CTRP-3 Regulates NOD1-mediated Inflammation and NOD1 Expression in Adipocytes and Adipose Tissue

The anti-inflammatory adipokine CTRP-3 might affect innate immune reactions such as NOD1. The impact of CTRP-3 on NOD1-mediated inflammation in adipocytes and monocytic cells as well as on NOD1 expression was investigated. Murine 3T3-L1 pre-adipocytes and adipocytes as well as human THP-1 monocyte-like cells were co-stimulated with the synthetic NOD1 agonist Tri-DAP and recombinant CTRP-3. Gonadal adipose tissue and primary adipocytes were obtained from a murine model carrying a knockout (KO) of CTRP-3 in adipocytes but not in stroma-vascular cells. Wildtype mice with lipopolysaccharide (LPS)-induced elevated NOD1 expression were treated with CTRP-3. Secreted inflammatory cytokines in cell supernatants were measured by ELISA and mRNA levels were quantified by RT-PCR. Pro-inflammatory chemokine and cytokine secretion (MCP-1, RANTES, TNFα) was induced by NOD1 activation in adipocytes and monocyte-like cells, and MCP-1 and RANTES release was effectively inhibited by pre-incubation of cells with CTRP-3. CTRP-3 also antagonized LPS-triggered induction of NOD1 gene expression in murine adipose tissue, whereas adipocyte CTRP-3 deficiency upregulated NOD1 expression in adipose tissue. CTRP-3 is an effective antagonist of peptidoglycan-induced, NOD1-mediated inflammation and of LPS-induced NOD1 expression. Since basal NOD1 expression is increased by adipocyte CTRP-3 deficiency, there have to be also inflammation-independent mechanisms of NOD1 expression regulation by CTRP-3.

It represents an adipokine with pleiotropic functions, including regulation of cell proliferation in different cell types [3,4] and predominantly beneficial metabolic and immunomodulatory effects [5][6][7]. Of note, previous studies have revealed the role of CTRP-3 as an endogenous LPS antagonist in adipose tissue and inhibitor of toll-like receptor (TLR)4-mediated inflammation in adipocytes and monocytes in vitro [6,8]. Furthermore, application of exogenous, recombinant CTRP-3 attenuated both local (adipose tissue) and systemic inflammation in a murine model of LPS-induced systemic inflammatory response syndrome (SIRS) [9]. Due to its inhibitory impact on pro-inflammatory TLR4 signaling, CTRP-3 has to be regarded as an adipokine linking metabolism, inflammation, and i m m u n i t y [ 10 ] , e s p e c i a l l y i n t h e c o n t e x t o f "metaflammation" [11].
In innate immunity, mechanisms of host defense against tissue damage and exogenous pathogens are largely based on various classes of pattern recognition receptors (PRRs) [12]. These receptors recognize microbial and pathogen-associated molecular patterns (PAMPs) as well as endogenous signals released by damaged cells, the socalled damage-associated molecular patterns (DAMPs) [13,14]. PRRs include TLRs, C-type lectin receptors (CLRs), retinoid acid-inducible gene-I-like receptors (RLRs), and NOD-like receptors (NLRs). While TLRs and CLRs represent transmembrane receptors with predominant plasma membrane and endosomal localization, RLRs and NLRs are mainly localized in cytoplasm [14]. Upon recognition of PAMPs or DAMPs, PRRs in immune cells induce a predominantly pro-inflammatory gene expression profile, including cytokines, chemokines, interferons, and anti-microbial peptides.
Most of the TLRs are expressed in adipocytes, and recent research has also provided evidence of functional TLR signaling with impact on adipocyte expression of immunomodulatory factors [15][16][17]. Besides TLRs, the role of the NLR nucleotide binding oligomerization domain containing 1 (NOD1) in fat cell biology and adipose tissue physiology has recently gained increasing attention. NOD1 recognizes fragments of bacterial peptidoglycans [18] and upon stimulation it mediates chemokine expression with subsequent immune cell recruitment [19]. Previous studies have shown promotion of adipose tissue inflammation, cellular insulin resistance [20], and impaired adipocyte differentiation [21] upon NOD1 activation. NOD1 stimulation by peptidoglycans has also been reported to induce adipocyte lipolysis [22], subsequently promoting inflammation by diacylglycerol accumulation [23]. Furthermore, Zhou et al. described increased NOD1 activity to be associated with the metabolic syndrome, thus representing a potential machinery of metabolic inflammation and development of insulin resistance [24].
Data on potential regulatory interrelations between immunomodulatory adipokines and NOD1 signaling are scarce. The hypothesis of the present study is based on the question whether CTRP-3 regulates adipocyte NOD1 expression and NOD1-mediated inflammatory gene expression. Therefore, it was the aim of the present study:

Y
to investigate the effects of NOD1 activation on MCP-1/RANTES/TNFα expression in murine pre-adipocytes and mature adipocytes as well as in human monocyte-like THP-1 cells, Y to study the effect of recombinant CTRP-3 on LPSinduced NOD1 expression in vivo, Y to elaborate whether deficiency of CTRP-3 in adipocytes affects basal NOD1 expression in total adipose tissue and in primary adipocytes.

Quantification of Secretory Protein Concentrations in Cell Culture Supernatants
Concentrations of cytokines in cell supernatants were measured in duplicates by ELISA (murine MCP-1: Biolegend; San Diego, USA; murine RANTES, human MCP-1, human TNFα: DuoSet ELISA development systems, R&D Systems, Wiesbaden, Germany). In Figs. 1 and 2, chemokine/cytokine concentrations in cell culture supernatants are expressed as relative values, and mean values ± standard error of the mean (SEM) are given. Measurement was generally repeated for exceptional samples exceeding a duplicate variation of 20%. The lower detection limits were 15.6 pg/ml for human MCP-1 and TNFα, 31.2 pg/ml for murine RANTES, and 62.5 pg/ml for murine MCP-1.

Adipose Tissue and Adipocyte mRNA Extraction
Total RNA was isolated from adipose tissue and cells as reported recently [26]. Briefly, cells and tissue were homogenized in TRIzol® Reagent (Life Technologies GmbH, Darmstadt, Germany) in combination with gentleMACS dissociator and M-tubes (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) for dissociation and RNA was isolated applying RNeasy® Mini Kit (Qiagen, Hilden, Germany) including DNase digestion (RNase-Free DNase Set, Qiagen, Hilden, Germany).

Quantitative Real-time PCR Analysis of NOD1 mRNA Expression
For gene expression analysis, reverse transcription of RNA (QuantiTect Reverse Transcription Kit from Qiagen, Hilden, Germany) was performed to generate corresponding cDNA for real-time PCR (RT-PCR) (iTaq Universal SYBR Green Supermix, CFX Connect RT-PCR system; Bio-Rad, Munich, Germany). NOD1 mRNA levels in murine adipocytes and adipose tissue were quantified using the following primer sequences: Expression levels of NOD1 were normalized to gene expression of murine GAPDH. All oligonucleotides used were purchased from Metabion, Martinsried, Germany.

Recombinant Expression of CTRP-3
Recombinant CTRP-3 protein expression was performed in H5 insect cells (Invitrogen, Karlsruhe, Germany) using the BacPak Baculovirus Expression System (BD Biosciences, Palo Alto, CA, USA) as published earlier [33]. Unlike prokaryotic expression systems, recombinant expression in insect cells usually maintains glycosylation and phosphorylation. Our expression system was proven to generate trimeric CTRP-3 [33]. High purity of recombinant CTRP-3 protein in the preparation was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis.

LPS-induced Systemic Inflammatory Response Syndrome (SIRS) Model
In male C57BL/6 wildtype mice (age 8-12 weeks; from Charles River, Sulzfeld, Germany), moderate inflammation was induced by intraperitoneal (i.p.) injection of lipopolysaccharide (LPS; 1 μg per animal) as described recently [26]. The animals were euthanized 2 h after LPS injection and intra-abdominal adipose tissue specimens were resected for gene expression analysis. This animal study was performed at the University of Regensburg, Germany, and was approved by the local government agency (Regierungspraesidium Oberpfalz, No. 54-2532.1-14/10).

