Allele NSCLC HC OR br p for all
p for all
Note: (1) Abbreviations: NSCLC, non-small cell lung cancer; HC, healthy controls; OR, odds ratio; CI, confidence interval; N, number of genotype or allele gene. (2) a Fisher’s exact test; * p < 0.05.
Fig. 2. IRF5 protein levels in the WBCs of the NSCLC patients. Peripheral WBCs from the NSCLC patients (NSCLC) or the healthy controls (HC) were collected and stained with surface or intracellular fluorescence-labelled anti-CD45, CD14, CD19, and IRF5. After gating for diﬀerent group cells, the percentages of IRF5+ Gilteritinib were analysed. (A) % of IRF5+ cells in the peripheral WBCs and % of leukocyte subsets including neutrophil (NE), monocyte (MO), and lymphocyte (LY) in the IRF5+ cells. (B) % of IRF5+ cells in the peripheral monocytes. (C) % of IRF5+ cells in the peripheral neutrophils. (D) % of IRF5+ cells in the peripheral B cells. NSCLC, non-small cell lung cancer; ES, early stage; PS, progressive stage.
this genotype was significantly positively associated with higher levels of IRF5 mRNA (p < 0.0001) (Fig. 1E), indicating that the genetic background influenced the IRF5 response. Consistent with the mRNA expression, the IRF5 protein levels in the WBCs of the NSCLC patients were significantly higher compared to those in the healthy controls (p < 0.0001); furthermore, they were much higher in the patients with early stage NSCLC than in those with progressive stage NSCLC (p = 0.0130); in the IRF5+ WBCs, neutrophils accounted for the highest proportion, followed by monocytes, while the proportion of lympho-cytes was the lowest (p < 0.0001) (Fig. 2A). Analysis of the WBC sub-populations indicated that the IRF5 expression was elevated in the neutrophils, B cells, and especially in the NSCLC patient monocytes, particularly in those at the early cancer stage (Fig. 2B-D). These results suggest that increased IRF5 expression in peripheral WBCs may be an early warning sign of NSCLC development.
3.2. Correlation between IRF5 and inflammatory cytokine/chemokine expression in peripheral circulation of NSCLC patients
To determine whether IRF5 upregulation in the peripheral WBCs of the NSCLC patients aﬀected the expression of inflammatory cytokines/ chemokines downstream of IRF5, we examined the levels of IL-6, IP-10, TNF-α, and IL-10 in the peripheral WBCs and plasma. The mRNA levels of TNF-α and IL-10 were significantly elevated in the NSCLC patients compared to those in the healthy controls (p = 0.0177 and p < 0.0001, respectively), but those of IL-6 and IP-10 did not show significant dif-ferences between the two groups (Fig. 3A-D). However, stratification according to the NSCLC stage revealed that the IP-10 mRNA levels were higher in the patients with progressive NSCLC (p = 0.0199; Fig. 3C), whereas the IL-10 mRNA levels were higher in those with early stage NSCLC (p = 0.0016; Fig. 3D). The expression of the other cytokines, TNF-α and IL-6, was similar in the patients with early and progressive NSCLC (Fig. 3A-B). Because mRNA levels are not always the same as
protein levels, we detected the plasma cytokines/chemokines of the same NSCLC patients and healthy donors using a CBA assay and found that IL-6 and IP-10 were significantly increased, whereas TNF-α levels were significantly decreased in the NSCLC patients compared to the healthy controls (p < 0.0001; Fig. 3E-G). The higher IL-6 levels corre-lated with progressive NSCLC (56.3%) rather than with early NSCLC (20.6%) (p = 0.0066; Fig. 3F), whereas the higher IP-10 levels were more characteristic for early stage NSCLC (52.9%) than for progressive disease (28.1%) (p = 0.0063; Fig. 3G). Although the IL-10 levels were not significantly diﬀerent between the NSCLC patients and the healthy controls, they were higher in the progressive NSCLC patients (56.3%) compared to those in early disease (29.4%) (p = 0.0263; Fig. 3H). We next analysed the correlation between the IRF5 expression in the WBCs and plasma levels of the inflammatory cytokines/chemokines in the NSCLC patients. The results revealed that the plasma levels of IP-10 positively correlated with the IRF5 protein expression in the WBCs (p < 0.0001, r = 0.7221; Fig. 3I) and monocytes (p < 0.0001, r = 0.7345; Fig. 3J), whereas those of IL-10 negatively correlated with the IRF5 protein expression in the WBCs (p = 0.0028, r = -0.3618; Fig. 3K) and monocytes (p = 0.0033, r = -0.3568; Fig. 3L), indicating that IRF5 can directly aﬀect the expression of downstream inflammatory cyto-kines and chemokines. Overall, these findings indicate that the levels of IRF5 and its downstream targets IL-10 and IP-10 in the peripheral cir-culation may be valuable early warning signs of NSCLC development.