Anti-inflammatory iron chelator, DIBI, reduces leukocyte-endothelial adhesion and clinical symptoms of LPS-induced interstitial cystitis in mice

BACKGROUND: Interstitial cystitis (IC) is a prevalent and debilitating chronic inflammatory disease of the urinary bladder. Currently there are no fully effective therapeutic agents available, in part due to the still obscure pathogenesis of IC. Lipopolysaccharide (LPS) also known as endotoxin from Gram negative bacteria elicits IC in mice and has formed the basis of model systems for investigation. Excess free iron plays an important role in inflammation through generation of reactive oxygen species (ROS). The novel iron chelator DIBI has been shown to sequester excess free iron and dampen excess inflammatory responses to systemic LPS administration and also to Gram negative bacterial infections. OBJECTIVE: The overall objective of this study was to evaluate the effects of DIBI on LPS induced IC in mice. Leukocyte activation, endothelial adhesion and functional capillary density were assessed by intravital microscopy of the bladder microcirculation following a single intravesical LPS administration with or without intravesical DIBI treatment. Clinical IC symptoms were also assessed through behavioral and pain threshold force measurements. METHODS: Four groups of female BALB/c mice (n = 5–6/group) were randomized in this study: control group, IC group without therapy, IC group with DIBI therapy and control group with DIBI therapy. The groups were examined using intravital microscopy (IVM) of the bladder for leukocyte-endothelial interactions (adherent leukocytes, temporarily interacting leukocytes) and functional capillary density (FCD). A modified behavioral score by Boucher et al. and Von-Frey-Aesthesiometry were used to evaluate key behavioral indices related to pain and visceral pain perception. RESULTS: LPS introduced intravesically induced an early (≤2h) inflammation of the bladder evidenced by leukocyte activation and adhesion to bladder capillary walls. Intravesical DIBI therapy of mice 30min following LPS administration and assessed after 1.5h treatment showed a significant decrease in the number of adherent leukocytes compared to IC animals without DIBI treatment. DIBI treated mice showed a significantly lowered increase in behavioral distress scores compared to IC mice without therapy. Untreated IC mice exhibited a significantly decreased threshold force value for evoked pain response and DIBI treatment improved the threshold pain response. A significant inverse correlation was found for the two pain and suffering evaluation methods results. CONCLUSION: DIBI reduced inflammatory endothelial leukocyte adhesion and key indices related to pain and suffering over those observed in untreated IC mice. Our findings suggest a potential therapeutic role for DIBI for IC treatment.

Inhibition of Factor XII Attenuates Prothrombotic Complications in Sickle Cell Mice

