Pancreatic islet cell transplantation is an important treatment option for brittle type 1 diabetes and as an adjunct procedure after total pancreatectomy to prevent brittle type 3c diabetes.1,2 Research into multiple facets of islet transplantation, such as anti-inflammatory regimens, has significantly improved islet transplant outcomes.3,4 Yet, despite improvements made in the past 2 decades, early graft loss and sustained islet function remain a challenge.5
Evidence suggests that islet transplant outcomes are compromised by inflammation sustained before transplantation.6 The organ procurement process subjects tissues to sterile stressors and damage which may promote acute rejection posttransplant, but also provides a potential window for therapeutic interventions before transplantation which remains largely unexplored.7,8 The process of isolating pure islets generates an environment of sterile inflammation mediated by ischemia, hypoxia, enzymatic and mechanical digestion, oxidative stress, and the release of endogenous damage-associated molecular patterns from stressed and damaged cells.9,10 Therefore, reducing sterile inflammation pretransplant may be an important step for improving transplant outcomes.
Although the upregulation and activation of inflammatory markers in tissues is commonly observed after organ procurement and islet isolation, the mechanisms by which this inflammation occurs remain understudied.11,12 Previously, studies highlighted the roles of mitogen-activated protein kinases (MAPKs) and the nuclear factor κB (NFkB) pathway in upregulated stress responses in islets during and after isolation.13-15 Tyrosine-activated pathways may also play a role through ischemia/reperfusion injury.16 Targeting these pathways has demonstrated protection of islets against apoptosis posttransplant.17-19 However, these treatments either require sustained exposure to agents that inhibit vital signaling pathways or require systemic administration of compounds with broad off-target toxic side effects. This makes the 2 approaches inappropriate for clinical use.20-22 Thus, selectively targeting a nonvital innate inflammatory pathway with a clinically safe compound is necessary to avoid these complications and provide a rapidly translatable methodology.
Recent evidence has elucidated the role of Toll-like receptors (TLRs) in poor transplant outcomes.23,24 Of particular interest is TLR4, the canonical receptor for Gram-negative bacterial lipopolysaccharide. Activation of TLR4 triggers a robust inflammatory cascade that ultimately results in the production of inflammatory cytokines and possibly cell death.25 TLR4 has been described as a promiscuous, noncanonical receptor for damage-associated molecular patterns and certain cytokines, such as high-mobility group box 1 and C-X-C motif chemokine 10 (CXCL10).26,27 Moreover, TLR4 is observed to be highly upregulated in multiple organs by ischemia/reperfusion injury after transplant.28-30 TLR4-deficient murine models and treatments targeting TLR4 and its endogenous ligands posttransplant have shown positive results in preventing aberrant inflammation and acute graft rejection.31,32 Therefore, we hypothesized that TLR4 was complicit in mediating islet inflammation during the isolation process and propose that TLR4 blockade before transplantation could reduce this inflammation and improve transplant outcomes.
For this study, TAK-242, a small molecular inhibitor of TLR4, was identified as an ideal candidate due to its immunity to enzymatic degradation and its safety as demonstrated by a phase II clinical trial.33,34 We investigated the therapeutic potential of early TLR4 blockade in the islet isolation process by incorporating soluble TAK-242 with collagenase to inhibit sterile inflammation sustained during and after isolation. The novelty of our approach is that it can be easily translated into a clinical setting. We used a syngeneic murine model to determine whether a single early treatment is sufficient to significantly improve transplant outcomes. Then, we examined the expression of key proteins and cytokines known to compromise transplant outcomes, as well as elucidated the downstream signaling pathways differentially activated by early TLR4 blockade.
MATERIALS AND METHODS
Male wild-type C57BL/6 mice 6 to 7 weeks old were purchased from Envigo (Houston, TX) and housed at the Institute of Metabolic Disease at Baylor University Medical Center for at least 5 days before use. Animal experiments were approved by the Institutional Animal Care and Use Committee at Baylor Scott & White Research Institute.
