Neutrophil Adhesion Molecules Research Articles

Neutrophil Activation Is a Diagnostic and Candidate Prognostic Biomarker of Acute GvHD

Background High-frequency longitudinal monitoring may help identifying GVHD and GVT and intercepting these events before their occurrence. Methods We implemented a targeted multiplex microfluidics q-PCR based transcriptional assay, able to measure the fluctuations of 264 transcripts (TFA, Chaussabel Nat Rev, 2014) with high frequency. This transcripts panel is not tailored to GVHD-known biomarkers, rather it is representative of a large selection of whole-blood transcripts, identified via co-expression analyses and annotated for function. Samples consist of 50 ul whole blood, self-collected by patients through fingerstick, and stored in an RNA-preservative solution ( Mathew, BMC Res Notes, 2020). Twenty-two patients were followed weekly from engraftment until 149 days after allogeneic hematopoietic stem cell transplantation (allo-HCT) (19-149, median 72) with this method in a discovery cohort (DC). Additionally, 55 patients were studied in an independent validation cohort (VC) with the same transcripts panel, but using Ficoll gradient-separated blood samples collected at fixed post allo-HCT time-points (days +14/+21/+28 and +100) and live-frozen. Transcriptome results were correlated with the event of acute GVHD (aGVHD), graded according to Glucksberg modified criteria, and further sub-classified as “active” when clearly measurable in a GVHD clinical score (not “silenced” by therapeutic intervention). In both cohorts, samples were analyzed by longitudinal statistical analyses, performed with 3 different Linear Mixed Models (LMM): Random Intercept, Random Slope and both Random Intercept+Slope. Additionally, each post allo-HCT outcome was studied by Functional PCA (FPCA) for individual transcripts. Results A total of 19 samples pre- and 234 post allo-HCT were suitable for analyses in the DC, (median 11 samples/pt; 4-18) and 32 samples pre- and 165 post allo-HCT in the VC (median 4 samples/pt; 1-5). Overall, 13 out of 22 patients in the DC and 29/55 in the VC developed grade 1-3 aGVHD. Patients without any sign of GVHD in 149 days (“Never GVHD”; n=85 in DC, n=72 in VC) were compared with patients having GVHD at any time point (“GVHD”; n=149 in DC, n=93 in VC) and with the subset of “active GVHD” samples within each cohort. As expected, different gene sets significantly changed over time in GVHD patients and in the active GVHD subset in the DC (including whole fresh blood cells and plasma) with respect to the VC (including Ficoll-processed cells). IFN-, cytotoxic T cell- and erythroid cells-related transcripts were mostly significant in the DC GVHD patients, while B cells and monocytes transcripts were more represented in the VC GVHD selected PBMCs. Cytokines and chemokines were represented in both cohorts, probably reflecting cellular and plasmatic RNAs in the DC and cells' internal RNA reservoirs in the VC. Most strikingly, only one set of transcripts, all related to neutrophil activation was robustly validated in both cohorts. CEACAM6 (CD66c) and CEACAM8 (CD66b) linked to the expression of neutrophil adhesion molecules, the defensin DEFA4 and the neutrophil elastase ELANE distinguished longitudinally the post allo-HCT outcome of patients affected by aGVHD of any grade. The FPCA analysis confirmed a clear distinction of aGVHD patients by these 4 genes (Figure 1). We studied then if this signature could prognosticate aGVHD. In the DC GVHD cohort, the earliest timepoint (tp2) pre-GVHD onset was available for 8 patients (median 22.5 days; 22-23). The same timepoint was available for 8 Never GVHD patients. CEACAM8 was significantly higher in the GVHD vs the Never GVHD cohort at this timepoint, thus preceding GVHD. Differently, in the VC the early day +14 timepoint was available for all patients and preceded the GVHD onset in all cases. All 4 neutrophil transcripts in this cohort were significantly higher in the GVHD vs the Never GVHD group. Thus, neutrophil activation anticipates the onset of GVHD and may be considered for future studies as a candidate aGVHD prognostic biomarker, especially in early post allo-HCT stage. In conclusion our generic blood immune profiling assay revealed that neutrophil activation is a robust diagnostic marker, consistently identifying aGVHD patients in their post-transplant course, regardless of the sample source (whole blood or Ficoll-separated PBMCs, fresh or frozen/thawed). Moreover, CEACAM8 (CD66b) may likely represent a prognostic biomarker of aGVHD.

Synergism between obesity and HFpEF on neutrophils phenotype and its regulation by adipose tissue-molecules and SGLT2i dapagliflozin.