Sickle Cell Disease (SCD) is the most common inherited hemoglobinopathy, affecting millions worldwide. Although characterized by chronic hemolytic anemia and recurrent vaso-occlusive episodes, SCD is increasingly recognized as a hypercoagulable state. Indeed, SCD patients have an 11-25% incidence of venous thromboembolism at a median age of 30 years, associated with a 3-fold increased risk of mortality. Moreover, ischemic stroke and silent cerebral infarctions occur in 7-13% of SCD patients. We have previously shown that tissue factor, an initiator of the extrinsic coagulation pathway, contributes to thrombo-inflammation and microvascular cerebral thrombosis in mouse models of SCD . Recently, the intrinsic coagulation pathway, including Factor XII (FXII), has received significant attention because targeting components of this pathway reduces thrombosis without affecting primary hemostasis. We have shown that FXII deficiency reduces plasma markers of thrombin generation and inflammation in sickle mice. However, the contribution of FXII to thrombosis and prothrombotic complications in SCD is not known. In this study we evaluated the effects of blocking FXII activity on venous thrombosis and ischemia/reperfusion (IR)-induced brain injury in SCD mice. First, Townes HbSS mice (SS) and non-sickle Townes HbAA controls (AA) (male and female, 16 weeks) received anti-FXII antibody or control IgGκ1 (10 mg/kg, IV) 30 minutes prior to subjecting them to venous thrombosis, initiated by applying positive current (3 volts, 90 sec) to the femoral vein. To visualize platelet and fibrin accumulation, mice were injected with rhodamine 6G and anti-fibrin antibody 59D8 labeled with Alexa Fluor 647, respectively. The femoral vein thrombi were imaged by intravital fluorescence microscopy using time-lapse capture every 10 seconds, to acquire images of fibrin and platelets over 60 min. The accumulation of platelets and fibrin was quantified for relative intensity of each fluorophore over the region of the observed thrombus. As previously shown, thrombi of SS/IgG mice showed an increased fibrin and platelet accumulation compared to AA/IgG group. Importantly, 15D10 treatment significantly attenuated both fibrin (p<0.001) and platelet (p<0.05) deposition over time in SS mice compared to SS/IgG group. The same effect of 15D10 treatment was observed in AA mice. At the end of experiment, clots were collected and stained with hematoxylin and eosin, and clot volume was assessed histomorphometrically (Nikon Ti-2, FIJI Software). Surprisingly, despite higher fibrin content, clots from SS/IgG mice had significantly smaller volume than clots from AA/IgG group (0.32 ± 0.04 versus 0.60 ± 0.11 mm 3, p<0.05). Importantly, administration of 15D10 significantly reduced clot volume in both SS (0.086 ± 0.01 mm 3, p<0.05) and AA mice (0.1 ± 0.02 mm 3, p<0.05). Next, AA and SS mice (male and female, 8-10 weeks) were subjected to brain IR injury induced by middle cerebral artery occlusion for 60 minutes followed by 24 hours of reperfusion (mouse model of ischemic stroke). 15D10 or control IgGκ1 (10 mg/kg, IV) were injected 30 minutes before occlusion and again at 6 hours into the reperfusion period to generate 3 experimental groups: AA/IgG, SS/IgG and SS/15D10. All analyzed parameters of brain IR injury were significantly worse in the SS/IgG group compared to the AA/IgG group. Compared to IgG, pre-treatment of SS mice with 15D10 significantly attenuated neuronal damage determined by volume of brain infarction (11.7 ± 3.7 vs 24.9 ± 2.4%, p<0.001) and improved behavioral deficit assessed by mean stroke score (9.0 ± 0.9 vs 14.6 ± 0.9, p<0.01). These changes were accompanied by a significant increase in leukocytes rolling (1978.0 ± 93.5 vs 1517.0 ± 180.3 rolling leukocytes/sec/mm 2, p<0.001), and significant reduction in the number of adherent leukocytes (367.2 ± 49.0 vs 723.4 ± 48.5, adherent leukocytes/mm 2, p<0.001) observed in the brain microvasculature of SS mice treated with 15D10 compared to SS/IgG group. Together, our data indicates that in the mouse model of SCD FXII contributes to the experimental venous thrombosis and ischemic stroke. Given that targeting the intrinsic pathway can reduce thrombosis without affecting hemostasis, our data suggest that targeting FXII might be a beneficial treatment in reducing inflammatory and thrombotic complications in SCD patients without a risk of bleeding. Disclosures Wallisch: Aronora Inc,: Current Employment. Key: Grifols: Research Funding; Takeda: Research Funding; BioMarin: Honoraria, Other: Participation as a clinical trial investigator; Sanofi: Consultancy; Uniqure: Consultancy, Other: Participation as a clinical trial investigator. Gruber: Aronora Inc.: Current Employment, Current equity holder in publicly-traded company; Oregon Health and Science University: Current Employment.

Platelet-Adherent Leukocytes Associated With Cutaneous Cross-Reactive Hypersensitivity to Nonsteroidal Anti-Inflammatory Drugs

Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most highly consumed drugs worldwide and the main triggers of drug hypersensitivity reactions. The most frequent reaction, named cross-reactive NSAID-hypersensitivity, is due to the pharmacological activity of these drugs by blocking the cyclooxygenase-1 enzyme. Such inhibition leads to cysteinyl-leukotriene synthesis, mainly LTE4, which are responsible for the reaction. Although the complete molecular picture of the underlying mechanisms remains elusive, the participation of platelet-adherent leukocytes (CD61+) and integrins have been described for NSAID-exacerbated respiratory disease (NERD). However, there is a lack of information concerning NSAID-induced urticaria/angioedema (NIUA), by far the most frequent clinical phenotype. Here we have evaluated the potential role of CD61+ leukocytes and integrins (CD18, CD11a, CD11b, and CD11c) in patients with NIUA, and included the other two phenotypes with cutaneous involvement, NSAID-exacerbated cutaneous disease (NECD) and blended reactions (simultaneous skin and airways involvement). A group NSAID-tolerant individuals was also included. During the acute phase of the reaction, the three clinical phenotypes showed increased frequencies of CD61+ neutrophils, eosinophils, and monocytes compared to controls, which correlated with urinary LTE4 levels. However, no correlation was found between these variables at basal state. Furthermore, increased expressions of CD18 and CD11a were found in the three CD61+ leukocytes subsets in NIUA, NECD and blended reactions during the acute phase when compared with CD61−leukocyte subpopulations. During the acute phase, CD61+ neutrophils, eosinophils and monocytes showed increased CD18 and CD11a expression when compared with CD61+ leukocytes at basal state. No differences were found when comparing controls and CD61+ leukocytes at basal state. Our results support the participation of platelet-adherent leukocytes and integrins in cutaneous cross-hypersensitivity to NSAIDs and provide a link between these cells and arachidonic acid metabolism. Our findings also suggest that these reactions do not involve a systemic imbalance in the frequency of CD61+ cells/integrin expression or levels of LTE4, which represents a substantial difference to NERD. Although further studies are needed, our results shed light on the molecular basis of cutaneous cross-reactive NSAID-hypersensitivity, providing potential targets for therapy through the inhibition of platelet-leukocyte interactions.

