Regulation of Cathelicidin Antimicrobial Peptide (CAMP) Gene Expression by TNFα and cfDNA in Adipocytes

Understanding the complex interactions between metabolism and the immune system (“metaflammation”) is crucial for the identification of key immunomodulatory factors as potential therapeutic targets in obesity and in cardiovascular diseases. Cathelicidin antimicrobial peptide (CAMP) is an important factor of innate immunity and is expressed in adipocytes. CAMP, therefore, might play a role as an adipokine in metaflammation and adipose inflammation. TNFα, cell-free nucleic acids (cfDNA), and toll-like receptor (TLR) 9 are components of the innate immune system and are functionally active in adipose tissue. The aim of the present study was to investigate the impact of TNFα and cfDNA on CAMP expression in adipocytes. Since cfDNA acts as a physiological TLR9 agonist, we additionally investigated TLR9-mediated CAMP regulation in adipocytes and adipose tissue. CAMP gene expression in murine 3T3-L1 and human SGBS adipocytes and in murine and human adipose tissues was quantified by real-time PCR. Adipocyte inflammation was induced in vitro by TNFα and cfDNA stimulation. Serum CAMP concentrations in TLR9 knockout (KO) and in wildtype mice were quantified by ELISA. In primary adipocytes of wildtype and TLR9 KO mice, CAMP gene expression was quantified by real-time PCR. CAMP gene expression was considerably increased in 3T3-L1 and SGBS adipocytes during differentiation. TNFα significantly induced CAMP gene expression in mature adipocytes, which was effectively antagonized by inhibition of PI3K signaling. Cell-free nucleic acids (cfDNA) significantly impaired CAMP gene expression, whereas synthetic agonistic and antagonistic TLR9 ligands had no effect. CAMP and TLR9 gene expression were correlated positively in murine and human subcutaneous but not in intra-abdominal/visceral adipose tissues. Male TLR9 knockout mice exhibited lower systemic CAMP concentrations than wildtype mice. CAMP gene expression levels in primary adipocytes did not significantly differ between wildtype and TLR9 KO mice. These findings suggest a regulatory role of inflammatory mediators, such as TNFα and cfDNA, in adipocytic CAMP expression as a novel putative molecular mechanism in adipose tissue innate immunity.


Introduction
The worldwide prevalence of obesity and metabolic syndrome is continuously increasing [1][2][3].Metabolic syndrome is one of the main risk factors for cardiovascular diseases [4] that are the number one cause of death in public health statistics [5].Its underlying complex interactions have not been fully elucidated until now.In particular, chronic low-grade inflammatory state, adipose inflammation, and metaflammation in obesity are assumed to play a crucial role in the development of local and systemic insulin resistance, cardiovascular diseases, and impaired immune responses [4].Recently published data argue for a role of adipose tissue innate immunity in regulating the molecular interface of metabolism and inflammation [6,7].
Cathelicidin antimicrobial peptide (CAMP) represents an immunomodulatory peptide mainly secreted by immune cells such as monocytes, macrophages, and lymphocytes [8] with a crucial role in the innate immune system.CAMP enhances monocyte [9] and granulocyte [10] infiltration at sites of local inflammation by chemoattractant action and alters the immune response in the course of infection via toll-like receptor (TLR) modulation [11].In bacteria, CAMP causes a loss of membrane integrity by increased permeability, leading to the death of microbial cells [12].
In 2015, for the first time, Zhang et al. described CAMP as being produced by adipocytes [13], thereby introducing CAMP as a novel adipokine.During subcutaneous Gram-positive infection with Staphylococcus aureus, CAMP secreted by adipocytes inhibits bacterial growth and is an important protective component of subdermal adipose tissue against bacterial infection [13].In mice, diet-induced obesity results in the loss of a dermal pool of preadipocytes and thus inhibits the capacity to initiate reactive adipogenesis and to express CAMP [14].In humans, CAMP gene expression is higher in subcutaneous than in visceral adipose tissues [15].However, further data on the precise function and regulation of CAMP in adipocytes are sparse.
In obesity, the death of adipocytes leads to an increase of cell-free nucleic acids (cfDNA) in adipose tissue and plasma [18], and adipose tissue-resident macrophages are involved in the recognition of these elevated cfDNA levels via TLR9 [18].TLR9 is a well-characterized intracellular receptor [19] recognizing non-methylated CpG DNA motifs derived from bacteria [20], viral double-stranded DNA (dsDNA) [21], and cfDNA as a signal for severe tissue damage inside the organism [18].Activation of TLR9 leads to an increased production of proinflammatory cytokines [22], and TLR9 signaling in macrophages is involved in obesity-induced insulin resistance [23].Of note, Nakagawa et al. demonstrated that CAMP is required for normal TLR9 function in dendritic cells and macrophages [24].Interestingly, adipocytes themselves express functionally active TLR9 [25].Whether CAMP plays an additional role in TLR9 modulation in adipocytes has not been elucidated so far.A possible interaction between CAMP expression, TNFα, and cfDNA in adipocytes and adipose tissues might provide a novel molecular interface of obesity-related inflammation with adipocyte function.
Therefore, the primary aim of the present study was to investigate: 1.
The underlying mechanisms involved in TNFα-mediated regulation of CAMP expression in adipocytes with respect to components of established proinflammatory signaling pathways; 2.
The potential effects of cfDNA, representing an inflammatory mediator in adipose tissue and a physiological TLR9-agonist, on CAMP gene expression in murine 3T3-L1 adipocytes; 3.
Circulating CAMP concentrations as well as local CAMP gene expression in adipose tissue and primary adipocytes of TLR9 knockout mice compared to wildtype mice.

