Monocyte TREM-1 Levels Associate With Anti-TNF Responsiveness in IBD Through Autophagy and Fcγ-Receptor Signaling Pathways

The expression of Triggering Receptor Expressed on Myeloid cells (TREM)-1 has been described as a predictive marker for anti-Tumor Necrosis Factor (TNF)-α monoclonal antibody (mAb) therapy responsiveness in patients with inflammatory bowel disease (IBD). Here we investigated expression of TREM-1 specifically in CD14+ monocytes in relation to anti-TNF response. The pretreatment TREM-1 expression levels of CD14+ monocytes of Crohn’s disease (CD) patients were predictive of outcome to anti-TNF mAb therapy, with low TREM-1 expression associated with response to anti-TNF. FACSorting of CD14+ monocytes with different TREM-1 levels showed that differentiation towards regulatory CD206+ M2 type macrophages by anti-TNF was suppressed in CD14+ monocytes with high TREM-1 expression. Activity of the Fcγ-Receptor and autophagy pathway, both necessary for M2 type differentiation and the response to anti-TNF, were decreased in CD14+ monocytes with high expression of TREM-1. We confirmed that the activity of the Fcγ-Receptor pathway was decreased in the CD patients that did not respond to anti-TNF therapy and that it was negatively correlated with TREM-1 expression levels in the CD patient cohort. In conclusion, our results indicate that TREM-1 expression levels in CD14+ monocytes associate with decreased autophagy and FcγR activity resulting in decreased differentiation to M2 type regulatory macrophages upon anti-TNF mAb treatment, which may explain anti-TNF non-response in IBD patients with high expression levels of TREM-1.


INTRODUCTION
Despite the considerable efficacy of anti-Tumor Necrosis Factor (TNF)-a monoclonal antibody (mAb) therapy in the treatment of inflammatory bowel disease (IBD), about one third of patients do not respond (1). The reason for this primary non-response is not entirely understood, therefore the search for predictive markers has been hampered considerably (2). In latest years, several research groups described the association of the expression levels of several genes in the intestine in relation to anti-TNF response in IBD (3)(4)(5)(6)(7)(8). However due to the invasiveness of intestinal biopsies and patient's preference prognostic tests on blood samples would be favorable for clinical practice. Therefore, after determining the association of the intestinal expression of Triggering Receptor Expressed on Myeloid cells (TREM)-1 with response to anti-TNF therapy, Gaujoux et al. (3) investigated the association of whole blood TREM-1 expression and anti-TNF response, and reported that low whole blood TREM-1 levels predict anti-TNF non-responsiveness. In contrast, Verstockt et al. (9,10) reported exactly the opposite, high whole blood TREM-1 levels predict anti-TNF non-responsiveness. Also an association was found between the soluble TREM-1 (sTREM-1) levels in serum and anti-TNF response, with high levels being associated with anti-TNF non-response (9). Although the studies defined response to anti-TNF differently, the explanation for the opposite association with anti-TNF response was not completely clear.
Over the last decade we have shown that anti-TNF mAbs interact with Fc Receptors on myeloid cells, and that this interaction is crucial for the therapeutic efficacy in IBD (11)(12)(13). Through this Fc-Receptor interaction anti-TNF mAbs skew monocytes towards CD206+ macrophages (13,14), exhibiting increased levels of autophagy (15). As TREM-1 is selectively expressed on monocytes and neutrophils in whole blood (16), and monocytes/myeloid cells are critical in the response to anti-TNF (11,13,14,17), we investigated whether the TREM-1 expression in CD14+ monocytes is related to future anti-TNF response, and what could be the functional link between the two.
Upon the addition of anti-TNF or isotype control (10 µg/ml) the LR12 was refreshed and every 24 hours afterwards until a total of 5 days of culture. 6-thioguanin (6TG, 25 µM) was dissolved in dimethylsulfoxide (DMSO) and added together with the anti-TNF with DMSO as control as reported before (18). Monocytes were cultured for 24 hours with or without LPS (100 ng/ml) in combination with LR12 or the agonistic TREM-1 antibody and TNFa production was measured in supernatant by ELISA (DY210, R&D systems).

