RESUMEN
Klebsiella pneumoniae is a common cause of Gram-negative pneumonia. The spread of antibiotic-resistant and hypervirulent strains has made treatment more challenging. This study sought to determine the immunomodulatory, antibacterial, and therapeutic potential of purified murine stem cell Ag-1+ (Sca-1+) lung mesenchymal stem cells (LMSCs) using in vitro cell culture and an in vivo mouse model of pneumonia caused by K pneumoniae. Sca-1+ LMSCs are plastic adherent, possess colony-forming capacity, express mesenchymal stem cell markers, differentiate into osteogenic and adipogenic lineages in vitro, and exhibit a high proliferative capacity. Further, these Sca-1+ LMSCs are morphologically similar to fibroblasts but differ ultrastructurally. Moreover, Sca-1+ LMSCs have the capacity to inhibit LPS-induced secretion of inflammatory cytokines by bone marrow-derived macrophages and neutrophils in vitro. Sca-1+ LMSCs inhibit the growth of K pneumoniae more potently than do neutrophils. Sca-1+ LMSCs also possess the intrinsic ability to phagocytize and kill K. pneumoniae intracellularly. Whereas the induction of autophagy promotes bacterial replication, inhibition of autophagy enhances the intracellular clearance of K. pneumoniae in Sca-1+ LMSCs during the early time of infection. Adoptive transfer of Sca-1+ LMSCs in K. pneumoniae-infected mice improved survival, reduced inflammatory cells in bronchoalveolar lavage fluid, reduced inflammatory cytokine levels and pathological lesions in the lung, and enhanced bacterial clearance in the lung and in extrapulmonary organs. To our knowledge, these results together illustrate for the first time the protective role of LMSCs in bacterial pneumonia.
Asunto(s)
Infecciones por Klebsiella , Células Madre Mesenquimatosas , Neumonía Bacteriana , Animales , Klebsiella , Klebsiella pneumoniae , Pulmón , RatonesRESUMEN
Neutrophil migration to the site of bacterial infection is a critical step in host defense. Exclusively produced in the bone marrow, neutrophil release into the blood is tightly controlled. Although the chemokine CXCL1 induces neutrophil influx during bacterial infections, its role in regulating neutrophil recruitment, granulopoiesis, and neutrophil mobilization in response to lung infection-induced sepsis is unclear. Here, we used a murine model of intrapulmonary Streptococcus pneumoniae infection to investigate the role of CXCL1 in host defense, granulopoiesis, and neutrophil mobilization. Our results demonstrate that CXCL1 augments neutrophil influx to control bacterial growth in the lungs, as well as bacterial dissemination, resulting in improved host survival. This was shown in Cxcl1 -/- mice, which exhibited defective amplification of early neutrophil precursors in granulocytic compartments, and CD62L- and CD49d-dependent neutrophil release from the marrow. Administration of recombinant CXCL2 and CXCL5 after infection rescues the impairments in neutrophil-dependent host defense in Cxcl1 -/- mice. Taken together, these findings identify CXCL1 as a central player in host defense, granulopoiesis, and mobilization of neutrophils during Gram-positive bacterial pneumonia-induced sepsis.
