Defective Neutrophil Research Articles

High Incidence of Serologic Markers of Inflammatory Bowel Disease in Asymptomatic Patients with Glycogen Storage Disease Type Ia.

Most patients with glycogen storage disease (GSD) type Ib show features related to inflammatory bowel disease (IBD). The development of IBD seems to be associated with the defect of neutrophil function in GSD Ib. Patients with GSD Ia were not recognized to have similar gastrointestinal complaints until recently and are not associated with a neutrophil defect. Fifty consecutive GSD Ia inpatients over the age of 2 years without a diagnosis of IBD were screened using serologic and genetic markers via the Prometheus IBD sgi Diagnostic test. Eleven patients were tested positive for IBD (22%), with five fitting the pattern for Crohn's disease, five for ulcerative colitis, and one with nonspecific IBD. Only 2 out of the 11 patients had any gastrointestinal complaints. No pattern could be distinguished from individual inflammatory markers, genetics, inflammation antibodies, age, complications, or metabolic control. Of note, 9 out of 11 patients testing positive were female. Patients with GSD Ia were found to have a higher rate of serologically indicated IBD when compared with the general population. While these subjects will need to be followed to determine if these serologic markers correlate with clinical disease, this study supports that IBD may be more common in the GSD Ia population. Further studies are warranted to explain the relationship between IBD and GSD I since it may provide clues regarding the pathogenesis of IBD development in the general population.

Leptin receptor mutation results in defective neutrophil recruitment to the colon during Entamoeba histolytica infection.

ABSTRACTAmebiasis is an enteric infection caused by Entamoeba histolytica, with symptoms ranging in severity from asymptomatic colonization to dysentery. Humans with the Q223R leptin receptor mutation have increased susceptibility to amebiasis, but the mechanism has been unclear. Using a mouse model expressing the mutation, we tested the impact of the Q223R mutation on the innate immune response to E. histolytica infection. The 223R mutation resulted in delayed clearance of amebae from the cecum, as had been previously observed. We found that neutrophil influx to the site of the infection was reduced 12 h after infection in 223R mice. Depletion of neutrophils with anti-Ly6G monoclonal antibody increased susceptibility of wild-type mice to infection, supporting the importance of neutrophils in innate defense. Leptin expression was increased in the cecum by E. histolytica infection, suggesting that leptin could serve as a homing signal for neutrophils to the gut. Interestingly, neutrophils from mice with the 223R mutation had diminished chemotaxis toward leptin. This impaired chemotaxis likely explained the reduced gut infiltration of neutrophils. The newly recognized effect of the leptin receptor Q223R mutation on neutrophil chemotaxis and the impact of this mutation on multiple infectious diseases suggest a broader impact of this mutation on susceptibility to disease.

MyD88-, but not Nod1- and/or Nod2-deficient mice, show increased susceptibility to polymicrobial sepsis due to impaired local inflammatory response.

Pathogen recognition and triggering of the inflammatory response following infection in mammals depend mainly on Toll-like and Nod-like receptors. Here, we evaluated the role of Nod1, Nod2 and MyD88-dependent signaling in the chemokine production and neutrophil recruitment to the infectious site during sepsis induced by cecal ligation and puncture (CLP) in C57Bl/6 mice. We demonstrate that Nod1 and Nod2 are not involved in the release of chemokines and recruitment of neutrophils to the infectious site during CLP-induced septic peritonitis because these events were similar in wild-type, Nod1-, Nod2-, Nod1/Nod2- and Rip2-deficient mice. Consequently, the local and systemic bacterial loads were not altered. Accordingly, neither Nod1 nor Nod2 was involved in the production of the circulating cytokines and in the accumulation of leukocytes in the lungs. By contrast, we showed that MyD88-dependent signaling is crucial for the establishment of the local inflammatory response during CLP-induced sepsis. MyD88-deficient mice were susceptible to sepsis because of an impaired local production of chemokines and defective neutrophil recruitment to the infection site. Altogether, these data show that Nod1, Nod2 and Rip2 are not required for local chemokine production and neutrophil recruitment during CLP-induced sepsis, and they reinforce the importance of MyD88-dependent signaling for initiation of a protective host response.

