RESUMO
BACKGROUND: In children, the acute pyelonephritis that can result from urinary tract infections (UTIs), which commonly ascend from the bladder to the kidney, is a growing concern because it poses a risk of renal scarring and irreversible loss of kidney function. To date, the cellular mechanisms underlying acute pyelonephritis-driven renal scarring remain unknown. METHODS: We used a preclinical model of uropathogenic Escherichia coli-induced acute pyelonephritis to determine the contribution of neutrophils and monocytes to resolution of the condition and the subsequent development of kidney fibrosis. We used cell-specific monoclonal antibodies to eliminate neutrophils, monocytes, or both. Bacterial ascent and the cell dynamics of phagocytic cells were assessed by biophotonic imaging and flow cytometry, respectively. We used quantitative RT-PCR and histopathologic analyses to evaluate inflammation and renal scarring. RESULTS: We found that neutrophils are critical to control bacterial ascent, which is in line with previous studies suggesting a protective role for neutrophils during a UTI, whereas monocyte-derived macrophages orchestrate a strong, but ineffective, inflammatory response against uropathogenic, E. coli-induced, acute pyelonephritis. Experimental neutropenia during acute pyelonephritis resulted in a compensatory increase in the number of monocytes and heightened macrophage-dependent inflammation in the kidney. Exacerbated macrophage-mediated inflammatory responses promoted renal scarring and compromised renal function, as indicated by elevated serum creatinine, BUN, and potassium. CONCLUSIONS: These findings reveal a previously unappreciated outcome for neutrophil-macrophage imbalance in promoting host susceptibility to acute pyelonephritis and the development of permanent renal damage. This suggests targeting dysregulated macrophage responses might be a therapeutic tool to prevent renal scarring during acute pyelonephritis.
Assuntos
Cicatriz/fisiopatologia , Rim/fisiopatologia , Macrófagos/citologia , Neutrófilos/citologia , Pielonefrite/metabolismo , Animais , Escherichia coli , Feminino , Fibrose/microbiologia , Fibrose/fisiopatologia , Inflamação , Rim/microbiologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Fagocitose , Pielonefrite/microbiologia , Pielonefrite/fisiopatologia , Infecções Urinárias/microbiologia , Infecções Urinárias/fisiopatologiaRESUMO
The signaling networks regulating antimicrobial activity during urinary tract infection (UTI) are incompletely understood. Interleukin-6 (IL-6) levels increase with UTI severity, but the specific contributions of IL-6 to host immunity against bacterial uropathogens are unknown. To clarify this we tested whether IL-6 activates the Stat3 transcription factor, to drive a program of antimicrobial peptide gene expression in infected urothelium during UTI. Transurethral inoculation of uropathogenic Escherichia coli led to IL-6 secretion, urothelial Stat3 phosphorylation, and activation of antimicrobial peptide transcription, in a Toll-like receptor 4-dependent manner in a murine model of cystitis. Recombinant IL-6 elicited Stat3 phosphorylation in primary urothelial cells in vitro, and systemic IL-6 administration promoted urothelial Stat3 phosphorylation and antimicrobial peptide expression in vivo. IL-6 deficiency led to decreased urothelial Stat3 phosphorylation and antimicrobial peptide mRNA expression following UTI, a finding mirrored by conditional Stat3 deletion. Deficiency in IL-6 or Stat3 was associated with increased formation of intracellular bacterial communities, and exogenous IL-6 reversed this phenotype in IL-6 knockout mice. Moreover, chronic IL-6 depletion led to increased renal bacterial burden and severe pyelonephritis in C3H/HeOuJ mice. Thus, IL-6/Stat3 signaling drives a transcriptional program of antimicrobial gene expression in infected urothelium, with key roles in limiting epithelial invasion and ascending infection.
