[MUSIC] Talking about liver inflammation in NASH, the macrophages play a pivotal role and in the inflammatory milieu a transition towards activation of Kupffer cells as well as recruitment of monocytes and all inflammatory cells also play a role. Macrophages are turned into a proinflammatory state and stimulating the stellate cells. Part of the excavation is caused by apoptotic cells caused by oxidative stress and endoplasmatic reticulum stress, and as assumed before genes are also involved in the transition from steatosis to inflammation. Recently my research team with consultant, Constantin Kosenko reviewed the role of macrophages in NAFLD, development and progression. As you see here, macrophages play a central role in the inflammatory process and may be activated in a number of different ways. There is a specific crosstalk between adipose tissue and the liver due to the secretion of TNF, IL 6 but also CCL2. Further, the macrophages may be activated directly by free fatty acids from the adipose tissue. It is also known that there is a change in the gut microbiome with dysbiosis and a leaky gut with increased intestinal permeability that may result in the release of toxic substances including deeper polysaccharides (LPS) and pathogen-associated molecular patterns (PAMPS). In the liver, damaged hepatocytes due to apoptosis, may activate the macrophages through damaged-associated molecular patterns or the DAMPS. Finally, the macrophages are activated, they attract other inflammatory cells like monocytes and lymphocytes, propagating an inflammatory process. With further production of inflammatory molecules and mediators like the TNF, IL-6, radical oxygen species and so on. In addition, they produced TGF-Beta that activate and stimulate hepatic stellate cells to be responsible for the fibrogenesis. Of interest is that when the macrophages are activated, they ship the lineage specific CD163 receptor, which can be measured in the circulation as Soluble CD163 and macrophage activation marker. Just recently we investigated the association between Macrophage activation and insulin resistance. In this study published in 2019, we investigated insulin resistance in adipose tissue and in the liver in NAFLD patients using specific clamp techniques. We then use soluble CD163 as a MARCO macrophage activation and the association with insulin resistance and liver disease severity with steatosis and fibrosis. We showed significant association with adipose tissue insulin resistance, and liposis, and fibrosis states. Further, we observed significant associations with liver steatosis. There was no association with hepatic insulin resistance and fibrosis. We also showed significant associations with soluble CD163 as a MARCO macrophage activation and liver fat and fibrosis. Solule CD163 levels were associated with adipose tissue insulin resistance and both soluble CD163 levels, and adipose tissue insulin resistance was associated with free fatty acids levels and liver disease severity and fibrosis. Further, CD163 levels were associated with the hepatic fat but not with visceral and subcutaneous fat. So the conclusion from this study is that in NAFLD patients, hepatic macrophage activity is associated with adipose tissue insulin resistance, and we therefore hypothesize that in NAFLD patients, adipose tissue insulin resistance stimulates hepatic macrophages through increased influx of free fatty acids resulting in liver inflammation and fibrosis. Previously we also investigated soluble CD163 levels in biopsy-verified NASH patients. In this study published in 2016, we demonstrated a stepwise increase in CD163 levels in association both with the NAFLD activity score and the fibrosis score into independent in NAFLD cohorts. Further soluble CD163 levels were good predictors for significant fibrosis, with a receiver operating curves or ROC of 0.73, and combining this with the NAFLD fibrosis score improved the prediction further. In another study from 2015, we observed a similar stepwise increase in soluble CD163 levels with liver disease severity as determined by the non-alcoholic fatty liver disease activity score, the NAS score and the fibrosis score. But more importantly, soluble CD163 decreased following bariatric surgery both in patients with a high and a low NAS score with higher degrees of fibrosis. And most importantly, we also discovered a gradient across the liver, suggesting that soluble CD163 actually comes from hepatic production by liver macrophages. During the recent years a number of studies have focused on the role of the gut microbiome and dysbiosis in NAFLD development and progression. Small intestinal bacterial overgrowth is well described in NASH patients and specific microbiota signatures are associated with NAFLD severity. There are several mechanisms involved, from disruption of intestinal barrier and increased permeability, as shown previously, along with endotoxins (PAMPS) that induce inflammation and macrophage activation. Also, metabolic mediators have been in focus with ethanol production from gut bacteria, production of short chain fatty acids and amino acid derived metabolites, like glutamine and cryptophane. Further, other bacterial components may be involved. And regarding the microbiome NAFLD severity has been associated with abundance of ruminococcus species and fibrosis and NASH severity with bacterias. In a study by Loomba and co-workers, they constructed a random forest classifier model that included 40 features, including 37 bacterial species, and this model was able to distinguish mild/moderate NAFLD from patient with more than F2 fibrosis. Gut derived metabolize may also play a role. As shown in the left panel dietary fibers are digested by gut bacteria, producing short chain fatty acids, especially butyrate, acetate and propionate. These may induce local inflammation in the intestinal wall accompanied by proinflammatory cytokines and also GLP-1 production. These aberrations may be involved in both triglyceride synthesis and lipid accumulation assumed for short-chain fatty acids, or (inaudible) by GLP-1. Recently, an emergent role for bile acids has been proposed as they are involved in hepatic glucose cholesterol and triglyceride homeostasis. The primary bile acids activate the bile acid receptor known as the farnesoid x receptor or FXR while secondary bile acids seem to inhibit FXR signaling. Thus the disregulation of primary and secondary bile acids may be involved in NAFLD pathogenesis. This is also now being investigated in novel treatment using FXR agonist that stimulate the FXR receptor to treat NAFLD patients. As shown in this video, we have moved forward from the two-hit hypothesis back in 1998 to the multiple hit hypothesis in 2010. And further information has derived during the recent years, especially with the advancement in new technologies, especially omics-techniques. Also employing artificial intelligence and machine learning techniques. It is predicted that the combination of these techniques with a deep and accurate phenotyping of human cohorts, including both children and adults, will provide novel pathophysiological information, and this may provide or procure other methods for the prediction and prognostication, including new treatment targets for NAFLD patients.