CAMP Gene Expression Is Induced during Differentiation in Murine 3T3-L1 Adipocytes and in Human SGBS Cells
During the differentiation process, 3T3-L1 adipocytes phenotypically turn from fibroblastic preadipocytes into mature adipocytes with multiple lipid vacuoles within 8 days.The pattern of adipokine expression and secretion varies depending on the stage of differentia-tion.In early stage 3T3-L1 preadipocytes, at days 0 and 3 of differentiation, no significant CAMP gene expression was detected.In later stages, at day 6 and in mature adipocytes (day 8), a strong and significant increase of CAMP expression was observed (Figure 1C).Human SGBS preadipocytes were differentiated over 14 days into mature adipocytes.In early preadipocytes, there was no significant CAMP expression.During differentiation, CAMP expression increased strongly until day 8, remaining at an elevated level during final adipocyte maturation (Figure 1F).

CAMP Gene Expression Is Induced during Differentiation in Murine 3T3-L1 Adipocytes and in Human SGBS Cells
During the differentiation process, 3T3-L1 adipocytes phenotypically turn from fibroblastic preadipocytes into mature adipocytes with multiple lipid vacuoles within 8 days.The pattern of adipokine expression and secretion varies depending on the stage of differentiation.In early stage 3T3-L1 preadipocytes, at days 0 and 3 of differentiation, no significant CAMP gene expression was detected.In later stages, at day 6 and in mature adipocytes (day 8), a strong and significant increase of CAMP expression was observed (Figure 1C).Human SGBS preadipocytes were differentiated over 14 days into mature adipocytes.In early preadipocytes, there was no significant CAMP expression.During differentiation, CAMP expression increased strongly until day 8, remaining at an elevated level during final adipocyte maturation (Figure 1F).murine 3T3-L1 adipocytes human SGBS adipocytes

Cell-Free Nucleic Acids (cfDNA) Impair CAMP Expression in 3T3-L1 Adipocytes
Adipocytes release cfDNA as a response to inflammatory stress [18] and cfDNA acts as an endogenous ligand for TLR9 in immune cells [26].In the present study, we investigated whether cfDNA from adipocytes has an impact on CAMP expression in adipocytes.In 3T3-L1 adipocytes, cfDNA reduced CAMP expression with an effective dosage of 1 µg/mL cfDNA (p = 0.002), whereas 100 ng/mL proved to be ineffective (Figure 3).

Cell-Free Nucleic Acids (cfDNA) Impair CAMP Expression in 3T3-L1 Adipocytes
Adipocytes release cfDNA as a response to inflammatory stress [18] and cfDN as an endogenous ligand for TLR9 in immune cells [26].In the present study, we gated whether cfDNA from adipocytes has an impact on CAMP expression in adip In 3T3-L1 adipocytes, cfDNA reduced CAMP expression with an effective dosa µg/mL cfDNA (p = 0.002), whereas 100 ng/mL proved to be ineffective (Figure 3).

CAMP and TLR9 mRNA Levels Are Positively Correlated in Human Subcutaneous Tissue
cfDNA is increased in the adipose tissue and plasma of obese patients [18 cfDNA represents a physiological TLR9 agonist [27] and cfDNA inhibited CAM expression in adipocytes in vitro (Figure 3), gene expression levels of CAMP an were analyzed using quantitative real-time PCR in subcutaneous and visceral adip sue specimens obtained from obese individuals undergoing bariatric surgery.I ingly, TLR9 and CAMP gene expression levels in subcutaneous adipose tissue we tively correlated (rho = +0.282;p = 0.008; n = 88) (Figure 4A).No correlation of TL CAMP expression was detected in human visceral adipose tissue (rho = +0.034;p n = 90).Furthermore, CAMP mRNA quantities in human visceral and subcutaneo pose tissue compartments correlated positively (rho = +0.224;p = 0.005; n = 157) 4B).

