RESUMO
BACKGROUND: Type 1 angiotensin (AT1) receptors are expressed on immune cells, and we previously found that bone marrow-derived AT1 receptors protect against Ang (angiotensin) II-induced hypertension. CD11c is expressed on myeloid cells derived from the bone marrow, including dendritic cells (DCs) that activate T lymphocytes. Here, we examined the role of AT1 receptors on CD11c+ cells in hypertension pathogenesis. METHODS: Mice lacking the dominant murine AT1 receptor isoform, AT1a, on CD11c+ cells (dendritic cell [DC] AT1aR knockout [KO]) and wild-type (WT) littermates were subjected to Ang II-induced hypertension. Blood pressures were measured by radiotelemetry. RESULTS: DC AT1aR KO mice had exaggerated hypertensive responses to chronic Ang II infusion with enhanced renal accumulation of effector memory T cells and CD40+ DCs. CCL5 (C-C motif chemokine ligand 5) recruits T cells into injured tissues, and CCR7 (C-C motif chemokine receptor 7) facilitates DC and T cell interactions in the kidney lymph node to allow T cell activation. DCs from the hypertensive DC AT1aR KO kidneys expressed higher levels of CCL5 and CCR7. mRNA expressions for CCR7 and tumor necrosis factor-α were increased in CD4+ T cells from the renal lymph nodes of DC AT1aR KO mice. During the second week of Ang II infusion when blood pressures between groups diverged, DC AT1aR KO mice excreted less sodium than WTs. Expressions for epithelial sodium channel subunits were increased in DC AT1aR KO kidneys. CONCLUSIONS: Following activation of the renin angiotensin system, AT1aR stimulation on DCs suppresses renal DC maturation and T cell activation with consequent protection from sodium retention and blood pressure elevation.
Assuntos
Hipertensão , Receptor Tipo 1 de Angiotensina , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Animais , Células Dendríticas/metabolismo , Hipertensão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores CCR7/metabolismo , Sódio/metabolismo , Linfócitos T/metabolismoRESUMO
A vast evolution of drug modalities has occurred over the last several decades. Novel modalities such as cell and gene therapies have proven to be efficacious for numerous clinical indications-primarily in rare disease and immune oncology. Because of this success, drug developers are heavily investing in these novel modalities. Given the complexity of these therapeutics, a variety of bioanalytical techniques are employed to fully characterize the pharmacokinetics of these therapies in clinical studies. Industry trends indicate that quantitative PCR (qPCR) and multiparameter flow cytometry are both valuable in determining the pharmacokinetics, i.e. cellular kinetics, of cell therapies. This manuscript will evaluate the pros and cons of both techniques and highlight regulatory guidance on assays for measuring cellular kinetics. Moreover, common considerations when developing these assays will be addressed.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Terapia Genética , Cinética , Citometria de FluxoRESUMO
Intestinal immunity is coordinated by specialized mononuclear phagocyte populations, constituted by a diversity of cell subsets. Although the cell subsets constituting the mononuclear phagocyte network are thought to be similar in both small and large intestine, these organs have distinct anatomy, microbial composition, and immunological demands. Whether these distinctions demand organ-specific mononuclear phagocyte populations with dedicated organ-specific roles in immunity are unknown. Here we implement a new strategy to subset murine intestinal mononuclear phagocytes and identify two novel subsets which are colon-specific: a macrophage subset and a Th17-inducing dendritic cell (DC) subset. Colon-specific DCs and macrophages co-expressed CD24 and CD14, and surprisingly, both were dependent on the transcription factor IRF4. Novel IRF4-dependent CD14+CD24+ macrophages were markedly distinct from conventional macrophages and failed to express classical markers including CX3CR1, CD64 and CD88, and surprisingly expressed little IL-10, which was otherwise robustly expressed by all other intestinal macrophages. We further found that colon-specific CD14+CD24+ mononuclear phagocytes were essential for Th17 immunity in the colon, and provide definitive evidence that colon and small intestine have distinct antigen presenting cell requirements for Th17 immunity. Our findings reveal unappreciated organ-specific diversity of intestine-resident mononuclear phagocytes and organ-specific requirements for Th17 immunity.
