ICAM-1 nanoclusters regulate hepatic epithelial cell polarity by leukocyte adhesion-independent control of apical actomyosin.

Epithelial intercellular adhesion molecule (ICAM)-1 is apically polarized, interacts with, and guides leukocytes across epithelial barriers. Polarized hepatic epithelia organize their apical membrane domain into bile canaliculi and ducts, which are not accessible to circulating immune cells but that nevertheless confine most of ICAM-1. Here, by analyzing ICAM-1_KO human hepatic cells, liver organoids from ICAM-1_KO mice and rescue-of-function experiments, we show that ICAM-1 regulates epithelial apicobasal polarity in a leukocyte adhesion-independent manner. ICAM-1 signals to an actomyosin network at the base of canalicular microvilli, thereby controlling the dynamics and size of bile canalicular-like structures. We identified the scaffolding protein EBP50/NHERF1/SLC9A3R1, which connects membrane proteins with the underlying actin cytoskeleton, in the proximity interactome of ICAM-1. EBP50 and ICAM-1 form nano-scale domains that overlap in microvilli, from which ICAM-1 regulates EBP50 nano-organization. Indeed, EBP50 expression is required for ICAM-1-mediated control of BC morphogenesis and actomyosin. Our findings indicate that ICAM-1 regulates the dynamics of epithelial apical membrane domains beyond its role as a heterotypic cell-cell adhesion molecule and reveal potential therapeutic strategies for preserving epithelial architecture during inflammatory stress.

The heavy subunit of ferritin stimulates NLRP3 inflammasomes in hepatic stellate cells through ICAM-1 to drive hepatic inflammation.

Serum ferritin concentrations increase during hepatic inflammation and correlate with the severity of chronic liver disease. Here, we report a molecular mechanism whereby the heavy subunit of ferritin (FTH) contributes to hepatic inflammation. We found that FTH induced activation of the NLRP3 inflammasome and secretion of the proinflammatory cytokine interleukin-1ß (IL-1ß) in primary rat hepatic stellate cells (HSCs) through intercellular adhesion molecule-1 (ICAM-1). FTH-ICAM-1 stimulated the expression of Il1b, NLRP3 inflammasome activation, and the processing and secretion of IL-1ß in a manner that depended on plasma membrane remodeling, clathrin-mediated endocytosis, and lysosomal destabilization. FTH-ICAM-1 signaling at early endosomes stimulated Il1b expression, implying that this endosomal signaling primed inflammasome activation in HSCs. In contrast, lysosomal destabilization was required for FTH-induced IL-1ß secretion, suggesting that lysosomal damage activated inflammasomes. FTH induced IL-1ß production in liver slices from wild-type mice but not in those from Icam1-/- or Nlrp3-/- mice. Thus, FTH signals through its receptor ICAM-1 on HSCs to activate the NLRP3 inflammasome. We speculate that this pathway contributes to hepatic inflammation, a key process that stimulates hepatic fibrogenesis associated with chronic liver disease.

Advanced Glycation End Products Upregulate CD40 in Human Retinal Endothelial and Müller Cells: Relevance to Diabetic Retinopathy.

CD40 induces pro-inflammatory responses in endothelial and Müller cells and is required for the development of diabetic retinopathy (DR). CD40 is upregulated in these cells in patients with DR. CD40 upregulation is a central feature of CD40-driven inflammatory disorders. What drives CD40 upregulation in the diabetic retina remains unknown. We examined the role of advanced glycation end products (AGEs) in CD40 upregulation in endothelial cells and Müller cells. Human endothelial cells and Müller cells were incubated with unmodified or methylglyoxal (MGO)-modified fibronectin. CD40 expression was assessed by flow cytometry. The expression of ICAM-1 and CCL2 was examined by flow cytometry or ELISA after stimulation with CD154 (CD40 ligand). The expression of carboxymethyl lysine (CML), fibronectin, and laminin as well as CD40 in endothelial and Müller cells from patients with DR was examined by confocal microscopy. Fibronectin modified by MGO upregulated CD40 in endothelial and Müller cells. CD40 upregulation was functionally relevant. MGO-modified fibronectin enhanced CD154-driven upregulation of ICAM-1 and CCL2 in endothelial and Müller cells. Increased CD40 expression in endothelial and Müller cells from patients with DR was associated with increased CML expression in fibronectin and laminin. These findings identify AGEs as inducers of CD40 upregulation in endothelial and Müller cells and enhancers of CD40-dependent pro-inflammatory responses. CD40 upregulation in these cells is associated with higher CML expression in fibronectin and laminin in patients with DR. This study revealed that CD40 and AGEs, two important drivers of DR, are interconnected.

