Relatively large expression differences were detected in PBMC between two strains of rats commonly used in models of organ transplantation. Conversely, only a small number of differentially expressed genes in heart tissue were related to rat strain. The larger effect of strain on gene expression in PBMC compared to heart tissue may in part reflect the abundance of strain-specific, self-recognition molecules that are expressed by lymphocytes. As such, strain specifically affected genes involved with the immune response, cell motility, and cell death, functional categories that might be misconstrued as ACR-related. Moreover, the magnitude of the strain effect in PBMC (FC ≥ 3 for 265 probesets) substantially obscured the gene expression signature of rejection. Potential markers specific to ACR could only be identified after measuring and accounting for the strain effect. Similar variability attributable to genotypic heterogeneity has also been documented in human populations [16
] and therefore has major implications for experimental design and power calculations in patient biomarker studies.
Approximately 85 to 95% of human genetic variation is attributable to individual heterogeneity within a population while the remaining 5 to 15% can be ascribed to differences between populations [16
]. In addition to external influences and epigenetic factors, this background genetic variation contributes to differences in gene expression [15
] that may add unwanted noise to results from high – throughput methodologies such as microarrays. Genotypic effects in human PBMC were found to significantly alter the expression of more than 300 transcripts [38
]. Major histocompatibility complex-associated and interferon-regulated genes were among those most affected by genotype in human PBMC preparations. Investigations performed in inbred animals reduce this source of variability and thereby serve as a "best case" scenario or "proof of principle." However, genetic background may still negatively impact the results and interpretation of animal experiments, a well recognized concern in gene targeting studies [39
]. At particular risk are investigations that require hybrid animals or multiple animal strains.
Natural variation in gene expression has been well documented among laboratory strains of fruit flies [19
] and mice [20
]. Expression profiling of brain [20
], spleen [21
], and liver [22
] have determined that 1 to 3% of mouse transcripts are significantly affected by animal strain. In addition to these baseline effects, the gene expression response to seizure was shown to be significantly different comparing the brains of two inbred strains of mice [20
]. Even more relevant to immunity and transplant medicine, bone marrow derived macrophages from 5 mouse strains displayed unique transcriptional phenotypes in response to lipopolysaccharide challenges [40
]. Allogeneic animal transplant models typically employ two immunologically distinct strains to serve as donor and recipient. Therefore background genotypic effects on transcript abundance have a real potential to confound the search for biomarkers and new therapeutic targets. In the current study, the impact of rat strain on PBMC gene expression was unexpectedly large and primarily affected transcripts associated with the immune response, effects that could be misinterpreted as ACR-related. These results underscore the importance of experimental designs and analytical approaches in expression profiling studies that control for strain effects in animal models and genotypic heterogeneity in patient populations.
Measuring and then adjusting post hoc
for large strain effects on gene expression identified 30 genes with the potential to be differentially regulated during rejection in circulating PBMC. A thematic analysis of this list suggested biological plausibility. However, homologues of only two of these genes, S100 calcium binding protein A9 and granzyme B have been previously linked to cardiac rejection in humans [8
]. Two S100 like binding proteins, myeloid related protein 8 (MRP8) and MRP14, have been shown to be increased in the serum of patients relatively early in acute renal allograft rejection [41
]. Granzyme B has also been found to be over-expressed in the peripheral blood of patients with acute renal allograft rejection [42
]. Another potentially rejection-related gene from the current study, high mobility group box 1 (Hmgb1) has been investigated in a previous animal study of acute rejection [43
]. In a murine cardiac transplant model, Hmgb1 expression was significantly increased in allogeneic compared to isogeneic transplants. Increased expression of Hmgb1 in allografts was associated with active secretion of Hmgb1 by infiltrating immune cells. Blockade of extracellular Hmgb1 significantly delayed acute allograft rejection [43
This study has several limitations. PBMC from transplanted animals differed by both transplant type (isogeneic vs. allogeneic) and animal strain. If strain and ACR interacted in a non-additive manner then the selection of rejection-related genes might have been unreliable. These genes require confirmation using an experimental design that avoids the need for cross strain comparisons. Likewise, it would be prudent to validate any results in more than one rat strain. Robust genes that identify rejection within multiple rat strains might be more likely to serve as reliable biomarkers in patients with their inherent heterogeneity. Another potential limitation of our analysis was the assumption that systemic effects of ACR on native hearts were minimal and that expression differences in native hearts were almost entirely due to strain. Nonetheless, the heart tissue strain effect, examined only in native hearts, was relatively small. Finally, in our study and others, some gene expression differences attributed to ACR in heart tissue may reflect the detection of strain-associated gene expression attributable to recipient lymphocytes that have infiltrated the donor allograft.
Despite these limitations, our results have implications for recent efforts to identify biomarkers of ACR in peripheral blood [8
]. Human genetic variation has a substantial impact on gene expression independent of exogenous factors and conditions [15
]. Notably, the influence of genetic variation on transcript abundance appears to be particularly strong in cells of the immune system [17
]. Baseline genetic differences between human transplant recipients may make it difficult to identify a universally applicable set of biomarkers for noninvasively detecting acute cellular rejection. Complex interactions between genetic background (polymorphisms), comorbidities, and immunosuppressive regimens may further degrade the performance characteristics of biomarkers in this heterogeneous patient population.