Assuntos
Colo/imunologia , Células Dendríticas/imunologia , Macrófagos/imunologia , Fagócitos/imunologia , Células Th17/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígeno CD24/imunologia , Antígeno CD24/metabolismo , Colo/citologia , Colo/metabolismo , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Células Dendríticas/metabolismo , Expressão Gênica/imunologia , Fatores Reguladores de Interferon/imunologia , Fatores Reguladores de Interferon/metabolismo , Intestino Delgado/imunologia , Receptores de Lipopolissacarídeos/imunologia , Receptores de Lipopolissacarídeos/metabolismo , Macrófagos/metabolismo , Camundongos da Linhagem 129 , Camundongos Knockout , Camundongos Transgênicos , Fagócitos/metabolismo , Receptor da Anafilatoxina C5a/imunologia , Receptor da Anafilatoxina C5a/metabolismo , Células Th17/metabolismoRESUMO
Activated T lymphocytes that infiltrate blood pressure control organs make a critical contribution to the pathogenesis of hypertension. Dendritic cells act as potent antigen-presenting cells to stimulate prohypertensive T cells. However, the mechanisms that facilitate the recruitment of prohypertensive T cells and dendritic cells into the kidney's draining lymph node during hypertension require elucidation. As CCR7 (C-C motif chemokine receptor type 7) directs the homing of lymphocytes and dendritic cells into lymph nodes, we posited that dendritic cell-mediated T lymphocyte stimulation in the renal lymph node is CCR7 dependent and required for a full hypertensive response. We found that CCR7-deficient (CCR7 KO) mice had a blunted hypertensive response in our model of chronic Ang II (angiotensin II) infusion. Ang II-infused CCR7 KO animals had exaggerated accumulation of CD8+ T cells in the kidney but reduced numbers of CD4+ and CD8+ T cells in the kidney's draining lymph node. To understand whether CCR7-dependent homing of T lymphocytes or dendritic cells into the lymph node regulates the hypertensive response, we injected CCR7 KO or wild-type T cells or dendritic cells into CCR7 KO recipients, neither of which restored the full hypertensive response to Ang II infusion. However, adoptive transfer of wild-type but not CCR7 KO T lymphocytes into RAG1 (recombination-activating gene 1)-deficient mice that lack a lymphocyte niche restored full blood pressure elevation during Ang II infusion. Thus, CCR7-dependent interactions between T lymphocytes and dendritic cells are essential for T lymphocyte stimulation and hypertension accruing from inappropriate activation of the renin-angiotensin system.
Assuntos
Quimiotaxia de Leucócito/fisiologia , Hipertensão/imunologia , Receptores CCR7/fisiologia , Subpopulações de Linfócitos T/imunologia , Imunidade Adaptativa , Transferência Adotiva , Angiotensina II/toxicidade , Animais , Células Dendríticas/transplante , Genes RAG-1 , Hipertensão/fisiopatologia , Rim/imunologia , Rim/fisiopatologia , Linfonodos/imunologia , Linfonodos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nefrectomia , Receptores CCR7/deficiência , Receptores CCR7/genéticaRESUMO
FLT3L (Fms-like tyrosine kinase 3 ligand) stimulates the development of classical dendritic cells (DCs). Here we tested the hypothesis that classical DCs drive blood pressure elevation by promoting renal fluid retention. FLT3L-deficient (FLT3L-/-) mice that lack classical DCs in the kidney had mean arterial pressures similar to wild-types (WTs) at baseline but had blunted hypertensive responses during 4 weeks of chronic Ang II (angiotensin II) infusion. In FLT3L-/- mice, the proportions of effector memory T cells in the kidney were similar to those in WTs at baseline. However, after Ang II infusion, proportions of effector memory T cells were dramatically lower in the FLT3L-/- kidneys versus WTs, indicating that classical DCs augment the renal accumulation of effector T cells after renin-angiotensin system activation. Consistent with their lower blood pressures, the Ang II-infused FLT3L-/- mice had attenuated cardiac hypertrophy and lower renal mRNA expression for pro-hypertensive cytokines. Moreover, the Ang II-infused FLT3L-/- mice had lower urinary excretion of the oxidative stress marker 8-isoprostane and lower renal mRNA levels of nicotinamide adenine dinucleotide phosphate oxidase 2. In an intraperitoneal saline challenge test at day 7 of Ang II, FLT3L-/- mice excreted higher proportions of the injected volume and sodium than WTs. Consistent with this enhanced diuresis, mRNA expressions for the sodium chloride cotransporter and all 3 subunits of the epithelial sodium channel were diminished by >40% in FLT3L-/- kidneys compared with the WTs. Thus, classical FLT3L-dependent DCs promote renal T-cell activation with consequent oxidative stress, fluid retention, and blood pressure elevation.
