Impaired Neutrophil Chemotaxis Research Articles

Dissecting thrombus-directed chemotaxis and random movement in neutrophil near-thrombus motion in flow chambers.

Thromboinflammation is caused by mutual activation of platelets and neutrophils. The site of thromboinflammation is determined by chemoattracting agents release by endothelium, immune cells, and platelets. Impaired neutrophil chemotaxis contributes to the pathogenesis of Shwachman-Diamond syndrome (SDS). In this hereditary disorder, neutrophils are known to have aberrant chemoattractant-induced F-actin properties. Here, we aim to determine whether neutrophil chemotaxis could be analyzed using our previously developed ex vivo assay of the neutrophils crawling among the growing thrombi. Adult and pediatric healthy donors, alongside with pediatric patients with SDS, were recruited for the study. Thrombus formation and granulocyte movement in hirudinated whole blood were visualized by fluorescent microscopy in fibrillar collagen-coated parallel-plate flow chambers. Alternatively, fibrinogen, fibronectin, vWF, or single tumor cells immobilized on coverslips were used. A computational model of chemokine distribution in flow chamber with a virtual neutrophil moving in it was used to analyze the observed data. The movement of healthy donor neutrophils predominantly occurred in the direction and vicinity of thrombi grown on collagen or around tumor cells. For SDS patients or on coatings other than collagen, the movement was characterized by randomness and significantly reduced velocities. Increase in wall shear rates to 300-500 1/s led to an increase in the proportion of rolling neutrophils. A stochastic algorithm simulating leucocyte chemotaxis movement in the calculated chemoattractant field could reproduce the experimental trajectories of moving neutrophils for 72% of cells. In samples from healthy donors, but not SDS patients, neutrophils move in the direction of large, chemoattractant-releasing platelet thrombi growing on collagen.

Esculin alleviates LPS-induced acute lung injury via inhibiting neutrophil recruitment and migration.

Acute lung injury (ALI) poses a serious threat to human health globally, particularly with the Coronavirus 2019 (COVID-19) pandemic. Excessive recruitment and infiltration of neutrophils is the major etiopathogenesis of ALI. Esculin, also known as 6,7-dihydroxycoumarin, is a remarkable compound derived from traditional Chinese medicine Cortex fraxini. Accumulated evidence indicates that esculin has potent anti-inflammatory effects, but its pharmaceutical effect against ALI and potential mechanisms are still unclear. This study evaluated the protective effect of esculin against ALI by histopathological observation and biochemical analysis of lung tissues and bronchoalveolar lavage fluid (BALF) in lipopolysaccharide (LPS)-challenged ALI mice in vivo. The effects of esculin on N-formyl-met-leu-phe (fMLP)-induced neutrophil migration and chemotaxis were quantitatively assessed using a Transwell assay and an automated cell imaging system equipped with a Zigmond chamber, respectively. The drug affinity responsive target stability (DARTS) assay, in vitro protein binding assay and molecular docking were performed to identify the potential therapeutic target of esculin and the potential binding sites and pattern. Esculin significantly attenuated LPS-induced lung pathological injury, reduced the levels of pro-inflammatory cytokines in both BALF and lung, and suppressed the activation of NF-κB signaling. Esculin also significantly reduced the number of total cells and neutrophils as well as myeloperoxidase (MPO) activity in the BALF. Esculin impaired neutrophil migration and chemotaxis as evidenced by the reduced migration distance and velocity. Furthermore, esculin remarkably inhibited Vav1 phosphorylation, suppressed Rac1 activation and the PAK1/LIMK1/cofilin signaling axis. Mechanistically, esculin could interact with β2 integrin and then diminish its ligand affinity with intercellular adhesion molecule-1 (ICAM-1). Esculin inhibits β2 integrin-dependent neutrophil migration and chemotaxis, blocks the cytoskeletal remodeling process required for neutrophil recruitment, thereby contributing to its protective effect against ALI. This study demonstrates the new therapeutic potential of esculin as a novel lead compound.

The role of G protein-coupled receptor in neutrophil dysfunction during sepsis-induced acute respiratory distress syndrome.

