Fig 1: The CD177 molecule may be related to ENL severity. (A) CD177 levels in sera from non-reactional lepromatous leprosy (LL; n=21) and ENL patients classified according to the ENLIST ENL Severity Scale: mild ENL reaction (n=5) and moderate-severe reaction (n=16). Statistical analysis was performed using a two-tailed Welch’s t-test. (B) Whole blood cells from healthy donors (n=7) were stimulated with 10 µg/mL of M. leprae sonicate (MLs) or not (NS, non-stimulated) for 24 h and the CD177 protein levels in the plasma were determined by ELISA. Statistical analysis was performed using a two-tailed paired t-test (*p<0.05).
Fig 2: Neutrophil-related genes predict febrile UTI and APN.(A) Receiver operating characteristic (ROC) curve of CD177 expression values, in acute compared with follow-up RNA samples in Cohort II. The area under the curve was 0.996 (95% confidence interval 0.985–1.000). (B) ROC curves of MCEMP1, GPR84, VNN1, HP, and MMP8 expression values, in acute compared with follow-up RNA samples in Cohort I. (C) ROC curves of MAP2K2, PGM2, DSP, ABCA13, SVIP, and FLG2, comparing first DMSA+ and DMSA− in acute RNA samples, Cohort I.
Fig 3: Neutrophil-related genes predict febrile UTI and APN.(A) Visualization of the acute regulation of neutrophil degranulation genes in the two study cohorts (cutoff FC > 1.5, adj. P < 0.05 compared with follow-up; red, up-regulation; blue, down-regulation; black, non-significant). (B) Strong up-regulation of gene expression was apparent in all patients with febrile UTI, in both cohorts (n = 138 genes). In addition, a subset of genes was specifically regulated in the first DMSA+ group compared with the first DMSA− group (n = 37 DMSA+ specific genes). Top regulated genes included CD177 (NB1) a specific marker of neutrophil adhesion and transmigration, MCEMP1 a transmembrane protein expressed by mast cells, HP (haptoglobin) secondary granule molecule released by activated neutrophils, VNN1 (vanin 1) involved in hematopoietic cell trafficking, GPR84 (neutrophil chemotaxis), MMP9 (neutrophil activation and migration). Expression of those genes was higher in patients with APN than in patients with febrile UTI without renal involvement. (C) Details of neutrophil biology genes compiled by Naranbhai et al and their regulation in APN patients (Naranbhai et al, 2015). Red are up-regulated, and blue are down-regulated genes. Asterisk mark genes with identified SNPs in the genome sequencing data. (D) ELISA assay of CD177 responses in urine, in acute compared with follow-up samples in Cohort II. Lines represent paired samples (P < 0.001, Wilcoxon matched-pairs signed rank test). (E) Receiver operating characteristic curve of CD177 expression values, in acute compared with follow-up RNA samples. The area under the curve was 0.980 (95% confidence interval 0.963–0.996). (F) The Boruta feature selection method was used to identify genes predictive of disease outcome. Boruta method plot showing neutrophil genes that are associated with first DMSA outcome. 34 genes were classified as important, including MAP2K2, FLG2, SVIP, and PGM2. Confirmed relevant (green), tentative (yellow), and rejected (red) predictor boxplots are shown. Reference levels are shown in blue. (G) Receiver operating characteristic curve of a four-gene signature associated with APN, comparing first DMSA+ and DMSA− in acute RNA samples. The combined MAP2K2, FLG2, SVIP, and PGM2 expression values were used, the resulting area under the curve was 0.944. (H) Boruta method plot showing no neutrophil gene significantly associated with second DMSA outcome. Eight interferon-related genes were classified as important, however, including GBP6, HLA, IFNG, and IFNA4.
Fig 4: Neutrophil genes are enriched in the blood transcriptomics of patients experiencing ENL. (A) Normalized expression levels from RNAseq differential gene expression (DGE) analysis of patients with lepromatous leprosy who were non-reactional (LL; purple, n=7) or had active erythema nodosum leprosum (ENL; red, n=15) for neutrophil genes: CD177, CHIT1, OLFM4, S100A12, S100A8, and S100A9. (B) Log normalized expression levels of the neutrophil genes CD177 (LL=6; ENL=8), CHIT1 (LL=5; ENL=6), OLFM4 (LL=4; ENL=8), S100A12 (LL=6; ENL=8); S100A8 (LL=7; ENL=8) and S100A9 (LL=7; ENL=8) obtained by RT-qPCR in a new cohort of LL (purple) and ENL patients (red). The genes RPL13 and RPS16 were used as reference controls for gene expression normalization. Statistical analysis was performed by applying Welch’s t-test. (C) Spearman’s correlation test of normalized expression log2 data from RT-qPCR of ENL patients between CD177 and S100A8 (left panel), CD177 and S100A9 (middle panel), and CD177 and S100A12 (right panel). Positive correlation and significance are considered when the r-value is > 0.7 and the p-value is < 0.05, respectively.
Fig 5: Neutrophil activation and renal CD177 expression in the murine pyelonephritis model.(A) Total renal RNA was isolated and was subjected to genome-wide transcriptomic analysis. Neutrophil degranulation genes were activated in infected mice compared with uninfected controls. The neutrophil degranulation pathway was identified as the top regulated pathway (Z-score = 8.85, P = 8.6 × 10−32). Neutrophil genes were strongly activated, including Cd177, Mcemp1, Hp, Mmp8, S100a8/9, and Lcn2. n = 2 per group, cutoff FC ≥ 2, P < 0.05 compared with uninfected mice, red: up-regulated, blue: down-regulated. (B) Staining of renal tissue sections for CD177 (green), 24 h and 7 d after infection. Nuclei were counterstained with DAPI (blue). (C) Quantification of CD177 staining. CD177 staining was increased in infected renal tissues compared with uninfected controls (ordinary one-way ANOVA followed by Dunnett’s multiple comparison). (D) Staining of renal tissue sections for CD177 in uninfected controls and secondary antibody controls. Scale bars represent 100 μm.
Supplier Page from Thermo Fisher Scientific for Human CD177 ELISA Kit