Assuntos
Células Dendríticas/metabolismo , Hipertensão/metabolismo , Rim/metabolismo , Estresse Oxidativo/fisiologia , Linfócitos T/metabolismo , Angiotensina II , Animais , Modelos Animais de Doenças , Hipertensão/induzido quimicamente , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos KnockoutRESUMO
Tubulointerstitial disease in the kidney culminates in renal fibrosis that portents organ failure. Twist1, a basic helix-loop-helix protein 38 transcription factor, regulates several essential biological functions, but inappropriate Twist1 activity in the kidney epithelium can trigger kidney fibrogenesis and chronic kidney disease. By contrast, Twist1 in circulating myeloid cells may constrain inflammatory injury by attenuating cytokine generation. To dissect the effects of Twist1 in kidney tubular versus immune cells on renal inflammation following toxin-induced renal injury, we subjected mice with selective deletion of Twist1 in renal epithelial cells or macrophages to aristolochic acid-induced chronic kidney disease. Ablation of Twist1 in the distal nephron attenuated kidney damage, interstitial fibrosis, and renal inflammation after aristolochic acid exposure. However, macrophage-specific deletion of Twist1 did not impact the development of aristolochic acid-induced nephropathy. In vitro studies confirmed that Twist1 in renal tubular cells underpins their susceptibility to apoptosis and propensity to generate pro-fibrotic mediators in response to aristolochic acid. Moreover, co-culture studies revealed that Twist1 in renal epithelia augmented the recruitment and activation of pro-inflammatory CD64+ macrophages. Thus, Twist1 in the distal nephron rather than in infiltrating macrophages propagates chronic inflammation and fibrogenesis during aristolochic acid-induced nephropathy.
Assuntos
Túbulos Renais Distais/patologia , Macrófagos/imunologia , Nefrite Intersticial/imunologia , Insuficiência Renal Crônica/imunologia , Proteína 1 Relacionada a Twist/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Ácidos Aristolóquicos/toxicidade , Técnicas de Cocultura , Modelos Animais de Doenças , Células Epiteliais , Feminino , Fibrose , Técnicas de Silenciamento de Genes , Receptor Celular 1 do Vírus da Hepatite A/metabolismo , Humanos , Túbulos Renais Distais/citologia , Túbulos Renais Distais/imunologia , Túbulos Renais Distais/metabolismo , Lipocalina-2/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Transgênicos , Nefrite Intersticial/induzido quimicamente , Nefrite Intersticial/patologia , Cultura Primária de Células , Insuficiência Renal Crônica/induzido quimicamente , Insuficiência Renal Crônica/patologia , Proteína 1 Relacionada a Twist/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologiaRESUMO
Nephrotoxic serum nephritis (NTN) models immune-mediated human glomerulonephritis and culminates in kidney inflammation and fibrosis, a process regulated by T lymphocytes. TNF-α is a key proinflammatory cytokine that contributes to diverse forms of renal injury. Therefore, we posited that TNF-α from T lymphocytes may contribute to NTN pathogenesis. Here, mice with T cell-specific deletion of TNF-α (TNF TKO) and wild-type (WT) control mice were subjected to the NTN model. At 14 days after NTN, kidney injury and fibrosis were increased in kidneys from TNF TKO mice compared with WT mice. PD1+CD4+ T cell numbers and mRNA levels of IL-17A were elevated in NTN kidneys of TNF TKO mice, suggesting that augmented local T helper 17 lymphocyte responses in the TNF TKO kidney may exaggerate renal injury and fibrosis. In turn, we found increased accumulation of neutrophils in TNF TKO kidneys during NTN. We conclude that TNF-α production in T lymphocytes mitigates NTN-induced kidney injury and fibrosis by inhibiting renal T helper 17 lymphocyte responses and infiltration of neutrophils.
