Dermatitis herpetiformis (DH) is an autoimmune blistering skin disease associated with gluten-sensitive enteropathy (CD). In order to investigate the pathogenesis of skin lesions at molecular level, we analysed the gene expression profiles in skin biopsies from 6 CD patients with DH and 6 healthy controls using Affymetrix HG-U133A 2.0 arrays. 486 genes were differentially expressed in DH skin compared to normal skin: 225 were upregulated and 261 were downregulated. Consistently with the autoimmune origin of DH, functional classification of the differentially expressed genes (DEGs) indicates a B- and T-cell immune response (LAG3, TRAF5, DPP4, and NT5E). In addition, gene modulation provides evidence for a local inflammatory response (IL8, PTGFR, FSTL1, IFI16, BDKRD2, and NAMPT) with concomitant leukocyte recruitment (CCL5, ENPP2), endothelial cell activation, and neutrophil extravasation (SELL, SELE). DEGs also indicate overproduction of matrix proteases (MMP9, ADAM9, and ADAM19) and proteolytic enzymes (CTSG, ELA2, CPA3, TPSB2, and CMA1) that may contribute to epidermal splitting and blister formation. Finally, we observed modulation of genes involved in cell growth inhibition (CGREF1, PA2G4, and PPP2R1B), increased apoptosis (FAS, TNFSF10, and BASP1), and reduced adhesion at the dermal epidermal junction (PLEC1, ITGB4, and LAMA5). In conclusion, our results identify genes that are involved in the pathogenesis of DH skin lesions.
Dermatitis herpetiformis (DH) is an autoimmune subepidermal blistering skin disease characterized by intense pruritic papulovesicular eruptions mainly localized on extensor surfaces [
The key feature of DH is a granular deposition of IgA within the tips of dermal papillae and along the basement membrane of perilesional skin. eTG has been shown to colocalize with such IgA deposits [
Moreover, a perivascular cellular infiltrate composed mainly by CD4+ lymphocytes is also present [
In DH, blister formation is associated with epidermal splitting due to destruction of basement membrane components and proteolysis of adhesion molecules at the dermal epidermal junction. A comprehensive analysis of the molecular mechanisms that coordinate the initiation and progression of the pathological process is still lacking. Our approach consists in the use of a gene array strategy that allows the simultaneous detection of thousands of genes in a given sample. We have examined gene expression directly in the skin tissue of patients with DH to analyze the transcriptional events that culminate in the skin lesion formation. We report here patterns of transcripts in 6 DH patients using DNA microarrays that characterize injured skin and identify signatures of gene expression that are involved in the pathogenesis of blister formation. The analysis of modulated genes provides evidence for the intervention of genes involved in immune activation, inflammation, impaired adhesion and cell death, considered key features in the pathogenesis of the disease.
Six adult patients (3 men and 3 females; mean age 51 years, median age 52 years, and age range 36–59 years) with DH and CD, showing all clinical and immunopathological features of the diseases, were included in this study. All patients had the typical clinical features of DH, with erythematous papules and vesicles symmetrically distributed on the extensor surfaces of the upper and/or lower extremities and buttocks. The duodenal histological damage of the 6 patients at diagnosis ranged from grade 2 to 3b, according to Marsh’s classification [
Skin biopsies presented classical histopathologic features of DH, including subepidermal cleft with neutrophils and/or eosinophils at the tips of the dermal papillae and granular deposits of IgA at the tips of derma papillae on direct immunofluorescence.
Serologically, five out of six patients had serum anti-tTG and antiendomysium (EMA) IgA antibodies without gluten-free diet. The seronegative patient had a duodenal biopsy with a grade 3b histological damage and was affected by IgA deficiency. Indeed anti-tTG IgG were detected in this patient.
