The Prerequisites for Central Tolerance Induction against Citrullinated Proteins in the Mouse.

To assess the prerequisites for negative selection of peptidylcitrulline-specific T cells in the thymus. In detail, we here analyzed murine medullary thymic epithelial cells for the expression of peptidylarginine deiminases (PAD) and subsequent citrullination.Medullary thymic epithelial cells were sorted, their mRNA was isolated and the expression of Pad genes was analyzed by quantitative PCR. Citrullination was detected by Western Blot in lysates of sorted medullary thymic epithelial cells and histologically by immunofluorescence of thymic thin sections.Pad2 and Pad4 are the main Pad isoforms expressed in mature medullary thymic epithelial cells of the mouse and their levels of expression are comparable to that of insulin (Ins2), another highly and promiscuously expressed protein in the thymus. Citrullination was detected in medullary thymic epithelial cells as shown by Western Blot and immunofluorescence.Even though we here show that the murine thymus harbors the prerequisites for central tolerance to PAD and citrullinated peptides, it remains an open question whether the emergence of peptidylcitrulline-specific T cells and of autoantibodies recognizing citrullinated epitopes is caused by a failure of central or peripheral tolerance mechanisms

Isolation of mature and immature mTECs from murine thymi.

<p>(A) Flow cytometric analyses of thymic cells from NMRI mice, magnetically depleted of CD45-positive cells. TECs are identified as EpCAM-positive and CD45-negative cells (left panel). mTECs were considered as Ly-51^-/low cells (middle panel). According to their CD80-expression, we further discriminated mature mTECs (m-mTEC) and immature mTECs (im-mTEC) (right panel). (B) Re-analyses showed high purities of m-mTEC (right panel) and im-mTEC (middle panel). (C) mRNA expression of <i>Aire</i> and <i>Ins2</i> was higher in mature compared to immature mTEC. <i>Gapdh</i> was used as the reference gene. Boxes show medians and quartiles. Data shown are pooled from C57BL/6, DBA/1, MRL/MpJ and SKG mice.</p

<i>Pad</i>-RNA is expressed in mTECs.

<p>(A) mRNA expression of <i>Aire</i>, the tissue-restricted gene <i>Ins2</i> as well as five <i>Pad</i> isoforms was analyzed in mature mTECs performing qPCR. <i>Gapdh</i> was used as a reference gene. Bars show mean +/- SEM (n = 20). p-values were calculated by t-test. (B) <i>Aire</i> expression is correlated to the expression of <i>Ins2</i> and all five <i>Pad</i> isoforms. Open and closed circles indicate the expression in immature and mature mTEC, respectively. The correlation coefficients were calculated by Spearman test. All p-values were below 0.001. The results shown are pooled data derived from experiments performed with C57BL/6, DBA/1, MRL/MpJ and SKG mice.</p

Citrullination is present in isolated mTEC and within medullary areas of the thymus.

<p>(A) Western blot to detect citrullination (clone F95) and the reference protein (GAPDH) in lysates from FACS sorted mTECs, muscle (positive control) and liver (negative control). The arrow indicates the band present in all samples that was used for a relative expression analysis with respect to GAPDH (arrowhead): muscle: 21-fold, mTEC: 28-fold and liver: 0.75-fold. Numbers within the band of the protein marker lane indicate protein sizes in kDa. (B) Identification of mTEC in thin sections of murine thymi by dual immunofluorescence staining with anti-Aire (green) and anti-cytokeratin 5 (red) or UEA-1 (red). (C) Detection of citrullination in mTEC areas of thymic thin sections. mTECs were stained with UEA-1 (red) and RA-specific epitopes are labeled with an ACPA-high serum mixture consisting of three RA patient's sera (green) (upper right panel). A mix of two age-matched healthy control sera was used as negative control (upper left panel). Staining with a rabbit anti-citrulline serum (green) shows citrullination within mTEC areas (lower right panel). Rabbit normal serum was used as a negative control (lower left panel). All results shown were derived from NMRI thymi.</p