Assuntos
Fibrose/metabolismo , Glomerulonefrite/metabolismo , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Modelos Animais de Doenças , Fibrose/genética , Fibrose/patologia , Glomerulonefrite/genética , Glomerulonefrite/patologia , Interleucina-17/genética , Interleucina-17/metabolismo , Rim/metabolismo , Rim/patologia , Camundongos , Camundongos Knockout , Linfócitos T/patologia , Fator de Necrose Tumoral alfa/genéticaRESUMO
RATIONALE: The ubiquitin-editing protein A20 in dendritic cells (DCs) suppresses NF-κB (nuclear factor-κB) signaling and constrains DC-mediated T-cell stimulation, but the role of A20 in modulating the hypertensive response requires elucidation. OBJECTIVE: Here, we tested the hypothesis that A20 in CD11c-expressing myeloid cells mitigates Ang II (angiotensin II)-induced hypertension by limiting renal T-cell activation. METHODS AND RESULTS: Mice with heterozygous deletion of A20 in CD11c-expressing myeloid cells (DC ACT[Cd11c-Cre+A20flox/wt]) have spontaneous DC activation but have normal baseline blood pressures. In response to low-dose chronic Ang II infusion, DC ACT mice compared with WT (wild type) controls had an exaggerated hypertensive response and augmented proportions of CD62LloCD44hi effector memory T lymphocytes in the kidney lymph node. After 10 days of Ang II, DC ACT kidneys had increased numbers of memory effector CD8+, but not CD4+ T cells, compared with WTs. Moreover, the expressions of TNF-α (tumor necrosis factor-α) and IFN-γ (interferon-γ) were upregulated in the DC ACT renal CD8+ T cells but not CD4+ T cells. Saline challenge testing revealed enhanced renal fluid retention in the DC ACT mice. DC ACT kidneys showed augmented protein expression of γ-epithelial sodium channel and NHE3 (sodium-hydrogen antiporter 3). DC ACT mice also had greater reductions in renal blood flow following acute injections with Ang II and enhanced oxidant stress in the vasculature as evidenced by higher circulating levels of malondialdehyde compared with WT controls. To directly test whether enhanced T-cell activation in the DC ACT cohort was responsible for their exaggerated hypertensive response, we chronically infused Ang II into lymphocyte-deficient DC ACT Rag1 (recombination activating protein 1)-deficient (Rag1-/-) mice and WT (Cd11c-Cre-A20flox/wt) Rag1-/- controls. The difference in blood pressure elevation accruing from DC activation was abrogated on the Rag1-/- strain. CONCLUSIONS: Following stimulation of the renin-angiotensin system, A20 suppresses DC activation and thereby mitigates T-cell-dependent blood pressure elevation.
Assuntos
Células Dendríticas/metabolismo , Hipertensão/metabolismo , Rim/metabolismo , Células Mieloides/metabolismo , Linfócitos T/metabolismo , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/deficiência , Animais , Células Cultivadas , Células Dendríticas/imunologia , Hipertensão/imunologia , Hipertensão/prevenção & controle , Rim/citologia , Rim/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células Mieloides/imunologia , Linfócitos T/imunologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/imunologiaRESUMO
Wnt/ß-catenin signaling is essential in the pathogenesis of renal fibrosis. We previously reported inhibition of the Wnt O-acyl transferase porcupine, required for Wnt secretion, dramatically attenuates kidney fibrosis in the murine unilateral ureteral obstruction model. Here, we investigated the tissue-specific contributions of porcupine to renal fibrosis and inflammation in ureteral obstruction using mice with porcupine deletion restricted to the kidney tubular epithelium or infiltrating myeloid cells. Obstruction of the ureter induced the renal mRNA expression of porcupine and downstream targets, ß-catenin, T-cell factor, and lymphoid enhancer factor in wild type mice. Renal tubular specific deficiency of porcupine reduced the expression of collagen I and other fibrosis markers in the obstructed kidney. Moreover, kidneys from obstructed mice with tubule-specific porcupine deficiency had reduced macrophage accumulation with attenuated expression of myeloid cytokine and chemokine mRNA. In co-culture with activated macrophages, renal tubular cells from tubular-specific porcupine knockout mice had blunted induction of fibrosis mediators compared with wild type renal tubular cells. In contrast, macrophages from macrophage-specific porcupine deficient mice in co-culture with wild type renal tubular cells had markedly enhanced expression of pro-fibrotic cytokines compared to wild type macrophages. Consequently, porcupine deletion specifically within macrophages augmented renal scar formation following ureteral obstruction. Thus, our experiments suggest a benefit of interrupting Wnt secretion specifically within the kidney epithelium while preserving Wnt O-acylation in infiltrating myeloid cells during renal fibrogenesis.