All patients were on normal gluten-containing diet and were not taking Dapsone at the moment of skin biopsy. Two punch biopsies of 6 mm each were performed at the diagnosis on each one of the 6 patients from early lesional skin (grouped erythematous papules surmounted by vesicles) following local anaesthesia (1% lidocaine with 1/100,000 epinephrine). Skin specimens for biopsy were obtained from elbows (2 patients) and from buttocks (4 patients).
Normal skin biopsies were obtained from 6 sex- and age-matched healthy adult subjects (3 males and 3 females, mean age 50 years, median age 53 years, age range 34–60 years) with no evidence of gastrointestinal or skin disease. Specimens were snap-frozen in liquid nitrogen immediately after biopsy.
All the subjects (patients and controls) were of Caucasian origin from Northwestern Italy.
The patients included showed no evidence of other coexisting autoimmune diseases.
Written informed consent was obtained in each case. The study was conducted according to the Declaration of Helsinki Principles and was approved by the local ethical committee.
Tissue samples from every single patient were frozen in liquid nitrogen immediately after dissection and stored at −70°C until homogenization. Frozen samples were homogenized in TRI REAGENT (1 mL per 50–100 mg of tissue) in a Potter-type mechanical homogenizer with Teflon pestle. RNA extraction, preparation of cRNA hybridization, and scanning of probe arrays for each samples were performed according to the protocols of the manufacturer (Affymetrix, Santa Clara, CA, United States) by Cogentech Affymetrix microarray unit (Campus IFOM-IEO, Milan, Italy) using the human genome U133A 2.0 gene chip (Affymetrix). The human genome U133A gene chip is a single array representing 14,500 well-characterized human genes and including more than 22,000 probe sets and 500,000 distinct oligonucleotide features.
The different gene expression patterns were analyzed by using Gene Spring software, version 11.0 (Agilent Technologies, Santa Clara, CA, United States).
The normalized background-corrected data were transformed to the log2 scale. A signal log2 ratio of 1.0 indicates an increase of the transcript level by twofold change (2 F.C.), and −1.0 indicates a decrease by twofold (−2 F.C.). A signal log2 ratio of zero would indicate no change.
The unpaired
Finally, statistically significant genes were selected for final consideration when their expression was at least 1.5-fold different in the test sample versus control sample.
Genes that passed both the
In order to identify genes involved in the pathogenesis of the typical skin lesions of DH, the gene expression patterns of 6 skin biopsies from 6 patients affected by DH were compared with 6 skin biopsies from 6 healthy controls.
A
For statistical comparison, an unpaired
Among these transcripts, 486 also fulfilled the fold change criterion, since they were differentially expressed 1.5 fold or more; in particular 225 and 261 transcripts resulted, respectively, to be up- and downregulated.
Such transcripts were classified in functional categories according to Gene Ontology annotations, including immune response, apoptosis, cell growth, proliferation and differentiation, inflammatory response, production and remodelling of the extracellular matrix, and metabolism.
Table
Annotated genes differentially expressed in DH versus healthy controls grouped according to their function.
