RESUMO
Intestinal macrophages and fibroblasts act as microenvironmental sentinels mediating inflammation and disease progression in Crohn's disease (CD). We aimed to establish the effects of fecal supernatants (FSs) from patients with CD on macrophage and fibroblast phenotype and function. FS were obtained by ultracentrifugation, and the metabolites were analyzed. Monocyte-derived M2 macrophages and fibroblasts were conditioned with FS, and secreted proteins, surface proteins and gene expression were analyzed. M2 macrophage efferocytosis was evaluated. Patients with CD (n = 15) had a skewed fecal metabolite profile compared to healthy subjects (HS, n = 10). FS from CD patients (CD-FS) induced an anti-inflammatory response in M2 macrophages with higher expression of IL-10, IL1RA and CD206 as compared to healthy FS (HS-FS) while the efferocytotic capacity was unaltered. CD-FS did not affect extracellular matrix production from fibroblasts, but increased expression of the pro-inflammatory proteins IL-6 and MCP-1. Conditioned media from M2 macrophages treated with CD-FS modulated gene expression in fibroblasts for TGFß superfamily members and reduced IL-4 expression compared to HS-FS. We show that M2 macrophages and fibroblasts react abnormally to the fecal microenvironment of CD patients, resulting in altered protein expression related to inflammation but not fibrosis. This implies that the gut microbiota and its metabolites have an important role in the generation and/or perpetuation of inflammation in CD.
Assuntos
Doença de Crohn , Humanos , Inflamação , Meios de Cultivo Condicionados/farmacologia , Progressão da Doença , FibroblastosRESUMO
Inflammasomes are intracellular protein complexes whose activation results in proinflammatory cytokines. Inflammasomes are implicated in Crohn´s disease (CD) pathogenesis, yet the contribution of inflammasomes in intestinal epithelial cells (IECs) versus lamina propria (LP) macrophages is poorly understood. Whether inflammasome expression in intestinal tissue reflects the serum inflammatory protein profile of patients is also not known. We aimed to determine the intestinal cell types where inflammasome expression is increased in CD and if they correlate with the serum protein profile. RT-PCR and NanoString nCounter technology were used to characterize inflammasome gene expression in CD patients and controls. The mucosa, LP and IEC cell fractions and FACS-sorted cells were analyzed. Proximity extension assay with a 92-protein panel was used to determine the serum inflammatory protein profile. Compositional analysis was used to correlate ileum inflammasome gene expression with intestinal mononuclear phagocyte populations. We show that NLRP3 and MEFV inflammasome sensors and downstream effector expression including IL-1ß are increased in inflamed mucosa of IBD patients and correlate with disease activity. Inflammasome gene expression increased with the abundance of immature intestinal macrophages, and increased IL-1ß released by CD LP cells correlated with immature macrophage frequency. Inflammasome gene expression was also increased in circulating monocytes, the precursors of immature intestinal macrophages. Finally, the serum inflammatory profile of CD patients correlates with ileal expression of genes related to NLRP3 and MEFV inflammasomes. Overall, we show that MEFV and NLRP3 inflammasome expression in CD intestine is attributed to the accumulation of immature macrophages and correlates with serum inflammatory proteins.
Assuntos
Doença de Crohn , Inflamassomos , Macrófagos , Proteínas Sanguíneas/metabolismo , Doença de Crohn/metabolismo , Humanos , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Pirina/genética , Pirina/metabolismoRESUMO
BACKGROUND: Molecular profiles change in response to perturbations. These changes are coordinated into functional modules via regulatory interactions. The genes and their products within a functional module are expected to be differentially expressed in a manner coherent with their regulatory network. This perspective presents a promising approach to increase precision in detecting differential signals as well as for describing differential regulatory signals within the framework of a priori knowledge about the underlying network, and so from a mechanistic point of view. RESULTS: We present Coherent Network Expression (CoNE), an effective procedure for identifying differentially activated functional modules in molecular interaction networks. Differential gene expression is chosen as example, and differential signals coherent with the regulatory nature of the network are identified. We apply our procedure to systematically simulated data, comparing its performance to alternative methods. We then take the example case of a transcription regulatory network in the context of particle-induced pulmonary inflammation, recapitulating and proposing additional candidates to previously obtained results. CoNE is conveniently implemented in an R-package along with simulation utilities. CONCLUSION: Combining coherent interactions with error control on differential gene expression results in uniformly greater specificity in inference than error control alone, ensuring that captured functional modules constitute real findings.