CAMP and TLR9 mRNA Levels Are Positively Correlated in Human Subcutaneous Adipose Tissue
cfDNA is increased in the adipose tissue and plasma of obese patients [18].Since cfDNA represents a physiological TLR9 agonist [27] and cfDNA inhibited CAMP gene expression in adipocytes in vitro (Figure 3), gene expression levels of CAMP and TLR9 were analyzed using quantitative real-time PCR in subcutaneous and visceral adipose tissue specimens obtained from obese individuals undergoing bariatric surgery.Interestingly, TLR9 and CAMP gene expression levels in subcutaneous adipose tissue were positively correlated (rho = +0.282;p = 0.008; n = 88) (Figure 4A).No correlation of TLR9 and CAMP expression was detected in human visceral adipose tissue (rho = +0.034;p = 0.747; n = 90).Furthermore, CAMP mRNA quantities in human visceral and subcutaneous adipose tissue compartments correlated positively (rho = +0.224;p = 0.005; n = 157) (Figure 4B).

CAMP and TLR9 Gene Expression Levels Are Positively Correlated in Murine Subcutaneous Adipose Tissue
It is well known that subcutaneous and intra-abdominal adipose tissues differ in the numerical ratio of adipocytes to stromal vascular cells as well as in functional characteristics [28].Therefore, we additionally investigated both adipose tissue compartments separately in murine samples.Subcutaneous and intra-abdominal adipose tissue specimens were resected from wildtype C57BL/6J mice.Similar to human adipose tissue samples, CAMP and TLR9 gene expression levels in subcutaneous adipose tissue were found to be positively correlated (rho = +0.657;p = 0.011; n = 14) (Figure 5).In contrast, there was no significant correlation of TLR9 and CAMP mRNA levels in intra-abdominal adipose tissue (rho = −0.178;p = 0.543; n = 14).

CAMP and TLR9 Gene Expression Levels Are Positively Correlated in Murine Subcutaneous Adipose Tissue
It is well known that subcutaneous and intra-abdominal adipose tissues differ in the numerical ratio of adipocytes to stromal vascular cells as well as in functional characteristics [28].Therefore, we additionally investigated both adipose tissue compartments separately in murine samples.Subcutaneous and intra-abdominal adipose tissue specimens were resected from wildtype C57BL/6J mice.Similar to human adipose tissue samples, CAMP and TLR9 gene expression levels in subcutaneous adipose tissue were found to be positively correlated (rho = +0.657;p = 0.011; n = 14) (Figure 5).In contrast, there was no significant correlation of TLR9 and CAMP mRNA levels in intra-abdominal adipose tissue (rho = −0.178;p = 0.543; n = 14).

CAMP and TLR9 Gene Expression Levels Are Positively Correlated in Murine Subcutaneous Adipose Tissue
It is well known that subcutaneous and intra-abdominal adipose tissues differ in the numerical ratio of adipocytes to stromal vascular cells as well as in functional characteristics [28].Therefore, we additionally investigated both adipose tissue compartments separately in murine samples.Subcutaneous and intra-abdominal adipose tissue specimens were resected from wildtype C57BL/6J mice.Similar to human adipose tissue samples, CAMP and TLR9 gene expression levels in subcutaneous adipose tissue were found to be positively correlated (rho = +0.657;p = 0.011; n = 14) (Figure 5).In contrast, there was no significant correlation of TLR9 and CAMP mRNA levels in intra-abdominal adipose tissue (rho = −0.178;p = 0.543; n = 14).

TLR9 Deficiency Is Associated with Reduced Systemic CAMP Serum Concentrations in Male Mice
To further investigate a possible interrelation between CAMP and TLR9 expression levels, we analyzed blood serum samples from wildtype mice and TLR9 KO mice.Serum samples were collected at the age of 5-8 months and circulating CAMP levels were quantified via ELISA.Of note, CAMP serum concentrations were significantly lower (p = 0.011) in male TLR9 KO mice when compared to wildtype mice (WT: 6122.70 ± 1962.79 pg/mL vs. TLR9 KO: 4445.01 ± 826.42 pg/mL) (Figure 6A).This difference between genotypes was not observed in female mice (WT: 3735.38 ± 973.51 pg/mL vs. TLR9 KO: 3937.66 ± 1847.37 pg/mL) (Figure 6B).

Knockout of TLR9 Does Not Affect CAMP Gene Expression in Murine Primary Subcutaneous and Intra-Abdominal Adipocytes
We detected significant correlations of CAMP and TLR9 gene expression in human and murine subcutaneous adipose tissue, and TLR9 knockout in male mice resulted in reduced CAMP serum levels.However, synthetic TLR9 ligands-as opposed to cfDNA-did not modify CAMP gene expression in murine adipocytes in vitro.In order to further investigate the impact of TLR9 on CAMP expression in adipocytes, we tested the cellular effect of TLR9 knockout on CAMP gene expression in primary adipocytes isolated from wildtype mice and TLR9 KO mice.Due to the diverging constitution of subcutaneous and intra-abdominal adipose tissue, we evaluated adipocytes from both compartments separately (Figure 8A,B).Of note, we detected no difference in CAMP gene expression in subcutaneous (Figure 8A) and intra-abdominal (Figure 8B) adipocytes of wildtype and TLR9 knockout mice.

