Circulating TREM2 as a noninvasive diagnostic biomarker for NASH in patients with elevated liver stiffness.

BACKGROUND AND AIMS: Reliable noninvasive biomarkers are an unmet clinical need for the diagnosis of NASH. This study investigates the diagnostic accuracy of the circulating triggering receptor expressed on myeloid cells 2 (plasma TREM2) as a biomarker for NASH in patients with NAFLD and elevated liver stiffness. APPROACH AND RESULTS: We collected cross-sectional, clinical data including liver biopsies from a derivation ( n = 48) and a validation cohort ( n = 170) of patients with elevated liver stiffness measurement (LSM ≥ 8.0 kPa). Patients with NAFLD activity scores (NAS) ≥4 were defined as having NASH. Plasma TREM2 levels were significantly elevated in patients with NASH of the derivation cohort, with an area under the receiver operating characteristics curve (AUROC) of 0.92 (95% confidence interval [CI], 0.84-0.99). In the validation cohort, plasma TREM2 level increased approximately two-fold in patients with NASH, and a strong diagnostic accuracy was confirmed (AUROC, 0.83; 95% CI, 0.77-0.89; p < 0.0001). Plasma TREM2 levels were associated with the individual histologic features of NAS: steatosis, lobular inflammation, and ballooning ( p < 0.0001), but only weakly with fibrosis stages. Dual cutoffs for rule-in and rule-out were explored: a plasma TREM2 level of ≤38 ng/ml was found to be an optimal NASH rule-out cutoff (sensitivity 90%; specificity 52%), whereas a plasma TREM2 level of ≥65 ng/ml was an optimal NASH rule-in cutoff (specificity 89%; sensitivity 54%). CONCLUSIONS: Plasma TREM2 is a plausible individual biomarker that can rule-in or rule-out the presence of NASH with high accuracy and thus has the potential to reduce the need for liver biopsies and to identify patients who are eligible for clinical trials in NASH.

Haptoglobin-related protein in human plasma correlates to haptoglobin concentrations and phenotypes.

Haptoglobin-related protein (Hpr) is a plasma protein with high sequence similarity to haptoglobin (Hp). Like Hp, Hpr also binds hemoglobin (Hb) with high affinity, but it does not bind to the Hb-Hp receptor CD163 on macrophages. The Hpr concentration is markedly lower than Hp in plasma and its regulation is not understood. In the present study, we have developed non-crossreactive antibodies to Hpr to analyze the Hpr concentration in 112 plasma samples from anonymized individuals and compared it to Hp. The results show that plasma Hpr correlated with Hp concentrations (rho = 0.46, p = .0001). Hpr accounts for on average 0.35% of the Hp/Hpr pool but up to 29% at low Hp levels. Furthermore, the Hpr concentrations were significantly lower in individuals with the Hp2-2 phenotype compared to those with the Hp2-1 or Hp1-1 phenotypes. Experimental binding analysis did not provide evidence that Hpr associates with Hp and in this way is removed via CD163. In conclusion, the Hpr concentration correlates to Hp concentrations and Hp-phenotypes by yet unknown mechanisms independent of CD163-mediated removal of Hb-Hp complexes.

Re-evalution of the measurement of haptoglobin in human plasma samples.

Haptoglobin (Hp) is an abundant plasma protein scavenging hemoglobin (Hb) via CD163 on macrophages. This process consumes Hp, which therefore negatively correlates to hemolysis. However, exact measurements of Hp plasma levels are complicated by different phenotypes (Hp1-1, Hp2-1, and Hp2-2) forming different oligomeric states with differences in immunoreactivity. In addition, humans have an immune-cross-reactive Hp-related protein. In the present study, we developed Hp-specific monoclonal antibodies for an accurate Hp analysis of the different Hp phenotypes in a panel of 112 anonymous samples from hospitalized individuals subjected to routine Hp immunoturbidimetric measurements. The data revealed immunoturbidimetry as a reliable method in most cases but also that the use of non-phenotype-specific calibrators leads to substantial bias in the measurement of the Hp-concentration, non at least in Hp1-1 individuals. Furthermore, analysis of the Hb-dependence of the CD163 interaction with Hp1-1 and Hp2-2 showed that a higher 'cost-effectiveness' in the consumption of dimeric Hp1-1 versus multimeric Hp phenotypes is a likely contribution to the observed differences in the plasma levels of the Hp phenotypes. In conclusion, the determination of Hp phenotype and the use of phenotype-specific calibrators are essential to obtain a precise estimate of the Hp level in healthy and diseased individuals.