Assuntos
Aciltransferases/metabolismo , Proteínas de Membrana/metabolismo , Nefroesclerose/metabolismo , Via de Sinalização Wnt , Animais , Quimiocinas/metabolismo , Feminino , Fibrose , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Células Mieloides/metabolismo , Nefroesclerose/etiologia , Obstrução UreteralRESUMO
BACKGROUND: Polarized macrophage populations can orchestrate both inflammation of the kidney and tissue repair during CKD. Proinflammatory M1 macrophages initiate kidney injury, but mechanisms through which persistent M1-dependent kidney damage culminates in fibrosis require elucidation. Krüppel-like factor 4 (KLF4), a zinc-finger transcription factor that suppresses inflammatory signals, is an essential regulator of macrophage polarization in adipose tissues, but the effect of myeloid KLF4 on CKD progression is unknown. METHODS: We used conditional mutant mice lacking KLF4 or TNFα (KLF4's downstream effector) selectively in myeloid cells to investigate macrophage KLF4's role in modulating CKD progression in two models of CKD that feature robust macrophage accumulation, nephrotoxic serum nephritis, and unilateral ureteral obstruction. RESULTS: In these murine CKD models, KLF4 deficiency in macrophages infiltrating the kidney augmented their M1 polarization and exacerbated glomerular matrix deposition and tubular epithelial damage. During the induced injury in these models, macrophage-specific KLF4 deletion also exacerbated kidney fibrosis, with increased levels of collagen 1 and α-smooth muscle actin in the injured kidney. CD11b+Ly6Chi myeloid cells isolated from injured kidneys expressed higher levels of TNFα mRNA versus wild-type controls. In turn, mice bearing macrophage-specific deletion of TNFα exhibited decreased glomerular and tubular damage and attenuated kidney fibrosis in the models. Moreover, treatment with the TNF receptor-1 inhibitor R-7050 during nephrotoxic serum nephritis reduced damage, fibrosis, and necroptosis in wild-type mice and mice with KLF4-deficient macrophages, and abrogated the differences between the two groups in these parameters. CONCLUSIONS: These data indicate that macrophage KLF4 ameliorates CKD by mitigating TNF-dependent injury and fibrosis.
Assuntos
Nefropatias/etiologia , Rim/patologia , Fatores de Transcrição Kruppel-Like/fisiologia , Macrófagos/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Fibrose/etiologia , Fator 4 Semelhante a Kruppel , Masculino , Camundongos , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
BACKGROUND: Following an acute insult, macrophages regulate renal fibrogenesis through the release of various factors that either encourage the synthesis of extracellular matrix synthesis or the degradation of matrix via endocytosis, proteolysis, or both. However, the roles of infiltrating versus resident myeloid cells in these opposing processes require elucidation. The transcription factor Twist1 controls diverse essential cellular functions through induction of several downstream targets, including matrix metalloproteinases (MMPs). In macrophages, Twist1 can influence patterns of cytokine generation, but the role of macrophage Twist1 in renal fibrogenesis remains undefined. METHODS: To study Twist1 functions in different macrophage subsets during kidney scar formation, we used two conditional mutant mouse models in which Twist1 was selectively ablated either in infiltrating, inflammatory macrophages or in resident tissue macrophages. We assessed fibrosis-related parameters, matrix metallopeptidase 13 (MMP13, or collagen 3, which catalyzes collagen degradation), inflammatory cytokines, and other factors in these Twist1-deficient mice compared with wild-type controls after subjecting the animals to unilateral ureteral obstruction. We also treated wild-type and Twist1-deficient mice with an MMP13 inhibitor after unilateral ureteral obstruction. RESULTS: Twist1 in infiltrating inflammatory macrophages but not in resident macrophages limited kidney fibrosis after ureteral obstruction by driving extracellular matrix degradation. Moreover, deletion of Twist1 in infiltrating macrophages attenuated the expression of MMP13 in CD11b+Ly6Clo myeloid cells. Inhibition of MMP13 abrogated the protection from renal fibrosis afforded by macrophage Twist1. CONCLUSIONS: Twist1 in infiltrating myeloid cells mitigates interstitial matrix accumulation in the injured kidney by promoting MMP13 production, which drives extracellular matrix degradation. These data highlight the complex cell-specific actions of Twist1 in the pathogenesis of kidney fibrosis.
