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Non-alcoholic steatohepatitis (NASH) and alcoholic steatohepatitis (ASH) are the leading causes of liver disease worldwide. To identify disease-specific pathomechanisms, we analyzed the lipidome, metabolome and immune cell recruitment in livers in both diseases. Mice harboring ASH or NASH had comparable disease severities regarding mortality rate, neurological behavior, expression of fibrosis marker and albumin levels. Lipid droplet size was higher in NASH than ASH and qualitative differences in the lipidome were mainly based on incorporation of diet-specific fatty acids into triglycerides, phosphatidylcholines and lysophosphatidylcholines. Metabolomic analysis showed downregulated nucleoside levels in both models. Here, the corresponding uremic metabolites were only upregulated in NASH suggesting stronger cellular senescence, which was supported by lower antioxidant levels in NASH as compared to ASH. While altered urea cycle metabolites suggest increased nitric oxide synthesis in both models, in ASH, this depended on increased L-homoarginine levels indicating a cardiovascular response mechanism. Interestingly, only in NASH were the levels of tryptophan and its anti-inflammatory metabolite kynurenine upregulated. Fittingly, high-content immunohistochemistry showed a decreased macrophage recruitment and an increased polarization towards M2-like macrophages in NASH. In conclusion, with comparable disease severity in both models, higher lipid storage, oxidative stress and tryptophan/kynurenine levels were seen in NASH, leading to distinct immune responses.
Behind the Wall - Compartment-Specific Neovascularisation during Post-Stroke Recovery in Mice
(2022)
Ischemic stroke is a highly prevalent vascular disease leading to oxygen- and glucose deprivation in the brain. In response, ischemia-induced neovascularization occurs, which is supported by circulating CD34+ endothelial progenitor cells. Here, we used the transient middle cerebral artery occlusion (tMCAO) mouse model to characterize the spatio-temporal alterations within the ischemic core from the acute to the chronic phase using multiple-epitope-ligand cartography (MELC) for sequential immunohistochemistry. We found that around 14 days post-stroke, significant angiogenesis occurs in the ischemic core, as determined by the presence of CD31+/CD34+ double-positive endothelial cells. This neovascularization was accompanied by the recruitment of CD4+ T-cells and dendritic cells as well as IBA1+ and IBA1− microglia. Neighborhood analysis identified, besides pericytes only for T-cells and dendritic cells, a statistically significant distribution as direct neighbors of CD31+/CD34+ endothelial cells, suggesting a role for these cells in aiding angiogenesis. This process was distinct from neovascularization of the peri-infarct area as it was separated by a broad astroglial scar. At day 28 post-stroke, the scar had emerged towards the cortical periphery, which seems to give rise to a neuronal regeneration within the peri-infarct area. Meanwhile, the ischemic core has condensed to a highly vascularized subpial region adjacent to the leptomeningeal compartment. In conclusion, in the course of chronic post-stroke regeneration, the astroglial scar serves as a seal between two immunologically active compartments—the peri-infarct area and the ischemic core—which exhibit distinct processes of neovascularization as a central feature of post-stroke tissue remodeling. Based on our findings, we propose that neovascularization of the ischemic core comprises arteriogenesis as well as angiogenesis originating from the leptomenigeal vasculature.
Bacterial and fungal toll-like receptor activation elicits type I IFN responses in mast cells
(2021)
Next to their role in IgE-mediated allergic diseases and in promoting inflammation, mast cells also have antiinflammatory functions. They release pro- as well as antiinflammatory mediators, depending on the biological setting. Here we aimed to better understand the role of mast cells during the resolution phase of a local inflammation induced with the Toll-like receptor (TLR)-2 agonist zymosan. Multiple sequential immunohistology combined with a statistical neighborhood analysis showed that mast cells are located in a predominantly antiinflammatory microenvironment during resolution of inflammation and that mast cell-deficiency causes decreased efferocytosis in the resolution phase. Accordingly, FACS analysis showed decreased phagocytosis of zymosan and neutrophils by macrophages in mast cell-deficient mice. mRNA sequencing using zymosan-induced bone marrow-derived mast cells (BMMC) revealed a strong type I interferon (IFN) response, which is known to enhance phagocytosis by macrophages. Both, zymosan and lipopolysaccharides (LPS) induced IFN-β synthesis in BMMCs in similar amounts as in bone marrow derived macrophages. IFN-β was expressed by mast cells in paws from naïve mice and during zymosan-induced inflammation. As described for macrophages the release of type I IFNs from mast cells depended on TLR internalization and endosome acidification. In conclusion, mast cells are able to produce several mediators including IFN-β, which are alone or in combination with each other able to regulate the phagocytotic activity of macrophages during resolution of inflammation.
Inflammation is a crucial host defense mechanism activated in response to injury or infection. Its primary goal is to eliminate the source of the disturbance, repair the damaged tissue, and restore homeostasis. Inflammatory processes can be recognized through increased blood flow, higher vascular permeability, and the recruitment of leukocytes and plasma proteins to the tissue. A pathogen-induced inflammation triggers various pro- and anti-inflammatory processes. Local tissue cells and Toll-like receptors call upon innate immune cells like neutrophils, dendritic cells (DCs), and monocytes to respond to the intruder. They move across the endothelium and respond to local signals by releasing mediators or cytotoxic compounds, phagocytosing, or polarizing. To study local pathogen-induced inflammation, a zymosan-induced inflammation model was used in the hind paws of mice, which caused a Toll-like receptor 2 mediated inflammation. Multi-Epitope-Ligand-Cartography (MELC) was used for multiple sequential immunohistochemistry with 40 different antibodies on the same tissue. Bioinformatic analysis and graphical representation revealed a specific inflammatory architecture consisting of three major areas based on macrophage polarization and their cellular neighborhoods: a core region containing the pathogen, a pro-inflammatory region containing M1-like macrophages, and a region containing anti-inflammatory cells. This discovery highlights the coexistence of pro- and antiinflammatory processes during an ongoing inflammation and challenges the concept of a gradual temporal transition from pro- to anti-inflammation. Flow cytometry of the whole paw was performed to support and refine the MELC results. Eosinophils were used as a specific immune cell population to investigate their role in the inflammatory structure. They were found to be present in all three inflammatory regions, adapting their cytokine profile according to their localization. Depleting eosinophils reduced Interleukin 4 (IL-4)- levels, increased edema formation, and mechanical and thermal hypersensitivities during inflammation resolution. In the absence of eosinophils, pro- and anti-inflammatory region could not be determined in the inflammatory architecture, neutrophil numbers increased, and efferocytosis and M2-macrophage polarization were reduced. IL-4 administration restored these regions, normalized neutrophil numbers, efferocytosis, M2-macrophage polarization, and resolution of zymosan-induced hypersensitivity. The results show that eosinophils expressing IL-4 support the resolution of inflammation by enabling the development of an anti-inflammatory framework that encloses pro-inflammatory regions.