Knockout of TLR9 Does Not Affect CAMP Gene Expression in Murine Primary Subcutaneous and Intra-Abdominal Adipocytes
We detected significant correlations of CAMP and TLR9 gene expression in human and murine subcutaneous adipose tissue, and TLR9 knockout in male mice resulted in reduced CAMP serum levels.However, synthetic TLR9 ligands-as opposed to cfDNAdid not modify CAMP gene expression in murine adipocytes in vitro.In order to further investigate the impact of TLR9 on CAMP expression in adipocytes, we tested the cellular effect of TLR9 knockout on CAMP gene expression in primary adipocytes isolated from wildtype mice and TLR9 KO mice.Due to the diverging constitution of subcutaneous and intra-abdominal adipose tissue, we evaluated adipocytes from both compartments separately (Figure 8A,B).Of note, we detected no difference in CAMP gene expression in subcutaneous (Figure 8A) and intra-abdominal (Figure 8B) adipocytes of wildtype and TLR9 knockout mice.

Discussion
During adipocyte differentiation, the expression and secretion of various adipokines increase [30].Zhang et al. reported that CAMP gene expression in mature murine 3T3-L1

Discussion
During adipocyte differentiation, the expression and secretion of various adipokines increase [30].Zhang et al. reported that CAMP gene expression in mature murine 3T3-L1 adipocytes is increased when compared to preadipocytes [13], which was confirmed by our group [31].In the experiments presented here, we could further demonstrate a strong and persistent induction of CAMP gene expression during differentiation of human SGBS preadipocytes into mature adipocytes, a finding comparable to the expression of CAMP during in vitro differentiation of primary human preadipocytes [13].SGBS adipocytes [32] therefore might represent an applicable cell culture model for the analysis of CAMP regulation in human adipocytes, improving the translational potential of in vitro findings for future clinical and therapeutical applications.
The capacity of host defense in subcutaneous bacterial infection depends on metabolic factors, which is demonstrated by impaired host defense in obesity [14].Importantly, adipocyte-derived CAMP plays a crucial role in the local host defense of subcutaneous adipose tissue [13].As an antimicrobial peptide, CAMP is part of the innate immune system.The regulation of CAMP in adipocytes by other factors of the innate immune system is unknown so far.The proinflammatory cytokine TNFα is expressed in adipose tissue and affects adipose tissue-resident macrophage activation [33].In obesity, increased levels of TNFα are observed, and TNFα levels correlate with the extent of adiposity and associated insulin resistance [33].Remarkably, we found that TNFα significantly induces CAMP gene expression in 3T3-L1 adipocytes.This inflammatory elevation of CAMP expression was antagonized by the inhibition of PI3K signaling.Of note, pharmacological antagonists of other classical signal transduction pathways exerted either no (NF-κB, MAPK) or even contrary effects (STAT3, MEK-1/2).Basal, non-stimulated CAMP gene expression levels were not affected by the inhibition of these signal transduction pathways, suggesting a specific TNFα-/inflammation-related mechanism.
In obesity, an increased quantity of visceral adipose tissue and adipocyte hypertrophy are associated with systemic low-grade inflammation, representing a key aspect of the metabolic syndrome [6].Macrophage infiltration into visceral adipose tissue induces local inflammatory stress in adipocytes [18].Subsequent adipocyte degeneration results in the release of nucleic acids (cell-free DNA, cfDNA) [18], presumably exerting paracrine effects on adipocytes [34].In obesity, levels of cfDNA in circulating blood are increased and positively correlated with the quantity of visceral adipose tissue [18].cfDNA has even been suggested as a biomarker for obesity [35].Local cfDNA acts as an endogenous ligand of TLR9 on immune cells leading to an increased recruitment of these cells to the site of inflammation [26].Of note, CAMP is required for normal TLR9 function in dendritic cells and macrophages [24].Our group demonstrated previously that there is significant and functional TLR9 expression in adipocytes [25].The activation or inhibition of TLR9 activity affects the pattern of adipokine secretion in 3T3-L1 adipocytes in vitro [25].We therefore aimed to investigate whether adipocyte CAMP gene expression is modulated by cfDNA and if TLR9 is involved in this context.
Interestingly, stimulation with cfDNA significantly reduced CAMP gene expression, suggesting that cfDNA represents a yet unknown paracrine factor in adipose tissue inhibiting CAMP expression in adipocytes.As CAMP gene expression was regulated by the physiological TLR9 agonist cfDNA, we investigated a possible association between CAMP and TLR9 expression in vivo.Considering the differing cellular composition and function of subcutaneous and intra-abdominal adipose tissues, we comparatively investigated these compartments in mice and in human individuals.In subcutaneous adipose tissue of wildtype mice, TLR9 and CAMP mRNA levels were positively correlated, whereas no significant correlation of these parameters was observed in intra-abdominal adipose tissue.In accordance with our findings in mice, TLR9 and CAMP expression were positively correlated in human subcutaneous but not in visceral adipose tissue.Thus, our data indicate a putatively compartment-specific association of TLR9 and CAMP expression in subcutaneous fat.Future studies elaborating on these data should focus on primary adipocytes and immune cells residing in adipose tissue-such as macrophages and T cells-in order to elucidate which cell types predominantly contribute to the observed correlation, and also whether the differences between subcutaneous and intra-abdominal/visceral adipose tissue might depend on the diverging cellular composition of both compartments.
