapy and urgent approaches are needed to boost the efficacy of these agents. Targeting the liver endothelial cells, as the orchestrators of immune cell recruitment, within the tumour microenvironment of this highly vascular cancer could potentially boost immune cell infiltration. We demonstrate the successful culture of primary human liver endothelial cells in organ-on-a-chip technology followed by perfusion of peripheral blood mononuclear cells. We confirm, with confocal and multiphoton imaging, the capture and adhesion of immune cells in response to pro-inflammatory cytokines in this model. This multicellular platform sets the foundation for testing the efficacy of new therapies in promoting leukocyte infiltration across liver endothelium as well as a model for testing cell therapy, such as chimeric antigen receptor (CAR)-T cell, capture and migration across human liver endothelium.

Regulation of von Willebrand factor by ADAMTS13 ameliorates lipopolysaccharide-induced lung injury in mice.

The relationship between von Willebrand factor (VWF) and inflammation has attracted considerable attention in recent years. VWF, which is stored in the Weibel-Palade bodies (WPBs) of endothelial cells (ECs), is released from WPBs in response to inflammatory stimuli and is thought to contribute to inflammation by promoting leukocyte extravasation. In this study, lung injury model mice were produced by intratracheal injection with lipopolysaccharides. The severity of lung inflammation was evaluated in mice with different genotypes (wild-type, Vwf-/-, Adamts13-/-) and mice treated with drugs that inhibit VWF function. Lung inflammation was significantly ameliorated in Vwf-/- mice compared with wild-type mice. Furthermore, inflammation was significantly suppressed in wild-type mice treated with anti-VWF A1 antibody or recombinant human ADAMTS13 compared with the untreated control group. The underlying mechanism appears to be an increased VWF/ADAMTS13 ratio at the site of inflammation and the interaction between blood cell components, such as leukocytes and platelets, and the VWF A1 domain, which promotes leukocyte infiltration into the lung. This study suggested that ADAMTS13 protein and other VWF-targeting agents may be a novel therapeutic option for treatment of pulmonary inflammatory diseases.

TSG6 hyaluronan matrix remodeling dampens the inflammatory response during colitis

In response to tissue injury, changes in the extracellular matrix (ECM) can directly affect the inflammatory response and contribute to disease progression or resolution. During inflammation, the glycosaminoglycan hyaluronan (HA) becomes modified by tumor necrosis factor stimulated gene-6 (TSG6). TSG6 covalently transfers heavy chain (HC) proteins from inter-α-trypsin inhibitor (IαI) to HA in a transesterification reaction and is to date is the only known HC-transferase. By modifying the HA matrix, TSG6 generates HC:HA complexes that are implicated in mediating both protective and pathological responses. Inflammatory bowel disease (IBD) is a lifelong chronic disorder with well-described remodeling of the ECM and increased mononuclear leukocyte influx into the intestinal mucosa. Deposition of HC:HA matrices is an early event in inflamed gut tissue that precedes and promotes leukocyte infiltration. However, the mechanisms by which TSG6 contributes to intestinal inflammation are not well understood. The aim of our study was to understand how the TSG6 and its enzymatic activity contributes to the inflammatory response in colitis. Our findings indicate that inflamed tissues of IBD patients show an elevated level of TSG6 and increased HC deposition and that levels of HA strongly associate with TSG6 levels in patient colon tissue specimens. Additionally, we observed that mice lacking TSG6 are more vulnerable to acute colitis and exhibit an aggravated macrophage-associated mucosal immune response characterized by elevated pro-inflammatory cytokines and chemokines and diminished anti-inflammatory mediators including IL-10. Surprisingly, along with significantly increased levels of inflammation in the absence of TSG6, tissue HA levels in mice were found to be significantly reduced and disorganized, absent of typical “HA-cable” structures. Inhibition of TSG6 HC-transferase activity leads to a loss of cell surface HA and leukocyte adhesion, indicating that the enzymatic functions of TSG6 are a major contributor to stability of the HA ECM during inflammation. Finally, using biochemically generated HC:HA matrices derived by TSG6, we show that HC:HA complexes can attenuate the inflammatory response of activated monocytes. In conclusion, our data suggests that TSG6 exerts a tissue-protective, anti-inflammatory effect via the generation of HC:HA complexes that become dysregulated in IBD.

