Loss of function of ENT3 leads to inflammatory cytokine secretion through activation of a signaling cascade involving TLR and MAPK. (A) Gene set enrichment analysis using MSigDB KEGG data set. The top 5 enriched pathways ordered by normalized enrichment score (NES) in H syndrome classical (left) and nonclassical (right) monocytes are shown. A false discovery rate adjusted P value (q value) of <.25 was considered significant. (B) Bar graph depicting the mean fluorescence intensity (MFI; mean + standard error of the mean [SEM]) of monocytes after incubation with a self-quenched substrate (controls: n = 3; patients: n = 3). Statistical significance was determined using unpaired 2-tailed t test; ∗∗P < .01. Original flow cytometry histograms are shown in supplemental Figure 5A. (C) Bar graph depicting the mean + SEM of phospho-ERK–stained monocytes (from healthy donors, n = 3), untreated or after incubation for 1 hour with 5 μg/mL ssRNA40 alone or in combination with 5 μM Cu-CPT9a (TLR8 inhibitor). Values were collected from 3 separate experiments and are normalized to MFI of the control in each experiment. Statistical significance was determined using 1-way analysis of variance (ANOVA) with post hoc Tukey multiple comparisons test; ∗∗P < .01. Original flow cytometry histograms are shown in supplemental Figure 5B. (D) Bar graph depicting the MFI (mean + SEM) of phospho-ERK–stained monocytes (healthy controls: n = 5; patients with H syndrome: n = 6). Values were collected from 3 separate experiments and are normalized to MFI of the control(s) in each experiment. Statistical significance was determined using unpaired 2-tailed t test; ∗P < .05. Original flow cytometry histograms are shown in supplemental Figure 5C. (E) Dot plots depicting the concentrations of chemokine ligand 2 (CCL2), CCL4, chemokine (C-X-C motif) ligand 8 (CXCL8), and interleukin 6 (IL-6) (pg/mL) secreted in vitro by monocytes (healthy controls: n = 5, patients with H syndrome: n = 5). Statistical significance was determined using unpaired 2-tailed t tests; ∗P < .05. (F) Dot plots depicting the concentrations of CCL2, CCL4, CXCL8, and IL-6 (pg/mL) secreted in vitro by untreated monocytes (as depicted in panel E) and by monocytes incubated in the presence of 5 μM Cu-cpt9a (TLR8 inhibitor). (G) Dot plots depicting the concentrations of CCL2, CCL4, CXCL8, and IL-6 (pg/mL) secreted in vitro by untreated monocytes (as depicted in panel E) and by monocytes incubated in the presence of 25 μM PD98059 (MEK inhibitor). Statistical significance was determined by 2-way ANOVA with Šidák multiple comparisons test. ns, not significant.