Sepsis is defined as a life-threatening dysfunction due to a dysregulated host response to infection. It is a common and complex syndrome and is the leading cause of death in intensive care units. The lungs are most vulnerable to the challenge of sepsis, and the incidence of respiratory dysfunction has been reported to be up to 70%, in which neutrophils play a major role. Neutrophils are the first line of defense against infection, and they are regarded as the most responsive cells in sepsis. Normally, neutrophils recognize chemokines including the bacterial product N-formyl-methionyl-leucyl-phenylalanine (fMLP), complement 5a (C5a), and lipid molecules Leukotriene B4 (LTB4) and C-X-C motif chemokine ligand 8 (CXCL8), and enter the site of infection through mobilization, rolling, adhesion, migration, and chemotaxis. However, numerous studies have confirmed that despite the high levels of chemokines in septic patients and mice at the site of infection, the neutrophils cannot migrate to the proper target location, but instead they accumulate in the lungs, releasing histones, DNA, and proteases that mediate tissue damage and induce acute respiratory distress syndrome (ARDS). This is closely related to impaired neutrophil migration in sepsis, but the mechanism involved is still unclear. Many studies have shown that chemokine receptor dysregulation is an important cause of impaired neutrophil migration, and the vast majority of these chemokine receptors belong to the G protein-coupled receptors (GPCRs). In this review, we summarize the signaling pathways by which neutrophil GPCR regulates chemotaxis and the mechanisms by which abnormal GPCR function in sepsis leads to impaired neutrophil chemotaxis, which can further cause ARDS. Several potential targets for intervention are proposed to improve neutrophil chemotaxis, and we hope that this review may provide insights for clinical practitioners.

TPPU treatment of burned mice dampens inflammation and generation of bioactive DHET which impairs neutrophil function

Oxylipins modulate the behavior of immune cells in inflammation. Soluble epoxide hydrolase (sEH) converts anti-inflammatory epoxyeicosatrienoic acid (EET) to dihydroxyeicosatrienoic acid (DHET). An sEH-inhibitor, TPPU, has been demonstrated to ameliorate lipopolysaccharide (LPS)- and sepsis-induced inflammation via EETs. The immunomodulatory role of DHET is not well characterized. We hypothesized that TPPU dampens inflammation and that sEH-derived DHET alters neutrophil functionality in burn induced inflammation. Outbred mice were treated with vehicle, TPPU or 14,15-DHET and immediately subjected to either sham or dorsal scald 28% total body surface area burn injury. After 6 and 24 h, interleukin 6 (IL-6) serum levels and neutrophil activation were analyzed. For in vitro analyses, bone marrow derived neutrophil functionality and mRNA expression were examined. In vivo, 14,15-DHET and IL-6 serum concentrations were decreased after burn injury with TPPU administration. In vitro, 14,15-DHET impaired neutrophil chemotaxis, acidification, CXCR1/CXCR2 expression and reactive oxygen species (ROS) production, the latter independent from p38MAPK and PI3K signaling. We conclude that TPPU administration decreases DHET post-burn. Furthermore, DHET downregulates key neutrophil immune functions and mRNA expression. Altogether, these data reveal that TPPU not only increases anti-inflammatory and inflammation resolving EET levels, but also prevents potential impairment of neutrophils by DHET in trauma.

Anti-Inflammatory and Healing Activity of the Hydroalcoholic Fruit Extract of Solanum diploconos (Mart.) Bohs

Background Solanum diploconos (Mart.) Bohs is a native Brazilian plant belonging to the Solanaceae family, popularly known as “tomatinho do mato” and poorly investigated. Herein, we presented for the first time evidence for the anti-inflammatory and wound healing activities of S. diploconos fruit hydroalcoholic extract. Material and Methods. In vitro fMLP-induced chemotaxis, LPS-induced inflammatory mediator levels (cytokines by ELISA and NO release by Griess reaction), and adhesion molecule expression (CD62L, CD49d, and CD18, by flow-cytometry) were assessed in neutrophils treated with different concentrations of the extract. Inflammation resolution was measured by the efferocytosis assay and the healing activity by in vivo and in vitro assays. The air pouch model of carrageenan-induced inflammation in Swiss mice was used to investigate the in vivo anti-inflammatory effects of the extract. Leukocyte influx (by optical microscopy) and cytokine release were quantified in the pouch exudates. Additionally, the acute and subacute toxic and genotoxic effects of the extract were evaluated. Results In vitro, the extract impaired neutrophil chemotaxis and its ability to produce and/or release cytokines (TNFα, IL-1β, and IL-6) and NO upon LPS stimuli (p < 0.01). LPS-treated neutrophils incubated with the extract presented increased CD62L expression (p < 0.01), indicating a reduced activation. An enhanced efferocytosis of apoptotic neutrophils by macrophages was observed and accompanied by higher IL-10 and decreased TNFα secretion (p < 0.01). In vivo, similar results were noted, including reduction of neutrophil migration, protein exudation, and cytokine release (p < 0.01). Also, the extract increased fibroblast proliferation and promoted skin wound healing (p < 0.01). No signs of toxicity or genotoxicity were observed for the extract. Conclusion S. diploconos fruit extract is anti-inflammatory by modulating neutrophil migration/activation as well macrophage-dependent efferocytosis and inflammatory mediator release. It also indicates its potential use as a healing agent. Finally, the absence of acute toxic and genotoxic effects reinforces its possible use as medicinal product.

