P119 Type I and III interferon are attenuated in a human in vitro model of alternatively activated macrophages and is mediated by IRF4

Abstract

Introduction Human monocytes may be polarized in vitro into classically or alternatively activated macrophages (CAM and AAM) after treatment with IFN-g or IL-4, respectively [1] . In vivo, AAM are closely associated with amplifying Th2 responses, and are implicated in the pathogenesis of allergic diseases and asthma. A current impediment to investigating the biology of human AAM is wide donor-to-donor variability and the quantity of primary macrophages that survive in vitro polarization. To overcome this impediment, we have successfully established a THP-1 derived -CAM and -AAM populations demonstrating typical macrophage-oriented morphological characteristics, gene and protein expression profile in vitro. The THP1-AAM model preferentially expressed interferon alpha/beta pathway genes, type I and III interferon genes, in addition to IRF4 compared to THP1-CAM. Methods THP-1 cells were activated with PMA for 48 h and polarized for 96 h with either IFN-g (500 IU/mL) or IL-4 (100 IU/mL). After polarization, the THP1-CAM and THP1-AAM were treated with LPS (100 ng/mL) and expression of IFN and inflammatory genes were measured by qRT-PCR or ELISA. Expression of IRF4 in THP1-AAM and -CAM was determined by qRT-PCR. To determine the role of IRF4 in THP1-AAM, the gene for IRF4 was silenced with siRNA prior to polarization of the THP-1 cells, and expression of interferon and inflammatory genes were measured in the IRF-silenced vs. control cells. Results THP1-AAM preferentially expressed mannose receptor and select chemokines including CCL18 and CCL22 while THP1-CAM expressed CXCL10 and TNF-alpha among other phenotype dependent markers. Upon LPS treatment, THP1-AAM preferentially expressed IL-10 in the supernatant while THP1-CAM expressed IFN-g, and IL-1Beta. Using THP1-AAM to understand interferon subtype expression in response to TLR4 challenge, we demonstrated that THP1-AAM express significantly attenuated level of interferon alpha/beta pathway, IFN-Beta, and IFN-Lambda1 genes compared to THP1-CAM upon LPS treatment. Expression of phenotype-oriented genes and IFN subtype expression profile were reproducible in primary human monocyte derived macrophages. To further investigate the mechanism for IFN attenuation in THP1-AAM, we demonstrate that IRF4 is up-regulated only in THP1-AAM. Silencing of IRF4 resulted in attenuation of expression of multiple AAM-oriented genes. Moreover, IRF4 silencing led to an enhanced expression of IFN-Beta and IFN-Lambda1 in response to LPS treatment pointing towards its critical role in mediating the AAM phenotype and its response to infection. Conclusion THP-1 derived -CAM and -AAM populations are a reliable and reproducible in vitro models to investigate a spectrum of molecular mechanisms governing classic, and alternative phenotypes in macrophage populations and their role in pathologic processes. THP1-AAM express a unique pattern of anti-inflammatory and type I and III interferon genes that may provide a signature response to TLR4 insult. Furthermore, IRF4 is a critical factor in mediating human monocytes during the alternatively activated polarization process and may be a potential therapeutic target for treatment of allergic inflammation of the upper airways.