Inactivation of DAP12 in PMN inhibits TREM1-mediated activation in rheumatoid arthritis

Abstract

Rheumatoid arthritis (RA) is an autoimmune disease characterized by dysregulated and chronic systemic inflammatory responses that affect the synovium, bone, and cartilage causing damage to extra-articular tissue. Innate immunity is the first line of defense against invading pathogens and assists in the initiation of adaptive immune responses. Polymorphonuclear cells (PMNs), which include neutrophils, are the largest population of white blood cells in peripheral blood and functionally produce their inflammatory effect through phagocytosis, cytokine production and natural killer-like cytotoxic activity. TREM1 (triggering receptor expressed by myeloid cells) is an inflammatory receptor in PMNs that signals through the use of the intracellular activating adaptor DAP12 to induce downstream signaling. After TREM crosslinking, DAP12's tyrosines in its ITAM motif get phosphorylated inducing the recruitment of Syk tyrosine kinases and eventual activation of PI3 kinases and ERK signaling pathways. While both TREM1 and DAP12 have been shown to be important activators of RA pathogenesis, their activity in PMNs or the importance of DAP12 as a possible therapeutic target have not been shown. Here we corroborate, using primary RA specimens, that isolated PMNs have an increased proportion of both TREM1 and DAP12 compared to normal healthy control isolated PMNs both at the protein and gene expression levels. This increased expression is highly functional with increased activation of ERK and MAPKs, secretion of IL-8 and RANTES and cytotoxicity of target cells. Importantly, based on our hypothesis of an imbalance of activating and inhibitory signaling in the pathogenesis of RA we demonstrate that inhibition of the DAP12 signaling pathway inactivates these important inflammatory cells.

Figure 1. Increased expression of TREM-1 in PMN from RA patients.

(A) PMN cells isolated from the synovial fluid of RA patients and healthy donors were analyzed by flow cytometry for the cell surface expression of TREM-1. A representative dot plot of CD66/TREM-1 expression is shown. (B) Representative figure of the expression of TREM-1 on the isotype stained cells, healthy PMN and the PMN from a primary RA specimen analyzed by flow cytometry. Legend shows the percent of total TREM 1 cells from the gate shown. (C) The same analysis shown in B was used to determine the relative levels of TREM-1 in isolated PMN from RA primary specimens compared with healthy controls (n = 19 RA and n = 6 controls). The MFI of all the samples was also obtained and averaged. However there were not significant changes (numbers shown). (D) Quantitative RT-PCR was used to determine the relative levels of TREM-1 in isolated PMN from RA primary specimens compared with healthy controls (n = 19 RA and n = 6 controls). (E) Surface expression of TREM-1 was compared between PMNs (blue), NK cells (red), T cells (green) and monocytes through flow cytometric analysis of isolated (n = 6). Error bars denote the standard error of duplicate determinations of 5 samples per group and p values are shown between figures except in E where it is shown as an asterisk to denote p = 0.0013 of normal versus RA while the other ones were not significant.

Figure 2. PMN cells from RA patients maintain constitutively activated ERK1/2.

(A) PMN cell lysates were prepared from RA patients and healthy donors and used to measure the activity of ERK by Western blot using antibodies specific for: phosphorylated or total ERK1/2. Concentrations of IL-8 (B), RANTES (C), IL-6 (D) and TNFα (E) were determined by ELISA from either the synovial fluid of RA patients or the serum of healthy donors. Error bars denote the SEM triplicate determinations of n = 19 RA samples and n = 6 controls, p values as shown. (F) Healthy PMN (n = 4) were isolated and cultured in the presence of either TNFα (1ng/ml), IL-6 (1ng/ml), or IL-17 (20ng/ml) for 24 and 48 hours before TREM1 analysis by flow cytometry (G) Healthy PMN (n = 4) were isolated and cultured in the presence of TNFα (0.01ug/ml to 0.1ug/ml) after the addition of TNFα blocking antibody for 24 and 48 hours before TREM1 analysis by flow cytometry. For both F and G error bars denote the SEM of duplicate readings of 4 treated primary healthy specimens. Asteriks denote p<0.05 against control untreated cells.

Figure 3. Increased activation of PMN cells from RA patients.

(A) Primary RA PMN were incubated with pre-fixed (1% paraformaldehyde) HTB-93 cells and collected at 0, 5, 15, 30 minutes and the lysate was run on a western blot and probed for the activity of ERK and AKT or total ERK/AKT as a loading control. (B) PMN cells isolated from the synovial fluid of RA patients and healthy blood donors were used in a cytotoxic assay with ⁵¹Cr-labeled HTB-93 cells. Percentage specific lysis of HTB-93 cells was compared using at effector to target (E:T) ratios of 50:1, 25:1,12.5:1, and 6.25:1. The mean and SD of triplicate determinations are shown.

Figure 4. Blocking DAP12 reduces the increased signaling and cytokine production brought on by increased expression of DAP12 in PMN from RA patients.

(A) Quantitative RT-PCR was used to determine the relative levels of DAP12 in isolated PMN from RA primary specimens (n = 19) compared with healthy controls (n = 6). Error bars denote the SEM of duplicate determinations per sample. P value shown. (B) Healthy PMN (n = 4) were isolated and cultured in the presence of either TNFα (1ng/ml), IL-6 (1ng/ml), or IL-17 (20ng/ml) for 24 and 48 hours before DAP12 analysis by flow cytometry. (C) Healthy PMN (n = 4) were isolated and cultured in the presence of TNFα after the addition of TNFα blocking antibody (0.01ug/ml to 0.1ug/ml) for 24 and 48 hours before DAP12 analysis by flow cytometry. For both B and C error bars denote the SEM of duplicate readings of 4 treated primary healthy specimens. Asterisk denote p<0.05 against control untreated cells. (D) HEK293 cells co-transfected with TREM-1 and either of the FLAG-tagged DAP12 constructs: wild type (WT, lanes 5, 6), or dnDAP12 (lane 7) were cross-linked with anti-TREM-1 antibody followed by western blot analysis for the activation of p42/p44 MAPK and compared against total ERK. Single transfections with TREM-1 and WTDAP12 are included as negative controls (lanes 3 and 4). Supernatants from this experiment were also used to analyze (E) IL-8 and (F) RANTES by ELISA were error bars denote the SEM of triplicate measurements of each sample and asterisk p<0.05 measured against * normal, ** Mock or ***CD56.

Figure 5. Expression of dnDAP12 in PMN cells from RA patients reduces adaptor signaling, cytotoxicity toward HTB-93 cells, and granule redistribution.

(A) The cytotoxic properties of PMN cells from RA patients infected with recombinant vaccinia virus encoding CD56 (negative control), dnDAP12, or dnMEK (positive control) were admixed at an E/T ratio of 50:1, 25:1, 12.5:1 and 6.25:1 with HTB-93 target cells and evaluated in 5-hour ⁵¹Cr release assays. Mock-infected PMN cells from RA patients and healthy controls are included for comparison. The mean and SEM of triplicate wells per sample are shown (n = 5). Asterisk denote a p value of less than 0.05 against normal untreated cells. (B) PMN and HTB-93 cells were mixed at 1:1 E/T ratio and examined for granule redistribution. Green shows granzyme B, red shows pre-stained HTB target cells with Cell-Tracker Orange, blue reflects nuclear staining with DAPI. Results using untreated PMN RA cells are shown at zero and 10 minutes after mixing. Representative results using PMN RA patients cells expressing CD56 (negative control), dnDAP12 are shown. Representative experiment shown out of a total of 5 with similar results.