Kefir peptides alleviate particulate matter <4 μm (PM4.0)-induced pulmonary inflammation by inhibiting the NF-κB pathway using luciferase transgenic mice

Kefir peptides, generated by kefir grain fermentation of milk proteins, showed positive antioxidant effects, lowered blood pressure and modulated the immune response. In this study, kefir peptide was evaluated regarding their anti-inflammatory effects on particulate matter <4 μm (PM4.0)-induced lung inflammation in NF-κB-luciferase+/+ transgenic mice. The lungs of mice under 20 mg/kg or 10 mg/kg PM4.0 treatments, both increased significantly the generation of reactive oxygen species (ROS) and inflammatory cytokines; increased the protein expression levels of p-NF-κB, NLRP3, caspase-1, IL-1β, TNF-α, IL-6, IL-4 and α-SMA. Thus, we choose the 10 mg/kg of PM4.0 for animal trials; the mice were assigned to four treatment groups, including control group (saline treatment), PM4.0 + Mock group (only PM4.0 administration), PM4.0 + KL group (PM4.0 + 150 mg/kg low-dose kefir peptide) and PM4.0 + KH group (PM4.0 + 500 mg/kg high-dose kefir peptide). Data showed that treatment with both doses of kefir peptides decreased the PM4.0-induced inflammatory cell infiltration and the expression of the inflammatory mediators IL-lβ, IL-4 and TNF-α in lung tissue by inactivating NF-κB signaling. The oral administrations of kefir peptides decrease the PM4.0-induced lung inflammation process through the inhibition of NF-κB pathway in transgenic luciferase mice, proposing a new clinical application to particulate matter air pollution-induced pulmonary inflammation.


Results
Effect of PM 4.0 exposure on pulmonary inflammation in NF-κB-luciferase +/+ transgenic mice. PM 4.0 in both doses (10 and 20 mg/kg) increased the luminescent signals in the total chest cavity and ex vivo lung tissue compared to control group (without treatment) when quantified using the In Vivo Imaging System (IVIS) (Fig. 1a.b). Interestingly, the deposition of PM 4.0 particles increased in the pulmonary tissue and BALF in a dose-dependent manner (Fig. 1c). PM 4.0 in both administrations (10 and 20 mg/kg of dose) increased the total protein levels, total and relative cell counts of macrophages and neutrophils compared to those in the control group (p < 0.01 and p < 0.001) (Fig. 1d). Data showed that PM 4.0 significantly increased the levels of IL-1β and TNF-α, in BALF and serum, compared to those in the control group (p < 0.05). A significant increase in the generation of extracellular ROS in the pulmonary tissue was observed in the PM 4.0 -induced groups compared with the control group (p < 0.001) when analyzed using DCF-DA fluorescence without difference between high and low doses of PM 4.0 (Fig. 1d). The balance between the production of ROS and the antioxidant defense system, which includes SOD, determines the degree of oxidative stress. PM 4.0 in both doses (10 and 20 mg/kg) decreased the total SOD activity compared to those in the control group (p < 0.01 and p < 0.001) (Fig. 1d).
Effect of PM 4.0 exposure on inflammatory mediator expression in NF-κB-luciferase +/+ transgenic mice. To determine whether exposure to PM 4.0 induced pulmonary inflammatory responses in transgenic mice, the levels of the inflammatory mediators p-NF-κB, NLRP3, caspase-1, IL-1β, TNF-α, IL-6 and IL-4 in pulmonary tissue were quantified (Fig. 2). The expression of NLRP3, p-NF-κB, caspase 1, IL-4 and TNF-α were significantly increased in the PM 4.0 groups, without differences between them, compare to control group (p < 0.001). The inflammatory cytokines IL-1β and IL6 increase the expression in both group with PM 4.0 group respect to control group (p < 0.001) being mayor in high dose group (p < 0.05) (Fig. 2).

Effect of PM 4.0 exposure on histopathological changes in NF-κB-luciferase +/+ transgenic mice.
