Bone Marrow Transplantation (2013) 48, 284–293 & 2013 Macmillan Publishers Limited All rights reserved 0268-3369/13 www.nature.com/bmt ORIGINAL ARTICLE Early-phase GVHD gene expression profile in target versus non-target tissues: kidney, a possible target? B Sadeghi1, H Al-Chaqmaqchi1, S Al-Hashmi1, D Brodin2, Z Hassan1,3, M Abedi-Valugerdi4, A Moshfegh5 and M Hassan1,3 GVHD is a major complication after allo-SCT. In GVHD, some tissues like liver, intestine and skin are infiltrated by donor T cells while others like muscle are not. The mechanism underlying targeted tropism of donor T cells is not fully understood. In the present study, we aim to explore differences in gene expression profile among target versus non-target tissues in a mouse model of GVHD based on chemotherapy conditioning. Expression levels of JAK–signal transducers and activators of transcription (STAT), CXCL1, ICAM1 and STAT3 were increased in the liver and remained unchanged (or decreased) in the muscle and kidney after conditioning. At the start of GVHD the expression levels of CXCL9, ITGb2, SAA3, MARCO, TLR and VCAM1 were significantly higher in the liver or kidney compared with the muscle of GVHD animals. Moreover, biological processes of inflammatory reactions, leukocyte migration, response to bacterium and chemotaxis followed the same pattern. Our data show that both chemotherapy and allogenicity exclusively induce expression of inflammatory genes in target tissues. Moreover, gene expression profile and histopathological findings in the kidney are similar to those observed in the liver of GVHD mice. Bone Marrow Transplantation (2013) 48, 284–293; doi:10.1038/bmt.2012.120; published online 23 July 2012 Keywords: gene expression; GVHD; kidney; target tissues; mouse model INTRODUCTION none of these reports describe any changes in gene expression Allo-SCT is the treatment of choice for a variety of malignant and profile of non-target tissues like muscle. non-malignant diseases, especially in the field of hematology.1 We hypothesized that comprehensive gene expression analysis However, its application is limited owing to several complications, of target versus non-target tissues after conditioning regimen and especially GVHD.2 in the early phases of GVHD might help us to understand the It is assumed that conditioning induces tissue injury and underlying mechanisms of T-cell trafficking. Hence, in the present subsequent lipopolysaccharides (LPS) extravasation promotes a report, we classified the liver as affected (target) and muscle as cytokine storm, leading to activation of donor T cells by host/ less-affected (non-target) organs, and explored the role of the donor APCs.3,4 Finally, allo-reactive donor T cells migrate and kidney in aGVHD. Then we compared gene expression profiles infiltrate peripheral tissues. Hypothetically all organs should be before and after conditioning, as well as in the early phase of possible targets for donor T cells; nevertheless, the liver, aGVHD within and between groups (allo- and syngeneic SCT). gastrointestinal tract, skin and lungs3,5,6 are the principal target organs.7 Although damage to other organs (for example, kidney) has been described,8,9 most clinicians do not consider the kidney a MATERIALS AND METHODS target organ for acute GVHD (aGVHD). Several hypotheses have Animals been discussed, but clear alterations of target versus non-target Female BALB/c and male C57BL/6 mice, 10–14 weeks old, were purchased tissues have not been explored yet. Inflammatory cytokine from Scanbur (Sollentuna, Sweden). They were maintained under specific secretion and chemokine expression have an important role in pathogen-free conditions in an animal facility with individually ventilated cages. Animals were fed autoclaved mouse chow and tap water ad libitum lymphocyte activation and migration to inflammation sites.10–13 14 15 and were allowed to acclimatize to their surroundings for 1 week. All Mapara et al. ,Maet al. and others have shown higher experiments described here were approved by the southern Stockholm expression of recruiting chemokines on the target tissues of GVHD ethics committee for animal research in accordance with Swedish Animal 16 mice. Overexpression or aberrant expression of MHC molecules Welfare law. has been suggested as a possible cause for the targeted invasion 3 of donor T cells. These reports shed light on the principal Induction of aGVHD molecular changes in the process of GVHD; however, owing GVHD induction was performed as previously described.17 Briefly, recipient to elevated levels of cytokines in the serum and systemic mice received BU (80 mg/kg) followed by CY (200 mg/kg). At day 0, inflammation following GVHD, these findings cannot be recipient mice were injected via the lateral tail vein with 2 Â 107 and considered exclusive changes in target organs—especially when 3 Â 107 viable cells of bone marrow cell (BMC) and splenocyte (SP) from compared with syngeneically transplanted animals. Moreover, C57BL/6 (allogeneic) or BALB/c (syngeneic) as donor, respectively. 