CD14(+)S100A9(+) inflammatory monocytes in patients with NSCLC are a distinct subset of MDSCs, which suppress T cells by arginase, iNOS, and the IL-13/IL-4Rα axis. The amount of these inflammatory monocytes is associated with poor response to chemotherapy. Clinical trial registered with www.clinicaltrials.gov (NCT 01204307).

Our study investigated whether tumor-associated macrophages (TAMs) in advanced non-small cell lung cancer (NSCLC) are related to treatment response to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) and may be a predictor of survival. Of 206 advanced NSCLC patients treated (first-line) with an EGFR-TKI at the study hospital from 2006 to 2009, 107 with adequate specimens for assessing CD68 immunohistochemistry as a marker of TAMs were assessed. After EGFR-TKI treatment, response was observed in 55 (51%) patients, and the median follow-up period was 13.5 months. Most TAMs were located in the tumor stroma (>95%) and positively costained with the M2 marker CD163. TAM counts were significantly higher in patients with progressive disease than in those without (p < 0.0001), a trend that remained in patients with known EGFR mutation status (n 5 59) and those with wild-type EGFR (n 5 20). High TAM counts, among other factors (e.g., wild-type EGFR), were significantly related to poor progression-free survival (PFS) and overall survival (OS) (all p < 0.0001 for TAMs). Multivariate Cox analyses showed that high TAM counts and EGFR mutations were both independent factors associated with PFS [odds ratio (OR), 8.0; 95% confidence interval (CI), 2.87-22.4; p 5 0.0001 and OR, 0.03; 95% CI, 0.003-0.31; p 5 0.003, respectively] and OS (OR, 2.641; 95% CI, 1.08-6.5; p 5 0.03 and OR, 0.14; 95% CI, 0.03-0.56; p 5 0.006, respectively). TAMs are related to treatment response irrespective of EGFR mutation and can independently predict survival in advanced NSCLC treated with an EGFR-TKI.

NF-κB repressing factor (NRF), a nuclear inhibitor of NF-κB, is constitutively expressed and is implicated in the basal silencing of specific NF-κB targeting genes, including IFN-β, IL-8/CXCL8, and iNOS. Little is known about the regulation of NRF and its role in response to stimuli. Airway smooth muscle (ASM) is a rich source of inflammatory mediators that may regulate the development and progression of airway inflammation. We have previously reported that NE activates NF-κB in primary human ASM (hASM), leading to induction of TGF-β1. In this study, we describe that, instead of inducing the NF-κB response gene IL-8/CXCL8, NE suppressed IL-8/CXCL8 release and mRNA expression in hASM cells. Transcriptional blockade studies using actinomycin D revealed a similar degradation rate of IL-8/CXCL8 mRNA in the presence or absence of NE, suggesting an involvement at the transcription level. Mechanistically, the NE repressive effect was mediated by inducing NRF, as shown by RT-PCR and Western blotting, which was subsequently recruited to the native IL-8/CXCL8 promoter leading to removal of RNA polymerase II from the promoter, as demonstrated by chromatin immunoprecipitation assays. Knockdown of NRF by small interfering RNA prevented NE-induced suppression of IL-8/CXCL8 expression. In contrast, NE did not induce NRF expression in A549 and Beas-2B cells, where NE only stimulates NF-κB activation and IL-8/CXCL8 induction. Forced expression of NRF in A549 cells by an NRF expression plasmid suppressed IL-8/CXCL8 expression. Hence, we describe a novel negative regulatory mechanism of NE-induced NRF, which is restricted to hASM and mediates the suppression of IL-8/CXCL8 expression.

Chronic systemic inflammation is implicated in the systemic manifestations and, probably, the excess mortality risk of chronic obstructive pulmonary disease (COPD). The role of nuclear factor (NF)-kB repressing factor (NRF), a DNA-binding, protein-inhibiting NF-kB response gene, in human diseases has not been explored. We hypothesised that the NRF-negative regulatory mechanism is impaired in COPD peripheral blood mononuclear cells (PBMCs) leading to excessive interleukin (IL)-8/CXCL8 production.NRF expression, NF-kB activation, IL-8/CXCL8 release and intracellular oxidative stress were assessed in PBMCs of normal subjects and stable COPD patients. Primary PBMCs with NRF overexpression, NRF knockdown and exposure to H 2 O 2 were used to elucidate the mechanisms.Stable COPD patients, especially those with severe COPD, showed decreased NRF expression, enhanced NF-kB activation and increased IL-8/CXCL8 release in PBMCs compared with normal subjects. This was associated with reduced NRF and increased RNA polymerase II occupancy at the IL-8/CXCL8 promoter. NRF knockdown enhanced IL-8/CXCL8 production in normal PBMCs, whilst NRF overexpression attenuated IL-8/CXCL8 production. Intracellular oxidative stress was increased in COPD PBMCs. H 2 O 2 -decreased NRF expression and -enhanced IL-8/CXCL8 production was augmented in COPD PBMCs.NRF expression is reduced in PBMCs of stable COPD patients, probably through oxidative stress, leading to increased production of IL-8/CXCL8 and potentially chronic systemic inflammation.