Lung histopathology was examined and showed pulmonary edema and alveolar infiltration of neutrophils in the PM 4.0 groups (Fig. 3a). After PM 4.0 administrations (10 and 20 mg/kg of dose), the amount of collagen was significantly increased compared to the control group (Fig. 3b,c). Furthermore, analysis of α-smooth muscle actin (α-SMA) in lung tissues by Western blotting also showed significant increases in the groups exposed to low and high doses of PM 4.0 compared to the control group, without difference between them (p < 0.001). The results suggested that the lung inflammation and fibrosis in the group exposed to the low dose (10 mg/kg) of PM 4.0 daily were sufficient, so we choose the low dose of PM 4.0 to further evaluate the pulmonary inflammation status after kefir peptides treatment in a preventive animal trial.
Effect of kefir peptides on PM 4.0 -induced NF-κB activation in NF-κB-luciferase +/+ transgenic mice. PM 4.0 stimulated the luminescence signal in the chest and lung tissue, but the luciferase signals in the PM 4.0 + KL and PM 4.0 + KH groups were significantly lower than that in the PM 4.0 + Mock group (Fig. 4a,b). The deposition of PM 4.0 particles was significantly increased in the pulmonary tissue and BALF in the PM 4.0 + Mock group; however, treatments with kefir peptides significantly decreased the PM 4.0 deposition compared to that in the PM 4.0 + Mock group (Fig. 4c). Bioluminescence imaging of the chest cavity and lung tissue of transgenic NF-κB +/+ mice after exposure to saline solution, 10 and 20 mg/Kg of PM 4.0 for 4 weeks. (c) Both PM 4.0 -exposed transgenic mice had more black particle deposition in the BALF and lung tissue than the control group. Red arrow: PM 4.0 aggregation. (d) PM 4.0 exposure increased the total cell and total protein levels, the macrophage and neutrophil cell counts, the inflammatory cytokine (IL-1β, TNF-α) levels, and the DCF (ROS) levels in BALF; decreased of total SOD activity in lung tissue; increased the circulating inflammatory cytokine (IL-1β, TNF-α) levels in serum compared to those in the control group. n = 8 per group. Data are expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control group. www.nature.com/scientificreports www.nature.com/scientificreports/ control group (p < 0.01). However, treatments with kefir peptides (KH and KL dose) led to a significant decrease in ROS, inflammatory cells and cytokines compared to those in the PM 4.0 + Mock group (p < 0.01) (Fig. 4d). In addition, the total SOD activity in pulmonary tissue were significantly lower in the PM 4.0 + Mock group than in the control group (p < 0.001), and treatments with kefir peptides significantly increased the SOD activity compared to that in the PM 4.0 + Mock group (p < 0.01), without differences between them (Fig. 4d).
Effect of kefir peptides on inflammatory mediator expression in PM 4.0 -treated NF-κBluciferase +/+ transgenic mice. The ratio of p-NF-κB/NF-κB protein expression was significantly increased in the PM 4.0 + Mock group compared to that in the control group (p < 0.001), and treatments with kefir peptides significantly decreased the p-NF-κB level and p-NF-κB/NF-κB ratio compared to that of the PM 4.0 + Mock group (Fig. 5a,b). In this study, we observed that treatments with either low dose or high dose of kefir peptides could reduce NF-κB expression and thus subsequently decrease NLRP3, caspase-1, IL-1β, IL-6, TNF-α and IL-4 expression ( Fig. 5a-d).  Fig. 6a-c). However, the groups treated with either low-dose (KL) or high-dose (KH) kefir peptides exhibited lower amounts of neutrophil infiltration, lung edema and lung fibrosis, including collagen deposition and collagen fibers (Fig. 6b,c). In addition, the expression of α-SMA protein was significantly higher in the PM 4.0 + Mock group than in the control group (p < 0.001), and treatments with kefir peptides at either the low dose or the high dose significantly decreased the α-SMA level compared to that of the PM 4.0 alone/Mock group (p < 0.01) (Fig. 6d). www.nature.com/scientificreports www.nature.com/scientificreports/ www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