Additionally, CAMP serum levels were measured in C57BL/6J wildtype and TLR9 knockout mice.Systemic CAMP concentrations were significantly lower in TLR9-deficient mice when compared to wildtype animals.Of note, this difference between genotypes was exclusively observed in male but not in female mice.This unexpected sexual dimorphism needs to be further elucidated, bearing in mind that testosterone or estradiol did not exert a significant impact on CAMP expression in adipocytes in previous experiments in vitro [15].In accordance with the present findings in mice, male patients in this previous study exhibited higher serum concentrations and subcutaneous adipose tissue mRNA levels of CAMP when compared to women [15].Taken together, these data strongly argue for a sexual dimorphism in adipose tissue CAMP gene expression that might also affect circulating CAMP levels.With the underlying regulatory processes remaining to be elucidated, future studies should address this important issue at the molecular level of sexual hormones, potentially affecting the transcriptional and/or secretory regulation of CAMP in more detail.In particular, future experiments should investigate CAMP gene expression in primary murine adipocytes isolated from males versus females to further elaborate on a potential sexual dimorphism and on a potential impact of impaired adipocyte differentiation.
Since we were able to demonstrate a strong association between the gene expression of CAMP and TLR9 in (subcutaneous) adipose tissue and on systemic levels in vivo, we asked if this association might be mediated by TLR9-dependent CAMP regulation in adipocytes.Thus, we investigated the effect of synthetic TLR9 ligands on CAMP gene expression in adipocytes in vitro.Representing well-established TLR9 ligands, the oligodeoxynucleotides ODN1585 (ODNA), ODN1826 (ODNB), ODN2395 (ODNC), and ODN2087 are known to modify immune responses by modulating TLR9 activity [36].Of note, CAMP gene expression was affected neither by ODNA, ODNB, ODNC, nor by ODN2087 in adipocytes.Additionally, we analyzed CAMP gene expression in primary adipocytes from wildtype and TLR9 knockout mice.Importantly, we observed no significant difference between CAMP gene expression levels in primary adipocytes from wildtype mice as compared to TLR9 knockout mice, neither for subcutaneous nor intra-abdominal adipose tissue.Together, these findings suggest that-although cfDNA impairs CAMP gene expression in adipocytes in vitro-the association of CAMP and TLR9 expression levels in adipose tissues presumably mainly depends on other cell types that are different from adipocytes, like monocytes or macrophages in the stromal vascular fraction of adipose tissue, which are a known source of CAMP [37].Furthermore, the observed inhibitory effect of cfDNA on CAMP gene expression in adipocytes might be mediated via additional receptors, for example TLR7, as cfDNA is a putative TLR7 agonist as well [34].Future studies on the putative regulatory interactions of CAMP and TLR7 in adipocytes are necessary to verify this hypothesis and to further elucidate the mechanisms underlying CAMP expression in adipose tissue.
Overall, we focused on a gene expression analysis of CAMP in adipocytes and adipose tissue in order to gain a basic insight into the regulatory relations between the inflammatory factors of innate immunity and CAMP.The predominant analysis on the level of transcriptional regulation represents a limitation concerning the physiological conclusions drawn from the present work.Therefore, future research elaborating on the provided data should apply immunological techniques such as Western blot and immunocytochemistry in order to analyze CAMP regulation in adipocytes and adipose tissue at the protein level and to confirm and further elucidate the biological relevance of these processes.
All applied stimulatory doses were within the concentration range recommended by the manufacturer and had been determined either by preliminary tests or previous experiments in adipocyte culture with respect to dose effects and cell viability.Furthermore, LDH (lactate dehydrogenase) concentration was measured in the cell supernatants (Cytotoxicity Detection Kit, Roche, Mannheim, Germany) of all experiments in order to exclude any unintended cytotoxic effects.

SGBS Cell Culture
The Simpson-Golabi-Behmel syndrome (SGBS) preadipocyte cell strain represents primary human cells originated from adipose tissue specimens of a patient suffering from SGBS [32].They were kindly provided by Prof. Martin Wabitsch (University of Ulm, Germany).The cells were differentiated into mature adipocytes within 14 days of culture following the provider's established protocol [32].SGBS preadipocytes were cultured in DMEM/F12 (1:1) (Invitrogen, Darmstadt, Germany) supplemented with 10% FCS (all purchased from Invitrogen, Darmstadt, Germany).Differentiation into mature adipocytes was induced at confluence.After 3 washing steps with phosphate-buffered saline (PBS), cells were cultured in serum-free medium supplemented with 0.01 g/mL transferrin, 20 nM insulin, 0.2 nM triiodothyronine, and 100 nM cortisol (all purchased from Sigma-Aldrich, Deisenhofen, Germany).During the initial 4 days of differentiation, 2 µM rosiglitazone (BRL 49653) (Cayman, Tallinn, Estonia), 250 µM isobutylmethylxanthine (IBMX), and 25 nM dexamethasone (all purchased from Sigma-Aldrich, Deisenhofen, Germany) were also added to the medium.The culture medium was replaced every third or fourth day.Adipocyte differentiation was completed after 14 days.The characteristic adipocyte morphology was visually controlled by light microscopy.

