S. Aureus Lipoteichoic Acid Failed to Activate NLRP3, but Downregulated the Transduction of NLRP3/MAPK and NF-κB Signaling Pathway in Mouse Mastitis Induced by S. Aureus


 Background It was confirmed that S. aureus infection activated the expression of NLRP3. As a major component of S. aureus cell wall, what about LTA? Firstly, in this study a mouse model was built following LTA intramammary infection for 0, 12, 24, 48 and 72 h, the characterization of mammary inflammatory response induced by LTA was observed. Subsequently, mouse mammary preconditioned with LTA was modelling. The effect of LTA treatment on the activation of NLRP3/ MAPK and NF-κB signaling pathways induced by S. aureus were detected.Results Results shown that the LTA intramammary infection induce mild but rapid recovery inflammation in mammary gland whereas failed to stimulate NLRP3. LTA treatment protected mammary gland against S. aureus infection by suppressed the activation of NLRP3/ MAPK and NF-κB signaling pathways. Meanwhile it is noteworthy that the expression of NLRP3/MAPK JNK induced by S. aureus was not regulated by LTA treatment.Conclusions These results suggested LTA induced mammary gland inflammation was mild and self-limiting. LTA treatment revealed a good anti-inflammation effect through downregulating the transduction of NLRP3/MAPK and NF-κB signaling pathways in mouse mastitis induced by S. aureus. Our research highlights the importance of an LTA during the innate immune response of the mammary gland and offer novel insights for new approaches concerning effective immunomodulation against a local bacterial infection.


Background
Mastitis is a serious problem in dairy industry worldwide which has a profound impact on animal health [1,2]. S. aureus is a major representative pathogenic bacterium causing bovine mastitis [3,4]. A number of studies have performed to explore the host/S. aureus interactions in the mammary gland. Jully et al. have demonstrated that S. aureus used complex regulatory networks to sense diverse signals that enable it to internalize into host cell and adapt to different environments [5]. As a result, mastitis induced by S. aureus is usually characterized by persistent infection. It was proved that S. aureus mastitis was extremely di cult to control by treatment alone. Up to 40% of mastitis cases induced by S. aureus may be recurrences of previous infections after therapy [6]. With further research, more and more mechanisms were found involved in the process of S. aureus survival in host cells and adaptive immune responses such as autophagy, pyroptosis, etc. [7,8]. Thus, prevention and control of the S. aureus mastitis is facing very serious challenges and further study on the underlying mechanisms of S. aureus infection will be helpful.
LTA can be found at the cell surface of most gram-positive. For a long time, LTA is thought to act as an adhesion. For example, LTA secreted by Streptococcus pyogenes will act as an adapter by binding the M protein to receptors on host tissues. The attachment of bacteria to host tissues allows colonization of those tissues. As the progress in the knowledge and the availability of pathogen-associated molecular patterns (PAMP) for gram-positive bacteria and in particular S. aureus makes it feasible to research the effects of LTA on immune and in ammatory response [9]. S. aureus LTA was proved to trigger in ammation in the lactating bovine mammary gland [10]. This makes LTA a potential substitute for S. aureus mastitis in scienti c research. Until recently, LTA was proposed as an immunostimulatory component of S. aureus which was involved in the activation of TLRs and release of in ammatory cytokines [11]. However, in spite of some certain immunostimulatory effects, the biological role of LTA is still unclear and even controversial. For example, it has been shown that, bovine mammary epithelial cells could express TLR2, however NF-κB is not activated by LTA in these cells [12]. Even more in some cases live bacteria and bacterial soluble factors might induce different transcriptional [13]. So, S. aureus has been considered a major pathogenic bacterium of bovine mastitis, it was thus far been di cult to identify the exact role of the individual bacterial cell components in relation to the onset of mastitis.
Nod-like receptors (NLRs) are critical for the host immune response [14]. As intracellular pattern recognition receptors (PRRs), NLRs are capable to form in ammasomes [15]. NLRP3 is one of the most researched NLRs. The NLRP3 in ammasome is assembled by NLRP3, apoptosis-associated speck-like protein containing (ASC) and caspase-1 [16]. Moreover, the NLRP3 in ammasome was proven to be activated by S. aureus in monocytes/macrophages [17]. The activation of the NLRP3 in ammasome promotes the maturation of caspase-1, which then processes pro-IL-1β into IL-1β [18,19]. On the other hand, NF-κB and MAPK are involved in the activation of NLRP3 in ammasome, furthermore activated NF-κB and MAPK can recruit a series of cytokines and chemokines, including TNF-α and IL-1α [20]. Although these molecules have the potential to protect against in ammation, however overreaction of proin ammatory cytokines will also injure cells and tissues [21]. Given the fact that LTA or S. aureus might have different effects on NLRP3/MAPK and NF-κB signaling pathways. LTA was de ned as different functions: infection function or treatment function, the characterization of mammary in ammatory response induced by LTA and the regulation effect of LTA on the NLRP3/MAPK and NF-κB signaling pathways induced by S. aureus were studied. Abcam (Cambridge, UK). Caspase-1, P65, P-P65, IκBα, P-IκBα, P38, P-P38, Jnk, P-Jnk, Erk, P-Erk and β-actin rabbit mAb and HRP-conjugated goat anti-rabbit antibodies were purchased from Cell Signaling Technology (CST; Danvers, MA, US).

