Fig 2: OPN promotes CD11c+ microglial proinflammatory responses via an interaction with the aVß3 integrin receptor and impairs Aß plaque compaction via suppression of the TREM2/TAM-lysosomal phagocytic pathway. (A) Flow cytometric analysis of CD11c+ microglial TNF-a production in 9-mo-old 5XFAD and OPN-KO.5XFAD mice (n=6). ***P < 0.001 by two-tailed Student’s t test. (B) Expression of TNF-a in CD11c+ aVß3+ vs. CD11c+ aVß3- microglia in 9-mo-old 5XFAD and OPN-KO.5XFAD mice (n=3). ****P < 0.0001, ns: not significant by two-tailed Student’s t test. (C) The impact of OPN-aVß3 interaction on CD11c+ microglial production of TNF-a. Up-regulated TNF-a production induced by rmOPN was fully abrogated by aVß3 inhibitor in CD11c+ microglia of 9-mo-old OPN-KO.5XFAD mice (n=3). ****P < 0.0001, ns: not significant by one-way ANOVA with Bonferroni’s multiple comparisons test. (D and E) The impact of OPN-aVß3 interaction on microglial caspase-1 activation and IL-1ß production. Activation of caspase-1 and IL-1ß by provision of rmOPN was blocked following provision of aVß3 inhibitor to microglia from 9-mo-old 5XFAD and OPN-KO.5XFAD mice (n=3). ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, ns: not significant by one-way ANOVA with Bonferroni’s multiple comparisons test. U.D. undetectable. (F) Aß engulfment (Me-XO4+) by CD11c+ microglia in 9-mo-old 5XFAD and OPN-KO.5XFAD mice was analyzed by flow cytometry. Increased Me-XO4+ microglia were observed in OPN-deficient 5XFAD mice compared with 5XFAD mice (n=6). ****P < 0.0001 by two-tailed Student’s t test. (G) Flow cytometric analysis of TREM2 expression by CD11c+ microglia from 9-mo-old 5XFAD mice and OPN-KO.5XFAD mice (n=3). **P < 0.01 by two-tailed Student’s t test. (H) Flow cytometric analysis of Axl and Mer expression by CD11c+ microglia from 9-mo-old 5XFAD mice and OPN-KO.5XFAD mice (n=6). ****P < 0.0001 by two-tailed Student’s t test. (I and J) Representative immunofluorescent images and quantification of CD68+ area in CD11c+ microglia located within 25 µm of Aß plaques (defined as plaque-associated CD11c+ microglia) in 9-mo-old 5XFAD and OPN-KO.5XFAD mice. (n=20 ROIs from 3 mice/group). *P < 0.05 by two-tailed Student’s t test. (Scale bar, 25 µm.) (K and L) Representative immunofluorescent images and quantification of cathepsin B+ area in plaque-associated CD11c+ microglia of 9-mo-old 5XFAD and OPN-KO.5XFAD mice (n=22 ROIs from 3 mice/group). ****P < 0.0001 by two-tailed Student’s t test. (Scale bar, 25 µm.) All data are presented as mean ± SEM.

