Fig 2: Increased IL-17 production in arthritic paws from Sparc-/- mice(A-L) (A) Intracellular staining for IL-17, IFN?, and TNF in CD4+ T cells from draining LN of naive and pristane-treated mice (130 days after treatment). Results are representative of three independent experiments (*p < 0.05, Student's t test; error bars are mean ± SD). (B) Representative dot plots for the intracellular staining. (C) qPCR for IL-17A performed by digesting the whole paws of the indicated mice; results are normalized to GAPDH; representative of two other independent experiments; (*p < 0.05, Student's t test; error bars are mean ± SD). (D) Intracellular staining for IL-17 in Th cells (CD45+CD4+), PMN (CD45+CD11b+Gr1hi), and other infiltrating cells (CD45+CD11b-CD4-) of paws isolated and digested from mice 130 days after pristane injection. (*p < 0.05, Student's t test; error bars are mean ± SD). (E) Production of IL-17 by ?dTCR and CD4 cells. Representative plots. (F) IHC analysis for Gr-1 performed on paws obtained from WT and Sparc-/- mice. Scale bar, 500 µm. (G) Frequency of proliferating ?dT cells when co-cultured with DC and PMN either from WT or Sparc-/- mice. This analysis highlights a direct capacity of Sparc-/- PMN (either naive or undergoing NETosis) of stimulating ?dT cells, independent of the presence of DCs (*p < 0.05; **p < 0.01, Student's t test, mean ± SEM). (H) Arthritis score obtained evaluating arthritis-inducing antibody cocktail (ArthritoMab)-treated mice every 2 weeks (n = 17 mice/group pooled from two independent experiments, *p< 0.05) (I) Representative pictures of paws from WT and Sparc-/- at day 200 treated with ArthritoMab. (J) Frequency of PMN (mean ± SEM), (K) ?dT cells (mean ± SEM), and (L) IL-17+ ?dT cells (mean ± SEM) in mice treated with ArthritoMab (*p < 0.05; ***p < 0.001; Student's t test).

Fig 3: MnNP Biodistribution in ID8 Tumor Model. C57Bl/6 mice bearing ID8 ovarian tumors were treated IP with Cy5-MnNPs. After 24 hours, the ascites, tumors, and spleens were collected for flow cytometry analysis. A The general cell population gated on FSC vs. SSC revealed a high percentage of MnNP+ cells in the ascites and almost 20% of cells in the tumor, but negligible delivery to any cells in the spleen. B Gating on CD45+/F4/80+ macrophages revealed that about 60% of macrophages in the ascites and ~ 75% of TAMs were MnNP+. Additional gating for (C) Gr-1- mature macrophages and (D) Gr-1+ myeloid cells also revealed high percentages of MnNP uptake in the ascites and tumor (n = 3, *p < 0.05 **p < 0.001)

Fig 4: Increased Therapeutic Benefit in Extended MnNP Treatment Model. A Treatment schematic for extended MnNP treatment in the TBR5 tumor model. Changes in (B) ascites volume and (C) tumor weight at endpoint (n = 4, *p < 0.05, **p < 0.001). Flow cytometry gating on CD45-/SSChi cells revealed significant changes in the percent of tumor cells in the (D) ascites and (E) tumors. Cells gated on F4/80int/CD206+ revealed significant decreases in (F) the percent of M2-like TAMs and (G) the ratio of M2 (CD206+):M1 (CD206-) TAMs. The cells also revealed significant increases in (H) classical monocytes (F4/80int/Ly6C+/Ly6G-) in the ascites and (I) NK cells (CD3-/B220-/NKp46+) in the tumor (n = 4, *p < 0.05, **p < 0.001)

Fig 5: Administration of antibodies against Ly6G ameliorated the postictal CBF reduction(A) Representative CBF and LFP signals over time with treatment of either isotype control antibody (cIgG) or anti-Ly6G (aLy6G) antibody in 4-AP-injected mice.(B and C) Time course traces of heart rate and CBF monitored during the antibody injections (cIgG: n = 5; aLy6G: n = 6). CBF signals were normalized to those measured during 10 min before the antibody injections (pre-injection baseline).(D) Magnitude of recurrent seizures, calculated by summation of coastline lengths of LFP signals (SEI: 53,713.48 ± 29,392.96, n = 7; cIgG+SEI: 49,715.37 ± 33,218.34, n = 6; aLy6G + SEI: 32,587.9 ± 16,306.22, n = 6; F(2,16) = 0.885, p = 0.432, one-way ANOVA test).(E) Duration of recurrent seizures (SEI: 97.82 ± 21.25, n = 7; cIgG+SEI: 95.24 ± 33.60, n = 6; aLy6G + SEI: 85.58 ± 19.28, n = 6; F(2,16) = 0.344, p = 0.714, one-way ANOVA test).(F) Averages of summed coastline lengths of LFP signals per second (SEI: 8.73 ± 3.30, n = 7; cIgG+SEI: 7.91 ± 2.53, n = 6; aLy6G + SEI: 6.13 ± 2.23, n = 6; F(2,16) = 1.243, p = 0.315, one-way ANOVA test).(G) Examples of LFP traces during the 60–80 min of the postictal periods in the cIgG- and aLy6G-treated groups.(H) Power spectra of LFPs recorded during 60–80 min of the postictal periods (cIgG+SEI, n = 12; aLy6G + SEI, n = 13). “n.s.” indicates no statistical significance. Delta (1–4 Hz): U(12,13) = 63, p = 0.415, Mann-Whitney U test; theta (4–8 Hz): t(23) = -1.156, p = 0.260, independent t test; alpha (8–14 Hz): U(12,13) = 76, p = 0.913, Mann-Whitney U test; beta (14–30 Hz): U(12,13) = 77, p = 0.957, Mann-Whitney U test; gamma (30–100 Hz): t(20.610) = -0.896, p = 0.380, independent t test.(I) Postictal CBF changes in 4-AP-injected animals that had received isotype control antibody (cIgG+SEI, n = 6) or anti-Ly6G (aLy6G + SEI, n = 6) antibody. The time when the last seizure ended was set “0” to define the beginning of postictal periods in individual mice. The colored lines represent the averaged trace of individual data, which are depicted by the gray lines.(J) CBF changes in each 20-min epoch of the postictal periods that had received isotype control antibody (cIgG+SEI, n = 6) or anti-Ly6G (aLy6G + SEI, n = 6) antibody. [0–20 min]: 62.65 ± 10.91 (cIgG+SEI) vs. 63.12 ± 12.29 (aLy6G + SEI), t(10) = -0.063, p = 0.951, independent t test; [20–40 min]: 64.62 ± 14.18 (cIgG+SEI) vs. 72.44 vs. 14.11 (aLy6G + SEI), t(10) = -0.875, p = 0.402, independent t test; [40–60 min]: 64.12 ± 14.01 (cIgG+SEI) vs. 89.57 ± 20.11 (aLy6G + SEI), t(10) = -2.323, *p = 0.043, independent t test; [60–80 min]: 59.25 ± 12.23 (cIgG+SEI) vs. 101.87 ± 15.21 (aLy6G + SEI), t(9) = -4.570, **p = 0.001, independent t test.(K) Scatter plots showing the relationship between the magnitude of seizures and the 60–90 min postictal CBF changes in the cIgG- and aLy6G-treated groups (cIgG+SEI: Pearson’s r = -0.898, *p = 0.015, R2 = 0.807, n = 6; aLy6G + SEI: Pearson’s r = -0.132, p = 0.803, R2 = 0.017, n = 6).