Supplementary information for the article: Dimitrijević, M.; Arsenović-Ranin, N.; Bufan, B.; Nacka-Aleksić, M.; Kosec, D.; Pilipović, I.; Kotur-Stevuljević, J.; Simić, L.; Sopta, J.; Leposavić, G. Sex-Based Differences in Monocytic Lineage Cells Contribute to More Severe Collagen-Induced Arthritis in Female Rats Compared with Male Rats. Inflammation 2020, 43 (6), 2312–2331. https://doi.org/10.1007/s10753-020-01302-0.

Abstract

Supplementary Fig. 1 Sex differences in the clinical and histological presentation of CIA. (a) A line graph indicates daily arthritic score (mean ± SEM) from the 12th to the 39th day post-immunization (d.p.i.) in male (n = 9) and female (n = 10) CIA rats. Mann–Whitney U test: * p ≤ 0.05, from the 17th to the 39th d.p.i. (b) Line graph indicates daily arthritic score (mean ± SEM) from the 13th to the 21st day post-immunization (d.p.i.) in male and female CIA rats. Mann–Whitney U test: n = 8 rats/sex. * p ≤ 0.05. Photographs show representative arthritic joints (arrows) of hind paws from male and female CIA rats. (c) Photomicrographs of HE-stained sections of paraffin-embedded joints from male and female CIA rats show replacement of the normal bone marrow cell populations by inflammatory cells. In females, numerous multinuclear giant cells (red arrows) are present as opposed to male CIA rats. Original magnification × 400. The bar indicates 100 μm (PNG 2723 kb) Supplementary Fig. 2 Fluorescence minus one controls for flow cytometry analysis of CD11b/CCR2/CX3CR1 staining of splenocytes. For setting cutoff boundaries, gates were controlled using fluorescence minus one (FMO) controls obtained by omitting a single antibody from the labeling antibody cocktail. Flow cytometry dot plots represent FMO controls without anti-CX3CR1 or anti-CCR2 Abs within CD11b+ splenocytes (gated as shown in Fig. 3) isolated from CIA rats on the 21st day post-immunization (PNG 98 kb). Supplementary Fig. 3 Fluorescence minus one controls for flow cytometry analysis of CD11b/CD43/CCR2/CX3CR1 staining of peripheral blood cells. For setting cutoff boundaries, gates were controlled using fluorescence minus one (FMO) controls obtained by omitting a single antibody from the labeling antibody cocktail. Flow cytometry dot plots represent FMO controls without (upper) CD43 mAb within CD11b+ peripheral blood cells (gated as shown in Fig. 4a) and (lower) anti-CX3CR1 or anti-CCR2 Abs within CD11b+CD43+ peripheral blood cells isolated from CIA rats on the 21st day post-immunization (PNG 157 kb). Supplementary Fig. 4 Sex differences in the activation of Th cells, Th17 cell function, and frequency of CD40+CD11b+ antigen presenting cells in draining lymph nodes from CIA rats, popliteal draining lymph nodes (DLNs) were retrieved from male and female CIA rats on the 21st day post-immunization. (a) Scatter plots with bar indicate the frequencies of activated Th cells (CD25+Foxp3-CD4+) and Th17 cells (IL-17+CD4+TCRαβ+) in DLNs from CIA rats and the concentration of IL-17 in supernatants of collagen type II-stimulated and unstimulated (medium) DLN cell cultures from male and female rats (see MATERIAL AND METHODS). Linear graph shows the correlation between the frequency of activated Th cells (CD25+Foxp3-CD4+) and the frequency of Th17 cells (IL-17+CD4+TCRαβ+) in DLNs from CIA rats. Pearson’s r value is shown in the graph. (b) Representative flow cytometry dot plots show (upper) CD11b staining and (lower) CD40 vs CD11b staining of DLN cells from male and female rats. Number indicates percent in the region. Scatter plots with bar indicate the frequency and the number of (upper) CD11b+ cells and (lower) CD40+CD11b+ cells in DLNs of male and female rats. The Number indicates percent in the region. Results are expressed as mean ± SEM. (c) The linear graph shows the correlation between the frequency of activated Th cells (CD25+Foxp3-CD4+) and the frequency of CD40+CD11b+ cells in DLNs from CIA rats. Pearson’s r value is shown in the graph. n = 8 rats/sex. * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001 (PNG 590 kb). Supplementary Fig. 5 Gating strategy for activated Th cells and Th17 cells, popliteal draining lymph nodes (DLNs) were retrieved from CIA rats on the 21st day post-immunization. Flow cytometry dot plots show gating strategy for (a) activated The cells (CD25+Foxp3-CD4+) and (b) Th17 cells (IL-17+CD4+TCRαβ+) (PNG 101 kb).