Impact of TNF and IL-33 Cytokines on Mast Cells in Neuroinflammation.

Mast cells (MCs) are derived from hematopoietic progenitors, mature in vascularized tissues, and participate in innate and acquired immunity. Neuroinflammation is a highly debated topic in the biomedical literature; however, the impact of tumor necrosis factor (TNF) and IL-33 on MCs in the brain has not been widely addressed. MCs can be activated by IgE binding to FcεRI, as well as by different antigens. After activation, MCs mediate various immunological and inflammatory responses through TNF and IL-33. TNF has two receptors: TNFR1, a p55 molecule, and TNFR2, a p75 molecule. This cytokine is the only one of its kind to be stored in the granules of MCs and can also be generated by de novo synthesis via mRNA. In the central nervous system (CNS), TNF is produced almost exclusively by microglial cells, neurons, astrocytes, and, minimally, by endothelial cells. After its release into brain tissue, TNF rapidly induces the adhesion molecules endothelial leukocyte adhesion molecule 1 (ELAM-1), intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1) in endothelial cells. TNF causes the chemoattraction of neutrophils by inducing several molecules, including CXC chemokines (IL-8). Both MCs and microglial cells act as a primary barrier against foreign molecules in the CNS, producing pro-inflammatory cytokines such as IL-33. IL-33 belongs to the IL-1 family, is activated through the ST2L/IL1-RAcP receptor complex, and mediates both the innate and adaptive immune response. IL-33 is a nuclear transcription factor expressed in the brain, where it induces pro-inflammatory cytokines (TNF and IL-1) and chemokines (CCL2, CCL3, CCL5, and CXCL10). Therefore, MCs and microglia in the CNS are a source of pro-inflammatory cytokines, including TNF and IL-33, that mediate many brain diseases. The inhibition of TNF and IL-33 may represent a new therapeutic approach that could complement existing neuroinflammatory therapies.

Amiodarone inhibits the Toll-like receptor 3-mediated nuclear factor κB signaling pathway by blocking organelle acidification.

Toll-like receptor (TLR) agonists or pro-inflammatory cytokines converge to activate the nuclear factor κB (NF-κB) signaling pathway, which provokes inflammatory responses. In the present study, we identified amiodarone hydrochloride as a selective inhibitor of the TLR3-mediated NF-κB signaling pathway by screening the RIKEN NPDepo Chemical Library. In human umbilical vein endothelial cells (HUVEC), amiodarone selectively inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) induced by polyinosinic-polycytidylic acid (Poly(I:C)), but not tumor necrosis factor-α, interleukin-1α, or lipopolysaccharide. In response to a Poly(I:C) stimulation, amiodarone at 20 µM reduced the up-regulation of mRNA expression encoding ICAM-1, vascular cell adhesion molecule-1, and E-selectin. The nuclear translocation of the NF-κB subunit RelA was inhibited by amiodarone at 15-20 µM in Poly(I:C)-stimulated HUVEC. Amiodarone diminished the fluorescent dots of LysoTracker® Red DND-99 scattered over the cytoplasm of HUVEC. Therefore, the present study revealed that amiodarone selectively inhibited the TLR3-mediated NF-κB signaling pathway by blocking the acidification of intracellular organelles.

Targeting aberrant glycosylation to modulate microglial response and improve cognition in models of Alzheimer's disease.

Altered glycosylation profiles have been correlated with potential drug targets in various diseases, including Alzheimer's disease (AD). In this area, the linkage between bisecting N-acetylglucosamine (GlcNAc), a product of N-acetylglucosaminyltransferase III (GnT-III), and AD has been recognized, however, our understanding of the cause and the causative role of this aberrant glycosylation in AD are far from completion. Moreover, the effects and mechanisms of glycosylation-targeting interventions on memory and cognition, and novel targeting strategies are worth further study. Here, we showed the characteristic amyloid pathology-induced and age-related changes of GnT-III, and identified transcription factor 7-like 2 as the key transcription factor responsible for the abnormal expression of GnT-III in AD. Upregulation of GnT-III aggravated cognitive dysfunction and Alzheimer-like pathologies. In contrast, loss of GnT-III could improve cognition and alleviate pathologies. Furthermore, we found that an increase in bisecting GlcNAc modified ICAM-1 resulted in impairment of microglial responses, and genetic inactivation of GnT-III protected against AD mechanistically by blocking the aberrant glycosylation of ICAM-1 and subsequently modulating microglial responses, including microglial motility, phagocytosis ability, homeostatic/reactive state and neuroinflammation. Moreover, by target-based screening of GnT-III inhibitors from FDA-approved drug library, we identified two compounds, regorafenib and dihydroergocristine mesylate, showing pharmacological potential leading to modulation of aberrant glycosylation and microglial responses, and rescue of memory and cognition deficits.