Differential Effects of Ultraviolet Irradiation in Neonatal versus Adult Mice Are Not Explained by Defective Macrophage or Neutrophil Infiltration

Epidemiological studies suggest that ultraviolet B exposure (UVR) during childhood is the most important environmental risk factor for melanoma. In accordance, neonatal, but not adult, UVR exacerbates melanoma incidence in mouse models. The inability of neonates, as opposed to adults, to mount a proper neutrophil inflammatory response in the skin upon UVR exposure has been one of the driving hypotheses explaining this observation for the past decade. However, this aspect remains controversial. Here, we evaluated the UVR-induced inflammatory response in neonatal versus adult mice. In neonates, a significant neutrophil infiltration could be identified and quantified using three different antibodies by flow cytometry or immunohistochemistry. On day 1 after UVR, neutrophils were increased by 84-fold and on day 4 macrophages increased by 37-fold compared with nonexposed age-matched skin. When compared with adults, neonatal skin harbored a higher proportion of neutrophils in the myeloid compartment without significant differences in absolute counts. This response was reproduced with different kinetics in C57Bl/6 and FVB mice with a more rapid attenuation of neutrophil counts in the latter. Overall, our results suggest that the greatly increased sensitivity to melanomagenesis in neonates does not result from their incompetence in terms of myeloid inflammatory response to UVR.

Poster Sessions

Neutrophils infiltrate lung grafts early after reperfusion and are important mediators of primary graft dysfunction. Immune pathways that regulate neutrophil recruitment to lungs during ischemia reperfusion injury remain poorly understood. The purpose of this study was to examine the role of DAP12, an adaptor molecule that regulates innate immune responses, in mediating ischemia reperfusion injury after lung transplantation. We used a mouse model of orthotopic vascularized lung transplantation. Grafts were assessed functionally (arterial blood gases), immunohistochemically, by quantitative PCR, flow cytometry and intravital two-photon imaging. Chemokine production by macrophages in vitro was assessed by ELISA. We found that macrophages in mouse (qPCR and immunohistochemistry) and human (immunohistochemistry) lungs that have been subjected to cold ischemia express DAP12. Donor, but not recipient deficiency in DAP12 protected against pulmonary ischemia reperfusion injury as evidenced by improved graft function (WTàWT, mean paO2=180.8+/-18.9, n=14, DAP12KOàWT, mean paO2=410.8 +/- 36.9, n=8, WTàDAP12KO mean paO2=217.9 +/-55.9, n=7). Compared to wild-type (WT) B6 cells, DAP12KO bone marrow-derived macrophages produced lower amounts of the neutrophil chemoattractant CXCL2 after in vitro stimulation (17 hours) with lysates derived from syngeneic B6 lung transplants (n=2). Similarly, qPCR of sorted donor macrophages 2 hours after reperfusion in vivo revealed significantly lower expression of CXCL2 when DAP12 was absent (n=4). Intravital two-photon imaging using LysM-GFP neutrophil reporter mice as recipients demonstrated a transendothelial migration defect of neutrophils into DAP12KO lungs at 4 hours after reperfusion (percentage of extravascular neutrophils: WTàLysM-GFP=69.3%, n=3; DAP12KOàLysM-GFP=25.6%, n=3). Neutrophil entry into DAP12-deficient lungs was restored when WT bone-marrow derived macrophages (76.8%, n=3) or recombinant CXCL2 (63.3%, n=3) was administered into the donor bronchus prior to reperfusion. Our study has uncovered a previously unrecognized role for DAP12 expression in tissue-resident macrophages in mediating ischemia reperfusion injury after lung transplantation through regulation of neutrophil trafficking.

