Human β-Defensin 3 Inhibition of P. gingivalis LPS-Induced IL-1β Production by BV-2 Microglia through Suppression of Cathepsins B and L

Cathepsin B (CatB) is thought to be essential for the induction of Porphyromonas gingivalis lipopolysaccharide (Pg LPS)-induced Alzheimer’s disease-like pathologies in mice, including interleukin-1β (IL-1β) production and cognitive decline. However, little is known about the role of CatB in Pg virulence factor-induced IL-1β production by microglia. We first subjected IL-1β-luciferase reporter BV-2 microglia to inhibitors of Toll-like receptors (TLRs), IκB kinase, and the NLRP3 inflammasome following stimulation with Pg LPS and outer membrane vesicles (OMVs). To clarify the involvement of CatB, we used several known CatB inhibitors, including CA-074Me, ZRLR, and human β-defensin 3 (hBD3). IL-1β production in BV-2 microglia induced by Pg LPS and OMVs was significantly inhibited by the TLR2 inhibitor C29 and the IκB kinase inhibitor wedelolactonne, but not by the NLRPs inhibitor MCC950. Both hBD3 and CA-074Me significantly inhibited Pg LPS-induced IL-1β production in BV-2 microglia. Although CA-074Me also suppressed OMV-induced IL-1β production, hBD3 did not inhibit it. Furthermore, both hBD3 and CA-074Me significantly blocked Pg LPS-induced nuclear NF-κB p65 translocation and IκBα degradation. In contrast, hBD3 and CA-074Me did not block OMV-induced nuclear NF-κB p65 translocation or IκBα degradation. Furthermore, neither ZRLR, a specific CatB inhibitor, nor shRNA-mediated knockdown of CatB expression had any effect on Pg virulence factor-induced IL-1β production. Interestingly, phagocytosis of OMVs by BV-2 microglia induced IL-1β production. Finally, the structural models generated by AlphaFold indicated that hBD3 can bind to the substrate-binding pocket of CatB, and possibly CatL as well. These results suggest that Pg LPS induces CatB/CatL-dependent synthesis and processing of pro-IL-1β without activation of the NLRP3 inflammasome. In contrast, OMVs promote the synthesis and processing of pro-IL-1β through CatB/CatL-independent phagocytic mechanisms. Thus, hBD3 can improve the IL-1β-associated vicious inflammatory cycle induced by microglia through inhibition of CatB/CatL.


Introduction
Microglia-mediated neuroinflammation is an important component of Alzheimer's disease (AD) pathogenesis and has been implicated in neurodegeneration [1][2][3].Cells 2024, 13 (IL-1β) is a potent proinflammatory cytokine involved in many important cellular functions.The release of IL-1β is a critical step in inflammation through the induction of other proinflammatory cytokines and chemokines [4,5].IL-1β is chronically upregulated in AD and believed to play a role in the vicious inflammatory cycle that drives AD pathology [6].
A two-step process is generally necessary for IL-1β production.The first step is the synthesis of pro-IL-1β, and the second step is the processing of synthesized pro-IL-1β.The TLR-dependent signals first activate nuclear factor-κB (NF-κB) to lead pro-IL-1β synthesis.
The Nod-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome then undergoes post-translational modifications that license its activation.The activation signals activate the NLRP3 inflammasome with subsequent activation of pro-caspase-1, which in turn catalyzes the cleavage of pro-IL-1β.Activation signals are provided by a variety of stimuli and multiple molecular or cellular events, including K + efflux, mitochondrial dysfunction with reactive oxygen species (ROS) generation, and lysosomal damage with cathepsin B (CatB; EC 3.4.22.1)leakage.Several studies have suggested a CatB/NLRP3/caspase-1-dependent pathway for pro-IL-1β processing in BV-2 microglia [7] and THP-1 cells [8], and the roles of multiple cathepsins in NLRP3 activation have also been implicated in macrophages/microglia [9,10].However, we previously reported a potential NLRP3-independent role of autolysosomal CatB in pro-caspase-1 activation and subsequent IL-1β secretion by microglia following stimulation with chromogranin A [11,12].Therefore, there are at least three different pathways for pro-IL-1β processing, with a special focus on the role of CatB, depending on the stimulating reagents and cell types: (1) a CatB/NLRP3/caspase-1-dependent pathway; (2) a CatB/caspase-1-dependent, but NLRP3-independent pathway; and (3) a CatB and other cysteine cathepsins/NLRP3/caspase-1-dependent pathway.In addition to pro-IL-1β processing, there is some evidence demonstrating that CatB is also involved in pro-IL-1β synthesis through the degradation of IκBα, an endogenous inhibitor of NF-κB in macrophages/microglia at the late stage of inflammation [9,[13][14][15].
The emerging role of microbes and innate immune pathways in AD pathology suggests that antimicrobial peptides may be effective as early therapeutic intervention in future clinical trials [16,17].The salivary proteome contains a complex mixture of over 45 antimicrobial proteins and peptides, including human defensins, histatins, and cathelicidin hCAP18/LL-37 [18].Human β-defensins (hBDs) are small, cationic antimicrobial peptides produced by the oral mucosa and salivary glands.We previously reported that hBD3 strongly suppresses the delayed type of inflammatory responses by microglia following treatment with a lipopolysaccharide (LPS) derived from Porphyromonas gingivalis (Pg), a major pathogen of chronic periodontitis.hBD3 suppresses Pg LPS-induced NF-κB activation through the inhibition of CatB and cathepsin L (CatL; EC 3.4.22.15)[19].Furthermore, we first reported that chronic systemic exposure to Pg LPS induces AD-like pathologies, including microglia-mediated neuroinflammation and cognitive decline in middle-aged mice, but not in CatB-deficient mice [20].
However, whether or not hBD3 and CatB inhibitors can suppress Pg virulence factorinduced IL-1β production by microglia remains unclear.In addition to Pg LPS, chronic oral gavage with outer membrane vesicles (OMVs) secreted from Pg also induced AD-like pathologies in middle-aged mice [21].In the present study, we have thus attempted to clarify the effects of hBD3 and CatB inhibitors on IL-1β production by microglia following stimulation with Pg LPS and OMVs.