Islets were isolated from C57BL/6 mice using a previously described method with minor modifications.35 Briefly, pancreases were perfused with collagenase containing either 0.01% DMSO or 3 μM TAK-242 (MedChemExpress, Monmouth Junction, NJ) via common bile duct injection and kept on ice for at least 30 minutes after excision. Pancreases were digested at 37 °C for 18 to 20 minutes, and purified via discontinuous density gradient. After isolation, islets were either used immediately or cultured in Roswell Park Memorial Institute medium supplemented with 1% penicillin/streptomycin, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and 1% bovine serum albumin (Sigma-Aldrich) at 37 °C and 5% CO2.
Islets used for transplantation experiments and Western blot analysis were isolated with collagenase type V (1 mg/mL; Sigma-Aldrich, St. Louis, MO). Islets used for proinflammatory cytokine analysis were isolated with collagenase type V or Liberase TL (0.125 mg/mL; Roche Diagnostics, Indianapolis, IN). Islets used for viability and function studies were isolated with Liberase TL.
Recipient mice were made diabetic by a single intraperitoneal injection of streptozotocin (STZ) (200 mg/kg). Diabetic mice are defined as having a nonfasting blood glucose greater than 450 mg/dL for 2 consecutive days. To study transplant outcomes, immediately after isolation, 150 islets were transplanted intraportally via a 27-G winged infusion set into diabetic recipient mice under general anesthesia. Blood glucose was measured 3 times per week for 8 weeks. Euglycemia was achieved on the first day of 2 consecutive glucose measurements less than 200 mg/dL For biomarker analysis, 200 islets were transplanted into the kidney subcapsular space of diabetic mice. See Supplemental Digital Content, SDC, http://links.lww.com/TP/B583 for details.
Intraperitoneal Glucose Tolerance Test
Intraperitoneal glucose tolerance test (IPGTT) was performed 45 days posttransplant. Mice were fasted for 6 hours by placing them in fresh cages with no food but access to water ad libitum. Then, a 2-mg/kg bolus of glucose was injected intraperitoneally into the mice as a 20% w/v glucose solution. Blood glucose was measure before injection (time 0) and then every 30 minutes for 150 minutes by tail-vein prick.
Serum Biomarker Analysis
Serum was isolated from kidney-capsule transplanted mice pretransplant, 4 hours posttransplant, and days 1, 2, 3, and 7. MicroRNA was isolated and assayed by RT-PCR using commercially available hsa-miR-375 and UniSp6 primers (Exiqon, Inc., Woburn, MA) on a CFX Connect (Bio-rad, Hercules, CA) with the following program: 95°C 10 minutes, then 40 cycles of 95 °C, 10 seconds; 60 °C, 1 minutes. Relative expression was calculated using the 2-ΔΔCT method normalized to UniSp6. See Supplemental Digital Content (SDC, http://links.lww.com/TP/B583) for details.
Serum cytokines for IL-6, CXCL1, and CXCL10 were measured by multiplex analysis with a Milliplex MCYTOMAG-70 K kit (EMD Millipore Corporation, Billerica, MA) according to the manufacturer's instructions with undiluted serum. Samples were incubated overnight at 4°C with shaking and analyzed on a Luminex 200 (Luminex Corporation, Austin, TX).
Islet qRT-PCR Analysis
Total RNA was isolated from samples using TRIzol (Invivogen, San Diego, CA) and converted to cDNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Waltham, MA) following the manufacturer's instructions. Quantitative expression of genes of interest was determined using SsoAdvanced Universal SYBR Green Supermix (Bio-rad) on a Bio-Rad CFX Connect with the following program: 95°C, 10 minutes; 40 cycles of 95°C, 15 seconds; and then 60°C, 1 minute. Primers for qRT-PCR analysis were purchased from commercially available stock (Integrated DNA Technologies, Coralville, IA). Relative gene expression was calculated using the 2-ΔΔCT method normalized to 18S mRNA.