Assuntos
Matriz Extracelular/metabolismo , Nefropatias/metabolismo , Rim/metabolismo , Rim/patologia , Macrófagos/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Actinas/metabolismo , Animais , Benzofuranos/farmacologia , Receptor 1 de Quimiocina CX3C/metabolismo , Colágeno Tipo I/metabolismo , Modelos Animais de Doenças , Fibrose , Expressão Gênica , Hidroxiprolina/metabolismo , Nefropatias/etiologia , Nefropatias/patologia , Macrófagos Peritoneais/metabolismo , Masculino , Metaloproteinase 13 da Matriz/genética , Inibidores de Metaloproteinases de Matriz/farmacologia , Camundongos , Morfolinas/farmacologia , Células Mieloides/enzimologia , Proteína 1 Relacionada a Twist/genética , Obstrução Ureteral/complicaçõesRESUMO
Most forms of chronic kidney disease culminate in renal fibrosis that heralds organ failure. In contrast to the protective effects of globally blocking type 1 angiotensin (AT1) receptors throughout the body, activating AT1 receptors directly on immune cells may serve protective functions. However, the effects of stimulating the T-cell AT1 receptor on the progression of renal fibrosis remain unknown. In this study, mice with T-cell-specific deletion of the dominant murine AT1 receptor isoform Lck-Cre Agtraflox/flox [total knockout (TKO)] and wild-type (WT) controls were subjected to the unilateral ureteral obstruction model of kidney fibrosis. Compared with WT controls, obstructed kidneys from TKO mice at day 14 had increased collagen 1 deposition. CD4+ T cells, CD11b+Ly6Chi myeloid cells, and mRNA levels of Th1 inflammatory cytokines are elevated in obstructed TKO kidneys, suggesting that augmented Th1 responses in the TKO mice may exaggerate renal fibrosis by driving proinflammatory macrophage differentiation. In turn, T-bet deficient (T-bet knockout) mice lacking Th1 responses have attenuated collagen deposition after unilateral ureteral obstruction. We conclude that activating the AT1 receptor on T cells mitigates renal fibrogenesis by inhibiting Th1 differentiation and renal accumulation of profibrotic macrophages.
Assuntos
Fibrose/prevenção & controle , Inflamação/prevenção & controle , Nefropatias/prevenção & controle , Receptor Tipo 1 de Angiotensina/fisiologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Fibrose/imunologia , Fibrose/metabolismo , Fibrose/patologia , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Nefropatias/imunologia , Nefropatias/metabolismo , Nefropatias/patologia , Masculino , Camundongos , Camundongos Knockout , Linfócitos T/patologiaRESUMO
Aims: The actin-binding protein Drebrin is up-regulated in response to arterial injury and reduces smooth muscle cell (SMC) migration and proliferation through its interaction with the actin cytoskeleton. We, therefore, tested the hypothesis that SMC Drebrin inhibits angiotensin II-induced remodelling of the proximal aorta. Methods and results: Angiotensin II was administered via osmotic minipumps at 1000 ng/kg/min continuously for 28 days in SM22-Cre+/Dbnflox/flox (SMC-Dbn-/-) and control mice. Blood pressure responses to angiotensin II were assessed by telemetry. After angiotensin II infusion, we assessed remodelling in the proximal ascending aorta by echocardiography and planimetry of histological cross sections. Although the degree of hypertension was equivalent in SMC-Dbn-/- and control mice, SMC-Dbn-/- mice nonetheless exhibited 60% more proximal aortic medial thickening and two-fold more outward aortic remodelling than control mice in response to angiotensin II. Proximal aortas demonstrated greater cellular proliferation and matrix deposition in SMC-Dbn-/- mice than in control mice, as evidenced by a higher prevalence of proliferating cell nuclear antigen-positive nuclei and higher levels of collagen I. Compared with control mouse aortas, SMC-Dbn-/- aortas demonstrated greater angiotensin II-induced NADPH oxidase activation and inflammation, evidenced by higher levels of Ser-536-phosphorylated NFκB p65 subunits and higher levels of vascular cell adhesion molecule-1, matrix metalloproteinase-9, and adventitial macrophages. Conclusions: We conclude that SMC Drebrin deficiency augments angiotensin II-induced inflammation and adverse aortic remodelling.