Fig 3: Increased microglial OPN production correlates with AD severity in human AD brains. (A) Analysis of OPN protein expression in brain (middle frontal gyrus) homogenates from AD patients (CDR = 1, n=11) with mild cognitive impairment (MCI, CDR 0.5, n=10) and cognitively normal controls (CDR 0, n=11). **P < 0.01, ns: not significant by one-way ANOVA with Bonferroni’s multiple comparisons test. (B) Pearson’s correlation analysis of OPN expression levels in brain tissues from AD patients with CDR scores (CDR = 1, n=11), MCI patients (CDR 0.5, n=10), and controls (CDR 0, n=11). r=0.5046, P=0.0032. (C and D) Pearson’s correlation analysis of brain OPN concentrations correlated with neuritic plaque rating (r=0.4919, P=0.0043) and tangle rating (r=0.4884, P=0.0046). (E and F) Representative immunofluorescent images from middle frontal gyrus of AD patients and normal controls stained for Iba-1 (microglia, red), CD11c (green) and OPN (cyan) are indicated by white arrows. Percent of CD11c+OPN+ microglia in brains of AD patients (CDR =1, n=8) compared with MCI (CDR 0.5, n=9) and control subjects (CDR 0, n=5). Each dot represents the average percentage of CD11c+OPN+ microglia over total Iba-1+ cells in 10 to 12 fields of each brain sample. (Scale bar, 25 µm.) ****P < 0.0001, ns: not significant by one-way ANOVA with Bonferroni’s multiple comparisons test. (G) Pearson’s correlation analysis of the percentage of CD11c+OPN+ microglia in human brain tissues correlated with CDR scores in AD patients (CDR =1, n=8), MCI patients (CDR 0.5, n=9) and controls (CDR 0, n=5). r=0.8383, P < 0.0001. (H and I) Pearson’s correlation analysis of the percentage of CD11c+OPN+ microglia in human brain tissues correlated with neurite plaque rating (r=0.8226, P < 0.0001) and tangle rating (r=0.7434, P < 0.0001). All data are presented as mean ± SEM.

Fig 4: OPN production divides CD11c+ microglia into a pathogenic and protective subset. (A) The percentage of CD11c+ OPN+ and CD11c+ OPN- microglial subsets in whole brain of 9-mo-old 5XFAD mice was determined by flow cytometry and calculated as the number of each subset within the number of total microglia of the brain (n=3). ***P < 0.001 by two-tailed Student's t test. (B) Bar plot showing the fraction of CD11c+ OPN+ and CD11c+ OPN- microglial subsets in periplaque area (within 25 µm of Aß plaque core) and the plaque-distal area (>25 µm of Aß plaque core) in the brain of 9-mo-old 5XFAD mice. Aß ingested or noningested cells of each microglial subset was calculated as the number of Aß engulfed (Aß+) or Aß nonengulfed (Aß-) microglia within the total number of each microglial subset resided in the periplaque area (33 ROIs from three mice were analyzed). The bar plot was a representative result of three independent experiments. (C) Enrichment of DAM gene signature was performed by mapping previously reported DAM signature genes with our CD11c+ microglial differentially expressed genes (DEGs) from OPN-KO.5XFAD compared with 5XFAD mice. Heatmap displaying OPN-deficient CD11c+ microglia highly resembled DAM-like gene signature with a 2.6-fold higher enrichment score (P=6.35e-15) compared with CD11c+ microglia from 5XFAD mice. Gene expression was considered significant if log2FC > 1 or log2FC < -1 with a FDR-adjusted P value < 0.05. (D) Volcano plot displaying CD11c+ microglial DEGs from 9-mo-old OPN-KO.5XFAD mice compared with 5XFAD mice. OPN deletion in CD11c+ microglia showed downregulation of a series of proinflammatory response-related genes (highlighted in red, i.e., Tnfrsf9, Il1b, Ifng) and upregulation of phagocytic/lysosomal activation-related genes essential for Aß uptake and lysosomal degradation (highlighted in blue, i.e., Mertk, Trem2, Cd68, Ctsb). Gene expression was considered significant if log2FC > 1 or log2FC < -1 with a FDR-adjusted P value < 0.05. (E) Flow cytometric analysis of TNF-a expression in CD11c+ OPN+ and CD11c+ OPN- microglia from 9-mo-old 5XFAD mice (n=6). ****P < 0.0001 by two-tailed Student’s t test. (F) Flow cytometric analysis of Aß engulfment (Me-XO4+) by CD11c+ OPN+ and CD11c+ OPN- microglial cell subsets from 9-mo-old 5XFAD mice (n=6). ****P < 0.0001 by two-tailed Student’s t test. (G) Flow cytometric analysis of TREM2 expression in CD11c+ OPN+ and CD11c+ OPN- microglia from 9-mo-old 5XFAD mice (n=3). **P < 0.01 by two-tailed Student’s t test. (H and I) Flow cytometric analysis of Axl and Mer expression by CD11c+ OPN+ and CD11c+ OPN- microglial subsets from 9-mo-old 5XFAD mice (n=6). ****P < 0.0001, **P < 0.01 by two-tailed Student’s t test. All data are presented as mean ± SEM.

