Cytokine gene transcription in the trachea, Harderian gland, and trigeminal ganglia of chickens inoculated with virulent infectious laryngotracheitis virus (ILTV) strain

Abstract

The objective of this study was to determine how cytokine transcription profiles correlate with patterns of infectious laryngotracheitis virus (ILTV) replication in the trachea, Harderian gland, and trigeminal ganglia during the early and late stages of infection after intratracheal inoculation. Viral genomes and transcripts were detected in the trachea and Harderian gland but not in trigeminal ganglia. The onset of viral replication in the trachea was detected at day one post-infection and peaked by day three post-infection. The peak of pro-inflammatory (CXCLi2, IL-1β, IFN-γ) and anti-inflammatory (IL-13, IL-10) cytokine gene transcription, 5 days post-infection, coincided with the increased recruitment of inflammatory cells, extensive tissue damage, and limiting of virus replication in the trachea. In contrast, transcription of the IFN-β gene in the trachea remained unaffected suggesting that ILTV infection blocks type I interferon responses. In the Harderian gland, the most evident transcription change was the early and transient upregulation of the IFN-γ gene at 1 day post-infection, which suggests that the Harderian gland is prepared to rapidly respond to ILTV infection. Overall, results from this study suggest that regulation of Th1 effector cells and macrophage activity by Th1/2 cytokines was pertinent to maintain a balanced immune response capable of providing an adequate Th1-mediated protective immunity, while sustaining some immune homeostasis in preparation for the regeneration of the tracheal mucosa.

Immune Responses in the Eye-Associated Lymphoid Tissues of Chickens after Ocular Inoculation with Vaccine and Virulent Strains of the Respiratory Infectious Laryngotracheitis Virus (ILTV)

“…Briefly, immediately after the homogenization of CALT and HG samples in 1 mL of TRIzol (Thermo Fisher, Waltham, MA, USA), 200 μL of chloroform was added and the samples were centrifuged at 4 °C for 15 min at 12,000× g . Total RNA was further purified from the upper aqueous phase using the RNeasy kit (QIAGEN, Valencia, CA, USA) as previously described [12]. To eliminate DNA contamination, the eluted RNA was treated with TURBO DNA-free™ (2 to 4 units/per reaction) (Ambion, Carlsbad, CA, USA) for 30 min at 37 °C.…”

“…To assess the purity and concentration of RNA per sample, optical density ratios (260/280 and 260/230) were obtained using the NanoDrop™ 2000c (Thermo Fisher Scientifics, Waltham, MA, USA). Previous to cDNA synthesis, a real time PCR reaction that amplifies the chicken β-actin gene was performed to ensure the absence of residual DNA in the eluted RNA using primers previously described [12]. The β-actin gene amplification reaction was performed in a 20 μL volume—10 μL of SYBR ® Select Master Mix (2X) (Life Technologies, Carlsbad, CA, USA), 1 μL of 5 μM of each primer, 5 μL of template and 3 μL of nuclease free water.…”

“…Primers, amplification reaction parameters and efficiencies for each reaction are shown in Table 1. The IL-12p40 , IFN-γ and Granzyme A genes relative transcription changes were determined as compared to the mock-inoculated group of chickens, using the chicken β-actin transcript as an endogenous control, and presented as median fold change (log 10 2 −ΔΔ C t ) and 95% confidence interval [12].…”

“…More recent studies suggest that the local inflammatory processes contribute to the pathology of the disease, and eventually modulate the responses of the adaptive immune system to facilitate viral replication [10]. Furthermore, ILTV infection appears to down-regulate type I interferon transcription and delays the early transcription of inflammatory genes to favor initial virus replication in the trachea [11,12].…”

Host Responses Following Infection with Canadian-Origin Wildtype and Vaccine Revertant Infectious Laryngotracheitis Virus

“…The experiments using bursectomized chickens showed that ILTV antibodies are not protective [ 28 , 29 ], and it has been demonstrated in thymectomized chickens that cell-mediated immunity is critical against ILT [ 30 ]. Additionally, a correlation has been observed between increased pro-inflammatory and anti-inflammatory cytokine gene transcription and enhanced inflammatory cell recruitment, significant tissue damage, and decreased ILTV replication in the trachea [ 32 ].…”