QOL-36. USE OF CANNABINOIDS IN THE PEDIATRIC CENTRAL NERVOUS SYSTEM TUMOR POPULATION

BACKGROUNDCannabinoids, including cannabidiol (CBD) and tetrahydrocannabinol (THC), are a class of compounds found in marijuana. Numerous studies in adults have examined cannabinoid use in management of cancer-related symptoms such as nausea, anorexia, and pain. Less is known about the use in the pediatric oncology population.METHODSA prospective observational study has been ongoing since 2016 at Children’s Hospital Colorado to evaluate cannabinoids’ impact using PedsQL™ modules on quality of life of pediatric patients with central nervous system (CNS) tumors who are 2–18 years old. Laboratory assessments of T-cell activity and pharmacokinetics of CBD, THC and associated metabolites are in process. Diaries with exploratory information on cannabinoid use patterns are being collected.RESULTSThirty-three patients (14:19; male:female) have been enrolled with a median age of 6.4 years (range, 2.9–17.7 years). The most common tumor type in enrolled patients is embryonal tumors (13/33; 39%). Nine (27%) patients have low-grade glial/glioneuronal tumors, and eight (24%) had high-grade/diffuse midline gliomas. The remaining patients had ependymoma or craniopharyngioma. The median time on cannabinoids is 9 months. Most (n=20) patients have used oral products with CBD and THC. One patient continues on cannabinoid therapy in follow up. Preliminary immune function analyses identified impaired neutrophil superoxide anion production and chemotaxis in patients taking cannabinoids at early time points on therapy.CONCLUSIONSFamilies of children with various CNS tumors are pursuing cannabinoid therapy for both antitumor and supportive care purposes. Analysis of the impact of cannabinoids on patients’ quality of life is ongoing.

Burn Injury Impairs Neutrophil Chemotaxis Through Increased Ceramide.

Infection is a common and often deadly complication after burn injury. A major underlying factor is burn-induced immune dysfunction, particularly with respect to neutrophils as the primary responders to infection. Temporally after murine scald injury, we demonstrate impaired bone marrow neutrophil chemotaxis toward CXCL1 ex vivo. Additionally, we observed a reduced recruitment of neutrophils to the peritoneal after elicitation 7 days after injury. We demonstrate that neutrophil ceramide levels increase after burn injury, and this is associated with decreased expression of CXCR2 and blunted chemotaxis. A major signaling event upon CXCR2 activation is Akt phosphorylation and this was reduced when ceramide was elevated. In contrast, PTEN levels were elevated and PTEN-inhibition elevated phospho-Akt levels and mitigated the burn-induced neutrophil chemotaxis defect. Altogether, this study identifies a newly described pathway of ceramide-mediated suppression of neutrophil chemotaxis after burn injury and introduces potential targets to mitigate this defect and reduce infection-related morbidity and mortality after burn.

Mechanisms and Points of Control in the Spread of Inflammation: A Mathematical Investigation

Understanding the mechanisms that control the body’s response to inflammation is of key importance, due to its involvement in myriad medical conditions, including cancer, arthritis, Alzheimer’s disease and asthma. While resolving inflammation has historically been considered a passive process, since the turn of the century the hunt for novel therapeutic interventions has begun to focus upon active manipulation of constituent mechanisms, particularly involving the roles of apoptosing neutrophils, phagocytosing macrophages and anti-inflammatory mediators. Moreover, there is growing interest in how inflammatory damage can spread spatially due to the motility of inflammatory mediators and immune cells. For example, impaired neutrophil chemotaxis is implicated in causing chronic inflammation under trauma and in ageing, while neutrophil migration is an attractive therapeutic target in ailments such as chronic obstructive pulmonary disease. We extend an existing homogeneous model that captures interactions between inflammatory mediators, neutrophils and macrophages to incorporate spatial behaviour. Through bifurcation analysis and numerical simulation, we show that spatially inhomogeneous outcomes can present close to the switch from bistability to guaranteed resolution in the corresponding homogeneous model. Finally, we show how aberrant spatial mechanisms can play a role in the failure of inflammation to resolve and discuss our results within the broader context of seeking novel inflammatory treatments.

