Memory-like Liver Natural Killer Cells are Responsible for Islet Destruction in Secondary Islet Transplantation

We previously demonstrated the pivotal role of natural killer (NK) cells in islet graft loss during the early phase after intraportal syngeneic islet transplantation (IT). Liver-resident DX5− NK cells were reported to possess memory-like properties, distinguishing them from conventional DX5+ NK cells. Here, we investigated the impact of primary IT-induced liver DX5− NK cells on the engraftment of secondary-transplanted islets in mice. The culture of liver NK cells isolated from naive mice with TNF-α, IFN-γ, and IL-lβ, mimicking instant blood-mediated inflammatory reaction, led to significantly increased DX5− NK cell percentage among total liver NK cells. Consistently, the prolonged expansion of DX5− CD69+ TRAIL+ CXCR3+ NK cells was observed after intraportal IT of 300 syngeneic islets (marginal mass). In most diabetic mice, 400 syngeneic islets of primary IT were sufficient to achieve normoglycaemia, whereas the same mass after secondary IT failed to induce normoglycaemia in mice that received 200 syngeneic islets during primary IT. These findings indicated that liver-resident DX5− NK cells significantly expanded even after syngeneic IT, and that these memory-like NK cells may target both originally engrafted and secondary-transplanted islets. Furthermore, anti-TNF-α treatment suppressed the expansion of liver-resident DX5− NK cells, resulting in successful islet engraftment after sequential ITs.

Accumulating evidence suggests that NK cells also exhibit memory properties and are divided into several subsets according to the nature of their inducers 24,[27][28][29][30] . Specifically, liver-resident NK cells lack DX5, the α2 integrin chain CD49b (a classical NK cell marker), and express TRAIL 29 . These DX5 − NK cells are involved in the immunological memory response and their hematopoietic progenitor and precursor cells can be found in the liver 29 . Several investigators reported that immune cells are involved in islet destruction 7,11,31 ; however, few studies have investigated multiple ITs using clinically relevant approaches in a mouse model, and the immune status following multiple ITs is not well characterised. Therefore, to evaluate the mechanism of NK cell activation, we investigated the involvement of liver-resident DX5 − NK cells in islet destruction in both early and late phases after intraportal ITs. Furthermore, we developed an in vivo model, which allowed us to compare the outcomes of the primary and secondary syngeneic ITs, and investigated the effects of the primary intraportal IT on the secondary IT by defining the population dynamics of liver resident DX5 − memory-like NK cells.

Results
Naive liver DX5 − NK cells express CD69, TRAIL, and CXCR3, which target islet grafts. MNCs were isolated from the livers or spleens of naive B6 mice. As previously reported, liver NK cells contained numerous DX5 − NK cells compared to splenic NK cells (p < 0.001) ( Supplementary Fig. S1) 29,32 . CD69, TRAIL, and CXCR3 expression on liver DX5 − NK cells was significantly higher than that on DX5 + NK cells (p < 0.001, for all) ( Supplementary Fig. S1) 32 . CD69 is known as an early activation marker induced in NK, T, and B cells in response to inflammatory stimuli 33 . TRAIL has already been shown to induce apoptosis through binding its respective receptors, death receptor (DR) 4 and DR5 34 . We have previously confirmed that dissociated islets express the TRAIL receptor DR5 6 . It has been reported that CXCL10 secreted from β cells activates and attracts autoreactive T cells and macrophages to the islets via CXCR3 after viral infection in human autoimmune type 1 diabetes [35][36][37] . All these findings, together with the remarkable expression of CXCR3 on DX5 − NK cells, demonstrate the possibility that the chemotaxis of liver resident NK cells, induced by the CXCL10-CXCR3 pathway, contributes to islet graft survival after IT. To address this issue, we further investigated the role of the CXCR3 molecule in IT. We used B6 wild-type (WT) or B6 CXCR3 −/− mice. In in vitro transwell migration assays, liver NK cells from WT compared with CXCR3 −/− mice displayed significantly increased migration to islets (p = 0.015) ( Supplementary  Fig. S2). In B6 WT recipients of 200 islets, hyperglycaemia could not be ameliorated after IT (Fig. 1A). However, normoglycaemia was achieved in B6 CXCR3 −/− recipients of the same dose of islets (Fig. 1B). A significant difference in the rate of normoglycaemia achievement in B6 WT mice (0%, 0/6) or B6 CXCR3 −/− mice (100%, 6/6) was observed (p = 0.0022).
