Fig 1: Increased expression of matrix metalloproteinase 8 (Mmp8) in brain-infiltrating Ly6Chigh monocytes of susceptible (SUS) mice.a, Outline of iDISCO+ brain clearing and light sheet imaging, and single-cell RNA-sequencing experiments. b, Representative images of a mouse brain before (upper image) and after (lower image) iDISCO+ clearing. c, Number of Ccr2rfp+ monocytes in whole brain of control (CON), SUS and resilient (RES) mice (One-way ANOVA with Bonferroni post hoc test). d, Top ten most significant correlations between brain-infiltrating monocytes and social interaction ratio (lower correlation coefficients (red) indicate that higher numbers of infiltrating monocytes are associated with greater social avoidance) (Pearson correlation). e, Reconstruction of immunofluorescence images in the nucleus accumbens (NAc), blood vessels (red), Ccr2rfp+ monocytes (green, white arrows). Scale bar: 10 µm. f, Uniform Manifold Approximation and Projection (UMAP) representation of 4 clusters identified using Seurat clustering. g, Relative (%) abundance of number of cells per cluster in CON, SUS and RES mice. h, Heat map of top ten cluster-defining protein coding genes. i, Gene ontology (GO) terms of significantly (adjusted p-value < 0.05) upregulated genes of Cluster 0. j, Genes of GO terms “extracellular space” and “extracellular matrix” compared between SUS vs. CON, RES vs. CON and SUS vs. RES mice. Feature plots of normalized gene expression of Mmp8 in k, brain-infiltrating Ccr2rfp+ monocytes and l, brain-resident immune cells in the NAc. ** p < 0.01. Data are shown as means ± s.e.m.
Fig 2: Dose response study for intra-peritoneal (i.p.) injection of recombinant matrix metalloproteinase 8 (rMMP8).Different doses of rMMP8 or vehicle (4-aminophenylmercuric acetate, APMA) were injected i.p. and blood was collected 20 min or 18 h after the injection followed by MMP8 measurement in plasma.
Fig 3: Stress increases matrix metalloproteinase 8 (MMP8) in both male and female mice.a, Social interaction ratio and b, locomotion of unstressed control (CON), susceptible (SUS) and resilient (RES) male mice (One-way ANOVA with Bonferroni post hoc test). Defeat characteristics of female mice after 10 days of chronic social defeat stress (CSDS). (c, social interaction ratio, d, locomotion) (One-way ANOVA with Bonferroni post hoc test). e, Plasma levels of MMP8 in SUS compared to CON female mice after 10 days of CSDS. (One-way ANOVA with Bonferroni post hoc test). f, MMP8 plasma levels of female mice that underwent a 21-day paradigm of chronic variable stress (CVS) compared to non-stressed control mice (two-tailed Student’s t-test). Plasma levels of g, MMP2, h, MMP3, i, proMMP9 and j, MMP12 in CON, SUS and RES male mice after 10 days of CSDS. * p < 0.01, ** p < 0.01, *** p < 0.001. Data are shown as means ± s.e.m.
Fig 4: Defeat characteristics of mice assessed for brain matrix metalloproteinase 8 (MMP8) and alterations in brain extracellular space.a, Social interaction ratio and b, locomotion of unstressed control (CON), susceptible (SUS) and resilient (RES) mice from which levels of MMP8 in the nucleus accumbens (NAc) were analysed (One-way ANOVA with Bonferroni post hoc test, each data point represents three pooled mouse brains). c, Social interaction ratio and d, locomotion of CON, SUS and RES mice analysed for the transmission electron microscopy experiment. (One-way ANOVA with Bonferroni post hoc test). ** p < 0.01, *** p < 0.001. Data are shown as means ± s.e.m.
Fig 5: Peripheral matrix metalloproteinase 8 (MMP8) is causally linked to stress-induced social avoidance behaviour, alterations in brain extracellular space and neurophysiology.a, Experimental outline of mice receiving either 100 µg/kg recombinant (r)MMP8 or vehicle (VEH) followed by a subthreshold stress. b, Social interaction (SI) ratio of mice injected with rMMP8 followed by a subthreshold defeat. c, Outline of social conditioned place preference (CPP) experiment. d, VEH injected mice show CPP towards a chamber that was paired with a juvenile mouse, e, while this was abolished in mice injected with rMMP8 during a subthreshold defeat. f, Outline of bone marrow chimera experiment, with bone marrow transplantation (BMT) on day 0, followed by 6 weeks of recovery and then chronic social defeat stress (CSDS). After CSDS, mice were tested in a comprehensive behaviour battery, consisting of a SI test with a novel CD-1 mouse, a juvenile interaction (JI) test with a novel same-sex juvenile mouse, sucrose preference test, elevated plus maze (EPM) and splash test. On the day of sacrifice, chimerism was validated and brains of a subset of stressed mice were processed for transmission electron microscopy (TEM) imaging of the extracellular space. g, Plasma levels of MMP8. h, SI ratio and i, time spent in the corner as a measure of social avoidance. j, Representative heat maps of behaviour during social interaction test. k, Time spent in the interaction zone during a JI test when the novel juvenile mouse was present. l, Quantification of the extracellular space relative to total brain area in WT compared to Mmp8-/- chimeras. m, Outline of ex vivo patch clamp experiment in nucleus accumbens (NAc) medium spiny neurons. n, Action potentials in Mmp8-/- and WT mice after 10 days of CSDS. o, Representative traces of action potentials evoked upon a +120 pA step of depolarizing current. p, Frequencies of spontaneous excitatory postsynaptic currents (sEPSCs) in Mmp8-/- and WT mice after 10 days of CSDS. q, Representative traces of sEPSCs. (Two-way ANOVAs followed by Tukey’s post hoc testing (for panels b, d, g, h, i, k, n, p) or two-sided Student’s t-test (for panel l). * p < 0.05, ** p < 0.01, *** p < 0.001. Data are shown as means ± s.e.m.
Supplier Page from R&D Systems, a Bio-Techne Brand for Recombinant Mouse MMP-8 Protein, CF