The adiposity invokes innate immune activity, coronary microvascular dysfunction and consequently heart failure preserved ejection fraction (HFpEF). Our aim was to study the neutrophils profile on obesity and cardiovascular disease and its regulation by adipose tissue‐secretome and dapagliflozin. We have isolated neutrophils from patients undergoing open heart surgery (19 women and 51 men). Its migration activity was performed with culture‐transwell, transcriptional studies of proteolytic enzymes, adhesion molecules or receptors were analysed by real‐time PCR and proteomics (from 20 patients) analysis by TripleTOF mass spectrometer. Differentiated HL‐60 (dHL‐60) was used as a preclinical model on microfluidic for endothelial cells attaching assays and genes regulation with epicardial and subcutaneous fat secretomes from patients (3 women and 9 men) or dapagliflozin 1–10 μM treatments. The transcriptional and proteomics studies have determined higher levels of adhesion molecules in neutrophils from patients with obesity. The adhesion molecule CD11b levels were higher in those patients with the combined obesity and HFpEF factors (1.70 ± 0.06 a.u. without obesity, 1.72 ± 0.04 a.u. obesity or HFpEF without obesity and 1.79 ± 0.08 a.u. obesity and HFpEF; p < .01). While fat‐secretome induces its upregulation, dapagliflozin can modulated it. Because CD11b upregulation is associated with higher neutrophils migration and adhesion into endothelial cells, dapagliflozin might modulate this mechanism on patients with obesity and HFpEF.

Galectin-9 mediates neutrophil capture and adhesion in a CD44 and β2 integrin-dependent manner.

Neutrophil trafficking is a key component of the inflammatory response. Here, we have investigated the role of the immunomodulatory lectin Galectin-9 (Gal-9) on neutrophil recruitment. Our data indicate that Gal-9 is upregulated in the inflamed vasculature of RA synovial biopsies and report the release of Gal-9 into the extracellular environment following endothelial cell activation. siRNA knockdown of endothelial Gal-9 resulted in reduced neutrophil adhesion and neutrophil recruitment was significantly reduced in Gal-9knockout mice in a model of zymosan-induced peritonitis. We also provide evidence for Gal-9 binding sites on human neutrophils; Gal-9 binding induced neutrophil activation (increased expression of β2 integrins and reduced expression of CD62L). Intra-vital microscopy confirmed a pro-recruitment role for Gal-9, with increased numbers of transmigrated neutrophils following Gal-9 administration. We studied the role of both soluble and immobilized Gal-9 on human neutrophil recruitment. Soluble Gal-9significantly strengthened the interaction between neutrophils and the endothelium and inhibited neutrophil crawling on ICAM-1. When immobilized, Gal-9 functioned as an adhesion molecule and captured neutrophils from the flow. Neutrophils adherent to Gal-9 exhibited a spread/activated phenotype that was inhibited by CD18 and CD44 neutralizing antibodies, suggesting a role for these molecules in the pro-adhesive effects of Gal-9. Our data indicate that Gal-9 is expressed and released by the activated endothelium and functions both in soluble form and when immobilized as a neutrophil adhesion molecule. This study paves the way for further investigation of the role of Gal-9 in leukocyte recruitment in different inflammatory settings.

Sevoflurane Exerts Protective Effects in Murine Peritonitis-induced Sepsis via Hypoxia-inducible Factor 1α/Adenosine A2B Receptor Signaling

Sepsis is one of the leading causes of mortality in intensive care units, and sedation in the intensive care unit during sepsis is usually performed intravenously. The inhalative anesthetic sevoflurane has been shown to elicit protective effects in various inflammatory studies, but its role in peritonitis-induced sepsis remains elusive. The hypothesis was that sevoflurane controls the neutrophil infiltration by stabilization of hypoxia-inducible factor 1α and elevated adenosine A2B receptor expression. In mouse models of zymosan- and fecal-induced peritonitis, male mice were anesthetized with sevoflurane (2 volume percent, 30 min) after the onset of inflammation. Control animals received the solvent saline. The neutrophil counts and adhesion molecules on neutrophils in the peritoneal lavage of wild-type, adenosine A2B receptor -/-, and chimeric animals were determined by flow cytometry 4 h after stimulation. Cytokines and protein release were determined in the lavage. Further, the adenosine A2B receptor and its transcription factor hypoxia-inducible factor 1α were evaluated by real-time polymerase chain reaction and Western blot analysis 4 h after stimulation. Sevoflurane reduced the neutrophil counts in the peritoneal lavage (mean ± SD, 25 ± 17 × 105vs. 12 ± 7 × 105 neutrophils; P = 0.004; n = 19/17) by lower expression of various adhesion molecules on neutrophils of wild-type animals but not of adenosine A2B receptor -/- animals. The cytokines concentration (means ± SD, tumor necrosis factor α [pg/ml], 523 ± 227 vs. 281 ± 101; P = 0.002; n = 9/9) and protein extravasation (mean ± SD [mg/ml], 1.4 ± 0.3 vs. 0.8 ± 0.4; P = 0.002; n = 12/11) were also lower after sevoflurane only in the wild-type mice. Chimeric mice showed the required expression of the adenosine A2B receptor on the hematopoietic and nonhematopoietic compartments for the protective effects of the anesthetic. Sevoflurane induced the expression of hypoxia-inducible factor 1α and adenosine A2B receptor in the intestine, liver, and lung. Sevoflurane exerts various protective effects in two murine peritonitis-induced sepsis models. These protective effects were linked with a functional adenosine A2B receptor.

