The Opposite Effects of Kynurenic Acid and Different Kynurenic Acid Analogs on Tumor Necrosis Factor-α (TNF-α) Production and Tumor Necrosis Factor-Stimulated Gene-6 (TSG-6) Expression

Purpose: The investigation of anti-inflammatory and immunosuppressive functions of Kynurenic acid (KYNA) is now in focus. There is also substantial evidence that TSG-6 has an anti-inflammatory activity. Therefore, in the present study, we compared the effects of newly synthetized KYNA analogs on the TNF-α production in U-937 monocytic cells in correlation with the effects on the TSG-6 expression. Methods: TNF-α production was measured by ELISA, the TSG-6 expression was determined by RTqPCR method. As cytokine inducers Staphylococcus aureus and Chlamydia pneumoniae were used. Results: KYNA and KYNA analogs attenuated TNF-α production and increased TSG-6 mRNA expression in U-937 cells stimulated by heat inactivated Staphylococcus aureus. In contrast, KYNA and some of the KYNA analogs increased the TNF-α production of C. pneumoniae infected U-937 cells; however, the newly synthetized analogs (SZR104, SZR 105, and SZR 109) exerted significant inhibitory effects on the TNF-α synthesis. The inhibitory and stimulatory effects correlated inversely with the TSG-6 expression. Conclusions: TSG-6 expression following activation with bacterial components could participate in the suppression of inflammatory cytokines, such as TNF-α, We suppose that the elevation of the TSG-6 expression by KYNA and especially by new KYNA analogs might be one of the mechanisms that are responsible for their suppressive effect on TNF-α production as a feedback mechanism. KYNA and KYNA analogs have an important role in influencing TSG-6 expression, and there is a possible benefit of targeting TSG-6 expression by kynurenines in inflammatory conditions following infections.


INTRODUCTION
There is an increasing interest in the role of kynurenines in the immune function. The kynurenine pathway is a regulator of both innate and adaptive immune responses, and the tryptophan metabolism kynurenine and production reflect a crucial interface between the immune and nervous systems (1,2). Kynurenic acid (KYNA) is one of the products of the kynurenine pathway of tryptophan metabolism (3)(4)(5). KYNA as an antagonist of ionotropic glutamate receptors N-methyl-D-aspartate (NMDA) and the α7 nicotinic acetylcholine receptor (α7nAchR) exert neuroprotective effects (2,(4)(5)(6)(7)(8)(9)(10). KYNA acts both as a blocker of the glycine co-agonistic site of the NMDA receptor and as a non-competitive inhibitor of the α7 nicotinic acetylcholine receptor (11). The investigation of anti-inflammatory and immunosuppressive functions of KYNA is now in focus. It has been proved that these immunomodulatory properties are based on the signaling by G-protein-coupled receptor 35 (GP35) and aryl hydrocarbon receptor (AHR)-mediated pathways ys (2,(12)(13)(14).
Several studies have revealed that KYNA can attenuate inflammation induced by different stimuli (2,15,16). Previously, we demonstrated that KYNA and a KYNA analog reduced the TNF-α secretion from human mononuclear cells (17). In the present study, we compared the effects of newly synthetized KYNA analogs on the α TNF-α production in U-937 monocytic cell line. We focused on the potential correlation between the effects on the TSG-6 (TNFα-stimulated gene 6) expression and the influence, i.e., the suppression, of TNF-α production by different KYNA analogs.
Therefore, the aim of the present study is to evaluate a possible connection between the capacity of KYNA and KYNA analogs on the TSG-6 expression and the inhibition of TNF-α production first of all in U-937 monocytic cells. Our hypothesis was that activation of TSG-6 expression might be at least partially responsible for the TNF-α inhibitory effect of KYNA. TNF-α induction in U-937 cells was performed with heat killed Staphylococcus aureus, and the effects were compared with Chlamydia pneumoniae (C. pneumoniae). Staphylococcus aureus is a Gram-positive pyogenic coccus and a good inducer of TNF in mononuclear cells, and it mimics natural conditions (29,30). Chlamydia pneumoniae is a Gram-negative bacterium, growing intracellularly, and it is responsible for different inflammatry conditions, especially in the lungs and in atherosclerosis. Chlamydia pneumoniae attach monocytes and multiply in them (31).The main question was, whether the production of TNF-α, and TSG-6 could be induced by these criteriae in U-937 cells. It was demonstrated in a previous study, that C.pneumoniae upregulated. numerous inflammatory genes in U-937 cells (32).
Chlamydia pneumoniae (C. pneumoniae) CWL029 strain from American Types Culture Collection (ATCC) was propagated in HEp-2 cells. Infective chlamydiae were quantitated by indirect immunofluorescent method applying anti-Chlamydia lipopolysaccharide (cLPS) monoclonal antibody (AbD Serotec, Oxford, United Kingdom) and FITC-labeled anti-mouse IgG (Sigma-Aldrich, St. Louis, MO). The concentration of infective elementary bodies (EB)-s was expressed as inclusion forming units/mL (IFU/mL).  (17), these concentrations proved to be optimal in reducing cytokine production. Cell supernatants were tested for TNF-α and TSG-6 content by ELISA and cell lysates for TSG-6 mRNA by RT qPCR. (b) U-937 cells were seeded in 24-well plates (5 × 10 5 cells/well), and the cells were then infected with C. pneumoniae at a multiplicity of infection (MOI) of 5 in complete RPMI with 0.5% glucose and centrifuged at 800 × g for 1 h RT. The growth medium was replaced in the wells with a medium containing KYNA analogs at a concentration of 250-500 µM. The culture plates were incubated for 24 h in CO 2 incubator at 37 • C. Cell supernatants were tested for TNF-α and TSG-6 content by ELISA and cell lysates for TSG-6 mRNA by RT qPCR.

