Mild Iron Overload as Seen in Individuals Homozygous for the Alpha-1 Antitrypsin Pi*Z Variant Does Not Promote Liver Fibrogenesis in HFE Knockout Mice

The presence of the homozygous ‘Pi*Z’ variant of alpha-1 antitrypsin (AAT) (‘Pi*ZZ’ genotype) predisposes to liver fibrosis development, but the role of iron metabolism in this process remains unknown. Therefore, we assessed iron metabolism and variants in the Homeostatic Iron Regulator gene (HFE) as the major cause of hereditary iron overload in a large cohort of Pi*ZZ subjects without liver comorbidities. The human cohort comprised of 409 Pi*ZZ individuals and 254 subjects without evidence of an AAT mutation who were recruited from ten European countries. All underwent a comprehensive work-up and transient elastography to determine liver stiffness measurements (LSM). The corresponding mouse models (Pi*Z overexpressors, HFE knockouts, and double transgenic [DTg] mice) were used to evaluate the impact of mild iron overload on Pi*Z-induced liver injury. Compared to Pi*Z non-carriers, Pi*ZZ individuals had elevated serum iron, transferrin saturation, and ferritin levels, but relevant iron overload was rare. All these parameters were higher in individuals with signs of significant liver fibrosis (LSM ≥ 7.1 kPa) compared to those without signs of significant liver fibrosis. HFE knockout and DTg mice displayed similar extent of iron overload and of fibrosis. Loss of HFE did not alter the extent of AAT accumulation. In Pi*ZZ individuals, presence of HFE mutations was not associated with more severe liver fibrosis. Taken together, Pi*ZZ individuals display minor alterations in serum iron parameters. Neither mild iron overload seen in these individuals nor the presence of HFE mutations (C282Y and H63D) constitute a major contributor to liver fibrosis development.


HFE mutation detection in human samples
To detect the H63D and C282Y mutations, a 393 base pair (bp) and 208 bp products were amplified with previously described primers (Table S1 ) [27,28]. PCR amplification was carried out with a commercial master mix (BioBudget Technologies GmbH, Germany) and 0,1-1 µm of each primer. Subsequently, the amplicons were digested with RsaI restriction endonuclease (R0167, New England Biolabs,Germany; detection of C282Y variant) [38] or Bcll restriction endonuclease (R0160, New England Bioloabs; detection of H63D variant) [28,39] as recommended by the manufacturer. RsaI cleavage of wild-type allele produces 247-bp and 146-bp fragments, whereas the presence of the C282Y mutation results in an additional cleavage of the 146-bp fragment into 117 bp and 29 bp products. BcII cleaves the 208-bp PCR product into two fragments of 138 and 70 bp length in the wild-type, but does not digest in the mutant allele. The fragments were resolved by electrophoresis in a 3% agarose gel. Table S1. Mouse and human genotyping primers.

Tissue stainings
For histological staining, the harvested liver tissues were kept in 4% buffered formalin at room temperature overnight. Afterwards, they were removed, dehydrated, embedded in paraffin, cut onto 2 µm thick sections and stained with Hematoxylin and Eosin, Perls Prussian Blue (iron staining), Sirius Red staining, and Periodic acid -Schiff diastase staining as specified below.
Hematoxylin and eosin staining (H&E) was used to analyze the extent of liver injury. Sections were first deparaffinized with xyline and hydrated with a serial dilution of ethanol following washing steps with water. The basophilic nuclei were stained with a hematoxylin (blue color) and the acidophilic cytoplasm was stained with eosin (pink) [40].

