Defective Neutrophil Chemotaxis Research Articles

Myelodysplastic neoplasm-associated U2AF1 mutations induce host defense defects by compromising neutrophil chemotaxis

Myelodysplastic neoplasm (MDS) is a hematopoietic stem cell disorder that may evolve into acute myeloid leukemia. Fatal infection is among the most common cause of death in MDS patients, likely due to myeloid cell cytopenia and dysfunction in these patients. Mutations in genes that encode components of the spliceosome represent the most common class of somatically acquired mutations in MDS patients. To determine the molecular underpinnings of the host defense defects in MDS patients, we investigated the MDS-associated spliceosome mutation U2AF1-S34F using a transgenic mouse model that expresses this mutant gene. We found that U2AF1-S34F causes a profound host defense defect in these mice, likely by inducing a significant neutrophil chemotaxis defect. Studies in human neutrophils suggest that this effect of U2AF1-S34F likely extends to MDS patients as well. RNA-seq analysis suggests that the expression of multiple genes that mediate cell migration are affected by this spliceosome mutation and therefore are likely drivers of this neutrophil dysfunction.

Sex-dependent effect of aging on calcium signaling and expression of TRPM2 and CRAC channels in human neutrophils

The vulnerability of older adults to bacterial infections has been associated with age-related changes in neutrophils. We analyzed the consequences of aging on calcium (Ca2+) mobilization and TRPM2 and CRAC channels expression in human neutrophils. The percentages of granulocytes, mature neutrophils, and neutrophil precursors were equivalent between young and older adults. However, neutrophil chemotaxis towards IL-8, C5a, or fMLP was lower in older adults of both sexes. Interestingly, a stronger Ca2+ transient followed by an identical Ca2+ influx to IL-8 was observed in older adult females. In addition, the Ca2+ response to LPS was delayed and prolonged in neutrophils of older adult males. There was no significant difference in Ca2+ response to fMLP, C5a, or store-operated Ca2+ entry in the older adults. There were also no differences in the expression of CXCR2, CD88, FPLR1, and TLR4. Interestingly, TRPM2- and ORAI1-mRNA expression was lower in neutrophils of older adults, mainly in females. Both channels were detected intracellularly in the neutrophils. TRPM2 was in late endosomes in young adults and in lysosomes in older adult neutrophils. In summary, defective neutrophil chemotaxis in aging seemed not to stem from alterations in Ca2+ signals; nevertheless, the low TRPM2 and ORAI1 expression may affect other functions.

Фактори імунодефіциту у дітей з трисомією за 21-ю хромосомою (синдром Дауна): сучасна парадигма імунних і неімунних механізмів

Синдром Дауна — найчастіше генетичне захворювання. Діти з трисомією за 21-ю хромосомою часто хворіють на тяжкі, тривалі респіраторні інфекції, що є ознакою дефекту імунної системи. Анатомічні та функціональні дефекти респіраторної, серцево-судинної, травної системи у дітей із синдромом Дауна можуть порушувати природні бар’єри та сприяти розвитку інфекційного захворювання, що необхідно враховувати при лікуванні пацієнтів. Природжені аномалії імунної системи, що асоціюються з трисомією за 21-ю хромосомою, включають легку або середньотяжку Т- та В-клітинну лімфопенію зі значним зниженням нативних лімфоцитів, мітогеніндукованої Т-клітинної проліферації, специфічної відповіді антитіл на імунізацію та дефект хемотаксису нейтрофілів. На сьогодні доведена роль підвищеної експресії генів, зокрема SOD1 та RCAN1, у розвитку первинного імунодефіциту, дефіциту цинку та метаболічних факторів, що роблять різний внесок в імунологічні аномалії, підвищуючи ризик інфекції, та потребують подальших досліджень при синдромі Дауна.

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.

