RESUMO
Expression of the activating transcription factor 3 (ATF3) gene is induced by Toll-like receptor (TLR) signaling. In turn, ATF3 protein inhibits the expression of various TLR-driven proinflammatory genes. Given its counter-regulatory role in diverse innate immune responses, we defined the effects of ATF3 on neutrophilic airway inflammation in mice. ATF3 deletion was associated with increased lipopolysaccharide (LPS)-driven airway epithelia production of CXCL1, but not CXCL2, findings concordant with a consensus ATF3-binding site identified solely in the Cxcl1 promoter. Unexpectedly, ATF3-deficient mice did not exhibit increased airway neutrophilia after LPS challenge. Bone marrow chimeras revealed a specific reduction in ATF3(-/-) neutrophil recruitment to wild-type lungs. In vitro, ATF3(-/-) neutrophils exhibited a profound chemotaxis defect. Global gene expression analysis identified ablated Tiam2 expression in ATF3(-/-) neutrophils. TIAM2 regulates cellular motility by activating Rac1-mediated focal adhesion disassembly. Notably, ATF3(-/-) and ATF3-sufficient TIAM2 knockdown neutrophils, both lacking TIAM2, exhibited increased focal complex area, along with excessive CD11b-mediated F-actin polymerization. Together, our data describe a dichotomous role for ATF3-mediated regulation of neutrophilic responses: inhibition of neutrophil chemokine production but promotion of neutrophil chemotaxis.
Assuntos
Fator 3 Ativador da Transcrição/fisiologia , Doenças do Sistema Imunitário/genética , Transtornos Leucocíticos/genética , Fator 3 Ativador da Transcrição/genética , Animais , Células Cultivadas , Quimiocina CXCL1/metabolismo , Lipopolissacarídeos/farmacologia , Pulmão/citologia , Pulmão/imunologia , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos/genética , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismoRESUMO
All three cytochrome P450 1 (CYP1) monooxygenases are believed to participate in lipid mediator biosynthesis and/or their local inactivation; however, distinct metabolic steps are unknown. We used multiple-reaction monitoring and liquid chromatography-UV coupled with tandem mass spectrometry-based lipid-mediator metabololipidomics to identify and quantify three lipid-mediator metabolomes in basal peritoneal and zymosan-stimulated inflammatory exudates, comparing Cyp1a1/1a2/1b1(â»/â») C57BL/6J-background triple-knockout mice with C57BL/6J wild-type mice. Significant differences between untreated triple-knockout and wild-type mice were not found for peritoneal cell number or type or for basal CYP1 activities involving 11 identified metabolic steps. Following zymosan-initiated inflammation, 18 lipid mediators were identified, including members of the eicosanoids and specialized proresolving mediators (i.e., resolvins and protectins). Compared with wild-type mice, Cyp1 triple-knockout mice exhibited increased neutrophil recruitment in zymosan-treated peritoneal exudates. Zymosan stimulation was associated with eight statistically significantly altered metabolic steps: increased arachidonic acid-derived leukotriene B4 (LTB4) and decreased 5S-hydroxyeicosatetraenoic acid; decreased docosahexaenoic acid-derived neuroprotectin D1/protectin D1, 17S-hydroxydocosahexaenoic acid, and 14S-hydroxydocosahexaenoic acid; and decreased eicosapentaenoic acid-derived 18R-hydroxyeicosapentaenoic acid (HEPE), 15S-HEPE, and 12S-HEPE. In neutrophils analyzed ex vivo, elevated LTB4 levels were shown to parallel increased neutrophil numbers, and 20-hydroxy-LTB4 formation was found to be deficient in Cyp1 triple-knockout mice. Together, these results demonstrate novel contributions of CYP1 enzymes to the local metabolite profile of lipid mediators that regulate neutrophilic inflammation.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Mediadores da Inflamação/metabolismo , Inflamação/metabolismo , Transdução de Sinais/imunologia , Animais , Sistema Enzimático do Citocromo P-450/imunologia , Humanos , Inflamação/imunologia , Mediadores da Inflamação/imunologia , Lipídeos/imunologia , Metaboloma , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/metabolismoRESUMO
INTRODUCTION: A large proportion of clinical islet transplant recipients fail to initially achieve or sustain meaningful independence from exogenous insulin use. We hypothesized that immediate allograft injury is a key constraint on independence from exogenous insulin use. METHODS: Standard human leukocyte antigen genotyping was reviewed to identify nonshared polymorphisms between 21 prospectively recruited islet transplant recipients from a single institution and their respective donors. Human leukocyte antigen polymorphism-specific quantitative polymerase chain reaction was used to quantify donor DNA shed into blood by injured islets from serial sera acquired over the first 10 days postprocedure and examined for correlation with achievement of insulin independence. RESULTS: Nearly fourfold higher serum concentrations of donor DNA were detected in subjects whose grafts failed to generate insulin independence. The median for the average area under the curve in recipients who did and did not achieve insulin independence was 12 (range, 1-61) and 45 (range, 14-255) donor genome equivalents (gEq)-day/mL (p=0.03), respectively. CONCLUSIONS: These findings represent the first direct testing of allograft injury in humans undergoing islet cell transplantation. Injury to donor islets very soon after transplantation may represent an important barrier to achieving insulin independence other than adaptive immune responses targeting allografts at later times. In addition, soluble donor DNA merits further development as a quantifiable biomarker to evaluate new interventions aimed at mitigating immediate islet injury.
Assuntos
DNA/sangue , Transplante das Ilhotas Pancreáticas/efeitos adversos , Ilhotas Pancreáticas/lesões , Doadores de Tecidos , Adulto , Idoso , Biomarcadores/sangue , DNA/genética , Diabetes Mellitus Tipo 1/cirurgia , Feminino , Genótipo , Antígenos HLA/genética , Humanos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/etiologia , Estudos Prospectivos , SolubilidadeRESUMO
BACKGROUND: There is no reliable serum marker available to monitor incipient pancreas or islet-cell rejection. We tested the hypothesis that quantification of donor-specific genomic DNA in serum (from tissue damage) can serve as a marker of rejection. METHODS: Using a recently developed panel of HLA-specific quantitative PCR assays (Q-PCR), we tested 158 sera from 42 pancreas-kidney transplant recipients. Temporally related biopsies for 65 sera permitted analysis for correlation of donor DNA concentrations with rejection. RESULTS: Donor DNA concentrations were higher in sera from recipients who had experienced allograft rejection (n = 31) than from those who had not (n = 34). Median concentrations, expressed as the genome-equivalent (gEq) number of donor cells per 10(6) host cells, were 2613 and 59 gEq/10(6), respectively (P = 0.03). CONCLUSION: Q-PCR for donor-specific genetic polymorphisms merits further investigation as a noninvasive approach to monitor pancreas-kidney as well as other types of allograft rejection.