Apolipoproteins L1 and L3 control mitochondrial membrane dynamics.

Apolipoproteins L1 and L3 (APOLs) are associated at the Golgi with the membrane fission factors phosphatidylinositol 4-kinase-IIIB (PI4KB) and non-muscular myosin 2A. Either APOL1 C-terminal truncation (APOL1Δ) or APOL3 deletion (APOL3-KO [knockout]) reduces PI4KB activity and triggers actomyosin reorganization. We report that APOL3, but not APOL1, controls PI4KB activity through interaction with PI4KB and neuronal calcium sensor-1 or calneuron-1. Both APOLs are present in Golgi-derived autophagy-related protein 9A vesicles, which are involved in PI4KB trafficking. Like APOL3-KO, APOL1Δ induces PI4KB dissociation from APOL3, linked to reduction of mitophagy flux and production of mitochondrial reactive oxygen species. APOL1 and APOL3, respectively, can interact with the mitophagy receptor prohibitin-2 and the mitophagosome membrane fusion factor vesicle-associated membrane protein-8 (VAMP8). While APOL1 conditions PI4KB and APOL3 involvement in mitochondrion fission and mitophagy, APOL3-VAMP8 interaction promotes fusion between mitophagosomal and endolysosomal membranes. We propose that APOL3 controls mitochondrial membrane dynamics through interactions with the fission factor PI4KB and the fusion factor VAMP8.

Obese Patients With Nonalcoholic Fatty Liver Disease Have an Increase in Soluble Plasma CD163 and a Concurrent Decrease in Hepatic Expression of CD163.

Background and Aims: Macrophages play an important role in the development of nonalcoholic fatty liver disease (NAFLD) and its progression to nonalcoholic steatohepatitis (NASH). In this study, we investigated the hepatic expression of the macrophage scavenger receptor CD163 and the plasma level of its shed soluble form (sCD163) in patients with obesity and NASH, non-NASH NAFLD (NAFL), or healthy livers (no NAFLD). Methods: Paired liver biopsies and plasma samples were collected from 61 patients with obesity (body mass index ≥35). Hepatic expression of CD163 was analyzed by immunohistochemistry and data-independent acquisition mass spectrometry, whilst plasma levels of sCD163 were determined by enzyme-linked immunosorbent assay and data-independent acquisition mass spectrometry. NAFLD stage and activity were assessed using the Kleiner fibrosis and NASH Clinical Research Network (NAS-CRN) scoring system. Results: sCD163 turned out as a promising predictor of NASH with an area under the receiver-operating characteristic curve of 0.78 [0.65;0.92] (P = .0008). sCD163 increased with more severe NAFLD both in univariate (odds ratio [OR] = 3.31[1.80;6.11], P < .001) and multivariable ordinal logistic regression adjusting for NAFLD risk factors (OR = 2.02 [1.03;3.97], P = .042). On the other hand, hepatic expression of CD163 was negatively associated with more severe NAFLD in univariate ordinal logistic regression determined by immunohistochemistry (OR = 0.91[0.84;0.98], P = .015) and proteomics (OR = 0.13[0.02;0.80], P = .028). Taking NAFLD risk factors into account, hepatic expression of CD163 was only associated with the fibrosis stage (OR = 0.01 [0.0003;0.21], P = .004). Accordingly, hepatic CD163 surface expression and sCD163 were negatively correlated (rho = -0.478, P = .0001). Conclusion: An increased plasma sCD163 and a concurrent decreased hepatic expression of CD163 are strongly associated with NAFLD in obese patients.