Dynamic Radioimaging Application And Regulation Mechanism Of TLR5 And Autophagy In Allogenic Transplant Model

Background:As treatment alternatives are limited and transplantations are ofter the only therapeutic option for thoses patients with end-stage organ failure. modern immunosuppressive therapies have allowed important progress in short-term outcomes. However, long-term graft and patient survival have not improved significantly for some decades, with ongoing immune injury to the graft and chronic toxicity of immunosuppressive treatments. Thus, a compelling need for post-transplantation dynamic grafts monitoring and new mechanisms of immune regulation exists. In this article, we committed to identify an immunosuppressant related target by a non-invasive molecular imaging method and evaluated the poteintial application and mechanism in immune rejection regulation.Except for the biopsy, the current traditional allograft detection methods mostly rely on the early detection, quantification, and monitoring of cellular or humoral inflammatory events during episodes of acute rejection, which is impacted by the immunosuppressant application. For example, 18F-fluorodeoxyglucose (18F-FDG), a radiopharmaceutical used in the medical imaging modality positron emission tomography (PET), was investigated in transplant rejection and evaluated immunosuppressive treatment efficacyThe 18F-FDG uptake in allograft was increased when acute rejection occurred, but high radioactive signal of PET images disappeared while the immunosuppressive drug cyclosporine was applied. It remained low and stable in the graft during treatment after renal transplantation, suggesting that F-FDG can be used to detect the efficacy of immunosuppressive therapy, but may not be a good tracer for follow-up surveillance. As the induction and maintenance of donor-specific tolerance is a central aim in solid organ transplantation, a reliably biomarker is essential for clinicians to identify and monitor tolerance accurately.Recent interest is focused on the role of a group of pattern recognition receptors, Toll-like receptors (TLRs), expressed on leukocytes and on tissue parenchymal cells as important sensors of infection and tissue damage. TLR5 recognizes flagellin and initiates a signaling cascade through recruitment of MyD88 and activation of NF-kB.High TLR5 expression has been observed in CD4+CD25+Treg cells, and such high expression potently increases the suppressive capacity of these cells via enhanced Foxp3 expression。activation of TLR5 by flagellin reduces GvHD (graftversus-host disease) following allogeneic haematopoietic stem cell transplantation。Our prior laboratory research found that use of an immunosuppressive drug (rapamycin) in vivo promoted the expression of TLR5 and Foxp3 in a mouse allogeneic transplantation model。Recombinant flagellin (rFlic) collaborates to strengthen tolerance induction by immunosuppressive drug rapamycin。Macroautophagy (termed autophagy herein) is a cellular catabolic process that is involved in cytoplasmic renewal and turnover of long-lived proteins and cellular organelles. Autophagy participates in immune responses at both the innate and adaptive levels. Autophagy also plays a critical role in both central and peripheral tolerance to self-antigens. Beclin 1-deficient CD4+T cells, which lack autophagy, are prone to apoptosis upon TCR stimulation due to greatly elevated levels of latent forms of cell deathrelated proteins, such as procaspase-3, procaspase-8 and Bim. Whether autophagy participates in tolerance induction to foreign antigens, including allografts is unknown.In our research, we clarified the dynamic imaging application and mechanisms of TLR5 and autophagy in rapamycin treated allogenic transplant model from the following three parts:1. targeting TLR5 expression serves as a means for rapamycin-treated graft detection. We employed radioiodinated 131Ⅰ-anti-TLR5 monoclonal antibody (mAb) as a radiotracer in an allogeneic skin transplant model; 2. Compared with the non-specific radioactive indicator 18F-FDG, we evaluate the advantages of TLR5 as a superior target for nuclear imaging of rapamycin-treated allogeneic graft; 3. Preliminary research of autophagy influenced the allogenic transplant rejection response through comparing with syngeneic transplantation and allogeneic transplantation.Part One Allograft imaging by targeting Toll-like receptor 5 in rapamycin-treated mouse allogeneic skin transplantation modelMethods1. Allogeneic skin transplant modelFull-thickness skin grafts from donor C57BL/6 (H-2b) female mice were transplanted onto the prepared graft beds on the right shoulders of recipient BALB/c (H-2d) female mice. Surgeries were performed under anaesthesia with 0.6% pentobarbital