We next investigated the cytotoxicity of liver DX5 − NK cells against islets. Cytotoxicity assays revealed that the isolated DX5 − exhibited a higher cytotoxicity against islets than DX5 + NK cells, even in the steady state (Fig. 1E). The cytotoxicity of DX5 − NK cells against islets was partially inhibited by either anti-TRAIL mAb or anti-IFN-γ mAb, but not by concanamycin A (CMA) (Fig. 1F). The cytotoxicity was almost completely inhibited by the combination of the antibodies. To investigate the direct evidence of the involvement of liver DX5 − NK cells in islet destruction, we first attempted to establish an experimental model to evaluate islet engraftment in B6 Rag-2 −/− γ chain −/− mice, which completely lack T, B, NKT, and NK cells, reconstructed with the liver resident NK cells. On day 14 after adoptive transfer of the total liver NK cells containing both DX5 − and DX5 + NK cells, DX5 − NK cells were predominantly engrafted in the liver (p < 0.000001) ( Supplementary Fig. S3). The expression of CD69, TRAIL, and CXCR3 on liver NK cells in the recipients considerably increased compared with that of those on transferred NK cells (p < 0.000001, p < 0.000001, p < 0.01, respectively) ( Supplementary Fig. S3). These data were consistent with a previous report that DX5 − NK cells preferentially traffic to the liver 29 . Hence, it is less feasible to accurately compare islet engraftment in the liver between DX5 − NK-and DX5 + NK cell-transferred hosts owing to their differences in chemotactic responses and the spontaneous activation of NK cells in consequence of adoptive transfer. Instead of an adoptive transfer study, we employed Tbx21 −/− mice lacking immature TRAIL + DX5 − NK cells 32 . In B6 Tbx21 −/− recipients of 200 islets, normoglycaemia was almost achieved (83%, 5/6), whereas hyperglycaemia could not be ameliorated after IT in B6 WT recipients of the same dose of islets (0%, 0/6) (p = 0.0152) (Fig. 1C,D). Taken together, our findings suggest that liver DX5 − NK cells have cytotoxic activity against islets via the TRAIL-TRAIL DR5 and CXCR3-CXCL10 pathways.

IBMIR induces the expansion of liver DX5 − NK cells in vitro. Consistent with predominant and
transient TNF-α, IFN-γ, and IL-1β production during IBMIR 8,12 , such cytokine mRNA expression significantly increased in the liver with peak value at 24 h after syngeneic IT ( Fig. 2A). Serum TNF-α was detected in mouse after IT as well ( Supplementary Fig. S4). To investigate the possibility that IBMIR triggers NK cell activation, liver NK cells from B6 mice were cultured for 24 h in the presence of the three cytokines. The proportion of DX5 − NK cells among total NK cells increased after the incubation (p < 0.01) (Fig. 2B), and CD69 expression on liver NK cells considerably increased after the culture (p < 0.001) (Fig. 2C). Liver NK cell CD69 mean fluorescence intensity (MFI) also increased following the incubation (without vs. with cytokines; 6.8 ± 1.3 vs. 68.7 ± 9.4, p < 0.01, not shown in Figure). To define the predominant cytokine activating NK cells, each cytokine was added to or removed from the culture. CD69-expressing NK cell proportion increased when cultured with TNF-α alone, but not with either IFN-γ or IL-1β (Fig. 2D). Thus, TNF-α was the predominant cytokine activating DX5 − NK cells which express both TNF-α 1 and 2 receptors ( Supplementary Fig. S5). When combined with the other two cytokines, the absolute DX5 − NK cell number further increased ( Supplementary Fig. S6).