Crohn's Disease Patient Study
All patients had given written consent to participate in the Institutional Review Board approved IBD Biobank of University Hospitals Leuven, Belgium (B322201213950/ S53684), Twenty-four anti-TNF naïve patients with Crohn's disease (CD), initiating anti-TNF therapy because of active endoscopic disease (presence of ulcerations), were enrolled. Prior to treatment initiation, a 20 ml blood sample was taken, and PBMCs were isolated by density centrifugation. After cell isolation, samples were cryopreserved with DMSO using Mr Frosty (Thermo Fisher Scientific, Waltham, Massachusetts, USA) for 24 hours and afterwards stored in liquid nitrogen. Endoscopic remission was assessed at 6 months after therapy initiation, and defined as a complete absence of ulcerations (19). Patient characteristics (17 responders, 7 non-responders) are depicted in Table 1.

Microarray
For microarray analysis of the TREM-1 low and TREM-1 high CD14+ monocytes the RNA quality (integrity and concentration) was measured on an Agilent 2100 Bioanalyzer. All samples had a RNA integrity number (RIN) above 9,0 and concentration above 250 pg/µl. Microarray analysis was performed using a Clariom S Pico Human Assay (#902929, Thermo Fisher Scientific) using a Gene Titan MC system (Affymetrix) according to the standard protocols of the Dutch Genomics Service and Support Provider (MAD, University of Amsterdam, Netherlands). The data was normalized using robust multiarray analysis and analyzed by Transcriptome Analysis Console software (Thermo Fisher Scientific).

TREM-1 Expression in CD14+ Monocytes Predicts Anti-TNF Response
Pretreatment TREM-1 expression levels of CD14+ monocytes in peripheral blood of CD patients were predictive of outcome to anti-TNF mAb therapy, with low TREM-1 expression being associated with response to anti-TNF ( Figure 1A). When analyzing the expression of the different TREM-1 isoforms we found that TREM-1_MB, the full TREM-1 receptor isoform expressed on the cell membrane, was significantly increased in CD14+ monocytes of anti-TNF non-responders compared to responders, while the other TREM-1 isoforms,TREM-1_SV and TREM-1_X2, tended to be increased significantly ( Figure 1B). The serum sTREM-1 levels of these CD patients [partly published in (9)] were not significantly different between anti-TNF non-responders compared to responders (Supplementary Figure S1A). The serum sTREM-1 levels tended to correlate with the expression levels of TREM-1_SV, the specific splice variant that encodes sTREM-1 (20) (Supplementary Figure S1B). The absence of a correlation with total TREM-1 or TREM-1_MB expression levels (Supplementary Figure S1C), that were predictive of anti-TNF response, indicate that circulating sTREM-1 levels do not directly contribute to the found association with anti-TNF response.

TREM-1 Expression Associates With Regulatory Macrophage Differentiation In Vitro
Differentiation of CD14+ monocytes to regulatory CD206+ M2 type macrophages, mediated through Fcg-Receptor signaling, is part of the mechanism of action of anti-TNF mAbs in IBD (11,13,14). As especially the full TREM-1 receptor isoform, TREM-1_MB, was increased in CD14+ monocytes of anti-TNF nonresponders compared to responders we hypothesized this full receptor form might be involved in the suppression of differentiation towards CD206+ M2 macrophages, hence suppress the response to anti-TNF. The anti-TNF induced differentiation of CD14+ monocytes can be studied quite simple in vitro by using a mixed lymphocyte reaction (MLR), an allogenic immune reaction created by mixing peripheral blood mononuclear cells (PBMCs) of two different donors, as extensively reported earlier (11,13,14). To investigate the relation between TREM-1 levels and CD14+ monocyte differentiation, we FACSorted CD14+ monocytes with different TREM-1 cell surface levels (low vs high, see Figure 1C), and cultured them with T-cells of a different donor, creating a allogenic immune reaction. As expected, in these sorted populations the TREM-1 expression levels were significantly decreased in the low versus high population, especially the full receptor form TREM-1_MB ( Figure 1D). The CD14+ monocytes with low TREM-1 expression levels were most capable of polarizing towards CD206+ regulatory macrophages in the presence of anti-TNF mAbs ( Figures 1E, F).

