Innate immune phenotype variation in bovine health and disease

Abstract

Infectious diseases are estimated to cost the global agriculture industry $300 billion annually and present significant challenges to animal welfare. Disease burden in cattle and other livestock species also threaten human health, with 75% of new emerging diseases zoonotic in nature. Currently, there is an overreliance on antimicrobials to treat infectious disease, with antimicrobial resistance predicted to result in 10 million deaths globally every year by 2050. New approaches are dependent on an improved understanding of the bovine immune system, which underpins all health and production traits of importance in agriculture. The innate immune response is the ‘non-specific’ component of the immune system and determines both susceptibility to infection and immunopathology. However, the natural variation and boundaries of healthy bovine innate immune activity remain unclear, as do the immune changes associated with early stage disease. The innate immune response during the early life of calves is of particular interest, as this period is associated with elevated disease susceptibility and mortality. While underdeveloped adaptive immune mechanisms have been described, innate immune responses associated with early life disease susceptibility remain unexplored. Here, we hypothesize variation in specific innate immune phenotypes underpin bovine health, pathology and disease susceptibility in early life. Our aims were to (1) optimise a standardised assay that can reliably capture variation in bovine innate immune phenotypes, (2) apply this assay to demonstrate the inter-individual and temporal variation of innate immune phenotypes associated with early life, and (3) identify innate immune phenotypes associated with disease pathology and susceptibility in young calves. Health and disease associated innate immune phenotypes can ultimately enable preventative health assessments to improve diagnostics, prognostics and reduce dependence on antimicrobials. Firstly, our ability to reliably measure innate immune phenotypes in cattle is currently limited by the lack of standardized assays. Here, we optimised a whole blood pathogen-associated molecular pattern (PAMP) stimulation system called ImmunoChek. By minimizing cell manipulation, our low-blood volume, standardized ex-vivo system reduces the potential for artefactual results and enables comparative temporal immune response analysis in cattle. ImmunoChek successfully captured biological variation in innate cytokine (IL-1β and IL-6) and chemokine (IL-8) responses to Gram-negative (LPS), Gram-positive (Pam3CSK4) bacterial or viral (R848) PAMPs across a 4-month time window in adult cattle (n=10). Significant and repeatable patterns of inter-individual variation in cytokine and iv chemokine responses, as well as consistent high innate immune responder individuals were identified at both baseline and induced levels. This standardized assay can capture variation in bovine innate immune phenotypes in a high-throughput manner. We then characterized temporal variation in innate immune phenotypes during early life of calves (n=10) using ImmunoChek. Increased inflammatory responses (IL-1β and IL- 8) to PAMPs were found from 3 to 5 months of age. Next, we carried out comparative analysis of innate immune phenotypes between this cohort and young calves that were supplemented with vitamin D from birth to 7 months of age (n=12). Calves are vitamin D insufficient in early life which may predispose them to infection and disease. In 24-hour unstimulated blood immune cells, vitamin D supplementation increased gene expression of type I interferons, chemokines and pattern recognition receptors, while decreasing IL-1 and inflammasome gene expression. In response to PAMPs, vitamin D supplementation increased IL-1 and inflammasome gene expression to LPS and Pam3CSK4 and IL-1β protein production to Pam3CSK4 and R848. In contrast, vitamin D supplementation decreased both chemokine gene expression in response to R848 and IL-8 protein expression in response to LPS, Pam3CSK4 and R848. Pan-genomic bulk RNA-sequencing of whole blood was carried out 1 month after the cessation of vitamin D supplementation to determine if the immunomodulatory effects of vitamin D persisted. This revealed 36 genes differentially expressed between groups. Vitamin D supplementation significantly increased gene expression of nine immunoglobulin domain genes. Overall, we identified early life changes in innate immune responses as well as a significant effect of vitamin D supplementation that could have important implications for disease susceptibility in cattle. Finally, using ImmunoChek we investigated the innate immune phenotypes in young calves naturally infected with bovine respiratory disease (BRD) [n=41], with healthy controls recruited from the same farms (n=20). BRD is the leading cause of mortality and compromised animal welfare in young calves. Calves were diagnosed with BRD using clinical assessment procedures thoracic ultrasonography scoring (TUS) and Wisconsin health scoring (WHS). Hyper-induction of IL-6 protein expression in response to PAMPs was found in calves with BRD, particularly in response to Pam3CSK4. Receiver operator characteristic (ROC) analysis highlighted IL-6 responses to Pam3CSK4 as a potential diagnostic for BRD. An optimal IL-6 threshold of >1780pg/ml had a 71% true positive rate and 5% false positive rate in diagnosis of BRD. Gene expression analysis in a subset of stimulations revealed calves with BRD had significantly increased expression of IL-6 receptor gene IL6R, IFN-γ receptor gene IFNGR1, and vitamin D pathway transcription factor RXRA in response to Pam3CSK4. Calves with BRD also had significantly decreased expression of IL-1 family genes IL1A and IL1RN, and vitamin D pathway enzyme CYP27B1 in response to Pam3CSK4. These results show altered immune responsiveness to PAMP stimulation is associated with BRD pathology and identified induced IL-6 as a potential diagnostic biomarker for BRD. Overall, ImmunoChek assay demonstrated important inter-individual and temporal variation in innate immune phenotypes in bovine health and disease. This study has shed light on the early life changes in innate immune responses in calves. We characterised a role for vitamin D supplementation in bovine health via modulation of innate immune signalling. Supplementation with vitamin D may offer a low cost mechanism to support the developing immune system and reduce neonatal mortality and morbidity. Finally, we identified that hyper-induction of IL-6 is an innate immune phenotype associated with BRD pathology and potentially susceptibility. ImmunoChek also has considerable potential for future high throughput innate immune phenotyping in cattle.