Circulating Reelin promotes inflammation and modulates disease activity in acute and long COVID-19 cases.

Thromboembolic complications and excessive inflammation are frequent in severe COVID-19, potentially leading to long COVID. In non-COVID studies, we and others demonstrated that circulating Reelin promotes leukocyte infiltration and thrombosis. Thus, we hypothesized that Reelin participates in endothelial dysfunction and hyperinflammation during COVID-19. We showed that Reelin was increased in COVID-19 patients and correlated with the disease activity. In the severe COVID-19 group, we observed a hyperinflammatory state, as judged by increased concentration of cytokines (IL-1α, IL-4, IL-6, IL-10 and IL-17A), chemokines (IP-10 and MIP-1β), and adhesion markers (E-selectin and ICAM-1). Reelin level was correlated with IL-1α, IL-4, IP-10, MIP-1β, and ICAM-1, suggesting a specific role for Reelin in COVID-19 progression. Furthermore, Reelin and all of the inflammatory markers aforementioned returned to normal in a long COVID cohort, showing that the hyperinflammatory state was resolved. Finally, we tested Reelin inhibition with the anti-Reelin antibody CR-50 in hACE2 transgenic mice infected with SARS-CoV-2. CR-50 prophylactic treatment decreased mortality and disease severity in this model. These results demonstrate a direct proinflammatory function for Reelin in COVID-19 and identify it as a drug target. This work opens translational clinical applications in severe SARS-CoV-2 infection and beyond in auto-inflammatory diseases.

Increased Caveolin-2 Expression in Brain Endothelial Cells Promotes Age-Related Neuroinflammation.

Aging is a major risk factor for common neurodegenerative diseases. Although multiple molecular, cellular, structural, and functional changes occur in the brain during aging, the involvement of caveolin-2 (Cav-2) in brain ageing remains unknown. We investigated Cav-2 expression in brains of aged mice and its effects on endothelial cells. The human umbilical vein endothelial cells (HUVECs) showed decreased THP-1 adhesion and infiltration when treated with Cav-2 siRNA compared to control siRNA. In contrast, Cav-2 overexpression increased THP-1 adhesion and infiltration in HUVECs. Increased expression of Cav-2 and iba-1 was observed in brains of old mice. Moreover, there were fewer iba-1-positive cells in the brains of aged Cav-2 knockout (KO) mice than of wild-type aged mice. The levels of several chemokines were higher in brains of aged wild-type mice than in young wild-type mice; moreover, chemokine levels were significantly lower in brains of young mice as well as aged Cav-2 KO mice than in their wild-type counterparts. Expression of PECAM1 and VE-cadherin proteins increased in brains of old wild-type mice but was barely detected in brains of young wild-type and Cav-2 KO mice. Collectively, our results suggest that Cav-2 expression increases in the endothelial cells of aged brain, and promotes leukocyte infiltration and age-associated neuroinflammation.

Serum VCAM-1 and ICAM-1 measurement assists for MACE risk estimation in ST-segment elevation myocardial infarction patients.

BackgroundVascular cell adhesion molecule‐1 (VCAM‐1) and intercellular adhesion molecule‐1 (ICAM‐1) modulate atherosclerosis by promoting leukocyte infiltration, neutrophil recruitment, endothelial cell proliferation, etc., which may directly or indirectly facilitate the occurrence of major adverse cardiac events (MACE). This study intended to investigate the value of VCAM‐1 and ICAM‐1 for predicting MACE in ST‐segment elevation myocardial infarction (STEMI) patients.MethodsTotally, 373 STEMI patients receiving the percutaneous coronary intervention and 50 health controls (HCs) were included. Serum VCAM‐1 and ICAM‐1 were detected by ELISA. Meanwhile, MACE was recorded during a median follow‐up of 18 (range: 1–46) months in STEMI patients.ResultsVascular cell adhesion molecule‐1 and ICAM‐1 were raised in STEMI patients compared with HCs (both p < 0.001). VCAM‐1 (p = 0.002) and ICAM‐1 (p = 0.012) high were linked with raised accumulating MACE rate in STEMI patients. Notably, VCAM‐1 high (hazard ratio [HR] = 2.339, p = 0.031), age ≥ 65 years (HR = 2.019, p = 0.039), history of diabetes mellitus (DM) (HR = 2.395, p = 0.011), C‐reactive protein (CRP) ≥ 5 mg/L (HR = 2.550, p = 0.012), multivessel disease (HR = 2.561, p = 0.007) independently predicted MACE risk in STEMI patients. Furthermore, a nomogram‐based prediction model combining these factors was established, exhibiting an acceptable value for estimating 1, 2, and 3‐year MACE risk, with AUC of 0.764, 0.716, and 0.778, respectively, in STEMI patients.ConclusionThis study confirms the value of VCAM‐1 and ICAM‐1 measurement in predicting MACE risk in STEMI patients. Moreover, VCAM‐1 plus other traditional prognostic factors (such as age, history of DM, CRP, and multivessel disease) cloud further improve the predictive accuracy of MACE risk in STEMI patients.