Macrophage AHR-TLR4 cross-talk drives p-STAT3 (Ser727)-mediated mitochondrial oxidative stress and upregulates IDO/ICAM-1 in the steatohepatitis induced by aflatoxin B1.

Aflatoxin B1 (AFB1) is a major mycotoxin contaminant showing in the environment and foods. In this study, the molecular initiating events (MIEs) of AFB1-induced steatohepatitis were explored in mice and human cell model. We observed dose-dependent steatohepatitis in the AFB1-treated mice, including triglyceride accumulation, fibrotic collagen secretion, enrichment of CD11b + and F4/80+ macrophages/Kupffer cells, cell death, lymphocytes clusters and remarkable atrophy areas. The gut barrier and gut-microbiota were also severely damaged after the AFB1 treatment and pre-conditioned colitis in the experimental mice aggravated the steatohepatitis phenotypes. We found that macrophages cells can be pro-inflammatorily activated to M1-like phenotype by AFB1 through an AHR/TLR4/p-STAT3 (Ser727)-mediated mitochondrial oxidative stress. The phenotypes can be rescued by AHR inhibitors in the mice model and human cell model. We further showed that this signaling axis is based on the cross-talk interaction between AHR and TLR4. Gene knock-up experiment found that the signaling is dependent on AFB1 ligand-binding with AHR, but not protein expressions of TLR4. The signaling elevated NLRP3 and two immune metabolic enzymes ICAM-1 and IDO that are associated with macrophage polarization. Results from intervention experiments with natural anti-oxidant and AHR inhibitor CH223191 suggest that the macrophage polarization may rely on AHR and ROS. Our study provides novel and critical references to the food safety and public health regulation of AFB1.

Functional roles of CD26/DPP4 in lipopolysaccharide-induced lung injury.

Acute respiratory distress syndrome (ARDS) is characterized by dysregulated inflammation and increased permeability of lung microvascular cells. CD26/dipeptidyl peptidase-4 (DPP4) is a type II membrane protein that is expressed in several cell types and mediates multiple pleiotropic effects. We previously reported that DPP4 inhibition by sitagliptin attenuates lipopolysaccharide (LPS)-induced lung injury in mice. The current study characterized the functional role of CD26/DPP4 expression in LPS-induced lung injury in mice, isolated alveolar macrophages, and cultured lung endothelial cells. In LPS-induced lung injury, inflammatory responses [bronchoalveolar lavage fluid (BALF) neutrophil numbers and several proinflammatory cytokine levels] were attenuated in Dpp4 knockout (Dpp4 KO) mice. However, multiple assays of alveolar capillary permeability were similar between the Dpp4 KO and wild-type mice. TNF-α and IL-6 production was suppressed in alveolar macrophages isolated from Dpp4 KO mice. In contrast, in cultured mouse lung microvascular endothelial cells (MLMVECs), reduction in CD26/DPP4 expression by siRNA resulted in greater ICAM-1 and IL-6 expression after LPS stimulation. Moreover, the LPS-induced vascular monolayer permeability in vitro was higher in MLMVECs treated with Dpp4 siRNA, suggesting that CD26/DPP4 plays a protective role in endothelial barrier function. In summary, this study demonstrated that genetic deficiency of Dpp4 attenuates inflammatory responses but not permeability in LPS-induced lung injury in mice, potentially through differential functional roles of CD26/DPP4 expression in resident cellular components of the lung. CD26/DPP4 may be a potential therapeutic target for ARDS and warrants further exploration to precisely identify the multiple functional effects of CD26/DPP4 in ARDS pathophysiology.NEW & NOTEWORTHY We aimed to clarify the functional roles of CD26/DPP4 in ARDS pathophysiology using Dpp4-deficient mice and siRNA reduction techniques in cultured lung cells. Our results suggest that CD26/DPP4 expression plays a proinflammatory role in alveolar macrophages while also playing a protective role in the endothelial barrier. Dpp4 genetic deficiency attenuates inflammatory responses but not permeability in LPS-induced lung injury in mice, potentially through differential roles of CD26/DPP4 expression in the resident cellular components of the lung.