TREM-1 Signaling Does Not Suppress Regulatory Macrophage Differentiation
As low TREM-1 levels are associated with increased induction of M2 differentiation by anti-TNF mAbs we wondered whether TREM-1 signaling would interfere with the induction of differentiation by anti-TNF mAbs. To test this we added a TREM-1 antibody with agonistic activity (16,21) to anti-TNF mAbs in the MLR. There was no effect on the induction of CD206+ macrophages by anti-TNF by the agonist TREM-1 antibody (Figure 2A). The expression of TREM-1 was increased in the CD206+ macrophages that were induced by anti-TNF, and the detection of TREM-1 by flow cytometry was blocked by binding of the agonistic TREM-1 antibody ( Figure  2B). To functionally show this antibody has TREM-1 agonistic activity monocytes were stimulated and released TNFa upon stimulation ( Figure 2C), as reported (16,21). The opposite experiment, in which we inhibited TREM-1 signaling, by the TREM-1 inhibitor LR12 (22, 23) also did not affect the anti-TNFinduced differentiation of CD14+ monocytes towards CD206+ macrophages in the MLR ( Figure 2D). In this experiment 6thioguanin (6TG) synergized with anti-TNF in the induction of CD14+CD206+ cells (Figure 2A), as we reported before (18), showing that the effect of anti-TNF could be enhanced in this experimental set-up. To show LR12 was effective in blocking TREM-1 signaling in the tested dose we again investigated TNFa production of monocytes and found LR12 was capable of decreasing TNFa production upon stimulation with LPS ( Figure 2E) as reported (24). These experiments show that it is not TREM-1 signaling itself that is responsible for the different anti-TNF-induced differentiation of low versus high TREM-1 CD14+ monocytes, indicating something underlying discriminates these different populations of monocytes.

TREM-1 Expression Associates With Autophagy
We tried to elucidate which underlying pathway(s) is/are responsible for the found difference in anti-TNF-induced differentiation between low versus high TREM-1 CD14+ monocytes. Therefore we performed gene expression analysis on FACSorted TREM-1 low versus TREM-1 high CD14+ monocytes (Supplementary Figure S1). Gene Set Enrichment Analysis (GSEA) revealed that 14 of the 15 autophagy pathways were enriched in the low TREM-1 population versus the TREM-1 high population ( Figure 3A/Supplementary Figure S2). The relation between TREM-1 and autophagy in IBD has been described before (25), and as we found that autophagy levels are increased upon the induction of regulatory type macrophages by anti-TNF we decided to investigate this further by  (25 µM) or DMSO control. TNFa production by monocytes stimulated for 24 hours with the agonistic TREM-1 antibody (10 µg/ml) in (C) and with LPS (100 ng/ml) in combination with LR12 (50 µg/ml) in (E). Representative (n=3-5 per condition) of 3 to 4 different independent experiments using different donors. Significance was determined by ANOVA followed by Sidak's multiple comparison test in A/B/D and unpaired t-test in C/E with *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.
immunoblotting for LC3. Indeed we found that LC3II/LC3I ratios, indicative of autophagy, were significantly lower in TREM-1 high monocytes compared to their TREM-1 low counterparts ( Figures 3B-D). Levels of Cathepsin S, an autophagy related protein which was identified as an inducer of regulatory type macrophages by anti-TNF, was significantly lower in the TREM-1 high versus low monocytes ( Figures 3B, E), although the CTSS gene expression levels were not significantly different ( Figure 3F). Lower levels of autophagy and Cathepsin S in CD14+ monocytes with high levels of TREM-1 could explain the reason for lower induction of regulatory type macrophages by anti-TNF.

TREM-1 Expression Associates With Activation of the FcgR Pathway
Besides the autophagy pathway also the FcgR pathway was enriched in the TREM-1 low population (Supplementary Figure S3), which became even more apparent when we compared paired samples (so low vs high TREM-1 samples of the same individual, Figure 4A), We confirmed the differential FcgR protein levels in TREM1-low vs TREM-1 high CD14+ monocytes by flow cytometry ( Figure 4B). The activating FcgR gene transcript FCGR3A, essential for the induction of regulatory type macrophages by anti-TNF (13), was increased in low vs high TREM-1 expressing CD14+ monocytes ( Figure 4A), while the inhibitory receptor FCGR2B was decreased in low vs high TREM-1 expressing CD14+ monocytes. Recently Castro-Dopico et al. (26) used the expression levels of FCGR3A and FCGR2B, to calculate a FcgR activating:inhibitory (A:I) ratio, and showed that this FcgR A:I ratio is altered in IBD. Calculating this FcgR A:I ratio showed it was significantly increased in TREM-1 low versus TREM-1 high CD14+ monocytes (Figures 4C, D). Indeed when investigating this FcgR A:I ratio in the CD14+ monocytes of the CD patients we found it was significantly higher in the patients that responded to anti-TNF ( Figure 4E), and was negatively correlated with the TREM-1 levels in the CD14+ monocytes of the CD patients ( Figure 4F).