Proportion of DX5 − NK cells increases early after intraportal IT.
To investigate the role of TNF-α in NK cell activation, LMNCs were collected 24 h after IT in mice treated with/without anti-TNF-α antibody. The NK cell proportion in LMNCs significantly increased 24 h after IT, compared with that in non-transplanted naive mice (9.5 ± 0.6% vs. 8.1 ± 0.9%, respectively, p = 0.022, not shown in Figure). The absolute number of NK  cells obtained from the whole liver tended to increase 24 h after IT, compared with that in the nontransplanted naive mice (0.523 ± 0.068 × 10 6 vs. 0.463 ± 0.055 × 10 6 , respectively, p = 0.210, not shown in Figure). The proportion of DX5 − NK cells in total NK cells and the number of DX5 − NK cells increased 24 h after IT (p = 0.013, p = 0.009, respectively) ( Fig. 3A,B). Based on FCM histogram, DX5 − NK cells were clearly distinguished from DX5 + NK cells by the MFI value of DX5 expression even after IT ( Supplementary Fig. S7), suggesting that the increase of DX5 − NK cells was not consequent to reduced DX5 + NK cell DX5 expression. In contrast, the number of DX5 + NK cells did not increase 24 h after IT, compared with that in the nontransplanted naive mice (0.268 ± 0.085 × 10 6 vs. 0.315 ± 0.067 × 10 6 , respectively, p = 0.411, not shown in Figure). IT increased the expression of CD69 and TRAIL on liver NK cells, although it did not change their expression on either DX5 − and DX5 + NK cells ( Supplementary Figs S8 and 9). The treatment with the TNF-α-neutralising antibody prior to IT significantly inhibited the changes in DX5 − NK cell number (p = 0.048) (Fig. 3B). Similarly, the increase in CD69 and TRAIL expression on liver NK cells in IT recipients was inhibited by anti-TNF-α antibody as well (p = 0.012, p = 0.003, respectively) ( Fig. 3C,D). Anti-TNF-α antibody of IT recipients led to the reduction in the expression of CXCR3 and NKG2D on liver NK cells, although the former level did not reach statistical significance (p = 0.108, p = 0.037, respectively) ( Fig. 3E,F). Thus, TNF-α had key roles in DX5 − NK cell expansion early after IT even in an in vivo model. To investigate the difference in NK cell activation between syngeneic and allogeneic IT, LMNCs were also collected 24 h after IT in mice treated with allogeneic islets. The liver NK cell activation did not significantly differ between syngeneic and allogeneic IT ( Supplementary Fig. S10).  4B). Even after IT, the DX5 + NK cell proportion in LMNCs was constant, whereas DX5 − NK cells remained undetectable (Fig. 4C). As the counterpart, liver DX5 − NK cells were also adoptively transferred into B6 Rag-2 −/− γ chain −/− mice that received IT. In those mice, only DX5 − NK cells were detected even after IT as expected (Fig. 4D,E). Thus, conversion of DX5 + NK cells to a DX5phenotype was not observed after IT, speculating an alternative explanation, i.e. direct expansion of DX5 − NK cells. This speculation was supported by higher DX5 − NK cell numbers in the transferred mice with IT than in those without IT, although the difference did not reach a statistical significance ( Supplementary Fig. S11). The expansion of DX5 − NK cells after IT, observed in the WT mice described above (Fig. 3), might be due to the proliferation of these cells. To examine the proliferative potential of liver NK cells after IT, Ki-67 expression on liver DX5 − and DX5 + NK cells was assessed. Ki-67 expression on both liver DX5 − and DX5 + NK cells of mice that received islets did not differ from that on both liver DX5 − and DX5 + NK cells of naive mice, suggesting that the expansion of DX5 − NK cells was not consequent to the proliferation of DX5 − NK cells, potentially due to the recruitment of these cells/precursors from other sites ( Supplementary Fig. S12).

