Plasma vascular non-inflammatory molecule 3 is associated with gastrointestinal acute graft-versus-host disease in mice

Male BALB/C (H-2d) and C57BL/6 J (H-2b) mice were purchased from the Chinese Academy of Medical Sciences (HFK, Beijing, China) and housed in groups of five in individually specific pathogen-free barrier facilities. During procedures, animals were kept under a laminar flow hood. Animal diet was standardized pellet chow and UV decontaminated water. All animal work was performed under approved ethical guidelines.

Recipient mice (BALB/C, 8 to 11 weeks-of-age) received 8 Gy of total body irradiation (¹³⁷Cs) delivered in two fractions separated by 4 h to reduce GI toxicity. One day afterward, recipients were treated with injected 1 × 10⁷ BM cells (iv) supplemented with 3 × 10⁷ donor spleen cells from age-matched allogeneic donor C57BL/6 J or syngeneic donor BALB/C mice. The allogeneic group consisted of 9 mice undergoing allo-HSCT, and the auto group included 12 mice undergoing auto HSCT.

GVHD severity was assessed with a clinical GVHD scoring system [11] every 2 days and included weight loss, posture, activity, fur texture and skin integrity. At the time of analysis, mice from coded cages were evaluated and graded from 0 to 2 for each criterion. A clinical index was subsequently generated by summation of the five criteria scores (maximum index = 10). As for GI aGVHD, we added diarrhea to these five criteria for diagnosis. Survival was monitored on a daily basis.

Blood tests were carried out 3 times a week and evaluation of hematopoietic reconstruction, weight, leukocyte counts exceeding 0.5 × 10⁹/L, or the presence of donor-derived cells was done. Peripheral blood (PB) cells from mice in aGVHD and control groups were stained with PE anti-H-2d and PE-Cy5.5 anti-H-2b (eBioscience, San Diego, CA) as described [12]. FACS CantoII (BD Biosciences, San Jose, CA) were used to acquire data which were analyzed with BD FACSDiva software (BD Biosciences, San Jose, CA).

Small intestine biopsies were taken from mice at various time points after transplantation. Tissues were placed in 10% buffered formalin phosphate and fixed tissues were paraffin-embedded, sectioned, and stained with H&E. Slides were coded without reference to prior treatment and examined in a blinded fashion by two pathologists.

Plasma and intestinal biopsy specimens were collected from mice using a random approach (allogeneic and syngeneic groups) after animals were cervical dislocated at days 0 (after conditioning and prior to transplantation), 3, and 7 (allogeneic group developed aGVHD and CsA was given, ip), day 11 (syngeneic group PBS or LPS, ip), day 15 (CsA was discontinued in the allogeneic group), day 22 (CsA was given for mice with relapsed aGVHD in the allogeneic group), and day 30.

A Seppro IgY-M7 antibody SpinColumn (Sigma, MO, USA) was used to remove the 7 most highly abundant proteins in murine plasma, including albumin, IgG, fibrinogen, transferring, IgM, haptoglobin, and α-1 antitryptase. According to instructions, the low abundant protein samples were prepared from plasma samples.

Plasma protein was measured using a BCA Protein Assay Kit. After quantification, each sample containing 40 μg protein was preserved at −80 °C.

Plasma sample containing 20 μg protein were denatured with buffer 1 (8 M carbamide +100 mM Tris-HCL), and to this was added 4 μl reducing reagent (100 mM dithiothreitol, Amresco, OH, USA) at 37 °C for 2 h, alkylated with freshly prepared cysteine-blocking reagent (500 mM iodacetamide, Amresco, OH, USA) at room temperature for 15 min, and the protein solution was centrifuged at 12,000 rpm for 20 min and the solution at the bottom was collected. Then, to this was added 100 μl buffer 2 (500 mM triethylammonium bicarbonate buffer, Sigma, MO, USA), centrifuged at 12,000 for 10 min, supernatant was discarded and solution at the bottom was collected. This was repeated three times. Subsequently, protein was incubated with 40 μl sequencing grade modified trypsin (Promega, WI, USA) at 50 ng/μl and 100 μl buffer 2 at 37 °C for 16 h.

Digested samples were labeled with iTRAQ reagents (SCIEX, MA, USA) twice (positive and negative). Briefly, one vial of 8-plex iTRAQ labeling reagent (i.e., m/z 113, 114, 115, 116, 117, 118, 119, 121) was used for every 100 μg protein. Positive labeling and negative labeling were shown in Additional file 2. First, iTRAQ reagent was solubilized in 70 μl of isopropanol and then to this was added to 100 μg protein sample for 2 h. Next, labeling was terminated with 100 μl of water. Then, 1 μl of sample was withdrawn from every protein sample and desalinated with Ziptip and identified by MALDI-TOF/TOF to assure labeling was successful. After identification, samples were then mixed at equal ratios and dried with vacuum freeze-drying.

Labeled proteins were separated with high pH reversed phase chromatography using a C18 trapping column (ZORBAX Extend C18, 100 Å2.1 × 150 mm) at a flow rate of 0.3 ml/min and the wavelengths used were 215 and 280 nm. Lyophilized proteins were dissolved in Nano-RPLC buffer A (2% acetonitrile/water containing 0.1% formic acid), desalted onto a C18 precolumn (100 μm × 3 cm, C18, 3 μm, 150 Å) for 10 min at 2 μL/min, and analyzed on EksigentnanoLC-Ultra™2D system (SCIEX, Beijing, China). Subsequently, the protein were analyzed on a nano-reverse-phase LC system (RPLC), using a C18 reversed phase chromatograph column (75 μm × 15 cm C18- 3 μm 120 Å, ChromXPEksigent), and Nano-RPLC buffer B (98% acetonitrile/water containing 0.1% formic acid) was increased from 5~38% in 70 min. Data were calculated using Protein Pilot Software v. 4.5 (SCIEX, MA, USA).