Experimental design
The schematic of experimental design is shown in Fig. 1.

Characterization of in ammation induced by LTA
Eighty SPF BALB/c female mice and ten male mice (60 days old) were purchased from the Center of Experimental Animals of Linyi University and the protocol was approved by the Animal Care and Ethics Committee of the Linyi University. The mice were fed separately in an air-conditioned room maintained at a temperature of 24 ± 1 °C for 2 weeks to adapt to the environment. The diet was prepared consulting the Characterization of LTA treatment on the activation of NLRP3/NF-κB and NLRP3/MAPK signaling pathway induced by S. aureus Forty SPF pregnant mice were randomly divided into four group: Control group (CG, n = 10) were infected with 0.1 mL physiological saline from milk ducts; Model group (MG, n = 10) was infected with 0.1 mL S. aureus (10 5 CFU/mL); LTA control group (LCG, n = 10) was infected with 0.1 mL LTA (5 µg) for 72 h; LTA treatment group (LTG, n = 10) was infected with 0.1 mL LTA (5 µg) for 72 h and then infected with 0.1 mL S. aureus (10 5 CFU/mL). 12 h after stimulus, mammary gland (R4, L4) in each group were collected.

Histopathologic examination
The tissues were collected and xed in 10% formaldehyde solution, and then embedded in para n wax.
The mammary tissue sections were stained with hematoxylin eosin (H&E) staining and the pathological changes were observed under a microscope (Olympus DX45, Japan).

Immunohistochemistry analysis
The mammary tissues were collected and xed in 10% form-aldehyde solution, and then embedded in para n wax. For the immunohistochemistry staining, the mammary section was routinely processed. Tissue sections were depara nised, rehydrated and then antigen retrieval was applied by microwave heat for 10 minutes at medium voltage in a 10 mM citrate buffer, pH 6.0. After cooling at room temperature, the sections were incubated in 3% hydrogen peroxide (H2O2) for 30 minutes to block endogenous peroxidase. Nonspeci c staining was eliminated by 10 minutes incubation with normal goat serum at the room temperature. Excess normal serum was removed and slides were then incubated with primary antibody (1:100) at 4˚C overnight. After washing the slides, the sections were incubated with biotinylated secondary antibody for 15 minutes and replaced in the streptavidin, horseradish peroxidase (HRP) conjugate for 15 minutes at the room temperature. The colour was developed with DAB in PBS for 5 minutes. Slides were counterstained with Harris haematoxylin, dehydrated and mounted with Entellan. IPP6.0 software was used to analyze the optical density of immunohistochemical photos. Four 400-fold photos were selected from each section for optical density analysis.