A New Diterpene and Anti-inflammatory Sesquiterpene Lactones from Sigesbeckia orientalis.

Sigesbeckia orientalis, more commonly referred to as Herba Sigesbeckiae or Xi Xian Cao in traditional Chinese medicine and hy thiêm in traditional Vietnamese medicine, is used in China and Vietnam to treat inflammatory diseases such as arthritis, rheumatism, and joint pain. In initial investigations, the dichloromethane extract from the aerial parts of S. orientalis showed distinct inhibitory effects on the release of interleukin-8 in human neutrophils. Therefore, the purpose of the present study was the phytochemical investigation of the bioactive dichloromethane extract and the in vitro analysis of the effects of the isolated compounds on interleukin-8, interleukin-1β, tumor necrosis factor-α, and monocyte chemoattractant protein 1 release, and surface expression of adhesion molecules (CD11a, CD11b, and CD62L) in lipopolysaccharide-stimulated human neutrophils to identify the active principle(s). The separation of the bioactive dichloromethane extract using various chromatographic techniques led to the isolation of nine compounds. Their chemical structures were elucidated from nuclear magnetic resonance and mass spectrometry data. One diterpene, 17(13 → 14)-abeo-ent-3S*,13S*,16-trihydroxystrob-8(15)-ene, was identified as a new natural product. Three germacranolide sesquiterpene lactones inhibited interleukin-8 production with IC50 values between 1.6 and 6.3 µM, respectively, and tumor necrosis factor-α production with IC50 values between 0.9 and 3.3 µM, respectively. Furthermore, they significantly inhibited interleukin-1β and monocyte chemoattractant protein 1 production and diminished the effects of lipopolysaccharide on the surface expression of the adhesion molecules CD11a, CD11b, and CD62L. These findings support the traditional use of S.orientalis in the treatment of inflammatory diseases.

Kinin B1 Receptor Is Important in the Pathogenesis of Myeloperoxidase-Specific ANCA GN.

Myeloperoxidase-specific ANCA (MPO-ANCA) are implicated in the pathogenesis of vasculitis and GN. Kinins play a major role during acute inflammation by regulating vasodilatation and vascular permeability and by modulating adhesion and migration of leukocytes. Kinin system activation occurs in patients with ANCA vasculitis. Previous studies in animal models of GN and sclerosing kidney diseases have demonstrated protective effects of bradykinin receptor 1 (B1R) blockade via interference with myeloid cell trafficking. To investigate the role of B1R in a murine model of MPO-ANCA GN, we evaluated effects of B1R genetic ablation and pharmacologic blockade. We used bone marrow chimeric mice to determine the role of B1R in bone marrow-derived cells (leukocytes) versus nonbone marrow-derived cells. We elucidated mechanisms of B1R effects using in vitro assays for MPO-ANCA-induced neutrophil activation, endothelial adherence, endothelial transmigration, and neutrophil adhesion molecule surface display. B1R deficiency or blockade prevented or markedly reduced ANCA-induced glomerular crescents, necrosis, and leukocyte influx in mice. B1R was not required for in vitro MPO-ANCA-induced neutrophil activation. Leukocyte B1R deficiency, but not endothelial B1R deficiency, decreased glomerular neutrophil infiltration induced by MPO-ANCA in vivo. B1R enhanced ANCA-induced neutrophil endothelial adhesion and transmigration in vitro. ANCA-activated neutrophils exhibited changes in Mac-1 and LFA-1, important regulators of neutrophil endothelial adhesion and transmigration: ANCA-activated neutrophils increased surface expression of Mac-1 and increased shedding of LFA-1, whereas B1R blockade reduced these effects. The leukocyte B1R plays a critical role in the pathogenesis of MPO-ANCA-induced GN in a mouse model by modulating neutrophil-endothelial interaction. B1R blockade may have potential as a therapy for ANCA GN and vasculitis.

Frontline Science: A flexible kink in the transmembrane domain impairs β2 integrin extension and cell arrest from rolling.