Flow Cytometric Assay of Respiratory Burst Capacity in Peripheral Blood Monocytes of Gaucher Type 1 Patients

Abstract Background: There is an apparently increased tendency toward infections in patients with Gaucher disease, possibly due to defective neutrophil function rather than a decreased neutrophil count. Since macrophages are the main cell type affected in Gaucher disease, our aim was to determine the contribution of these cells to the susceptibility of Gaucher patients to infection by studying the respiratory burst capacity of peripheral blood monocytes. Methods: The study was performed in eleven Gaucher type 1 patients and eleven sex and age matched control subjects by measuring peripheral blood monocytes’ respiratory burst capacity using flow cytometry. The respiratory burst capacity was measured as dihydrorhodamine-123 median fluorescence in patients and respective controls. Results: There was no statistical difference in the median fluorescence among the patients and respective controls (p>0.05) after phorbol 12-myristate 13-acetate stimulation. Also, statistical difference was not reached among patients treated with enzyme replacement therapy at the time and those untreated. Conclusions: Flow cytometry might represent a more accurate and more reliable measure of respiratory burst compared to the methods of other researchers. Respiratory burst disturbance in monocytes does not seem to contribute to increased susceptibility to infection in Gaucher patients.

PWE-091 Crohn’s Disease Monocyte-derived Macrophages Exhibit Equivalent Responses To Intramacrophage Bacterial Infection Relative To Healthy Controls

IntroductionPatients with Crohn’s disease exhibit an attenuated inflammatory response after trauma or Escherichia coli infection and have delayed clearance of subcutaneous bacteria compared to healthy controls. Adherent, invasive E. coli,...

Diabetes exacerbates infection severity due to hyperinflammation rather than neutrophil defects (INM7P.425)

Abstract Patients with diabetes mellitus are at increased risk of dying from Gram negative bacterial infections. This excess mortality is often attributed to immune suppression, including neutrophil dysfunction. However, diabetic hosts also exhibit increased inflammatory responses to LPS. Here we show that lethal infections with Acinetobacter baumannii in diabetic mice are driven by LPS interactions with TLR4, as opposed to neutrophil defects. Mice made diabetic either by streptozotocin (Type 1) or diet induced obesity (Type 2) were hypersusceptible to lethality from A. baumannii bacteremia, whereas mice made neutropenic by anti-Ly6G administration were not more susceptible to infection. Diabetic infected mice developed severe septic shock, with hypothermia, medical acidosis, and marked elevations in inflammatory cytokines, compared to non-diabetic mice. Diabetic mice with impaired or absent TLR4 function, however, were protected against lethal infection and did not develop sepsis. Collectively, these results suggest that enhanced lethality of A. baumannii infection in diabetic mice is not driven by diabetes-related abrogation of neutrophil function. Rather, diabetic mice are more prone to progression to severe sepsis syndrome and are protected from this progression by impairment of TLR4 function. Short term abrogation of TLR4 signaling is a promising therapeutic approach to the treatment of A. baumannii, and possibly other Gram negative infections in diabetic hosts.

Altered neutrophil homeostasis and DARC regulate CXCL5-mediated pulmonary immune responses (INM2P.435)

Abstract CXCL5 is a neutrophil chemokine, predominantly expressed by epithelial cells and platelets. We recently demonstrated the critical role of CXCL5 in chemokine scavenging and innate immunity to severe bacterial pneumonia, and in regulating neutrophil homeostasis through IL-17/G-CSF axis. Here we show that CXCL5-inhibited chemokine scavenging and innate immune response to severe E.coli pneumonia is completely dependent on G-CSF-mediated increases in BM neutrophils and CXCR2 concentrations, but not on DARC. However, DARC functions as a chemokine reservoir rather than a sink in this pneumonia model. Darc-/- mice showed increased neutrophils in the BM, with normal blood neutrophil counts under basal conditions, and defective neutrophil release from BM upon systemic CXCL2 stimulation. Furthermore, DARC deficiency altered neutrophil homeostasis independently of G-CSF and exhibited additive effect with CXCL5 deficiency on increasing BM neutrophil percentages in Cxcl5-/-Darc-/- mice. These data provide the first evidence for a beneficial role of delicate regulation of neutrphil homeostasis in chemokine scavenging and innate immunity to bacterial pneumonia. Moreover, these results substantiate a mechanistic role for DARC deficiency in neutrophil homeostasis, which may provide insight into benign ethnic neutropenia and other inflammatory diseases seen in Duffy null humans.

