S. M. Langness1, B. Eliceiri1, R. Coimbra1, A. Baird1, T. W. Costantini1 1University Of California – San Diego,Trauma, Surgical Critical Care, Burns And Acute Care Surgery,San Diego, CA, USA
Introduction: In humans, the alpha-7 nicotinic acetylcholine receptor (a7nAChR) is encoded by the CHRNA7 gene and mediates an anti-inflammatory response to injury and infection. However, the human genome also encodes a uniquely human gene called CHRFAM7A that is an antagonist to a7nAChR-mediated signaling. Several studies have suggested that in humans, the ratio of CHRNA7 to CHRFAM7A modulates receptor activity. We have previously shown that: a) CHRFAM7A is highly expressed in human leukocytes; b) the ratio of CHRNA7 to CHRFAM7A gene expression in leukocytes varies widely amongst individuals; and c) CHRFAM7A is biologically active in the human THP1 pre-monocyte cell line. Here, we postulated that PMA differentiation of these monocyte precursors to monocytes might alter the CHRNA7 to CHRFAM7A ratio and alter monocyte gene expression. The effects of preventing a change in differentiation-induced CHRFAM7A gene expression was evaluated by its constitutive expression using gene delivery.
Methods: THP-1 cells were used in all experiments and monocyte differentiation was induced by incubation with PMA for 3 days in culture. Cell appearance and viability was assessed by light microscopy and flow cytometry respectively. CHRNA7 and CHRFAM7 gene expression was measured by quantitative RT-PCR. Constitutive expression of CHRFAM7A was achieved with lentiviral transfection prior to PMA-induced differentiation. Gene expression in transfected CHRFAM7A and GFP-transduced differentiated monocytes were compared using RNA-seq with subsequent pathway analyses.
Results: CHRFAM7A expression was decreased by 70% in PMA-differentiated monocytes compared to untreated THP-1 cells. In contrast, there was no difference in CHRNA7 expression resulting in a net 5-fold increase in the expression of CHRNA7 compared to CHRFAM7A (see Figure). Over-expression of CHRFAM7A in differentiated monocytes produced viable cells with monocytic phenotype based on microscopic appearance and the presence of monocyte cell surface markers like CD14, CD22 and CD163. In contrast, the constitutive expression of CHRFAM7A in differentiated monocytes changed the expression of genes in pathways traditionally associated with regulating inflammatory response signaling and antigen presentation.
Conclusions: The ratio of CHRNA7 to CHRFAM7A gene expression in THP1 cells changes after differentiation to monocytes. Preventing this change by gene transfection, CHRFAM7A has no effect on differentiation but alters the expression of genes that mediate the inflammatory response. Because CHRFAM7A is unique to the human genome, these data support the existence of a human-specific mechanism that regulates a7nAChR anti-inflammatory signaling and hence, the human immune response to injury and infection.