In the present study, we note three major findings indicating that kefir peptides alleviate PM 4.0 -induced pulmonary inflammation in NF-κB-luciferase +/+ transgenic mice through NF-κB pathway inhibition. First, exposure to PM 4.0 through intratracheal instillation once a day for 4 weeks successfully induces pulmonary inflammation in transgenic mice. PM 4.0 exposure induces inflammatory cell infiltration, oxidative stress and inflammatory mediator overexpression in lung tissue by activating the NF-κB pathway. Second, kefir peptides reduce the ROS www.nature.com/scientificreports www.nature.com/scientificreports/ levels; decrease NF-κB activation, proinflammatory cytokine production and inflammatory cell infiltrates; and increase the total SOD activity in lungs. Third, the antioxidant effect and the subsequent reduction in activation of the NF-κB, NLRP3-dependent inflammasome and caspase-1 pathways contribute to the complex molecular anti-inflammatory mechanisms of kefir peptides. The medical imaging system using NF-κB-luciferase +/+ transgenic mice carrying the luciferase gene driven by the NF-κB promoter is a potential animal model for monitoring inflammation and the effects of treatments. The best advantage is that this method provides noninvasive, real-time and whole-body screening. The results from the medical imaging agree with our expectations to some extent; the most evident organ expressing luciferase in vivo was the lung. The present study demonstrated, for the first time, the anti-inflammatory effect of kefir peptides on PM 4.0 -induced lung inflammation in NF-κB-luciferase +/+ transgenic mice, as shown in bioluminescence images obtained by the In Vivo Imaging System (IVIS) 22,23 . The imaging results show us that the lung is one of the organs with oxidative stress and inflammatory responses after exposure to PM 4.0 (Fig. 1a,b). PM 2.5 is well known to induce prooxidant and proinflammatory actions 9,24,25 , but the PM 4.0 -induced effects on inflammatory responses in mice were not known. Previous reports demonstrated that PM 2.5 could be internalized into cells through endocytosis processes and have potentials to activate NLRP3 inflammasome through activation of NF-κB-dependent cascade and assembly of inflammasome complex (including cathepsin B release, ROS production and potassium efflux), as a result of pulmonary fibrosis 26,27 . To our knowledge, this is the first report showing that PM 4.0 exposure leads to inflammatory responses in the lung and the occurrence of systematic inflammation, resulting in the release of inflammatory cytokines, which can induce lung inflammation through mechanisms that are similar to those for PM 2.5 [28][29][30] . In this study, PM 4.0 activated p-NF-κB, leading to activation of the NLRP3 inflammasome, which induced caspase-1 activation and thus the production of proinflammatory IL-1β. The elevated IL-1β simultaneously activated the expression of TNF-α, IL-6 and IL-4 (Fig. 2), which play a crucial role in the inflammatory pathway [31][32][33] . Nevertheless, treatments with kefir peptides significantly decreased the protein expression of p-NF-κB, NLRP3, caspase-1, IL-1β, TNF-α, IL-6 and IL-4 and increased the SOD activity in NF-κB-luciferase +/+ transgenic mice (Figs 4 and 5). Taken together, these results indicate that kefir peptides affects p-NF-κB, NLRP3, caspase-1, IL-1β, TNF-α, IL-6 and IL-4, all of which reduce the inflammatory response, by inactivating NF-κB signaling (luciferase expression, phosphorylated NF-κB, NLRP3-dependent inflammasome and caspase-1). A hypothetical scheme of the kefir peptides regulatory pathway against PM 4.0 -induced lung inflammation is shown in Fig. 7.
The lungs are susceptible to damage by airborne particles, as observed in the histological sections, BALF and serum samples. Following PM exposure, inflammatory responses are stimulated and numerous inflammatory cytokines are released from the lung parenchyma 34,35 . Several pulmonary diseases including asthma, acute lung injury, COPD and acute respiratory distress syndrome (ARDS), are associated with abnormal TNF-α, IL-1β and IL-6 expression 36 . To investigate whether the protective effect of kefir peptides against PM 4.0 -induced pulmonary injury was associated with inflammation, TNF-α and IL-1β levels were measured. The present study revealed that kefir peptides treatment significantly decreased TNF-α and IL-1β protein levels in BALF and serum. These results indicate that kefir peptides treatment can ameliorate PM 4.0 -induced damage by suppressing inflammation. In addition, pulmonary inflammation and fibrosis, including inflammatory cell infiltration, interstitial edema, collagen deposition, collagen fibers and overexpression of α-SMA protein (Fig. 3), were observed in PM 4.0 -treated mice, suggesting that the pulmonary biofilm and parenchymal cells were damaged. This damage was significantly attenuated by kefir peptides treatment (Fig. 6). These results indicate that kefir peptides could exert protective effects against PM 4.0 -induced pulmonary inflammation.