Whole islet protein lysates were resolved by SDS-PAGE and electroblotted onto a polyvinylidene difluoride membrane by semidry transfer using standard techniques. Immunoblotting was performed according to standard methods. See Supplemental Digital Content, SDC, http://links.lww.com/TP/B583 for details. All antibodies were purchased from Cell Signaling Technology (Danvers, MA) and used at a 1:1000 dilution unless otherwise stated. Antibodies used included β-actin (1:2000, #4970), phospho-extracellular signal-regulated kinase (ERK)1/2 (1:2000, #4370), ERK1/2 (#9102), phospho-P38 (#4511), P38 (#9212), phospho-P65 (#3033), P65 (#8242), phospho–stress-activated protein kinase (SAPK) (#4668), SAPK (#9252), and α-rabbit IgG HRP-linked (1:2000, #7074). Band density was measured using Fiji (http://fiji.sc/).36 Relative densities were calculated and compared using the ([phospho]:[actin])/([total]:[actin]) method to normalize relative activation across membranes.
Islet Viability Assessment
Islet viability was determined by staining with Hoechst 33342 and propidium iodide. See Supplemental Digital Content, SDC, http://links.lww.com/TP/B583 for details.
Glucose-stimulated Insulin Secretion
Islets were incubated with low glucose (2 mM) and then high glucose (20 mM). Insulin was measured using a mouse insulin ELISA kit (ALPCO Diagnostics, Salem, NH) according to manufacturer instructions. See Supplemental Digital Content (SDC, http://links.lww.com/TP/B583) for details.
All data were presented as means ± SEM. Survival was compared by log-rank (Mantel-Cox) test. Single pairwise comparisons were performed using a 2-tailed unpaired t test with Welch correction. Multiple t test with Holm-Sidak post hoc test was used for multiple comparisons. Statistical analysis was performed in GraphPad Prism 6 (San Diego, CA). Graphs were made in either GraphPad Prism 6 or Microsoft Office 2016 (Redmond, WA). Statistical significance was achieved when P value is less than 0.05.
Early TLR4 Blockade Promotes Successful Intraportal Islet Transplantation Outcomes
To determine whether early blockade of TLR4 affects islet transplant outcomes, we used a syngeneic transplant model. A marginal dose of 150 islets for intraportal transplant was chosen based on previous islet dose titrations.31 Islets were isolated from male C57BL/6 mice with enzyme containing 0.01% DMSO (control group) or with enzyme containing 3 μM TAK-242 (TAK group). Immediately after isolation, islets were transplanted intraportally into STZ-induced diabetic mice and followed for 8 weeks (Figure 1A). Mice receiving TAK islets had superior transplant outcomes, with 75% (6/8) achieving euglycemia, compared with 29% (2/7) of those receiving control islets (Figure 1B; P < 0.05), with reduced glycemic lability (Figure S1A,B, SDC, http://links.lww.com/TP/B583). The time to euglycemia was also significantly reduced by early TLR4 blockade, with the TAK group having a mean time to euglycemia of 21.2 ± 3.7 days compared with the control group with 35.0 ± 1.0 days (Figure 1C; P < 0.05).
Long-term function of the islet grafts in vivo was assessed by IPGTT on day 45 posttransplant (Figure 1D). Area-under-the-curve analysis revealed superior islet function in mice from the TAK group compared to the control group (Figure 1E; P < 0.05). Lastly, because insulin deficiency hinders weight gain, the mean body weight of both groups over the monitoring period was compared, and mice in the TAK group gained significantly more weight than mice in the control group (Figure 1F; P < 0.0001). The results demonstrate that simple early TLR4 blockade during islet isolation is sufficient to significantly improve transplant outcomes.
Serum Markers of Islet Stress Are Reduced Posttransplant by Early TLR4 Blockade
To examine islet damage posttransplant between the TAK and control groups, we analyzed serum for MiR-375 and proinflammatory cytokines. Two hundred islets isolated with (TAK, n = 3) or without (control, n = 3) early TLR4 blockade were transplanted into the kidney subcapsular space of diabetic mice. Serum was collected from recipient mice pretransplant and at 4 HR, 1, 2, 3, and 7 days posttransplant for analysis. We detected significantly higher levels of serum MiR-375 in control animals (20.6 ± 7.4-fold) compared with TAK animals (3.0 ± 0.6-fold) only at 4 HR posttransplant (P < 0.0001), but not on days 1, 2, 3, or 7 (Figure 2A).