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Quimiocina CXCL1/fisiología , Homeostasis , Pulmón/inmunología , Infiltración Neutrófila/inmunología , Infecciones Neumocócicas/complicaciones , Neumonía Bacteriana/complicaciones , Sepsis/inmunología , Animales , Quimiocina CXCL2/genética , Quimiocina CXCL2/metabolismo , Quimiocina CXCL5/genética , Quimiocina CXCL5/metabolismo , Femenino , Pulmón/microbiología , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones Neumocócicas/microbiología , Infecciones Neumocócicas/patología , Neumonía Bacteriana/microbiología , Neumonía Bacteriana/patología , Sepsis/metabolismo , Sepsis/microbiología , Serogrupo , Streptococcus pneumoniae/fisiologíaRESUMEN
Gram-positive bacteria, including Staphylococcus aureus are endemic in the U.S., which cause life-threatening necrotizing pneumonia. Neutrophils are known to be critical for clearance of S. aureus infection from the lungs and extrapulmonary organs. Therefore, we investigated whether the NLRP6 inflammasome regulates neutrophil-dependent host immunity during pulmonary S. aureus infection. Unlike their wild-type (WT) counterparts, NLRP6 knockout (KO) mice were protected against pulmonary S. aureus infection as evidenced by their higher survival rate and lower bacterial burden in the lungs and extrapulmonary organs. In addition, NLRP6 KO mice displayed increased neutrophil recruitment following infection, and when neutrophils were depleted the protective effect was lost. Furthermore, neutrophils from the KO mice demonstrated enhanced intracellular bacterial killing and increased NADPH oxidase-dependent ROS production. Intriguingly, we found higher NK cell-mediated IFN-γ production in KO mouse lungs, and treatment with IFN-γ was found to enhance the bactericidal ability of WT and KO neutrophils. The NLRP6 KO mice also displayed decreased pyroptosis and necroptosis in the lungs following infection. Blocking of pyroptosis and necroptosis in WT mice resulted in increased survival, reduced bacterial burden in the lungs, and attenuated cytokine production. Taken together, these novel findings show that NLRP6 serves as a negative regulator of neutrophil-mediated host defense during Gram-positive bacterial infection in the lungs through regulating both neutrophil influx and function. These results also suggest that blocking NLRP6 to augment neutrophil-associated bacterial clearance should be considered as a potential therapeutic intervention strategy for treatment of S. aureus pneumonia.
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Infiltración Neutrófila/inmunología , Neumonía Estafilocócica/inmunología , Receptores de Superficie Celular/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Femenino , Interacciones Huésped-Patógeno/inmunología , Humanos , Inflamasomas/inmunología , Interferón gamma/biosíntesis , Células Asesinas Naturales/inmunología , Pulmón/inmunología , Pulmón/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neumonía Necrotizante/inmunología , Neumonía Necrotizante/microbiología , Neumonía Estafilocócica/microbiología , Piroptosis/inmunología , Especies Reactivas de Oxígeno/metabolismo , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Staphylococcus aureus/inmunología , Regulación hacia ArribaRESUMEN
Polymicrobial sepsis is the result of an exaggerated host immune response to bacterial pathogens. Animal models and human studies demonstrate that alcohol intoxication is a key risk factor for sepsis-induced mortality. Multiple chemokines, such as CXCL1, CXCL2 and CXCL5 are critical for neutrophil recruitment and proper function of neutrophils. However, it is not quite clear the mechanisms by which acute alcohol suppresses immune responses and whether alcohol-induced immunosuppression can be rescued by chemokines. Thus, we assessed whether acute ethanol challenge via gavage diminishes antibacterial host defense in a sepsis model using cecal ligation and puncture (CLP) and whether this immunosuppression can be rescued by exogenous CXCL1. We found acute alcohol intoxication augments mortality and enhances bacterial growth in mice following CLP. Ethanol exposure impairs critical antibacterial functions of mouse and human neutrophils including reactive oxygen species production, neutrophil extracellular trap (NET) formation, and NET-mediated killing in response to both Gram-negative (E. coli) and Gram-positive (Staphylococcus aureus) pathogens. As compared with WT (C57Bl/6) mice, CXCL1 knockout mice display early mortality following acute alcohol exposure followed by CLP. Recombinant CXCL1 (rCXCL1) in acute alcohol challenged CLP mice increases survival, enhances bacterial clearance, improves neutrophil recruitment, and enhances NET formation (NETosis). Recombinant CXCL1 (rCXCL1) administration also augments bacterial killing by alcohol-treated and E. coli- and S. aureus-infected neutrophils. Taken together, our data unveils novel mechanisms underlying acute alcohol-induced dysregulation of the immune responses in polymicrobial sepsis, and CXCL1 is a critical mediator to rescue alcohol-induced immune dysregulation in polymicrobial sepsis.