Assuntos
Cistite/metabolismo , Infecções por Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Interleucina-6/metabolismo , Fator de Transcrição STAT3/metabolismo , Bexiga Urinária/metabolismo , Infecções Urinárias/metabolismo , Urotélio/metabolismo , Animais , Linhagem Celular , Cistite/genética , Cistite/microbiologia , Modelos Animais de Doenças , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Feminino , Hepcidinas/genética , Hepcidinas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas a Pancreatite/genética , Proteínas Associadas a Pancreatite/metabolismo , Fosforilação , Fator de Transcrição STAT3/deficiência , Fator de Transcrição STAT3/genética , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Bexiga Urinária/microbiologia , Infecções Urinárias/genética , Infecções Urinárias/microbiologia , Urotélio/microbiologiaRESUMO
Acquired renal scarring occurs in a subset of patients following febrile urinary tract infections and is associated with hypertension, proteinuria, and chronic kidney disease. Limited knowledge of histopathology, immune cell recruitment, and gene expression changes during pyelonephritis restricts the development of therapies to limit renal scarring. Here, we address this knowledge gap using immunocompetent mice with vesicoureteral reflux. Transurethral inoculation of uropathogenic Escherichia coli in C3H/HeOuJ mice leads to renal mucosal injury, tubulointerstitial nephritis, and cortical fibrosis. The extent of fibrosis correlates most significantly with inflammation at 7 and 28 days postinfection. The recruitment of neutrophils and inflammatory macrophages to infected kidneys is proportional to renal bacterial burden. Transcriptome analysis reveals molecular signatures associated with renal ischemia-reperfusion injury, immune cell chemotaxis, and leukocyte activation. This murine model recapitulates the cardinal histopathological features observed in humans with acquired renal scarring following pyelonephritis. The integration of histopathology, quantification of cellular immune influx, and unbiased transcriptional profiling begins to define potential mechanisms of tissue injury during pyelonephritis in the context of an intact immune response. The clear relationship between inflammatory cell recruitment and fibrosis supports the hypothesis that acquired renal scarring arises as a consequence of excessive host inflammation and suggests that immunomodulatory therapies should be investigated to reduce renal scarring in patients with pyelonephritis.
Assuntos
Cicatriz/metabolismo , Escherichia coli/isolamento & purificação , Inflamação/microbiologia , Rim/microbiologia , Pielonefrite/microbiologia , Refluxo Vesicoureteral/imunologia , Animais , Modelos Animais de Doenças , Feminino , Fibrose/imunologia , Fibrose/microbiologia , Inflamação/imunologia , Inflamação/patologia , Rim/patologia , Camundongos , Camundongos Endogâmicos C3H , Nefrite Intersticial/imunologia , Nefrite Intersticial/microbiologia , Nefrite Intersticial/patologia , Pielonefrite/imunologia , Traumatismo por Reperfusão/microbiologia , Traumatismo por Reperfusão/patologia , Refluxo Vesicoureteral/microbiologiaRESUMO
Urinary tract infections (UTIs) commonly afflict people with diabetes. To better understand the mechanisms that predispose diabetics to UTIs, we employ diabetic mouse models and altered insulin signaling to show that insulin receptor (IR) shapes UTI defenses. Our findings are validated in human biosamples. We report that diabetic mice have suppressed IR expression and are more susceptible to UTIs caused by uropathogenic Escherichia coli (UPEC). Systemic IR inhibition increases UPEC susceptibility, while IR activation reduces UTIs. Localized IR deletion in bladder urothelium promotes UTI by increasing barrier permeability and suppressing antimicrobial peptides. Mechanistically, IR deletion reduces nuclear factor κB (NF-κB)-dependent programming that co-regulates urothelial tight junction integrity and antimicrobial peptides. Exfoliated urothelial cells or urine samples from diabetic youths show suppressed expression of IR, barrier genes, and antimicrobial peptides. These observations demonstrate that urothelial insulin signaling has a role in UTI prevention and link IR to urothelial barrier maintenance and antimicrobial peptide expression.
