Cell-Free Supernatant from Lactobacillus and Streptococcus Strains Modulate Mucus Production via Nf-κB/CREB Pathway in Diesel Particle Matter-Stimulated NCI-H292 Airway Epithelial Cells

Airway epithelial cells are a major site of airway inflammation and may play an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Diesel particulate matter (DPM) is associated with mucus hypersecretion and airway inflammation and has been reported to overexpress airway mucin in the NCI-H292 airway epithelial cells. Therefore, regulation of mucin hypersecretion is essential for developing novel anti-inflammatory agents. This study aimed to investigate the effects of cell-free supernatant (CFS) from Lactobacillus and Streptococcus on nitro oxide (NO) production in RAW264.7 and proteins associated with mucus production in NCI-H292 cells. We observed that NO production was reduced by CFS from Lactobacillus and Streptococcus in RAW 264.7, and MUC4, MUC5AC, and MUC5B gene expression was increased by phosphorylation of nuclear factor kappa B (NF-κB) p65 and cAMP response element-binding protein (CREB) in DPM-stimulated NCI-H292 cells. However, CFS from L. paracasei MG4272, MG4577, L. gasseri MG4247, and S. thermophilus MG5140 inhibited mRNA expression related to mucus production by downregulating the CREB/NfκB signaling pathway. These results suggest that CFS from L. paracasei MG4272, MG4577, L. gasseri MG4247, and S. thermophilus MG5140 can contribute as a strategic candidate to the prevention of airway inflammatory diseases caused by DPM.


Introduction
Airway inflammation is a major cause of chronic obstructive pulmonary disease (COPD), a non-communicable disease that causes pulmonary and extra-pulmonary symptoms [1]. Inhalation toxicity pollutants cause chronic airway inflammation by activating epithelial cells and macrophages in the lungs, and systemic inflammation occurs when these inflammatory mediators are exposed to the circulatory system [2,3]. Airway inflammation increases the production of a mixture of sputum by excess mucin and other glycoproteins [4]. Mucin production is regulated by nine membrane-tethered genes and seven gel-forming genes. Among them, MUC5AC and MUC5B, which are gel-forming genes, account for 90% of mucins [5]. Activation of the mucin transcription factor mainly involves the expression of the nuclear factor kappa B (NfκB) p65/cAMP response elementbinding protein (CREB) pathway via phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinases (p38) [6][7][8]. COPD is one of the five leading causes of death worldwide, with an increase in the prevalence of 15.6% between 2007 and 2017 [9]. According to recent reports, the mortality rate has been increasing due to respiratory diseases, such as COPD caused by DPM, in recent years [10]. There are various drug therapies to treat airway inflammation such as COPD, but the early mortality rate is high because it imposes a significant economic burden [11]. Thus, there is

Inhibition of NO Production by CFS from Lactobacillus and Streptococcus Strains in LPS-induced RAW 264.7 Cells
The cytotoxicity of 2.5 and 5% CFS in RAW 264.7 was established by MTT assay ( Figure 1A). The results showed that 5% CFS of Lactobacillus and Streptococcus strains showed no cytotoxicity (≥100%) in RAW 264.7 cells. Moreover, LDH release of 5% CFS on LPS-induced RAW 264.7 cells were measured, shown in Figure 1B. All CFS decreased LDH release compared to LPS-induced control. Therefore, NO production was evaluated at 5% CFS, a concentration without cytotoxicity. As shown in Figure 1C, the CFS of all probiotics remarkably inhibited NO production compared to the LPS-induced controls.

CFS from Lactobacillus and Streptococcus Strains Suppress Cytotoxicity in DPM-induced NCI-H292 Cells
First, cell morphology was observed to set the treatment time of DPM on NCI-H292 cells ( Figure 2A). As a result, it was confirmed that DPM completely penetrated the NCI-H292cells after 24 h of treatment, so subsequent experiments were conducted at 24 h treatment. Based on the results of RAW 264.7 cells, the cytotoxicity of 5 and 10% CFS in NCI-H292 cells was assessed by MTT assay. In Figure 2B,C, 5% CFS of Lactobacillus and Streptococcus strains showed no cytotoxicity (≥86.74%) in the presence or absence of DPM for 24 h. Moreover, LDH release was measured at 5% CFS. As a result, the 5% CFS of all probiotics significantly inhibited LDH release compared to the DPM-induced controls.   The RAW 264.7 cells were pretreated with 2.5 and/or 5% of CFS for 1 h and then incubated with or without only LPS (0.5 µg/mL) for 24 h. The results indicate the mean ± SEM of three separate experiments. ### p < 0.001 compared with control and * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with LPS alone. , and production of nitro oxide (NO) with LPS (C) in RAW 264.7 cells. The RAW 264.7 cells were pretreated with 2.5 and/or 5% of CFS for 1 h and then incubated with or without only LPS (0.5 µ g/mL) for 24 h. The results indicate the mean ± SEM of three separate experiments. ### p < 0.001 compared with control and * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with LPS alone.