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Quimiocina CXCL1/inmunología , Etanol/toxicidad , Trampas Extracelulares/inmunología , Inmunidad Innata/inmunología , Sepsis/inmunología , Animales , Western Blotting , Quimiocina CXCL1/farmacología , Modelos Animales de Enfermedad , Trampas Extracelulares/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Humanos , Inmunidad Innata/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de TransmisiónRESUMEN
NLRP3 inflammasome is a critical player in innate immunity. Neutrophil recruitment to tissues and effective neutrophil function are critical innate immune mechanisms for bacterial clearance. However, the role of NLRP3 in neutrophil-dependent bacterial clearance in polymicrobial sepsis is unclear. In this study, we evaluated the role of NLRP3 in polymicrobial sepsis induced by cecal ligation and puncture (CLP). Our results showed protection from death in NLRP3-deficient (Nlrp3-/-) and NLRP3 inhibitor-treated wild-type (C57BL/6) mice. Nlrp3-/- and NLRP3 inhibitor-treated mice displayed lower bacterial load but no impairment in neutrophil recruitment to peritoneum. However, neutrophil depletion abrogated protection from death in Nlrp3-/- mice in response to CLP. Intriguingly, following CLP, Nlrp3-/- peritoneal cells (primarily neutrophils) demonstrate decreased autophagy, augmented phagocytosis, and enhanced scavenger receptor (macrophage receptor with collagenous structure) and mannose-binding leptin expression. These findings enhance our understanding of the critical role of NLRP3 in modulating autophagy and phagocytosis in neutrophils and suggest that therapies should be targeted to modulate autophagy and phagocytosis in neutrophils to control bacterial burden in tissues during CLP-induced polymicrobial sepsis.
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Autofagia , Proteína con Dominio Pirina 3 de la Familia NLR/fisiología , Fagocitosis , Sepsis/mortalidad , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR/deficiencia , Infiltración Neutrófila , Peritoneo/patología , Receptores Inmunológicos/análisis , Sepsis/microbiologíaRESUMEN
Objective: To perform a preliminary study on the quality evaluation of compound Danshen preparations based on the xCELLigence Real-Time Cell-based Assay (RTCA) system and make a pharmacodynamics verification. Methods: The compound Danshen was discussed as a methodological example, and the bioactivity of the compound Danshen preparations were evaluated by real-time cell electronic analysis technology. Meanwhile, an in vivo experiment on an acute blood stasis rat model was performed in order to verify this novel evaluation through the curative effect of dissipating blood stasis. Results: We determined the cell index (CI) and IC50 of the compound Danshen preparations and produced time/dose-dependent cell response profiles (TCRPs). The quality of the three kinds of compound Danshen preparations was evaluated through the RTCA data. The trend of CI and TCRPs reflected the effect of drugs on the cell (promoting or inhibiting), and it was verified that the results correlated with the biological activity of the drugs using a pharmacodynamics experiment. Conclusion: The RTCA system can be used to evaluate the quality of compound Danshen Preparations, and it can provide a new idea and new method for quantitatively characterizing the biological activity of traditional Chinese medicines (TCMs).