Tissues from CTRP-3 Knockout Mice
Transgenic mice with an adipocyte-specific CTRP-3 k n o c k o u t ( f u l l n o m e n c l a t u r e : B 6 N T a c . C g -C1qtnf3 tm3113Arte Tg(Fabp4-cre)1Rev; abbreviation: CTRP-3 KO) together with littermate control mice (B6NTac.Cg-C1qtnf3 tm3113Arte ) were bred under standard conditions and were euthanized for organ and tissue resection. In this novel mouse model, CTRP-3 is completely lacking in mature adipocytes but not in the stromavascular cell fraction of adipose tissue. Intra-abdominal adipose tissue specimens were resected and were either used for primary cell isolation or otherwise shock-frosted in liquid nitrogen. Deletion of exon 4 and subsequent frame-shift mutations within the C1qtnf3 gene were introduced applying the Cre/loxP system, resulting in a dysfunctional gene product. Cell-type specificity of the knockout was achieved by transcriptional control of the Cre recombinase encoding transgene by the aP2-promotor (from Fabp4 gene). B6NTac.Cg-C1qtnf3 tm3113Arte mice carrying C1qtnf3 gene alleles with loxP-flanked exon 4 were created in collaboration with Taconic Artemis (Cologne, Germany). B6.Cg-Tg(Fabp4-cre)1Rev/J mice (purchased from Jackson Laboratories) were backcrossed to C57BL/6NTac genetic background applying the Speed Congenics method for at least 6 generations, resulting in the strain B6.Cg-Tg(Fabp4-cre)1Rev/N. This o f f s p r i n g w a s c r o s s e d w i t h C 5 7 B L / 6 N T a c -C1qtnf3tm3113Arte mice to generate B6NTac.Cg-C1qtnf 3tm3113Arte Tg(Fabp4-cre)1Rev mice with adipocyte CTRP-3 KO.

Isolation and Cell Culture of Primary Murine Adipocytes
For primary cell culture, fresh intra-abdominal adipose tissue obtained from CTRP-3 KO mice was cut into small pieces and treated with 0.225 U/ml collagenase NB6 (Serva) at 37°C for a maximum of 60 min. Digestion process was stopped by adding twice the amount of buffer (PBS containing 0.5% BSA and 2 mM EDTA). Cell suspension was filtered by 120-μm nylon mesh to eliminate undissolved tissue. Pre-adipocytes were separated from adipocytes by 10 min centrifugation at 300 g and 4°C.
Magnetic labeling plus depletion of non-adipocyte progenitor cells was done according to the manufacturer's instructions (Adipose Tissue Progenitor Isolation Kit mouse, MACS Miltenyi Biotec, Bergisch Gladbach, Germany), as well as magnetic labeling and positive selection of adipocyte progenitor cells. Isolated pre-adipocytes were seeded at a density of 2.03 × 10 4 cells/cm 2 in DMEM (Dulbecco's Modified Eagle Medium, Biochrom AG, Berlin, Germany) that was supplemented with 10% newborn calf serum (NCS; from Sigma-Aldrich, Deisenhofen, Germany) and were cultured at 37°C and 5% CO 2 . Adipocyte differentiation was initiated after cells had reached 85 % confluency. Media for hormonal differentiation were supplemented as described above for 3T3-L1 cell line. Cellular phenotype during adipocyte differentiation was monitored by light microscopy.

Statistical Analysis
For explorative data analysis, a statistical software package (SPSS 26.0) was used. Non-parametric numerical parameters were analyzed by the Mann-Whitney U-test (for 2 unrelated samples) and the Kruskal-Wallis test (>2 unrelated samples). A p-value below 0.05 (two-tailed) was considered as statistically significant. In the figures, the bars are showing the mean values and the whiskers are giving the standard error of the mean (SEM).