Functional class | Probe set ID | F.C. | Regulation | Gene symbol | Gene title | Accession number |
---|---|---|---|---|---|---|
Immune response | 206486_at | 1.5 | Up | LAG3 | Lymphocyte-activation gene 3 | NM_002286 |
204352_at | 1.6 | Up | TRAF5 | TNF receptor-associated factor 5 | NM_004619 | |
205821_at | 1.7 | Up | KLRK1 | Killer cell lectin-like receptor subfamily K, member 1 | NM_007360 | |
203717_at | 2.4 | Up | DPP4 | Dipeptidyl peptidase 4 | NM_001935 | |
203939_at | 3.8 | Up | NT5E | 5′-nucleotidase, ecto (CD73) | NM_002526 | |
204502_at | 2.0 | Up | SAMHD1 | SAM domain and HD domain 1 | NM_015474 | |
| ||||||
Inflammation | 206332_s_at | 3.1 | Up | IFI16 | Interferon, gamma-inducible protein 16 | NM_005531 |
217738_at | 2.0 | Up | NAMPT | Nicotinamide phosphoribosyltransferase | NM_005746 | |
203176_s_at | 2.2 | Up | TFAM | Transcription factor A, mitochondrial | NM_003201 | |
205870_at | 2.2 | Up | BDKRB2 | Bradykinin receptor B2 | NM_000623 | |
204655_at | 2.2 | Up | CCL5 | Chemokine (C-C motif) ligand 5 | NM_002985 | |
209392_at | 2.3 | Up | ENPP2 | Ectonucleotidepyrophosphatase | L35594 | |
202859_x_at | 2.3 | Up | IL8 | Interleukin 8 | NM_000584 | |
211272_s_at | 2.4 | Down | DGKA | Diacylglycerol kinase, alpha 80 kDa | AF064771 | |
207177_at | 2.5 | Up | PTGFR | Prostaglandin F receptor | NM_000959 | |
208782_at | 2.9 | Up | FSTL1 | Follistatin-like 1 | BC000055 | |
204563_at | 7.3 | Up | SELL | Selectin L | NM_000655 | |
206211_at | 4.3 | Up | SELE | Selectin E | NM_000450 | |
217800_s_at | 1.9 | Up | NDFIP1 | Nedd4 family interacting protein 1 | NM_030571 | |
214475_x_at | 2.8 | Down | CAPN3 | Calpain 3, (p94) | AF127764 | |
201859_at | 3.1 | Up | SRGN | Serglycin | NM_002727 | |
201110_s_at | 5.2 | Up | THBS1 | Thrombospondin 1 | NM_003246 | |
| ||||||
Apoptosis | 202558_s_at | 1.5 | Up | STCH | Stress 70 protein chaperone | NM_006948 |
217786_at | 1.5 | Down | PRMT5 | Protein arginine methyltransferase 5 | NM_006109 | |
204781_s_at | 1.5 | Up | FAS | TNF receptor superfamily, member 6 | NM_000043 | |
202693_s_at | 1.7 | Up | STK17A | Serine/threonine kinase 17a | NM_004760 | |
201912_s_at | 2.6 | Up | GSPT1 | G1 to S phase transition 1 | NM_002094 | |
202887_s_at | 2.6 | Down | DDIT4 | DNA-damage-inducible transcript 4 | NM_019058 | |
202687_s_at | 2.9 | Up | TNFSF10 | Tumor necrosis factor (ligand) superfamily, member 10 | NM_003810 | |
202411_at | 3.1 | Up | IFI27 | Interferon, alpha-inducible protein 27 | NM_005532 | |
202391_at | 3.1 | Up | BASP1 | Brain abundant, membrane-attached signal protein 1 | NM_006317 | |
| ||||||
Cell proliferation | 208676_s_at | 1.5 | Up | PA2G4 | Proliferation-associated 2G4, 38 kDa | U87954 |
205937_at | 1.5 | Up | CGREF1 | Cell growth regulator with EF-hand domain 1 | NM_006569 | |
1773_at | 1.5 | Down | FNTB | Farnesyltransferase, CAAX box, beta | L00635 | |
202886_s_at | 2.2 | Up | PPP2R1B | Protein phosphatase 2, regulatory subunit A, beta isoform | M65254 | |
202167_s_at | 1.9 | Down | MMS19 | MMS19 nucleotide excision repair homolog | NM_022362 | |
203108_at | 2.1 | Up | GPRC5A | G protein-coupled receptor, family C, group 5, member A | NM_003979 | |
202454_s_at | 2.7 | Down | ERBB3 | v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 | NM_001982 | |
| ||||||
204798_at | 1.6 | Up | MYB | v-myb myeloblastosis viral oncogene homolog | NM_005375 | |
218717_s_at | 1.7 | Up | LEPREL1 | Leprecan-like 1 | NM_018192 | |
209765_at | 1.8 | Up | ADAM19 | ADAM metallopeptidase domain 19 | AF311317 | |