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Medicamentos Herbarios Chinos/química , Medicamentos Herbarios Chinos/farmacología , Medicina Tradicional China/normas , Salvia miltiorrhiza/química , Animales , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Coagulación Sanguínea/efectos de los fármacos , Viscosidad Sanguínea/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Índices de Eritrocitos/efectos de los fármacos , Humanos , RatasRESUMEN
Severe bacterial sepsis leads to a proinflammatory condition that can manifest as septic shock, multiple organ failure, and death. Neutrophils are critical for the rapid elimination of bacteria; however, the role of neutrophil chemoattractant CXCL1 in bacterial clearance during sepsis remains elusive. To test the hypothesis that CXCL1 is critical to host defense during sepsis, we used CXCL1-deficient mice and bone marrow chimeras to demonstrate the importance of this molecule in sepsis. We demonstrate that CXCL1 plays a pivotal role in mediating host defense to polymicrobial sepsis after cecal ligation and puncture in gene-deficient mice. CXCL1 appears to be essential for restricting bacterial outgrowth and death in mice. CXCL1 derived from both hematopoietic and resident cells contributed to bacterial clearance. Moreover, CXCL1 is essential for neutrophil migration, expression of proinflammatory mediators, activation of NF-κB and MAPKs, and upregulation of adhesion molecule ICAM-1. rIL-17 rescued impaired host defenses in cxcl1(-/-) mice. CXCL1 is important for IL-17A production via Th17 differentiation. CXCL1 is essential for NADPH oxidase-mediated reactive oxygen species production and neutrophil extracellular trap formation. This study reveals a novel role for CXCL1 in neutrophil recruitment via modulating T cell function and neutrophil-related bactericidal functions. These studies suggest that modulation of CXCL1 levels in tissues and blood could reduce bacterial burden in sepsis.
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Movimiento Celular/inmunología , Quimiocina CXCL1/inmunología , Sistema de Señalización de MAP Quinasas/inmunología , Neutrófilos/inmunología , Sepsis/inmunología , Células Th17/inmunología , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Movimiento Celular/genética , Quimiocina CXCL1/sangre , Quimiocina CXCL1/genética , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/inmunología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Interleucina-17/sangre , Interleucina-17/genética , Interleucina-17/inmunología , Sistema de Señalización de MAP Quinasas/genética , Ratones , Ratones Noqueados , FN-kappa B/genética , FN-kappa B/inmunología , FN-kappa B/metabolismo , Neutrófilos/metabolismo , Neutrófilos/patología , Sepsis/sangre , Sepsis/genética , Sepsis/microbiología , Células Th17/metabolismo , Células Th17/patologíaRESUMEN
Introduction: Sepsis remains a major cause of death in Intensive Care Units. Sepsis is a life-threatening multi-organ dysfunction caused by a dysregulated systemic inflammatory response. Pattern recognition receptors, such as TLRs and NLRs contribute to innate immune responses. Upon activation, some NLRs form multimeric protein complexes in the cytoplasm termed "inflammasomes" which induce gasdermin d-mediated pyroptotic cell death and the release of mature forms of IL-1ß and IL-18. The NLRP6 inflammasome is documented to be both a positive and a negative regulator of host defense in distinct infectious diseases. However, the role of NLRP6 in polymicrobial sepsis remains elusive. Methods: We have used NLRP6 KO mice and human septic spleen samples to examine the role of NLRP6 in host defense in sepsis. Results: NLRP6 KO mice display enhanced survival, reduced bacterial burden in the organs, and reduced cytokine/chemokine production. Co-housed WT and KO mice following sepsis show decreased bacterial burden in the KO mice as observed in singly housed groups. NLRP6 is upregulated in CD3, CD4, and CD8 cells of septic patients and septic mice. The KO mice showed a higher number of CD3, CD4, and CD8 positive T cell subsets and reduced T cell death in the spleen following sepsis. Furthermore, administration of recombinant IL-18, but not IL-1ß, elicited excessive inflammation and reversed the survival advantages observed in NLRP6 KO mice. Conclusion: These results unveil NLRP6 as a negative regulator of host defense during sepsis and offer novel insights for the development of new treatment strategies for sepsis.