β2 integrins are the main adhesion molecules in neutrophils and other leukocytes and are rapidly activated by inside-out signaling, which results in conformational changes that are transmitted through the transmembrane domain (TMD). Here, we investigated the biologic effect of introducing a proline mutation in the β2 integrin TMD to create a flexible kink that uncouples the topology of the inner half of the TMD from the outer half and impairs integrin activation. The β2 integrin alpha chains, αL, αM, αX, and αD, all contain an inserted (I) domain with homology to von Willebrand factor A domain. β2 activation was monitored in a homogenous binding assay of 2reporter monoclonal antibodies: KIM127 reporting extension (E+ ) and mAb24 reporting the high-affinity (H+ ) conformation of the β2 I-like domain. The proline mutation partially diminished chemokine-induced extension, but not the high-affinity conformation. The proline mutation in the TMD of β2 completely inhibited arrest of rolling HL-60 cells in response to the chemokine IL-8. TMD mutant HL-60 cells rolling on P-selectin and ICAM-1 were unable to reduce their rolling velocity in response to IL-8. Quantitative dynamic footprinting live-cell imaging showed that blocking TMD topology transmission impaired the chemokine-induced activation of β2, limiting the appearance of extended high-affinity (E+ H+ ) β2. This also resulted in a defect in early spreading (3min after arrest), which could be overcome by forced integrin activation using Mn2+ . We conclude that the TMD proline mutation severely impairs β2 integrin extension, cell arrest, and early spreading.

Anti-Inflammatory and Neuroprotective Effects of DIPOPA (N,N-Diisopropyl-2-Oxopropanamide), an Ethyl Pyruvate Bioisoster, in the Postischemic Brain.

Ethyl pyruvate (EP) is a simple aliphatic ester of pyruvic acid and has been shown to have protective properties, which have been attributed to its anti-inflammatory, anti-oxidative, and anti-apoptotic functions. In an effort to develop better derivatives of EP, we previously synthesized DEOPA (N,N-diethyl-2-oxopropanamide, a novel isoster of EP) which has greater neuroprotective effects than EP, probably due to its anti-inflammatory and anti-excitotoxic effects. In the present study, we synthesized 3 DEOPA derivatives, in which its diethylamino group was substituted with diisopropylamino, dipropylamino, or diisobutylamino groups. Among them, DIPOPA (N,N-diisopropyl-2-oxopropanamide) containing diisopropylamino group had a greater neuroprotective effect than DEOPA or EP when administered intravenously to a rat middle cerebral artery occlusion (MCAO) model at 9h after MCAO. Furthermore, DIPOPA had a wider therapeutic window than DEOPA and a marked reduction of infarct volume was accompanied by greater neurological and behavioral improvements. In particular, DIPOPA exerted robust anti-inflammatory effects, as evidenced by marked suppressions of microglia activation and neutrophil infiltration in the MCAO model, in microglial cells, and in neutrophil-endothelial cocultures at lower concentration, and did so more effectively than DEOPA. In particular, DIPOPA remarkably suppressed neutrophil infiltration into brain parenchyma, and this effect was attributed to the expressional inhibitions of cell adhesion molecules in neutrophils of brain parenchyma and in circulating neutrophils via NF-κB inhibition. Together, these results indicate the robust neuroprotective effects of DIPOPA are attributable to its anti-inflammatory effects and suggest that DIPOPA offers a potential therapeutic means of ameliorating cerebral ischemic injury and other inflammation-related pathologies.

Inhibition of Pro-Inflammatory Functions of Human Neutrophils by Constituents of Melodorum fruticosum Leaves.

In an initial screening, the dichloromethane extract from the leaves of Melodorum fruticosum showed distinct inhibitory effects on the release of interleukin‐8 (IL‐8) in human neutrophils. Therefore, the aim of the present study was the phytochemical and pharmacological investigation of this extract, to better understand which compounds might be responsible for the anti‐inflammatory effect. Phytochemical analysis led to the isolation of 12 known compounds and two new natural products, 5‐hydroxy‐6‐(2‐hydroxybenzyl)‐4′,7‐dimethoxyflavanone (13) and 2′,4′‐dihydroxy‐3′‐(2‐hydroxybenzyl)‐4,6′‐dimethoxychalcone (14). The influence of the isolated compounds on the production and release of the pro‐inflammatory factors IL‐8, tumor necrosis factor alpha (TNF‐α), reactive oxygen species (ROS), and adhesion molecules (CD62L and CD11b) in human neutrophils was evaluated. Three constituents, melodamide A, 2′,4′‐dihydroxy‐4,6′‐dimethoxychalcone, and 2′,6′‐dihydroxy‐4′‐methoxychalcone, showed significant inhibition of IL‐8 release (IC50=6.6, 8.6, and 11.6 μm, respectively) and TNF‐α production (IC50=4.5, 13.3, and 6.2 μm, respectively).