Effects of Intraperitoneal LPS and CTRP-3 Application on NOD1 Gene Expression in Intra-abdominal and Subcutaneous Adipose Tissue in Mice In Vivo
In C57BL/6 wildtype mice, intraperitoneal (i.p.) LPS injection (1 μg/animal) caused a significant increase of gonadal adipose tissue NOD1 gene expression (p = 0.001; Fig. 3A). This effect was significantly attenuated by pre-treatment of mice with recombinant CTRP-3 (10 μg/animal, i.p. injection) 30 min prior to LPS application (p = 0.048; Fig. 3B). In contrast to these findings, neither LPS alone (Fig. 3C) nor co-treatment with LPS and CTRP-3 (Fig. 3D) had a significant impact on NOD1 expression in subcutaneous adipose tissue.

Elevated NOD1 Gene Expression in Adipose Tissue and Primary Adipocytes from Mice with Adipocytespecific CTRP-3 Deficiency
Gene expression analysis in intra-abdominal adipose tissue of CTRP-3 KO mice and CTRP-3 positive control animals (littermates) revealed higher NOD1 mRNA levels in KO animals (p = 0.018; Fig. 4A). The promotion of NOD1 transcription in total adipose tissue by adipocyte CTRP-3 deficiency might indicate inhibitory effects of CTRP-3 on basal NOD1 gene expression.
Primary pre-adipocytes were isolated from intraabdominal adipose tissue of CTRP-3 KO and control mice and were differentiated into fat cells in vitro. After 9 days, when cells exhibited a mature adipocyte phenotype, mRNA was isolated and NOD1 expression was quantified. In adipocytes derived from CTRP-3 deficient cells, a considerable, yet non-significant trend of elevated NOD1 mRNA levels was observed (p = 0.065; Fig. 4B).

DISCUSSION
The role of adipocytes and their secretory proteins in various processes of innate immunity such as TLRs and PRRs is a novel and complex aspect of adipose inflammation or even metaflammation. Regarding the immunomodulatory functions of the adipokine CTRP-3, its role as endogenous LPS antagonist and anti-inflammatory adipokine-at least in adipose tissue-has been described [6,10]. Outside adipose tissue physiology, general and supra-physiological overexpression of CTRP-3 failed-in contrast-to demonstrate an anti-inflammatory capacity of CTRP-3 [34]. Recent studies have provided evidence of active and functional NOD1 signaling pathways in adipocytes affecting fat cell differentiation, metabolism, and inflammation [20][21][22], whereas an impact of the closely related NLR NOD2 on adipocyte biology and adipose inflammation seems rather questionable so far [21,24,35] and requires further research applying adequate clinical studies and experimental/genetic models in the context of metaflammation. To the best of our knowledge, the present study is the first to investigate CTRP-3 effects on peptidoglycan-triggered and NOD1-mediated inflammation as well as NOD1 gene expression. NOD1 activation by the synthetic diaminopimelic acid derivative Tri-DAP in murine 3T3-L1 fibroblasts/pre-adipocytes as well as in mature adipocytes strongly induced the secretion of the chemokines MCP-1 and RANTES in vitro. This proinflammatory reaction was significantly attenuated by costimulation with exogenous and recombinant CTRP-3. It is reasonable to build the hypothesis that inhibition of NOD1mediated expression and secretion of chemoattractant proteins by adipocytes might antagonize immune cell recruitment to adipose tissue as well as pro-inflammatory activation of tissue-resident macrophages. In a subsequent experimental approach, human THP-1 cells were stimulated with Tri-DAP, resulting in increased MCP-1 and TNFα release. Similar to the observations in 3T3-L1 adipocytes, the strong induction of MCP-1 secretion was significantly attenuated by co-stimulation of these monocyte-like cells with CTRP-3, whereas TNFα release was not significantly affected. Previous studies have suggested CTRP-3 as an important paracrine, immunomodulatory factor in the context of an inflammatory adipocyte-macrophage cross-talk [10,36]. The present data suggest that CTRP-3 also affects analogous cellular interactions in peptidoglycan-induced, NOD1-dependent adipose tissue inflammation. Although primarily descriptive, our findings are new and consistent and have been evaluated in human and murine cell lines in vitro and in vivo by using two sophisticated murine models (LPS-induced SIRS model and a model of CTRP-3 knockout in adipocytes). However, subsequent mechanistic studies are needed to investigate the involved intracellular signal transduction components and pathways of both, NOD1 activation and CTRP-3-induced inhibition.
Besides NOD1 activation, NOD1 expression in adipocytes and adipose tissue has been poorly described so far. During LPS-induced moderate inflammation, mice exhibited strongly increased NOD1 mRNA expression in intra-abdominal adipose tissue. This effect was absent in subcutaneous adipose tissue and therefore appears to be specific for the intra-abdominal fat compartment. In animals that had been treated with recombinant CTRP-3 30 min prior to LPS treatment, elevated intra-abdominal adipose tissue NOD1 expression was significantly attenuated. Vice versa and in accordance with this observation, transgenic mice with adipocyte CTRP-3 knockout exhibited significantly elevated basal NOD1 mRNA levels in intra-abdominal total adipose tissue. Furthermore, there was a considerable, yet non-significant trend of increased Based on the present data, future experimental approaches will have to gain a more precise insight in mechanisms underlying the antagonizing effects exerted by CTRP-3, including detailed analysis of involved signaling pathway components and genetic models, especially for cell-type-specific NOD1 deficiency. Furthermore, a potential immune-regulatory role and regulatory interaction with CTRP-3 should be investigated for NOD1-related NLRs, such as NOD2, as well as a putative specificity of the observed effects for different adipose tissue compartments.