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Inmunidad Innata , Péptidos y Proteínas de Señalización Intracelular , Sepsis , Animales , Femenino , Humanos , Masculino , Ratones , Citocinas/metabolismo , Modelos Animales de Enfermedad , Inmunidad Innata/genética , Inflamasomas/metabolismo , Inflamasomas/inmunología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Superficie Celular , Sepsis/inmunología , Sepsis/microbiología , Bazo/inmunologíaRESUMEN
During acute infectious and inflammatory conditions, a large number of neutrophils are in high demand as they are consumed in peripheral organs. The hematopoietic system rapidly responds to the demand by turning from steady state to emergency granulopoiesis to expedite neutrophil generation in the bone marrow (BM). How the hematopoietic system integrates pathogenic and inflammatory stress signals into the molecular cues of emergency granulopoiesis has been the subject of investigations. Recent studies in the field have highlighted emerging concepts, including the direct sensing of pathogens by BM resident or sentinel hematopoietic stem and progenitor cells (HSPCs), the crosstalk of HSPCs, endothelial cells, and stromal cells to convert signals to granulopoiesis, and the identification of novel inflammatory molecules, such as C/EBP-ß, ROS, IL-27, IFN-γ, CXCL1 with direct effects on HSPCs. In this review, we will provide a detailed account of emerging concepts while reassessing well-established cellular and molecular players of emergency granulopoiesis. While providing our views on the discrepant results and theories, we will postulate an updated model of granulopoiesis in the context of health and disease.
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Células Endoteliales , Interleucina-27 , Hematopoyesis , Leucopoyesis , Especies Reactivas de OxígenoRESUMEN
Bacterial pneumonia is a significant cause of morbidity, mortality, and health care expenditures. Optimum neutrophil recruitment and their function are critical defense mechanisms against respiratory pathogens. The nucleotide-binding oligomerization domain-like receptor (NLRP) 6 controls gut microbiota and immune response to systemic and enteric infections. However, the importance of NLRP6 in neutrophil homeostasis following lung infection remains elusive. To investigate the role of NLRs in neutrophil homeostasis, we used Nlrp6 gene-deficient (Nlrp6-/-) mice in a model of Klebsiella pneumoniae-induced pneumonia-derived sepsis. We demonstrated that NLRP6 is critical for host survival, bacterial clearance, neutrophil influx, and CXC-chemokine production. Kp-infected Nlrp6-/- mice have reduced numbers of hematopoietic stem cells and granulocyte-monocyte progenitors but increased retention of matured neutrophils in bone marrow. Neutrophil extracellular trap (NET) formation and NET-mediated bacterial killing were also impaired in Nlrp6-/- neutrophils in vitro. Furthermore, recombinant CXCL1 rescued the impaired host defense, granulopoietic response, and NETosis in Kp-infected Nlrp6-/- mice. Using A/J background mice and co-housing experiments, our findings revealed that the susceptible phenotype of Nlrp6-/- mice is not strain-specific and gut microbiota-dependent. Taken together, these data unveil NLRP6 as a central regulator of neutrophil recruitment, generation, and function during bacterial pneumonia followed by sepsis.
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Infecciones por Klebsiella/metabolismo , Klebsiella pneumoniae/fisiología , Macrófagos/inmunología , Neutrófilos/inmunología , Neumonía Bacteriana/metabolismo , Receptores de Superficie Celular/metabolismo , Sepsis/metabolismo , Animales , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Trampas Extracelulares/metabolismo , Homeostasis , Humanos , Infecciones por Klebsiella/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Activación Neutrófila , Neumonía Bacteriana/inmunología , Receptores de Superficie Celular/genética , Sepsis/inmunología , Células THP-1RESUMEN
NACHT, LRR (leucine-rich repeat), and PYD (pyrin domain) domain-containing 6 (Nlrp6) is a member of the NLR (nucleotide-oligomerization domain-like receptor) family that patrols the cytosolic compartment of cells to detect pathogen- and damage-associated molecular patterns. Because Nlrp6 is a recently discovered inflammasome, details of its signaling mechanism, structural assembly, and roles in host defense have yet to be determined. To date, Nlrp6 has been proposed to perform a multitude of functions ranging from control of microbiota, maintenance of epithelial integrity, and regulation of metabolic diseases to modulation of host defense during microbial infections, cancer protection, and regulation of neuroinflammation. While recent studies have questioned some of the proposed functions of Nlrp6, Nlrp6 has been shown to form an inflammasome complex and cleaves interleukin-1ß (IL-1ß) and IL-18 during microbial infection, indicating that it is a bonafide inflammasome. In this review, we summarize the recent advancements in knowledge of the signaling mechanisms and structure of the Nlrp6 inflammasome and discuss the relevance of NLRP6 to human disease.