Distinct neutrophil counts and functions in newly diagnosed type 1 diabetes, latent autoimmune diabetes in adults, and type 2 diabetes

Recent discoveries from animal models demonstrated that neutrophils can induce type 1 diabetes (T1D) through infiltrating into the islets. However, the evidence of their actions in T1D patients is relatively rare, and the change trend of neutrophil numbers and functions in different subtypes of diabetes has not been investigated. Patients with newly diagnosed T1D (n=189), latent autoimmune diabetes in adults (LADA) (n=86), T2D (n=235), and healthy controls (n=709) were enrolled. Circulating neutrophil counts were measured, and their correlations with clinical parameters were analysed. Neutrophils were isolated by density gradient centrifugation and magnetic bead cell sorting method. Neutrophil migration rate and chemokine levels in the blood were explored by trans-well and ELISA, respectively. Neutrophil phagocytosis rate, adhesion molecules and chemokine receptors expression were investigated by flow cytometry. Compared with controls, neutrophil counts decreased in T1D patients but increased in T2D patients, with no change in LADA patients. The numbers showed a gradual increase trend from T1D, LADA to T2D. In autoimmune diabetes, neutrophil counts were associated with the number and titre of positive autoantibodies against β-cell antigens. No difference was found in neutrophil phagocytosis rate, but neutrophil migration in T1D patients was impaired and associated with CD62L expression, which was related closely to the titre of autoantibody. Neutrophil numbers and migration abilities displayed distinct levels in different types of diabetes. In T1D, CD62L seems to play an important role in the migration of neutrophils and β-cell autoimmunity.

Neutrophil Migration and Adhesion Molecule Expression after Acute High-Intensity Street Dance Exercise.

The physical demands of street dancing may result in inflammation and changes in leukocyte numbers/function, impairing the health of dancers. Herein, we investigated the effect of street dancing on inflammation, adhesion molecules, and neutrophil function. Fifteen amateur dancers (mean ± SE: age 22.4 ± 1.08 years, BMI 24.8 ± 0.69 kg/m2, and body fat 12.3 ± 1.52%) participated in a single high-intensity street dance class. Blood samples were taken before and after the class. The dance class had no effect on the plasma concentration of CRP, TNF-α, IL-6, IL-10, and IL-8; however, we noted an increase in levels of IL-1β (4.06%) and sL-selectin (17.67%). The dance class resulted in a 12.36% increase in neutrophil counts, while neutrophil CD62L expression and migration were reduced (25.27% and 78.92%, resp.). After the dance class, neutrophil production of IL-8 and TNF-α increased, respectively, by 59.75% and 49.23%, in the control condition, and 43.55% and 32.22%, after LPS stimulation. A single bout of street dancing induced inflammation and reduced neutrophil migration and adhesion molecule expression. These findings may contribute to a better understanding of the susceptibility to infection after acute dance exercise.

Toll-Like Receptor 3 Signaling Contributes to Regional Neutrophil Recruitment in Cultured Human Glomerular Endothelial Cells

Background: Given the importance of neutrophil recruitment in the pathogenesis of glomerulonephritis (GN), the representative neutrophil chemoattractant C-X-C motif chemokine 1 (CXCL1)/GROα and the adhesion molecule E-selectin in glomerular endothelial cells (GECs) play a pivotal role in the development of GN. Endothelial Toll-like receptor 3 (TLR3) is thought to be involved in the inflammatory response via innate immunity. However, the role of endothelial TLR3 signaling in the expression of neutrophil chemoattractants and adhesion molecules remains to be elucidated. Thus, we aimed to examine this issue. Methods: We treated normal human GECs with polyinosinic-polycytidylic acid (poly IC), an authentic double-stranded RNA, and analyzed the expressions of CXCL1 and E-selectin using quantitative real-time reverse transcription-polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay. To further elucidate the poly IC-induced signaling pathway, we subjected the cells to RNA interference against TLR3, interferon (IFN)-β, nuclear factor (NF)-κB p65, and IFN regulatory factor (IRF) 3. We also used immunofluorescence to examine the endothelial expression of CXCL1 in biopsy specimens from patients with crescentic and non-crescentic purpura nephritis (PN). Results: We found that the activation of TLR3 induced the endothelial expression of CXCL1 and E-selectin, and that this involved TLR3, ­NF-κB, IRF3, and IFN-β. Intense endothelial CXCL1 expression was observed in biopsy specimens from patients with crescentic PN. Conclusion: These findings support a role for glomerular antiviral innate immunity in the pathogenesis of GN. Intervention of glomerular TLR3 signaling may therefore be a suitable therapeutic strategy for treating GN in the future.