Activation of the sympathetic nervous system modulates neutrophil function.

Emerging evidence has revealed that noradrenaline (NA), the main neurotransmitter of the sympathetic nervous system (SNS), regulates a variety of immune functions via binding to adrenergic receptors present on immune cells. In this study, we examined the role of NA in the regulation of neutrophil functions. Neutrophils were isolated from the bone marrow of naïve mice and treated with NA at various concentrations to assess the effect on various neutrophil functions. Additionally, we performed cremaster intravital microscopy to examine neutrophil‐endothelial cell interactions following NA superfusion in vivo. In a separate group of animals, mice were subjected to an experimental model of stroke and at 4 and 24 h neutrophils were isolated for assessment on their ability to migrate toward various chemokines. Treatment of neutrophils with NA for 4 h significantly impaired neutrophil chemotaxis and induced an N2 neutrophil phenotype with reduced expression of the genes critical for cytoskeleton remodeling and inflammation. Prolonged NA administration promoted neutrophils to release myeloperoxidase and IL‐6, but suppressed the production of interferon‐γ and IL‐10, reduced neutrophil activation and phagocytosis. Superfusion of NA over the cremaster muscle almost completely inhibited fMLP‐induced neutrophil adhesion/arrest and transmigration. Furthermore, using a mouse model of stroke, a pathological condition in which SNS activation is evident, neutrophils isolated from poststroke mice showed markedly reduced chemotaxis toward all of the chemokines tested. The findings from our study indicate that neutrophil chemotaxis, activation, and phagocytosis can all be negatively regulated in an NA‐dependent manner. A better understanding of the relationship between sympathetic activation and neutrophil function will be important for the development of effective antibacterial interventions.

385 IL-17E activates M2 macrophages to produce IL-8 in a p38 dependent manner and favor the recruitment of neutrophils in psoriatic skin

Psoriasis vulgaris is a common chronic recurrent immune-mediated skin disease. We have recently found that IL-17E (i.e IL-25), a member of the IL-17 cytokine family, is over-expressed in lesional psoriatic skin when compared to non lesional and healthy donors. Within the psoriatic plaque, macrophages having a mixed M1/M2 phenotype internalize IL-17E in a receptor induced clathrin-mediated mechanism. In this study we investigated the biological effects of IL-17E in psoriasis. In vitro M2-polarized macrophages, but not M1, responded to IL-17E by producing inflammatory cytokines (e.g. TNF and IL-6) and chemokines (e.g. IL-8 and MCP-1) typical expressed by M1 cells. Nuclear factor-kappa B (NF- κB), p38 and STAT3 were required for generating the IL-17E-dependent effects. Of note, IL-17E did not stimulate the production of cytokines/chemokines involved in T cell polarization and recruitment. Supernatants of IL-17E-stimulated M2 macrophages contained high levels of IL-8 and favored the attraction of neutrophils to a higher extent compared to supernatants of resting macrophages. Chemical p38 inhibition impaired IL-8 production and neutrophil chemotaxis in vitro. In vivo, intra-dermal injection of rmIL-17E in BALB/c mice induced severe dermal inflammation as assessed by immunohistological and FACS analysis, with increased neutrophil/eosinophil infiltration and reduced T-cell recruitment, compared to saline control group. Neutralization of IL-17E by intra-peritoneal injection of anti-IL-17E antibodies reduced the infiltration of neutrophils in a tape-stripping model. Consistent with the in vivo and in vitro data, the number of IL-17E+ cells in lesional skin correlated with the number of neutrophils, while being inversely proportional to the number of infiltrating T cells. Together, our data show that IL-17E favors the preferential recruitment of neutrophils via macrophage activation and define a novel pro-inflammatory role of IL-17E in psoriasis.