Chlamydial DNA Quantitation
For the quantitative assessment of chlamydial replication, a direct DNA quantitation method was used (36). The cells in the 96-well plates were infected with C. pneumoniae at a multiplicity of infection (MOI) of 5. After 24 and 48 h, the infected cells in 3 parallel wells were washed in the plates twice with 200 µL/well phosphate buffered saline (PBS). Then 100 µL Milli-Q water was added to the wells, and the plates were stored at −80 • C. In order to free the DNA from the cells, two freeze-thaw cycles were applied. Thoroughly mixed lysates were used as templates directly for quantitative PCR (qPCR) using SsoFast TM EvaGreen R Supermix (BioRad). For the detection of C. pneumoniae DNA, the following primers were used: ompA F: 5 ′ TGCGACGCTATTAGCTTACGT 3 ′ and ompA R: 5 ′ TAGTTTGCAGCAGCGGATCCA 3 ′ . A BLAST search was performed to check the specificity of the product target sequence of the primer sets. The primers were synthetized by Integrated DNA Technologies Inc. (Montreal, Quebec, Canada). During qPCR reaction, after the 10 min at 95 • C polymerase activation step, 40 PCR cycles of 20 s at 95 • C, and 1 min at 64 • C were performed. The fluorescence intensity was measured at the end of the annealing-extension step. The specificity of amplification was confirmed by the melting curve analysis. For each PCR, the cycle threshold (Ct) corresponding to the cycle, where the amplification curve crossed the base line, was determined. The difference in Ct values detected in the samples incubated with KYNA and the analogs at a concentration of 250 and 500 µM compared to that of the untreated samples was calculated.

TNF-α ELISA
The TNF-α concentrations in the supernatants were quantified by using the TNF-α ELISA kit (Legend Max BioLegend San Diego) according to the instructions of the manufacturer.

TSG-6 ELISA
The TSG-6 concentrations in the supernatants were quantified by using the TSG-6 ELISA kit (SIGMA U.S.A. St. Louis) according to the instructions of the manufacturer.

TSG-6 mRNA Quantification by Reverse Transcription Quantitative PCR (RT qPCR)
Total RNA was extracted from the samples by using TRI Reagent (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's protocol. The quality and the quantity of the extracted RNA were assessed by a NanoDrop Lite spectrophotometer (Thermo Scientific, Waltham, MA, USA). First-strand cDNA was synthesized by using 2 µg of total RNA with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) strictly adhering to the manufacturer's recommendations. The qPCR was conducted with cDNA, 1 µL of primers (10 µM) and SensiFast SYBR R No-ROX Mix (Bioline GmbH, Luckenwalde, Germany) in a total volume of 10 µL. The primers used in the assay were the following: TSG-6 sense 5 ′ -ACT CAA GTA TGG TCA GCG TAT TC−3 ′ , TSG-6 antisense 5 ′ -GCC ATG GAC ATC ATC GTA ACT−3 ′ ; β-actin sense 5 ′ -TTC TAC AAT GAG CTG CGT GTG GCT−3 ′ , and β-actin antisense 5 ′ -TAG CAC AGC CTG GAT AGC AAC GTA−3 ′ . All primers were synthetized by Integrated DNA Technologies Inc. (Montreal, Quebec, Canada). The RT-qPCR was performed in a CFX96 Touch PCR detection system (Bio-Rad, Hercules, CA, USA). Thermal cycling was initiated with a denaturation step of 2 min at 95 • C followed by 40 cycles each of 10 s at 95 • C and 1 min at 60 • C. The fluorescence intensity was detected at the end of the annealing-extension steps. The specificity of amplification was confirmed by carrying out a melting curve analysis. The cycle threshold (C t ) corresponding to the cycle, where the amplification curve crossed the base line, was determined. The Ct of target transcripts was compared with that of β-actin, the difference being referred to as C t . The relative expression level was given as 2 −( Ct) , where C t = C t for the experimental sample minus C t for the control sample. Increases in transcripts >2-fold compared to the control samples were considered to be significant. Uninfected cells were used as controls. All of the measurements were performed in duplicate from 3 biological repetitions.