Next, we analyzed the sera for the proinflammatory cytokine IL-6, neutrophil chemoattractant CXCL1, and the chemokine CXCL10. Serum concentrations of both IL-6 and CXCL1 were significantly higher in control animals than in TAK animals at 1 day posttransplant (Figure 2B, C; both P < 0.05). We also saw a nonstatistically significant increase in the level of IL-6 (P = 0.51) and CXCL1 (P = 0.21) at day 3 posttransplant in control islets. Serum levels of these 2 cytokines remained largely unchanged in TAK animals throughout the sampling period. Levels of serum CXCL10 were significantly higher in control animals at 4 HR posttransplant (P < 0.01) than in TAK animals (Figure 2D), but not at other time points. Graft-bearing kidneys were excised on day 7 for histological analysis. We observed reduced intragraft edema and necrosis in the TAK group, but no differences in macrophage infiltration between the groups (Figure S2, SDC, http://links.lww.com/TP/B583). Overall, these data suggest that a significant amount of islet damage normally observed after transplantation is inhibited by early TLR4 blockade, and that treatment ameliorates systemic inflammatory cytokine reactions.
TLR4 Blockade During Islet Isolation Reduces Expression of Key Inflammatory Proteins
To examine the immediate postisolation effects of early TLR4 blockade, we examined the expression of key proinflammatory genes known to compromise graft function and transplant outcomes (Figure 3A-F). Islets isolated with or without early TLR4 blockade were cultured for 4 hours postisolation in serum-free media to provide sufficient time to upregulate mRNA expression. In TAK islets, tissue factor (TF) upregulation was reduced by ~52% (Figure 3A; P < 0.05), CXCL10 by ~65% (Figure 3B; P < 0.01), intercellular adhesion molecule (ICAM)-1 by ~60% (Figure 3C; P < 0.01), chemokine (C-C motif) ligand (CCL2) by ~79% (Figure 3D; P < 0.01), TLR4 by ~47% (Figure 3E; P = 0.13), and IL-6 by ~63% (Figure 3F; P = 0.08).
We also examined the expression of IL-1β, TNF-α, and IFN-γ at 4 and 24 hours postisolation in control and TAK islets. TAK islets had reduced IL-1β expression at 4 hours (~ − 75%, P < 0.05) and 24 hours (~ − 50%, P = 0.15) after isolation (Figure 3G). TNF-α expression was slightly increased at both timepoints in both groups compared with cultured islets, but there were no statistically significant differences among the groups (Figure 3H). IFN-γ expression was below the detection threshold in all groups. Genes encoding the inflammasome proteins PYCARD, NLRP3, and Caspase-3 were also examined, but observed no significant upregulation or differences (Figure S3 A-C, SDC, http://links.lww.com/TP/B583). We also observed that early TLR4 blockade indeed inhibited inflammation mediated exclusively during, not after, the isolation process as determined by a significant reduction of IL-6 expression in islets treated with TAK-242 during and after islet isolation compared with islets treated only after isolation (Figure S4, SDC, http://links.lww.com/TP/B583). These data demonstrate that early TLR4 blockade effectively reduces the upregulation of many proinflammatory genes mediated by sterile inflammation which are detrimental to transplant outcomes.