Noninvasive Microscopic Imaging Reveals Increased Leukocyte Adhesion and Rolling in Skin of Acute Graft-Versus-Host Disease Patients Compared to Post-Transplant Controls

BACKGROUND. The nature and kinetics of leukocyte migration are intimately connected to the pathophysiology of acute graft-versus-host disease (aGVHD). Real-time visualization of leukocyte-endothelial interactions in patients post hematopoietic transplantation (HCT) may augment clinical diagnosis and patient management. In this cross-sectional pilot study, we aimed to test the feasibility of parameters characteristic of leukocyte motion in skin capillaries as potential imaging biomarkers to detect aGVHD. STUDY POPULATION. We enrolled 16 post-HCT patients: 8 patients with any organ aGVHD and 8 patients with no organ aGVHD on the day of the imaging. Diagnoses were retrospectively confirmed on day 100 by a transplant physician (MB), who was blinded to the results of the confocal imaging. 7 out of 8 aGVHD patients had only skin involvement: stage 1 (N=2 patients), stage 2 (N=2), and stage 3 (N=3) cutaneous aGVHD, whereas one patient had stage 1 gut aGVHD with no skin involvement. 6 out of 8 aGVHD group patients required systemic therapy with steroids. METHODS. To noninvasively visualize leukocyte motion in the topmost capillaries of post-HCT patients' skin, we used a clinical reflectance confocal microscope (Vivascope 1500, Caliber I.D.). It enables real-time en face view of individual cells at 9 frames per second. We took twenty 30-second videos of different capillaries in the left volar forearm and left upper chest. We counted the number of adherent and rolling leukocytes per 10 minutes of videos per patient. RESULTS. We found increased leukocyte rolling in the aGVHD group (average of 3 rolling leukocytes per patient), compared to post-HCT controls (average of 1 rolling leukocyte). Highest leukocyte rolling (>8 rolling leukocytes) was associated with increased all-cause mortality, but not predictive of transplant-related mortality (TRM). Similarly, we found increased leukocyte adhesion in the aGVHD group (average of 3 adherent leukocytes per patient), compared to post-HCT controls (average of 1 adherent leukocyte). Interestingly, we observed 7 adherent leukocytes in one patient who had isolated gut aGVHD and no skin involvement. DISCUSSION. Our preliminary results show altered leukocyte-endothelial interactions in the skin capillaries of aGVHD patients, suggesting that confocal microscopy may be an important tool in augmenting the clinical diagnosis and managing post-HCT patients. Future studies should test whether there is a difference in the pattern of change in parameter values between patients who did and who did not develop aGVHD. ACKNOWLEDGEMENT. This work was supported in part by Career Development Award Number IK2 CX001785 from the United Sates Department of Veterans Affairs Clinical Science R&D (CSRD) Service. Disclosures Byrne: Karyopharm: Research Funding. Jagasia:Janssen: Research Funding; Kadmon: Consultancy; Incyte: Consultancy. Tkaczyk:Incyte: Consultancy.

Neutrophils promote venular thrombosis by shaping the rheological environment for platelet aggregation

In advanced inflammatory disease, microvascular thrombosis leads to the interruption of blood supply and provokes ischemic tissue injury. Recently, intravascularly adherent leukocytes have been reported to shape the blood flow in their immediate vascular environment. Whether these rheological effects are relevant for microvascular thrombogenesis remains elusive. Employing multi-channel in vivo microscopy, analyses in microfluidic devices, and computational modeling, we identified a previously unanticipated role of leukocytes for microvascular clot formation in inflamed tissue. For this purpose, neutrophils adhere at distinct sites in the microvasculature where these immune cells effectively promote thrombosis by shaping the rheological environment for platelet aggregation. In contrast to larger (lower-shear) vessels, this process in high-shear microvessels does not require fibrin generation or extracellular trap formation, but involves GPIbα-vWF and CD40-CD40L-dependent platelet interactions. Conversely, interference with these cellular interactions substantially compromises microvascular clotting. Thus, leukocytes shape the rheological environment in the inflamed venular microvasculature for platelet aggregation thereby effectively promoting the formation of blood clots. Targeting this specific crosstalk between the immune system and the hemostatic system might be instrumental for the prevention and treatment of microvascular thromboembolic pathologies, which are inaccessible to invasive revascularization strategies.

