Analysis of host immune parameters during M. avium infection

Abstract

The goal of this study was to establish and optimize methods to analyze effector cytokine levels and other parameters in murine tissue and plasma samples. These methods should then be used to study the role of regulatory T (Treg) cells and innate immune protein lipocalin 2 (Lcn2) in the Mycobacterium avium (M. avium) infection model established by the group. We also wanted to identify factors that might contribute to explain why the number of Treg cells decreases in the mouse model 10-20 days post M. avium infection and why Lcn2 defective mice seem to have lower Treg cell numbers than wild-type mice.

We established and optimized ELISA assays for measurement of IFN-γ, TNF and TGF-β1 effector cytokine levels from spleen, liver and plasma samples. Further, we tested methods to find the optimal protocol for analyzing murine spleen and liver tissue by quantitative real-time PCR (qPCR). These techniques were used to analyze host response parameters in M. avium infected C57BL/6 wild-type (WT) and Lcn2 knock-out (KO) mice, undepleted and depleted in the Treg cell population.

ELISA analysis of tissue and plasma samples during M. avium infection revealed a strong Th1 response with a maximum of IFN-γ effector cytokine production 2 weeks post infection, but did not show clear differences in effector cytokine production between the different groups of mice. With the qPCR analysis we could confirm the ELISA results and identify factors differentially regulated in spleen and liver as well as between the groups during M. avium infection. M. avium infection in C57BL/6 mice resulted in upregulation of pro-inflammatory cytokines and chemokines. Our results might also indicate higher inflammation in Lcn2 KO than in WT mice. Further we detected downregulation of Treg cell attracting chemokine CCL22 in infected WT mice compared to uninfected control mice, pointing at less recruitment of Treg cells in infected mice. The findings made in this study could thus be valuable in further revealing the complex and only partly understood host response to M. avium infection in particular and mycobacterial infections in general.