| dc.description.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. | en |