Systemic Adenosine Triphosphate Impairs Neutrophil Chemotaxis and Host Defense in Sepsis.

Sepsis remains an unresolved clinical problem. Therapeutic strategies focusing on inhibition of neutrophils (polymorphonuclear neutrophils) have failed, which indicates that a more detailed understanding of the underlying pathophysiology of sepsis is required. Polymorphonuclear neutrophil activation and chemotaxis require cellular adenosine triphosphate release via pannexin-1 channels that fuel autocrine feedback via purinergic receptors. In the current study, we examined the roles of endogenous and systemic adenosine triphosphate on polymorphonuclear neutrophil activation and host defense in sepsis. Prospective randomized animal investigation and in vitro studies. Preclinical academic research laboratory. Wild-type C57BL/6 mice, pannexin-1 knockout mice, and healthy human subjects used to obtain polymorphonuclear neutrophils for in vitro studies. Wild-type and pannexin-1 knockout mice were treated with suramin or apyrase to block the endogenous or systemic effects of adenosine triphosphate. Mice were subjected to cecal ligation and puncture and polymorphonuclear neutrophil activation (CD11b integrin expression), organ (liver) injury (plasma aspartate aminotransferase), bacterial spread, and survival were monitored. Human polymorphonuclear neutrophils were used to study the effect of systemic adenosine triphosphate and apyrase on chemotaxis. Inhibiting endogenous adenosine triphosphate reduced polymorphonuclear neutrophil activation and organ injury, but increased the spread of bacteria and mortality in sepsis. By contrast, removal of systemic adenosine triphosphate improved bacterial clearance and survival in sepsis by improving polymorphonuclear neutrophil chemotaxis. Systemic adenosine triphosphate impairs polymorphonuclear neutrophil functions by disrupting the endogenous purinergic signaling mechanisms that regulate cell activation and chemotaxis. Removal of systemic adenosine triphosphate improves polymorphonuclear neutrophil function and host defenses, making this a promising new treatment strategy for sepsis.

Further Insights into the Hematological Disorders Observed in Shwachman-Diamond Syndrome: mTOR and STAT3 Hyper-Phosphorylation in CD4+ and CD8+ T Cells and Efficacy of Rapamycin Treatment

Shwachman-Diamond syndrome (SDS) is a rare autosomal recessive disease which affects 1/168,000 newborns in Italy with a mean of 3.0 new cases/year. SDS is caused by mutations in the Shwachman-Bodian-Diamond syndrome (SBDS) gene, which encodes for the homonymous protein SBDS, whose exact function is still unknown. SBDS protein has been reported to play a role in eukaryotic ribosome biogenesis. Thus, SDS is considered a ribosomopathy. The pathology is characterized by multiple-organ impairment involving bone marrow failure, exocrine pancreatic insufficiency, skeletal malformations, hepatic and cognitive disorders. Neutropenia and impaired neutrophil chemotaxis, which in turn cause recurrent infections, are reported in young children. Furthermore, 15-20% of SDS patients develop myelodysplastic syndrome (MDS), with increased risk of acute myeloid leukemia (AML) progression, which represent the main cause of mortality. However, the exact pathologic mechanism whereby loss of SDBS function could lead to the specific SDS hematological issues remains unclear. We recently reported, for the first time to the best of our knowledge, that the mammalian Target of Rapamycin (mTOR) and Signal Transducer and Activator of Transcription (STAT)-3 pathways are hyper-activated in B cells, PMNs and, mostly, in monocytes obtained from SDS patients (Bezzerri V et al, Sci Rep 2016, in press). Since mTOR and STAT3 activation are associated with neutrophil development and AML, this finding could at least partially explain the onset of the hematological issues. Here we show a further Phospho flow analysis of mTOR and STAT3 pathways activation in otherlymphocytes subsets,in particular in CD8+/CD4+ T cells and NK cells obtained from five SDS patients. We found that STAT3 S727 is the most phosphorylated site in CD8+ and CD4+ T cells (more than twice than the healthy control cells, each). Furthermore, mTOR (S2448) is hyper-phosphorylated in CD8+ and CD4+ T cells derived from SDS patients. Median fluorescence intensity shifted from 220 ± 25 (healthy controls) to 405 ± 29 (SDS patients) in CD8+ T cells and from 350 ± 132 (healthy controls) to 590 ± 150 (SDS patients) in CD4+ T cells, similarly to results obtained from Monocytes and B cells. NK seems to be less responsive to mTOR/STAT3 activation than B and T cells. Importantly, mTOR inhibitor rapamycin is able to reduce both mTOR and STAT3 activation, with different efficacy, in a cell type-dependent manner. In particular, rapamycin strongly reduces both mTOR and STAT3 S727 phosphorylation in CD8+ and CD4+ T cells. Thus, these results suggest a role of mTOR/STAT3 pathways in both myeloid and lymphoid lineages of SDS blood cells. Since several drugs approved by FDA and EMA targeting the JAK-STAT and mTOR pathways have been currently evaluated for the treatment of different forms of hematological malignancies, this work could open a wider scenario into the current SDS therapeutic approaches. DisclosuresNo relevant conflicts of interest to declare.