CONCLUSIONS
CTRP-3 is an effective antagonist of proinflammatory NOD1 activation in adipocytes as well as in monocyte-like cells. Furthermore, it appears to inhibit NOD1 expression in adipocytes and in adipose tissue ex vivo and in vitro. Future mechanistic studies will have to investigate in detail the pathways of NOD1 activation and CTRP-3 inhibition in adipose tissue in specific disease contexts.

ACKNOWLEDGEMENTS
The laboratory work of Lisa Knüpfer is highly appreciated. The technical and scientific advice of Frank Hanses (University of Regensburg, Germany) is highly appreciated.

FUNDING.
Open Access funding enabled and organized by Projekt DEAL. The present study was supported by grants of the German Research Association (DFG) (DFG Sachbeihilfe; SCHM 3261/3-1 and KA 1846/4-1).

AVAILABILITY OF DATA AND MATERIAL
All data and material included in the present study are available. Fig. 4. Impact of adipocyte CTRP-3 deficiency on NOD1 gene expression in adipose tissue and primary adipocytes. Intra-abdominal adipose tissue was obtained from control mice and mice with an adipocyte-specific CTRP-3 knockout. Primary pre-adipocytes were derived from intra-abdominal adipose tissue and were differentiated to mature adipocytes ex vivo. NOD1 gene expression levels were quantified via RT-PCR and normalized to GAPDH gene expression. Ctrl., control mice; CTRP-3, C1q/TNF-related protein-3; KO, knockout mice; TD, Tri-DAP. A NOD1 gene expression levels are elevated in intra-abdominal adipose tissue from female CTRP-3 KO mice (n = 17) when compared to littermate controls. B NOD1 gene expression levels are not significantly altered in primary intra-abdominal adipocytes from female CTRP-3 KO mice (n = 6).

N/A.DECLARATIONS
Ethics Approval. Concerning all analyses on CTRP-3 knockout mice performed within in the present study, an announcement was made at the local government agency (Regierungspraesidium Giessen) conformable to §4 Abs. 3 Tierschutzgesetz. Internal project identification code 544_M was assigned to the project at the University of Giessen.
Animal experiments on LPS-induced SIRS model were performed at the University of Regensburg, Germany, and were approved by the local government agency (Regierungspraesidium Oberpfalz, No. 54-2532.1-14/10).

Consent for Publication. N/A.
Conflict of Interest. The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.