Human Blood Samples
EDTA-anticoagulated peripheral blood samples from 10 healthy volunteers were obtained.
Samples (1 mL each) were incubated in the presence of heat inactivated S, aureus for 18 hr. Parallel blood samples were pretreated for 30 min with KYNA and KYNA analogs at a concentration of 500 µM. Following the incubation period, the blood samples were centrifuged at 300 × g, and the supernatants were tested for TNF-α and TSG-6 content by ELISA.
For the experiments performed with the human blood we have the approval of the ethics commitee of the Medical Faculty of the University. of Szeged (ETT-TUKEB 905/PI/09). This study was conducted in full accordance with the tenets of Declaration of Helsinki (1964).

Statistical Analysis
Data are expressed as means ± SD. Differences between group means were determined by the unpaired Student ttest. p-values <0.05 were considered significant. Data of box and whiskers analysis were evaluated by Mann-Whitney test. The correlation between the TNF-α production and expression of TSG-6 was evaluated by correlation analysis. All statistical calculations were performed with the Graph-Pad Prism 5 statistical program (GraphPad Software Inc., San Diego, CA, USA).

KYNA and KYNA Analogs Increase TSG-6 mRNA Expression in U-937 Cells
To gain further insight into the connection between the inhibition of TNF-α production and the induction of TSG-6 expression exerted by KYNA analoques, we determined the effects of KYNA analoques on TSG-6 mRNA expression. Both KYNA and KYNA analogs increased the TSG-6 relative expression at equimolar concentrations of 500 µM (Figure 2) significantly. SZR 73 was not effective in this respect, similarly as it was observed in the experiments with TNF-α production. Thus, we suspect that there is a connection between the attenuation of SA1-induced TNF protein synthesis and the TSG-6 gene transcription, which is elevated by KYNA and KYNA analoques.

KYNA and the KYNA Analogs Differently Influence TNF-α Production Induced by C. pneumoniae in U-937 Human Monocytic Cells
We wanted to compare the effects of KYNA and KYNA derivates on TNF-α production when the inducer is a Gramnegative, intracellular bacterium, i.e., Chlamydia pneumoniae (C. pneumoniae). Our results were unexpected; instead of having inhibitory effects, KYNA and some of the KYNA analoques increased the TNF-α production of C. pneumoniae infected U-937 cells. In contrast, the newly synthetized analogs (SZR104, SZR 105, and SZR 109) exerted a significant inhibitory effect on the cytokine synthesis (Figure 3).

KYNA and KYNA Analogs Differently
Influence TSG-6 mRNA Expression in U-937 Cells Infected With Chlamydia pneumoniae C. pneumoniae induced a considerable TSG-6 expression in U-937 cells. KYNA, SZR72, and SZR81 inhibited the rate of expression (Figure 4A). Interestingly, the same chemicals enhanced the TNF-α production of C. pneumoniae-induced U-937 cells (Figure 3). On the other hand, further KYNA analoques (SZR 104, SZR 105, and SZR 109) with different chemical structure (see Table 1) stimulated TSG-6 expression ( Figure 4B). It is also noteworthy that only these analoques inhibited significantly the TNF-α production of C. pneumoniaeinduced U-937 cells (Figure 3). Considering the variable effects of KYNA analogs on the TSG-6 expression and also on the TNF-α production, we checked the correlation between the two effects. As it was expected, a significant inverse correlation was found between the effects on the TNF-α secretion and the TSG-6 expression exerted by different KYNA analogs (Figure 5). KYNA, SZR72, and SZR81 induced higher TNF-α secretion by U-937 cells after C. pneumoniae infection, but they decreased the TSG-6 expression compared to the cells that were infected only with C. peumomiae, without any of the compounds (i.e., Cpn in Figure 5). In contrast, in the case of the highest rate of TSG-6 expression (SZR 105), a maximal rate of inhibition of TNF-α production was observed. Therefore, we suppose that the different effects of KYNA analoques on the TSG-6 expression in C. pneumoniae infected cells might explain the difference in their effects on the secretion of TNF-α.
Altogether, from these data, it seems that inhibition of TNFα is not only in correlation with the antiinflammatory effect of TSG-6, but in this situations, KYNA analogs are able to increase or even decrease the expression of TSG-6.

