RESUMO
While the treatment of inflammatory disorders is generally based on inhibiting factors that drive onset of inflammation, these therapies can compromise healing (NSAIDs) or dampen immunity against infections (biologics). In search of new antiinflammatories, efforts have focused on harnessing endogenous pathways that drive resolution of inflammation for therapeutic gain. Identification of specialized pro-resolving mediators (SPMs) (lipoxins, resolvins, protectins, maresins) as effector molecules of resolution has shown promise in this regard. However, their action on inflammatory resolution in humans is unknown. Here, we demonstrate using a model of UV-killed Escherichia coli-triggered skin inflammation that SPMs are biosynthesized at the local site at the start of resolution, coinciding with the expression of receptors that transduce their actions. These include receptors for lipoxin A4 (ALX/FPR2), resolvin E1 (ChemR23), resolvin D2 (GPR18), and resolvin D1 (GPR32) that were differentially expressed on the endothelium and infiltrating leukocytes. Administering SPMs into the inflamed site 4 hours after bacterial injection caused a reduction in PMN numbers over the ensuing 6 hours, the phase of active resolution in this model. These results indicate that in humans, the appearance of SPMs and their receptors is associated with the beginning of inflammatory resolution and that their therapeutic supplementation enhanced the resolution response.
Assuntos
Anti-Inflamatórios/farmacologia , Escherichia coli/imunologia , Inflamação/imunologia , Inflamação/metabolismo , Pele/imunologia , Pele/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adolescente , Adulto , Vesícula/imunologia , Vesícula/metabolismo , Quimiocinas/metabolismo , Citocinas/metabolismo , Ácidos Docosa-Hexaenoicos/farmacologia , Eicosanoides/imunologia , Eicosanoides/farmacologia , Ácido Eicosapentaenoico/análogos & derivados , Ácido Eicosapentaenoico/farmacologia , Escherichia coli/efeitos da radiação , Humanos , Inflamação/tratamento farmacológico , Leucócitos/imunologia , Leucócitos/metabolismo , Lipoxinas/farmacologia , Masculino , Pessoa de Meia-Idade , Neutrófilos/efeitos dos fármacos , Receptores de Quimiocinas/metabolismo , Receptores de Formil Peptídeo/metabolismo , Receptores Acoplados a Proteínas G , Receptores de Lipoxinas/metabolismo , Pele/efeitos dos fármacos , Pele/patologia , Voluntários , Adulto JovemRESUMO
Anabasum is a synthetic analog of Δ8 -tetrahydrocannabinol (THC)-11-oic acid that in preclinical models of experimental inflammation exerts potent anti-inflammatory actions with minimal central nervous system (CNS) cannabimimetic activity. Here we used a novel model of acute inflammation driven by i.d. UV-killed E. coli in healthy humans and found that anabasum (5 mg) exerted a potent anti-inflammatory effect equivalent to that of prednisolone in terms of inhibiting neutrophil infiltration, the hallmark of acute inflammation. These effects arose from the inhibition of the neutrophil chemoattractant LTB4 , while the inhibition of antiphagocytic prostanoids (PGE2 , TxB2 , and PGF2 α) resulted in enhanced clearance of inflammatory stimulus from the injected site. Anabasum at the higher dose of 20 mg possessed the additional properties of triggering the biosynthesis of specialized pro-resolving lipid mediators including LXA4 , LXB4 , RvD1, and RvD3. Collectively, we demonstrate for the first time a striking anti-inflammatory and pro-resolution effects of a synthetic analog of THC in healthy humans.