IgG1 phosphorylcholine ameliorates plaque stability via reduced intraplaque angiogenesis and intraplaque haemorrhage in a murine atherosclerosis model

Abstract Background Phosphorylcholine, (PC) the polar headgroup of the dominating membrane phospholipid phosphatidylcholine, is one of the main oxLDL epitopes and an important pro-inflammatory damage associated molecular pattern. Experimental and epidemiologic data show that natural anti-PC IgM protect against cardiovascular disease. Within atherosclerotic lesions, inflammatory and angiogenesis processes are interdependent and contribute to plaque destabilization. Atherosclerotic lesion resident CD163+ macrophages promote leukocyte infiltration but also induce angiogenesis and vessel permeability by secreting VEGFA. PC antibodies are recognized for their anti-inflammatory properties. However, the effect of PC antibodies on intraplaque angiogenesis (IPA) and intraplaque hemorrhage (IPH), the main entrance route for inflammatory cells in advanced lesions, is unknown. Purpose To investigate the therapeutic effect of a new IgG1 PC antibody (PCmAB) on lesion development, IPA and IPH in murine vein graft atherosclerosis. Methods All animal experiments were performed in compliance with Dutch government guidelines and the Directive 2010/63/EU of the European Parliament. Hypercholesterolemic male ApoE3*Leiden mice received a (donor) caval vein interposition in the carotid artery. Mice received weekly ip injections of (5mg/kg) PCmAb (n=11) or vehicle (n=12) until sacrifice at day 28. Immunohistochemistry was used to evaluate vein graft morphometry and lesion composition including IPA and IPH. PCmAB isolated effects on pro-angiogenic and pro-inflammatory behaviour was investigated in vitro in HUVECs and Hemoglobin (Hb):Haptoglobin (Hp)-cultured THP-1 macrophages. Results PCmAB treatment decreased vein graft media area (13%) and intima lesion (25%), but more importantly increased lumen area with 53% when compared to vehicle treatment. PCmAb improved lesion stability by increasing collagen content (18%) and by decreasing macrophages presence (31%). VCAM-1 and ICAM-1 expression in the vessel wall were also reduced (resp.29% and 36%) by PCmAb. PCmAb improved IPA by a significant reduction in neovessel density of 34%. This was supported in vitro by significant reduced EC proliferation and migration upon PCmAB with and without oxLDL stimulation. Moreover, PCmAb enhanced maturation of intraplaque angiogenic vessels by increasing neovessel pericyte coverage in vivo (31%). Together, this resulted in a reduction of IPH of 62% in the PCmAB group. PCmAb resulted in decreased macrophages CD163+ content in vein grafts by 23% whereas CD163 expression was reduced by PCmAb in Hb:Hp stimulated macrophages. Conclusion PCmAB is an effective inhibitor of atherosclerotic lesion formation in ApoE3*Leiden mice. PCmAb reduces IPA and IPH by decreased neovessel density and (CD163+) macrophages influx via reduced expression of VCAM-1 and ICAM-1, and increased neovessel maturation in vein graft atherosclerosis. PCmAB holds a promise as a new therapeutic approach for plaque stability. Funding Acknowledgement Type of funding source: Public hospital(s). Main funding source(s): Leiden University Medical Center

