Autophagy in antigen-presenting cells results in presentation of citrullinated peptides to CD4 T cells

Abstract

Antibody responses to citrullinated self-proteins are found in autoimmunities, particularly in rheumatoid arthritis, where they serve as a diagnostic indicator. We show here that processing of the protein hen egg-white lysozyme (HEL) resulted in citrullination of peptides presented on class II MHC molecules by antigen-presenting cells. The presentation of the citrullinated peptides but not of the unmodified peptides was associated with autophagy. Dendritic cells (DCs), macrophages, and thymic DCs presented citrullinated peptides constitutively. Their treatment with 3-methyladenine (3MA) blocked presentation of citrullinated HEL peptides, but presentation of unmodified peptides was not affected. Presentation of citrullinated peptides was not detected on B cells or B lymphoma cells under normal culture conditions. In B cells, engagement of the B cell antigen receptor was required for presentation of the citrullinated peptides, also inhibited by 3MA. B lymphoma-expressing HEL cells presented citrullinated peptides only after brief serum starvation. This presentation was reduced by 3MA or by reduction in Atg5 expression. Presentation of the unmodified peptides was not changed. The findings indicate a linkage between autophagy and autoreactivity through the generation of this neo-epitope.

Figure 1.

Presentation of citrullinated peptides by freshly harvested APCs. (A–F) IL-2 production from CD11c⁺ cells isolated from thymi (A and B), spleen (C and D), and splenic CD19 ⁺ cells (E and F). A, C, and E show response to the 48–62-Cit61 peptide by Granny, whereas B, D, and F show response to the unmodified 48–62 by 3A9. (G and H) The response to CD11c⁺, CD11b⁺ cells purified from the popliteal lymph nodes of mice 24 h after immunization with 10 nmol HEL and tested against (G) Granny or (H) 3A9 is shown. Data from each panel are representative of at least two independent experiments, and each data point represents duplicate wells. At least three mice were used in each experiment. Error bars indicate SEM.

Figure 2.

C3.F6.mHEL cells present citrullinated peptides after serum starvation–induced autophagy. (A–C) T cell responses to C3.F6.mHEL cultured in DME supplemented with 5% FCS (D5F), no serum (DOF), or no serum in the presence of 10 mM 3MA (D0F 3MA) for 4 h; the culture medium was then changed to 5% FCS, and the T cell hybridomas Granny (A and B) or 3A9 (C) were added. (D) Western blot of actin and LC3II in C3.F6.mHEL cultured in D10F, D0F, and D0F with 3MA. Data are representative of three independent experiments; each data point represents duplicate wells. Targeted knockdown of Atg5 expression inhibited citrullination of HEL peptides. (E–H) Responses of T cells to C3.F6.mHEL cells expressing either shRNA targeting Atg5 expression (E and G) or luciferase expression (F and H). Cells were cultured in media supplemented with 5% FCS, 0.5% FCS, or no FCS for 4 h and then replaced with media supplemented with 5% FCS. (I) Western blot of actin, Atg5 levels, and LC3II in the treated C3.F6.mHEL cells shows that Atg5-deficient cells had reduced levels of LC3II conversion after serum starvation. Data are representative of three independent experiments. Error bars indicate SEM.

Figure 3.

PAD expression in APCs alone is not sufficient for citrullination of HEL; PAD activity can be detected in autophagosomes. (A and B) PAD2 (A) and PAD4 (B) mRNA was measured by RT-PCR and quantified using DNA standards. Data are presented as the mean of quadruplicate reaction replicates and are representative of at least two independent experiments. (C) PAD activity was measured biochemically in lysates of C3.F6.P4 cells after the addition of tetracycline. Data are the mean of triplicate reactions and are representative of three independent experiments. (D) Presentation of HEL or HEL peptide to Granny by C3.F6.P4 with or without tetracycline to induce PAD4 expression in normal culture conditions or after culture in DME without FCS (SS in the panel). Data are representative of three independent experiments. (E) PAD activity as measured by conversion of an artificial substrate was assessed in fractions of elicited PECs. The fractions are represented with numbers as follows: 1, whole cell lysate; 2, postnuclear supernatant; 3, nuclei; 4, second pellet enriched in mitochondria and peroxisomes; 5, light membrane fraction; 6, complex heavy fraction enriched in the ER; and 7, final autophagosome pellet. The negative control (Neg) contained substrate without protein. The data presented are pooled from three independent experiments. 20–60 mice were used in each experiment. (F) The fractions were analyzed by Western blot for LC3II enrichment. The data are representative three independent experiments. Error bars indicate SEM.

Figure 4.

Treatment with 3MA inhibits presentation of citrullinated peptides by DCs or PECs. (A and B) Presentation of HEL to Granny (left panel labeled 48-62Cit61) or 3A9 (right panel labeled 48–62) with and without 10 mM 3MA to DCs (A) or PECs (B). (C and D) Presentation of the synthetic peptide with and without 3MA to DCs (C) or PECs (D). (E–H) Presentation of 30 µM HEL or 3 µM of the synthetic peptide to DCs (E and G) or PECs (F and H) examining different concentrations of 3MA. (I and J) Presentation of citrullinated peptide was examined after a 1-h culture in Krebs-Ringer bicarbonate buffer (KRB) after a short 2-h pulse with HEL in DCs (I) or PECs (J). Data are representative of two to three experiments. Error bars indicate SEM.

Figure 5.

BCR engagement induces presentation of citrullinated peptides by primary B cells. (A and B) Presentation by CD19⁺ cells isolated from spleens of mHEL mice. (A) Effects of various concentrations of anti-IgM or anti-Ig on Granny ± 3MA. (B) The same as in A but on 3A9 ± 3MA. (C) Presentation of HEL by CD19⁺ cells from anti-HEL transgenic mice or B10.BR mice to Granny. (D) The same as in C but to 3A9. (E) The effect of 3MA on presentation by CD19⁺ cells from anti-HEL transgenic mice after processing HEL to Granny. (F) The same as in E but to 3A9. (G) Presentation of HEL by B cells from B10.BR mice to Granny after treatment with 40 µg/ml anti-IgM ± 3MA. (H) The same as in G but treating the cells with anti-IgG ± 3MA. (I and J) Western blots of actin and LC3II from mHEL B cells cultured overnight with anti-Ig (I) or anti-HEL B cells cultured with 30 µM HEL ± 3MA (J). (K) B cells from GFP-LC3 mice were labeled on ice with anti-IgM and fixed immediately or after 1 h at 37°C and stained to label MHC class II. All data are representative of at least two independent experiments.