PDE4 Inhibitor Rolipram Mitigates Liver Ischemia Reperfusion Injury in a Rat Model by Preventing Neutrophil Accumulation

BACKGROUND/INTRODUCTIONIschemia‐reperfusion (IR) injury is caused by a transient loss of blood flow (ischemia) followed by return of blood flow (reperfusion), which initiates an inflammatory response. IR injury occurs in organ transplantation, sepsis, respiratory failure, hemorrhage, trauma, stroke, cardiac arrest, and shock. Ischemia reperfusion, as a primary cause of post graft non‐function, is a limiting factor in the success of liver transplantation. Due to a crucial shortage of donor livers, there are about 17,000 people on the waiting list for a liver transplant, and 1,500 a year will die while waiting for an organ (American Liver Foundation). Pharmacological attenuation of the IR‐induced inflammatory response could improve graft function and survival. We investigated the potential of using the phosphodiesterase (PDE) inhibitor, rolipram, in hepatic ischemia reperfusion injury. PDEs are the only enzymes that can hydrolyze cyclic adenosine monophosphate (cAMP). Higher cAMP levels can initiate and sustain anti‐inflammatory signaling. Therefore, we hypothesized that suppression of the inflammatory response by rolipram would reduce liver damage following IR injury.METHODSMale Sprague‐Daley rats were randomized to six groups (n=6–8 per group): 1) sham, 2) sham + 3 mg/kg rolipram, 3) 40% ischemia reperfusion, 4) 40% ischemia reperfusion + 3 mg/kg rolipram, 5) 70% ischemia reperfusion, and 6) 70% ischemia reperfusion + 3 mg/kg rolipram. Two different levels of severity of IR injury were achieved by clamping either 40% (left lobe) or 70% (left and median lobes) of the liver. In both cases, occlusion was for 1 hour followed by 3 hours of reperfusion. Treatment was given I.V. at the end of the ischemic period. Blood and tissue were collected. Blood chemistry was assayed on a Vetscan 2 (Abaxis). Paraffin embedded liver sections were stained for H&amp;E (morphology), intercellular adhesion molecule 1 (ICAM‐1) (neutrophil adhesion molecule), and chloroacetate esterase (CAE, stains neutrophils). Liver myeloperoxidase (MPO, from neutrophils) and tumor necrosis factor alpha (TNF‐α) and serum hyaluronate (a marker of vascular damage) were assessed by ELISA.RESULTSBlood chemistry revealed that IR significantly (p=0.01) elevated serum alanine aminotransferase (ALT), an indicator of liver damage, in both the 40% and 70% models. Rolipram lowered serum ALT levels in the 40% model. Inflammation‐induced neutrophil infiltration is a major source of IR damage. Histology showed areas of coagulative necrosis and increased neutrophil infiltration, which were ameliorated by rolipram, in both 40% and 70% models. Liver ICAM‐1 was reduced by rolipram treatment in both models. MPO was significantly increased by 70% but not 40% IR, and rolipram did not lower it. Liver TNF‐α showed no difference between sham and IR groups, but rolipram seemed to lower it independently of IR. Rolipram treatment showed a trend towards decreasing serum hyaluronan in both models.CONCLUSIONMechanisms still remain to be determined, but overall, these data suggest that inhibition of PDE reduces hepatic IR injury by decreasing neutrophil recruitment. We conclude that rolipram might have therapeutic potential in ischemia reperfusion injury.Support or Funding InformationSupported by grants from NIH and Veterans AdministrationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Novel anti-tumour necrosis factor receptor-1 (TNFR1) domain antibody prevents pulmonary inflammation in experimental acute lung injury

BackgroundTumour necrosis factor alpha (TNF-α) is a pleiotropic cytokine with both injurious and protective functions, which are thought to diverge at the level of its two cell surface receptors, TNFR1...

GRPR antagonist protects from drug-induced liver injury by impairing neutrophil chemotaxis and motility.

Drug-induced liver injury (DILI) is a major cause of acute liver failure (ALF), where hepatocyte necrotic products trigger liver inflammation, release of CXC chemokine receptor 2 (CXCR2) ligands (IL-8) and other neutrophil chemotactic molecules. Liver infiltration by neutrophils is a major cause of the life-threatening tissue damage that ensues. A GRPR (gastrin-releasing peptide receptor) antagonist impairs IL-8-induced neutrophil chemotaxis in vitro. We investigated its potential to reduce acetaminophen-induced ALF, neutrophil migration, and mechanisms underlying this phenomenon. We found that acetaminophen-overdosed mice treated with GRPR antagonist had reduced DILI and neutrophil infiltration in the liver. Intravital imaging and cell tracking analysis revealed reduced neutrophil mobility within the liver. Surprisingly, GRPR antagonist inhibited CXCL2-induced migration in vivo, decreasing neutrophil activation through CD11b and CD62L modulation. Additionally, this compound decreased CXCL8-driven neutrophil chemotaxis in vitro independently of CXCR2 internalization, induced activation of MAPKs (p38 and ERK1/2) and downregulation of neutrophil adhesion molecules CD11b and CD66b. In silico analysis revealed direct binding of GRPR antagonist and CXCL8 to the same binding spot in CXCR2. These findings indicate a new potential use for GRPR antagonist for treatment of DILI through a mechanism involving adhesion molecule modulation and possible direct binding to CXCR2.

Acute Respiratory Distress Syndrome Neutrophils Have a Distinct Phenotype and Are Resistant to Phosphoinositide 3-Kinase Inhibition.