DNA Methylation-Governed Gene Expression in Autoimmune Arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease hallmarked by progressive and irreversible joint destruction. RA pathogenesis is a T cell-regulated and B cell-mediated process in which activated lymphocyte-produced chemokines and cytokines promote leukocyte infiltration that ultimately leads to destruction of the joints. There is an obvious need to discover new drugs for RA treatment that have different biological targets or modes of action than the currently employed therapeutics. Environmental factors such as cigarette smoke, certain diet components, and oral pathogens can significantly affect gene regulation via epigenetic factors. Epigenetics opened a new field for pharmacology, and DNA methylation and histone modification-implicated factors are feasible targets for RA therapy. Exploring RA pathogenesis involved epigenetic factors and mechanisms is crucial for developing more efficient RA therapies. Here we review epigenetic alterations associated with RA pathogenesis including DNA methylation and interacting factors. Additionally, we will summarize the literature revealing the involved molecular structures and interactions. Finally, potential epigenetic factor-based therapies will be discussed that may help in better management of RA in the future.

Chemokine Expression in Neutrophils and Subcutaneous Adipose Tissue Cells Obtained during Abdominoplasty from Patients with Obesity and Normal Body Weight.

The study was aimed at assessing the role of chemokines in inflammatory changes in tissue following abdominoplasty. The levels of neutrophil-coupled chemokines and their receptors in the serum and blood cells, as well as in cells isolated from the subcutaneous adipose tissue sampled during abdominoplasty were compared in patients with obesity and normal body weight. The levels of chemokines CCL3, CCL3, and CCL5 in blood serum and expression of chemokine receptor CXCR2 and CXCR6 on blood neutrophils were significantly higher (p<0.05) in obese patients in comparison with patients with normal body weight. Elevated expression of chemokines CCL2, CCL3, CCL4, CCL5, CCL18, and CCL20 (p<0.05) was detected in subcutaneous adipose tissue cells isolated obese patients in comparisons with persons with normal body weight. These findings attest to favorable conditions for enhanced neutrophil migration to the adipose tissue in patients with obesity, which can promote leukocyte infiltration of the suture site after abdominoplasty and serves as additional risk factor for the development of postoperative complications associated with activity of neutrophil-derived proteolytic enzymes.

SAT-141 DISTINCT MECHANISMS OF ACTION OF CYCLOPHILIN A VERSUS CYCLOPHILIN D IN PROMOTING ISCHAEMIA/REPERFUSION INJURY

Cyclophilins are enzymes that regulate protein folding. Within this enzyme family, cyclophilin A (CypA) promotes leukocyte recruitment and inflammation, while cyclophilin D (CypD) facilitates mitochondrial-dependent cell death. CypD-/- mice are known to be protected from ischaemia/reperfusion injury (IRI). We investigated: (1) whether a novel pan-cyclophilin inhibitor (CYPi), which does not block calcineurin function, could prevent anticipated renal IRI, (2) the contributions of CypA and CypD in renal IRI. Aims: To determine if cyclophilin (Cyp) inhibition can prevent renal IRI, and examine the specific contributions of CypA and CypD in promoting renal IRI. Groups of 10 mice underwent bilateral renal ischaemia and were killed 24 hours after reperfusion: (1) C57BL/6J mice were treated with 30mg/kg/BID CYPi or vehicle by oral gavage; (2) CypA-/- versus wild type (WT) mice on the 129 background. Controls were sham operated. (3) CypD-/- vs WT cultured proximal tubular epithelial cells (PTEC) were exposed to 0.5 mM of H2O2 for 24hr, with or without CYPi. Renal IRI caused acute renal failure in C57BL/6J mice (179.0±19.1 vs 12.2±1.4umol/L serum creatinine (sCr) in sham; P<0.0001). CYPi protected against renal failure (sCr 36.4±6.7umol/L; P<0.0001). CYPi treatment reduced histologic tubular damage (P<0.0001). CYPi treatment also reduced apoptotic tubular cells (TUNEL+ cells), TNF-α mRNA levels and neutrophil and macrophage infiltration (all P<0.005 vs vehicle). Renal IRI induced acute renal failure in 129 mice (sCr 41±13.76 vs 6.25±1.66umol/L in sham; P<0.0001). CypA-/- mice were protected from renal dysfunction (sCr 20.7±3.53umol/L; P<0.0001). CypA-/- mice had less histologic tubular damage (P<0.005) and lower KIM-1 mRNA levels (P<0.005). CypA-/- mice also had less tubular cell death (TUNEL+ cells; P<0.001), inflammatory cytokines (TNF-α and IL-36-α PCR; P<0.05), and reduced neutrophil infiltration (P<0.001). In addition, cell culture studies showed that CYPi treatment significantly reduced H2O2 induced cell death in WT PTEC (P<0.0001). CypD-/- PTEC also showed reduced H2O2 induced cell death which was not further reduced by CYPi treatment, indicating that it is CypD and not the other cyclophilins that mediate oxidant-induced cell death. Pan-cyclophilin inhibition prevented anticipated IRI-induced acute renal failure by suppressing tubular cell death and inflammation. Our data suggest that CypA promotes leukocyte infiltration and inflammation leading to acute kidney injury following renal IRI, whilst CypD specifically contributes to cell death from the ischaemic/hypoxic insult.