Assuntos
Receptor de Insulina , Transdução de Sinais , Bexiga Urinária , Infecções Urinárias , Urotélio , Receptor de Insulina/metabolismo , Infecções Urinárias/microbiologia , Infecções Urinárias/metabolismo , Infecções Urinárias/patologia , Animais , Urotélio/metabolismo , Urotélio/patologia , Urotélio/microbiologia , Humanos , Bexiga Urinária/microbiologia , Bexiga Urinária/patologia , Bexiga Urinária/metabolismo , Camundongos , Escherichia coli Uropatogênica/patogenicidade , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Feminino , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Insulina/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , MasculinoRESUMO
INTRODUCTION: The ribonuclease (RNase) A superfamily encodes cationic antimicrobial proteins with potent microbicidal activity toward uropathogenic bacteria. Ribonuclease 6 (RNase6) is an evolutionarily conserved, leukocyte-derived antimicrobial peptide with potent microbicidal activity toward uropathogenic Escherichia coli (UPEC), the most common cause of bacterial urinary tract infections (UTIs). In this study, we generated Rnase6-deficient mice to investigate the hypothesis that endogenous RNase 6 limits host susceptibility to UTI. METHODS: We generated a Rnase6EGFP knock-in allele to identify cellular sources of Rnase6 and determine the consequences of homozygous Rnase6 deletion on antimicrobial activity and UTI susceptibility. RESULTS: We identified monocytes and macrophages as the primary cellular sources of Rnase6 in bladders and kidneys of Rnase6EGFP/+ mice. Rnase6 deficiency (i.e., Rnase6EGFP/EGFP) resulted in increased upper urinary tract UPEC burden during experimental UTI, compared to Rnase6+/+ controls. UPEC displayed increased intracellular survival in Rnase6-deficient macrophages. CONCLUSION: Our findings establish that RNase6 prevents pyelonephritis by promoting intracellular UPEC killing in monocytes and macrophages and reinforce the overarching contributions of endogenous antimicrobial RNase A proteins to host UTI defense.
Assuntos
Infecções por Escherichia coli , Macrófagos , Camundongos Knockout , Ribonucleases , Infecções Urinárias , Escherichia coli Uropatogênica , Animais , Infecções Urinárias/imunologia , Infecções Urinárias/microbiologia , Camundongos , Escherichia coli Uropatogênica/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Infecções por Escherichia coli/imunologia , Ribonucleases/metabolismo , Ribonucleases/genética , Camundongos Endogâmicos C57BL , Humanos , Monócitos/imunologia , Modelos Animais de Doenças , Feminino , Células CultivadasRESUMO
Mounting evidence suggests that antimicrobial peptides and proteins (AMPs) belonging to the RNase A superfamily have a critical role in defending the bladder and kidney from bacterial infection. RNase 6 has been identified as a potent, leukocyte-derived AMP, but its impact on urinary tract infection (UTI) in vivo has not been demonstrated. To test the functional role of human RNase 6, we generated RNASE6 transgenic mice and studied their susceptibility to experimental UTI. In addition, we generated bone marrow-derived macrophages to study the impact of RNase 6 on antimicrobial activity within a cellular context. When subjected to experimental UTI, RNASE6 transgenic mice developed reduced uropathogenic Escherichia coli (UPEC) burden, mucosal injury, and inflammation compared to non-transgenic controls. Monocytes and macrophages were the predominant cellular sources of RNase 6 during UTI, and RNASE6 transgenic macrophages were more proficient at intracellular UPEC killing than non-transgenic controls. Altogether, our findings indicate a protective role for human RNase 6 during experimental UTI.
Assuntos
Ribonucleases , Infecções Urinárias , Animais , Humanos , Camundongos , Endorribonucleases/genética , Rim , Camundongos Transgênicos , Ribonucleases/genética , Bexiga Urinária/microbiologiaRESUMO
Chemokines induce calcium (Ca(2+)) signaling and chemotaxis in dendritic cells (DCs), but the molecular players involved in shaping intracellular Ca(2+) changes remain to be characterized. Using siRNA and knockout mice, we show that in addition to inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release and store-operated Ca(2+) entry (SOCE), the transient receptor potential melastatin 2 (TRPM2) channel contributes to Ca(2+) release but not Ca(2+) influx in mouse DCs. Consistent with these findings, TRPM2 expression in DCs is restricted to endolysosomal vesicles, whereas in neutrophils, the channel localizes to the plasma membrane. TRPM2-deficient DCs show impaired maturation and severely compromised chemokine-activated directional migration as well as bacterial-induced DC trafficking to the draining lymph nodes. Defective DC chemotaxis is due to perturbed chemokine-receptor-initiated Ca(2+) signaling mechanisms, which include suppression of TRPM2-mediated Ca(2+) release and secondary modification of SOCE. DCs deficient in both TRPM2 and IP(3) receptor signaling lose their ability to perform chemotaxis entirely. These results highlight TRPM2 as a key player regulating DC chemotaxis through its function as Ca(2+) release channel and confirm ADP-ribose as a novel second messenger for intracellular Ca(2+) mobilization.