Defective Neutrophil Recruitment in Leukocyte Adhesion Deficiency Type I Disease Causes Local IL-17–Driven Inflammatory Bone Loss

Leukocyte adhesion deficiency type I (LAD-I), a disease syndrome associated with frequent microbial infections, is caused by mutations on the CD18 subunit of β₂ integrins. LAD-I is invariably associated with severe periodontal bone loss, which historically has been attributed to the lack of neutrophil surveillance of the periodontal infection. We provide an alternative mechanism by showing that the cytokine interleukin-17 (IL-17) plays a major role in the oral pathology of LAD-I. Defective neutrophil recruitment in LAD-I patients or in LFA-1 (CD11a/CD18)-deficient mice--which exhibit the LAD-I periodontal phenotype--was associated with excessive production of predominantly T cell-derived IL-17 in the periodontal tissue, although innate lymphoid cells also contributed to pathological IL-17 elevation in the LFA-1-deficient mice. Local treatment with antibodies to IL-17 or IL-23 in LFA-1-deficient mice not only blocked inflammatory periodontal bone loss but also caused a reduction in the total bacterial burden, suggesting that the IL-17-driven pathogenesis of LAD-I periodontitis leads to dysbiosis. Therefore, our findings support an IL-17-targeted therapy for periodontitis in LAD-I patients.

Galectin-9 Signaling through TIM-3 Is Involved in Neutrophil-Mediated Gram-Negative Bacterial Killing: An Effect Abrogated within the Cystic Fibrosis Lung

The T cell Ig and mucin domain-containing molecule (TIM) family of receptors have emerged as potential therapeutic targets to correct abnormal immune function in chronic inflammatory conditions. TIM-3 serves as a functional receptor in structural cells of the airways and via the ligand galectin-9 (Gal-9) can modulate the inflammatory response. The aim of this study was to investigate TIM-3 expression and function in neutrophils, focusing on its potential role in cystic fibrosis (CF) lung disease. Results revealed that TIM-3 mRNA and protein expression values of circulating neutrophils were equal between healthy controls (n = 20) and people with CF (n = 26). TIM-3 was detected on resting neutrophil membranes by FACS analysis, and expression levels significantly increased post IL-8 or TNF-α exposure (p < 0.05). Our data suggest a novel role for TIM-3/Gal-9 signaling involving modulation of cytosolic calcium levels. Via TIM-3 interaction, Gal-9 induced neutrophil degranulation and primed the cell for enhanced NADPH oxidase activity. Killing of Pseudomonas aeruginosa was significantly increased upon bacterial opsonization with Gal-9 (p < 0.05), an effect abrogated by blockade of TIM-3 receptors. This mechanism appeared to be Gram-negative bacteria specific and mediated via Gal-9/ LPS binding. Additionally, we have demonstrated that neutrophil TIM-3/Gal-9 signaling is perturbed in the CF airways due to proteolytic degradation of the receptor. In conclusion, results suggest a novel neutrophil defect potentially contributing to the defective bacterial clearance observed in the CF airways and suggest that manipulation of the TIM-3 signaling pathway may be of therapeutic value in CF, preferably in conjunction with antiprotease treatment.

Defective stromal remodeling and neutrophil extracellular traps in lymphoid tissues favor the transition from autoimmunity to lymphoma.

