Dual islet transplantation modeling of the instant blood-mediated inflammatory reaction

Abstract

Islet xenotransplantation is a potential treatment for diabetes without the limitations of tissue availability. Although successful experimentally, early islet loss remains substantial and attributed to an instant blood-mediated inflammatory reaction (IBMIR). This syndrome of islet destruction has been incompletely defined and characterization in pig-to-primate models has been hampered by logistical and statistical limitations of large animal studies. To further investigate IBMIR, we developed a novel in vivo dual islet transplant model to precisely characterize IBMIR as proof-of-concept that this model can serve to properly control experiments comparing modified xenoislet preparations. WT and α1,3-galactosyltransferase knockout (GTKO) neonatal porcine islets were studied in nonimmunosuppressed rhesus macaques. Inert polyethylene microspheres served as a control for the effects of portal embolization. Digital analysis of immunohistochemistry targeting IBMIR mediators was performed at 1 and 24 h after intraportal islet infusion. Early findings observed in transplanted islets include complement and antibody deposition, and infiltration by neutrophils, macrophages and platelets. Insulin, complement, antibody, neutrophils, macrophages and platelets were similar between GTKO and WT islets, with increasing macrophage infiltration at 24 h in both phenotypes. This model provides an objective and internally controlled study of distinct islet preparations and documents the temporal histology of IBMIR.

Keywords: animal models: nonhuman primate; animal models: porcine; basic (laboratory) research / science; islet transplantation; islets of Langerhans; xenotransplantation.

Figure 1. The dual islet transplant model

A. Islets of a different phenotype (denoted by colors) are infused into contralateral hemilivers. Islets are separated by the independent right and left portal venous distribution. B. At the experimental time point, the liver is divided by lobe and sectioned by predetermined margins. A small central margin is eliminated to maintain phenotypic purity for analysis. C. Sections then are processed and converted into digital format. D. Digital pathology software analyzes the staining of the image and computes a quantitative value for the positivity of each immunohistochemical stain. In this representative image, red means strong staining positivity, orange medium staining positivity, yellow weak staining positivity, and blue is negative.

Figure 2. Islets preparations are separated by the anatomic distribution of portal venous blood flow

A. PBMC flow cytometry confirming loss of Gal expression in GTKO animals. B. Immunohistochemical staining of galactose-alpha-1,3-galactose was analyzed to determine adequate segregation of islets by hemiliver. At both 1 hour (p=0.05) and 24 hours (p=0.01) staining between islet phenotypes was significantly different. There was no difference in the positivity of staining from 1 to 24 hours within each phenotype (WT p=0.97, GTKO p=0.96). C. Representative Image of α-Gal positive WT islet cluster at 24 hours. D. Representative image of α-Gal negative GTKO islet cluster at 24 hrs.

Figure 3. Antibody binding similar in both islet phenotypes

A. At 1 hour there was no difference in the presence of IgG (p=0.20) or IgM (p=0.36) between WT and GTKO NPIs. B. At 24 hours there was no difference in the presence of IgG (p=0.68) or IgM (p=0.49) between WT and GTKO NPIs. Over 24 hours, there was a significant decrease in IgG (WT: p=0.01, GTKO: p=0.05) and IgM (WT: p<0.01, GTKO: p=0.01) in both phenotypes.

Figure 4. Increasing macrophage infiltration over 24 hours is independent of gal presence

A. At 1 and 24 hours, there is no significant difference in macrophage presence between islet phenotypes (1h p=0.52, 24h p=0.36). However, there was a significant increase in both WT (p<0.01) and GTKO (p<0.01) islets from 1 to 24 hours. B. Representative group of GTKO islets surrounded by macrophages with minimal infiltration at one hour. C. GTKO islets from two different animals demonstrating infiltration of macrophages within the islets clusters by 24 hours.

Figure 5. Temporal summary of WT and GTKO NPIs within the dual islet transplantation model

Findings over 24 hours for WT (a) and GTKO (b) NPIs, an asterisk (*) denotes a statistically significant difference observed from 1 to 24 hours. Both islet phenotypes demonstrated slightly diminished insulin release, decrease in antibody binding, minimal changes in complement deposition and platelet aggregation, a slight decrease in neutrophil infiltration and an increase in macrophage infiltration.

Figure 6. GTKO NPIs initiate coagulation

A. Platelet deposition around microspheres was significantly lower compared to GTKO islets (1hr p=0.03, 24h p=0.02). Over the 24 hour period there was little change in both microspheres (p= 0.13) and GTKO islets (p=0.74). B. Obstructive embolus consisting of multiple microspheres with platelet clot formation at one hour. C. A single microsphere at the proximal end of a sinusoid with minimal platelet involvement at 24 hours. D. GTKO islet cluster with surrounding platelets at one hour.

Figure 7. GTKO NPIs promote neutrophil adhesion

A. Neutrophils are increased in GTKO islets (1h p=0.08, 24h p=0.05). There was no significant change over 24 hours: MS (p=0.15), GTKO (p=0.77). B. Representative histology depicting neutrophils around a microsphere embolus at one hour. C. Neutrophils surrounding a GTKO islet cluster at one hour.

Figure 8. GTKO NPIs stimulate macrophage infiltration

A. Macrophage staining is similar at one hour (p=0.83), at 24 hours GTKO islets have an increased involvement of macrophages over MS that approach significance (p=0.06). There is minimal change in microsphere staining of macrophages (p=0.33) with GTKO islets approaching a significant increase over 24 hours (p=0.09). B. Representative histology of macrophages around a microsphere embolus at one hour. C. Macrophages surrounding a GTKO islet cluster at one hour.