Human neutrophils activated by TLR8 agonists, with or without IFNγ, synthesize and release EBI3, but not IL-12, IL-27, IL-35, or IL-39

Abstract

The IL-12 family of cytokines plays crucial functions in innate and adaptive immunity. These cytokines include heterodimers sharing distinct (IL-12A, IL-23A, and IL-27A) with two (IL-12B and Epstein-Barr virus induced gene 3 [EBI3]) chains, respectively, IL-12 (IL-12B plus IL-12A) and IL-23 (IL-12B plus IL-23A) sharing IL-12B, IL-27 (EBI3 plus IL-27A), IL-35 (EBI3 plus IL-12A), and IL-39 (EBI3 plus IL-23A) sharing EBI3. In this context, we have recently reported that highly pure neutrophils incubated with TLR8 agonists produce functional IL-23. Previously, we showed that neutrophils incubated with LPS plus IFN for 20 h produce IL-12. Herein, we investigated whether highly pure, TLR8-activated, neutrophils produce EBI3, and in turn IL-27, IL-35, and IL-39, the IL-12 members containing it. We report that neutrophils incubated with TLR8 ligands, TNF and, to a lesser extent, LPS, produce and release remarkable amounts of EBI3, but not IL-27A, consequently excluding the possibility for an IL-27 production. We also report a series of unsuccessful experiments performed to investigate whether neutrophil-derived EBI3 associates with IL-23A to form IL-39. Furthermore, we show that neutrophils incubated with IFN in combination with either TLR8 or TLR4 ligands express/produce neither IL-12, nor IL-35, due to the inability of IFN , contrary to previous findings, to activate IL12A transcription. Even IL-27 was undetectable in supernatants harvested from IFN plus R848-treated neutrophils, although they were found to accumulate IL27A transcripts. Finally, by immunohistochemistry experiments, EBI3-positive neutrophils were found in discrete pathologies only, including diverticulitis, cholecystitis, Gorham disease, and Bartonella Henselae infection, implying a specific role of neutrophil-derived EBI3 in vivo.

Induction of OCT2 contributes to regulate the gene expression program in human neutrophils activated via TLR8

“…To date, TLR8 ligands, have been shown to trigger or modulate a variety of effector functions of neutrophils, including the survival, the respiratory burst, the phagocytic activities, and the formation of NETs (Thomas and Schroder, 2013). In our research, TLR8 agonists, including R848 and other compounds, such as CL075 or VTX-2337, have been found to very potently activate expression of multiple inflammatory genes (including interleukin 6 [IL6], tumor necrosis factor [TNF], CSF3, IL12B, IL23A, EBI3, and CCL23) and production of the corresponding products (Arruda-Silva et al, 2017;Cassatella et al, 2020;Tamassia et al, 2019;Zimmermann et al, 2015Zimmermann et al, , 2016) by human neutrophils. In particular, we found that the induction of IL6 gene transcription via TLR8 activation depends on active remodeling of chromatin at the IL6 genomic locus in human neutrophils, turning it from an ''inactive'' to an ''active'' configuration (Zimmermann et al, 2015).…”

“…No expression of ISGs was observed in R848-treated neutrophils (as illustrated by Figures 1F and S1D) because they do not upregulate type I IFN expression (Zimmermann et al, 2015(Zimmermann et al, , 2016. Furthermore, cluster 7 was found to also include genes encoding for chemokines specifically expressed in monocytes, such as CCL7, CCL8, and CCL15, as well as immunoregulatory cytokines, such as IL7, IL15, IL10, and IL27 (Figure 1F), the latter two molecules already known as not transcribed in R848-stimulated neutrophils (Cassatella et al, 2020;Zimmermann et al, 2015). Finally, the genes included in clusters 8 and 9 were found to be related to ''response to molecule of bacterial origin'' GO terms (Figure S1C) and listed many of the cytokines recently shown to be produced by R848-stimulated neutrophils (e.g., IL6, CSF3, EBI3, IL12B, IL23A, CCL4, and CCL23) (Cassatella et al, 2020;Tamassia et al, 2019;Tamassia et al, 2018;Zimmermann et al, 2015) other than monocytes.…”

“…Consequently, several clinical trials are currently ongoing to test the efficacy of TLR7/TLR8 ligands, either alone or in combination with monoclonal antibodies or chemotherapeutic agents, for the treatment of cancer or as adjuvants for vaccines (Chi et al, 2017). Our laboratory has recently demonstrated that R848, CL075 (Arruda-Silva et al, 2017;Zimmermann et al, 2015;Zimmermann et al, 2016), and VTX-2337 (Cassatella et al, 2020) act as very potent activators of human neutrophils, particularly for the production of proinflammatory cytokines. However, although important for a better comprehension of the activities of these drugs at a systemic level, accurate analysis of the transcriptional response of neutrophils to TLR8 ligands and its related molecular regulation has not yet been performed.…”

“…Furthermore, cluster 7 was found to also include genes encoding for chemokines specifically expressed in monocytes, such as CCL7, CCL8, and CCL15, as well as immunoregulatory cytokines, such as IL7, IL15, IL10, and IL27 (Figure 1F), the latter two molecules already known as not transcribed in R848-stimulated neutrophils (Cassatella et al, 2020;Zimmermann et al, 2015). Finally, the genes included in clusters 8 and 9 were found to be related to ''response to molecule of bacterial origin'' GO terms (Figure S1C) and listed many of the cytokines recently shown to be produced by R848-stimulated neutrophils (e.g., IL6, CSF3, EBI3, IL12B, IL23A, CCL4, and CCL23) (Cassatella et al, 2020;Tamassia et al, 2019;Tamassia et al, 2018;Zimmermann et al, 2015) other than monocytes. Other cytokine genes emerging from these latter clusters include CCL1, CCL2, CCL20, CXCL2, CXCL3, IL1B, IL1RN, IL36G, and TNF in cluster 8, and CCL18, CCL3, CCL3L1, CCL4L2, CXCL1, CXCL8, EDN1, and OSM in cluster 9.…”

Lack of evidence for expression and function of IL-39 in human immune cells

“…While it was shown in the literature that overexpressed murine p19 can be secreted as a standalone protein, the human p19 subunit could only be detected in the conditioned medium if it was complexed with IL-23p40 [17]. However, recent papers underscore the possibility that the IL-23A-p19 subunit can be secreted as a standalone protein as shown that activated neutrophils and monocytes not only secrete the IL-23 p19/p40 heterodimer in cell supernatants, but also the IL-23A-p19 subunit which was not complexed with EBI3 [20]. In addition, a recent publication described that epthelial cancer cells could secrete the p19 subunit without being complexed to p40 beta subunit [21].…”