RESUMO
Nuclear factor-κB (NF-κB) is a crucial pro-inflammatory transcription factor whose activation is of immense interest to immunology research. Estimation of NF-κB activation through flow cytometry is not possible due to the unavailability of robust flow cytometry antibodies that can bind to its phosphorylated, active, nuclear form. In this protocol, we describe a flow cytometry assay that measures the activation of the pro-inflammatory transcription factor NF-κB in stimulated immune cells by quantifying the degradation of its upstream regulator IκBα. We demonstrate the utility of this protocol by assessment of intracellular IκBα in human primary regulatory T cells experiencing TNFR2 agonism, a process previously reported to activate NF-κB in these cells. We also show that this assay may be applied to study NF-κB activation in other cell types, such as human primary T cells and THP-1 cell-derived macrophages, when induced by their corresponding inflammatory cues. Thus, this robust and reproducible protocol will be of interest to a wide range of scientists who aim to measure NF-κB activity in medium-to-high-throughput assays. © 2024 Wiley Periodicals LLC. Basic Protocol: Quantifying inflammatory activation by flow cytometry of IκBα degradation Support Protocol 1: Isolating and expanding human regulatory T cells Support Protocol 2: Calculating IC50 from flow cytometry data using Excel.
Assuntos
Citometria de Fluxo , Inibidor de NF-kappaB alfa , NF-kappa B , Humanos , Citometria de Fluxo/métodos , NF-kappa B/metabolismo , Inibidor de NF-kappaB alfa/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Proteólise , Células THP-1 , Macrófagos/metabolismo , Macrófagos/imunologiaRESUMO
BACKGROUND: The first-in-class treatment PF-06480605 targets the tumor necrosis factor-like ligand 1A (TL1A) molecule in humans. Results from the phase 2a TUSCANY trial highlighted the safety and efficacy of PF-06480605 in ulcerative colitis. Preclinical and in vitro models have identified a role for TL1A in both innate and adaptive immune responses, but the mechanisms underlying the efficacy of anti-TL1A treatment in inflammatory bowel disease (IBD) are not known. METHODS: Here, we provide analysis of tissue transcriptomic, peripheral blood proteomic, and fecal metagenomic data from the recently completed phase 2a TUSCANY trial and demonstrate endoscopic improvement post-treatment with PF-06480605 in participants with ulcerative colitis. RESULTS: Our results revealed robust TL1A target engagement in colonic tissue and a distinct colonic transcriptional response reflecting a reduction in inflammatory T helper 17 cell, macrophage, and fibrosis pathways in patients with endoscopic improvement. Proteomic analysis of peripheral blood revealed a corresponding decrease in inflammatory T-cell cytokines. Finally, microbiome analysis showed significant changes in IBD-associated pathobionts, Streptococcus salivarius, S. parasanguinis, and Haemophilus parainfluenzae post-therapy. CONCLUSIONS: The ability of PF-06480605 to engage and inhibit colonic TL1A, targeting inflammatory T cell and fibrosis pathways, provides the first-in-human mechanistic data to guide anti-TL1A therapy for the treatment of IBD.
Assuntos
Colite Ulcerativa , Colite Ulcerativa/tratamento farmacológico , Fibrose/tratamento farmacológico , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Ligantes , Necrose , Proteômica , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/antagonistas & inibidores , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genéticaRESUMO
Noninfectious lung injury and acute graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) are associated with significant morbidity and mortality. Azithromycin is widely used in allogeneic HCT recipients for pulmonary chronic GVHD, although current data appear controversial. We induced GVHD and noninfectious lung injury in lethally irradiated B6D2F1 mice by transplanting bone marrow and splenic T cells from allogeneic C57BL/6 mice. Experimental groups were treated with oral azithromycin starting on day 14 until the end of week 6 or week 14 after transplantation. Azithromycin treatment resulted in improved survival and decreased lung injury; the latter characterized by improved pulmonary function, reduced peribronchial and perivascular inflammatory cell infiltrates along with diminished collagen deposition, and a decrease in lung cytokine and chemokine expression. Azithromycin also improved intestinal GVHD but did not affect liver GVHD at week 6 early after transplantation. At week 14, azithromycin decreased liver GVHD but had no effect on intestinal GVHD. In vitro, allogeneic antigen-presenting cell (APC)- dependent T cell proliferation and cytokine production were suppressed by azithromycin and inversely correlated with relative regulatory T cell (Treg) expansion, whereas no effect was seen when T cell proliferation occurred APC independently through CD3/CD28-stimulation. Further, azithromycin reduced alloreactive T cell expansion but increased Treg expansion in vivo with corresponding downregulation of MHC II on CD11c(+) dendritic cells. These results demonstrate that preventive administration of azithromycin can reduce the severity of acute GVHD and noninfectious lung injury after allo-HCT, supporting further investigation in clinical trials.