Sepsis-induced impairment of neutrophil chemotaxis on a microfluidic chip

This study aimed to design a microfluidic chip to measure neutrophil chemotaxis, which is a convenient assay to assess the severity and prognosis of sepsis, and to study the mechanisms involved in the variation of neutrophil chemotaxis. Neutrophil chemotaxis was investigated in this microfluidic device by measuring the migration speed of neutrophils following the LPS concentration gradient stimulus. Neutrophils of 32 sepsis patients were divided into three groups according to the seriousness of physician-diagnosed sepsis, and 12 healthy individuals served as controls. Statistical significance was set at an alpha value of P<0.05. Neutrophil chemotaxis was significantly decreased following the seriousness of sepsis. By contrast, in septic neutrophils, the expression of TLR2 was significantly increased, whereas the expression of CXCR2 was significantly decreased. Neutrophil chemotaxis in sepsis was significantly reduced as compared to healthy individuals. We speculated that impaired neutrophil chemotaxis in sepsis was probably mediated by the TLR2-CXCR2 pathway.

MTOR and differential activation of mitochondria orchestrate neutrophil chemotaxis.

Neutrophils use chemotaxis to locate invading bacteria. Adenosine triphosphate (ATP) release and autocrine purinergic signaling via P2Y2 receptors at the front and A2a receptors at the back of cells regulate chemotaxis. Here, we examined the intracellular mechanisms that control these opposing signaling mechanisms. We found that mitochondria deliver ATP that stimulates P2Y2 receptors in response to chemotactic cues, and that P2Y2 receptors promote mTOR signaling, which augments mitochondrial activity near the front of cells. Blocking mTOR signaling with rapamycin or PP242 or mitochondrial ATP production (e.g., with CCCP) reduced mitochondrial Ca(2+) uptake and membrane potential, and impaired cellular ATP release and neutrophil chemotaxis. Autocrine stimulation of A2a receptors causes cyclic adenosine monophosphate accumulation at the back of cells, which inhibits mTOR signaling and mitochondrial activity, resulting in uropod retraction. We conclude that mitochondrial, purinergic, and mTOR signaling regulates neutrophil chemotaxis and may be a pharmacological target in inflammatory diseases.

Killing of Escherichia coli by Crohn's Disease Monocyte-derived Macrophages and Its Enhancement by Hydroxychloroquine and Vitamin D.

Crohn's disease (CD) is associated with defective innate immunity, including impaired neutrophil chemotaxis, and mucosal invasion by bacteria, particularly adherent and invasive Escherichia coli that replicate inside macrophage phagolysosomes. We compared CD and healthy control (HC) macrophages for their abilities to kill E. coli and generate neutrophil chemoattractants and also assessed the effects of hydroxychloroquine (HCQ) and vitamin D on killing of phagocytosed E. coli. Peripheral blood monocyte-derived macrophages from CD and HC were compared for bacterial killing and generation of neutrophil chemoattractants in response to CD-derived E. coli. Escherichia coli replication was also assessed in the presence and absence of HCQ, alone and with antibiotics, and vitamin D. Monocyte-derived macrophages from patients with CD were similar to HC in allowing replication of phagocytosed CD-derived E. coli: HM605 {CD: N = 10, mean fold replication in 3 hr = 1.08 (95% confidence interval [CI], 0.39-1.78); HC: N = 9, 1.50 (95% CI, 1.02-1.97); P = 0.15} and also in generation of neutrophil chemoattractants in response to E. coli (mean fold chemotaxis relative to control: CD = 2.55 [95% CI, 2.31-2.80]; HC = 2.65 [95% CI, 2.46-2.85], P = 0.42). HCQ and 1,25 OH2-vitamin D3 both caused dose-dependent inhibition of intramacrophage E. coli replication 3-hour postinfection; HCQ: 73.9% inhibition (P < 0.001) at 1 μg/mL, accompanied by raised intraphagosomal pH, and 1,25 OH2-vitamin D3: 80.7% inhibition (P < 0.05) at 80 nM. HCQ had synergistic effects with doxycycline and ciprofloxacin. CD and HC macrophages perform similarly in allowing replication of phagocytosed E. coli and generating neutrophil chemoattractants. Replication of phagocytosed E. coli was substantially decreased by HCQ and vitamin D. These warrant further therapeutic trials in CD in combination with relevant antibiotics.