Assuntos
Angiotensina II , Doenças da Aorta/metabolismo , Hipertensão/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Neuropeptídeos/metabolismo , Remodelação Vascular , Animais , Aorta/metabolismo , Aorta/patologia , Aorta/fisiopatologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Doenças da Aorta/fisiopatologia , Pressão Arterial , Proliferação de Células , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Células HEK293 , Humanos , Hipertensão/genética , Hipertensão/patologia , Hipertensão/fisiopatologia , Mediadores da Inflamação/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/patologia , NADPH Oxidases/metabolismo , Neuropeptídeos/deficiência , Neuropeptídeos/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Acute kidney injury (AKI) is a leading cause of morbidity and mortality. Drug-induced/toxic AKI can be caused by a number of therapeutic agents. Cisplatin is an effective chemotherapeutic agent whose administration is limited by significant nephrotoxicity. Therapies to prevent cisplatin-induced AKI are lacking. Although tumor necrosis factor-α (TNF) plays a key role in the pathogenesis of cisplatin nephrotoxicity, the innate immune signaling pathways that trigger TNF generation in this context require elucidation. In this regard, sterile injury triggers the release and activation of both isoforms of interleukin(IL)-1, IL-1α and IL-1ß. In turn, stimulation of the interleukin-1 receptor (IL-1R1) by these ligands engages a proinflammatory signaling cascade that induces TNF induction. We therefore hypothesized that IL-1R1 activation exacerbates cisplatin-induced AKI by inducing TNF production, thereby augmenting inflammatory signals between kidney parenchymal cells and infiltrating myeloid cells. IL-1R1+/+ (WT) and IL-1R1-/- (KO) mice were subjected to cisplatin-induced AKI. Compared with WT mice, IL-1R1 KO mice had attenuated AKI as measured by serum creatinine and BUN, renal NGAL mRNA levels, and blinded histological analysis of kidney pathology. In the cisplatin-injured kidney, IL-1R1 KO mice had diminished levels of whole kidney TNF, and fewer Ly6G-expressing neutrophils. In addition, an unbiased machine learning analysis of intrarenal immune cells revealed a diminished number of CD11bint/CD11cint myeloid cells in IL-1R1 KO injured kidneys compared with IL-1R1 WT kidneys. Following cisplatin, IL-1R1 KO kidneys, compared with WTs, had fewer TNF-producing: macrophages, CD11bint/CD11cint cells, and neutrophils, consistent with an effect of IL-1R1 to polarize intrarenal myeloid cells toward a proinflammatory phenotype. Interruption of IL-1-dependent signaling pathways warrants further evaluation to decrease nephrotoxicity during cisplatin therapy.
Assuntos
Injúria Renal Aguda/metabolismo , Cisplatino , Rim/metabolismo , Receptores Tipo I de Interleucina-1/metabolismo , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/genética , Injúria Renal Aguda/imunologia , Animais , Comunicação Celular , Separação Celular/métodos , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Mediadores da Inflamação/metabolismo , Rim/patologia , Aprendizado de Máquina , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/metabolismo , Receptores Tipo I de Interleucina-1/deficiência , Receptores Tipo I de Interleucina-1/genética , Transdução de Sinais , Processos Estocásticos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The link between inappropriate salt retention in the kidney and hypertension is well recognized. However, growing evidence suggests that the immune system can play surprising roles in sodium homeostasis, such that the study of inflammatory cells and their secreted effectors has provided important insights into salt sensitivity. As part of the innate immune system, myeloid cells have diverse roles in blood pressure regulation, ranging from prohypertensive actions in the kidney, vasculature, and brain, to effects in the skin that attenuate blood pressure elevation. In parallel, T lymphocyte subsets, as key constituents of the adaptive immune compartment, have variable effects on renal sodium handling and the hypertensive response, accruing from the functions of the cytokines that they produce. Conversely, salt can directly modulate the phenotypes of myeloid and T cells, illustrating bidirectional regulatory mechanisms through which sodium and the immune system coordinately impact blood pressure. This review details the complex interplay between myeloid cells, T cells, and salt in the pathogenesis of essential hypertension.
