Isolation and high‐dimensional phenotyping of gastrointestinal immune cells

David, Bruna Araújo; Rubino, Stephen; Moreira, Thaís Garcias; Freitas-Lopes, Maria Alice; Araújo, Arnaldo de Albuquerque; Paul, Nicole; Rezende, Rafael M.; Menezes, Gustavo B.

doi:10.1111/imm.12706

Cited by 17 publications

(17 citation statements)

References 30 publications

(35 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In fact, a reliable gating strategy to differentially assess KCs and liver DCs is still under debate. 20 , 21 , 28 , 39 Importantly, an overt infiltration of neutrophils occurred at later time points (9 weeks), which was previously described as a key factor for enhanced collateral liver damage upon insults, 14 , 23 , 24 , 40 , 41 suggesting that such early alterations in these subpopulations may constitute a hallmark in the evolution from simple steatosis to steatohepatitis. However, it is reasonable to hypothesize that such overt neutrophil infiltration during NAFLD onset might also function as a backup strategy for bacterial clearance and phagocytosis while KCs are relatively absent.…”

Section: Discussionmentioning

confidence: 62%

See 1 more Smart Citation

Imaging and immunometabolic phenotyping uncover changes in the hepatic immune response in the early phases of NAFLD

et al. 2020

View full text Add to dashboard Cite

Background & Aims The precise determination of non-alcoholic fatty liver disease (NAFLD) onset is challenging. Thus, the initial hepatic responses to fat accumulation, which may be fundamental to our understanding of NAFLD evolution and clinical outcomes, are largely unknown. Herein, we chronologically mapped the immunologic and metabolic changes in the liver during the early stages of fatty liver disease in mice and compared this with human NAFLD samples. Methods Liver biopsies from patients with NAFLD (NAFLD activity score [NAS] 2–3) were collected for gene expression profiling. Mice received a high-fat diet for short periods to mimic initial steatosis and the hepatic immune response was investigated using a combination of confocal intravital imaging, gene expression, cell isolation, flow cytometry and bone marrow transplantation assays. Results We observed major immunologic changes in patients with NAS 2–3 and in mice in the initial stages of NAFLD. In mice, these changes significantly increased mortality rates upon drug-induced liver injury, as well as predisposing mice to bacterial infections. Moreover, deletion of Toll-like receptor 4 in liver cells dampened tolerogenesis, particularly in Kupffer cells, in the initial stages of dietary insult. Conclusion The hepatic immune system acts as a sentinel for early and minor changes in hepatic lipid content, mounting a biphasic response upon dietary insult. Priming of liver immune cells by gut-derived Toll-like receptor 4 ligands plays an important role in liver tolerance in initial phases, but continuous exposure to insults may lead to damage and reduced ability to control infections. Lay summary Fatty liver is a very common form of hepatic disease, leading to millions of cases of cirrhosis every year. Patients are often asymptomatic until becoming very sick. Therefore, it is important that we expand our knowledge of the early stages of disease pathogenesis, to enable early diagnosis. Herein, we show that even in the early stages of fatty liver disease, there are significant alterations in genes involved in the inflammatory response, suggesting that the hepatic immune system is disturbed even following minor and undetectable changes in liver fat content. This could have implications for the diagnosis and clinical management of fatty liver disease.

show abstract

Section: Discussionmentioning

confidence: 62%

“…Primary hepatocyte purification was performed as described previously. 21 Liver non-parenchymal cells (LNPCs) from mice were isolated as reported previously. 17 For flow cytometry, cells were stained with antibodies described in the supplementary CTAT table .…”

Section: Methodsmentioning

confidence: 99%

Imaging and immunometabolic phenotyping uncover changes in the hepatic immune response in the early phases of NAFLD

et al. 2020

View full text Add to dashboard Cite

show abstract

“…Normalized data were imported to Cytobank, where gating was performed as described in Supplementary Fig. 5 and ViSNE analysis was performed as described previously 64 .…”