sodium (0.1 ml/10 g body weight) intraperitoneally (i.p.) during the entire operation to control analgnesia. The mice were divided into 2 groups: rapamycin treated group (1.5 mg/kg/day, i.p.) and allogeneic rejection group (equivalent volume of PBS i.p.). Observe the allograft skin survival from day 7 after transplantation.2. Radioiodination and quality controlAnti-TLR5 mAb was radiolabeled with 131Ⅰ. Radioligand-based binding assays (RBA) were carried out to evaluate the Kd, Ki and Bmax values. An aliquot of 2 radiotracers were mixed in mouse serums or NS (Normal Saline) respectively for the stability analysis in vitro.3. Ex vivo biodistribution studiesTo validate of the quantity of 131Ⅰ-anti-TLR5 mAb uptake and confirm the results of the imaging study, a set of mice from the rapamycin-treated group was injected with 131Ⅰ-anti-TLR5 mAb intravenously into the tail vein for ex vivo biodistribution studies. Mice were sacrificed at 1 h,12 h,24 h,48 h and 72 h after 131Ⅰ-anti-TLR5 mAb injection. Organs and tissues were excised, rinsed for residual blood, and weighed. Samples and primed sandards were evaluated for radioactivity in the gamma counter and corected for physical decay. Tissues activities were expressed as the percent injected dose per gram (%ID/g). The target-to-non traget ratio was defined as the radioactivity of allograft to opposite native skin ratio.4. Dynamic in vivo whole-body phosphor-autoradiography of 131Ⅰ-anti TLR5 mAbAnimals in the 2 groups were injected intravenously via the tail vein with a PBS solution of 131Ⅰ-anti-TLR5 mAb (0.37 MBq) at day 7 after transplantation. For binding specificity studies,5 mice randomly chosen from the rapamycin-treated group received a blocking dose of anti-TLR5 mAb (10 mg/kg) via the tail vein at 30 min before injection with 131Ⅰ-anti-TLR5 mAb. Dynamic in vivo whole-body phosphor-autoradiography was acquired at 1 h,12 h,24 h,48 h, and 72 h.5. Correlation between TLR5 expression and 131Ⅰ-anti-TLR5 mAb uptake in allograftTwo groups of animals which injected 131Ⅰ-anti-TLR5 mAb were sacrificed after 72 h imaging. The grafts were harvested to be paraffin section for TLR5 staining. Correlation between TLR5 expression and 131Ⅰ-anti-TLR5 mAb uptake in allografts were performed by Graph Pad Prism 5 software.Results1.131Ⅰ-anti-TLR5 mAb were successfully radioiodinated. Radiochemical purity of 131Ⅰ-anti-TLR5 mAb was 96.9% with specific radioactivity 458.6 MBq/mg. 131Ⅰ-anti-TLR5 mAb showed good immunoactivity, with Kd value 3.672 nM, Bmax value 5.388 μM, Ki value 8.89 nM and IC50 value 40.38 nM. Even after 96 h after radioiodination, the stability of 131Ⅰ-anti-TLR5 was still higher than 88%.2. Ex vivo biodistribution studies revealed that 131Ⅰ-anti-TLR5 mAb uptake in allo-treated graft reached a maximum of 12.05 ± 1.86%ID/g (the percent injected dose) at 1 h and graft-to-native skin ratio reached 8.10 ± 0.10 at 72 h after injection. Although 131Ⅰ-anti-TLR5 mAb also accumulated in allogeneci rejection group, but it is much lower than that in rapamycin treated group (p<0.001).%ID/g of grafts in block group was reduced about 92% to the rapamycin treated group. These results demonstrated that 131Ⅰ-anti-TLR5 mAb was specific to the rapamycin-treated graft.3. Dynamic phosphor-autoradiography imaging showed clear graft localization from 12 h onward and highest contrast images were acquired at 72 h after 131Ⅰ-anti-TLR5 injection. Graft uptake quantified from image was 26448 ± 904 DLU/mm2 (Digital light units) for rapamycin reated group compared with 9176 ± 576 DLU/mm2 for allogeneic rejection group and 3881.5 ± 62.6 DLU/mm2 for block group (p<0.01). These were equivalent to organ biodistribution study at 72 h after injection. The inhibited 131Ⅰ-anti-TLR5 mAb accumulation also confirmed a specific graft uptake.4.131Ⅰ-anti-TLR5 mAb uptake in the grafts from the biodistribution study displayed a significant correlation with the percentage of TLR5 expression (r2=0.93, P< 0.0001).Part Two Evaluation of 131Ⅰ-anti-TLR5 mAb/131Ⅰ-IgG/18F-FDG for allograft monitor in rapamycin-treated allogeneic toleranceMethods1. Allogeneic skin transplant modelAccordance withe the part one, full thickness skin grafts from donor C57BL/6 (H-2b) mice were transplanted onto the prepared graft bed on the right shoulders of recipient BALB/c (H-2d) mice. The mice were divided into 2 groups:rapamycin treated group (1.5 mg/kg/day, i.p.) and allogeneci rejection group (equivalent volume of PBS i.p.). Observe the allograft skin survival from day 7 after transplantation.2. Radioiodination and quality controlAnti-TLR5 mAb radiolabeled method was same as part one. Mouse isotype IgG was radiolabeled with 131Ⅰ. An aliquot of 131Ⅰ-IgG were mixed in mouse serums or NS (Normal Saline) respectively