Early TLR4 Blockade Reduces MAPK Activation in Islets
The TLR4/MyD88 signaling axis activates the MAPK and NFkB pathways.37 Inhibition of the MyD88 pathway has been shown to promote graft survival and tolerance posttransplant.38 To investigate if these pathways are upregulated following islet isolation, and to see if early TLR4 blockade differentially inhibits the activation of these pathways, we performed Western blot analysis on islets isolated with or without early TLR4 blockade. Islets cultured for 2 days were used for baseline reference. Immediately after isolation, the activation of P65 and MAPK family proteins ERK1/2, P38, and SAPK were significantly upregulated compared with baseline (Figure 4). Activation of ERK1/2 was moderately inhibited in early TLR4 blockade islets compared with control islets at time 0 and remained below control activation levels for 24 hours. Both groups increased slightly at 6 hours, then rapidly became dephosphorylated by 24 hours (Figure 4A, B). Relatively equal levels of P38 activation were observed in both groups immediately after isolation; however, P38 activation in control islets increased at 6 hours postisolation, but decreased in the early TLR4 blockade group (Figure 4C, D). Stress-activated protein kinase activation began trending downward after isolation in both groups but was slightly reduced by early TLR4 blockade immediately postisolation. By 6 hours, SAPK was near undetectable levels in both groups (Figure 4E, F). We saw no major differences in P65 activation between both groups at any time point (Figure 4G, H). These observations support the reduction of inflammatory gene expression seen in Figure 3 and suggest that this reduction is primarily due to reduced MAPK activation during and after isolation.
TLR4 Blockade Rescues Islet Viability Immediately After Isolation
To examine if early TLR4 blockade has any effects on islet viability after isolation, we examined viability immediately and on days 1, 2, 3, and 7 postisolation by Hoechst 33342/propidium iodide staining (Figure 5A). Immediately after isolation, we observed a significant reduction in viability in the control islets compared with TAK islets (P < 0.05), but no significant differences on days 1, 2, 3, or 7 (Figure 5B). We saw no toxic effects of acute TAK-242 treatment, but to determine if long-term treatment impaired viability, we cultured islets in media containing 3 μM or 0.3 μM TAK-242 or 0.01% DMSO. After 5 days of culture, we saw no reductions in viability in any group (Figure S6, SDC, http://links.lww.com/TP/B583).
Finally, we examined if early TLR4 blockade had any impact on islet function. A glucose-stimulated insulin secretion assay was performed on islets isolated with or without early TLR4 blockade at 4 hours (Figure 5C, D) and 24 hours (Figure 5E, F) postisolation. In low (2 mM) glucose at 4 hours, we saw higher basal insulin secretion in TAK islets than in control islets (7.87 ± 0.19 pg/mL vs 5.86 ± 0.15 pg/mL, respectively; P ≤ 0.01) but similar insulin levels in high (20 mM) glucose (27.32 ± 1.81 pg/mL vs 28.66 ± 0.36 pg/mL, respectively; P = 0.54), resulting in a technically lower stimulation index (3.46 ± 0.16 vs 4.90 ± 0.17; P < 0.01). No significant differences were measured at 24 hours for insulin secretion or stimulation index (Figure 5E, F), but the stimulation index for TAK was slightly higher than that of control (4.64 ± 0.44 vs 3.74 ± 0.20, respectively; P = 0.17).
The Protective Effects of Early TLR4 Blockade Are Independent of Enzyme Blend
The enzyme primarily used in this study (collagenase type V) is commonly used in research to isolate islets from murine pancreases. However, its composition differs from clinically used enzyme blends and we detected relatively high levels of endotoxin (~220 EU/mL) at working concentrations. Therefore, to investigate if the reduction in inflammation postisolation would be similarly observed using an enzyme blend related to clinical formulations, islets were isolated as described above except with Liberase enzyme from Roche instead of collagenase type V. After isolation, the islets were analyzed for expression of CCL2 and CXCL10. CCL2 upregulation was reduced by ~66% (Figure S5A, SDC, http://links.lww.com/TP/B583; P = 0.15), and CXCL10 was reduced by ~92% (Figure S5B, SDC, http://links.lww.com/TP/B583; P < 0.05). We did not observe any inhibition of enzymatic activity or islet yield by the addition of TAK-242 during the isolation process. These data demonstrate that the reduction of inflammation by early TLR4 blockade is independent of enzyme blend.
Because collagenase preparations used for islet isolation are produced in Gram-positive Clostridia bacteria, we believe the endotoxin found in the enzyme preparation is lipoteichoic acid, a TLR2/6 agonist. Moreover, lipoteichoic acid from Clostridia histolyticum has been found to be minimally immunogenic.39 Thus, we do not believe this had a major impact on our findings.
