Sitaoxacin Inhibits TNF α Release from Monocytic THP-1 Cells Stimulated by LPS

Sepsis is a systemic reaction to infection and excessive production of inflammatory cytokines and chemokines. It sometimes results in septic shock. The present study was designated to find out which quinolone antibiotic reduces TNF  production the most and to elucidate its mechanisms. We examined which quinolone antibiotic reduced TNF  production from THP-1 cells stimulated by lipopolysaccharide (LPS). Then, we examined the mechanism of inhibition of TNF  production by the antibiotic. STFX most effectively reduced TNF  concentrations within LPS-stimulated THP-1 cells supernatant. STFX suppressed TNF  production in a dose-dependent manner. We found that STFX did not inhibit the NF-  B, ERK, or p38 pathways, nor did it inhibit the production of TNF  mRNA. The percentage of intracellular TNF  was increased in cells stimulated by LPS and with STFX compared to that of cells stimulated by LPS alone. In conclusion, one of the mechanisms reducing TNF  production from LPS-stimulated THP-1 cells treated with STFX involves inhibition of TNF  release from these cells. STFX has a broad antimicrobial spectrum for gram-positive, gram-negative, and anaerobic bacteria , and may be effective for treating sepsis by both killing bacteria and suppressing inflammation.


Introduction
Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection [1]. The first definition of sepsis was that of sepsis-1 in 1991, and in 2016 its definition was redefined as sepsis-3. Sepsis was further defined as the systemic inflammatory response syndrome caused by an infection [2]. Particularly during gram-negative bacterial infection, lipopolysaccharide (LPS) stimulates cells to produce inflammatory cytokines and chemokines which can sometimes result in septic shock. Inflammatory cytokines cause decreased blood pressure via dilation of blood vessels and blood clotting within the capillaries of organs. These effects can aid the immune system in the process of fighting infection, but can also be harmful. Thus, drugs that not only fight bacteria but also reduce inflammatory cytokines are required to avoid such harmful effects.
Quinolones, such as garenoxacin or moxifloxacin, have also been reported to reduce inflammatory cytokines [10,11]. But there remain few reports as to whether sitafloxacin (STFX), a quinolone antimicrobial agent, can lead to reduction in these cytokines.
STFX exhibits a broad antimicrobial spectrum for gram-positive, gram-negative, and anaerobic bacteria [12]. STFX is an effective agent against pneumococcal infections and incidence of drug-resistant mutants is low in vitro [13].
STFX was shown to be effective against Haemophilus influenzae pneumonia in a murine model [14].
In a clinical study, STFX was also proven effective and safe for the treatment of nursing and healthcare-associated pneumonia, including aspiration pneumonia, in elderly patients [15]. STFX treatment was effective for patients with both acute complicated urinary tract infection and pyelonephritis caused by Escherichia coli producing extended spectrum beta-lactamase (ESBL) and producing non-ESBL [16]. In another report, STFX was effective against the E. coli producing ESBL following 3 days of carbapenem therapy [17].
STFX, a broad-spectrum oral fluoroquinolone, has been approved in Japan for the treatment of respiratory and urinary tract infections. However, it is unknown whether STFX can be used for treating patients with sepsis, or whether it suppresses the production of inflammatory cytokines and chemokines, which we aimed to determine in the current study.

STFX inhibited TNF production significantly compared with other quinolones
We examined which quinolones exhibited inhibition upon TNF production by determining the concentration of TNF within the supernatant of LPS-stimulated TNF concentrations following LPS and STFX exposure were significantly lower than those of LPS plus LVFX, GRNX, MFLX, or CPFX (p < 0.01), with STFX reducing TNF concentration the most (Fig. 1).

STFX inhibited the production of chemokines
STFX inhibited not only TNF production but also that of chemokines, as shown by additional experiments with LPS-stimulated THP-1 cells. Concentration of IL-8 in supernatants of cells treated with 50 µg/ml STFX was significantly decreased to 10472.00 ± 474.67 pg/mL compared with that of LPS alone 17802.33 ± 190.07 (p < 0.01) (Fig. 3a). Concentrations of IP-10 in supernatants of cells exposed to 50 µg/ml STFX was significantly decreased to 77.83 ± 9.70 pg/mL compared with that of LPS alone 3649.00 ± 377.59 (p < 0.01) (Fig. 3b). The concentration of MCP-1 in cell supernatants in the presence of 50 µg/ml STFX was also significantly decreased, to 161.67 ± 11.59 pg/mL compared with that of LPS alone 3453.00 ± 148.55 (p < 0.01) (Fig. 3c). Furthermore, MIP-1 concentrations in the supernatants of cells followed by treatment with 50 µg/ml STFX were significantly decreased to 9336.67 ± 206.50 pg/mL compared with that of LPS alone 20859.33 ± 196.41 (p < 0.01) (Fig. 3d). The supernatant concentration of MIP-1 from cells exposed to 50 µg/ml STFX were additionally significantly decreased to 2844.67 ± 135.43 pg/mL compared with that of LPS alone 12950.67 ± 409.62 (p < 0.01) (Fig. 3e).