P84: COMBINED IMMUNODEFICIENCY DUE TO DEFICIENCY OF ACTINRELATED PROTEIN COMPLEX 1B (ARPC1B)

A number of cytoskeletal defects arising from perturbations in actin polymerisation and resulting in combined primary immunodeficiency (PID) have been described. Defects include Wiskott Aldrich Syndrome (WAS), WAS Interacting Protein (WIP) and Dedicator of Cytokinesis 8 (DOCK-8) deficiencies. A newly reported cytoskeletal defect is Actin-related Protein Complex 1B (ARPC1B) deficiency. Here we report the first Australian, youngest yet diagnosed, and 5th only reported case of ARPC1B deficiency. A female infant born to Nepalese parents presented with bloody diarrhoea and metabolic acidosis at 2 months of age. A diagnosis of cow’s milk enteritis was made and she was commenced on an elemental formula with resolution of the hematochezia and improvement in the diarrhoea. Subsequently she developed an eczematous rash which progressed to an erosive purpuric dermatitis with ulceration, and later took on a psoriasiform appearance. Skin biopsy showed a leucocytoclastic vasculitis. Her progress was complicated by cytomegalovirus infection, recurrent suppurative otitis media, twice by periorbital cellulitis, persistent oral candidiasis, urinary tract infection and failure to thrive. Investigations showed defective neutrophil chemotaxis and random migration, reduced Natural Killer cell cytotoxicity, decreased naïve T-cells, variable thrombocytopenia, eosinophilia, and raised IgA and IgE. Whole exome sequencing showed her to be homozygote for an exon 2 splice site mutation in ARPC1B (c.64+2T>A) which is predicted to cause loss of gene function. Western blot analysis showed low expression of the ARPC1B protein. ARPC1B is a regulatory subunit of the actin-related protein 2/3 complex critical for F-actin polymerisation in haemopoetic cells. Deficiency of ARPC1B results in defective actin polymerisation and a combined immunodeficiency similar but distinct to WAS.

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,...

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.

TRPC6 Regulates CXCR2-Mediated Chemotaxis of Murine Neutrophils

Unraveling the mechanisms involved in chemotactic navigation of immune cells is of particular interest for the development of new immunoregulatory therapies. It is generally agreed upon that members of the classical transient receptor potential channel family (TRPC) are involved in chemotaxis. However, the regulatory role of TRPC channels in chemoattractant receptor-mediated signaling has not yet been clarified in detail. In this study, we demonstrate that the TRPC6 channels play a pronounced role in CXCR2-mediated intermediary chemotaxis, whereas N-formyl-methionine-leucine-phenylalanine receptor-mediated end-target chemotaxis is TRPC6 independent. The knockout of TRPC6 channels in murine neutrophils led to a strongly impaired intermediary chemotaxis after CXCR2 activation which is not further reinforced by CXCR2, PI3K, or p38 MAPK inhibition. Furthermore, CXCR2-mediated Ca(2+) influx but not Ca(2+) store release was attenuated in TRPC6(-/-) neutrophils. We demonstrate that the TRPC6 deficiency affected phosphorylation of AKT and MAPK downstream of CXCR2 receptor activation and led to altered remodeling of actin. The relevance of this TRPC6-depending defect in neutrophil chemotaxis is underscored by our in vivo findings. A nonseptic peritoneal inflammation revealed an attenuated recruitment of neutrophils in the peritoneal cavity of TRPC6(-/-) mice. In summary, this paper defines a specific role of TRPC6 channels in CXCR2-induced intermediary chemotaxis. In particular, TRPC6-mediated supply of calcium appears to be critical for activation of downstream signaling components.

The Fcγ receptor III genotype as a risk factor for aggressive periodontitis in Korean patients

Aggressive periodontitis (AgP) represents an inflammatory disease with pathogenic features, such as onset in the juvenile or early adults years and severe and rapid destruction of periodontal tissue. It is now recognized that microbial factors can not solely be held responsible for AgP, although these are important in the initiation of periodontitis. 1 Genetically determined variance in host immune response bacteria has been identified in individuals with AgP. Many AgP (especially those with localized form) patients display defective neutrophil chemotaxis due to changes in chemotactic receptors or activation pathways. 2,31) It was after only the Kormann’s study4 on the genetic polymorphism of interleukin (IL) that many researchers were really being interested in the genetic factors and trying to find the scientific evidences of genetic factors that affect the pathogenesis of periodontitis. Since then, the wide spectrum of genes that might affect the progression of the peridontal disease, such as IL, tumor necrosis factor-α (TNF-α), Fcγ receptor (Fcγ R), vitamine D receptor, Nformyl receptor, matrix metalloproteinase (MMP) promoter, Toll-like receptor 2 (TLR2) and TLR4 have been studied for their role in the various types of periodontitis. Leukocyte receptors for the constant (or Fc-) part of immunoglobulins (FcR) link cellular and humoral branches of the immune systems, which are considered essential for host defense against periodontopathic bacteria. FcR for IgG (FcγR) may play a crucial role in the host defense against periodontopathic bacteria. 6-7 Three receptor