CFS from Lactobacillus and Streptococcus Strains Suppress Cytotoxicity in DPM-induced NCI-H292 Cells
First, cell morphology was observed to set the treatment time of DPM on NCI-H292 cells ( Figure 2A). As a result, it was confirmed that DPM completely penetrated the NCI-H292cells after 24 h of treatment, so subsequent experiments were conducted at 24 h treatment. Based on the results of RAW 264.7 cells, the cytotoxicity of 5 and 10% CFS in NCI-H292 cells was assessed by MTT assay. In Figures 2B and 2C, 5% CFS of Lactobacillus and Streptococcus strains showed no cytotoxicity (≥86.74%) in the presence or absence of DPM for 24 h. Moreover, LDH release was measured at 5% CFS. As a result, the 5% CFS of all probiotics significantly inhibited LDH release compared to the DPM-induced controls.  The results indicate the mean ± SEM of three separate experiments. ## p < 0.01, and ### p < 0.001 compared with control and * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with DPM alone.
(1, 2, 4, 6, and 24 h) of DPM, was observed by the microscope (×20, A). Red arrows indicate intracel-103 lular accumulation of DPM. Cell viability of CFS (B) and CFS with DPM (C) was measured by MTT 104 assay. Cytotoxicity was assessed by lactate dehydrogenase (LDH) release (D). The NCI-H292 cells 105 were pretreated with 5% CFS and then incubated with or only DPM (20 µ g/mL) for 24 h. The results 106 indicate the mean ± SEM of three separate experiments. ##p < 0.01, and ###p < 0.001 compared with 107 control and *p < 0.05, **p < 0.01, and ***p < 0.001 compared with DPM alone. 108 119 and MUC5B (C) mRNA expression in DPM-induced NCI-H292 cells. The expression of mRNA was 120 determined by qRT-PCR. The NCI-H292 cells were pretreated with 5% CFS and then incubated with 121 or only DPM (20 µ g/mL) for 24 h. The mRNA expression was normalized to GAPDH as the internal 122 control. The results indicate the mean ± SEM of four separate experiments. #p < 0.05, and ###p < 0.001 123 compared with control and *p < 0.05, **p < 0.01, and ***p < 0.001 compared with DPM alone. 124  The NCI-H292 cells were pretreated with 5% CFS and then incubated with or only DPM (20 µg/mL) for 24 h. The mRNA expression was normalized to GAPDH as the internal control. The results indicate the mean ± SEM of four separate experiments. # p < 0.05, and ### p < 0.001 compared with control and * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with DPM alone.

Effect on Phosphorylation of Nf-κB by CFS from Lactobacillus and Streptococcus Strains in DPM-induced NCI-H292 cells
Protein expression of NF-κB phosphorylation in NCI-H292 cells treated with DPM at various time points was measured using western blotting ( Figure 5A). Before 8 h of treatment with DPM, there was no change in NF-κB phosphorylation in NCI-H292 cells; however, at 18 and 24 h, there was a gradual increase. As shown in Figure 5B, treatment with CFS Lactobacillus and Streptococcus strains reduced the phosphorylation of NF-κB, except for MG4604, in DPM-induced NCI-H292 cells. L. paracasei MG4272, MG4577, L. gasseri MG4247, and S. thermophilus MG5140 markedly inhibited the protein expression of p-NF-κB (0.54-, 0.57-, 0.32-, and 0.49-fold, respectively) in DPM NCI-H292 cells.

