Genome-wide DNA methylation profiling is able to identify prefibrotic PMF cases at risk for progression to myelofibrosis

Background!#!Patients suffering from the BCR-ABL1-negative myeloproliferative disease prefibrotic primary myelofibrosis (pre-PMF) have a certain risk for progression to myelofibrosis. Accurate risk estimation for this fibrotic progression is of prognostic importance and clinically relevant. Commonly applied risk scores are based on clinical, cytogenetic, and genetic data but do not include epigenetic modifications. Therefore, we evaluated the assessment of genome-wide DNA methylation patterns for their ability to predict fibrotic progression in PMF patients.!##!Results!#!For this purpose, the DNA methylation profile was analyzed genome-wide in a training set of 22 bone marrow trephines from patients with either fibrotic progression (n = 12) or stable disease over several years (n = 10) using the 850 k EPIC array from Illumina. The DNA methylation classifier constructed from this data set was validated in an independently measured test set of additional 11 bone marrow trephines (7 with stable disease, 4 with fibrotic progress). Hierarchical clustering of methylation β-values and linear discriminant classification yielded very good discrimination between both patient groups. By gene ontology analysis, the most differentially methylated CpG sites are primarily associated with genes involved in cell-cell and cell-matrix interactions.!##!Conclusions!#!In conclusion, we could show that genome-wide DNA methylation profiling of bone marrow trephines is feasible under routine diagnostic conditions and, more importantly, is able to predict fibrotic progression in pre-fibrotic primary myelofibrosis with high accuracy

Splenectomy Prior to Experimental Induction of Autoimmune Hepatitis Promotes More Severe Hepatic Inflammation, Production of IL-17 and Apoptosis

Autoimmune hepatitis (AIH) is detected at a late stage in the course of the disease. Therefore, induction and etiology are largely unclear. It is controversial if the induction of autoimmunity occurs in the liver or in the spleen. In our experimental murine AIH model, the induction of autoimmunity did not occur in the spleen. Instead, a protective role of the spleen could be more likely. Therefore, we splenectomized mice followed by induction of experimental murine AIH. Splenectomized mice presented more severe portal inflammation. Furthermore, these mice had more IL-17, IL-23 receptor (IL-23R) and caspase 3 (casp3) and a decreased amount of erythropoietin in serum, while intrahepatic T cell compartments were unaffected. These results indicate that the spleen is not necessary for induction of AIH, and splenectomy disrupts the ability to immune regulate the intensity of hepatic inflammation, production of IL-17 and apoptosis

Pediatric autoimmune hepatitis shows a disproportionate decline of regulatory T cells in the liver and of IL-2 in the blood of patients undergoing therapy

<div><p>Background & Aims</p><p>The autoimmune hepatitis (AIH) is a chronic hepatitis driven by the adaptive immunity that affects all age groups. A functional and numerical regulatory T cell (Treg) defect has been reported in pediatric AIH (pAIH), while an intrahepatic increase in adult AIH (aAIH) patients has been detected in current research findings.</p><p>Methods</p><p>Therefore, we quantified the intrahepatic numbers of Treg, T and B cells, as well as serum cytokine levels before and during therapy in pAIH.</p><p>Results</p><p>We found a disproportional intrahepatic enrichment of Tregs in untreated pAIH compared to pediatric non-alcoholic fatty liver disease. The increase of Treg/total T cells was even more pronounced than in aAIH due to fewer infiltrating T and B cells. Portal densities of Treg, as well as total T and B cells, declined significantly during therapy. However, portal Treg densities decreased disproportionately, leading to even decreasing ratios of Treg to T and B cells during therapy. Out of 28 serum cytokines IL-2 showed the strongest (10fold) decrease under therapy. This decline of IL-2 was associated with decreasing intrahepatic Treg numbers under therapy. None of the baseline T and B cell infiltration parameters were associated with the subsequent treatment response in pAIH.</p><p>Conclusions</p><p>Intrahepatic Tregs are rather enriched in untreated pAIH. The disproportional decrease of Tregs during therapy may be caused by a decrease of IL-2 levels. New therapies should, therefore, aim in strengthening intrahepatic immune regulation.</p></div

Association of intrahepatic baseline immunophenotyping and subsequent treatment response.

<p>Association of intrahepatic baseline immunophenotyping and subsequent treatment response.</p

IL-2 associated decline of portal Treg infiltration under therapy for pAIH.