Cell Culture
The BV-2 cells, a murine microglial cell line [22], and a well-accepted alternative to primary microglia [23,24], were used in this study.BV-2 microglia were cultured in Dulbecco's modified Eagle's medium (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 5% fetal bovine serum (FBS), penicillin, and streptomycin.To establish NanoLuc (Nluc) probe-expressing cells (Nluc reporter BV-2 microglia), we infected the cells with a lentiviral vector carrying the Nluc probe and selected EGFP-positive cells, as described previously [25].The luciferase activity was only induced when proteolytic pro-IL-1β processing occurred because the Nluc luciferase was fused with the sequence of IL-1β (Il1b 17-216), which has been subjected to proteolytic processing of proteases including caspase-1 [26].Furthermore, the C-terminus of Il1b 17-216 was fused with two protein destabilization sequences (hCL1 and hPEST) (Figure 1A).Thus, the Nluc luciferase protein was retained only when the proteolytic IL-1β processing was successful.

Cell Culture
The BV-2 cells, a murine microglial cell line [22], and a well-accepted alternative to primary microglia [23,24], were used in this study.BV-2 microglia were cultured in Dulbecco's modified Eagle's medium (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 5% fetal bovine serum (FBS), penicillin, and streptomycin.To establish NanoLuc (Nluc) probe-expressing cells (Nluc reporter BV-2 microglia), we infected the cells with a lentiviral vector carrying the Nluc probe and selected EGFP-positive cells, as described previously [25].The luciferase activity was only induced when proteolytic pro-IL-1β processing occurred because the Nluc luciferase was fused with the sequence of IL-1β (Il1b 17-216), which has been subjected to proteolytic processing of proteases including caspase-1 [26].Furthermore, the C-terminus of Il1b 17-216 was fused with two protein destabilization sequences (hCL1 and hPEST) (Figure 1A).Thus, the Nluc luciferase protein was retained only when the proteolytic IL-1β processing was successful.

The Measurement of the Luciferase Activity (RLU)
Nluc reporter BV-2 microglia were plated in 96-well culture plates at a density of 3 × 10 4 cells per well.After overnight culture, drug treatments were performed, and luciferase activity following treatment with Pg LPS (10 µg/mL) or OMVs (150 µg/mL) for 1 h was measured using a luminometer (GloMax; Promega Corp., Madison, WS, USA) with a Nano-Glo ® luciferase assay system (N1110; Promega Corp.) according to the manufacturer's protocol.The luciferase activity (RLU) in BV-2 microglia induced by Pg LPS or OMVs was then measured.Each treatment was repeated in triplicate on the same plate, and at least three independent experiments were performed.