Experimental Cannabinoid 2 Receptor-Mediated Immune Modulation in Sepsis

Sepsis is a complex condition that results from a dysregulated immune system in response to a systemic infection. Current treatments lack effectiveness in reducing the incidence and mortality associated with this disease. The endocannabinoid system offers great promise in managing sepsis pathogenesis due to its unique characteristics. The present study explored the effect of modulating the CB2receptor pathway in an acute sepsis mouse model. Endotoxemia was induced by intravenous injection of lipopolysaccharide (LPS) in mice and intestinal microcirculation was assessed through intravital microscopy. We found that HU308 (CB2receptor agonist) reduced the number of adherent leukocytes in submucosal venules but did not restore muscular and mucosal villi FCD in endotoxemic mice. AM630 (CB2receptor antagonist) maintained the level of adherent leukocytes induced by LPS but further reduced muscular and mucosal villi FCD. URB597 (FAAH inhibitor) and JZL184 (MAGL inhibitor) both reduced the number of adherent leukocytes in submucosal venules but did not restore the mucosal villi FCD. Using various compounds we have shown different mechanisms of activating CB2receptors to reduce leukocyte endothelial interactions in order to prevent further inflammatory damage during sepsis.

Effect of Estragole on Leukocyte Behavior and Phagocytic Activity of Macrophages

Estragole, a chemical constituent of the essential oils of many aromatic plants, is used as flavoring in beverage and food industries.In vivoandin vitroexperimental assays have shown that EST has sedative, anticonvulsant, antioxidant, antimicrobial, and anesthetic activity. In this work, we evaluate the effect of EST on leukocyte behavior and phagocytic activity of macrophages. In the peritonitis model, EST (500 and 750 mg/kg) decreased the infiltration of peritoneal exudate leukocytes.In vitrochemotaxis assay showed that EST (3, 10, 30, and 60 μg/mL) inhibited neutrophil migration toward fMLP. In thein vivomicrocirculation assay, EST at doses of 250, 500, and 750 mg/kg significantly reduced the number of rolling and adherent leukocytes and at doses of 250 and 500 mg/kg decreased number of leukocyte migrated to perivascular tissue. The results showed that EST (3, 10, and 30 μg/mL) was able to stimulate the macrophages phagocytosis but only at concentration of 10 μg/mL promoted an increase in nitric oxide (NO) production. In conclusion, this study showed that EST had potential anti-inflammatory effects, likely by inhibiting leukocyte migration and by stimulating macrophages phagocytosis.

Caveolin-1 scaffolding domain promotes leukocyte adhesion by reduced basal endothelial nitric oxide-mediated ICAM-1 phosphorylation in rat mesenteric venules

Exogenously applied caveolin-1 scaffolding domain (CAV) has been shown to inhibit inflammatory mediator-induced nitric oxide (NO) production and NO-mediated increases in microvessel permeability. However, the effect of CAV on endothelial basal NO that prevents leukocyte adhesion remains unknown. This study aims to investigate the roles of exogenously applied CAV in endothelial basal NO production, leukocyte adhesion, and adhesion-induced changes in microvessel permeability. Experiments were conducted in individually perfused rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (Lp). NO was quantified with fluorescence imaging in DAF-2-loaded vessels. Perfusing venules with CAV inhibited basal NO production without affecting basal Lp. Resuming blood flow in CAV-perfused vessels significantly increased leukocyte adhesion. The firmly adherent leukocytes altered neither basal Lp nor adherens junction integrity. Increases in Lp occurred only upon formyl-Met-Leu-Phe application that induces release of reactive oxygen species from the adherent leukocytes. The application of NO synthase inhibitor showed similar results to CAV, and NO donor abolished CAV-mediated leukocyte adhesion. Immunofluorescence staining showed increases in binding of ICAM-1 to an adhesion-blocking antibody concurrent with a Src-dependent ICAM-1 phosphorylation following CAV perfusion. Pre-perfusing vessels with anti-ICAM-1 blocking antibody or a Src kinase inhibitor attenuated CAV-induced leukocyte adhesion. These results indicate that the application of CAV, in addition to preventing excessive NO-mediated permeability increases, also causes reduction of basal NO and promotes ICAM-1-mediated leukocyte adhesion through Src activation-mediated ICAM-1 phosphorylation. CAV-induced leukocyte adhesion was uncoupled from leukocyte oxidative burst and microvessel barrier function, unless in the presence of a secondary stimulation.