Total RNA from mice recipients in different groups at different interval times were extracted by TRIzol (Invitrogen, Carlsbad, CA, USA). RNA concentration and quality were quantified by measuring the absorbance at 260 nm with Thermo Scientific™ NanoDrop™ spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and gel analysis. Relative expression was calculated using the 2^-△△CT method. The primers of VNN3 and other gene transcripts were designed by Primer Express Software v2.0. β- actin housekeeping gene was used for normalization.

Experiments were repeated at least 2 times with 3 sample replicates; To analyze one variable in one BMT experiment, at least two groups of 5 recipients are required.

HSCs from donor mice were transplanted into irradiated recipient mice as described and mice were divided into allogeneic and syngeneic groups. Seven days after transplantation, mice in the allogeneic group had characteristic aGVHD, including hair loss, skin lesions, hunched postures, diarrhea and weight loss. Significant differences in bodyweight and aGVHD scores relative to syngeneic mice were observed from day 4 onward. The bodyweights of mice in the allogeneic group were significantly lower than those in the control group at day 7 (P < 0.05). Similarly, the aGVHD scores of allogeneic transplantation mice differed from the scores of the syngeneic transplantation mice; the gap between the aGVHD scores in the allogeneic and syngeneic groups was largest at day 7 (P < 0.001) (Fig. 1a and b). WBC and Neutrophil counts at days 6 and 22 were significantly elevated compared with that at other time points in allogeneic group. Especially at day 6, WBC and Neutrophil counts reached the peak (Fig. 4a and b). Flow cytometry data (Additional file 1) showed nearly 100% of PB cells from mice in the allogeneic group were donor-derived at day 7, which confirmed successful reconstruction of hematopoiesis with donor HSCs. Additionally, histological examinations of intestines demonstrated lymphocyte infiltration in the aGVHD group (day 7) but not in the control group (Additional file 1) and histological scoring analysis revealed different impairment patterns of intestine (Fig. 2) [13, 14]. Then, CsA was administered (ip) daily to aGVHD mice until the allogeneic group was in remission at day 15. The aGVHD relapsed at day 22, and mice received CsA and went into remission again. In contrast, the syngeneic group had a significantly lower aGVHD score than the allogeneic group during the entire observation period of 30 days (Fig. 1b). Mice that received LPS or PBS had no aGVHD symptoms. Thus, we successfully constructed the GI aGVHD murine models after allogeneic HSCT.

We used 8-plex iTRAQ to identify candidate biomarkers in murine plasma samples taken before and after the onset of aGVHD. We identified and quantified 712 proteins of which 5 were increased at least 2-fold in the allogeneic group at day 7 compared with days 0, 3 and 15 (after CSA treatment) and the syngeneic group at day 7 (Additional file 2). These 5 proteins were VNN3, ZNF746, C4BP, KNG1 and FETUB, and they were consistent with results from negative labeling with 8-plex iTRAQ (Additional file 2).

These five plasma proteins in the allogeneic group increased at the onset of GI aGVHD (day 7), and decreased with remission of GI GVHD after CSA treatment. Furthermore, proteins peaked at day 7, whereas aGVHD symptoms were the most evident at day 7. Compared to the allogeneic group, the five plasma proteins in the syngeneic group did not change appreciably.

In addition, LPS was given to the auto group at day 11 to simulate infectious intestinal milieus. Similar protein was observed in LPS treated and PBS control mice. Protein in the allogeneic group at day 7 during GI aGVHD was greater than that in the syngeneic group treated with LPS at day 11. Thus, these five elevated proteins may not be caused by pathogens such as LPS.

To determine whether the five plasma proteins of interest agreed with gene expression data in intestinal epithelial cells, we obtained intestinal epithelial specimens to measure VNN3, ZNF746, C4BP, KNG1 and FETUB gene expression using Real-Time PCR. Kinetic changes in VNN3 expression in the intestinal epithelial cells were consistent with protein expression in plasma (Fig. 3a). [15] According to the results of Real-Time PCR, the VNN3 level of allogeneic group fluctuated as the occurrence and remission of GI aGVHD as well. It has a 2.7-fold increase at the time of aGVHD at day 7 in allogeneic group as compared with corresponding time point of control syngeneic group (p < 0.001). After CSA treatment at day 15 of allogeneic group, the VNN3 level decreased 1.8-fold (p < 0.001).When the GI aGVHD relapsed at day 22, the level of VNN3 increased to 2.6 times as against day 15 (p < 0.001). By contraries, the VNN3 level of syngeneic group at day 11(after LPS injection) was merely 0.26 times as against day 7 in auto-group (p > 0.05). Also, PBS could not cause significant increase of VNN3 level (0.2-fold) as compared with day 7 in syngeneic group (p > 0.05).

Besides, the expression of gene VNN3, ZNF746, C4BP, KNG1 and FETUB achieved the peak at day 22 when the GI aGVHD recurred and were more than 3 times as those at day 7(Fig. 3a-e). In our study, the count of WBC especially neutrophil at day 22 is much more than that at day 7 as well (Fig. 4a and b). However, kinetic changes of RNA ZNF746, C4BP, KNG1 and FETUB levels were not consistent with those changes in plasma (Fig. 3b-e).