Kefir, which originated in the North Caucasian mountains, is rich in protein complex, EPS and peptides 15,16 . Recent research showed that kefir products comprise many of the bacterial strains that may survive in the digestive process and actually reach the gut, that results in transient changes in the inflammatory cytokines and achieve long-term benefits through regulating the gut barrier and microbiota, in both in vivo and in vitro experiments 37 . In addition, analysis of the peptides in bovine kefir revealed 236 casein-derived unique peptides in kefir grains, including 16 peptides with angiotensin-converting enzyme-inhibitory, antimicrobial, immunomodulating, opioid, mineral-binding, antioxidant, and antithrombotic effects 38 .
Our previous in vivo animal study demonstrated that kefir peptides improve hyperlipidemia and obesity via inhibition of lipogenesis, modulation of oxidative damage, and stimulation of lipid oxidation in high-fatdiet-induced obese rats 17 . The mechanisms of kefir probiotic products to exhibit health benefits is through modulating the gut immune system. Many studies have proved that kefir involved in modulating the inflammatory responses possibly through regulating NF-κB signaling pathway in both of intestinal epithelial cells (in vitro) 39 and LPS-induced acute kidney injury mouse (in vivo) 40 . Lee et al. 41 mentioned that Lactobacillus acidophilus (main probiotics of kefir) modulates inflammatory activity by decreasing the levels of toll-like receptor-4 (TLR4)-induced NF-κB activity in peripheral blood mononuclear cells of LPS-challenged porcine model. Kefir regulates Th1-to-Th2 shift of immune responses and others mentioned that kefir increases in some pro-inflammatory cytokines such as TNF-α, IFN-γ, or IL-12 as an initial reaction of the immune system to TLR agonists present, which resulted in attenuating following further interaction with the immune cells 42,43 . Kefir peptides improved nonalcoholic fatty liver diseases via activation of Janus kinase 2 (JAK2) signal transduction through the JAK2/signal transducer and activator of transcription protein 3 (STAT3) and JAK2/AMP-activated protein kinase (AMPK) pathways in a high fructose-induced fatty liver animal model 44 . Kefir significantly improved the body weight, energy expenditure and basal metabolic rate in nonalcoholic fatty liver disease by inhibiting the lipogenesis pathway in leptin receptor-deficient ob/ob mice 45 . In addition, one study demonstrated that polysaccharides of Astragalus and Codonopsis pilosula improved the alveolar macrophage phagocytosis and inflammation in COPD mice exposed to PM 2.5 46 . Collectively, the present study demonstrated, for the first time, that treatment with 150 or 500 mg/kg body weight of kefir peptides in NF-κB-luciferase +/+ transgenic mice could be protecting against the lung inflammation and oxidative stress caused by PM 4.0 exposure. www.nature.com/scientificreports www.nature.com/scientificreports/

Conclusion
In summary, our results demonstrate that PM 4.0 -induced inflammatory cell infiltration, oxidative stress and overexpression of inflammatory mediators in lung tissue by activating the NF-κB pathway in NF-κB-luciferase +/+ transgenic mice. However, treatment with kefir peptides reduced the PM 4.0 -induced generation of ROS, suppressed p-NF-κB, NLRP3, caspase-1, IL-1β, TNF-α, IL-6, IL-4 and α-SMA expression and increased the SOD activity. Therefore, kefir peptides alleviated PM 4.0 -induced lung inflammation through inhibition of NF-κB signaling and may have the potential for clinical applications involving particulate matter air pollution. Methods PM 4.0 (SRM 2786) characterization. PM 4.0 , standard reference material (SRM) No. 2786, is a fine atmospheric particulate matter with a mean particle diameter <4 μm; PM 4.0 was purchased from the European Virtual Institute for Speciation Analysis (EVISA, Gaithersburg, MD, USA). SRM 2786 is an analytical method for the determination of selected polycyclic aromatic hydrocarbons (PAHs), nitro-substituted PAHs (nitro-PAHs), polybrominated diphenyl ether (PBDE) congeners, hexabromocyclododecane (HBCD) isomers, sugars, polychlorinated dibenzo-p-dioxin (PCDD) and dibenzofuran (PCDF) congeners, inorganic constituents, and particle-size characteristics in atmospheric particulate material and similar matrices [47][48][49] . Detailed information about the PAHs, trace elements and inorganic constituents of PM 4.0 can be found in Supplementary Tables S1, S2 and S3, respectively.