PGRN inhibits CD8+T cell recruitment and promotes breast cancer progression by up-regulating ICAM-1 on TAM.

BACKGROUND: Tumor microenvironment actually reduces antitumor effect against the immune attack by exclusion of CD8+T cells. Progranulin (PGRN) is a multifunctional growth factor with significant pathological effects in multiple tumors; however, its role in immunity evasion of breast cancer (BCa) is not completely understood. METHODS: We depleted GRN (PGRN gene) genetically in mice or specifically in PY8119 murine BCa cell line, and mouse models of orthotopic or subcutaneous transplantation were used. Chimeric mice-deficient of PGRN (Grn-/-) in bone marrow (BM) compartment was also generated. Association of PGRN expression with chemokine production or BCa development was investigated by histological and immunological assays. RESULTS: We found PGRN was involved in exhaustion of cytotoxic CD8+T cell in BCa with the increasing expressions of M2 markers and intercellular cell adhesion molecule-1 (ICAM-1) on macrophages. Specifically, ablation of PGRN in PY8119 cells reduced tumor burden, accompanied by the infiltrating of cytotoxic CD8+T cells into tumor nests. Moreover, our result revealed that blockade of PD-1 in PGRN-depleted tumors exhibited better antitumor effect in vivo and significantly decreased tumor burden. CONCLUSION: These findings suggest that inhibition of PGRN may act as a potential immune-therapeutic strategy by recovering infiltration of CD8+T cell in BCa tissue and thereby enhancing the response to anti-PD-1 therapy.

Multiple exposures to low-dose ionizing radiation induced the initiation and progression of pro-atherosclerotic phenotypes in mice and vascular endothelial cell damage.

OBJECTIVE: This study aimed to investigate the effects of low-dose radiation on the abdominal aorta of mice and vascular endothelial cells. METHODS: Wild-type and tumor-bearing mice were exposed to 15 sessions of low-dose irradiation, resulting in cumulative radiation doses of 187.5, 375, and 750 mGy. The effect on the cardiovascular system was assessed. Immunohistochemistry analyzed protein expressions of PAPP-A, CD62, P65, and COX-2 in the abdominal aorta. Microarray technology, Gene Ontology analysis, and pathway enrichment analysis evaluated gene expression changes in endothelial cells exposed to 375 mGy X-ray. Cell viability was assessed using the Cell Counting Kit 8 assay. Immunofluorescence staining measured γ-H2AX levels, and real-time polymerase chain reaction quantified mRNA levels of interleukin-6 (IL-6), ICAM-1, and Cx43. RESULTS: Hematoxylin and eosin staining revealed thickening of the inner membranes and irregular arrangement of smooth muscle cells in the media membrane at 375 and 750 mGy. Inflammation was observed in the inner membranes at 750 mGy, with a clear inflammatory response in the hearts of tumor-bearing mice. Immunohistochemistry indicated increased levels of PAPP-A, P65, and COX-2 post-irradiation. Microarray analysis showed 425 up-regulated and 235 down-regulated genes, associated with processes like endothelial cell-cell adhesion, IL-6, and NF-κB signaling. Cell Counting Kit 8 assay results indicated inhibited viability at 750 mGy in EA.hy926 cells. Immunofluorescence staining demonstrated a dose-dependent increase in γ-H2AX foci. Reverse transcription quantitative PCR results showed increased expression of IL6, ICAM-1, and Cx43 in EA.hy926 cells post 750 mGy X-ray exposure. CONCLUSION: Repeated low-dose ionizing radiation exposures triggered the development of pro-atherosclerotic phenotypes in mice and damage to vascular endothelial cells.

Ginsenoside Compound K Reduces Psoriasis-related Inflammation by Activation of the Glucocorticoid Receptor in Keratinocytes.