STFX did not inhibit the signaling pathway
THP-1 cells (2 × 10 5 /mL) were stimulated by LPS (0.1 g/mL) with or without the presence of STFX (50 g/mL) for 1 hr. The phosphorylated forms of NF-kB, ERK and p38 did not decrease within the cells treated with STFX plus LPS compared to LPS alone ( Fig. 4).

STFX did not influence TNF mRNA levels
Expression of TNF mRNA was measured in THP-1 cells stimulated with LPS (0.1 g/mL) for 1 or 2 hr with or without STFX (50 g/mL). TNF mRNA levels within cells treated with STFX did not significantly change compared with those of LPS alone after either 1 or 2 hrs of exposure (Fig. 5).

STFX inhibit TNF release from cells
THP-1 cells (2 × 10 5 /mL) were stimulated by LPS (0.1 g/mL) with or without STFX (50 g/mL). After 4 hr of incubation, intracellular TNF was stained by anti-TNF antibody PE. The percentage of intracellular TNF in cells treated with STFX and LPS was increased to 16.2% from 4.4% within cells treated with LPS alone (Fig. 6).

Discussion
TNF plays a very important role within sepsis. Blocking of TNF has been shown to protect mice from the symptoms of sepsis [18]. Some clinical studies investigating the monoclonal antibodies produced against TNFfor patients with sepsis or septic shock have been reported [19][20][21]. Modulation of TNF and other inflammatory cytokines and chemokines is considered very important in the treatment of severe infectious diseases, especially that of sepsis or septic shock.
In the present study, STFX significantly reduced the concentration of TNF in the supernatants of LPS-stimulated THP-1 cells as compared with other quinolone antibiotics; STFX also reduced the levels of some inflammatory chemokines.
Some types of antibiotics can modulate inflammatory cytokines, but the mechanisms of cytokine inhibition may vary. It has already been reported that minocycline (MINO) inhibits IkB kinase a/b phosphorylation of the NF-kB pathway in THP-1 cells [4]. It was also reported that clarithromycin (CAM) attenuates STAT6 phosphorylation. 5 Other groups reported that macrolide antibiotics decreased functions of the ERK and NF-kb signaling pathways [6,7]. GRNX and MFLX were reported to inhibit these signaling pathways to suppress the production of inflammatory cytokines.
Even when using similar quinolone antibacterial drugs, the mechanism of cytokine suppression differs depending upon the characteristics each drug. Previous studies reported that quinolones with a cyclopropyl group at the N1 position and/or a piperazinyl group at the C7 position, can modify inflammatory responses [22][23][24].
STFX consists of a fluorocyclopropene at the 1-position of the quinolone skeleton, a chlorine group at the 8-position, a spiroheptane group at the 7-position, and a quinolone with a chlorine group introduced at the 8-position. Such characteristics are considered to cause differences within the spectrum of antibacterial activity, but may also cause differences in anti-inflammatory effects.
In the present study, STFX suppressed TNF production more strongly than the other tested quinolone antibiotics. It did not suppress the signaling pathways that produced TNF, nor did it suppress TNF mRNA production. It is possible that STFX may inhibit the process of producing TNF from mRNA, or may inhibit the release of produced TNF from THP-1 cells. Flow cytometry analysis suggested that STFX inhibited the extracellular release of TNF, but this tendency is slight, and it is unknown whether this mechanism alone suppressed TNF production. One of the mechanisms inhibiting TNF production by STFX may not be due to inhibition of the signaling pathways but instead interference with TNF release from cells. However, a mechanism inhibiting TNF production from mRNA may also be involved and warrants further verification. STFX may be an effective drug for patients with bacterial infection due to its antimicrobial action and simultaneous reduction of TNF. STFX is approved as an oral antibacterial drug, and could be a candidate used to treat patients exhibiting sepsis or septic shock.

RNA extraction and quantification
Total RNA was extracted using Sepasol-RNA I Super G (Nacalai Tesque), cDNA was according to the manufacturer's protocol. Intracellular TNF was stained using anti-TNF antibody PE (BD Biosciences) for 1 h. Cells were washed and resuspended in PBS supplemented with 2% fetal bovine serum and 0.05% NaN 3 . Intracellular TNF was evaluated using a FACS Canto II (BD Biosciences).

Statistical analysis
Values are presented as average ± SD. Data were analyzed by using Student's t-test with statistical software (Microsoft Excel 2008; Microsoft Corporation, Redmond, WA, USA), in which a p-value of < 0.05 was considered statistically significant.