<p><b>(A)</b> Comparison of portal cell densities and portal cell ratios of CD4⁺FOXP3⁺ Treg in untreated pediatric AIH (n = 40) and under ongoing therapy (n = 13). <b>(B)</b> Fold changes of CD4⁺FOXP3⁺ (Treg; n = 11), CD4⁺+CD8⁺ (T cells; n = 11) and CD79a⁺ cells (B cells; n = 9) under therapy in paired samples. <b>(C)</b> Comparison of CD4⁺FOXP3⁺ Treg and total T cells (CD4⁺+CD8⁺) in treated pediatric AIH (n = 13) and treated adult AIH matched for the treatment response (n = 16). <b>(D)</b> Quantification of serum cytokine levels in untreated pAIH at diagnosis (D; n = 43) and during follow-up (FU; n = 28) under therapy. Depicted are all cytokine with significant changes in the follow-up in paired and unpaired non-parametric comparisons. The horizontal line and error bars represent the median and the interquartile range. The seventeen cytokines without significant changes are not shown. Cytokine levels below the detection threshold were set to the lowest detected concentration. <b>(E)</b> Serum IL-2 levels in pediatric AIH similar to (D) and adult AIH (diagnosis: n = 88; under therapy: n = 34), as well as age and gender, matched pediatric controls for cytokines (n = 34). (n.s.: not significant; * p<0.05; ** p<0.01; *** p<0.001)</p

Multicolor immunofluorescence of formalin-fixed and paraffin embedded liver biopsies from pAIH.

<p><b>(A)</b> T cell staining with CD4 (red), CD8 (green), FOXP3 (blue) in a single formalin fixed and paraffin embedded liver biopsy section with pediatric AIH and <b>(B)</b> B cell staining with CD79a (red) and CD4 (green, autofluorescence in blue) in subsequent liver biopsy sections. The white lines surround the evaluated area of the portal infiltrates and exclude lumen of veins, arteries and bile ducts. White bars represent 100 μm. <b>(C)</b> T cell staining of comparator liver biopsies with pediatric non-alcoholic fatty liver disease as in A. <b>(D)</b> Surface expression of CD4 (red) and CD8 (green) in comparison to nuclear colocalization of FOXP3 (blue) and DAPI (white) in pediatric AIH. White bars represent 50 μm.</p

Portal T cell infiltration pattern in untreated AIH-1.

<p><b>(A-C)</b> Comparison of size of portal infiltrates, portal cell densities and portal cell ratios, as well as absolute numbers per portal tract of T cells (CD4⁺+CD8⁺), B cells (CD79a⁺) and CD4⁺FOXP3⁺ Treg in untreated pediatric AIH (pAIH; n = 40), pediatric non-alcoholic liver disease (pNAFLD; n = 12) and untreated adult AIH (aAIH; n = 45). <b>(D)</b> Correlation analysis (n = 38; SR: Spearman rank correlation coefficient) of the portal CD4⁺FOXP3⁺ Treg cell densities and cell ratios as well as the absolute number per portal tract with the hepatitis activity index (mHAI). Horizontal line represents the median and error bars the interquartile range. (* p<0.05; ** p<0.01; *** p<0.001; n.s.: not significant)</p

Patient data.

<p>Patient data.</p

Hyperferritinemia and hypergammaglobulinemia predict the treatment response to standard therapy in autoimmune hepatitis.

Autoimmune hepatitis (AIH) is a chronic hepatitis with an increasing incidence. The majority of patients require life-long immunosuppression and incomplete treatment response is associated with a disease progression. An abnormal iron homeostasis or hyperferritinemia is associated with worse outcome in other chronic liver diseases and after liver transplantation. We assessed the capacity of baseline parameters including the iron status to predict the treatment response upon standard therapy in 109 patients with untreated AIH type 1 (AIH-1) in a retrospective single center study. Thereby, a hyperferritinemia (> 2.09 times upper limit of normal; Odds ratio (OR) = 8.82; 95% confidence interval (CI): 2.25-34.52) and lower immunoglobulins (<1.89 times upper limit of normal; OR = 6.78; CI: 1.87-24.59) at baseline were independently associated with the achievement of complete biochemical remission upon standard therapy. The predictive value increased when both variables were combined to a single treatment response score, when the cohort was randomly split into a training (area under the curve (AUC) = 0.749; CI 0.635-0.863) and internal validation cohort (AUC = 0.741; CI 0.558-0.924). Patients with a low treatment response score (<1) had significantly higher cumulative remission rates in the training (p<0.001) and the validation cohort (p = 0.024). The baseline hyperferritinemia was accompanied by a high serum iron, elevated transferrin saturations and mild hepatic iron depositions in the majority of patients. However, the abnormal iron status was quickly reversible under therapy. Mechanistically, the iron parameters were not stringently related to a hepatocellular damage. Ferritin rather seems deregulated from the master regulator hepcidin, which was down regulated, potentially mediated by the elevated hepatocyte growth factor. In conclusion, baseline levels of serum ferritin and immunoglobulins, which are part of the diagnostic work-up of AIH, can be used to predict the treatment response upon standard therapy in AIH-1, although confirmation from larger multicenter studies is pending