202381_at | 1.8 | Up | ADAM9 | ADAM metallopeptidase domain 9 | NM_003816 | |
203044_at | 2.1 | Up | CHSY1 | Chondroitin sulfate synthase 1 | NM_014918 | |
Extracellular matrix | 205479_s_at | 2.1 | Up | PLAU | Plasminogen activator, urokinase | NM_002658 |
210845_s_at | 2.1 | Up | PLAUR | Plasminogen activator, urokinase receptor | U08839 | |
201995_at | 2.2 | Up | EXT1 | Exostoses (multiple) 1 | NM_000127 | |
205828_at | 3.4 | Up | MMP3 | Matrix metallopeptidase 3 (stromelysin 1) | NM_002422 | |
203936_s_at | 2.2 | Up | MMP9 | Matrix metallopeptidase 9 | NM_004994 | |
202620_s_at | 2.4 | Up | PLOD2 | Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 | NM_000935 | |
207316_at | 2.8 | Up | HAS1 | hyaluronan synthase 1 | NM_001523 | |
203343_at | 3.2 | Up | UGDH | UDP-glucose dehydrogenase | NM_003359 | |
204620_s_at | 4.0 | Up | VCAN | Versican | NM_004385 | |
202766_s_at | 5.6 | Up | FBN1 | Fibrillin 1 | NM_000138 | |
202404_s_at | 4.1 | Up | COL1A2 | Collagen, type I, alpha 2 | NM_000089 | |
201852_x_at | 2.9 | Up | COL3A1 | Collagen, type III, alpha 1 | NM_000090 | |
211980_at | 2.4 | Up | COL4A1 | Collagen, type IV, alpha 1 | NM_001845 | |
221730_at | 2.7 | Up | COL5A2 | Collagen, type V, alpha 2 | NM_000393 | |
207134_x_at | 2.2 | Up | TPSB2 | Tryptase beta 2 | NM_024164 | |
210084_x_at | 2.1 | Up | TPSAB1 | Tryptase alpha/beta 1 | AF206665 | |
214533_at | 3.5 | Up | CMA1 | Chymase 1, mast cell | NM_001836 | |
205624_at | 2.1 | Up | CPA3 | Carboxypeptidase A3 (mast cell) | NM_001870 | |
206871_at | 3.3 | Up | ELA2 | Elastase 2, neutrophil | NM_001972 | |
205653_at | 5.0 | Up | CTSG | Cathepsin G | NM_001911 | |
202376_at | 1.7 | Down | SERPINA3 | Serpin peptidase inhibitor, clade A, member 3 | NM_001085 | |
201147_s_at | 1.8 | Down | TIMP3 | TIMP metallopeptidase inhibitor 3 | NM_000362 | |
206243_at | 2.8 | Down | TIMP4 | TIMP metallopeptidase inhibitor 4 | NM_003256 | |
| ||||||
Dermal-epidermal junction | 216971_s_at | 1.5 | Down | PLEC1 | Plectin 1, intermediate filament binding protein | Z54367 |
214292_at | 1.5 | Down | ITGB4 | Integrin, beta 4 | AA808063 | |
210150_s_at | 1.5 | Down | LAMA5 | Laminin, alpha 5 | BC003355 | |
| ||||||
Metabolism |
207786_at | 1.9 | Down | CYP2R1 | Cytochrome P450, family 2, subfamily R, polypeptide 1 | NM_024514 |
211019_s_at | 2.1 | Down | LSS | 2,3-oxidosqualene-lanosterol cyclase | D63807 | |
205676_at | 2.5 | Up | CYP27B1 | Cytochrome P450, family 27, subfamily B, polypeptide 1 | NM_000785 |
Among genes involved in the immune response, upregulated genes play a role in T lymphocyte activation, for example, lymphocyte-activation gene 3 (LAG3) [
Other upregulated genes involved in the immune response belong to the CD40 signalling pathways, including the TNF receptor-associated factor 5 (TRAF5) or play a role in innate immunity such as the killer cell lectin-like receptor subfamily K, member 1 (KLRK1, better known as NKG2D), or SAM domain and HD domain 1 (SAMHD1) [
Moreover, a cluster of genes that have a role in the inflammatory process was upregulated. This cluster encompasses the interferon, gamma-inducible protein 16 (IFI16), bradykinin receptor B2 (BDKRB2), chemokine (C-C motif) ligand 5 (CCL5), ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2, also called autotaxin), interleukin 8 (IL8), prostaglandin F receptor (PTGFR), follistatin-like 1 (FSTL1), selectin L (SELL), selectin E (SELE), thrombospondin 1 (THBS1), and serglycin (SRGN).