Trasfection of CatB shRNA
Nluc reporter BV-2 microglia were transfected to CatB shRNA or control shRNA lentiviral particle (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) according to the manufacture's transfection protocol.In brief, the Nluc reporter BV-2 microglia were cultured with complete optimal medium in a 12-well plate (3 × 10 5 cells/well) 24 h prior to lentiviral infection.Media were removed from the plate wells and replaced with 1 mL of polyberene/media mixture (5 µg/mL) per well.CatB or control shRNA lentiviral particles were then added and incubated overnight.The cells were cultured for an additional 48 h in complete medium without polybrene.Stable clones expressing CatB shRNA (Nluc reporter CatB-knockdown BV-2 microglia) or control shRNA were selected using puromycin dihydrochloride (6 µg/mL).

Immunoblotting Analyses
For detecting IκBα and β-actin, Nluc BV-2 microglia were used.WT and CatB-KD Nluc BV-2 microglia were used to detect CatB and GAPDH.Cells were seeded in a 6 cm petri dish at a density of 2.5-3.3 × 10 6 cells/dish for 1 day.After treatment with Pg LPS or OMVs in the absence or presence of hBD3 (1 µM) or CA-074Me (30 µM), the cells were subjected to immunoblotting analyses, as described previously [19,27].

Statistical Analyses
Data are presented as the mean ± standard error (SE).Statistical analyses of the results were performed using a one-way analysis of variance (ANOVA) with a post hoc Tukey's test and Student's t-test using the GraphPad Prism8 (GraphPad Software, Inc., La Jolla, CA, USA) software package.p < 0.05 was considered to indicate statistical significance.

Possible Involvement of Phagocytosis of OMVs by BV-2 Microglia in IL-1β Production
We next examined the possible phagocytosis and cellular localization of Cy5-labeled OMVs by BV-2 microglia.F-actin localized around the cell periphery.Cy5-labeled OMVs were phagocytosed by BV-2 microglia and accumulated as coarse granular aggregates, suggesting endosomal/ lysosomal localization (Figure 2A,C).Vertical optical sections of BV-2 microglia clearly showed the intracellular localization of OMVs.

Possible Involvement of Gingipains in IL-1β Production by BV-2 Microglia Following Treatment with OMVs
After secretion through the type IX system, Kgp and Rgp attach to anionic LPS located on the outer membrane surface of Pg [31].Therefore, in addition to LPS, gingipains attached to the surface of OMVs may also be associated with IL-1β production.KYT-1 (10 µM, an Rgp inhibitor), KYT-36 (10 µM, a Kgp inhibitor), and the combination thereof had no effect on OMV-induced luciferase activity in BV-2 microglia (Figure 2B).Furthermore, OMVs prepared from KDP129, a Kgp-deficient mutant strain, induced luciferase activity in BV-2 microglia to a level similar to that of the WT.In contrast, OMVs prepared from KDP136, a Kgp-and Rgp-deficient mutant strain, did not induce luciferase activity in BV-2 microglia.Furthermore, it was also noted that BV-2 micoglia did not phagocytose Cy5-labeled OMVs prepared from KDP136 (Figure 2C).

Possible Involvement of Gingipains in IL-1β Production by BV-2 Microglia Following Treatment with OMVs
After secretion through the type IX system, Kgp and Rgp attach to anionic LPS located on the outer membrane surface of Pg [31].Therefore, in addition to LPS, gingipains attached to the surface of OMVs may also be associated with IL-1β production.KYT-1 (10 µM, an Rgp inhibitor), KYT-36 (10 µM, a Kgp inhibitor), and the combination thereof had no effect on OMV-induced luciferase activity in BV-2 microglia (Figure 2B).Furthermore, OMVs prepared from KDP129, a Kgp-deficient mutant strain, induced luciferase activity in BV-2 microglia to a level similar to that of the WT.In contrast, OMVs prepared from KDP136, a Kgp-and Rgp-deficient mutant strain, did not induce luciferase activity in BV-2 microglia.Furthermore, it was also noted that BV-2 micoglia did not phagocytose Cy5labeled OMVs prepared from KDP136 (Figure 2C).
We examined the possible inhibitory effects of CA-074Me and ZRLR on the enzymatic activities of CatB and CatL in BV-2 microglia using cell-permeable, fluorescently labeled substrates, z-Arg-Arg-cresyl violet and z-Phe-Arg-cresyl violet, respectively.The fluorescent cresyl violet group was designed to be dequenched upon cleavage of dipeptides by CatB or CatL.CA-074Me (10 µM) markedly reduced the enzymatic activity of both CatB and CatL in BV-2 microglia.In contrast, ZRLR (10 µM) markedly reduced the fluorescent signal of CatB, but not that of CatL (Figure 4).
The effects of the inhibitors used in this study on IL-1β production, nuclear NF-κB p65 translocation, and IκBα degradation by BV-2 microglia are summarized in Table 1.