Altered expression of matricellular proteins can become pathogenic in the presence of persistent perturbations in tissue homeostasis. Here, we show that autoimmunity associated with Fas mutation was exacerbated and transitioned to lymphomagenesis in the absence of SPARC (secreted protein acidic rich in cysteine). The absence of SPARC resulted in defective collagen assembly, with uneven compartmentalization of lymphoid and myeloid populations within secondary lymphoid organs (SLO), and faulty delivery of inhibitory signals from the extracellular matrix. These conditions promoted aberrant interactions between neutrophil extracellular traps and CD5(+) B cells, which underwent malignant transformation due to defective apoptosis under the pressure of neutrophil-derived trophic factors and NF-κB activation. Furthermore, this model of defective stromal remodeling during lymphomagenesis correlates with human lymphomas arising in a SPARC-defective environment, which is prototypical of CD5(+) B-cell chronic lymphocytic leukemia (CLL).

Aggressive periodontitis: A clinico-hematological appraisal.

Background:Human leukocyte antigens (HLA) have been considered a candidate of genetic risk markers for aggressive periodontitis (AP). AP has also been associated with polymorphonuclear leukocyte (PMN) dysfunction. The role of monocyte subsets in AP has also not been completely explored. Therefore, the present study was undertaken to assess in, AP subjects, the possible association between defective PMN adhesion and β2-integrin expression; defective neutrophil migration and actin polymerization level; the expression of ABO blood group and HLA antigen; and the percentage of CD14+ CD16+ monocytes and CD45RA monocytes. All these parameters have been compared with the subjects of chronic periodontitis (CP) and healthy controls.Materials and Methods:A total of 45 subjects of the age group 20-50 years, free from any known systemic disease, were divided into three groups – Group I - periodontally healthy control (n = 15), Group II - CP (n = 15) and Group III - AP (n = 15). Peripheral blood samples were collected. ABO grouping and HLA typing were performed. β2-integrin expression, actin polymerization level and percentage of CD14+ CD16+ monocytes and CD45RA monocytes were estimated by fluorescence-activated cell sorter analysis.Results:Most of the subjects of AP belonged to the blood group AB, and an increased frequency of HLA-A30, CW1 and DR1 (P < 0.1) and B44 and DQ2 (P < 0.05) were also observed in this group. In the AP group, both average values (β2-integrin and actin level) were significantly less than those of normal subjects (P < 0.001). The mean percentage of CD14+ CD16+ monocytes was found to be maximum in CP, followed by AP, and then in healthy subjects, while the mean percentage of CD45RA was maximum in AP, followed by CP, and then in healthy subjects.Conclusions:With the present state of knowledge from this study, a definite association of ABO blood groups and HLA phenotypes with periodontal diseases is yet to be established. Leukocytic functional defects were found in AP subjects. A statistically significant percentage of CD14+ CD16+ and CD45RA monocytes were found in AP subjects as compared with the normal control and CP groups.