Conversion of DX5 + NK cells to a DX5
Intraportal IT consistently sustains the activation of liver DX5 − NK cells. As an additional clinically relevant model of islet engraftment, we performed IT of 300 syngeneic islets into STZ-induced diabetic mice. LMNCs isolated from the islet recipients were obtained at 14 and 35 days after IT. LMNCs from control diabetic mice who did not receive islets were obtained at the corresponding time points. NK cell numbers at both time points were significantly higher than those from non-IT controls (all p < 0.001) (Fig. 5A); the numbers of both subsets of NK cells were significantly higher than those from non-IT controls (DX5 − NK cells: p < 0.001, p < 0.001; DX5 + NK cells: p < 0.001, p < 0.01 for days 14 and 35, respectively) (Fig. 5C,D), although the DX5 − NK cell proportion in total NK cells of the islet recipients showed a tendency to increase (p = 0.119 at day 14, p = 0.086 at day 35) (Fig. 5B). Among liver NK cells, the CD69-, TRAIL-, and CXCR3-positive cell proportions in the IT recipients were significantly higher than those in the non-IT control (CD69: p = 0.017, p < 0.001; TRAIL: p = 0.004, p = 0.013; CXCR3: p = 0.136, p = 0.009 for days 14 and 35, respectively) ( Fig. 5E-G). CD69-, TRAIL-, and CXCR3-positive DX5 − NK cell proportions in the IT recipients were also significantly higher ( Supplementary  Fig. S13). Thus, intraportal IT consistently sustained liver NK cell activation.
Primary IT inhibits the reversal of diabetes in recipients with the sufficient mass of secondary transplanted islets. The sustained activation of liver DX5 − NK cells after the primary IT might interfere the engraftment of the secondary transplanted islets. We have previously shown that NK cell depletion leads to successful transplantation of 200 islets in STZ-induced diabetic CD1d −/− mice, indicating that liver NK cells are involved in islet destruction during transplantation of 200 islets 38 . Hence, 200 islets were used as priming dose during the IT to elucidate whether the activation of liver NK cells affects the engraftment of 400 secondary transplanted islets. In the control group that did not receive the priming dose, normoglycemia was nearly achieved in the recipients following the transplantation of 400 islets (Fig. 6A). However, hyperglycaemia could not be ameliorated in the recipients that received 400 islets during the secondary IT (Fig. 6B). A significant difference in the rate of normoglycemia achievement in diabetic recipient mice after the primary 400 IT (83%, 5/6) or the CD69 among total liver NK cells are shown in bar graphs as the means ± SD of 4 independent experiments (n = 5). *** p < 0.001. (D) Liver NK cells treated with the cytokine combinations for 24 h (n = 4-5). The data in bar graphs are presented as the means ± SD of 4 independent experiments. * p < 0.05; ** p < 0.01. # p < 0.001, compared with the results of liver NK cells without cytokines.  secondary 400 IT after priming (0%, 0/6) was observed (p = 0.015). In comparison with that in the control group, the proportion of liver DX5 − NK cells expressing CD69, TRAIL, and CXCR3 significantly increased after secondary IT (p = 0.008) (Fig. 6D). Administration of ASGM1 antibody, which significantly and exclusively reduced NK cells but barely influenced NKT cells ( Supplementary Fig. S14), improved the 400 secondary transplanted islet engraftment (83%, 5/6) (Fig. 6C), compared with that in the transplanted mice without antibody (0%, 0/6) (Fig. 6B) (p = 0.015). Thus, liver NK cells play a significant role in secondary islet rejection.