Prediction of hBD3 Binding to CatB and CatL
To examine the possibility that hBD3 inhibits the activities of CatB and CatL through direct binding, a high-quality model of hBD3-bound CatB was generated using AlphaFold (Figure 7A,B).The CatB model in the complex had a median predicted Local Distance Difference Test (pLDDT) score of 95.54 for 257 Cα atoms, except for 3 residues at C-terminus (Glu258-Ile260) (Figure 7A).The hBD3 model in the complex had a median pLDDT score of 87.48 for 45 Cα atoms, which included 2 residues with low confidence in the loop between the β1 and β2 strands, Arg36 (68.00) and Gly37 (66.69) (Figure 7A).In addition, the predicted alignment error (PAE) between CatB and hBD3 was extremely low (Figure 7B).Therefore, the complex model is reliable for analyzing the interactions between proteins.In the CatB model, an active-site cleft was formed on the molecular surface.At the bottom, the substrate-binding pocket, consisting of S1-S3, S1', and S2' sites, were lined across the catalytic Cys29 residue (Figure 7C).In the complex model, the first half (Cys18-Cys23) of the loop between the α1 helix and β1 strand of hBD3 was inserted into the cleft of CatB toward the S2 site, while the other half (Cys23-Lys26) was inserted toward the S3 site (Figure 7C).Therefore, the CatB:hBD3 complex model suggests that hBD3 may inhibit enzymatic activity by blocking the substratebinding pocket in the cleft of CatB.On the other hand, the interaction between hBD3 and CatL, the model of hBD3-bound CatL indicates that hBD3 binds to the active cleft formed on the molecular surface of CatL (Figure 8A,B).However, the confidence level was relatively low (Figure 8C).