Preparation of Cell-Free Nucleic Acids from 3T3-L1 Adipocytes
Prior to isolation of cell-free nucleic acids (cfDNA) using the QIAamp ® DNA Micro Kit according to the manufacturer's instructions (Qiagen, Hilden, Germany), murine 3T3-L1 adipocytes were exposed to inflammatory stress induced by stimulation with 50 ng/mL tumor necrosis factor α (TNFα) (Biomol, Hamburg, Germany).After 18 h, adipocytes were harvested and cfDNA was isolated from cell lysates as described earlier [34].Differing doses of cfDNA (100 ng/mL; 1 µg/mL) were applied for overnight (18 h) stimulation experiments in 3T3-L1, while treatment with solvent control containing no cfDNA served as a control setting.

Human Visceral and Subcutaneous Adipose Tissues for Gene Expression Analysis
Human adipose tissue specimens were obtained from the ROBS (Research in Obesity and Bariatric Surgery) study [46], an open-label, non-randomized, monocentric, prospective, and observational study of patients undergoing either bariatric surgery or dietary intervention at the University Hospital of Giessen, Germany.Subcutaneous and visceral adipose tissues were obtained intra-surgically from patients receiving bariatric surgery (gastric sleeve or Roux-en-Y gastric bypass).Detailed information about the ROBS study cohort was published previously and can be drawn from the literature [46].The study was approved by the local ethical committee at the University of Giessen, Germany (file: AZ 101/14).All patients gave informed consent for their participation in the study.Data anonymization and privacy policy were accurately applied.

Quantification of CAMP Gene Expression in Murine Intra-Abdominal and Subcutaneous Adipose Tissues and Primary Adipocytes
Intra-abdominal and subcutaneous adipose tissue compartments were resected from C57BL/6J-Tlr9 M7Btlr /Mmjax mice (TLR9-KO) [25] and wildtype C57BL/6J mice (WT) that were bred under standard conditions and fed normal chow (age 5-8 months).Animals were euthanized for the sampling of serum, subcutaneous and intra-abdominal adipose tissue, and primary adipocytes conformably to the German animal protection law ( §4 Abs. 3 Tierschutzgesetz).A specific announcement was made at the local ethical committee (Regierungspraesidium Giessen; internal registration number: 710_M) that was subsequently approved.

Isolation of mRNA and Quantitative Real-Time PCR Analysis of CAMP Gene Expression in Murine and Human Adipose Tissues and Adipocytes
Total mRNA was isolated from frozen human and murine total adipose tissues and from 3T3-L1, SGBS, and primary adipocytes following a protocol described previously [39].Tissues 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 All oligonucleotides used were purchased from Metabion, Martinsried, Germany.

Quantification of CAMP Concentrations in Murine Blood Serum
Healthy C57BL/6J (WT) and C57BL/6J-Tlr9 M7Btlr /Mmjax mice (TLR9-KO) were bred under standard conditions being fed a chow diet ad libitum until euthanasia for organ and tissue resection and blood serum collection.Concentrations of CAMP in blood serum from C57BL/6J-Tlr9 M7Btlr /Mmjax mice (TLR9-KO) and wildtype C57BL/6J mice (WT) (age 5-8 months) were measured in technical duplicates by enzyme-linked immunosorbent assay (ELISA) (kit purchased from Abbexa Ltd., Cambridge, UK) and are expressed as means ± standard deviation in the text and displayed as box plots in the figures.The test range of the applied ELISA kit was 1.56-100 ng/mL.All measurements exceeding an intra-duplicate variance of 20% were repeated.Animal studies were performed at the University of Giessen, Germany, and were approved by the local government agency.

Statistical Analysis
For explorative data analysis, a statistical software package (SPSS 28.0) was used.Non-parametric numerical parameters were analyzed using the Mann-Whitney U-test (for 2 unrelated samples) or the Kruskal-Wallis test (>2 unrelated samples).A correlation analysis was performed by using the Spearman-rho test.p values below 0.05 (two-tailed) were considered statistically significant.In the figures, the data are presented as box plots, with the box representing the second and third quartile of values, the whiskers giving the inter quartile range, and statistical outliers outside inter quartile range being indicated by dots and stars.