Hyper‐IgE Syndrome with Widespread Premalign Oral Papillomas Treated with Interferon α2b

We report a case of a 7-year-old girl with hyperimmunoglobulin-E syndrome presenting with widespread oral papillomas which were tested for human papilloma virus DNA and had shown to be at high/intermediate risk group for malignancy. She had elevated levels of IgE, recurrent sinopulmonary infections, atopic-like dermatitis, peripheral eosinophilia and defective neutrophil chemotaxis. Interferon alfa 2b therapy and chemoprophylaxis with sulfamethoxazole-trimethoprim was given. Although the papillomas partially improved with the treatment, sinopulmonary infections continue to occur.

RAC2 GTPase deficiency and myeloid cell dysfunction in human and mouse.

Rho GTPases including Rho, Rac, and Cdc42 act as intracellular molecular switches to control cellular responses such as actin cytoskeleton rearrangement, gene transcription, cell growth, and possibly transformation. Their roles have been increasingly implicated in activation of signaling processes in leukocytes including integrin-mediated signal transduction and growth factor-induced cell survival and proliferation pathways. In particular, functional disruption of Rac2, a hematopoietic-specific Rho GTPase, causes severe myeloid cell dysfunction in both mouse and man. Rac2-deficient mice and a human patient with a D57N Rac2 mutant share a phenotype of leukocytosis with defective neutrophil chemotaxis and superoxide production in response to some, but not all, agonists. Our studies also suggested that the phenotypic abnormalities associated with D57N may involve not only neutrophil cellular functions, but also abnormal cell survival in other hematopoietic cells. Together, these data demonstrate a critical and unique role for Rac2 in normal neutrophil function and define a new genetic immunodeficiency syndrome in humans.

Neutrophil-specific granule deficiency results from a novel mutation with loss of function of the transcription factor CCAAT/enhancer binding protein epsilon.

Neutrophil-specific granule deficiency (SGD) is a rare disorder characterized by recurrent pyogenic infections, defective neutrophil chemotaxis and bactericidal activity, and lack of neutrophil secondary granule proteins. CCAAT/enhancer binding protein (C/EBP)epsilon, a member of the leucine zipper family of transcription factors, is expressed primarily in myeloid cells, and its knockout mouse model possesses distinctive defects, including a lack of neutrophil secondary granule proteins. Sequence analysis of the genomic DNA of a patient with SGD revealed a five-basepair deletion in the second exon of the C/EBPepsilon locus. The predicted frame shift results in a truncation of the 32-kD major C/EBPepsilon isoform, with loss of the dimerization domain, DNA binding region, and transcriptional activity. The multiple functional defects observed in these early neutrophil progenitor cells, a consequence of C/EBPepsilon deficiency, define SGD as a defect in myelopoiesis and establish the requirement for C/EBPepsilon for the promyelocyte-myelocyte transition in myeloid differentiation.

Phosphorylation and the actin cytoskeleton in defective newborn neutrophil chemotaxis.

We have investigated the role of actin polymerization in the defective polymorphonuclear neutrophil (PMN) chemotaxis of the human newborn, and its regulation by protein kinase C and by phosphatases 1 and 2A. Isolated PMNs from adult volunteers and healthy term newborns, i.e. umbilical cord blood, were studied. Chemotaxis was measured by a modified micropore filter assay, and actin polymerization was assessed by flow cytometry. Chemotaxis of newborn PMNs (median 18 microm, range 9-21 microm) was significantly reduced compared with adult PMNs (median 23 microm, range 17-34 microm) (p < 0.001). Coincubation with the protein kinase C inhibitor bisindolylmaleimide GF109203X, did not significantly alter chemotaxis, whereas coincubation with the phosphatase inhibitors calyculin A or okadaic acid caused parallel dose-dependent inhibition of chemotaxis in adult and newborn PMNs. Peak actin polymerization was reduced in newborn compared with adult PMNs in response to stimulation with formyl-methionyl-leucyl-phenylalanine and zymosan-activated serum, but was normal in response to phorbol myristate acetate. Prior incubation for 5 min with bisindolylmaleimide GF109203X, calyculin A, or okadaic acid caused no significant alterations in the actin polymerization response to stimulation with formyl-methionyl-leucyl-phenylalanine. We conclude that: 1) newborn PMNs have reduced actin polymerization in response to stimulation with chemotactic agents which act via cell surface receptors, but not with phorbol myristate acetate, which acts directly in the cytoplasm. This suggests that a defect in cell signal transduction may be an underlying factor in defective newborn PMN chemotaxis. 2) Phosphatase inhibitors strongly inhibit chemotaxis but not actin polymerization, therefore phosphatases 1 and 2A may be important regulators of PMN chemotaxis, but this regulation takes place either at a point distal to actin polymerization or via another pathway. 3) Similar results in adult and newborn PMNs suggest that this is not the site of the underlying defect in newborn PMN chemotaxis.