Moreover, a downregulation of the diacylglycerol kinase, alpha 80 kDa (DGKA) [
Many genes coding for protein involved in apoptosis and/or in apoptosis regulation resulted to be modulated in pathological samples. Among these, several proapoptotic genes were upregulated such as TNF receptor superfamily, member 6 (FAS), tumour necrosis factor (ligand) superfamily, member 10 (TNFSF10) brain abundant, membrane-attached signal protein 1 (BASP1), stress 70 protein chaperone microsome associated (STCH) [
On the other hand, genes coding for the antiapoptotic protein arginine methyltransferase 5 (PRMT5) and DNA-damage-inducible transcript 4 (DDIT4) were downregulated.
Antiproliferative genes were upregulated in DH skin samples including the cell growth regulator with EF-hand domain 1 (CGREF1) and the tumor suppressor genes named proliferation-associated 2G4 (PA2G4/EBP1) [
Moreover positive regulators of cell growth, such as MMS19 nucleotide excision repair homolog (MMS19) and v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (ERBB3), resulted downregulated.
Several genes involved in extracellular matrix components synthesis as well as in wound healing and tissue repair were upregulated.
These genes are involved in the synthesis of collagen such as procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2), or in the production of hyaluronan as hyaluronan synthase 1 (HAS1) [
Four genes coding for different collagen molecules were also upregulated, and these are collagen, type I, alpha 2 (COL1A2), collagen, type III, alpha 1 (COL3A1), collagen, type IV, alpha 1 (COL4A1), and collagen, type V, alpha 2 (COL5A2).
Moreover, we also observed an up-regulation of versican (VCAN) [
When we analyzed genes involved in extracellular matrix remodeling, we observed an upregulation of several proteases such as matrix metallopeptidase 3 (MMP3) [
Moreover, among proteolytic enzymes, we found an increased expression of genes coding for proteins that belong to the neutrophil and mast cell secretory repertoire such as tryptase alpha/beta 1 (TPSAB1), tryptase beta 2 (TPSB2), chymase 1 (CMA1), carboxypeptidase A3 (CPA3), elastase 2 (ELA2), and cathepsin G (CTSG).
On the contrary, the alpha-1 antiproteinase (SERPINA3) and the metallopeptidase inhibitors 3 and 4 (TIMP3 and TIMP4) were downregulated.
Three genes coding for protein that are present at the dermal-epidermal junctions were downregulated. These transcripts are plectin 1, intermediate filament binding protein 500 kDa (PLEC1) [
Despite the huge effort in elucidating the pathogenesis of DH, a detailed understanding of the molecular events involved in DH lesion formation is still lacking. In the present work we provide for the first time a comprehensive analysis of the transcriptome within DH lesional skin.
First of all, we observed the modulation of genes, that are involved in the regulation of both immune response and inflammation.