Anti-TNF-α antibody regulates liver DX5 − NK cell expansion after secondary IT leading to enhanced secondary transplanted islet engraftment. On the basis of above described finding that TNF-α predominantly activated DX5 − NK cells, anti-TNF-α antibody administration might constitute a therapeutic strategy for improving either primary or secondary islet engraftment. On the study investigating this issue, most diabetic mice that received anti-TNF-α antibodies at the both time of primary and secondary ITs became normoglycemic (86%, 6/7) (Fig. 7A), whereas none of the mice treated with control goat IgG became normoglycemic (0%, 0/7) (Fig. 7B) (p = 0.0047). Consistently, anti-TNF-α antibody administration significantly reduced the DX5 − NK cell proportion after secondary IT, accompanying the CD69-and TRAIL-positive NK cell proportion decreases (Fig. 7E). Anti-TNF-α antibodies on secondary IT alone did not modulate NK cell activation ( Supplementary Fig. S15), and islet engraftment between recipient mice with anti-TNF-α (20%, 1/5) and control antibodies (0%, 0/5) did not significantly differ (Fig. 7C,D) (p = 1). Thus, anti-TNF-α antibody treatment may prevent NK cell activation after IT but may not calm primary IT-induced pre-activated NK cells.

Discussion
The clinical application of IT is limited mainly owing to the early loss of transplanted islets, resulting in low transplantation efficiency. It has been demonstrated that high-mobility group box 1 (HMGB1) proteins released from transplanted islets, regardless whether allogeneic or syngeneic, trigger the activation of liver resident innate immune cells causing the early loss of transplanted islets 39 . NKT cell-dependent IFN-γ production has been proven to be essential for this process 7 . In addition, we recently showed that NK cells play a role in the early islet graft loss after syngeneic intraportal IT in an NKT cell-independent manner; i.e., anti-NK1.1 mAbs were shown to improve the engraftment of intraportally transplanted syngeneic islets even in NKT-deficient CD1d −/− mice 38 . IBMIR is a nonspecific response mediated by the innate immune system including the liver resident lymphocytes, and it is characterised by coagulation, complement activation, platelet adhesion, and leukocyte infiltration into the islets. Previous reports have demonstrated that IBMIR plays significant roles in the damaging of allogeneic, xenogeneic, and even syngeneic transplanted islets during the peritransplant period 9,40,41 . Tissue factor, expressed by the islets, is considered a major IBMIR trigger, leading to the platelet activation and release of downstream inflammatory mediators, such as TNF-α, IFN-γ, and IL-lβ 8,12 .
In this study, we showed that proinflammatory cytokines, predominantly TNF-α, further induce the activation of liver DX5 − NK cells that express both TNF-α 1 and 2 receptors. TNF-α has been generally regarded as a toxic cytokine mediating islet injury after IT 42,43 , and its blockade has been proven to have beneficial effects on the engraftment of transplanted islets, which led to the inclusion of this procedure in the latest IT therapy protocol Clinical Islet Transplantation 07 (CIT-07) 3,44 . Despite the clinical use, the efficacy of anti-TNF-α antibody against liver immune cells is little known with certainty. In this study, we observed that the treatment with anti-TNF-α antibody prior to IT significantly inhibits the alterations in the absolute number of DX5 − NK cells and CD69/ TRAIL expression on liver NK cells. Therefore, we showed that TNF-α plays key roles in the activation of DX5 − NK cells early after IT, and that the inhibition of TNF-α has protective effects on the transplanted islets, at least partly by preventing the activation of liver NK cells during IBMIR.