ADAM9 disintegrin domain activates human neutrophils through an autocrine circuit involving integrins and CXCR2.

ADAM9 is a member of the ADAM family whose expression positively correlates with tumor progression. Besides the metalloprotease activity, ADAM9D interacts with different integrins, modulating cell-adhesion events. Previous studies pointed to an important role for neutrophils in tumor development, as the inhibition of neutrophil migration or depletion of this immune cell impairs tumor growth. However, our understanding of the molecular mechanisms involved in this process, as well as the main key players acting on neutrophils, is very limited. Here, we investigated the possible modulatory effects of ADAM9D on human neutrophil functions. Our results show that ADAM9D promotes neutrophil activation and chemotaxis in a process that depends on the engagement of αvβ3 and α9β1 integrins and on the activation of PI3K/Akt and MAPK signaling pathway. ADAM9D impairs migration of neutrophils toward fMLP, LTB4, and IL-8 as classic chemoattractants. This effect is blocked by PTX, a G(i)PCR inhibitor. Furthermore, CXCR2 antagonists RPTX and SB225002 also impaired neutrophil chemotaxis in response to ADAM9D, suggesting a hierarchical cross-talk of integrins with CXCR2. Our results indicate that ADAM9D activates neutrophil functions and may be implicated in the inflammatory events associated with cancer and other disorders.

Lipopolysaccharide-Induced Lung Injury Is Independent of Serum Vitamin D Concentration

Vitamin D deficiency is increasing in incidence around the world. Vitamin D, a fat-soluble vitamin, has documented effects on the innate and adaptive immune system, including macrophage and T regulatory (Treg) cell function. Since Treg cells are important in acute lung injury resolution, we hypothesized that vitamin D deficiency increases the severity of injury and delays injury resolution in lipopolysaccharide (LPS) induced acute lung injury. Vitamin D deficient mice were generated, using C57BL/6 mice, through diet modification and limited exposure to ultraviolet light. At 8 weeks of age, vitamin D deficient and sufficient mice received 2.5 g/kg of LPS or saline intratracheal. At 1 day, 3 days and 10 days, mice were anesthetized and lung elastance measured. Mice were euthanized and bronchoalveolar lavage fluid, lungs and serum were collected. Ex vivo neutrophil chemotaxis was evaluated, using neutrophils from vitamin D sufficient and deficient mice exposed to the chemoattractants, KC/CXCL1 and C5a, and to bronchoalveolar lavage fluid from LPS-exposed mice. We found no difference in the degree of lung injury. Leukocytes were mildly decreased in the bronchoalveolar fluid of vitamin D deficient mice at 1 day. Ex-vivo, neutrophils from vitamin D deficient mice showed impaired chemotaxis to KC but not to C5a. Vitamin D deficiency modestly impairs neutrophil chemotaxis; however, it does not affect lung injury or its resolution in an LPS model of acute lung injury.

Reactive Oxygen Species-Induced Actin Glutathionylation Controls Actin Dynamics in Neutrophils

The regulation of actin dynamics is pivotal for cellular processes such as cell adhesion, migration, and phagocytosis and thus is crucial for neutrophils to fulfill their roles in innate immunity. Many factors have been implicated in signal-induced actin polymerization, but the essential nature of the potential negative modulators are still poorly understood. Here we report that NADPH oxidase-dependent physiologically generated reactive oxygen species (ROS) negatively regulate actin polymerization in stimulated neutrophils via driving reversible actin glutathionylation. Disruption of glutaredoxin 1 (Grx1), an enzyme that catalyzes actin deglutathionylation, increased actin glutathionylation, attenuated actin polymerization, and consequently impaired neutrophil polarization, chemotaxis, adhesion, and phagocytosis. Consistently, Grx1-deficient murine neutrophils showed impaired invivo recruitment to sites of inflammation and reduced bactericidal capability. Together, these results present a physiological role for glutaredoxin and ROS- induced reversible actin glutathionylation in regulation of actin dynamics in neutrophils.