HLA-DR alleles are associated with IDDM, but not with impaired neutrophil chemotaxis in IDDM.

Insulin-dependent diabetes mellitus (IDDM) is a risk factor for periodontitis. Depressed neutrophil chemotaxis has been demonstrated in IDDM and in early-onset periodontitis (EOP). HLA-DR antigens are associated with both IDDM and periodontitis. This investigation sought to determine an association of HLA-DR3, -DR4, and -DR53 with impaired neutrophil chemotaxis in an IDDM sample. The neutrophil chemotaxis index of 41 diabetics and 27 controls was determined by a modified Boyden chamber method, and certain class II HLA genotypes were determined by polymerase chain-reaction amplification of genomic DNA by means of sequence-specific primers (PCR-SSP). The mean chemotaxis index of the diabetics was significantly less than that of the controls (p < or = 0.02). HLA-DR3 (p < or = 0.002), -DR4 (p < 0.003), and -DR53 (p < or = 0.001) were associated with IDDM. Neutrophil chemotaxis and glucose metabolism were not significantly correlated. None of the HLA-DR alleles was associated with impaired neutrophil chemotaxis. Therefore, the neutrophil chemotaxis defect of IDDM appears to be independent of these HLA-DR-associated genes.

Defective neutrophil actin polymerisation and chemotaxis in stressed newborns

Abnormalities of polymorphonuclear leukocyte (PMN) function contribute to high rates of postoperative infection in the newborn and to the vulnerability of newborns to overwhelming bacterial and fungal sepsis. The authors investigated (1) the effects of major surgery and sepsis of PMN chemotaxis in the newborn and (2) the role of cytoskeletal rearrangements in regulating chemotaxis. The subjects studied included newborns with sepsis (n = 16), newborns who underwent major surgery (n = 7), healthy full-term newborns (n = 21), and healthy adult volunteers (n = 28). Peak actin polymerisation was diminished in all newborns (relative to the adults) after stimulation with formyl methionyl leucyl phenylalanine (FMLP) (10 nmol/L), and with zymosan activated serum (ZAS) (10%). Major surgery and sepsis in newborns caused no further reduction in actin polymerisation. Changes in PMN shape after stimulation with FMLP were reduced in the newborn. PMN chemotaxis was significantly lower in healthy newborns than in adults ( 17 ± 4μmv 24 ± 5μm; P < .0001) and was even lower in septic newborns (11 ± 4 μm; P < .005). Surgery and anaesthesia did not alter chemotaxis.

TNFα and IL‐1β in serum of LJP patients with normal and defective neutrophil chemotaxis

TNFα and IL‐1β in serum of LJP patients with normal and defective neutrophil chemotaxis

Characterization of GP110, a neutrophil surface protein

Localized juvenile periodontosis is associated with a defect of neutrophil chemotaxis that is characterized by selective depletion of a surface protein with a molecular mass of 110 kDa (GP110). Data on partial characterization of GP110 suggest that it is a glycoprotein which is enriched in Lys, Glu, His, Leu, and Ala residues.