Consistently with the autoimmune origin of DH, we found an overexpression of genes involved in T and B immune response (LAG3, TRAF5, DPP4, and NT5E) [
Lymphocyte activation gene-3 (LAG-3; CD223) is a negative costimulatory receptor that modulates T-cell homeostasis, proliferation, and activation; it is a CD4 homolog that is required for maximal regulatory T-cell function and for the control of CD4(+) and CD8(+) T cell. Interestingly, it may be required for the control of autoimmunity [
Many proinflammatory genes were found to be upregulated in DH samples and some of them with high fold changes (Table
Particular attention deserves the upregulation of selectin-E (SELE) and IL8 (Table
Hall et al. [
Interestingly, we found overexpression of ENPP2/autotaxin, a molecule that exacerbates inflammation by increasing chemotaxis through the upregulation of neutrophil integrins [
The downregulation of the two anti-inflammatory genes, DGKA and CAPN3, may be also linked to increased neutrophil migration [
Apoptosis is thought to play a role in the pathogenesis of cutaneous lesions, and increased apoptotic events in basal and suprabasal keratinocytes were observed within lesional and perilesional skin of DH [
We noticed a remarkable modulation of genes coding for several components of the extracellular matrix such as collagen type III, IV, and V. An elevated level of collagen type III, IV, and V has been described in the DH blisters of the papillary derma [
Matrix degradation at the dermal-epidermal junction has been thought to contribute to DH blister formation [
We found an increased expression of neutrophil and mast cell enzymes such as TPSB2, TPSAB1, CMA1, CPA3, ELA2, and CTSG that are thought to be involved in the splitting up of epidermis from dermis [
Proteases secreted by granulocytes and mast cells could mediate the development of DH cutaneous lesions either directly or indirectly by the activation of metalloproteases [
Several genes coding for metalloproteases resulted upregulated in our DH skin samples including MMP3/stromelysin, MMP9/gelatinase B, ADAM9/meltrin gamma, and ADAM19/meltrin beta. It has been demonstrated that MMP3 participates to blister formation by degrading basement membrane components [
Airola et al. reported an increased secretion of this enzyme by basal keratinocytes surrounding neutrophil abscesses [
It has been suggested that the formation of blisters may be induced by an overexpression of local enzymes [
Interestingly we found a strong downregulation of genes coding for tissue inhibitors of proteases such as SERPINA3, TIMP3 and TIMP4.
Therefore, our gene analysis confirms that an important role in the maintenance and amplification of the immunological processes underlying blister formation may be played by an imbalance between the activities of MMPs and their tissue inhibitors, as previously hypothesised by Zebrowska et al. [
Another molecule involved in the degradation of basement membrane is the plasminogen activator urokinase (PLAU) that has been found to be highly expressed in keratinocytes in experimentally induced DH lesions [
It is tempting to speculate that an overexpression of PLAU may lead to increased production of plasmin that in turn activates MMP9, as seen in experiments carried out in mice [
We also observed a downregulation of genes coding for proteins involved in the network that anchor the keratin filaments of cells cytoskeleton to the underlying dermis at the dermal-epidermal junctions. These molecules are: PLEC/plectin 1, ITGB4, and LAMA5/laminin alpha 5.
Plectin is a large 200 nm long protein found in hemidesmosomes and whose function is to bind keratin intermediate filaments to the hemidesmosome, and specifically to transmembrane collagen XVII and
Laminin 5 is essential for adhesion of keratinocytes to basement membrane [
In DH skin lesions, proteins within the dermal epidermal junction are target of proteolytic enzymes released by neutrophils. In addition, the decreased expression of the above-mentioned molecules might worsen the damage induced by granulocytic enzymatic activity.
Overall, the results obtained support the hypothesis that during blister development, the inflammatory reaction evoked by the autoimmune response typical of the disease is associated to a local overexpression of proteolytic enzymes leading to the detachment of the dermal-epidermal junction. The consequent tissue damage may be amplified by a reduced production of protease inhibitors.
Moreover, our data suggest that an increased rate of apoptosis and a reduced expression of anchoring proteins at dermal-epidermal junction are key features in DH skin lesions.
In conclusion, we believe that our study on gene expression gives a better understanding of the molecular mechanisms involved in the pathogenesis of skin lesions in DH.
The authors declare that they have no conflict of interests.
There is no external funding.
The authors thank Massimo Drosera for excellent technical assistance.