Conclusions
As a part of the innate immune system, adipocytes, as well as adipose tissue as a whole organ, play a crucial role in immune-metabolism ("metaflammation", "adipose inflammation") [47].Here, we investigate and discuss a novel aspect of the inflammationmediated regulation of CAMP in adipocytes and adipose tissues.As visualized in Figure 9, our results implicate that CAMP expression in adipocytes is induced by TNFα involving the PI3K pathway.Furthermore, the physiological TLR7/TLR9 ligand cell-free nucleic acids (cfDNA) impairs CAMP gene expression in adipocytes.Further studies are necessary in order to investigate the molecular mechanisms and functional consequences of this novel relation within innate immunity and metaflammation in more detail.Most importantly, TNFα, CAMP, and adipose tissue-derived cfDNA might provide potential molecular targets in future anti-inflammatory drug therapies addressing metabolic inflammation and concomitant morbidities.
As a part of the innate immune system, adipocytes, as well as adipose tissue as a whole organ, play a crucial role in immune-metabolism ("metaflammation", "adipose inflammation") [47].Here, we investigate and discuss a novel aspect of the inflammationmediated regulation of CAMP in adipocytes and adipose tissues.As visualized in Figure 9, our results implicate that CAMP expression in adipocytes is induced by TNFα involving the PI3K pathway.Furthermore, the physiological TLR7/TLR9 ligand cell-free nucleic acids (cfDNA) impairs CAMP gene expression in adipocytes.Further studies are necessary in order to investigate the molecular mechanisms and functional consequences of this novel relation within innate immunity and metaflammation in more detail.Most importantly, TNFα, CAMP, and adipose tissue-derived cfDNA might provide potential molecular targets in future anti-inflammatory drug therapies addressing metabolic inflammation and concomitant morbidities.

Figure 1 .Figure 1 .
Figure1.CAMP gene expression is induced during differentiation in murine 3T3-L1 adipocytes (C) and in human SGBS cells (F).In 3T3-L1 preadipocytes, no significant CAMP expression is detectable.At day 6 and in mature adipocytes, a considerable increase of CAMP mRNA levels is observed (C).CAMP expression significantly increases during differentiation of SGBS cells (F).Light microscopyFigure 1. CAMP gene expression is induced during differentiation in murine 3T3-L1 adipocytes (C) and in human SGBS cells (F).In 3T3-L1 preadipocytes, no significant CAMP expression is detectable.At day 6 and in mature adipocytes, a considerable increase of CAMP mRNA levels is observed (C).CAMP expression significantly increases during differentiation of SGBS cells (F).Light microscopy of naïve (A,D) and Oil Red O stained (B,E) adipocytes is shown.During differentiation, 3T3-L1 fibroblasts on day 0 mature to adipocytes on day 8 (A,B).SGSB preadipocytes on day 0 mature to adipocytes on day 14 (D,E).CAMP expression was investigated by quantitative real-time PCR.The Kruskal-Wallis test was applied for calculation of p values and statistical significance (p < 0.05).N = 6-12 wells were investigated per experimental setting.Gene expression levels are given in relative gene expression as compared to day 0 in 3T3-L1 and day 4 in SGBS cells.(** p < 0.01).

Figure 3 .
Figure 3. Adipocytic cell-free nucleic acids (cfDNA) impair CAMP expression in 3T3-L1 adi cfDNA (1 µg/mL) reduces CAMP mRNA levels in 3T3-L1 adipocytes.CAMP expression w tigated by quantitative real-time PCR.Kruskal-Wallis test was applied for calculation of and statistical significance (p < 0.05).N = 16-18 wells were investigated per experimenta Gene expression levels are given in relative gene expression as compared to control.(** p <

Figure 4 .
Figure 4. CAMP and TLR9 mRNA levels are positively correlated in human subcutaneous adipose tissue (n = 88) (A).CAMP gene expression levels in subcutaneous and visceral adipose tissue are positively correlated (n = 157) (B).Specimens of human subcutaneous adipose tissue were collected during bariatric surgery.CAMP and TLR9 expression were investigated by quantitative real-time PCR and Spearman-rho test was applied for calculation of correlation coefficient and statistical significance (** p < 0.01).

Figure 5 .
Figure 5. CAMP and TLR9 gene expression levels are positively correlated in murine subcutaneous adipose tissue: TLR9 expression in subcutaneous adipose tissue is positively correlated with CAMP expression in subcutaneous adipose tissue of wildtype mice.Samples of subcutaneous adipose tissue from wildtype mice were collected at the age of 12 months.CAMP and TLR9 expression were investigated by quantitative real-time PCR.The Spearman-rho test was applied for calculation of

Figure 4 .
Figure 4. CAMP and TLR9 mRNA levels are positively correlated in human subcutaneous adipose tissue (n = 88) (A).CAMP gene expression levels in subcutaneous and visceral adipose tissue are positively correlated (n = 157) (B).Specimens of human subcutaneous adipose tissue were collected during bariatric surgery.CAMP and TLR9 expression were investigated by quantitative real-time PCR and Spearman-rho test was applied for calculation of correlation coefficient and statistical significance (** p < 0.01).