Assuntos
Cálcio/metabolismo , Quimiotaxia/fisiologia , Células Dendríticas/citologia , Células Dendríticas/fisiologia , Lisossomos/metabolismo , Canais de Cátion TRPM/metabolismo , Adenosina Difosfato Ribose , Animais , Sinalização do Cálcio/fisiologia , Quimiocinas/farmacologia , Células Dendríticas/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Inflamassomos/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente PequenoRESUMO
Infectious peritonitis is a common complication in patients undergoing chronic peritoneal dialysis (PD), limiting the duration of PD as a modality for renal replacement therapy and increasing patient morbidity and mortality. Antimicrobial peptides (AMPs) serve critical roles in mucosal defense, but their expression and activity during peritonitis are poorly understood. We hypothesized that AMPs belonging to the Ribonuclease (RNase) A Superfamily are present in peritoneal fluid and increase during peritonitis in patients undergoing chronic PD. In the absence of peritonitis, we detected RNase 3, RNase 6, and RNase 7 in cell-free supernatants and viable cells obtained from peritoneal fluid of chronic PD patients. The cellular sources of these RNases were eosinophils (RNase 3), macrophages (RNase 6), and mesothelial cells (RNase 7). During peritonitis, RNase 3 increased 55-fold and RNase 7 levels increased 3-fold on average, whereas RNase 6 levels were unchanged. The areas under the receiver-operating characteristic curves for RNase 3 and RNase 7 were 0.99 (95% confidence interval (CI): 0.96-1.0) and 0.79 (95% CI: 0.64-0.93), respectively, indicating their potential as biomarkers of peritonitis. Discrete omental reservoirs of these RNases were evident in patients with end stage kidney disease prior to PD initiation, and omental RNase 3 reactive cells increased in patients undergoing PD with a history of peritonitis. We propose that constitutive and inducible pools of antimicrobial RNases form a network to shield the peritoneal cavity from microbial invasion in patients undergoing chronic PD.
Assuntos
Diálise Peritoneal/efeitos adversos , Peritonite/metabolismo , Ribonuclease Pancreático/análise , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/metabolismo , Anti-Infecciosos/metabolismo , Líquido Ascítico/microbiologia , Criança , Pré-Escolar , Feminino , Humanos , Falência Renal Crônica/terapia , Masculino , Pessoa de Meia-Idade , Peptídeos/análise , Peptídeos/metabolismo , Diálise Peritoneal/métodos , Peritônio/metabolismo , Peritonite/etiologia , Ribonuclease Pancreático/metabolismo , Ribonucleases/análiseRESUMO
Helminths cause chronic infections and affect the immune response to unrelated inflammatory diseases. Although helminths have been used therapeutically to ameliorate inflammatory conditions, their anti-inflammatory properties are poorly understood. Alternatively activated macrophages (AAMÏs) have been suggested as the anti-inflammatory effector cells during helminth infections. Here, we define the origin of AAMÏs during infection with Taenia crassiceps, and their disease-modulating activity on the Experimental Autoimmune Encephalomyelitis (EAE). Our data show two distinct populations of AAMÏs, based on the expression of PD-L1 and PD-L2 molecules, resulting upon T. crassiceps infection. Adoptive transfer of Ly6C+ monocytes gave rise to PD-L1+/PD-L2+, but not PD-L1+/PD-L2- cells in T. crassiceps-infected mice, demonstrating that the PD-L1+/PD-L2+ subpopulation of AAMÏs originates from blood monocytes. Furthermore, adoptive transfer of PD-L1+/PD-L2+ AAMÏs into EAE induced mice reduced disease incidence, delayed disease onset, and diminished the clinical disability, indicating the critical role of these cells in the regulation of autoimmune disorders.