Liver NK cells differ from conventional NK cells. NK cells contribute to the early defence against virus-infected and neoplastic cells based on the absent self-markers; a missing-self hypothesis had been proposed to explain the protective effects of target cell MHC class I on NK cell-mediated lysis 45,46 . We have previously demonstrated that liver resident TRAIL-positive NK cells lack the expression of Ly-49 inhibitory receptors recognising self-MHC class I, which leads to the lowering of their capacity for the self-recognition of NK cells that possess cytotoxic activity against syngeneic hepatocytes expressing DRs, which recognise TRAIL in mice 47 . We further demonstrated that NK cell-mediated cytotoxicity represents a major obstacle to the engraftment of autologous hepatocytes during hepatocyte transplantation because the direct interaction of the hepatocytes transplanted via the portal vein with liver NK cells is inevitable 47 . This self-cytotoxic mechanism may contribute to the prevention of the aberrant implantation of self-cells undesirably peeled away from the gastrointestinal tract through the portal vein. In a similar way, the transplanted islets expressing DRs also become stacked at the portal vein radices, resulting in the deposition of some cells at the hepatic sinusoid in close contact with liver TRAIL + NK cells 12 . In addition to the anatomical features of sinusoids, the characteristic chemotaxis of liver DX5 − NK cells expressing CXCR3 contribute to the inhibition of the engraftment of transplanted islets secreting CXCL10.
Liver-resident NK cells were recently described to have the unique capacity to confer immunological memory in the form of hapten-specific contact hypersensitivity independent of T and B cells 29,48 . Peng et al. characterised this unique phenotype as CD49a + DX5and found that they display memory response in contact hypersensitivity models 29 . They have further demonstrated that liver DX5 − NK cells originate primarily from the liver and are not differentiated from other NK cell subsets, including both non-liver NK cells and liver DX5 + NK cells. Consistent with the previous study, the adoptively transferred liver DX5 + NK cells did not convert into liver DX5 − NK cells after IT in B6 Rag-2 −/− γ chain −/− mice. In addition, liver DX5 − NK cells did not show any proliferative potential after IT. This finding suggested that liver DX5 − NK cells are recruited from other sites after IT. We investigated whether the memory function of liver DX5 − NK cells activated after the primary IT affects the engraftment of secondary transplanted islets. We found that primary IT disturbed the reversal of diabetes in recipients that received a sufficient mass of secondary transplanted islets, and a significant increase in CD69, TRAIL, and CXCR3 expression was observed in liver DX5 − NK cells after secondary IT, in comparison with that in the control group that did not receive the primary IT, indicating their memory-like function. There may be a possibility that the inflammation owing to the primary IT remains exist at the time of secondary IT or that the other immune cell memory function is involved in secondary islet rejection. To investigate the direct evidence for the role of memory DX5 − NK cells in secondary islet rejection, we designed an experimental model to evaluate islet engraftment in the memory-like DX5 − NK cell-transferred new host. However, the difference in chemotactic responses between DX5 − NK and DX5 + NK cells and the spontaneous activation of NK cells in consequence of adoptive transfer did not allowed us to compare the islet engraftment in the liver between DX5 − NK-and DX5 + NK cell-transferred hosts. Hence, we assessed the role of memory NK cells in secondary islet rejection by depleting NK cells prior to secondary IT. NK cell depletion improved the engraftment of 400 secondary transplanted islets, suggesting that liver DX5 − NK cell activation was accelerated as a recall response after secondary IT. Considering the characteristic deployment of memory-like DX5 − NK cells in the liver, this organ may not be the optimal site for islet infusion. Alternative implantation sites, such as subcutaneous tissue, omentum, and intramuscular tissue, have been investigated 13,14,49 . The effects of innate immune response on islets transplanted into these anatomical sites should be further investigated.