Assuntos
Imunidade Adaptativa/fisiologia , Pressão Sanguínea/fisiologia , Hipertensão/fisiopatologia , Rim/fisiopatologia , Monócitos/imunologia , Cloreto de Sódio/metabolismo , Linfócitos T/imunologia , Animais , Citocinas/metabolismo , Humanos , Hipertensão/imunologia , Hipertensão/metabolismo , Rim/imunologia , Rim/metabolismo , Monócitos/metabolismo , Linfócitos T/metabolismoRESUMO
A variety of immune cell subsets contribute to the pathogenesis of hypertension and associated kidney damage following inappropriate activation of the renin-angiotensin system (RAS). These immune cell subsets often express common surface markers, which complicates their separation and characterization in vivo. Accordingly, flow cytometry has become an invaluable tool for parsing immune cell populations because this technique permits the simultaneous detection of up to 18 markers on a single cell. Below we describe a process by which one can determine the immune cell subsets in the kidney via flow cytometry.
Assuntos
Citocinas/metabolismo , Citometria de Fluxo/métodos , Rim/metabolismo , Sistema Renina-Angiotensina/fisiologia , Subpopulações de Linfócitos T/metabolismo , Animais , Rim/imunologia , Camundongos , Fenótipo , Subpopulações de Linfócitos T/imunologiaRESUMO
Immune cells infiltrate the kidney, vasculature, and central nervous system during hypertension, consequently amplifying tissue damage and/or blood pressure elevation. Mononuclear cell motility depends partly on chemokines, which are small cytokines that guide cells through an increasing concentration gradient via ligation of their receptors. Tissue expression of several chemokines is elevated in clinical and experimental hypertension. Likewise, immune cells have enhanced chemokine receptor expression during hypertension, driving immune cell infiltration and inappropriate inflammation in cardiovascular control centers. T lymphocytes and monocytes/macrophages are pivotal mediators of hypertensive inflammation, and these cells migrate in response to several chemokines. As powerful drivers of diapedesis, the chemokines CCL2 and CCL5 have long been implicated in hypertension, but experimental data highlight divergent, context-specific effects of these chemokines on blood pressure and tissue injury. Several other chemokines, particularly those of the CXC family, contribute to blood pressure elevation and target organ damage. Given the significant interplay and chemotactic redundancy among chemokines during disease, future work must not only describe the actions of individual chemokines in hypertension, but also characterize how manipulating a single chemokine modulates the expression and/or function of other chemokines and their cognate receptors. This information will facilitate the design of precise chemotactic immunotherapies to limit cardiovascular and renal morbidity in hypertensive patients.
Assuntos
Quimiocinas/imunologia , Hipertensão/complicações , Hipertensão/imunologia , Inflamação/complicações , Inflamação/imunologia , Animais , Quimiocinas/análise , Humanos , Hipertensão/patologia , Imunidade Celular , Inflamação/patologia , Receptores de Quimiocinas/análise , Receptores de Quimiocinas/imunologiaRESUMO
Inappropriate activation of the renin-angiotensin system (RAS) exacerbates renal and vascular injury. Accordingly, treatment with global RAS antagonists attenuates cardiovascular risk and slows the progression of proteinuric kidney disease. By reducing BP, RAS inhibitors limit secondary immune activation responding to hemodynamic injury in the target organ. However, RAS activation in hematopoietic cells has immunologic effects that diverge from those of RAS stimulation in the kidney and vasculature. In preclinical studies, activating type 1 angiotensin (AT1) receptors in T lymphocytes and myeloid cells blunts the polarization of these cells toward proinflammatory phenotypes, protecting the kidney from hypertensive injury and fibrosis. These endogenous functions of immune AT1 receptors temper the pathogenic actions of renal and vascular AT1 receptors during hypertension. By counteracting the effects of AT1 receptor stimulation in the target organ, exogenous administration of AT2 receptor agonists or angiotensin 1-7 analogs may similarly limit inflammatory injury to the heart and kidney. Moreover, although angiotensin II is the classic effector molecule of the RAS, several RAS enzymes affect immune homeostasis independently of canonic angiotensin II generation. Thus, as reviewed here, multiple components of the RAS signaling cascade influence inflammatory cell phenotype and function with unpredictable and context-specific effects on innate and adaptive immunity.