for the stability analysis in vitro (Method was same as Part one).3. Imaging comparing research of 131Ⅰ-anti-TLR5 mAb,131Ⅰ-IgG and 18F-FDG applicationTwo groups of animals were injected intravenously via the tail vein with a PBS solution of 131Ⅰ-anti-TLR5 mAb (0.37 MBq/150 μl) or 131Ⅰ-IgG (0.37 MBq/150 μl) at day 7 after transplantation. Dynamic in vivo whole-body phosphor-autoradiography was acquired at 1 h,12 h,24 h,48 h, and 72 h.Five mice from each group were injected intraperitoneally with 18F-FDG (5.55 MBq/150 μl) on day 10 after transplantation. Images were acquired at 30,60 and 90 min. after injection. OptiQuantTM image analysis software was employed to assess the radioactive accumulation in allograft area.Results:1. Radiochemical purity of 131Ⅰ-IgG was 96.6%, with specific radioactivity 407.2 MBq/mg. The stability analysis showed more than 90% after radioiodination both in mouse serum and NS.2. Although 131Ⅰ-IgG accumulated in rapamycin treated allograft area from 12 h after injection but the radioactivity was much lower than 131Ⅰ-anti-TLR5 mAb (p<0.01). It confirmed that 131Ⅰ-anti-TLR5 mAb accumulation was due to the specific binding of 131Ⅰ-anti-TLR5 mAb and TLR5 receptor, rather than Fc portion of IgG non-specific binding.2. High 18F-FDG uptake was not observed in the rapamycin treated group, but in allogeneic treated group.131Ⅰ-anti-TLR5 mAb had sustained high accumulation in rapamycin treated group. The highest allograft skin-to-native-skin ratio (A:N) of 131Ⅰ-anti-TLR5 mAb uptake was significantly higher than the ratio for 18F-FDG (7.68 versus 1.16, respectively, p<0.05), which showed significantly advantage of 131Ⅰ-anti-TLR5 mAb allograft imaging application.Part Three Autophagy expression and regulation mechanism during allogenic transplant rejectionMethods1. Allogeneic skin transplant model and rapamycin treated transplant modelThe method was same as part one.2. Autophagy related gene and protein evaluation among the groupsMice were sacrificed at day 4,8,12 and 16 post-transplantation. Skin and spleen were harvested; the total mRNA and protein were contracted. The autophagy related genes expression of Beclinl and ATG5 were determined by Quantitative Real-time PCR. The autophagy related protein expression of Beclinl, ATG5 and LC3-1, LC3-Ⅱ were determined by western blot. Autophagy level wasd determined as LC3-Ⅱ/LC3-Ⅰ3. Corelation analysis of TLR5 expression and autophagy level in allograftsCorelation between allograft TLR5 expression determined by TLR5 immunochemistry stain and autophagy level was measured by Graph Pad Prism 5 software.Results1. Quantitative Real-time PCR results:In allogenice rejection group, Beclinl and ATG5 genes expression was elevated from day 8, reached the peak at day 12 and declined at day 16. The same trend was happened at rapamycin-treated group, but thoese expressions were all much higher than allogeceic group (p<0.01) and the highest Beclin1 and ATG5 genes expression were in allograft skin area of rapamycin-treated group at day 12.2. Western bolt results:Beclinl and ATG5 protein expression were consistent with genes expression. Autophagy level determined by LC3-Ⅱ/LC3-Ⅰ value also confirmed that the pattern of autophagy overlapped with the progression of graft rejection in response to the allogeneic antigen. More important, those autophagy dynamic changing was more remarkable in allograft skin than spleen cells.3. Autophagy level was correlated with TLR5 expression in allograft area-(r2=0.79).Conclusions1. We constructed the 131Ⅰ-anti-TLR5 mAb with high specificity and biological activity to TLR5.2. In vivo biodistribution results showed that 131Ⅰ-anti-TLR5 mAb accumulated higher in allografts than opposite native skin. Comparing with allogeneic rejection group, T/NT ratio of 131Ⅰ-anti-TLR5 mAb accumulation in rapamycin treated group was significantly higher. Whole-body phosphor-autoradiography results were consistent with the biodistribution results, clear graft localization imgaes were acquired from 12 h after 131Ⅰ-anti-TLR5 mAb injection in rapamycin treated group.3. Anti-TLR5 mAb inhibited 131Ⅰ-anti-TLR5 mAb accumulation, which confired the specific combination to TLR5 expression in allografts.4. Allografts radioactivity had positive correlation with TLR5 expression in allografts5. Compared with 131Ⅰ-IgG and non-specific radiotracer 18F-FDQ 131Ⅰ-anti-TLR5 mAb showed significant advantages with highest T/NT ratio and specific immunoactivity.6. Real-time PCR and western blot confirmed that autophagy level correlated closely with the gradually increasing allograft injection, it might be a new mechanism used to regulate the allograft rejection.7. Autophagy and TLR5 may be the new regulation mechanisms in allogeneic rejection response.