DISCUSSION
The whole blood TREM-1 expression has been described as a predictive marker for anti-TNF mAb therapy responsiveness in patients with IBD (3, 9, 10) Interestingly, while both Gaujoux et al. (3) and Verstockt et al. (10) describe TREM-1 expression consistently being upregulated in intestinal biopsies of anti-TNF non-responders compared to anti-TNF responders, the high TREM-1 expression in whole blood was only found to associate with anti-TNF non-response in the study by Verstockt et al. (10), in contrast to the study by Gaujoux et al. (3) that showed exactly the opposite association. Although the difference in patient populations, difference in response criteria and the different primers used to determine TREM-1 expression could explain the found differences, mainly the lack of understanding the underlying biological mechanism of the association of whole blood TREM-1 expression specifically with anti-TNF response makes its use in clinical practice questionable.
Here we set out to investigate the reason for the predictive value of TREM-1 expression in whole blood for anti-TNF responsiveness in IBD. Therefore we first analyzed the sole expression of TREM-1 in CD14+ monocytes, one of the main cell types in blood that expresses TREM-1 abundantly (16) and is important in mediating efficacy of anti-TNF therapy (11,13,14,17). We found that low TREM-1 levels in CD14+ monocytes isolated before anti-TNF treatment were associated with response to anti-TNF. Deciphering the reason for this association we investigated the functional consequence for CD14+ monocytes to express low or high levels of TREM-1. TREM-1 protein levels on the cell surface of CD14+ monocytes were found to correlate with their ability to differentiate into M2 macrophages upon anti-TNF mAb treatment, while TREM-1 signaling was not involved. Characterization of underlying pathways revealed that the autophagy pathway was increased in the CD14+ monocytes with low TREM-1 levels which we confirmed on the protein level. The association between TREM-1 signaling and autophagy in IBD has been described (25), and is associated with the induction of regulatory macrophages by anti-TNF (15). In addition, the activating FcgR pathway was increased in the CD14+ monocytes with low TREM-1 levels, which we have identified to be crucial for the response to anti-TNF (13). Moreover, not only were the FcgR activity levels in CD14+ monocytes of the CD patients negatively correlated with the TREM-1 levels, also the high pretreatment FcgR activity levels were predictive of response to anti-TNF. As we identified both pathways earlier to be essential for the induction of M2 macrophages by anti-TNF mAb therapy, this might explain the found differences. It would indicate that CD14+ monocytes with high TREM-1 levels have a lower FcgR activation threshold signaling when an anti-TNF-TNF immune complex is encountered, important for the response (11,13), while decreased autophagy levels would contribute to the suppression of the subsequent differentiation towards regulatory type macrophages. Another mechanism of action of anti-TNF therapy that has been proposed is the induction of Tce ll a p o p t os i s thr o u gh v a ri ou s m ec h a nis m s ( 2 7 ). Transmembrane TNFa (mTNFa) expressed on macrophages can protect T-cells from undergoing apoptosis through interaction with the TNF-Receptor 2 (TNF-R2), and interference of this signal by anti-TNF induces T-cell apoptosis (17). Apoptosis resistant T-cells expressing both TNF-R2 and IL-23 Receptor were significantly more present the intestine of anti-TNF non-responders and could be activated by IL-23 produced by CD14+ macrophages (28). Although we have not specifically investigated these pathways we did not detect differences in TNFA or IL23 expression in relation to TREM-1 expression in the CD14+ monocytes (not shown). In conclusion, our results indicate that TREM-1 expression levels in CD14+ monocytes  Figure 1D in (D) (n=4). The FcgR A:I ratio of the CD14+ monocytes from CD patients (n=24) before the start of anti-TNF therapy (E) and in correlation to the TREM-1 expression levels (G). Mean + SEM is shown in (D), whiskers indicate the 10-90 percentiles in (F). Significance was determined by Wilcoxon signed rank tests (B, C), a paired t-test (D), and Mann Whitney (E) with *P < 0.05, ***P < 0.001. associate with both decreased FcgR activity and autophagy levels resulting in decreased differentiation to M2 type regulatory macrophages upon anti-TNF mAb treatment, which may explain anti-TNF non-response in IBD patients with high expression levels of TREM-1.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi. nlm.nih.gov/, GSE165522.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Institutional Review Board approved IBD Biobank of University Hospitals Leuven, Belgium (B322201213950/ S53684) and The Medical Ethical Committee of the Amsterdam UMC, METC 2009_113). The patients/participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
MP, BV, MW and PK performed experiments and analyzed and discussed data. MF and SV provided patient samples and discussed data. PK and MW supervised the study. MP and PK wrote the manuscript with input of all authors. All authors contributed to the article and approved the submitted version.