Western blot analysis
Total protein was extracted by using a total protein extraction kit and the protein concentrations were determined by BCA protein assay kit. 50 µg (5 µL) proteins were loaded into a 10% sodium dodecyl sulphate (SDS) polyacrylamide gel for electrophoresis and transferred to PVDF membranes. The membranes were blocked in 5% nonfat milk for 1 h to 2 h and incubated overnight with the primary monoclonal antibodies, including Caspase-1, NF-κB, MAPK and β-actin. Then, all of the membranes were subsequently incubated with HRP-conjugated goat anti-rabbit antibodies. The blots were washed with PBS-T and processed with supersignal west pico chemiluminescent substrate.

ELISA analysis
Mammary tissue samples were weighed and grinded with RIPA lysis buffer until homogenized. Then the homogenate was centrifuged at 12000 rpm for 15 min at 4 °C, and the supernatant was collected in − 20 °C until measurement. The expression of IL-1α, IL-1β and TNF-α were measured by ELISA and according to the manufacturer.

Statistical analysis
All data is shown as the mean ± S.E.M, and experiments were independently repeated at least 3 times. One-way ANOVA and Dunnett's test were used for statistical analyses. Values of p < 0.05 were considered statistically signi cant.  (Fig. 2E); White blood cell count shown that the WBC were above normal at 12, 24 and 48 h but at 72 h the WBC returns to normal (Fig. 2F).

LTA stimulus induce mild in ammation in mammary gland
The expression of IL-1α, IL-1β and TNF-α in serum and mammary gland induced by LTA back to normal within 72 h The qRT-PCR analysis and ELISA assay were used to detect the mRNA and release of IL-1α, IL-1β and TNF-α. The results are shown in Fig. 3. Compared with 0 h, the IL-1α mRNA expression in mammary gland increased signi cantly from 12 h to 24 h (p < 0.05), compared with 12 h and 24 h, the IL-1α mRNA expression decreased signi cantly (p < 0.05) at 48 h, there were not statistically difference in the IL-1α mRNA expression between 0 h and 72 h (Fig. 3A); compared with 0 h the mRNA expression of IL-1β increased signi cantly from 12 h to 48 h (p < 0.05), at 72 h the IL-1β mRNA expression subside and drop back to a normal level (Fig. 3B); compared with 0 h and 72 h, the TNF-α mRNA expression increased signi cantly from 12 h to 48 h (p < 0.05) (Fig. 3C); compared with 0 h the release of IL-1α in mammary gland increased signi cantly from 12 h to 48 h (p < 0.05) and at 72 h the release of IL-1α was back to normal level (Fig. 3D); the release level of IL-1β and TNF-α in mammary gland were both increased signi cantly from 12 h to 24 h (p < 0.05) and compared with 0 h no signi cant increase was detected at 72 h ( Fig. 3E and F); the release of IL-1α, IL-1β and TNF-α in serum were not as much as those in mammary gland (Fig. 3D, E, F and G, H, I), No signi cant changes in the release level of IL-1α was detected in serum from 0 h to 72 h (Fig. 3G); compared with 0 h the release of IL-1βincrease signi cantly at 12 h and 48 h (p < 0.05) in serum and at 72 h the release of IL-1β was back to normal level (Fig. 3H), compared with 0 h the release of TNF-αincrease signi cantly from 12 h to 24 h (p < 0.05) in serum and from 48 h to 72 h the release of TNF-α was back to normal level (Fig. 3I).
LTA stimulus activated the expression of Caspase-1 but failed to activate NLRP3 in mammary gland The expression of NLRP3 and Caspase-1 were detected by using immunohistochemistry and western blot analysis. The results are shown in Fig. 4. Compared with 0 h no statistically signi cant difference was observed on the expression of NLRP3 from 12 h to 72 h (Fig. 4A, B, C, D, E and F); compared with 0 h the expression of Caspase-1 increased signi cantly at 12 h and 48 h (p < 0.05) and reach peak at 48 h, compared with 48 h the expression of Caspase-1 decreased signi cantly at 72 h (p < 0.05), however compared with 0 h the expression of Caspase-1 was still increased signi cantly at 72 h (p < 0.05) (Fig. 4G and H).
LTA treatment decreased the expression of NLRP3 in ammasome induced by S. aureus Immunohistochemistry and western blot analysis were used to detect the effect of LTA treatment on the expression of NLRP3 in ammasome induced by S. aureus. The results are shown in Fig. 5. S. aureus infection signi cantly upregulate the expression of NLRP3 in mammary gland (p < 0.05), LTA treatment signi cantly decreased the expression of NLRP3 induced by S. aureus (p < 0.05), however compared with CG group and LCG group the expression of NLRP3 in LTG group was still markedly increased (p < 0.05) (Fig. 5A, B, C, D and E); compared with CG and LCG group the expression of Caspase-1 in MG and LTG group increased signi cantly by S. aureus infection(p < 0.05), LTA treatment decreased the expression of Caspase-1 but its distinction does not proved a statistical signi cance ( Fig. 5F and G).