Functional Defects In Neutrophils Derived From Ezh2 Null Mice

IntroductionWe investigate the role of Ezh2 in neutrophil function using murine progenitor cells differentiated into neutrophils lacking the Ezh2 gene. Ezh2 is the catalytic component of the polycomb repressive complex 2, which methylates lysine 27 of histone H3. It is frequently disrupted in myelodysplastic syndromes (MDS) leading to loss of function (Ernst et al., 2010). Mutations in EZH2 are found in 6% of MDS patients and while not strongly linked to cytopenias or blast proportion, they are independently associated with worse overall survival compared to patients with wildtype EZH2 (Bejar R. et al., 2011 and 2012). We hypothesize that Ezh2 mutations may cause qualitative defects in myeloid cells that impact their function and could contribute to the adverse prognosis observed in EZH2 mutant MDS. MethodsBone marrow from Ezh2 null (Ezh2-/-) and littermate control mice (WT) were transduced with HOXB8 fused to the estrogen receptor ligand-binding domain to produce immortalized myeloid progenitor cells. Removal of estrogen from the media allows these cells differentiate into mature neutrophils (Wang G.G., 2006). Differentiated cells were characterized for surface markers by flow cytometry and for gene expression by PCR of mRNA. Spontaneous cell death was measured by annexin/PI staining. Cell cycle patterns were determined by measuring the red emission of PI. Chemotactic function was assessed by counting cells that migrated across a transwell in presence/absence of the attractant zymosan. For phagocytosis experiments, cells were incubated with Fluoresbrite YG carboxylate beads at 37°C or 4°C. Reactive oxygen species (ROS) generation was measured by the oxidation of dihydrorhodamine 123 into fluorescent rhodamine 123. ResultsEstrogen withdrawal caused differentiation of both WT and Ezh2-/- lines into cells with mature neutrophil morphology after six days (Figure 1a). Both differentiated lines expressed the neutrophil surface markers CD11b and CD62L and the neutrophil-specific genes lactoferrin and Itgb2l.Ezh2-/- cells had an increased rate of spontaneous cell death compared to WT in undifferentiated (32.81% vs. 20.33%) and mature cells (32.82% vs. 14.23%). Nevertheless, both progenitor cell lines showed similar cell cycle patterns, demonstrating that Ezh2 absence had no other effect on cell cycle progression.Ezh2-/- neutrophils failed to migrate towards zymosan (Figure 1b). Expression of Tlr2, which binds zymosan, and other Toll-like receptors (Tlr4/5/9) were similar between the differentiated cell lines. Cells incubated with FITC-zymosan at 37°C showed no fluorescence differences between cell lines, indicating similar adherence. Experiments with neutrophils from an MDS patient with homozygous EZH2 mutations demonstrated a similar migration defect. Additional studies in MDS patient samples are ongoing and will be presented.Phagocytosis was reduced in Ezh2-/-cells. Unstimulated, the number of cells ingesting and adhering YG-beads was significantly greater with WT cells than with Ezh2-/-cells. When activated with fMLP, both lines showed increased adherence of YG-beads but the number of phagocytosing Ezh2-/- cells was reduced. The average number of beads ingested by each cell was lower for Ezh2-/- cells compared to WT (5.95 vs 2.94, p < 0.001) in resting cells, and 9.47 vs. 3.73 in fMLP-activated cells, p < 0.01. [Display omitted] The fraction of Ezh2-/- neutrophils generating ROS when stimulated with PMA is 2.4-fold higher than for WT cells. ROS production was greatly reduced in the presence of diphenyleneiodonium (DPI), confirming the role of NADPH oxidase in the generation of ROS. ConclusionOur results indicate impaired function of neutrophils derived from Ezh2-/- mice, demonstrating increased spontaneous cell death, impaired migration, decreased phagocytosis, and overproduction of ROS. Qualitative defects observed in neutrophils deficient for EZH2 may help explain the adverse prognosis associated with these mutations in MDS patients. Disclosures:Bejar:Genoptix: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Neutrophil Homeostasis and Periodontal Health in Children and Adults

This review summarizes the current state of knowledge on neutrophil basic biology and discusses how the breakdown of neutrophil homeostasis affects periodontal health. The homeostasis of neutrophils is tightly regulated through coordinated bone marrow production, release into the circulation, transmigration to and activation in peripheral tissues, and clearance of senescent neutrophils. Dysregulation of any of these homeostatic mechanisms at any age can cause severe periodontitis in humans and animal models. Accordingly, both impaired and excessive neutrophil activity (in terms of numbers or immune function) can precipitate periodontitis. Neutrophil defects of congenital origin (e.g., congenital neutropenia, leukocyte adhesion deficiency, and Chediak-Higashi syndrome) are associated with cutaneous and systemic infections and early-onset forms of periodontitis affecting both the primary and permanent dentitions of children. However, the strong association between congenital neutrophil disorders and early-onset periodontitis is not currently adequately explained mechanistically. This suggests the operation of as-yet-unknown molecular mechanisms, although the available body of evidence leaves no doubt that neutrophils are integral to periodontal tissue homeostasis and health.