Assuntos
Antibacterianos/farmacologia , Azitromicina/farmacologia , Transplante de Medula Óssea , Doença Enxerto-Hospedeiro/prevenção & controle , Lesão Pulmonar/prevenção & controle , Pulmão/efeitos dos fármacos , Doença Aguda , Animais , Citocinas/biossíntese , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Modelos Animais de Doenças , Feminino , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/mortalidade , Doença Enxerto-Hospedeiro/patologia , Intestinos/efeitos dos fármacos , Intestinos/imunologia , Fígado/efeitos dos fármacos , Fígado/imunologia , Pulmão/imunologia , Pulmão/patologia , Lesão Pulmonar/imunologia , Lesão Pulmonar/mortalidade , Lesão Pulmonar/patologia , Camundongos , Cultura Primária de Células , Testes de Função Respiratória , Baço/efeitos dos fármacos , Baço/imunologia , Análise de Sobrevida , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/transplante , Transplante Homólogo , Irradiação Corporal TotalRESUMO
Acute graft-versus-host disease (GVHD) considerably limits wider usage of allogeneic hematopoietic cell transplantation (allo-HCT). Antigen-presenting cells and T cells are populations customarily associated with GVHD pathogenesis. Of note, neutrophils are the largest human white blood cell population. The cells cleave chemokines and produce reactive oxygen species, thereby promoting T cell activation. Therefore, during an allogeneic immune response, neutrophils could amplify tissue damage caused by conditioning regimens. We analyzed neutrophil infiltration of the mouse ileum after allo-HCT by in vivo myeloperoxidase imaging and found that infiltration levels were dependent on the local microbial flora and were not detectable under germ-free conditions. Physical or genetic depletion of neutrophils reduced GVHD-related mortality. The contribution of neutrophils to GVHD severity required reactive oxygen species (ROS) because selective Cybb (encoding cytochrome b-245, beta polypeptide, also known as NOX2) deficiency in neutrophils impairing ROS production led to lower levels of tissue damage, GVHD-related mortality and effector phenotype T cells. Enhanced survival of Bcl-xL transgenic neutrophils increased GVHD severity. In contrast, when we transferred neutrophils lacking Toll-like receptor-2 (TLR2), TLR3, TLR4, TLR7 and TLR9, which are normally less strongly activated by translocating bacteria, into wild-type C57BL/6 mice, GVHD severity was reduced. In humans, severity of intestinal GVHD strongly correlated with levels of neutrophils present in GVHD lesions. This study describes a new potential role for neutrophils in the pathogenesis of GVHD in both mice and humans.