LTA treatment inhibited the activation of MAPK signaling pathway induced by S. aureus
Western blot analysis was used to detect the effect of LTA on the activation of MAPK signaling pathway induced by S. aureus. The results are shown in Fig. 6. Compared with CG and LCG group, S. aureus infection signi cantly increased the phosphorylation level of JNK in MG and LTG group (p < 0.05) however there was no statistical difference between MG group and LTG group ( Fig. 6A and B); compared with CG and LCG group the phosphorylation level of P38 and ERK were signi cantly increased by S. aureus in MG and LTG group (p < 0.05), LTA treatment in LTG group signi cantly decreased the phosphorylation level of P38 and ERK induced by S. aureus (p < 0.05) (Fig. 6A, C and D).

LTA treatment inhibited the activation of NF-κB signaling pathway induced by S. aureus
Western blot analysis was performed to detect the effect of LTA on the activation of NF-κB signaling pathway induced by S. aureus. The results are shown in Fig. 7. Compared with CG and LCG group the phosphorylation level of P65 and IκBα were signi cantly increased by S. aureus in MG and LTG group (p < 0.05), LTA treatment signi cantly decreased the phosphorylation level of P65 and IκBα induced by S. aureus (p < 0.05) (Fig. 7A, B and C). LTA treatment suppressed the expression of cytokines IL-1α, IL-1β and TNF-α in serum and mammary The qRT-PCR and ELISA analysis were used to detect the effect of LTA treatment on the mRNA and release of IL-1α, IL-1β and TNF-α. The results are shown in Fig. 8. S. aureus infection signi cantly increased the mRNA expression of IL-1α, IL-1β and TNF-α in mammary gland (p < 0.05) (Fig. 8A, B and C), LTA treatment signi cantly decreased the mRNA expression of IL-1α and TNF-α (p < 0.05) (Fig. 8A and C) and the mRNA expression of IL-1β was also decreased by LTA treatment but its distinction does not proved a statistical signi cance (Fig. 8B); compared with CG and LCG group the release of IL-1α, IL-1β and TNF-α increased signi cantly in MG group (p < 0.05) and LTA treatment signi cantly decreased the release of these three cytokines in mammary gland (p < 0.05) (Fig. 8D, E and F); compared with mammary gland there were less amount of cytokines in serum, S. aureus signi cantly increased the release of these three cytokines in serum (p < 0.05), however LTA shown no inhibitory effect on the release of these three cytokines in serum (Fig. 8G, H and I).

