Impact of Eimeria tenella Oocyst Dose on Parasite Replication, Lesion Score and Cytokine Transcription in the Caeca in Three Breeds of Commercial Layer Chickens

Eimeria species parasites infect the gastrointestinal tract of chickens, causing disease and impacting on production. The poultry industry relies on anticoccidial drugs and live vaccines to control Eimeria and there is a need for novel, scalable alternatives. Understanding the outcomes of experimental infection in commercial chickens is valuable for assessment of novel interventions. We examined the impact of different infectious doses of Eimeria tenella (one low dose, three high doses) in three commercial layer chicken lines, evaluating lesion score, parasite replication and cytokine response in the caeca. Groups of eight to ten chickens were housed together and infected with 250, 4,000, 8,000 or 12,000 sporulated oocysts at 21 days of age. Five days post-infection caeca were assessed for lesions and to quantify parasite replication by qPCR and cytokine transcription by RT-qPCR. Comparison of the three high doses revealed no significant variation between them in observed lesions or parasite replication with all being significantly higher than the low dose infection. Transcription of IFN-γ and IL-10 increased in all infected chickens relative to unchallenged controls, with no significant differences associated with dose magnitude (p > 0.05). No significant differences were detected in lesion score, parasite replication or caecal cytokine expression between the three lines of chickens. We therefore propose 4,000 E. tenella oocysts is a sufficient dose to reliably induce lesions in commercial layer chickens, and that estimates of parasite replication can be derived by qPCR from these same birds. However, more accurate quantification of Eimeria replication requires a separate low dose challenge group. Optimisation of challenge dose in an appropriate chicken line is essential to maximize the value of in vivo efficacy studies. For coccidiosis, this approach can reduce the numbers of chickens required for statistically significant studies and reduce experimental severity.

Keap1 binds cytokine promoters upon virus infection and moderates their induction by recruiting NFκB p50 and G9a-GLP

Innate immunity requires a balance of positive and negative regulators of cytokine transcription. Keap1 deletion in mice alters innate immunity and inflammation. We investigated the influence of Keap1 on cytokine gene induction by Sendai virus infection in mouse embryo fibroblasts (MEFs). Keap1 bound to the Ifnb1, Tnf and Il6 promoters upon virus infection, and moderated viral induction of their transcription. Keap1 was required for viral induction of NFκB p50 and G9a-GLP lysine methyltransferase binding to these genes. Keap1 formed BiFC complexes with NFκB p50 that were localized to the nuclei in a subset of cells. Nrf2 counteracted viral induction of Keap1 binding to the promoters, and the effects of Keap1 on NFκB p50 and on G9a-GLP recruitment. Lysine methyltransferase inhibitors enhanced viral induction of transcription of the genes that were bound by Keap1 only in MEFs with intact Keap1, and not in Keap1-/- MEFs. They also enhanced NFκB p50 and NFκB p65 recruitment to these genes only in MEFs with intact Keap1, whereas they inhibited G9a-GLP recruitment. The reciprocal effects of Keap1 and of G9a-GLP lysine methyltransferase activity on chromatin binding by each other constitute a feedback circuit that moderates viral induction of cytokine transcription.SummaryVirus infection induces Keap1 binding to cytokine promoters, which recruits NFκB p50 and G9a-GLP and moderates their transcription.

Intracellular antibody signalling is regulated by phosphorylation of the Fc receptor TRIM21

Cell surface Fc receptors activate inflammation and are tightly controlled to prevent autoimmunity. Antibodies also simulate potent immune signalling from inside the cell via the cytosolic antibody receptor TRIM21, but how this is regulated is unknown. Here we show that TRIM21 signalling is constitutively repressed by its B-Box domain and activated by phosphorylation. The B-Box occupies an E2 binding site on the catalytic RING domain by mimicking E2-E3 interactions, inhibiting TRIM21 ubiquitination and preventing immune activation. TRIM21 is derepressed by IKKβ and TBK1 phosphorylation of an LxxIS motif in the RING domain, at the interface with the B-Box. Incorporation of phosphoserine or a phosphomimetic within this motif relieves B-Box inhibition, promoting E2 binding, RING catalysis, NF-κB activation and cytokine transcription upon infection with DNA or RNA viruses. These data explain how intracellular antibody signalling is regulated and reveal that the B-Box is a critical regulator of RING E3 ligase activity.