Fig 2: Reduction of FRC progenitors in the aged lymph nodes. Skin-draining (sdLN) and mesenteric (mLN) lymph nodes from young and aged WT mice were depleted of CD45+ cells before analysis by flow cytometry. (A). Gating of CD45-CD35-B220-ICAM-1+ stroma into fibroblastic reticular cells (FRCs), lymphatic endothelial cells (LECs), blood endothelial cells (BECs), and double negative cells (DNs) in the sdLN and mLN of young and aged mice. (B). Frequency of FRCs, LECs, BECs, and DNs within young and aged sdLN and mLN stroma. (C). Number per lymph node of FRCs, LECs, BECs, and DNs within young and aged sdLN and mLN stroma. (D). Gating of MAdCAM-1- FRCs into VCAM-1lo, VCAM-1int, and VCAM-1hi subsets in the sdLN and mLN of young and aged mice. (E). Frequency of VCAM-1lo, VCAM-1int, and VCAM-1hi FRC subsets within young and aged sdLN and mLN stroma. Relative fluorescence intensity (RFI) of surface markers (F). LTBR and (G). SCA-1 on VCAM-1lo, VCAM-1int, and VCAM-1hi FRC subsets within young and aged sdLN and mLN stroma. RFI normalized to expression on young VCAM-1hi FRCs. Data is representative of lymph nodes retrieved from 6 young and 6 aged mice. Analyzed by two-way ANOVA with adjustment for multiple comparisons, p-values: ns, not significant; * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001.

Fig 3: Reduced expression of Ltbr and Ccl21 in aged stroma. (A). Lymph nodes from young and aged WT mice were depleted of CD45+ cells before qPCR measurement of Il7, Ltbr, Ccl19, and Ccl21 expression. Data were normalized to Actb expression for each mouse, then normalized to young transcript expression for the given experiment. Compiled data from 3 experiments, in which lymph node stroma from 3 young and 3 aged mice was pooled for each experiment. Analyzed by t tests, p-values: ns, not significant; *** < 0.001, **** < 0.0001. (B). Splenocytes from young and aged mice were analyzed by qPCR for Lta, Ltb, and LIGHT expression. Data were normalized to Actb expression for each mouse, then normalized to young transcript expression for the given experiment. Compiled data from 2 experiments, analyzed by t tests: ns, not significant.

Fig 4: Analysis of Inflammation in the Musculoskeletal Tissues of Mxra8?8/?8 Mice after CHIKV Infection(A–D) WT and Mxra8?8/?8 mice were inoculated subcutaneously in the foot with 103 FFU of CHIKV-AF15561.(A) At 3 dpi, ipsilateral feet were collected, fixed, decalcified, paraffin embedded, sectioned, and stained with H&E. Scale bar, 100 µm; representative images are shown from n = 4 mice from two experiments. Arrows indicate tissue edema. At days 0 (B) (uninfected), 3 (C), and 7 (D) post-infection, cells from ipsilateral feet were harvested and analyzed for numbers of leukocytes (CD45+), monocytes (CD11b+CD11c-Ly6G-Ly6C+), neutrophils (CD11b+CD11c-Ly6G+), natural killer(NK) cells (CD3-NK1.1+), macrophages (CD11b+CD11c-Ly6G-Ly6C-F4/80+), monocyte-derived dendritic cells (DCs) (CD11b+CD11c+Ly6G-Ly6C+MHCII+), CD4+ T cells (CD3+CD4+), and CD8+ T cells (CD3+CD8+) by flow cytometry (see Figure S4 for gating scheme, two experiments; n = 8–10; two-tailed Mann-Whitney test; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns, not significant).(E) At 3 and 7 dpi, ipsilateral ankles from WT and Mxra8?8/?8 mice were harvested, homogenized, and the indicated cytokines or chemokines were measured (two experiments; n = 8–10, mean ± SD; two-way ANOVA with Sidak’s test; *p < 0.05; **p < 0.01; ****p < 0.0001; ns, not significant).See also Figures S4 and S5.

Fig 5: Increase fibrosis in the aged T cell zone network. Lymph nodes from young (2-3MO) and aged (16-20MO) WT mice were harvested, fixed, and frozen for cryosectioning. Confocal 20× tile-scans of lymph node cross-sections stained with antibodies directed against (A). PDGFRß (magenta) and B220 (green), (B). collagen IV (magenta) and PDGFRß (green), (C). aSMA (magenta) and PDPN (green) in young and aged mice. Scale bar is 500 µm. (D). Maximum intensity projection of 12-µm thick confocal volumes, at 20× magnification, stained for aSMA (magenta) and PDPN (green) in young and aged mice. Scale bar is 100 µm. Data is representative of lymph nodes retrieved from 6 young and 6 aged mice. (E). Percentage of aSMAhi voxels within T cell zone confocal volumes (as in D) in young and aged lymph node fields of view (FOV). Analyzed by t test, p-value: ** < 0.01.