Discussion
COPD is an airway inflammatory disease caused by genetic predisposition, smoking, environmental factors such as DPM and cigarette smoke, and chronic inflammation. Immune responses play an important role in the progression of COPD [3]. In COPD, although probiotics are unlikely to act directly in the lungs via the circulatory system, systemically, metabolites from probiotics modulate inflammatory factors to activate immune cells and macrophages, thereby indirectly protecting against airway inflammation [13]. Metabolites, short-chain fatty acids (SCFA), and microbe-associated molecular patterns (MAMPs) derived from the probiotics suppress the loss of immune homeostasis and inflammatory response caused by excessive DPM exposure by promoting tight-junction proteins to repair the intestinal epithelial barrier function or prevents COPD by directly influencing pulmonary immune homeostasis [22]. Recently, it was reported that metabolites of probiotics carried through the circulation to the lungs might help with COVID-19 by inhibiting viral replication or enhancing the immune response [23]. This inhibition of inflammation has been demonstrated in animal studies that induced COPD with DPM and cigarette smoke; Bifidobacterium breve and Lactobacillus rhamnosus reduced gene expression related to mucin production and inflammatory factors, NF-κB, and cytokines [18,24]. Lactobacillus casei HY2782 and Bifidobacterium lactis HY8002 decreased oxidative stress and pro-inflammatory cytokines in the lung in vivo [25]. In addition, since DPM has been reported to cause intestinal imbalance, probiotics may contribute to the prevention of airway inflammation by altering the intestinal microflora [26]. In this study, we attempted to prove the efficacy of CFS from Lactobacillus and Streptococcus strains against inflammatory factors in macrophages, mucin production, and airway inflammation in NCI-H292 airway epithelial cells induced by DPM. Based on these results, it was intended to be presented as a candidate probiotic with COPD prevention efficacy.
As mentioned above, systemic inflammation can affect airway inflammation in the respiratory system [1]. LPS-induced RAW264.7, a macrophage-like cell line, is an in vitro model that can confirm systemic inflammation [27]. In our results, NO production in LPS-induced RAW 264.7 was decreased by 5% CFS from Lactobacillus and Streptococcus. NO plays a vital role in the immune response to inflammatory activity and is beneficial for the host's defense against pathogens and parasites [28]. Additionally, Lactobacillus and Streptococcus reduced pro-inflammatory cytokines, interleukin 6 (IL-6), and tumor necrosis factor (TNF)-α in our previous study and Figure S1 [29]. TNF and IL6 are recognized as major factors in the systemic inflammatory immune response, and overexpression of these factors leads to respiratory diseases, including COPD and COVID-19 [30,31]. These results reveal that CFS of Lactobacillus and Streptococcus can indirectly reduce airway inflammation by regulating various systemic inflammatory factors in macrophages.
In inflammatory airway diseases, such as COPD, cough and sputum symptoms due to excessive mucus secretion appear [32]. In 11 mucus samples mainly expressed in the lungs, MUC5AC and MUC5B mRNA are upregulated by inflammatory cytokines or DPM as major factors that produce airway mucus [33]. In addition, it has been reported that the expression of MUC4 mRNA can affect airway inflammation in human airway epithelial cells [5]. Our results showed that L. paracasei MG4272, MG4577, L. gasseri MG4247, and S. thermophilus MG5140 inhibited the mRNA expression of MUC4, MUC5AC, and MUC5B, which are mucin-producing genes, in NCI-H292 cells treated with DPM. Phosphorylation of CREB by p-ERK1/2 and/or p-p38 MAPK is a major intracellular mechanism for the expression of MUC4, MUC5AC, and MUC5B genes [8,34]. It has been reported that DPM-induced mucin production is mainly mediated by ERK and p38 MAPK [8]. In our results, the phosphorylation of ERK1/2 and p38 was significantly suppressed by L. paracasei MG4272, MG4577, L. gasseri MG4247, and S. thermophilus MG5140 CFS in DPM-induced NCI-H292 cells. In addition, DPM elevates NF-κB signaling pathways, which is an inflammatory/immune response mediator in human airway epithelial cells, and overexpression of NF-κB phosphorylation activated by p-p38 results in mucin overproduction [6,35]. The CFS from L. paracasei MG4272, MG4577, L. gasseri MG4247, and S. thermophilus MG5140 markedly reduced NF-κB phosphorylation in DPM-induced NCI-H292 cells. L. paracasei MG4272, MG4577, L. gasseri MG4247, and S. thermophilus MG5140, which were proven to inhibit mucus and anti-inflammatory effects in this study, can be used as safe probiotics because their hemolysis, cytotoxicity, and adhesion to intestinal epithelial cells have been confirmed [29]. In our previous reports, L. paracasei MG4272, MG4577, and L. gasseri MG4247 attenuate allergic inflammatory response in mast cells by modulating signal transducer and activator of transcription 6 (STAT6) phosphorylation [29]. Thus, these strains may effectively modulate allergic reactions and airway inflammation related to the respiratory tract.
In summary, CFS from L. paracasei MG4272, MG4577, L. gasseri MG4247, and S. thermophilus MG5140 downregulated mucin production by indirectly reducing pro-inflammatory factors in macrophages and directly inhibiting airway inflammation by modulating the CREB/Nf-κB signaling pathway, which is involved in the expression of MUC4, MUC5AC, and MUC5B in NCI-H292 airway epithelial cells treated with DPM. It seems that L. paracasei MG4272, MG4577, L. gasseri MG4247, and S. thermophilus MG5140 are potential functional foods that can prevent COPD-related airway inflammation; however, this must be verified through additional animal and clinical studies.