Evidence suggests, and we have shown here, that before transplant, islets are significantly stressed and inflamed due to the organ procurement and islet isolation process which may prime islets for dysfunction and apoptosis posttransplant.9,13,15,17 Therefore, early intervention targeting nonvital innate inflammatory pathways, such as TLR4, is an attractive therapeutic option. For our study, we added the TLR4 inhibitor TAK-242 directly to the collagenase used to isolate islets for analysis and transplantation. This compound is attractive for our study because it is immune to degradation by proteases and has been clinically tested for safety.34
In our analysis, the expression of TF in islets was significantly reduced by early TLR4 blockade, as were other key mediators of inflammation and innate immune responses (CXCL10, ICAM-1, CCL2, TLR4, IL-6, IL-1β). Our group demonstrated that CXCL10 is one of the key mediators of islet graft failure.40 CCL2 has also been investigated as a major chemokine implicit in early graft failure.41 The reduction in TLR4 is welcome, since this receptor is commonly upregulated after organ transplant and may act in a positive feedback mechanism to induce further inflammation.42 The significant reduction in IL-1β expression by early TLR4 blockade was not unexpected, because TLR4 activation is known to upregulate this cytokine.43 We observed no reduction in TNF-α expression, which partially explains the lack of P65 inhibition observed in our blots, because this cytokine could be functioning in an autocrine/paracrine fashion to activate NFkB.44 Expression of IFN-γ was not detected, suggesting the MyD88-independent IRF3 pathway does not participate in the early phases of sterile inflammation.
Our analysis of intracellular signaling pathways supports previous work showing that the MAP kinases, as well as P65, are upregulated during and after isolation.6,9,13,45 MAPK phosphorylation, which activate AP-1 family proteins c-Fos and c-Jun, was reduced by early TLR4 blockade, either immediately after isolation or after short culture, compared with untreated islets.46 Our observations support previous work correlating MAPK activation in isolated islets with impaired viability.47 Unexpectedly, no significant differences in P65 activity were observed, suggesting that other receptors, such as TNF receptors, may be the primary regulators of NFkB activation during islet isolation.
Using a syngeneic intraportal islet transplant model and a marginal mass of 150 islets, we were able to demonstrate that early TLR4 blockade alone was sufficient to significantly improve transplant outcomes. Recipients that received treated islets achieved euglycemia at a higher rate with a shorter time to euglycemia, have superior long-term insulin function, and gained more weight compared to recipients that received untreated islets. These results are important in the context of islet transplantations because oftentimes a single clinical auto- or allo-islet transplant is insufficient to reverse diabetes.
Posttransplant, islets are damaged and inflamed, which can be detected and measured in patient serum. We used MiR-375, a beta cell–specific microRNA, as an islet damage biomarker.48,49 Early blockade of TLR4 significantly inhibited serum MiR-375 levels posttransplant. Additionally, key inflammatory proteins (IL-6, CXCL1, CXCL10) were also inhibited. These findings suggest that more of the islet mass is preserved with treatment and that the posttransplant inflammatory response is ameliorated.
In conclusion, our findings demonstrate that early therapeutic intervention is a viable method for reducing tissue inflammation pretransplant, and that TLR4 is a major mediator of sterile inflammation during the islet isolation process and posttransplant. Including TAK-242 in the preservation solution and wash buffers may further increase the efficiency and protective effects of early TLR4 blockade. Additionally, incorporating this compound into perfusion solutions for solid-organ machine perfusion may also be a simple treatment option with profound effects. A TAK-242 concentration of 3 μM was used in this study, based on prior experience with this compound,35 but optimizing the dosage should be a priority for future studies and clinical usage. The results of our study support the use of TAK-242 during the islet isolation process to reduce TLR4-mediated sterile inflammation and to improve islet transplant outcomes.
The authors thank Dr. P. B. Saravanan for technical advice with microRNA isolation and analysis, and A. Rahman and Y. Liu for assistance with endotoxin testing.
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