Roles of Lung Epithelium in Neutrophil Recruitment during Pneumococcal Pneumonia

Epithelial cells line the respiratory tract and interface with the external world. Epithelial cells contribute to pulmonary inflammation, but specific epithelial roles have proven difficult to define. To discover unique epithelial activities that influence immunity during infection, we generated mice with nuclear factor-κB RelA mutated throughout all epithelial cells of the lung and coupled this approach with epithelial cell isolation from infected and uninfected lungs for cell-specific analyses of gene induction. The RelA mutant mice appeared normal basally, but in response to pneumococcus in the lungs they were unable to rapidly recruit neutrophils to the air spaces. Epithelial cells expressed multiple neutrophil-stimulating cytokines during pneumonia, all of which depended on RelA. Cytokine expression by nonepithelial cells was unaltered by the epithelial mutation of RelA. Epithelial cells were the predominant sources of CXCL5 and granulocyte-macrophage colony-stimulating factor (GM-CSF), whereas nonepithelial cells were major sources for other neutrophil-activating cytokines. Epithelial RelA mutation decreased whole lung levels of CXCL5 and GM-CSF during pneumococcal pneumonia, whereas lung levels of other neutrophil-recruiting factors were unaffected. Defective neutrophil recruitment in epithelial mutant mice could be rescued by administration of CXCL5 or GM-CSF. These results reveal a specialized immune function for the pulmonary epithelium, the induction of CXCL5 and GM-CSF, to accelerate neutrophil recruitment in the infected lung.

A Congenital Neutrophil Defect Syndrome Associated with Mutations in VPS45

BackgroundNeutrophils are the predominant phagocytes that provide protection against bacterial and fungal infections. Genetically determined neutrophil disorders confer a predisposition to severe infections and reveal novel mechanisms that control vesicular trafficking, hematopoiesis, and innate immunity.MethodsWe clinically evaluated seven children from five families who had neutropenia, neutrophil dysfunction, bone marrow fibrosis, and nephromegaly. To identify the causative gene, we performed homozygosity mapping using single-nucleotide polymorphism arrays, whole-exome sequencing, immunoblotting, immunofluorescence, electron microscopy, a real-time quantitative polymerase–chain-reaction assay, immunohistochemistry, flow cytometry, fibroblast motility assays, measurements of apoptosis, and zebrafish models. Correction experiments were performed by transfecting mutant fibroblasts with the nonmutated gene.ResultsAll seven affected children had homozygous mutations (Thr224Asn or Glu238Lys, depending on the child's ethnic origin) in VPS45, which encodes a protein that regulates membrane trafficking through the endosomal system. The level of VPS45 protein was reduced, as were the VPS45 binding partners rabenosyn-5 and syntaxin-16. The level of β1 integrin was reduced on the surface of VPS45-deficient neutrophils and fibroblasts. VPS45-deficient fibroblasts were characterized by impaired motility and increased apoptosis. A zebrafish model of vps45 deficiency showed a marked paucity of myeloperoxidase-positive cells (i.e., neutrophils). Transfection of patient cells with nonmutated VPS45 corrected the migration defect and decreased apoptosis.ConclusionsDefective endosomal intracellular protein trafficking due to biallelic mutations in VPS45 underlies a new immunodeficiency syndrome involving impaired neutrophil function. (Funded by the National Human Genome Research Institute and others.)