1Experimental Cancer Medicine, Institution for Laboratory Medicine, Karolinska Institute, Stockholm, Sweden; 2Bioinformatics and Expression Analysis core facility, Karolinska Institute, Stockholm, Sweden; 3Clinical Research Centre (KFC, Novum), Karolinska University Hospital-Huddinge, Stockholm, Sweden; 4Department of Biochemistry and Biophysics, Arrhenius Laboratories for the Natural Sciences, Stockholm University, Stockholm, Sweden and 5Department of Oncology and Pathology, Cancer Center Karolinska (CCK), Karolinska Hospital and Institute, Stockholm, Sweden. Correspondence: Dr B Sadeghi, Division of Clinical Immunology, F79, Karolinska Institutet, Karolinska University Hospital-Huddinge, SE 14186 Stockholm, Sweden. E-mail: [email protected] Received 27 February 2012; accepted 20 May 2012; published online 23 July 2012 Gene expression pattern in early phase of GVHD B Sadeghi et al 285 Assessment of GVHD Data analysis Recipient mice were examined daily from the start of conditioning until the Data analysis was performed with Affymetrix GeneChip Operating Software indicated sampling day. Animals were evaluated for five clinical symptoms (GCOS, v1.1) using the MAS5 method (TGT ¼ 100). A quantitative signal of GVHD: weight loss, posture, activity, fur texture and skin integrity as and a qualitative detection call was generated for each sample and described elsewhere.17 transcript. Within each tissue, pair-wise comparisons of treated samples versus the untreated sample were performed, generating a corresponding Histopathology quantitative signal log ratio and a qualitative change call. To confirm GVHD, histopathological evaluation was performed as described previously.17 Tissue samples were fixed in neutral buffered Enrichment analysis formalin for 24 h, transferred to 70% ethanol, dehydrated and embedded To investigate induced treatment effects in the different tissues in a in paraffin according to standard procedure. Sections of 4 mm were functional context, enrichment analysis was performed using WebGestalt prepared and stained by hematoxylin and eosin for histopathological (http://bioinfo.vanderbilt.edu/webgestalt/). Differently expressed genes evaluation. within each tissue were selected and tested for enrichment of the gene ontology subcategory (http://www.geneontology.org) and KEGG pathways Tissue preparation (http://www.genome.ad.jp/kegg/). Hypergeometric was considered for statistical method, significance level was 0.01, minimum number of genes At day À 7 (control), 0 (after conditioning) and þ 7 (start of GVHD) between three to six mice per group were killed. To exclude the effects of for a category considered 4 and P-value correction by Benjamini & circulatory cells in tissues, the entire mouse body was perfused through Hochberg, statistical method (BH). Principal component analysis plot sample similarities in expression the heart with 20–30-mL-ice-cold PBS containing 2% FBS and 10 mM EDTA. Then portions of the liver, kidney and muscle were isolated, snap frozen in patterns were visualized by three-dimensional principal component analysis plots. A set of gene ontology terms were selected for visualization liquid nitrogen and transferred to À 150 1C until analysis. and genes associated with the respective term and present in at least one sample were subjected to principal component analysis using Qlucore Microarray analysis Omics Explorer 2.0 (www.qlucore.com/). RNA extraction was done according to Qiagen RNeasy kit instructions manual (WVR, Stockholm, Sweden). The integrity of extracted RNA was confirmed using the Agilent 2100Bioanalyzer (Agilent Technologies, Palo RESULTS Alto, CA, USA). cRNA sysnthesis and microarray analysis were done according to Establishment of GVHD Affymetrix manual (Affymetrix Inc., Santa Clara, CA, USA). Scanned images In this model (C57BL/6 to BALB/c), preliminary signs of clinical and were inspected and analyzed using established quality control criteria. immunological manifestations of aGVHD emerge 1 week after allo- Skin allo Colon allo Skin syn Colon syn Figure 1. Development of GVHD in the allogeneically transplanted mice versus syngeneically transplanted mice. Female BALB/c mice were conditioned using Bu–Cy regimen and transplanted with the BM and SP cells from an allogeneic or syngeneic donor (Materials and methods). Twenty-one days after transplantation samples from the skin and intestine were collected, fixed and stained for histopathological evaluation. (a) Skin of allogeneically transplanted mice; (b) skin of syngeneically