Immunological stimuli change expression of genes and neutrophil function in fathead minnow Pimephales promelas Rafinesque

Fathead minnows Pimephales promelas were exposed to lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid [poly(I:C)] to observe immunological responses during simulated bacterial and viral challenge at the level of gene expression and granulocyte function. Complementary DNA libraries were created from LPS- and poly(I:C)-treated fish and c. 5000 expressed sequence tags (ESTs) were sequenced. The ESTs were subjected to BLASTx analysis and 1500 genes were annotated, grouped by function and 20 immune genes were selected for expression studies by real-time PCR. Lipopolysaccharide treatment significantly downregulated expression of interferon regulatory factor 2 binding protein 1 (nine-fold), Chemokine (C-X-C motif) ligand 12a (three-fold) and TNF-related apoptosis-inducing ligand, TRAIL (two-fold). In poly(I:C)-treated fish, a significant upregulation was observed for IFN-inducible and antiviral proteins belonging to the family of Mx proteins (73-fold) and chemokine CCL-C5a (28-fold). Blood neutrophil count was significantly increased in poly(I:C)-treated fish at 24 and 48 h post-injection. Neutrophil extracellular trap release and respiratory burst of kidney granulocytes were suppressed in poly(I:C)-treated fish, while degranulation of primary granules was not affected significantly by the treatment. The changes in gene expression and neutrophil function in P. promelas exposed to LPS and poly(I:C) support the use of this species as an alternative model for studies of pathogen effects on the innate immune system of fishes.     

Development of a genotype‐by‐sequencing immunogenetic assay as exemplified by screening for variation in red fox with and without endemic rabies exposure

Pathogens are recognized as major drivers of local adaptation in wildlife systems. By determining which gene variants are favored in local interactions among populations with and without disease, spatially explicit adaptive responses to pathogens can be elucidated. Much of our current understanding of host responses to disease comes from a small number of genes associated with an immune response. High‐throughput sequencing (HTS) technologies, such as genotype‐by‐sequencing (GBS), facilitate expanded explorations of genomic variation among populations. Hybridization‐based GBS techniques can be leveraged in systems not well characterized for specific variants associated with disease outcome to “capture” specific genes and regulatory regions known to influence expression and disease outcome. We developed a multiplexed, sequence capture assay for red foxes to simultaneously assess ~300‐kbp of genomic sequence from 116 adaptive, intrinsic, and innate immunity genes of predicted adaptive significance and their putative upstream regulatory regions along with 23 neutral microsatellite regions to control for demographic effects. The assay was applied to 45 fox DNA samples from Alaska, where three arctic rabies strains are geographically restricted and endemic to coastal tundra regions, yet absent from the boreal interior. The assay provided 61.5% on‐target enrichment with relatively even sequence coverage across all targeted loci and samples (mean = 50×), which allowed us to elucidate genetic variation across introns, exons, and potential regulatory regions (4,819 SNPs). Challenges remained in accurately describing microsatellite variation using this technique; however, longer‐read HTS technologies should overcome these issues. We used these data to conduct preliminary analyses and detected genetic structure in a subset of red fox immune‐related genes between regions with and without endemic arctic rabies. This assay provides a template to assess immunogenetic variation in wildlife disease systems.     

Chemical complementarity between immune receptors and cancer mutants,independent of antigen presentation protein binding,is associated with increased survival rates

Uterine cancer has been associated with a T-cell immune response that leads to increased survival. Therefore, we used several bioinformatics approaches to explore specific interactions between T-cell receptor (TCR) and tumor mutant peptide sequences. Using endometrioid uterine cancer exome files from the The Cancer Genome Atlas database, we obtained tumor resident V-J recombinations for the T-Cell Receptor alpha gene (TRA). The charged-based, chemical complementarity for each patient''s LRP2 or TTN mutant amino acids (AAs) and the recovered, TRA complementarity determining region-3 (CDR3) sequences was calculated, allowing a division of patients into complementary and noncomplementary groups. Complementary groups with TTN mutants had increased disease-free survival and increased expression of complement genes. Furthermore, the survival distinction based on CDR3-mutant peptide complementarity was independent of programmatically assessed HLA class II binding and was not observable based on the CDR3 AA chemical features alone. The above approach provides a potential, highly efficient method for identifying TCR targets in uterine cancer and may aid in the development of novel prognostic tools.