Acute respiratory distress syndrome is refractory to pharmacological intervention. Inappropriate activation of alveolar neutrophils is believed to underpin this disease's complex pathophysiology, yet these cells have been little studied. To examine the functional and transcriptional profiles of patient blood and alveolar neutrophils compared with healthy volunteer cells, and to define their sensitivity to phosphoinositide 3-kinase inhibition. Twenty-three ventilated patients underwent bronchoalveolar lavage. Alveolar and blood neutrophil apoptosis, phagocytosis, and adhesion molecules were quantified by flow cytometry, and oxidase responses were quantified by chemiluminescence. Cytokine and transcriptional profiling were used in multiplex and GeneChip arrays. Patient blood and alveolar neutrophils were distinct from healthy circulating cells, with increased CD11b and reduced CD62L expression, delayed constitutive apoptosis, and primed oxidase responses. Incubating control cells with disease bronchoalveolar lavage recapitulated the aberrant functional phenotype, and this could be reversed by phosphoinositide 3-kinase inhibitors. In contrast, the prosurvival phenotype of patient cells was resistant to phosphoinositide 3-kinase inhibition. RNA transcriptomic analysis revealed modified immune, cytoskeletal, and cell death pathways in patient cells, aligning closely to sepsis and burns datasets but not to phosphoinositide 3-kinase signatures. Acute respiratory distress syndrome blood and alveolar neutrophils display a distinct primed prosurvival profile and transcriptional signature. The enhanced respiratory burst was phosphoinositide 3-kinase-dependent but delayed apoptosis and the altered transcriptional profile were not. These unexpected findings cast doubt over the utility of phosphoinositide 3-kinase inhibition in acute respiratory distress syndrome and highlight the importance of evaluating novel therapeutic strategies in patient-derived cells.

Inhaled corticosteroids increase blood neutrophil count by decreasing the expression of neutrophil adhesion molecules Mac-1 and L-selectin

ObjectiveThe objective was to investigate the effect of commonly used inhaled corticosteroids on white blood cell count (WBC) and to examine the mechanisms involved. MethodsThis randomized comparative study comprised 60 healthy adults. We measured the effects of budesonide (by face mask inhalation or aerosol inhaler), fluticasone (by inhaler), and saline inhalation (control) on WBC and the differential leukocyte count, especially the absolute neutrophil count (ANC). To elucidate the mechanisms involved, we measured the expression of the adhesion neutrophil ligands Mac-1 (CD11b) and L-selectin (CD62L), and granulocyte colony-stimulating factor serum levels. ResultsSix hours after a single-dose inhalation of budesonide, mean increases of 23.4% in WBC (95% confidence interval [CI], 11.3-35.4) and 30.1% in ANC (95% CI, 7.2-53.0) were noted. The percentage of neutrophils increased from 54.6% to 58.1% (P< .001). Inhaled fluticasone increased WBC and ANC by 12.6% (95% CI, 1.5-23.7) and 22.7% (95% CI, 6.2-39.2), respectively (P< .01 for both). The absolute lymphocyte and eosinophil counts did not change significantly from baseline.The expression of Mac-1 and L-selectin decreased by 51.0% (P< .01) and 30.9% (P= .02), respectively, following face mask inhalation of budesonide and by 39.8% (P= .01) and 17.4% (P= .17), respectively, following inhalation of fluticasone. No significant changes in granulocyte colony-stimulating factor levels were noted. ConclusionsGlucocorticoid inhalation increases WBC by increasing ANC. Reduced neutrophil adhesion to the endothelial surface, mediated by decreased adhesion molecule expression on neutrophils, is a plausible mechanism. Physicians should be aware of the effect of inhaled corticosteroids on WBC, as it may influence clinical decisions, especially in the emergency department.

The role of MyD88- and TRIF-dependent signaling in monophosphoryl lipid A-induced expansion and recruitment of innate immunocytes.

Treatment with the TLR4 agonist MPLA augments innate resistance to common bacterial pathogens. However, the cellular and molecular mechanisms by which MPLA augments innate immunocyte functions are not well characterized. This study examined the importance of MyD88- and TRIF-dependent signaling for leukocyte mobilization, recruitment, and activation following administration of MPLA. MPLA potently induced MyD88- and TRIF-dependent signaling. A single injection of MPLA caused rapid mobilization and recruitment of neutrophils, a response that was largely mediated by the chemokines CXCL1 and -2 and the hemopoietic factor G-CSF. Rapid neutrophil recruitment and chemokine production were regulated by both pathways although the MyD88-dependent pathway showed some predominance. In further studies, multiple injections of MPLA potently induced mobilization and recruitment of neutrophils and monocytes. Neutrophil recruitment after multiple injections of MPLA was reliant on MyD88-dependent signaling, but effective monocyte recruitment required activation of both pathways. MPLA treatment induced expansion of myeloid progenitors in bone marrow and upregulation of CD11b and shedding of L-selectin by neutrophils, all of which were attenuated in MyD88- and TRIF-deficient mice. These results show that MPLA-induced neutrophil and monocyte recruitment, expansion of bone marrow progenitors and augmentation of neutrophil adhesion molecule expression are regulated by both the MyD88- and TRIF-dependent pathways.