Assuntos
Anti-Inflamatórios/administração & dosagem , Canabinol/análogos & derivados , Dermatite/tratamento farmacológico , Dronabinol/administração & dosagem , Infecções por Escherichia coli/tratamento farmacológico , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Dermatopatias Bacterianas/tratamento farmacológico , Pele/efeitos dos fármacos , Adolescente , Adulto , Anti-Inflamatórios/efeitos adversos , Canabinol/administração & dosagem , Canabinol/efeitos adversos , Citocinas/imunologia , Citocinas/metabolismo , Dermatite/imunologia , Dermatite/metabolismo , Dermatite/microbiologia , Relação Dose-Resposta a Droga , Dronabinol/efeitos adversos , Dronabinol/análogos & derivados , Escherichia coli/efeitos dos fármacos , Escherichia coli/patogenicidade , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Fagocitose/efeitos dos fármacos , Prednisolona/farmacologia , Pele/imunologia , Pele/metabolismo , Pele/microbiologia , Dermatopatias Bacterianas/imunologia , Dermatopatias Bacterianas/metabolismo , Dermatopatias Bacterianas/microbiologia , Adulto JovemRESUMO
Acute inflammation is an immediate response to infection and injury characterised by the influx of granulocytes followed by phagocytosing mononuclear phagocytes. Provided the antigen is cleared and the immune system of the host is fully functional, the acute inflammatory response will resolve. Until now it is considered that resolution then leads back to homeostasis, the physiological state tissues experienced before inflammation occurred. Using a human model of acute inflammation driven by intradermal UV killed Escherichia coli, we found that bacteria and granulocyte clearance as well as pro-inflammatory cytokine catabolism occurred by 72h. However, following a lag phase of about 4 days there was an increase in numbers of memory T cells and CD163+ macrophage at the post-resolution site up to day 17 as well as increased biosynthesis of cyclooxygenase-derived prostanoids and DHA-derived D series resolvins. Inhibiting post-resolution prostanoids using naproxen showed that numbers of tissue memory CD4 cells were under the endogenous control of PGE2, which exerts its suppressive effects on T cell proliferation via the EP4 receptor. In addition, we re-challenged the post-resolution site with a second injection of E. coli, which when compared to saline controls resulted in primarily a macrophage-driven response with comparatively fewer PMNs; the macrophage-dominated response was reversed by cyclooxygenase inhibition. Re-challenge experiments were also carried out in mice where we obtained similar results as in humans. Therefore, we report that acute inflammatory responses in both humans and rodents do not revert back to homeostasis, but trigger a hitherto unappreciated sequence of immunological events that dictate subsequent immune response to infection.
Assuntos
Inflamação/imunologia , Doença Aguda , Adulto , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Dinoprostona/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Escherichia coli/efeitos da radiação , Feminino , Humanos , Inflamação/microbiologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Viabilidade Microbiana/efeitos da radiação , Pessoa de Meia-Idade , Receptores de Superfície Celular/metabolismo , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Raios Ultravioleta , Adulto JovemRESUMO
Acute inflammation is characterized by granulocyte infiltration followed by efferocytosing mononuclear phagocytes, which pave the way for inflammatory resolution. Until now, it was believed that resolution then leads back to homeostasis, the physiological state tissues experience before inflammation occurred. However, we discovered that resolution triggered a prolonged phase of immune suppression mediated by prostanoids. Specifically, once inflammation was switched off, natural killer cells, secreting interferon γ (IFNγ), infiltrated the post-inflamed site. IFNγ upregulated microsomal prostaglandin E synthase-1 (mPGES-1) alongside cyclo-oxygenase (COX-1) within macrophage populations, resulting in sustained prostaglandin (PG)E2 biosynthesis. Whereas PGE2 suppressed local innate immunity to bacterial infection, it also inhibited lymphocyte function and generated myeloid-derived suppressor cells, the net effect of which was impaired uptake/presentation of exogenous antigens. Therefore, we have defined a sequence of post-resolution events that dampens the propensity to develop autoimmune responses to endogenous antigens at the cost of local tissue infection.
Assuntos
Ciclo-Oxigenase 1/imunologia , Dinoprostona/imunologia , Inflamação/imunologia , Proteínas de Membrana/imunologia , Prostaglandina-E Sintases/imunologia , Animais , Inflamação/enzimologia , Interferon gama/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Defects in neutrophil function have been implicated in a wide spectrum of clinical conditions. Several models are employed to study activated human neutrophils akin to those found at a site of inflammation. These include whole blood (WB) ex vivo stimulation with lipopolysaccharide (LPS) and in vivo techniques: cantharidin blister, skin windows and intra-dermal injection of UV-killed E.coli (UVKEc). Neutrophils obtained from these have never been compared. We compared the activation status of neutrophils from each technique in order to inform the optimal model for use in human studies. Healthy male volunteers were randomised to undergo one of the four techniques (n = 5/group). LPS: WB stimulated with 1ng/ml of LPS for 4 hours. Cantharidin: 12.5µl of 0.1% cantharidin elicited a single blister, aspirated at 24 hours. Skin windows: four 6mm mechanical-suction blisters created, de-roofed and an exudate-collection chamber placed over the windows for 4 hours before aspiration. UVKEc: 1.5 x 107 UVKEc injected intra-dermally. A single 10mm mechanical-suction blister formed and aspirated at 4 hours. Unstimulated WB used as the control. Flow cytometry was used to determine activation status using CD16, CD11b, CD54, CD62L and CD88. Functional status was assessed with a phagocytosis assay. The pattern of neutrophil activation was similar in all models. Neutrophil CD11b was elevated in all models, most markedly in UVKEc (p<0.0001), and CD54 was also elevated but only significant in the LPS model (p = 0.001). CD62L was significantly reduced in all 4 models (p<0.0001) and CD88 was also suppressed in all. There were no changes in CD16 in any model, neither was there any significant difference in the phagocytic capacity of the neutrophils. In summary, there are no significant differences in activation marker expression or phagocytic capacity in the neutrophils obtained from each technique. Therefore we believe whole blood stimulation is the best model in experimentally challenging inpatient populations.