Significant Modifications of the Salivary Proteome Potentially Associated with Complications of Down Syndrome Revealed by Top-down Proteomics

People with Down syndrome, a frequent genetic disorder in humans, have increased risk of health problems associated with this condition. One clinical feature of Down syndrome is the increased prevalence and severity of periodontal disease in comparison with the general population. Because saliva plays an important role in maintaining oral health, in the present study the salivary proteome of Down syndrome subjects was investigated to explore modifications with respect to healthy subjects. Whole saliva of 36 Down syndrome subjects, divided in the age groups 10-17 yr and 18-50 yr, was analyzed by a top-down proteomic approach, based on the high performance liquid chromatography-electrospray ionization-MS analysis of the intact proteins and peptides, and the qualitative and quantitative profiles were compared with sex- and age-matched control groups. The results showed the following interesting features: 1) as opposed to controls, in Down syndrome subjects the concentration of the major salivary proteins of gland origin did not increase with age; as a consequence concentration of acidic proline rich proteins and S cystatins were found significantly reduced in older Down syndrome subjects with respect to matched controls; 2) levels of the antimicrobial α-defensins 1 and 2 and histatins 3 and 5 were significantly increased in whole saliva of older Down syndrome subjects with respect to controls; 3) S100A7, S100A8, and S100A12 levels were significantly increased in whole saliva of Down syndrome subjects in comparison with controls. The increased level of S100A7 and S100A12 may be of particular interest as a biomarker of early onset Alzheimer's disease, which is frequently associated with Down syndrome.

Functional defect of peripheral neutrophils in mice with induced deletion of CXCR2

Type 2 CXC chemokine receptor CXCR2 plays roles in development, tumorigenesis, and inflammation. CXCR2 also promotes demyelination and decreases remyelination by actions toward hematopoietic cells and nonhematopoietic cells. Germline CXCR2 deficient (Cxcr2(-/-) ) mice reported in 1994 revealed the complexity of CXCR2 function and its differential expression in varied cell-types. Here, we describe Cxcr2(fl/fl) mice for which the targeting construct was generated by recombineering based on homologous recombination in E. coli. Without recombination Cxcr2(fl/fl) mice have CXCR2 expression on neutrophils in peripheral blood, bone marrow and spleen. Cxcr2(fl/fl) mice were crossed to Mx-Cre mice in which Cre recombinase is induced by Type I interferons, elicited by injection with polyinosinic-polycytidylic acid (poly(I:C)). CXCR2-deficient neutrophils were observed in poly(I:C) treated Cxcr2(fl/fl) ::Mx-Cre(+) (Cxcr2-CKO) mice, but not in poly(I:C) treated Cxcr2(f//+) ::Mx-Cre(+) mice. CXCR2 deletion was mainly observed peripherally but not in the CNS. Cxcr2-CKO mice showed impaired neutrophil migration in sterile peritonitis. Cxcr2-CKO mice reported here will provide a genetic reagent to dissect roles of CXCR2 in the neutrophil granulocyte lineage. Furthermore Cxcr2(fl/fl) mice will provide useful genetic models to evaluate CXCR2 function in varied cell populations.

Interferon-β Production via Dectin-1-Syk-IRF5 Signaling in Dendritic Cells Is Crucial for Immunity to C. albicans

Type I interferon (IFN) is crucial during infection through its antiviral properties and by coordinating the immunocompetent cells involved in antiviral or antibacterial immunity. Type I IFN (IFN-α and IFN-β) is produced after virus or bacteria recognition by cytosolic receptors or membrane-bound TLR receptors following the activation of the transcription factors IRF3 or IRF7. IFN-β production after fungal infection was recently reported, although the underlying mechanism remains controversial. Here we describe that IFN-β production by dendritic cells (DCs) induced by Candida albicans is largely dependent on Dectin-1- and Dectin-2-mediated signaling. Dectin-1-induced IFN-β production required the tyrosine kinase Syk and the transcription factor IRF5. Type I IFN receptor-deficient mice had a lower survival after C.albicans infection, paralleled by defective renal neutrophil infiltration. IFN-β production by renal infiltrating leukocytes was severely reduced in C.albicans-infected mice with Syk-deficient DCs. These data indicate that Dectin-induced IFN-β production by renal DCs is crucial for defense against C.albicans infection.