br Association of IRF expression in cancer tissues
3.3. Association of IRF5 (±)-Baclofen in cancer tissues with the NSCLC stage
To understand the role of IRF5 in NSCLC development, we analysed the IRF5 expression in the cancer tissues (CT) and para-carcinoma tis-sues (PCT) of the NSCLC patients (n = 102) using Western blotting. The results showed that in most of the NSCLC cases, the protein levels of IRF5 in the CT were lower than those in the PCT of the same patients (Fig. 4A, left). Comparing the data from all of the NSCLC patients, the
Fig. 3. Association of IRF5 with inflammatory cytokine/chemokine in the peripheral circulation of the NSCLC patients. (A–D) The mRNA levels of TNF-α, IL-6, IP-10, and IL-10 in the WBCs were detected by qRT-PCR. (E–H) The protein levels of TNF-α, IL-6, IP-10, and IL-10 in the plasma were analysed by cytometric bead array. (I–L) Correlation of the IRF5+ WBCs or monocytes with the protein levels of IP-10 and IL-10 in the plasma of the NSCLC patients. NSCLC, non-small cell lung cancer; HC, healthy controls; ES, early stage; PS, progressive stage.
IRF5 levels in the CT were significantly lower than those in the PCT (p
= 0.0345; Fig. 4A, right), and there was no diﬀerence between ade-nocarcinoma and squamous cell carcinoma (Fig. 4B). Analysis of the IRF5 expression according to the NSCLC stage indicated that it was significantly higher in the CT at the early stage than at the progressive stage (p = 0.0205). The average level (grey value) of the IRF5 ex-pression in the CT at the progressive stage was approximately 0.5, whereas it was 0.7 at the early stage. The ratio of NSCLC with an IRF5 level > 0.7 was 56.3% at the early stage and 25.7% at the progressive stage (Fig. 4C). To analyse the correlation between the IRF5 expression in the CT and NSCLC patient survival, we divided the NSCLC patients
tended to have longer survival times than those in the IRF5low group, although the diﬀerence was not significant (Fig. 4D). Further analysis demonstrated that the IRF5 expression was significantly increased in the PCT compared to the CT in the NSCLC patients younger than 45 years (p = 0.0227) or older than 60 years (p = 0.0195), but not in those between 45 and 59 years old (Fig. 4E), whereas the IRF5 levels in the CT were similar in the three age groups (Fig. 4F). Furthermore, only in the older NSCLC patients (≥ 60 years), the IRF5 levels in the CT were significantly higher at the early stage than at the progressive stage (p = 0.0457; Fig. 4G). These results suggest that higher IRF5 levels in the CT are associated with early stage NSCLC and that the IRF5 expression in the PCT is upregulated sooner than in the CT.
Inflammation plays an important role in cancer initiation, promo-tion, progression, and metastasis [10,36] and the inflammatory mi-croenvironment is an essential component of tumour niches . For
an established tumour, blood circulation is a link between the local tissues and the entire body: on the one hand, immune cells in the blood can be recruited to the tumour site; on the other hand, tumour-derived exosomes and inflammatory mediators can enter the circulation and activate innate immune cells. This study found that IRF5 expression was significantly higher in the peripheral blood cells of the NSCLC patients compared to the healthy controls, suggesting the activation of the IRF5 signalling pathway in the WBCs in response to NSCLC development. The activation manifested as the increased protein expression of IL-6 and IP-10, two downstream inflammatory factors of the IRF5 pathway, and the positive correlation between the IRF5 and IP-10 protein ex-pression in the peripheral blood of the NSCLC patients.
Research has found that N-glycan on lung carcinoma cells activates innate immune cells with C-type lectin receptors (CLRs) . More-over, N-glycan is a ligand of Dectin-1, a CLR family member, and Dectin-1 activation triggers the expression of IRF5 and its downstream cytokines . Based upon these findings, we speculate that NSCLC cells activate innate immune cells through the Dectin-1 pathway. No-tably, the IL-6 levels were elevated in the plasma of the NSCLC patients. Alternatively, the IRF5 and IL-6 protein expression levels were reversed in NSCLC development and there was no correlation between them, which could be partially due to IL-6 secretion from the cancer cells . The maintenance of high-\IL-6 levels in the peripheral blood re-sulted in monocyte polarisation to M2 macrophages , which rarely express IRF5, but rather produce IL-10 . In this study, the elevated IP-10 protein expression was also detected in the peripheral blood of the NSCLC patients, especially in the early stage patients. In mice, IP-10 can inhibit tumour growth by acting to inhibit angiogenesis and limit tumour metastasis by activating NK cells . In addition, the NSCLC severity was positively correlated with the plasma IL-10 levels, but negatively correlated with the IL-10 mRNA expression in the WBCs. This discrepancy could be due to the feedback eﬀects of IL-10, which