Figure 4 .
Figure 4. CAMP and TLR9 mRNA levels are positively correlated in human subcutaneous adipose tissue (n = 88) (A).CAMP gene expression levels in subcutaneous and visceral adipose tissue are positively correlated (n = 157) (B).Specimens of human subcutaneous adipose tissue were collected during bariatric surgery.CAMP and TLR9 expression were investigated by quantitative real-time PCR and Spearman-rho test was applied for calculation of correlation coefficient and statistical significance (** p < 0.01).

Figure 5 .
Figure 5. CAMP and TLR9 gene expression levels are positively correlated in murine subcutaneous adipose tissue: TLR9 expression in subcutaneous adipose tissue is positively correlated with CAMP expression in subcutaneous adipose tissue of wildtype mice.Samples of subcutaneous adipose tissue from wildtype mice were collected at the age of 12 months.CAMP and TLR9 expression were investigated by quantitative real-time PCR.The Spearman-rho test was applied for calculation of

Figure 5 .
Figure 5. CAMP and TLR9 gene expression levels are positively correlated in murine subcutaneous adipose tissue: TLR9 expression in subcutaneous adipose tissue is positively correlated with CAMP expression in subcutaneous adipose tissue of wildtype mice.Samples of subcutaneous adipose tissue from wildtype mice were collected at the age of 12 months.CAMP and TLR9 expression were investigated by quantitative real-time PCR.The Spearman-rho test was applied for calculation of correlation coefficient and statistical significance (p < 0.05).N = 14 samples were investigated.(* p < 0.05).

Figure 6 .
Figure 6.TLR9 KO is associated with reduced systemic CAMP concentrations in male mice: CAMP concentration in blood serum is significantly lower (p = 0.011) in male TLR9 KO mice than in wildtype mice (A), whereas this difference was not observed in female animals (B).Blood serum samples from wildtype and TLR9 KO mice were collected at the age of 5-8 months.Blood serum CAMP concentrations were measured by ELISA and Mann-Whitney U-test was applied for calculation of statistical significance (p < 0.05).Samples from n = 10-22 animals per group were investigated.n.s.: not significant, * p < 0.05; *: statistical outlier >2.5 interquartile range

Figure 6 .
Figure 6.TLR9 KO is associated with reduced systemic CAMP concentrations in male mice: CAMP concentration in blood serum is significantly lower (p = 0.011) in male TLR9 KO mice than in wildtype mice (A), whereas this difference was not observed in female animals (B).Blood serum samples from wildtype and TLR9 KO mice were collected at the age of 5-8 months.Blood serum CAMP concentrations were measured by ELISA and Mann-Whitney U-test was applied for calculation of statistical significance (p < 0.05).Samples from n = 10-22 animals per group were investigated.n.s.: not significant, * p < 0.05; *: statistical outlier >2.5 interquartile range.

Figure 7 .
Figure 7. Synthetic TLR9 ligands do not modify CAMP gene expression in murine adipocytes: Stimulation of murine 3T3-L1 adipocytes with various doses of ODNA (A), ODNB (B), ODNC (C), and ODN2087 (D) does not significantly affect CAMP gene expression.CAMP expression was investigated by quantitative real-time PCR.The Kruskal-Wallis test (A-C) and Mann-Whitney U-test (D) were applied for calculation of p values and statistical significance (p < 0.05).N = 11-20 wells were investigated per experimental setting.Gene expression levels are given in relative gene expression as compared to control.n.s.: not significant.

Figure 7 .Figure 8 .
Figure 7. Synthetic TLR9 ligands do not modify CAMP gene expression in murine adipocytes: Stimulation of murine 3T3-L1 adipocytes with various doses of ODNA (A), ODNB (B), ODNC (C), and ODN2087 (D) does not significantly affect CAMP gene expression.CAMP expression was investigated by quantitative real-time PCR.The Kruskal-Wallis test (A-C) and Mann-Whitney U-test (D) were applied for calculation of p values and statistical significance (p < 0.05).N = 11-20 wells were investigated per experimental setting.Gene expression levels are given in relative gene expression as compared to control.n.s.: not significant.Int.J. Mol.Sci.2023, 24, x FOR PEER REVIEW 9 of 17

Figure 8 .
Figure 8. CAMP gene expression in primary adipocytes from subcutaneous (A) and intra-abdominal adipose tissue (B) does not differ between wildtype (WT) and TLR9 knockout (KO) mice.CAMP expression was investigated by quantitative real-time PCR, and Mann-Whitney U-test was applied for calculation of statistical significance (p < 0.05).Samples from n = 5-17 animals per group were analyzed.n.s.: not significant; *: statistical outlier > 2.5 interquartile range.

Figure 9 .
Figure 9. Putative mechanisms of CAMP regulation in adipocytes.Adipocyte CAMP gene expression is induced by TNFα via the PI3K pathway.The impaired CAMP gene expression by cfDNA might be modulated via endosomal receptors, e.g., TLR7/9.ER: endoplasmatic reticulum; CN: cell nucleus.