Effects of KYNA Analoques on the Quantity of C. pneumoniae
To ascertain that the effects of KYNA analoques on the TNFα or TSG-6 induction is not simply due to their effects on the replication of C. pneumoniae, we performed experiments for quantitative assessment of chlamydial replication by a direct quantitative PCR method (36). C. pneumoniae ompA gene was detected in the lysate of U-937 cells infected with C. pneumoniae at a MOI of 5 in the presence or absence of KYNA analoques at a concentration of 250 or 500 µM, respectively. Direct detection of C. pneumoniae DNA in the lysate of infected cells was done at 24 and 48 h postinfection. There was no significant inhibition or even elevation in the quantity of chlamydial DNA in the presence of different KYNA analoques after the 24 h (open bars) or 48 h (filled bars) incubation period. The results of the samples tested at 24 and 48 h of incubation are presented in Figure 6. Therefore, FIGURE 5 | Correlation between the TSG-6 expression and TNF-α production by U-937 cells infected with C. pneumoniae at a MOI 5 in the presence of KYNA or the analogs. U-937 cells were pretreated for 30 min with KYNA or KYNA analogs at a concentration of 500 µM, and thereafter incubated for 24 h with 5 MOI of Chlamydia pneumoniae. The TNF-α levels in the supernatants were determined with ELISA assay, and the TSG-6 expression by RT qPCR reactions. The significance of correlation was calculated by correlation analysis with the Graph-Pad Prism 5 statistical program. Symbols and numbers represent the data obtained with KYNA or KYNA analogs. Cpn: incubation only with Chlamydia pneumoniae without compounds. The correlation coefficient, r value is-0.891, the p-value = 0.0072, the 95% confidence interval is −0.9838 to −0,4174.
we assume that KYNA analoques do not influence the replication or the quantity of C. pneumoniae.

Effects of KYNA Analogs on TGS-6 Protein Production in U-937 Human Monocytic Cells Stimulated With Heat Inactivated S. aureus or by Chlamydia pneumoniae
To ascertain whether the effects of KYNA and analogs on the TSG-6 expression influence parallelly the protein level, the TSG-6 concentrations in the supernatants of U-937 cells were determined.
At a concentration of 500 µM, KYNA and KYNA analogs increased the TGF-6 level significantly, except SZR 73 in SA1 induced cells (Figure 7A). The new analogs SZR 104, 105, and 109 exerted the most potent stimulatory effects (p < 0.001) in equimolar (500 µM) concentration. C. pneumoniae induced also TSG-6 production in U-937 cells, but KYNA, SZR72, and SZR81 decreased the level of TSG-6 protein expression ( Figure 7B). On the other hand, further KYNA analoques (SZR 104, SZR 105, and SZR 109) increased the TSG-6 concentration in the supernatants (7b). These experiments obtained with 500 µM of KYNA and KYNA analogs support the results obtained with RT PCR data demonstrating the effects of the chemicals on the TSG-6 RNA expression.

Staphylococcus aureus
Some of the results obtained by in vitro experiments with U-937 monocytic cells were repeated by "ex vivo" experimets  investigating the effects of two KYNA analogs in human peripheral blood.
There was big individual differences in the TNF-α concentrations and in TSG-6 concentrations in the supernatants in SA1-induced blood cultures (Figure 8), between 179 pg/ml and 850 pg/ml, and between 150 and 750 pg/ml, respectively. At a concentration of 500 µM, both SZR 72 and SZR 105 suppressed the TNF-α level significantly in the S. aureus induced blood cultures. Again, the new analog SZR 105 exerted more potent inhibitory effect (p = 0.001) in equimolar (500 µM) concentration. Similarly to the effects on U-937 cells, the KYNA analogs SZR72 and SZR 105 significantly increased the TSG-6 concentrations in SA1 induced blood samples (Figure 8).