Gene 5'-Sequence -3' Accession number
Transgenic mice Sirius Red staining was used to visualize the collagen fibers and thereby to quantify the extent of liver fibrosis. After deparaffinization in xylol, the sections were hydrated with serial dilutions of isopropanol. After washing shortly with deionized water, they were stained in Sirius red solution for 1 hour at room temperature [40]. Fibrosis scores were determined by modified Ishak scoring system: score 0-no obvious pathology; score 1-mild; periportal; score 2-moderate; portal fibrosis; score 3-distinct; septal fibrosis; score 4-complete bridging fibrosis. Iron distribution was assessed via modified Deugnier score as described previously [41]: 0-granules absent; 1-granules barely discernible; 1.5-granules discernible; 2masses visible.
Periodic acid -Schiff diastase staining (PASD) was used to visualize and analyze AAT inclusions. Firstly, the deparaffinized sections were hydrated with serial dilutions of isopropanol and washed shortly with deionized water. To degrade polysaccharides, the tissues were treated with saliva which contains abundant amount of α-amylase (also known as diastase) for 30 minutes at RT. After treatment with periodic acid (P0430; Sigma, Steinheim, Germany) solution, subsequent incubation with Schiff reagent (Sigma, Germany) for 15 minutes at RT resulted in positive staining of magenta colored-diastase resistant AAT globules that were analyzed microscopically. To quantify the extent of AAT inclusions, stained liver sections were scored by two independent observers as: 0, no inclusions; 1-mild; 1.5moderate; 2-distinct in periportal areas.

Protein analysis
Total liver lysates were prepared by homogenization in 3 % sodium dodecyl sulphate (SDS, Roth, Germany) containing sample buffer followed by centrifugation to remove non-soluble debris. The isolated protein extracts were diluted in 4x reducing Laemmli buffer and the proteins were separated by 10% SDS-polyacrylamide gel electrophoresis. Electrophoresed samples were either visualized by Coomassie staining or were transferred to PVDF membranes (GE Healthcare, Germany) for immunoblotting. Membranes were blocked and incubated with the appropriate primary and secondary antibodies, the resulting HRP signal was visualized by enhanced chemiluminescence (GE Healthcare, UK). Following primary antibodies were used: human AAT (D11, kindly provided by Prof. Dr. Janciauskiene [42]), mouse ferritin (FTH1; 3998, Cell signaling, Germany) and mouse GAPDH (NB300-221; NovusBio, UK)

Biochemical assays
Quantification of hepatic collagen content was performed with a hydroxyproline assay as described [43]. Briefly, liver tissue was homogenized in distilled water and hydrolyzed in 6N HCL at 110 °C for 18 hours. Lysates were filtered (55 mm, Macherey-Nagel, Germany) to remove debris and evaporated by speed vacuum centrifugation. The pellets or standards (trans-4-hydroxy-L-proline; H54409, Sigma, Steinheim, Germany) were dissolved in distilled water, then mixed with 0,6% of chloramines-T (31224, Sigma, Steinheim, Germany) for 10 minutes at RT in order to oxidize free hydroxyproline to pyrrole. Ehrlich solution (X867.1, Roth, Karlsruhe, Germany) was added to form the desired chromophore and measured at wavelength of 570 nm using a plate reader (BioTek Cytation 3, USA). Hydroxyproline content was normalized to wet liver weight and was presented as mg/g (hydroxyproline amount / wet liver weight).
To quantify the extent of iron accumulation, hepatic non-heme iron content was measured as described [43]. Briefly, samples were incinerated at 110 °C for 24 hours and hydrolysed in 100mM citric acid (C0759, Sigma, Steinheim, Germany) at 60 °C for 4 hours. After reduction with L-ascorbic acid (A5960, Sigma, Steinheim, Germany), bathophenanthroline disulfonic acid disodium salt (BPT, 146617, Sigma, Steinheim, Germany) was added that forms a red color complex upon reaction with iron. Absorbance was measured with a spectrophotometer at 535 nm via a plate reader (BioTek Cytation 3, USA). The normalization was done according to wet liver weight and was presented as µg/g (iron amount / wet liver weight). Figure S1. Parameters of iron metabolism in individuals homozygous for the alpha1-antitrypsin Pi*Z variant (Pi*ZZ) and Pi*Z non-carriers, both with low alcohol consumption (i.e. ≤30 for men and ≤20 for women). 237 non-carriers and 392 Pi*ZZ individuals were analyzed. Scatter plots depict serum iron (adjusted P value=0.0002) (A), serum ferritin (adjusted P value=0.00002) (B), serum transferrin (adjusted P value=0.0030) (C), and serum transferrin saturation (adjusted P value=1.2x10 -7 ) (D). Multivariable adjustments were performed for the covariates age, sex, BMI, presence of diabetes mellitus, and mean alcohol consumption.  . Immunoblotting and the corresponding morphometric quantification assessed hepatic AAT levels compared to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), that was used as a loading control (B). Results are shown as mean ± SD (n≥5 per group). n.s., not significant. Scale bar: 100 µm