In this study, it is notable that the liver DX5 − NK cell activation during IBMIR led to long-lasting NK cell cytotoxic activity and amplified their activation against secondary transplanted islets as a recall response even after syngeneic IT. Liver DX5 − NK cell activation after allogeneic IT was comparable to that in syngeneic IT, indicating that an NK cell innate immunity-induced barrier constitutes a common feature of both types of IT. However, based on the possible collaboration between innate and acquired immunity, NK cell activation might accelerate subsequent alloimmune responses. Further investigations are required to clarify this issue. The regulation of the activity of these memory-like NK cells by anti-TNF-α treatment may improve the rates of engraftment of sequentially transplanted islets. However, the selective depletion of liver DX5 − NK cells remains difficult in clinical settings. Thus, preventing direct interaction between liver NK cells and transplanted islets through the inhibition of TRAIL-DR and/or CXCR3-CXCL10 pathways may represent a promising approach. Liver NK cell isolation. LMNCs were prepared as previously described 50  Islet isolation and transplantation. Islets were isolated using our standard procedure 6 . Islets 100-to 200-µm in diameter were suspended in 200 µL Hank's balanced salt solution (Gibco) and transplanted into the liver via the portal vein 51 . Streptozotocin (STZ, 200 mg/kg; Sigma-Aldrich) was intraperitoneally administered to recipient mice to induce diabetes 7 days before IT. Blood glucose levels were measured using a GT-1830 glucose analyser (Arkray, Tokyo, Japan). Non-fasting blood glucose levels exceeded 500 mg/dL by day 3 after STZ injection and mice remained hyperglycaemic until IT. Blood glucose levels shown to decrease below 200 mg/dL by two consecutive measurements indicated the reversal of diabetes mellitus. We previously showed that transplanting 400 syngeneic islets into the liver was sufficient to reverse hyperglycaemia in diabetic recipient mice 38 whereas transplanting 300 or 200 syngeneic islets was considered as a marginal or insufficient mass of islets to achieve normoglycemia in diabetic recipient mice, respectively. Luminescent cytotoxicity assay for islets. NK cell cytotoxicity against islets was measured using the CytoTox-Glo Cytotoxicity Assay (Promega, Madison, WI, USA), which measures cell death through the release of dead-cell protease activity from dying cells that have lost membrane integrity. As previously described 6 , 0.2 × 10 6 isolated NK cells were used as effector cells and two islets served as target cells. After 8-h incubation, data was collected using the GloMax Discover System (Promega). In some experiments, the assay was performed in the pres- TNF-α neutralising antibody treatment. Specific neutralising antibodies against TNF-α (100 µg per mouse; R&D Systems) were administered by systemic intraperitoneal injection into B6 mice 24 h before IT. For secondary IT experiments, TNF-α neutralising antibodies were administered on days 0, 3, 7, and 10 of IT. Control mice were injected with normal goat IgG (R&D Systems).

Real-Time RT-PCR.
Real-time RT-PCR was performed for TNF-α, IFN-γ, IL-1β, and beta-2-microglobulin as the housekeeping gene. Liver samples were harvested after IT and stored until use. Total mRNA was extracted using an RNeasy Mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. cDNA was generated using a QuantiTect Reverse Transcription Kit (Qiagen) and amplified with a Rotor-Gene Q 2PLEX HRM Real-Time PCR system (Qiagen). TNF-α, IFN-γ, and IL-1β expressions were investigated using appropriate primers and probes (Taqman Universal PCR MasterMix, TaqmanGene Expression Assays, Applied Biosystems) with 32 ng of reverse transcribed total RNA in a total volume of 25 µl (Supplementary Table S1). The amplification protocol consisted of denaturation at 95 °C for 5 min, followed by 40 cycles of 95 °C for 5 s and 60 °C for 10 s.
Statistical analysis. Data were presented as the means ± standard deviation (SD). Continuous and dichotomous variables were compared using unpaired Student's t-tests and Fisher's exact tests, respectively. For three or more group comparisons, statistical significance was determined using one-way ANOVA with Tukey post hoc analysis. All statistical analyses were performed using statistical software JMP version 10 (SAS Institute, Cary, NC, USA). A p-value of < 0.05 was considered statistically significant.