Synthetic CpG-ODN rapidly enriches immune compartments in neonatal chicks to induce protective immunity against bacterial infections

Thushari Gunawardana,Suresh K Tikoo,Shelly Popowich,Marianna Foldvari,Betty Lockerbie,Susantha Gomis,Ashish Gupta,Shanika Kurukulasuriya,Philip Willson,Khawaja Ashfaque Ahmed,Ruwani Karunarathna,Kalhari Goonewardene

doi:10.1038/s41598-018-36588-6

Thushari Gunawardana, Suresh K Tikoo + Show 10 more

Open Access

https://doi.org/10.1038/s41598-018-36588-6

Copy DOI

Abstract

Oligodeoxynucleotides containing CpG motifs (CpG-ODN) induce innate immunity against bacterial infections. Despite recent advances, how CpG-ODN alone protects against bacterial infections remained elusive. Here, we report for the first time, to our knowledge, that CpG-ODN orchestrates anti-microbial protective immunity by inducing a rapid enrichment of various immune compartments in chickens. In this study, eighteen-day-old embryonated eggs were injected with either 50 µg of CpG-ODN or saline (~n = 90 per group). In the first experiment, four days after CpG-ODN treatment, chicks were challenged subcutaneously with a virulent strain of Escherichia coli (E. coli) and mortality was monitored for 8 days. We found significant protection, and reduced clinical scores in CpG-ODN treated chicks. To gain insights into mechanisms of protection induced by CpG-ODN, first we investigated cytokine expression kinetics elicited by CpG-ODN. The spleen and lung were collected from embryos or chicks (n = 3–4 per group) at 10 time points post-CpG-ODN inoculation. Multiplex gene analysis (interleukin (IL)-1, IL-4, IL-6, IL-10, IL-18, interferon (IFN)-γ, IFN-α, and lipopolysaccharide induced tumor necrosis factor (LITAF), revealed a significantly higher expression of pro-inflammatory cytokines following CpG-ODN treatment compared to the saline controls. In our study, LITAF stands out in the cytokine profiles of spleen and lungs, underscoring its role in CpG-ODN-induced protection. The third experiment was designed to examine the effects of CpG-ODN on immune cell populations in spleen, lungs, and thymus. Flow cytometry analysis was conducted at 24, 48 and 72 hrs (thymus only collected at 72 hr) after CpG-ODN administration to examine the changes in CD4+ and CD8+ T-cell subsets, monocyte/macrophage cell populations and their expression of maturation markers (CD40 and CD86). Flow cytometry data indicated a significant enrichment of macrophages, CD4+ and CD8+ T-cell subsets in both spleen and lungs of CpG-ODN treated embryos and chicks. Macrophages in spleen and lungs showed an upregulation of CD40 but not CD86, whereas thymocytes revealed significantly high CD4 and CD8 expression. Overall, the present study has demonstrated that CpG-ODN provides protection in neonatal chicks against E. coli infection not only by eliciting cytokine responses and stimulating immune cells but also through enriching immunological niches in spleen and lungs.

Highlights

Synthetic oligodeoxynucleotides containing CpG-motifs (CpG-ODNs) are short segments of DNA representing a type of pathogen-associated molecular pattern (PAMP) that stimulate immune cells and trigger immune www.nature.com/scientificreports/
Human toll-like receptors (TLR)-9 and avian TLR-21 are very similar in CpG-ODN motif recognition; and both respond to CpG-ODNs containing GTCGTT motifs, whereas murine but not human TLR-9 recognizes GACGTT motifs[15,16]
The cumulative clinical scores (CCS) of chicks following E. coli challenge with either 1 × 104 or 1 × 105 cfu of E. coli showed that the chicks that received 50 μg CpG-ODN had a significantly lower CCS compared to the saline group (P = 0.03) (Fig. 1b)

Summary

Introduction

Synthetic oligodeoxynucleotides containing CpG-motifs (CpG-ODNs) are short segments of DNA representing a type of pathogen-associated molecular pattern (PAMP) that stimulate immune cells and trigger immune www.nature.com/scientificreports/. Another study reported a significant increase in the heterophil degranulation and oxidative burst following an intraperitoneal administration of CpG-ODN that caused an enhanced resistance to Salmonella Enteritidis infection in neonatal chickens[33]. Several recent studies showed enhanced expression of cytokines and chemokines in response to CpG-ODN in chickens[30,35] Despite these advances, the mechanisms by which CpG-ODN alone provides protection in chickens against bacterial infections are not completely understood. We investigated immunity induced by in ovo administered CpG-ODN including response to E. coli challenge, cytokine kinetics by using QuantiGene Plex (multiplex assay for mRNA gene expression), and effects on the immunological niches[36] in spleen and lungs using flow cytometry

Methods

Results

Conclusion