Assuntos
Transferência Adotiva/métodos , Antígenos Ly/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Ativação de Macrófagos , Células Precursoras de Monócitos e Macrófagos/imunologia , Taenia/imunologia , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Células Cultivadas , Encefalomielite Autoimune Experimental/terapia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Proteína 2 Ligante de Morte Celular Programada 1/genética , Proteína 2 Ligante de Morte Celular Programada 1/metabolismoRESUMO
Despite the continually increasing rates of adverse perinatal outcomes across the globe, the molecular mechanisms that underlie adverse perinatal outcomes are not completely understood. Clinical studies report that 10% of pregnant women will experience a urinary tract infection (UTI) and there is an association of UTIs with adverse perinatal outcomes. We introduced bacterial cystitis into successfully outbred female mice at gestational day 14 to follow pregnancy outcomes and immunological responses to determine the mechanisms that underlie UTI-mediated adverse outcomes. Outbred fetuses from mothers experiencing localized cystitis displayed intrauterine growth restriction (20-80%) as early as 48 hours post-infection and throughout the remainder of normal gestation. Robust infiltration of cellular innate immune effectors was observed in the uteroplacental tissue following introduction of UTI despite absence of viable bacteria. The magnitude of serum proinflammatory cytokines is elevated in the maternal serum during UTI. This study demonstrates that a localized infection can dramatically impact the immunological status as well as the function of non-infected distal organs and tissues. This model can be used as a platform to determine the mechanism(s) by which proinflammatory changes occur between non-contiguous genitourinary organs.
Assuntos
Cistite/patologia , Infecções por Escherichia coli/etiologia , Infecções por Escherichia coli/patologia , Retardo do Crescimento Fetal , Infecções Urinárias/etiologia , Infecções Urinárias/patologia , Escherichia coli Uropatogênica/isolamento & purificação , Animais , Cistite/imunologia , Citocinas/sangue , Células Dendríticas/imunologia , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Feminino , Retardo do Crescimento Fetal/etiologia , Retardo do Crescimento Fetal/patologia , Humanos , Infiltração Leucêmica , Leucócitos Mononucleares/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos , Placenta/metabolismo , Gravidez , Complicações Infecciosas na Gravidez , Transcrição Gênica , Infecções Urinárias/imunologia , Infecções Urinárias/microbiologiaRESUMO
Growth hormone (GH) and insulin-like growth factor-I (IGF-I) are key links to nutritional condition and growth regulation in teleost. To understand the endocrine mechanism of growth regulation in grouper, we cloned the cDNAs for grouper GH and IGF-I and examined their mRNA expression during different nutritional status. Grouper GH cDNA is 936 base pairs (bp) long excluding the poly-A tail. It contained untranslated regions of 85 and 231bp in the 5'- and 3'-ends, respectively. It has an open reading frame of 612bp coding for a signal peptide of 17 amino acids (aa) and a mature hormone of 187aa residues. Based on the aa sequence of the mature hormone, grouper GH shows higher sequence identity (>76%) to GHs of perciforms than to GHs of cyprinids and salmonids (53-69%). Grouper preproIGF-I cDNA consisted of 558bp, which codes for 186aa. This is composed of 44aa for the signal peptide, 68aa for the mature peptide comprising B, C, A, and D domains, and 74aa for the E domain. Mature grouper IGF-I shows very high sequence identity to IGF-I of teleost fishes (84-97%) compared to advanced groups of vertebrates such as chicken, pig, and human (80%). Using DNA primers specific for grouper GH and IGF-I, the changes in mRNA levels of pituitary GH and hepatic IGF-I in response to starvation and refeeding were examined by a semi-quantitative RT-PCR. Significant elevation of GH mRNA level was observed after 2 weeks of food deprivation, and increased further after 3 and 4 weeks of starvation. GH mRNA level in fed-controls did not change significantly during the same period. Hepatic IGF-I mRNA level decreased significantly starting after 1 week of starvation until the 4th week. There was no significant change in IGF-I mRNA levels in fed-controls. One week of refeeding can restore the GH and IGF-I mRNA back to its normal levels. Deprivation of food for 1-4 weeks also resulted in cessation of growth and decrease in condition factor.