Assuntos
Doença Enxerto-Hospedeiro/imunologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Íleo/imunologia , Microbiota/imunologia , Neutrófilos/imunologia , Animais , Bussulfano , Ciclofosfamida , Citometria de Fluxo , Adjuvante de Freund , Doença Enxerto-Hospedeiro/fisiopatologia , Técnicas Histológicas , Íleo/microbiologia , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Luciferases , Imageamento por Ressonância Magnética , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise em Microsséries , NADPH Oxidase 2 , NADPH Oxidases/genética , Peroxidase , Espécies Reativas de Oxigênio/metabolismoRESUMO
The success of allogeneic (allo) hematopoietic cell transplantation (HCT) is limited by its treatment related complications, mostly graft versus host disease (GVHD) and fungal and viral infections. CMV reactivation after HCT has been associated with increased morbidity and mortality, and a causal relation between GVHD, immunosuppressive therapy and vice versa has been postulated. Using a low GVHD severity murine HCT model, we assessed the role of MCMV reactivation and GVHD development. BALB/c mice were infected with either murine CMV (MCMV) or mock and monitored for 25 weeks to establish latency, followed by sublethal irradiation conditioning and infusion of bone marrow plus splenocytes from either syngeneic (syn) BALB/c or allo B10.D2 donors. Engraftment of allo donor cells was confirmed by PCR for D2Mit265 gene product size. Day+100 mortality and overall GVHD severity in allo MCMV pre-infected recipients was higher than in allo mock controls. Pathologic changes of lung and liver GVHD in immediate-early gene 1 (IE1) positive recipients were significantly increased compared to mock controls, and were only slightly increased in IE1 negative. No significant gut injury was seen in any group. Aggravated lung injury in IE1 positive recipients correlated with higher BAL cell counts both for total cells and for CD4+ T cells when compared with mock controls, and also with protein expression of lung IFN-gamma and liver TNF. No evidence for CMV specific morphologic changes was seen on histopathology in any organ of IE1 positive recipients, suggesting that CMV reactivation is related to increased GVHD severity but does not require active CMV disease, strengthening the concept of a reciprocal relationship between CMV and GVHD.
Assuntos
Expressão Gênica , Genes Precoces/genética , Doença Enxerto-Hospedeiro/etiologia , Transplante de Células-Tronco Hematopoéticas , Muromegalovirus/genética , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Quimiocinas/metabolismo , Quimerismo , Feminino , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/mortalidade , Doença Enxerto-Hospedeiro/virologia , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/virologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Inflamação/metabolismo , Fígado/metabolismo , Fígado/patologia , Fígado/virologia , Pulmão/metabolismo , Pulmão/patologia , Pulmão/virologia , Camundongos , Polimorfismo Genético , Transplante Homólogo , Ativação Viral/genética , Latência ViralRESUMO
BACKGROUND: It is well known that enteric bacterial antigens drive the development of chronic colitis in a variety of different mouse models of the inflammatory bowel diseases (IBD). The objective of this study was to evaluate the role of gut-associated lymphoid tissue (GALT; Peyer's patches, isolated lymphoid follicles), mesenteric lymph nodes (MLNs) and spleen in the pathogenesis of chronic colitis in mice. METHODS: Surgical as well as genetic approaches were used to generate lymphopenic mice devoid of one or more of these lymphoid tissues. For the first series of studies, we subjected recombinase activating gene-1-deficient mice (RAG(-/-) ) to sham surgery (Sham), mesenteric lymphadenectomy (MLNx), splenectomy (Splx) or both (MLNx/Splx). In a second series of studies we intercrossed lymphotoxinß-deficient (LTß(-/-) ) mice with RAG(-/-) animals to generate LTß(-/-) x RAG(-/-) offspring that were anticipated to contain functional MLNs but be devoid of GALT and most peripheral lymph nodes. Flow purified naïve (CD4(+) CD45RB(high) ) T-cells were adoptively transferred into the different groups of RAG(-/-) recipients to induce chronic colitis. RESULTS: We found that at 3-5 wks following T-cell transfer, all four of the surgically-manipulated RAG(-/-) groups (Sham, MLNx, Splx and MLNx/Splx) developed chronic colitis that was similar in onset and severity. Flow cytometric analysis revealed no differences among the different groups with respect to surface expression of different gut-homing markers nor were there any differences noted in IFN-γ and IL-17 generation by mononuclear cells isolated among these surgically-manipulated mice. Although we anticipated that LTß(-/-) x RAG(-/-) mice would contain functional MLNs but be devoid of GALT and peripheral lymph nodes (PLNs), we found that LTß(-/-) x RAG(-/-) mice were in fact devoid of MLNs as well as GALT and PLNs. Adoptive transfer of CD45RB(high) T-cells into LTß(-/-) x RAG(-/-) mice or their littermate controls (LTß(+/+) x RAG(-/-) ) induced rapid and severe colitis in both groups. CONCLUSIONS: Taken together, our data demonstrate that: a) neither the GALT, MLNs nor PLNs are required for induction of chronic gut inflammation in this model of IBD and b) T-and/or B-cells may be required for the development of MLNs in LTß(-/-) mice.