DISCUSSION
In our experiments, KYNA and different KYNA derivates inhibited the TNF-α production of U-937 cells stimulated with heat inactivated Staphylococcus aureus. The rate of the inhibition was variable according the structure of the analoques (Figure 1). The effect of the analogs were compared in equimolar concentration on the TNF-α production when the inducer was Chlamydia pneumoniae, a Gram negative, intracellular bacterium. In these experiments, however, not all KYNA derivates inhibited TNF-α production by U-937 monocytic cells; moreover, KYNA itself, and SZR72 and SZR81 increased it (Figure 3). We hypothesized that the difference in the influence on the TNF-α production might be connected with the difference in the TSG-6 expression (Figure 4).
It is noteworthy, that TSG-6 itself does not only exert an antiinflammatory effect (20,26,27), but its expression might be under the influence of KYNA (37). It has been published that kynurenic acid controls TSG-6-mediated immunosuppression of the human mesenchymal stem cells (MSCs). In elegant experiments, it has been demonstrated that KYNA specifically regulates TSG-6 production by activating aryl hydrocarbon receptor (AHR). KYNA activates AHR, which directly binds to the TSG-6 promoter to enhance TSG-6 expression. Moreover, KYNA-pretreated MSCs can further boost TSG-6 production, and thus enhance the therapeutic capacity of human MSCs against lipopolysaccharide (LPS)-induced acute lung injury (37).
We found that in most experiments, TSG-6 expression was up-regulated in U-937 monocytic cells stimulated with bacterial components, and KYNA and KYNA analogs were able to influence the rate of expression of TSG-6. The elevation of the TSG-6 expression might be one of the mechanisms that are responsible for the suppression of TNF-α production as a feedback effect. This effect was clearly demonstrated in our experiments using heat inactivated S. aureus as a cytokine inducer. In the case of C. pneumoniae infection, however, KYNA and KYNA analoques did not exert this effect uniformly. Some of them increased TSG-6 expression with a concomitant inhibition of the production of TNF-α, but several compounds (KYNA, SZR72, and SZR 81) rather decreased the expression of TSG-6, and it is very likely that this could lead to an elevated TNF-α production compared to the TNF-α production of U-937 cells infected with C. pneumoniae without any KYNA analoque. We hypothesized that the explanation of the difference in the results might be due to the different chemical structure of the analoques (see Table 1). The examined substrates (SZR-72, SZR-73 SZR-81, SZR-104, SZR-105, and SZR-109) can be classified into two classes of compounds: the first are amide derivatives (SZR-72, SZR-73, SZR-81) containing cationic center at the amide side chain. The second class of compounds (SZR-104, SZR-105, and SZR-109) are the C-3 aminoalkylated amides, therefore they can be interpreted as derivatives with dual cationic centers.
They could differently influence the binding of C. pneumoniae to the Toll-like receptor 2 (TLR2), and especially, differently activate AHR in the presence of C. pneumoniae. It has to be highlighted that the newly synthetized analogs, SZR 105 and SZR 109, were the most potent inducers of TSG-6 expression, and the highest inhibitors of TNF-α production in both types of bacterial inducers. The study of the exact effect of Chlamydia pneumoniae on the interaction between AHR and some KYNA analogs needs to be further investigated and proved.
Whatever the explanation is, our results indicate that there is a close connection between TNF production and TSG-6 expression, and there is an inverse correlation between the TSG-6 expression and TNF-α production in the presence of KYNA and KYNA analogs.
This negative correlation was further demonstrated at the protein level of TSG-6 measured in the supernatants of U-937 cells. and also in unseparated human peripheral blood samples The stimulation of TSG-6 expression by KYNA and KYNA derivates might be one of the mechanisms that have an important role in their suppressive effect on TNF-α production. TSG-6 expression following activation with bacterial components could participate in the suppression of inflammatory cytokines, such as TNF-α, and it is noteworthy that KYNA and especially KYNA analogs are able to enhance this effect. Further studies are required to elucidate the different effects of KYNA derivates in the case of different bacterial inducers and the possible benefits of targeting TSG-6 expression by kynurenines in inflammatory conditions following infections.

DATA AVAILABILITY
All datasets generated for this study are included in the manuscript and/or the supplementary files.

ETHICS STATEMENT
For the experiments performed with the human blood we have the approval of the ethics commitee of the Medical Faculty of the University of Szeged (ETT-TUKEB 905/PI/09). This study was conducted in full accordance with the tenets of Declaration of Helsinki (1964).