Kefir peptide obtaining. Kefir peptides powder was purchased from Phermpep Co. (Taichung, Taiwan) and was produced via kefir grain fermentation at 20 °C for 20 h in sterilized milk. The grains were passed through a sieve and reinoculated (10%, wt/vol) into sterilized fresh milk, and the incubation was performed according to the previously described preparation methods 17,18,45 . After the grains were filtered, the fermented products were spray-dried into kefir peptides powder using a spray dryer. The peptide content was determined according to the OPA (O-phthaladehyde) method, using triglycine as the standard. The sample or standard solution (5 μL) was mixed with 200 μL of OPA reagent (50 mM borax, 1% SDS, 0.5% thiolactic acid and 1.25 mg/mL OPA). After 2 min of incubation at room temperature, the absorbance was measured at 340 nm. The total peptide content was expressed as triglycine equivalents in g per 100 g sample. The peptide content in the kefir peptides powder (Phermpep Co.) was 23.1 g/100 g. Animal and experimental model. NF-κB-luciferase +/+ transgenic mice carry the luciferase gene driven by the NF-κB promoter; thus, the luciferase activity reflects the NF-κB activity 22,23 . Female homozygous transgenic mice of 8 weeks old were given a standard laboratory diet and distilled water ad libitum and were kept www.nature.com/scientificreports www.nature.com/scientificreports/ on a 12-h light/dark cycle at 24 ± 2 °C. These mice were randomly assigned in three groups (n = 8): the first group without treatment (control group), second group (10 mg/kg of PM 4.0 ) and the last group (20 mg/kg of PM 4.0 ). PM 4.0 -induced lung inflammation was established via intratracheal instillation once a day for 4 weeks. Additionally, because the preliminary results did not showed differences between the low and high dose of PM 4.0 , the group with low dose was chosen for the treatment with kefir peptides. Therefore, homozygous transgenic mice were randomly assigned to four groups for treatment (n = 8): (1) a normal control group receiving no treatment (Control group), as a negative control; (2) a group treated with 10 mg/kg PM 4.0 alone (PM 4.0 + Mock group); (3) a group treated with 10 mg/kg PM 4.0 plus 150 mg/kg low-dose kefir peptides (PM 4.0 + KL group); and (4) a group treated with 10 mg/kg PM 4.0 plus 500 mg/kg high-dose kefir peptides (PM 4.0 + KH group) 50 . Two groups were fed kefir peptides one hour before the intratracheal administration of PM 4.0 (daily, 4 weeks). Mice were sacrificed at 12 weeks of age after 4 weeks of kefir peptides treatment. At the end of the experiment, each mouse was anesthetized, and pulmonary tissues were collected for bronchoalveolar lavage fluid (BALF), pathological histology, and protein extraction as described previously 51,52 . All animal experiments were performed according to the guidelines and were approved by the Institutional Animal Care and Utilization Committee of National Chung Hsing University, Taiwan (IACUC No. 104-077R).
Bioluminescence imaging. Imaging was performed with the IVIS Imaging System 200 Series (Xenogen Corp., Alameda, CA, USA) with the camera set at the highest sensitivity. NF-κB-luciferase +/+ transgenic mice were injected intraperitoneally with luciferin (Promega, Los Altos, CA, USA) at 150 mg/kg in a volume of 200 μL and anesthetized with isoflurane 53 . After 5 min, the mice were placed supine in the chamber and imaged for 90 sec by the IVIS Imaging System. Photons were quantified using Living Image ® software (Xenogen Corp., Alameda, CA, USA) and the intensity of the signal was expressed as photons/sec/cm 2 .