Antibody-cytokine fusion proteins: A novel class of biopharmaceuticals for the therapy of cancer and of chronic inflammation

Antibody-cytokine fusion proteins represent a novel class of biopharmaceuticals, with the potential to increase the therapeutic index of cytokine ‘payloads’ and to promote leukocyte infiltration at the site of disease. In this review, we present a survey of immunocytokines that have been used in preclinical models of cancer and in clinical trials. In particular, we highlight how antibody format, choice of target antigen and cytokine engineering, as well as combination strategies, may have a profound impact on therapeutic performance. Moreover, by using anti-inflammatory cytokines, antibody fusion strategies can conveniently be employed for the treatment of auto-immune and chronic inflammatory conditions.

Stimulation of Prostate Cells by the Senescence Phenotype of Epithelial and Stromal Cells: Implication for Benign Prostate Hyperplasia.

Hyperproliferation of prostate transition‐zone epithelial and stromal cells leads to benign prostate hyperplasia (BPH), a prevalent pathology in elderly men. Senescent cells in BPH tissue induce a senescence‐associated secretory phenotype (SASP) which, by generating inflamed microenvironment and reactive stroma, promotes leukocyte infiltration, cellular hyperproliferation, and nodular prostate growth. We examined human prostate epithelial (BPH‐1, PNT‐1α) and stromal (HPS‐19I) cells for SASP induction by ionizing radiation and assessed SASP's impacts on cell proliferation and on signal transducers that promote cellular growth, proliferation, and survival. Radiation‐induced DNA damage led to cellular senescence, evident from elevated expression of senescence‐associated β‐galactosidase and the cell‐cycle inhibitor p16/INK4a. Clinical BPH tissue showed p16 accumulation. SASP induced mRNA expression for inflammatory cytokines (IL‐1α, IL‐6, IL‐8, TNF‐α); chemokines (GM‐CSF, CXCL12); metalloproteases (MMP‐1, MMP‐3, MMP‐10); growth factor binding IGFBP‐3. Media from irradiated epithelial or stromal cells enhanced BPH‐1 proliferation. ERK1/2 and AKT, which enhance cell growth/survival and STAT5, which facilitates cell cycle progression and leukocyte recruitment to epithelial microenvironment, were activated by SASP components. The radiation‐induced cellular senescence model can be a platform for identification of individual SASP components and pathways that drive BPH etiology/progression in vivo and targeting them may form the basis for novel BPH therapy.

Antibody blockade of CD47‐SIRPα axis is protective in the murine model of Experimental Autoimmune Encephalomyelitis