Fig 5: OPN deletion diminishes AD pathology and rescues cognitive deficits in 5XFAD mice. (A) Flow cytometric analysis of OPN expression in CD11c+ and CD11c- microglia from 5XFAD mice (n=6) at different disease stages. ****P < 0.0001 by two-way ANOVA with Bonferroni’s multiple comparisons test. (B) Flow cytometric analysis showing the increased percentage in CD11c+ OPN+ microglia from 5XFAD mice compared with age-matched WT mice during disease progression (n=5 to 6). ****P < 0.0001, **P < 0.01 by two-way ANOVA with Bonferroni’s multiple comparisons test. (C) Ex vivo analysis of microglial tumor necrosis factor-alpha (TNF-a) expression in WT, 5XFAD, and OPN-KO.5XFAD mice (n=6) during disease progression according to flow cytometry. *: WT vs. 5XFAD, ***P < 0.001, ****P < 0.0001; #: 5XFAD vs. OPN-KO.5XFAD, ####P < 0.0001, ns: not significant by two-way ANOVA with Bonferroni’s multiple comparisons test. (D) Representative immunofluorescent images of brain sections from 9-mo-old 5XFAD mice and OPN-KO.5XFAD mice stained with 6E10 (red) and Thioflavin-S (green) displaying different forms of Aß plaques. Total plaques were defined as 6E10+. White arrows indicate diffuse plaques (6E10+ Thio-S-), while compact plaques (6E10+ Thio-S+) are indicated by yellow arrows. (Scale bar, 100 µm.) (E–G) Quantification of total area (6E10+), compact area (6E10+Thio-S+), and diffuse area (6E10+Thio-S-) of Aß plaques in 3-, 6-, and 9-mo-old 5XFAD mice and OPN-KO.5XFAD mice (n= 54 fields from 3 mice/group). ***P < 0.001, **P < 0.01, *P < 0.05, ns: not significant by two-way ANOVA with Bonferroni’s multiple comparisons test. (H) The compactness index of Aß plaques in 3-, 6-, and 9-mo-old 5XFAD mice and OPN-KO.5XFAD mice. The compactness index was calculated as 6E10+Thio-S+ area/ 6E10+ area (n =54 fields from three mice/group). **P < 0.01, ns: not significant by two-way ANOVA with Bonferroni’s multiple comparisons test. (I and J) Representative immunofluorescent images and quantification of dystrophic neurites (labeled with N terminus APP) per plaque (labeled with Thioflavin-S) in brain sections of 5XFAD and OPN-KO.5XFAD mice. The number of APP+ dystrophic neurites was quantified within 25 µm area of each plaque (n=30 to 40 plaques from three mice per group). ****P < 0.0001, ***P < 0.001, ns: not significant by two-way ANOVA with Bonferroni’s multiple comparisons test. (Scale bar, 200 µm.) (K) Spatial learning and memory (acquisition trials) and cognitive flexibility (reversal trials) were assessed by water T maze in 9-mo-old 5XFAD and OPN-KO.5XFAD mice. Age-matched WT mice were included as controls (n=3 to 5, #: WT vs. 5XFAD, #P < 0.05, ##P < 0.01, ###P < 0.001; *: 5XFAD vs. OPN-KO.5XFAD, **P < 0.01). Statistical analysis was performed by two-way ANOVA with Bonferroni’s multiple comparisons test. All data are presented as mean ± SEM.