Sex-specific regulation of chemokine Cxcl5/6 controls neutrophil recruitment and tissue injury in acute inflammatory states.

BackgroundTissue infiltration by neutrophils during acute inflammatory states causes substantial tissue injury. While the magnitude of tissue neutrophil accumulation in innate immune responses is profoundly greater in males than females, fundamental aspects of the molecular mechanisms underlying these sex differences remain largely unknown.MethodsWe investigated sex differences in neutrophil stimulation and recruitment in ischemia/reperfusion (I/R; mesenteric or renal) or carrageenan pleurisy in rats or mice, as well as skin injury in human volunteers. The induction of potent chemoattractive mediators (chemokines) and neutrophil adhesion molecules were measured by real-time PCR, flow cytometry, and protein assays.ResultsMesenteric I/R in age-matched Wistar rats resulted in substantially more neutrophil accumulation and tissue injury at 2 h reperfusion in males than females. Using intravital microscopy, we show that the immediate (<30 min) neutrophil response to I/R is similar in males and females but that prolonged neutrophil recruitment occurs in males at sites local and distal to inflammatory insult partly due to an increase in circulating neutrophil populations with elevated surface expression of adhesion molecules. Sex differences in neutrophil kinetics were correlated with sustained induction of chemokine Cxcl5 in the tissue, circulation, and bone marrow of males but not females. Furthermore, blockade of Cxcl5 in males prior to ischemia resulted in neutrophil responses that were similar in magnitude to those in females. Conversely, administration of Cxcl5 to males in the absence of I/R was sufficient to increase levels of systemic neutrophils. Cxcl5 treatment of bone marrow neutrophils in vitro caused substantial induction of neutrophil-mobilizing cytokine granulocyte colony-stimulating factor (GCSF) and expression of β2 integrin that accounts for sexual dimorphism in circulating neutrophil populations in I/R. Moreover, male Cxcl5-stimulated bone marrow neutrophils had an increased capacity to adhere to β2 integrin ligand ICAM-1, implicating a greater sensitivity of male leukocytes to Cxcl5-mediated activation. Differential induction of Cxcl5 (human CXCL6) between the sexes was also evident in murine renal I/R, rat pleurisy, and human skin blisters and correlated with the magnitude of neutrophil accumulation in tissues.ConclusionsOur study reveals that sex-specific induction of chemokine Cxcl5/CXCL6 contributes to sexual dimorphism in neutrophil recruitment in diverse acute inflammatory responses partly due to increased stimulation and trafficking of bone marrow neutrophils in males.Electronic supplementary materialThe online version of this article (doi:10.1186/s13293-015-0047-5) contains supplementary material, which is available to authorized users.

Apelin attenuates postburn sepsis via a phosphatidylinositol 3-kinase/protein kinase B dependent mechanism: A randomized animal study

IntroductionThis study aims to investigate whether apelin would regulate inflammatory response and promote survival in an experimental burn sepsis model through a phosphatidylinositol 3-kinase/protein kinase B dependent pathway. MethodsMale BALB/c mice were divided into the following groups: sham, burn, burn sepsis, burn sepsis treated with apelin, burn sepsis treated with apelin plus LY294002, and burn sepsis treated with LY294002 alone. Apelin level and inflammatory cytokines in serum were detected by enzyme-linked immuno sorbent assay. Apelin/APJ (apelin receptor, gene symbol APLNR) mRNA expression in spleen and adhesion molecules levels in lung was detected by real-time polymerase chain reaction. Neutrophil infiltration in lung was determined by myeloperoxidase assay. Phosphorylation of protein kinase B in lung was determined by western blot. Mortality rate was monitored. ResultsBurn sepsis induced decreased apelin/APJ mRNA expression in spleen and reduced apelin level in plasma, which were both restored by exogenous apelin treatment. Burn sepsis treated with apelin resulted in decreased interleukin-6, tumor-necrosis factor-alpha, interleukin -1β and monocyte chemotactic protein-1 levels in plasma. Mice with apelin treatment also showed decreased neutrophil infiltration and adhesion molecules expression, accompanied by a remarkable increased protein kinase B phosphorylation in lung tissue. The mortality rate in apelin treated animals was also significantly reduced. Importantly, the above effects of apelin were abolished by LY294002 treatment. ConclusionApelin regulates inflammatory response, diminishes inflammatory remote organ damage and improves survival in an experimental model of burn sepsis, which is at least partly mediated by a phosphatidylinositol 3-kinase/protein kinase B dependent pathway.