Figure 4. TNF- and IL-6-producing CX3CR1+CD301b/MGL2⁺ MNPs are enriched in inflamed cardiac valves and blockade of either cytokine prevents valve inflammation and fibrosis.

A, Gating strategy for identifying and discretizing CD301b/MGL2-expressing MNPs (Live, CD45.2⁺Thy1.2⁻B220⁻Ly6G⁻CX3CR1⁺) based on Ly6C expression to assess their TNF and IL-6 production capacity (positive gating for CD301b/MGL2, TNF, and IL-6 expression was generated from each respective fluorescence-minus-one [FMO] control). B, Quantification of TNF- and IL-6-producing CX3CR1⁺CD301b/MGL2⁺ MNPs in inflamed K/B.g7 MVs relative to the systemic circulation (pooled secondary lymphoid organs [SLO]: axillary, brachial, cervical, inguinal, popliteal, para-aortic, mesenteric lymph nodes and spleens) (*p<0.05, **p<0.01, n=3). C, Timeline for TNF and IL-6 blockade studies: twice-weekly, IP injections of 200 μg of either anti-TNF or anti-IL-6 (or rat IgG as a control) were given from weeks 4 to 8. D, IF staining for vascular cell adhesion molecule-1 (VCAM-1) following TNF or IL-6 blockade, relative to rat IgG. E, Quantification of VCAM-1 fluorescence, normalized to MV area, following TNF or IL-6 blockade, relative to isotype control-treated K/B.g7 animals (median fluorescence/area [arbitrary units]: 44.86 [n=4], 16.22 [n=4], 11.2 [n=4], rat IgG-, and anti-IL-6, anti-TNF, respectively, ***p<0.005). F, Quantification of MVs fibrotic thickening in the setting of TNF or IL-6 blockade, relative to isotype control-treated animals (median MV thicknesses at 8 weeks: 162.5 μm [n=6], 98.3 μm [n=5], 88.05 μm [n=4] rat IgG-, and anti-IL-6, anti-TNF respectively, *p<0.05, **p<0.01). Scale bar in D equals 50 microns and applies to all images. Statistical differences in E and F were assigned using one-way analysis of variance (ANOVA) with post-hoc Tukey’s test for multiple comparisons.