CD47 and its interaction with Signal Regulatory Protein alpha (SIRPα) play an important role in immune cell homeostasis. Our lab has previously demonstrated that CD47 on both endothelium and T cells plays an important role in T cell adhesion and diapedesis in vivo and in vitro, and that CD47−/− CD4+ T cells have impaired antigen‐mediated proliferation in the mouse model of Experimental Autoimmune Encephalomyelitis (EAE). Given the reports that CD47−/− mice and mice expressing a nonsignaling mutant SIRPα transgene are protected from developing EAE, suggests CD47 is a therapeutic target. However, a previous study found anti‐CD47 monoclonal antibody (mAb) MIAP301 treatment at the same time as myelin oligodendrocyte glycoprotein (MOG) immunization of mice delayed onset of disease and that treatment at peak of disease in animals resulted in a more severe disease. This “Janus like” effect has not been studied further. Using in vitro flow adhesion assays, here we show that mAb MIAP301 blocks both in cis (with LFA‐1 integrin) and in trans (with SIRPα) interactions of CD47 and that a different mAb MIAP410, blocks the SIRPα interaction and not LFA‐1‐ICAM‐1 interaction. We hypothesized that targeting only the “in trans” SIRPα interaction with MIAP410 in EAE would be protective. Thus we treated MOG immunized mice with MIAP410 or an isotype control IgG1 and investigated the leukocyte infiltration into the spinal cord using mass cytometry. Strikingly, treatment with MIAP410 prevented a more severe form of EAE when given at disease peak and was protective when given simultaneously with MOG. Mechanistically, MIAP410 had a profound inhibitory effect (68% inhibition) on the infiltration of IL‐23+ IFN‐γ producing γδ T cells into the spinal cord of the CNS with no significant differences observed on other immune cell infiltration such as Th17 cells. Additionally, we observed that there was decreased neutrophil and conventional dendritic cell infiltration in the spinal cord of MIAP410 as compared to IgG treated mice. Based on these findings, we conclude that CD47‐SIRPα interactions are necessary for the infiltration of pathogenic γδ T cells that express IL‐23R and promote leukocyte infiltration and neuronal damage in EAE.Support or Funding InformationT32 HL007627R01 HL125780RThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

An immune-stimulating proteoglycan from the medicinal mushroom Huaier up-regulates NF-κB and MAPK signaling via Toll-like receptor 4

Trametes robiniophila Murr. (Huaier) is a mushroom with a long history of use as a medicinal ingredient in China and exhibits good clinical efficacy in cancer management. However, the antitumor components of Huaier and the underlying molecular mechanisms remain poorly understood. Here, we isolated a proteoglycan with a molecular mass of ∼5.59 × 104 Da from Huaier aqueous extract. We named this proteoglycan TPG-1, and using FTIR and additional biochemical analyses, we determined that its total carbohydrate and protein compositions are 43.9 and 41.2%, respectively. Using biochemical assays and immunoblotting, we found that exposing murine RAW264.7 macrophages to TPG-1 promotes the production of nitric oxide (NO), tumor necrosis factor α (TNFα), and interleukin-6 (IL-6) through Toll-like receptor 4 (TLR4)-dependent activation of NF-κB and mitogen-activated protein kinase (MAPK) signaling. Of note, the TPG-1 treatment significantly inhibited the tumorigenesis of human hepatoma HepG2 cells likely at least in part by increasing serum levels of TNFα and promoting leukocyte infiltration into tumors in nude mice. TPG-1 also exhibited good antitumor activity in hepatoma H22-bearing mice and had no obvious adverse effects in these mice. We conclude that TPG-1 exerts antitumor activity partially through an immune-potentiating effect due to activation of the TLR4-NF-κB/MAPK signaling cassette. Therefore, TPG-1 may be a promising candidate drug for cancer immunotherapy. This study has identified the TPG-1 proteoglycan as an antitumor agent and provided insights into TPG-1's molecular mechanism, suggesting a potential utility for applying this agent in cancer therapy.

Role of mast cells in inflammatory and reactive pathologies of pulp, periapical area and periodontium.

Mast cells (MCs) have been discovered over 130 years ago; their function was almost exclusively linked to allergic affections. At the time being, it is well known that MCs possess a great variety of roles, in both physiologic and pathologic conditions. In the oral tissues, MCs release different pro-inflammatory cytokines and tumor necrosis factor-alpha that promote leukocyte infiltration in various inflammatory states of the oral cavity. These cells play a key role in the inflammatory process and, as a consequence, their number changes in different pathologic conditions of the oral cavity, such as gingivitis and periodontitis. By understanding the role of MCs in the pathogenesis of different inflammatory diseases of the oral cavity, these cells may become therapeutic targets that could possibly improve the prognosis. Therefore, this review summarizes the current understanding of the role of MCs in various inflammatory pulpal, periapical and periodontal pathophysiological conditions.

The Multifunctional Role of the Chemokine System in Arthritogenic Processes.

