*2.2. The Impact of CIA and Bone Injury in Secondary Lymphoid Organs*

Next, we wanted to ascertain if CIA and its combination with a bone defect correlated with acute systemic changes in secondary lymphoid organs. Spleen from CIA and CIA+FD groups were collected three days after bone injury and found to be enlarged when compared to control, with a significant increase in weight for CIA+FD animals (Figure 2A). Cell proliferation was assessed by Ki-67 staining, and was also increased in both CIA and CIA+FD groups, but a statistically significant increase was only observed for the CIA group (Figure 2B). Lymph nodes draining the hind paws were also collected and found to be enlarged in CIA and CIA+FD groups (Figure 2C). Cell proliferation in lymph nodes was quantified across the different animals and found significantly increased in CIA animals when compared to controls (Figure 2D). Overall, these results support that spleen and lymph nodes are responding to CIA induction with increased cell proliferation.

To further determine the impact of CIA and CIA+FD in systemic immune cell populations, we performed multicolor flow cytometry analysis of cells from blood, draining lymph nodes and spleen. The gating strategy is illustrated in Supplementary Figure S2. Results obtained are summarized in Figure 3 and show significant changes in the proportion of lymphoid and myeloid cells in blood, draining lymph nodes and spleen. Concerning the lymphoid lineage populations (Figure 3A), we observed a significant increase in the percentage of B lymphocytes in blood and lymph nodes from CIA animals relative to control animals, but no differences were observed in spleen. T cell percentage was significantly decreased in lymph nodes of CIA animals, while Natural Killer (NK) cells were increased (*p* = 0.0571) in the spleen of those animals. The proportions of CD8<sup>+</sup> and CD4<sup>+</sup> T cells were similar between all groups in blood and spleen, but significant differences were observed in CIA lymph nodes. Results showed an increased percentage of CD8<sup>+</sup> and decreased CD4<sup>+</sup> T cells in CIA animals when compared to control animals. Bone injury (CIA+FD) did not induce further significant changes in lymphoid cell proportion in CIA animals at three days post-injury, except for a significant increase in NK cells in blood at day 3 after bone defect.

**Figure 2.** Arthritis induction and the combination with femoral defect promotes alterations in rat secondary lymphoid organs. (**A**) Spleen weight from control, CIA, and CIA+FD animals. (**B**) Representative spleen paraffin-section showing immunohistochemistry (IHQ) staining for the proliferation marker Ki-67, and quantitative staining evaluation across different sections of all animals. (**C**) Draining lymph nodes general view of histological analysis. (**D**) Higher magnification showing Ki-67 IHQ staining, and quantitative staining evaluation across different sections of all animals. Box plots represent min-to-max distribution of *n* = 4 to 5 animals per group. \* *p* < 0.05 determined by Mann–Whitney test. Scale bar: 3 mm (black), 100 μm (white).

**Figure 3.** CIA and CIA+FD lead to alterations in systemic immune cell proportions. The percentages of lymphoid and myeloid populations at systemic level were analyzed in blood, draining lymph nodes and spleen of control, CIA, and CIA+FD animals, by multicolor flow cytometry. (**A**) Percentage of main lymphoid populations: B cells (TCR−/CD45+), T cells (TCR+/CD161−), NK cells (TCR−/CD161+), and CD4 (TCR+/CD4+) and CD8 (TCR+/CD8+) T cells. (**B**) Myeloid cells (CD11b/c+), and their activation status as determined by co-expression of the co-stimulatory markers CD40 and CD86. Box plots represent min-to-max distribution of *n* = 4 to 5 animals per group. \* *p* < 0.05, \*\*\* *p* < 0.001 determined by Kruskal–Wallis test and Dunn's multiple comparisons test.

Analyzing the myeloid cells (Figure 3B) revealed a significantly higher percentage of myeloid cells in blood of the CIA group, which did not change with bone injury. Conversely, in draining the lymph nodes and spleen, no increases were observed in the CIA group, but there were significant increases in percentages of myeloid cells 3 days after bone injury. To further explore these results, expression of co-stimulatory molecules CD40 and CD86 was analyzed to investigate myeloid cell activation. Cells in circulation from CIA animals present increased the percentage of cells expressing each molecule, albeit not significant, while lymph node cells showed a significant decrease of the percentage of myeloid cells expressing CD40, with CD86 following the same tendency. Importantly, 3 days after bone injury, myeloid cells showed increased expression of both co-stimulatory molecules CD40 and CD86, in lymph nodes, and for CD40 also in spleen, when compared to the CIA group. Overall, these evidences suggest a persistent systemic immune response in CIA animals that are nonetheless still able to respond to an acute injury, particularly increasing their myeloid cells proportion and activation.