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Susceptibilidad a Enfermedades , Homeostasis , Inflamasomas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Animales , Comunicación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Microbioma Gastrointestinal , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Inflamación/etiología , Inflamación/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Enfermedades Metabólicas/etiología , Enfermedades Metabólicas/metabolismo , Transducción de SeñalRESUMEN
Gram-positive pathogens, including Staphylococcus aureus, cause necrotizing pneumonia. The central feature of S. aureus pneumonia is toxin-induced necroptosis of immune and resident cells, which impedes host defense. However, the role of the NLRC4 in the lung following S. aureus infection remains elusive. Here, we demonstrate that S. aureus activates the NLRC4 to drive necroptosis and IL-18 production, which impaired IL-17A-dependent neutrophil-mediated host susceptibility. In particular, Nlrc4-/- mice exhibit reduced necroptosis, enhanced neutrophil influx into the lungs, decreased bacterial burden, and improved host survival. Loss of NLRC4 signaling in both hematopoietic and non-hematopoietic cells contributes to the host protection against S. aureus pneumonia. Secretion of IL-17A by γδ T cells is essential for neutrophil recruitment into the lungs of Nlrc4-/- mice following infection. Moreover, treatment of wild-type mice with necroptosis inhibitors or genetic ablation of MLKL and IL-18 improves host defense against S. aureus infection, which is associated with increased IL-17A+γδ T cells and neutrophils. Taken together, these novel findings reveal that S. aureus activates the NLRC4 to dampen IL-17A-dependent neutrophil accumulation through induction of necroptosis and IL-18. Thus, modulating the function of the NLRC4 may be an attractive therapeutic approach for treating S. aureus infections.
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Pulmón/inmunología , Neutrófilos/inmunología , Neumonía Estafilocócica/inmunología , Staphylococcus aureus/inmunología , Linfocitos T/inmunología , Animales , Apoptosis , Proteínas Adaptadoras de Señalización CARD/metabolismo , Proteínas de Unión al Calcio/metabolismo , Células Cultivadas , Humanos , Enfermedades del Sistema Inmune , Interleucina-18/genética , Interleucina-18/metabolismo , Trastornos Leucocíticos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Necrosis , Proteínas Quinasas/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Transducción de Señal , Regulación hacia ArribaRESUMEN
Macrophage inhibitory cytokine 1 (MIC-1/GDF15) has been characterized as a candidate biomarker for colorectal cancer (CRC) recently. However, the role of serum MIC-1 in screening patients with early stage CRC and monitoring therapeutic response have not been well-established, particularly in the combination with CEA for the screening and the prejudgment of occurrence with liver metastasis. In this study, we performed a retrospective blinded evaluation of 987 serum samples from 473 individuals with CRC, 25 with adenomatous polyps, and 489 healthy individuals using ELISA or immunoassay. The sensitivity of serum MIC-1 was 43.8% and 38.5% for CRC diagnosis and early diagnosis, respectively, which were independent of and comparatively higher than for CEA (36.6% and 27.3%) at comparable specificity. Serum MIC-1 after surgery were significantly elevated at the time of tumor recurrence, and notable increase were observed in 100% patients with liver metastasis. Besides the TNM classification and differentiation grade, MIC-1 was an independent prognostic factor contributing to overall survival. We conclude that MIC-1 can act as a candidate complementary biomarker for screening early-stage CRC by combination with CEA, and furthermore, for the first time, identify a promising prognostic indicator for monitoring recurrence with liver metastasis, to support strategies towards personalized therapy.