Abstract
To investigate the correlation between TLR3 and pro-inflammatory cytokines (TNFα, IL6) expression with the distribution of macrophage M2 and Treg on Epstein Barr virus-encoded RNAs (EBER+) nasopharyngeal carcinoma (NPC) tissues. A total of 23 FFPE NPC tissue samples were obtained from patients in Dr. Sardjito General Hospital, Yogyakarta, Indonesia in 2008-2010, which expressed EBER was collected. The expressions of TLR3, TNFα, and IL6 were examined using immunofluorescence assay. The distribution of macrophage M2 and Treg were examined by immunohistochemistry with anti-CD163 and -FOXP3 antibodies, respectively. The quantification of fluorescence intensity was analyzed by the RGB space method using ImageJ software. The M2 interpretation was done by the eyeballing method and the M2 scores were divided into 0 (negative), 1 (scant), 2 (focal), 3 (abundant). The average number of Treg FOXP3+ cells in five high power fields was counted. The relationship between variables were tested by the Spearman correlation test, and the coefficient correlation was used to see the correlation between variables. All EBER+ NPC specimens showed TLR3 expression intracellularly. The expression of TNFα could be observed in the cell membranes and secreted extracellularly, while IL6 was secreted to the extracellular area. The expression of TNFα was two times higher than IL6. Most specimens showed low M2 score (56.52%) and high Treg (52.17%). A positive correlation was found between TLR3 and IL6 (12.9%). TNFα was positively correlated with the M2 distribution of 13.7% and Treg distribution of 12.9%, while the rest were explained by other factors. TNFα has a positive correlation with M2 and Treg distribution,but mostly through a different mechanism other than EBER-TLR3 interaction. Possibly, other pro-inflammatory and anti-inflammatory cytokines are involved in the formation of the NPC microenvironment, especially related to the presence of M2 and Treg, which provide immunosuppressive effects in NPC tumors.<br />.
Highlights
Nasopharyngeal cancer (NPC) is a malignancy with unique characteristics related to epidemiology, clinical manifestations, biological markers, risk factors, and prognosis compared to other types of head and neck cancers
Positive staining for Toll-like receptor-3 (TLR3) and TNFα was indicated by a green fluorescent, while IL6 was indicated by red fluorescent (Figure 1)
The results show that the TLR3 expression has a negative correlation with the TNFα expression (r = -0.303), but has a positive correlation with IL6 (r = 0.129) (Table 2)
Summary
Nasopharyngeal cancer (NPC) is a malignancy with unique characteristics related to epidemiology, clinical manifestations, biological markers, risk factors, and prognosis compared to other types of head and neck cancers. NPC is endemic in southern China and Southeast Asia (Adham et al, 2012; Wang et al, 2017). NPC is the most common malignancy in the head and neck region and is one of the most common malignancies in Indonesia after breast, cervical, lung, and liver cancer. 98% of NPC cases in endemic areas are the non-keratinizing type and closely related to Epstein Barr virus (EBV) infection (Tsang et al, 2019). Persistent EBV infection of the premalignant nasopharyngeal epithelium initiates the transformation of NPC tumor development (Tsao et al, 2017). EBV has several oncogenes and viral proteins; six EBV-encoded nuclear antigens (EBNA-1, EBNA-2, EBNA-3A, 3B, 3C, and EBNA-LP) and three latent membrane proteins
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