The Effect of Granulocyte Colony-Stimulating Factor (G-CSF) upon Burn-induced Defective Neutrophil Chemotaxis

Severe thermal injury results in impairment of granulocyte production and function. The ability to improve the functional capacity of neutrophils could contribute to a reduced morbidity and mortality from sepsis following thermal injury. Previous studies from this laboratory have shown that rhG-CSF increases the number of femoral marrow granulocyte progenitor cells and circulating neutrophils as well as the survival rate following burn wound infection. The studies reported here examine the effect of in-vivo administration of rhG-CSF on neutrophil chemotaxis following a burn injury and also following superimposed Pseudomonas burn wound sepsis in mice. Casein-elicited peritoneal neutrophils were harvested 72 hours after burn injury and 24 hours after infection. Chemotaxis was assessed using microchemotaxis chambers and 10(-5) M fMet-Phe as a chemoattractant. The number of neutrophils that migrated into the filter was used as an index of directed chemotaxis. Burn injury resulted in depressed chemotaxis compared with sham or sham/G-CSF-treated animals (p less than 0.05). Administration of rhG-CSF to burned animals resulted in a level of neutrophil chemotaxis comparable with that in control animals. The presence of a burn wound infection caused no further impairment of chemotaxis. Administration of rhG-CSF to animals with a burn wound infection resulted in improved chemotaxis compared with sham, burned, and burned/infected animals. The beneficial effect of G-CSF following burn wound infections from this and previous studies appears to be a combination of expanded numbers of myeloid elements and preservation of their function.

Abnormal Neutrophil Chemotaxis after Successful Bone Marrow Transplantation.

Twenty patients with self-sustaining hematopoiesis were evaluated for neutrophil functions and bone marrow histology 7 to 34 months after bone marrow transplantation (BMT) (7 allogeneic, 13 autologous) performed for acute leukemia in complete remission (11 patients), Hodgkin's lymphoma (2 patients), chronic myeloid leukemia (6 patients) or severe aplastic anemia (1 patient). The chemotactic response toward zymosan-treated serum was severely depressed (<35% of normal) in peripheral neutrophils of 11 patients (2 allogeneic and 9 autologous BMT) and moderately defective (35-70% of normal) in 5 others (2 allogeneic and 3 autologous BMT). On the other hand, phagocytic activity, activation of the metabolic burst and surface expression of CD11/CD18 molecules were within normal limits or moderately increased. The chemotactic defect was independent of age, sex, conditioning regimen and the time period after marrow infusion. The incidence of defective chemotaxis was much greater in patients receiving an autologous BMT (92% of the patients) than in those who had an allogeneic BMT (57% of the patients). Simultaneous bone marrow biopsy studies showed significant stromal alterations in most of our patients; since the bone marrow microenvironment plays an essential role in the process of blood cell formation and release, these observations suggest that defective neutrophil chemotaxis may well serve as a marker of abnormal post-transplant hematopoiesis.

Moderate zinc deficiency in rhesus monkeys. An intrinsic defect of neutrophil chemotaxis corrected by zinc repletion.

Experimental animals fed zinc-deficient diets are well known for susceptibility to infections and impaired mitogen response and Ig production. However, the levels of zinc deficiency used have generally been severe, not comparable to human populations, and have not addressed neutrophil function. To address this issue we have studied the effect in rhesus monkeys of a well defined moderately zinc-deficient (MZ) diet on polymorphonuclear leukocyte (PMN) function. Female adult rhesus monkeys were fed either a control (100 micrograms Zn/g) or MZ (2 micrograms Zn/g) diet for 9 mo with quantitation of PMN chemotaxis, and phagocytosis of opsonized yeast. In addition, membrane potential and secretion responses (changes in 90 degrees light scatter) and changes in PMN shape (forward light scatter shifts) were also measured. When compared to the PMN of animals fed control diets, there was a significant reduction in chemotaxis to FMLP of MZ-fed monkey PMN. Although shape change, cell membrane depolarization, as well as phagocytosis were not significantly different among the two groups, the PMN of MZ animals had significantly lower relative loss of orthogonal light scatter (degranulation) due primarily to a lower resting orthogonal light scatter and also a smaller loss when stimulated with FMLP. In vitro addition of zinc to the cells (25 microM) did not improve chemotaxis, and in fact, was inhibitory for most control and zinc-deficient cells. However, after 2 wk of dietary zinc repletion (100 micrograms Zn/g), chemotaxis in the low zinc group was higher and comparable to the control response. These data indicate that zinc deficiency is associated with an intrinsic PMN defect that specifically affects chemotaxis and is corrected with dietary zinc repletion.