Impact of endogenous IL-27 on innate and adaptive responses during acute and chronic toxoplasmosis

In mice infected with the parasite Toxoplasma gondii the cytokine IL-27 limits the development of T cell mediated pathology but it is unclear if IL-27 acts early to constrain the expansion of pathological T cells or acts late to limits their activities. Analysis of infected mice revealed the production of IL-27p28 by a subset of inflammatory monocytes and sustained IL-27p28 production at sites of acute and chronic infection. Administration of anti-IL-27p28 prior to infection resulted in the development of immune pathology associated with increased levels of macrophage and granulocyte activation and enhanced effector T cell responses. However, IL-27 blockade that started on 4 dpi still resulted in enhanced numbers of parasite specific effector T cells but did not result in adverse systemic pathology. Neutralization of IL-27p28 during the chronic phase of infection did not alter peripheral T cell responses but manifested as enhanced T cell responses in the brain. Thus, IL-27 has distinct suppressive effects that impact innate and adaptive immunity during acute and chronic phases of infection. Mouse splenocytes were prepared by mechanical dissociation of whole spleens, followed by ACK lysis of red blood cells. After staining with antibody cocktails, total CD45+ cells (day 5; n=5 per treatment) and tetramer positive T-cells (day 10; n=5 per treatment) were sorted by FACS directly into lysis buffer. Total RNA was extracted from sorted cells with the RNeasy® Mini Kit (Qiagen, Cat. No: 74104) and adjusted to 20 ng/ul in nuclease free water (Qiagen, Cat. No: 19101). Gene expression profiling on was performed on Affymetrix GeneChip™ Mouse Gene 2.0 ST Arrays (Applied Biosystems, Cat. No: 902118). Processing of RNA samples, hybridization and array scanning were carried out using standard Affymetrix GeneChipTM protocols at the Boston University Microarray and Sequencing Resource (BUMSR). All CEL files were normalized by Robust Multi-array Average (RMA) (Irizarry et al., 2003) and gene expression data were preprocessed by removing unexpressed probes and discarding transcripts with high inter-replicate coefficient of variance. Subsequent analyses (mean expression, fold change, t.test) were performed in R.