AIM: To investigate the effects and mechanism of Ginsenoside Compound K (GCK) on psoriasis, focusing on the glucocorticoid receptor (GR) in keratinocytes. METHODS: An imiquimod (IMQ)-induced psoriasis-like dermatitis mouse model was generated to evaluate the anti-inflammatory effect of GCK. Hematoxylin and eosin (H&E) staining was used to assess skin pathological changes. Protein expression of K17 and p-p65 in mice skin was assayed by immunohistochemical. Protein expression and phosphorylation of p65 IκB were assayed by Western blot. Protein expression of K1, K6, K10, K16, K17, and GR were assayed by Western blot and immunofluorescence. Enzyme-linked immunosorbent assay (ELISA) was used to determine cytokine levels of TNF-α, IL-6, CXCL-8, and ICAM-1. Real-time polymerase chain reaction (RT-PCR) was used to quantify TNF-α, IL-6, IL-8, and ICAM-1 mRNA expression. Cell viability was determined by Cell Counting Kit-8(CCK-8) assay. A high-content cell-imaging system was used to assay cell proliferation. Nuclear translocation of p65 and GR was assayed by imaging flow cytometry and immunofluorescence microscopy. Small interfering RNA was used to confirm the role of GR in the anti-inflammatory and immunoregulatory effect of GCK in normal human epidermal keratinecytes (NHEKs). RESULTS: GCK reduced the psoriasis area, severity index, and epidermal thickening in IMQ-induced mice. GCK significantly attenuated the mRNA levels of IL-6, IL-8, TNF-α, and ICAM-1 and reduced epidermal hyperproliferation in the skin of IMQ-induced mice. GCK inhibited in vitro activation of NF-κB, leading to attenuated release of inflammatory mediators (IL-6, IL-8, TNF-α, and ICAM-1) and suppression of NHEK hyperproliferation and abnormal differentiation. These inhibitory effects of GCK were diminished by GR silencing in NHEKs. CONCLUSION: GCK suppressed psoriasis-related inflammation by suppressing keratinocyte activation, which may be related to promoting GR nuclear translocation and inhibiting NF-κB activation. In summary, GCK appears to be a GR activator and a promising therapeutic candidate for antipsoriatic agents.

Alantolactone derivatives inhibit the tumor necrosis factor α-induced nuclear factor κB pathway by a different mechanism from alantolactone.

Alantolactone is a eudesmane-type sesquiterpene lactone that exerts various biological effects, including anti-inflammatory activity. In the present study, screening using the RIKEN Natural Products Depository chemical library identified alantolactone derivatives that inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells stimulated with proinflammatory cytokines and Toll-like receptor ligands. In human lung adenocarcinoma A549 cells stimulated with tumor necrosis factor-α (TNF-α), six alantolactone derivatives inhibited ICAM-1 expression in a dose-dependent manner and at IC50 values of 13-21 µM, whereas that of alantolactone was 5 µM. Alantolactone possesses an α-methylene-γ-lactone moiety, whereas alantolactone derivatives do not. In the nuclear factor κB (NF-κB) signaling pathway, alantolactone prevented the TNF-α-induced phosphorylation and degradation of the inhibitor of NF-κB α (IκBα) protein, and its downstream signaling pathway. In contrast, alantolactone derivatives neither reduced TNF-α-induced IκBα degradation nor the nuclear translocation of the NF-κB subunit RelA, but inhibited the binding of RelA to the ICAM-1 promoter. The inhibitory activities of alantolactone and alantolactone derivatives were attenuated by glutathione. These results indicate that alantolactone derivatives inhibit the TNF-α-induced NF-κB pathway by a different mechanism from alantolactone.

Labetalol Ameliorates Experimental Colitis in Rat Possibly Through its Effect on Proinflammatory Mediators and Oxidative Stress.

BACKGROUND: Members of beta blockers drugs possess significant antioxidant activities. The current research is to assess the effect of the labetalol on acetic acid (AA-induced) colitis in rat model. METHODS: Forty adult Wistar rats were separated into 4 groups, including the negative control group, AA group, AA + sulfasalazine (100 mg/kg/day) group, and AA + labetalol (300 mg/kg/day) group. Colitis was induced in rats by the interrectal installation of 2 mL of 4% (v/v) AA. Sulfasalazine and labetalol were administered orally for 7 days after 2 hours of induction. The following parameters were measured: disease activity index (DAI), histopa-thological changes and colon tissue homogenate concentrations of proinflammatory mediators IL-1ß, adhesion molecules ICAM-1, and oxidative stress marker myeloperoxidase (MPO). RESULTS: The treatment with labetalol significantly reduced DAI and histopathological changes induced by AA. Also, labetalol markedly decreased the concentrations of IL-1ß, ICAM-1, and MPO in colonic tissue that were increased by AA. The effects of labetalol were significantly lower than that produced by sulfasalazine as standard drug. CONCLUSIONS: Labetalol exerts ameliorative effects on disease activity and histopathological features of AA-induced colitis in rats possibly through antioxidant effects and inhibition of inflammatory mediators.

