NFAM1 promotes pro-inflammatory cytokine production in mouse and human monocytes.

NFAM1 is an ITAM bearing-transmembrane receptor. Based on overexpression studies, NFAM1 is reported to play a role in B cell signaling and development. We performed expression analysis of NFAM1 in publicly available gene expression data sets and discovered that NFAM1 expression is significantly induced in intestinal biopsies from Crohn’s disease (CD) and ulcerative colitis (UC) patients. At the cellular level, we observed high expression of NFAM1 in monocytes and neutrophils, in addition to expression in B and T cells. To explore the role of NFAM1 in multiple immune cells and its potential role in IBD, we generated NFAM1-/- mice. Contrary to previous reports using NFAM1-transgenic mice, NFAM1-/- mice have no obvious defects in immune cell development, or in T cell-dependent or independent B cell responses. Interestingly, NFAM1-/- monocytes produce reduced levels of TNF-α in response to activation by multiple IBD-relevant stimuli, including CD40L, TLR ligands and MDP. Collectively, these findings indicate that NFAM1 promotes pro-inflammatory monocyte development, thereby amplifying the response to diverse stimuli. These findings are not mouse-specific, as we found that deletion of NFAM1 in human monocytes also reduced expression of CD40L-induced CCL4. To better understand the impact of NFAM1 deletion of monocyte activation, monocytes isolated from human PBMCs were nucleofected with CRISPR ribonucleoprotein (RNP) complexes using an Amaxa 4D nucleofection system according to manufacturer’s instructions (Lonza). Knockout efficiency was assessed by western blot detection of NFAM1 protein or sanger sequencing of PCR amplified target genomic loci. We performed whole transcriptome analysis on monocytes that were cultured in the presence or absence of IFN-γ plus CD40L for 6 and 24 hours. NFAM1+/+ and NFAM1-/- monocytes were left unstimulated, or stimulated with IFN-γ plus 1 ug/mL MegaCD40L. 6 or 24 hours later, supernatant was removed, cells were resuspended in RNAlater, and RNA was isolated using an RNeasy Plus Mini kit. Library construction and sequencing were performed at Beijing Genomics Institute as follows. RNA was then fragmented and cDNA was synthesized using the Illumina Truseq polyA mRNA library preparation protocol (non-stranded). RNA-sequencing was performed on an Illumina HiSeq2000 sequencer to generate 101x2 pair-end reads.