The involvement of chemokines and their receptors in the genesis and perpetuation of rheumatoid arthritis, spondyloarthritis, and osteoarthritis has been clearly recognized for a long time. Nevertheless, the complexity of their contribution to these diseases is now becoming evident and this review focuses on published evidence on their mechanism of action. Studies performed on patients and in vivo models have identified a number of chemokine-mediated pathways involved in various aspects of arthritogenic processes. Chemokines promote leukocyte infiltration and activation, angiogenesis, osteoclast differentiation, and synoviocyte proliferation and activation and participate to the generation of pain by regulating the release of neurotransmitters. A number of chemokines are expressed in a timely controlled fashion in the joint during arthropathies, regulating all the aspects of inflammation as well as the equilibrium between damage and repair and between relief and pain. Thus, the targeting of specific chemokine/chemokine receptor interactions is considered a promising tool for therapeutic intervention.

Versican Deficiency Significantly Reduces Lung Inflammatory Response Induced by Polyinosine-Polycytidylic Acid Stimulation

Viral infection is an exacerbating factor contributing to chronic airway diseases, such as asthma, via mechanisms that are still unclear. Polyinosine-polycytidylic acid (poly(I:C)), a Toll-like receptor 3 (TLR3) agonist used as a mimetic to study viral infection, has been shown to elicit inflammatory responses in lungs and to exacerbate pulmonary allergic reactions in animal models. Previously, we have shown that poly(I:C) stimulates lung fibroblasts to accumulate an extracellular matrix (ECM), enriched in hyaluronan (HA) and its binding partner versican, which promotes monocyte adhesion. In the current study, we aimed to determine the in vivo role of versican in mediating inflammatory responses in poly(I:C)-induced lung inflammation using a tamoxifen-inducible versican-deficient mouse model (Vcan-/- mice). In C57Bl/6 mice, poly(I:C) instillation significantly increased accumulation of versican and HA, especially in the perivascular and peribronchial regions, which were enriched in infiltrating leukocytes. In contrast, versican-deficient (Vcan-/-) lungs did not exhibit increases in versican or HA in these regions and had strikingly reduced numbers of leukocytes in the bronchoalveolar lavage fluid and lower expression of inflammatory chemokines and cytokines. Poly(I:C) stimulation of lung fibroblasts isolated from control mice generated HA-enriched cable structures in the ECM, providing a substrate for monocytic cells in vitro, whereas lung fibroblasts from Vcan-/- mice did not. Moreover, increases in proinflammatory cytokine expression were also greatly attenuated in the Vcan-/- lung fibroblasts. These findings provide strong evidence that versican is a critical inflammatory mediator during poly(I:C)-induced acute lung injury and, in association with HA, generates an ECM that promotes leukocyte infiltration and adhesion.

Regulatory Mechanism of Collagen Degradation by Keratocytes and Corneal Inflammation: The Role of Urokinase-Type Plasminogen Activator.

Keratocytes, corneal resident cells in the corneal stroma, exist between collagen lamellae and maintain the corneal stromal structure. When the corneal stroma is damaged, keratocytes are transformed to myofibroblasts to aid corneal wound healing by phagocytizing debris. Keratocytes and extracellular collagen influence each other because keratocytes cultured in a 3D collagen gel undergo morphological changes and keratocytes produce metalloproteases that degrade extracellular collagen. IL-1 and plasminogen are critical mediators for collagen degradation. The plasminogen system contributes to tissue repair by activating matrix metalloproteinases (MMPs), releasing growth factors from the extracellular matrix and extracellular matrix degradation. Urokinase-type plasminogen activator (uPA) is thought to be involved in corneal disorders and regulates corneal wound healing. uPA is a serine protease synthesized by various cells such as corneal epithelial cells, corneal fibroblasts, vascular endothelial cells, smooth muscle cells, monocytes, macrophages, and malignant tumor cells of different origins. Here, we review the role of uPA in corneal stromal wound healing. uPA is expressed in leukocytes and corneal fibroblasts in the corneas of patients with corneal ulcerations suggesting it is a key regulator of corneal stromal wound healing. uPA is directly involved in plasmin-mediated collagen degradation induced by IL-1. Moreover, uPA is critically involved in promoting leukocyte infiltration in corneal inflammation by activating MMP-9. This activation is presumably directly and indirectly mediated by the plasminogen/plasmin cascade. Moreover, uPA mediates the release of inflammatory cytokines from corneal fibroblasts to promote leukocyte infiltration.