Histological analysis. Pulmonary tissue was fixed with 10% formalin (Macron Fine Chemicals ™ , Avantor Performance Materials, Center Valley, PA, USA) and embedded in paraffin wax. Paraffin-embedded sections were examined using hematoxylin and eosin (H&E), Masson's trichrome and picrosirius red staining as previously described 51,54,55 . The severity of collagen deposition and lung fibrosis was assessed by measuring the Masson's trichrome and picrosirius red staining, respectively 56,57 . Western blot analysis. Expression of pulmonary tissue protein was measured by Western blotting as previously described 51 . Briefly, pulmonary tissues were homogenized in 500 μL of radioimmunoprecipitation assay (RIPA) buffer (EMD Millipore, Billerica, MA, USA). The homogenates were centrifuged at 12,000 rpm for 30 min at 4 °C. The protein (50 μg) was then separated by SDS-PAGE in a 10% polyacrylamide gel and electrotransferred onto a polyvinylidene difluoride membrane. The membranes were incubated in blocking solution (5% BSA) at room temperature for 1 h. The membranes were washed three times (5 min each) with 0.1% T-TBS and then incubated with primary antibody (NLRP3, NF-κB, p-NF-κB, caspase-1, IL-1β, IL-6, TNF-α, IL-4, α-SMA and β-actin; Cell Signaling Technology, Inc., Danvers, MA, USA) in 0.05% T-TBS containing 2.5% BSA at room temperature for 2 h. After washing, the membranes were incubated with peroxidase-conjugated anti-mouse/rabbit antibody (Abcam, Inc., Cambridge, MA, USA) in 0.01% T-TBS at room temperature for 1 h. The membranes were developed with an enhanced chemiluminescence (ECL, Millipore Corporation, Billerica, MA, USA) detection system. Superoxide dismutase (SOD) activity in lung extracts. Pulmonary tissues were homogenized in 300 μL of RIPA buffer. The homogenates were centrifuged at 12,000 rpm for 30 min at 4 °C. To quantify total SOD activity, a water-soluble tetrazolium monosodium salt (WST-1) assay (SOD Assay Kit-WST; Dojindo Molecular Technologies, Inc., Rockville, MD, USA) was performed in a 96-well plate, with bovine erythrocyte SOD1 as a standard. Aliquots of the solution were immediately pipetted into 96-well flat-bottom microtiter plates containing three empty blanks, a range of concentrations of the SOD standard, and a range of concentrations of each lung extract. The rates of WST-1 reduction were measured via the OD 450 value using a microplate reader (Thermo Scientific, Waltham, MA, USA). All determinations of SOD activity were made in triplicate 58 .

Bronchoalveolar lavage fluid (BALF).
The trachea was exposed with a midline incision and cannulated with a modified 21-gauge needle. After euthanization, the BALF was flushed 3 times with 500 µL of sterile endotoxin-free saline each time. An average of 80% BALF was recovered after each lavage. The BALF was combined and centrifuged at 500 rpm for 10 min at 4 °C. The cell pellets were resuspended in 1 mL of PBS, and cell counts were performed 59 . The total number of cells in BALF was determined by staining with Liu's stain to count the different cell types by using a hemocytometer. The supernatant was subjected to total protein analysis using a bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA).

Measurement of reactive oxygen species (ROS) generation.
The generation of ROS, including hydrogen peroxide (H 2 O 2 ), hydroxyl radicals ( • OH) and peroxynitrite (ONOO − /ONOOH), in the perfused lungs was monitored via 2′, 7′-dichlorodihydrofluorescein diacetate (H 2 DCF-DA) fluorescent probe (In Vitro ROS/RNS Assay Kit; Cell Biolabs, Inc., San Diego, CA, USA) as previously described 60 . After internalization, the acetate group of the nonfluorescent molecule is cleaved by intracellular esterases to form H 2 DCF, which serves as a substrate for intracellular ROS to generate the highly fluorescent DCF. Fluorescence was measured with a spectrofluorometer at 480 nm excitation and 530 nm emission wavelengths. Data are expressed in relative fluorescence units for each cell.
Measurement of cytokine levels. Blood samples were clotted at 4 °C for 60 min and then centrifuged for 10 min at 10,000 rpm. The serum levels of IL-1β and TNF-α were measured in the overnight fasting serum and assayed using commercially quantitative enzyme-linked immunosorbent assay (ELISA) kits (Abcam Inc., Cambridge, MA, USA) according to the manufacturer's instructions.