Endothelial marker profiles in cerebral radiation-induced vasculopathy: A comparative immunohistochemical analysis.

Radiation therapy results in radiation-induced vasculopathy, characterized by alterations in the vascular architecture stemming from radiation exposure. The exact molecular pathways and associated pathologies of this condition have yet to be comprehensively understood. This study aimed to identify specific markers' roles in cerebral vascular endothelial injury pathogenesis after radiosurgery and explore their unique expression patterns in diverse pathologies post-stereotactic radiosurgery. A retrospective cohort study was conducted to assess the expression profiles of endothelial markers via immunohistochemical analysis in 25 adult patients (13 males and 12 females) who had undergone neurosurgical resection for various central nervous system pathologies following stereotactic radiosurgery or radiotherapy from 2001 to 2015. Our findings revealed strong immunohistochemical expression of ICAM-1 and E-selectin across various disease states, while MMP-9, PAI-1, and eNOS exhibited moderate expression levels. In contrast, VCAM-1 and P-Selectin had the weakest expression across all groups. Notably, while individual markers showed significant variations in expression levels when comparing different diseases (P < .001), no substantial differences were found in the overall immunohistochemical expression patterns across the 5 distinct pathologies studied (P = .407, via 2-way ANOVA). Despite the varied long-term effects of radiotherapy on the vascular endothelium, a common thread of inflammation runs through the pathology of these conditions. The distinct patterns of marker expression identified in our study suggest that different markers play unique roles in the development of radiation-induced vasculopathy. These findings offer insights that could lead to the development of novel preventive strategies and treatments.

NGF facilitates ICAM-1-dependent monocyte adhesion and M1 macrophage polarization in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disorder in which monocytes adhering to synovial tissue differentiate into the pro-inflammatory M1 macrophage phenotype. Nerve growth factors (NGF) referred to as neurotrophins have been associated with inflammatory events; however, researchers have yet to elucidate the role of NGF in RA. Our examination of clinical tissue samples and analysis of data sourced from the Gene Expression Omnibus dataset unveiled elevated expression levels of M1 macrophage markers in human RA synovial tissue samples compared to normal tissue, with no such distinction observed for M2 markers. Furthermore, immunofluorescence data depicted increased expression levels of NGF and M1 macrophages in RA mice in contrast to normal mice. It appears that NGF stimulation facilitates macrophage polarization from the M0 to the M1 phenotype. It also appears that NGF promotes ICAM-1 production in human RA synovial fibroblasts, which enhances monocyte adhesion through the TrkA, MEK/ERK, and AP-1 signaling cascades. Our findings indicate NGF/TrkA axis as a novel target for the treatment of RA.

[Association of ITGA4 and ICAM-1 gene polymorphisms with the risk and clinicopathological characteristics of Crohn's disease].

OBJECTIVE: To assess the association between the polymorphism of integral protein α4 (ITGA4) and intercellular adhesion molecule 1 (ICAM-1) genes and the risk and clinicopathological characteristics of Crohn's disease (CD) among Chinese patients. METHODS: From January 2010 to January 2021, a total of 215 CD patients and 529 gender- and age-matched healthy controls were enrolled from the Second Affiliated Hospital of Wenzhou Medical University as the study subjects. Genotypes of ITGA4 (rs6740847, rs7562325) and ICAM-1 (rs5498) were determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Harvey-Bradshaw Index (HBI) was applied to assess the disease activity of CD, and the patients were further divided into subgroups based on the Montreal Classification Criteria of CD. Unconditional logistic regression was employed to analyze the distribution of ITGA4 (rs6740847, rs7562325) and ICAM-1 (rs5498) polymorphisms between the patients and healthy controls and their association with the clinicopathological characteristics of the patients. RESULTS: The frequencies of T allele and CT+TT genotypes of ITGA4 (rs7562325) were higher in CD patients than the healthy controls (40.70% vs. 31.57%, P = 0.001; 62.79% vs. 54.36%, P = 0.042). The G variant and AG+GG genotypes of ITGA4 (rs6740847) were less common in patients with moderately to severely active CD compared with those with mildly active CD (31.18% vs. 51.72%, P = 0.002; 55.91% vs. 75.86%, P = 0.042). However, the opposite conclusion was drawn for the G allele (G) and AG+GG genotypes of ICAM-1 (rs5498) (31.45% vs. 17.24%, P = 0.027; 54.30% vs. 31.04%, P = 0.020). Compared with patients with terminal ileal or ileocolic CD, G allele and AG+GG genotypes of ITGA4 (rs6740847) were more prevalent in patients with colonic CD (55.26% vs. 29.38%, P < 0.001; 84.21% vs. 53.11%, P<0.001). The same conclusion could also be drawn for the G allele and AG+GG genotypes of ICAM-1 (rs5498) (42.11% vs. 26.84%, P = 0.008; 73.69% vs. 46.33%, P = 0.002). The frequency of homozygous GG genotype of ICAM-1 (rs5498) was lower in patients with stricturing and penetrating CD than those with non-stricturing and non-penetrating CD (0.00% vs. 12.32%, P = 0.001). The G allele and AG+GG genotypes of the ITGA4 (rs6740847) were more common in patients with perianal lesions than those without (40.28% vs. 30.77%, P = 0.049; 72.22% vs. 51.75%, P = 0.004). CONCLUSION: Variants of the ITGA4 (rs7562325) may be a risk factor for CD, whilst those of the ITGA4 (rs6740847) may be associated with the decline of disease activity and risk for colon involvement and perianal lesions. Variants of the ICAM-1 (rs5498) may increase the risk of disease activity and colonic involvement in CD patients, however, it may be a protective factor for stenosis and penetration. In addition, variants of the ITGA4 (rs6740847) and ICAM-1 (rs5498) may be associated with the early onset of CD.