Discussion
S. aureus induced mastitis have become a serious threat to human health and the stockbreeding [23,24]. Thus, it is particular important to identify the underlying mechanism of S. aureus mastitis. A number of studies have performed to explore the host/pathogen interactions in the mammary gland by using animal model [25,26]. However, the experimental infections are costly furthermore the interactions between host defense and bacterial virulence factors are complex which make it di cult to analysis. As a result, a surrogate for live bacterial infection would be helpful. Studies have proved that LPS was a suitable substitute for mastitis [27]. However, the Escherichia coli and S. aureus would elicit differential innate immune responses whether in vivo or in vitro [28,29]. Recently, LTA has been suggested to contribute to S. aureus virulence and LTA induced mastitis has been reported by several studies also [30,31]. Data indicated that that LTA usually induced a weak in ammatory response in mammary epithelial cells. In this study characterization of LTA induced in ammatory response following intramammary infection was observed. Result shown at the early stage of infection (within 48 h), LTA would induce mild in ammation in mammary gland which highlights the morphologic changes of the acinar contour, the increase of white blood cell in peripheral blood, and the release of in ammatory cytokines mainly in mammary gland. However, as the immune response progressed, all of these indicators were back to normal which indicated that the in ammatory reaction disappeared. These results suggested that to some extent LTA used as a surrogate for S. aureus mastitis was suitable, however the in ammatory reaction induced by LTA were mild and rapid recovery. The results were interesting and further research is needed to understand the underling mechanisms. Here we hypothesized two main mechanisms may explain: "Mechanism one": LTA infection enhanced the pathogen recognition of innate immune system; "mechanism two": LTA treatment upregulated the host immune response. However which mechanism is a more sensitive one is not clear. Thus, a follow-up experiment was performed.
Innate immune system plays an important role in defensing against infection. Pattern recognition receptors (PRRs) act as the rst line in innate immune system [32]. There are two PRRs mainly response for S. aureus infection: Toll-like receptors family (TLRs) and Nod-like receptors family (NLRs) [33,34]. Most TLRs are located at the cell surface, which serve as important links between innate and adaptive immunity primarily by responding to extracellular bacteria including LTA [35]. Unlike TLRs, NLRs were capable to form in ammasomes in response to their speci c stimulators, which mainly mediate cytosolic recognition of intracellular stimulate. Research about NLRs are incomplete and whether LTA acts as a speci c ligand for NLRs remains to be further studied. Thus, in this study the LTA induced NLRP3 (one of the most characterized members of the NLRs) expression was detected. Result shown that LTA failed to active the expression of NLRP3 in mammary gland which indicated that LTA was not an excellent intracellular stimulator or there were others related mechanism to clear LTA in mammary gland.
MAPK and NF-κB are two main pathways that associated to in ammatory immune response. Experiments have veri ed that NF-κB and MAPK involved in the regulation of S. aureus mastitis [36,37]. Based on the important roles of MAPK and NF-κB in in ammation, another follow-up experiment was designed to investigate the regulation effect of LTA treatment on the NF-κB and MAPK signaling pathways during S. aureus mammary infection. Results shown that LTA preconditioning decreased the expression of NLRP3 and Caspase-1 induced by S. aureus and the protective effects of LTA treatment were achieved mainly through NF-κB, MAPK P38 and MAPK ERK signaling pathway. However, the inhibition effects of LTA treatment on MAPK JNK pathway was not obvious. During in ammation the release of cytokines such as IL-1α, IL-1β and TNF-α were regulated by NLRP3-Caspase-1, NF-κB and MAPK pathways [38,39]. Subsequently, the inhibitory effects of LTA treatment on the expression of IL-1α, IL-1β and TNF-α induced by S. aureus were examined. Result shown LTA played an inhibitory effect of LTA treatment on the mRNA and protein expression of cytokines induced by S. aureus in mammary gland however the inhibitory effects for serum cytokines were not obvious. These results revealed that the innate immune response was upregulated by LTA preconditioning prior to S. aureus infection.

Conclusion
In conclusion, the experiment proved LTA mouse intramammary stimulation failed to activate the expression of NLRP3 but induce mild and rapidly recovering in ammation, LTA treatment inhibited the S. aureus induced mastitis by suppressing the NLRP3/Caspase-1/NF-κB and MAPK P38/ERK signaling pathways.

Declarations
Ethical approval and consent to participate The protocol was approved by the Animal Care and Ethics Committee of Linyi University.

Consent for publication
Written informed consent for publication was obtained from all participants.

Availability of date and materials
All data used during the study appear in the submitted article.

Competing interests
The authors declare that they have no con ict of interest.

Funding
The investigation was supported by the National Natural Science Foundation of China (NO. 31802254). The science and technology project of Shandong Province higher education institutions (J18KB074).

Authors' contributions
Chong-Liang Bi contributed to the overall study design and performed the experiment. Hui Li analyzed the data and prepared Figures Figure 1 The schematic of experimental design