Section: Methodsmentioning

confidence: 99%

Acute microglia ablation induces neurodegeneration in the somatosensory system

et al. 2018

View full text Add to dashboard Cite

Previous studies have reported that microglia depletion leads to impairment of synapse formation and these cells rapidly repopulate from CNS progenitors. However, the impact of microglia depletion and repopulation in the long-term state of the CNS environment has not been characterized. Here, we report that acute and synchronous microglia depletion and subsequent repopulation induces gray matter microgliosis, neuronal death in the somatosensory cortex and ataxia-like behavior. We find a type 1 interferon inflammatory signature in degenerating somatosensory cortex from microglia-depleted mice. Transcriptomic and mass cytometry analysis of repopulated microglia demonstrates an interferon regulatory factor 7-driven activation state. Minocycline and anti-IFNAR1 antibody treatment attenuate the CNS type 1 interferon-driven inflammation, restore microglia homeostasis and reduce ataxic behavior. Neither microglia depletion nor repopulation impact neuropathology or T-cell responses during experimental autoimmune encephalomyelitis. Together, we found that acute microglia ablation induces a type 1 interferon activation state of gray matter microglia associated with acute neurodegeneration.

show abstract

“…Primary hepatocyte purification was performed as described previously [ 17 , 18 ]. Briefly, mice were treated with APAP and 6, 12, and 24 h after treatment mice were anesthetized and submitted to liver perfusion with collagenase (Sigma-Aldrich, C2139) through the portal vein.…”

Section: Methodsmentioning

confidence: 99%

Liver Immune Cells Release Type 1 Interferon Due to DNA Sensing and Amplify Liver Injury from Acetaminophen Overdose

Araújo

Antunes

Mattos

et al. 2018

Cells

View full text Add to dashboard Cite

Hepatocytes may rupture after a drug overdose, and their intracellular contents act as damage-associated molecular patterns (DAMPs) that lead to additional leukocyte infiltration, amplifying the original injury. Necrosis-derived DNA can be recognized as a DAMP, activating liver non-parenchymal cells (NPCs). We hypothesized that NPCs react to DNA by releasing interferon (IFN)-1, which amplifies acetaminophen (APAP)-triggered liver necrosis. We orally overdosed different knockout mouse strains to investigate the pathways involved in DNA-mediated amplification of APAP-induced necrosis. Mice were imaged under intravital confocal microscopy to estimate injury progression, and hepatocytes and liver NPCs were differentially isolated for gene expression assays. Flow cytometry (FACS) using a fluorescent reporter mouse estimated the interferon-beta production by liver leukocytes under different injury conditions. We also treated mice with DNase to investigate the role of necrosis DNA signaling in IFN-1 production. Hepatocytes released a large amount of DNA after APAP overdose, which was not primarily sensed by these cells. However, liver NPCs promptly sensed such environmental disturbances and activated several DNA sensing pathways. Liver NPCs synthesized and released IFN-1, which was associated with concomitant hepatocyte necrosis. Ablation of IFN-1 recognition in interferon α/β receptor (IFNAR−/−) mice delayed APAP-mediated liver necrosis and dampened IFN-1 sensing pathways. We demonstrated a novel loop involving DNA recognition by hepatic NPCs and additional IFN-1 mediated hepatocyte death.

show abstract

Isolation and high‐dimensional phenotyping of gastrointestinal immune cells

Cited by 17 publications

References 30 publications

Imaging and immunometabolic phenotyping uncover changes in the hepatic immune response in the early phases of NAFLD

Imaging and immunometabolic phenotyping uncover changes in the hepatic immune response in the early phases of NAFLD

Acute microglia ablation induces neurodegeneration in the somatosensory system

Liver Immune Cells Release Type 1 Interferon Due to DNA Sensing and Amplify Liver Injury from Acetaminophen Overdose

Contact Info

Product

Resources

About