Endothelial IL17RD promotes Western diet-induced aortic myeloid cell infiltration.

The Interleukin-17 (IL17) family is a group of cytokines implicated in the etiology of several inflammatory diseases. Interleukin-17 receptor D (IL17RD), also known as Sef (similar expression to fibroblast growth factor) belonging to the family of IL17 receptors, has been shown to modulate IL17A-associated inflammatory phenotypes. The objective of this study was to test the hypothesis that IL17RD promotes endothelial cell activation and consequent leukocyte adhesion. We utilized primary human aortic endothelial cells and demonstrated that RNAi targeting of IL17RD suppressed transcript levels by 83 % compared to non-targeted controls. Further, RNAi knockdown of IL17RD decreased the adhesion of THP-1 monocytic cells onto a monolayer of aortic endothelial cells in response to IL17A. Additionally, we determined that IL17A did not significantly enhance the activation of canonical MAPK and NFκB pathways in endothelial cells, and further did not significantly affect the expression of VCAM-1 and ICAM-1 in aortic endothelial cells, which is contrary to previous findings. We also determined the functional relevance of our findings in vivo by comparing the expression of endothelial VCAM-1 and ICAM-1 and leukocyte infiltration in the aorta in Western diet-fed Il17rd null versus wild-type mice. Our results showed that although Il17rd null mice do not have significant alteration in aortic expression of VCAM-1 and ICAM-1 in endothelial cells, they exhibit decreased accumulation of proinflammatory monocytes and neutrophils, suggesting that endothelial IL17RD induced in vivo myeloid cell accumulation is not dependent on upregulation of VCAM-1 and ICAM-1 expression. We further performed proteomics analysis to identify potential molecular mediators of the IL17A/IL17RD signaling axis. Collectively, our results underscore a critical role for Il17rd in the regulation of aortic myeloid cell infiltration in the context of Western diet feeding.

Annotation of CD8+ T-cell function via ICAM-1 imaging identifies FAK inhibition as an adjuvant to augment the antitumor immunity of radiotherapy.

Background: Radiotherapy (RT) may trigger systemic antitumor immunity, manifesting as regression of non-irradiated lesions (abscopal effect). Intracellular adhesion molecule-1 (ICAM-1) is a key molecule involved in the abscopal effect of RT. However, the specific function of ICAM-1 in CD8+ T cells during antitumor immune responses remains unclear. Herein, we investigated whether noninvasive imaging of ICAM-1 can be used to annotate CD8+ T-cell function, thereby better selecting combinational therapy to enhance the antitumor immunity induced by RT. Methods: Using knockout mouse models, we investigated the role of ICAM-1 expressed on CD8+ T cells in the antitumor immunity of RT and conducted drug screening guided by ICAM-1-targeted noninvasive imaging. Results: The systemic antitumor effect of RT relies on the expression of ICAM-1 on CD8+ T cells. ICAM-1 expression is essential for CD8+ T-cell activation, proliferation, and effector function. Noninvasive annotation of the proliferation and effector function of CD8+ T cells by ICAM-1-targeted imaging identified VS-6063, a focal adhesion kinase inhibitor, as a new adjuvant to augment systemic antitumor immunity of RT in an immunologically "cold" tumor model. Mechanistically, VS-6063 overcomes the physical barriers in tumors and promotes the migration and infiltration of CD8+ T cells primed by RT into distant tumors. Conclusion: Our findings highlight that molecular imaging of ICAM-1 levels provides a dynamic readout of the proliferation and effector function of tumor-infiltrating CD8+ T cells, which facilitates the high-throughput exploitation of new combinational drugs to maximize the systemic antitumor effect of RT.