Discussion
hBD3 significantly suppressed IL-1β production induced by Pg LPS but not by OMVs, while CA-074Me significantly inhibited the IL-1β production induced by both Pg LPS and OMVs.Furthermore, both hBD3 and CA-074Me significantly inhibited Pg LPS-induced nuclear NF-κB p65 translocation and IκBα degradation.In contrast, neither hBD3 nor CA-074Me blocked OMV-induced nuclear NF-κB p65 translocation and IκBα degradation.CA-074 could have non-cathepsin off-target effects [9,34], which may be responsible for the inhibitory effect of CA-074Me on OMV-induced IL-1β production.On the other hand, ZRLR, a specific CatB inhibitor, had no effect on Pg virulence factor-induced IL-1β production.This was consistent with the results obtained using shRNA-mediated knockdown of CatB expression.These results suggest that Pg LPS induces the synthesis and processing of pro-IL-1β through CatB/CatL-dependent mechanisms, without activation of the NLRP3 inflammasome.In contrast, OMVs may promote the synthesis and processing of pro-IL-1β through CatB/CatL-independent phagocytic mechanisms.
The structural models generated by AlphaFold in this study indicated that hBD3 can bind more strongly to the substrate-binding pocket of CatB than to that of CatL, suggesting that hBD3 can more potently inhibit the enzymatic activity of CatB than CatL.This is consistent with our previous observations that hBD3 (1 µM) inhibits the enzymatic activities of human recombinant CatB and CatL by approximately 60% and 10%, respectively [19].It was reported that hBD3 can be a substrate for cysteine cathepsins such as CatB and CatL [35].Therefore, the present findings extend our previous implication that hBD3 suppresses Pg LPS-induced oxidative and inflammatory responses in microglia through the suicide substrate-based inhibition of CatB and CatL.
The methyl ester of CA-074Me is considered to be hydrolyzed by intracellular esterases releasing the active inhibitor, CA-074.However, the amount of CA-074 released into BV-2 microglia after treatment with CA-074Me remains unclear.Activity-based cysteine protease labeling using DCG-04 showed that CA-074Me is not a CatB-specific inhibitor in murine fibroblasts [36].Therefore, CA-074Me may inhibit CatB and other cysteine cathepsins, especially CatL, at the concentrations used in previous studies (e.g., 10-30 µM).In contrast, ZRLR is a highly potent, irreversible, membrane-permeable, CatB-specific inhibitor.ZRLR is an azapeptide, which are peptide analogs in which the α-CH group of one amino acid resides in the peptide and is replaced by a nitrogen atom.The intrinsic structural prevalence of the N-terminal part of ZRLR is to remain bent, which allows a covalent attachment of ZRLR onto the active site Cys29 in CatB [37].ZRLR exclusively blocks CatB in living primary antigen-presenting cells, in contrast to CA-074Me, by use of DCG-4, which detects active cysteine cathepsins in an activity-based reaction [32].
Currently, OMVs are considered to be potent vehicles for transmitting virulence factors into the host cells [38].OMVs contain gingipains, which covalently bind to anionic LPS on their surface [31].Furthermore, gingipains can activate pro-caspase-1 [39].However, it is unlikely that the enzymatic activities of gingipains are involved in the IL-1β production, as pharmacological inhibition of both Kgp and Rgp did not block OMV-induced IL-1β production by BV-2 microglia.Our observations here showed that BV-2 microglia did not phagocytose OMVs prepared from KDP136, which is devoid of both Kgp and Rgp.It was reported that Rgp is necessary for the fimbriae formation [40,41].Therefore, BV-2 microglia could not phagocytose OMVs prepared from KDP136 because of a lack of fimbriae, which is necessary for a lipid raft-mediated endocytic pathway [29,42].Of particular note, OMVs prepared from KDP136 did not induce the IL-1β production by BV-2 microglia.
OMVs secreted by Gram-negative bacteria function as a vehicle that delivers LPS into the cytosol and induces caspase-11-dependent inflammatory responses [43,44].It has been suggested that a functional interaction between caspase-11 and the NLRP3 inflammasome, and perhaps involving additional partners, could promote noncanonical activation of the pro-IL-β processing [45].It has been reported that phagocytic machinery can activate NF-κB in macrophages [46] and monocytes [47].We previously reported that OMVs can deliver gingipains into the cytosol of hCMEC/D3 cells [27].However, whether or not Pg LPS released into the cytosol is involved in the IL-1β production by BV-2 microglia after phagocytosis of OMVs remains unclear.The precise CatB-/CatL-independent mechanisms involved in OMV-induced IL-1β production by BV-2 microglia should be elucidated in future studies.
The limitation of tis study is that all experimental findings were obtained from in vitro studies by use of BV-2 microglia.Futher studies are also necessary to examine the effects of hBD3 (e.g., through intranasary injection) on AD-like pathological changes induced by chronic systemic exposure to Pg LPS and OMVs in middle-aged mice.

Conclusions
The abnormal expression and/or regulation of salivary antimicrobial peptides has been suggested to be associated with AD [48].The present results suggest that hBD3, a salivary antimicrobial peptide, can improve the IL-1β-associated vicious inflammatory cycle induced by Pg LPS-stimulated microglia.Therefore, hBD3 may be a potential pharmacological intervention for the treatment of patients with sporadic AD, especially those with severe periodontitis.

Figure 1 .
Figure 1.Schematic illustration of the IL-1β probe construct and effects of inhibitors for TLR4, TLR2, IKK, and NLRP3 inflammasome on the luciferase activity of the IL-1β probe in BV-2 microglia following stimulation with Pg LPS and OMVs for 1 h.(A) Nluc luciferase gene was fused with the

Figure 1 .
Figure 1.Schematic illustration of the IL-1β probe construct and effects of inhibitors for TLR4, TLR2, IKK, and NLRP3 inflammasome on the luciferase activity of the IL-1β probe in BV-2 microglia following stimulation with Pg LPS and OMVs for 1 h.(A) Nluc luciferase gene was fused with the sequence of Il1b cleaving site (Il1b 17-216).The C-terminus of the Il1b cleaving site was fused with two protein destabilization sequences (hCL1 and hPEST).The fusion gene was ligated downstream of the mouse IL-1β promoter.(B,C) The mean relative luciferase activity of the IL-1β probe induced by PgLPS (B) and OMVs (C) in the absence or presence of the TLR4 inhibitor TAK-242 or the TLR2 inhibitor C29.(D) The mean relative luciferase activity of the IL-1β probe induced by Pg LPS and OMVs in the absence or presence of IKK inhibitor wedelolactone (WDL).(E).The mean relative luciferase activity of the IL-1β probe induced by Pg LPS and OMVs in the absence or presence of the NLRP3 inflammasome inhibitor MCC950 (MCC).The data relative to the values in Pg LPS or OMV-treated cells are presented as the mean ± SE of three independent experiments.