Cell Culture
The RAW264.7 cell (American Type Culture Collection, Manassas, VA, USA) were cultured in DMEM containing 10% heat-inactivated FBS and 1% P/S. NCI-H292 airway epithelial cells (Korean Cell Line Bank, Seoul, Republic of Korea) were cultured in RPMI1640 medium with 10% heat-inactivated FBS and 1% P/S. The cells were maintained at 37 • C in a 5% CO2 incubator and passaged after cells reached 70-80% confluency.

Assessment of NO Production
As previously described, NO production was measured using Griess reagent [37]. The RAW264.7 cells were seeded in 24-well plates (3 × 10 5 cells/mL). After treatment with CFS from Lactobacillus and Streptococcus strains and MRS (as control) for 1 h and treatment with LPS (0.5 µg/mL) for 24 h, the cell culture supernatant (50 µL) was mixed with Griess reagent (50 µL), followed by measurement at 550 nm with a microplate reader (BioTek).

Preparation of mRNA and Real-Time Polymerase Chain Reaction (qRT-PCR)
RAW 264.7 (7 × 10 5 cells/well) and NCI-H292 cells (4 × 10 5 cells/well) were seeded in 6-well plates. Cells were pretreated with CFS from Lactobacillus and Streptococcus strains and MRS (as control) for 1 h and then treated with LPS (0.5 µg/mL) in RAW 264.7 and/or DPM (20 µg/mL) in NCI-H292 cells. The purity of mRNA was measured using µDrop plates (Thermo Scientific, Waltham, MA, USA) and a microplate reader (BioTek). mRNA was isolated using NucleoZol, following the manufacturer's protocols. cDNA was prepared using the isolated mRNA and Maxime RT PreMix. qRT-PCR was performed using the CFX96™ System (Bio-Rad, Hercules, CA, USA) with AmfiSure qGreen Q-PCR Master Mix. The primer sequences used are listed in Tables 1 and S1. Relative quantitative expression was analyzed using the 2 −∆∆CT method and normalized using the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [38]. The relative mRNA expression was determined as the fold-change of LPS-or DPM-treated control.

Protein Extraction
For total protein extracts, cells were pretreated with CFS from Lactobacillus and Streptococcus strains and MRS (as control) for 1 h and then treated with LPS (0.5 µg/mL) in RAW 264.7 and/or DPM (20 µg/mL) in NCI-H292 cells. Protein lysates were obtained by RIPA lysis buffer containing phosphatase and protease inhibitors. Lysates were centrifuged at 13,000 rpm for 15 min at 4 • C, and the supernatants containing the extracted proteins were collected and stored at −80 • C. The extracted proteins were quantified at 1 µg/µL using Bradford reagent (Coomassie). The protein sample was prepared by diluting in 4X LDS sample buffer and heating at 70 • C for 10 min.

Western Blotting
Western blotting was performed as previously reported [39]. Briefly, protein samples were loaded onto 8 and 10% Tris-Bis gels and electrophoresed in MOPS buffer. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Middlesex County, MA, USA) and washed with TBS-Tween buffer (TBST). After blocking with Smart-Block™ 5 min Fast Blocking Buffer, the membranes were incubated with primary antibodies (1:1000) overnight at 4 • C. After washing three times with TBS-Tween buffer; the membranes were incubated with HRP-conjugated secondary antibodies (1:5000) for 1 h. The membrane was developed using LuminoGraph III Lite (ATTO, Tokyo, Japan) with West-Q Femto Clean ECL solution, and densitograph analysis was performed using CS Analyzer 4(ATTO).

Statistical Analysis
Results are expressed as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) and Tukey's multiple comparison test (GraphPad Software, Inc., San Diego, CA, USA) was considered statistically significant at p < 0.05.

Data Availability Statement:
The authors declare that all data and materials support published claims and comply with field standards.