Endoplasmic Reticulum Protein 72 Regulates Integrin Mac-1 Activity to Influence Neutrophil Recruitment.

BACKGROUND: Integrins mediate the adhesion, crawling, and migration of neutrophils during vascular inflammation. Thiol exchange is important in the regulation of integrin functions. ERp72 (endoplasmic reticulum-resident protein 72) is a member of the thiol isomerase family responsible for the catalysis of disulfide rearrangement. However, the role of ERp72 in the regulation of Mac-1 (integrin αMß2) on neutrophils remains elusive. METHODS: Intravital microscopy of the cremaster microcirculation was performed to determine in vivo neutrophil movement. Static adhesion, flow chamber, and flow cytometry were used to evaluate in vitro integrin functions. Confocal fluorescent microscopy and coimmunoprecipitation were utilized to characterize the interactions between ERp72 and Mac-1 on neutrophil surface. Cell-impermeable probes and mass spectrometry were used to label reactive thiols and identify target disulfide bonds during redox exchange. Biomembrane force probe was performed to quantitatively measure the binding affinity of Mac-1. A murine model of acute lung injury induced by lipopolysaccharide was utilized to evaluate neutrophil-associated vasculopathy. RESULTS: ERp72-deficient neutrophils exhibited increased rolling but decreased adhesion/crawling on inflamed venules in vivo and defective static adhesion in vitro. The defect was due to defective activation of integrin Mac-1 but not LFA-1 (lymphocyte function-associated antigen-1) using blocking or epitope-specific antibodies. ERp72 interacted with Mac-1 in lipid rafts on neutrophil surface leading to the reduction of the C654-C711 disulfide bond in the αM subunit that is critical for Mac-1 activation. Recombinant ERp72, via its catalytic motifs, increased the binding affinity of Mac-1 with ICAM-1 (intercellular adhesion molecule-1) and rescued the defective adhesion of ERp72-deficient neutrophils both in vitro and in vivo. Deletion of ERp72 in the bone marrow inhibited neutrophil infiltration, ameliorated tissue damage, and increased survival during murine acute lung injury. CONCLUSIONS: Extracellular ERp72 regulates integrin Mac-1 activity by catalyzing disulfide rearrangement on the αM subunit and may be a novel target for the treatment of neutrophil-associated vasculopathy.

Long-term abuse of caffeine sodium benzoate induces endothelial cells injury and leads to coagulation dysfunction.

Our hospital admitted a patient who had difficulty in coagulation even after blood replacement, and the patient had abused caffeine sodium benzoate (CSB) for more than 20 years. Hence, we aimed to explore whether CSB may cause dysfunction in vascular endothelial cells and its possible mechanism. Low, medium, and high concentrations of serum of long-term CSB intake patients were used to treat HUVECs, with LPS as the positive control. MTT and CCK8 were performed to verify CSB's damaging effect on HUVECs. The expression of ET-1, ICAM-1, VCAM-1, and E-selectin were measured by ELISA. TUNEL assay and Matrigel tube formation assay were carried out to detect apoptosis and angiogenesis of HUVECs. Flow cytometry was applied to analyze cell cycles and expression of CD11b, PDGF, and ICAM-1. Expression of PDGF-BB and PCNA were examined by western blot. The activation of MAPK signaling pathway was detected by qRT-PCR and western blot. Intracellular Ca2+ density was detected by fluorescent probes. CCK8 assay showed high concentration of CSB inhibited cell viability. Cell proliferation and angiogenesis were inhibited by CSB. ET-1, ICAM-1, VCAM-1, and E-selectin upregulated in CSB groups. CSB enhanced apoptosis of HUVECs. CD11b, ICAM-1 increased and PDGF reduced in CSB groups. The expression level and phosphorylation level of MEK, ERK, JUN, and p38 in MAPK pathway elevated in CSB groups. The expression of PCNA and PDGF-BB was suppressed by CSB. Intracellular Ca2+ intensity was increased by CSB. Abuse of CSB injured HUVECs and caused coagulation disorders.