Assuntos
Inflamação/patologia , Modelos Biológicos , Neutrófilos/metabolismo , Adolescente , Adulto , Vesícula/imunologia , Vesícula/metabolismo , Vesícula/patologia , Antígeno CD11b/metabolismo , Cantaridina/toxicidade , Citometria de Fluxo , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Lipopolissacarídeos/toxicidade , Masculino , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/citologia , Fagocitose/efeitos dos fármacos , Receptores de IgG/metabolismo , Pele/patologia , Adulto JovemRESUMO
Whilst numerous studies investigating the aetiology of inflammatory diseases have been performed in rodents, the applicability of these data to human pathophysiology is frequently debated. Regardless of the strengths and weaknesses of rodent models in biomedical research, there is a need to develop models of experimental inflammation in humans. Here, we describe a self-resolving acute inflammatory response triggered by the intradermal injection of UV-killed Escherichia coli into the forearm of healthy volunteers. Cells and exudates were harvested from onset to resolution by applying negative pressure over the inflamed site. Onset was characterized by high blood flow, neutrophilia and peak levels of pro-inflammatory cytokines, whilst resolution showed a decline in blood blow, reduction in neutrophils, increase in monocytes/macrophages and waning of classic pro-inflammatory cytokine levels. An anti-inflammatory effect, defined as suppression of onset phase events, was demonstrated by administering naproxen, a conventional non-steroidal anti-inflammatory drug. In summary, this model of resolving acute inflammation is minimally invasive, highly tractable and allows simultaneous investigation of the vascular response, cellular trafficking and chemical mediator profile of onset and resolution phases of acute inflammation in humans. It can serve as a translational platform to provide mechanistic insights and to test the clinical efficacy of novel anti-inflammatory and pro-resolving drugs, and also as a tool in patients to explore inherent defects in resolution pathways.
RESUMO
BACKGROUND: Tissue infiltration by neutrophils during acute inflammatory states causes substantial tissue injury. While the magnitude of tissue neutrophil accumulation in innate immune responses is profoundly greater in males than females, fundamental aspects of the molecular mechanisms underlying these sex differences remain largely unknown. METHODS: We investigated sex differences in neutrophil stimulation and recruitment in ischemia/reperfusion (I/R; mesenteric or renal) or carrageenan pleurisy in rats or mice, as well as skin injury in human volunteers. The induction of potent chemoattractive mediators (chemokines) and neutrophil adhesion molecules were measured by real-time PCR, flow cytometry, and protein assays. RESULTS: Mesenteric I/R in age-matched Wistar rats resulted in substantially more neutrophil accumulation and tissue injury at 2 h reperfusion in males than females. Using intravital microscopy, we show that the immediate (<30 min) neutrophil response to I/R is similar in males and females but that prolonged neutrophil recruitment occurs in males at sites local and distal to inflammatory insult partly due to an increase in circulating neutrophil populations with elevated surface expression of adhesion molecules. Sex differences in neutrophil kinetics were correlated with sustained induction of chemokine Cxcl5 in the tissue, circulation, and bone marrow of males but not females. Furthermore, blockade of Cxcl5 in males prior to ischemia resulted in neutrophil responses that were similar in magnitude to those in females. Conversely, administration of Cxcl5 to males in the absence of I/R was sufficient to increase levels of systemic neutrophils. Cxcl5 treatment of bone marrow neutrophils in vitro caused substantial induction of neutrophil-mobilizing cytokine granulocyte colony-stimulating factor (GCSF) and expression of ß2 integrin that accounts for sexual dimorphism in circulating neutrophil populations in I/R. Moreover, male Cxcl5-stimulated bone marrow neutrophils had an increased capacity to adhere to ß2 integrin ligand ICAM-1, implicating a greater sensitivity of male leukocytes to Cxcl5-mediated activation. Differential induction of Cxcl5 (human CXCL6) between the sexes was also evident in murine renal I/R, rat pleurisy, and human skin blisters and correlated with the magnitude of neutrophil accumulation in tissues. CONCLUSIONS: Our study reveals that sex-specific induction of chemokine Cxcl5/CXCL6 contributes to sexual dimorphism in neutrophil recruitment in diverse acute inflammatory responses partly due to increased stimulation and trafficking of bone marrow neutrophils in males.