Polymicrobial Multiple Intraabdominal Infections - A Rare Complication of Colonoscopy in a Cirrhotic Patient

Case Description: A 58-year-old male with a history of HCV and alcohol related advanced cirrhosis, presented with altered mental status and right upper quadrant pain, three days following upper and lower endoscopy. The endoscopy showed hemorrhoids and minimal gastritis and no interventions were performed. Diagnostic work-up upon admission showed elevated white blood cell count. CT abdomen showed multiple liver abscesses, which were partially drained and grew Streptococcus anginosus and Staphylococcus epidermidis. There were no other intra-abdominal fluid collections or abscesses, no features suggestive of bowel perforation on imaging. Blood cultures grew E. coli. His condition gradually deteriorated despite treatment with appropriate antibiotics. Repeat CT scan 10 days later showed right upper lobe pneumonia, bilateral pleural effusion along with persistent liver abscesses and new ascites. Paracentesis revealed an exudate with WBC count of 8125 with 94% neutrophils and grew Klebsiella pneumoniae. Antibiotics were adjusted according to sensitivities. However, his condition continued to deteriorate and he finally died of severe sepsis and multi-organ dysfunction. Discussion: There is a wide variation in the rate of bacteremia after various endoscopic procedures ranging from 2 to 45% based on type of procedure performed. They occur predominantly following interventional procedures. To our knowledge, there have only been two cases of bacterial peritonitis following colonoscopy in patients undergoing continuous ambulatory peritoneal dialysis and 2 other instances in patients with cirrhosis and ascites. Septicemia has been reported in only 4 cases after colonoscopy. These include 2 patients with cirrhosis and ulcerative colitis who developed gram negative septicemia; a patient who underwent splenectomy and was taking corticosteroids developed septicemia with Flavobacterium meningosepticum and Escherichia coli; and Listeria in a patient being treated with ACTH for ulcerative colitis. Conclusions: Patients with cirrhosis have a higher rate of infectious complications, likely due to decreased compliment levels, impaired neutrophil chemotaxis, lymphocyte dysfunction and immunoglobulin dysfunction, combined with portal hypertensive colopathy due to venous congestion. In summary, infectious complications following diagnostic endoscopies in cirrhotics are rare but can be severe and life threatening, even without obvious signs of bowel perforation.

Impaired neutrophil and monocyte chemotaxis in chronic and aggressive periodontitis and effects of periodontal therapy

Depressed chemotactic activity of polymorphoneutrophil (PMN) and monocyte (MN) appears to be one of the significant risk factors in the development of periodontal disease. Although bacteria are the primary etiologic factor in periodontal disease, the patient's host response is a determinant of disease susceptibility. Depressed chemotaxis of PMN and MN could lead to periodontal destruction by altering the host response i.e. impairment of the normal host response in neutralizing infection and alterations that result in destruction of the surrounding periodontal tissues. Thirty patients (10 healthy subjects, 10 chronic periodontitis, and 10 with aggressive periodontitis) participated in this study. Clinical parameters like plaque index, gingival index, probing pocket depth, and radiographic assessment were done. The peripheral blood PMNs and MNs were isolated from the patient and the chemotactic response was studied. Statistical analysis was performed using post-hoc Newman-Keul range test. PMN and MN chemotaxis was found to be statistically significant (P<0.05) at baseline and three months after periodontal therapy in chronic and aggressive periodontitis group compared to healthy subjects. However on comparison between chronic and aggressive periodontitis group statistical significance was not found (P>0.05).Comparision between chronic periodontitis, aggressive periodontitis with healthy subjects, PMN and MN chemotaxis showed statistical significance (P<0.05) at baseline and three months after periodontal therapy, Whereas statistically there was no difference when chronic periodontitis was compared with aggressive periodontitis. Depressed chemotaxis of PMN and MN results in increased periodontal destruction. In this study, depressed PMN and MN chemotaxis is seen in both aggressive periodontitis group and chronic periodontitis group and the response was altered although to a lesser degree after periodontal therapy in both groups indicating that effect of treatment does exist.