15 Figure 2 .
Figure 2. Phagocytosis of OMVs by BV-2 microglia and possible involvement of gingipains in OMVinduced IL-1β production.(A) CLSM images of BV-2 microglia after treatment with Cy5-labelled OMVs for 1 h prepared from wild-type strain (WT OMV).F-actin and nuclei were visualized with Acti-stain 488 phalloidin (green) and Hoechst 33342 (blue), respectively.Bottom and right rectangular panels represent z-stack images.Scale bar = 20 µm.(B) The mean relative luciferase activity of the IL-1β probe induced by OMVs for 1 h after pharmacological and genetic inhibition of gingipains.KYT-1: Rgp inhibitor; KYT-136: Kgp inhibitor; KDP129: OMVs prepared from Kgp mutant strain; KDP136: OMVs prepared from Rgp and Kgp mutant strains.The data relative to the values in WT OMV-treated cells are presented as the mean ± SE of three-six independent experiments.(C) CLSM images of BV-2 microglia after treatment with Cy5-labelled OMVs (red) prepared from wild-type (WT OMV) and gingipain-null mutant KDP136 strains for 1 h.F-actin and nuclei were visualized with Acti-stain 488 phalloidin (green) and Hoechst 33342 (blue), respectively.Scale bar = 20 µm.

Figure 2 .
Figure 2. Phagocytosis of OMVs by BV-2 microglia and possible involvement of gingipains in OMVinduced IL-1β production.(A) CLSM images of BV-2 microglia after treatment with Cy5-labelled OMVs for 1 h prepared from wild-type strain (WT OMV).F-actin and nuclei were visualized with Acti-stain 488 phalloidin (green) and Hoechst 33342 (blue), respectively.Bottom and right rectangular panels represent z-stack images.Scale bar = 20 µm.(B) The mean relative luciferase activity of the IL-1β probe induced by OMVs for 1 h after pharmacological and genetic inhibition of gingipains.KYT-1: Rgp inhibitor; KYT-136: Kgp inhibitor; KDP129: OMVs prepared from Kgp mutant strain; KDP136: OMVs prepared from Rgp and Kgp mutant strains.The data relative to the values in WT OMV-treated cells are presented as the mean ± SE of three-six independent experiments.(C) CLSM images of BV-2 microglia after treatment with Cy5-labelled OMVs (red) prepared from wild-type (WT OMV) and gingipain-null mutant KDP136 strains for 1 h.F-actin and nuclei were visualized with Acti-stain 488 phalloidin (green) and Hoechst 33342 (blue), respectively.Scale bar = 20 µm.

Figure 3 .
Figure 3. Effects of pharmacological and genetic inhibition of CatB on IL-1β production b microglia following stimulation with Pg LPS and OMVs.(A,B) The mean relative luciferase a of the IL-1β probe induced in BV-2 microglia following treatment with Pg LPS (A) and OM after treatment with hBD3 (1 µM), CA-074Me (10 µM) or ZRLR (10 µM).The data are presen the mean ± SE of 3-6 independent experiments.(C) The mean values of CatB intensity, whic detected by the immunoblot shown, were measured in Nluc reporter BV-2 microglia (Nluc and Nluc reporter CatB-knockdown BV-2 microglia (CatB-KD Nluc BV-2) and normalized a the signal of GAPDH.The data are presented as the mean ± SE of three independent experi and the p-value was calculated using Student's t-test.(D) The mean luciferase activity (RLU IL-1β probe induced by Pg LPS or OMV in CatB-KD Nuc BV-2 microglia.The data are prese the mean ± SE of 3 independent experiments.

Figure 3 .
Figure 3. Effects of pharmacological and genetic inhibition of CatB on IL-1β production by BV-2 microglia following stimulation with Pg LPS and OMVs.(A,B) The mean relative luciferase activity of the IL-1β probe induced in BV-2 microglia following treatment with Pg LPS (A) and OMVs (B) after treatment with hBD3 (1 µM), CA-074Me (10 µM) or ZRLR (10 µM).The data are presented as the mean ± SE of 3-6 independent experiments.(C) The mean values of CatB intensity, which were detected by the immunoblot shown, were measured in Nluc reporter BV-2 microglia (Nluc BV-2) and Nluc reporter CatB-knockdown BV-2 microglia (CatB-KD Nluc BV-2) and normalized against the signal of GAPDH.The data are presented as the mean ± SE of three independent experiments, and the p-value was calculated using Student's t-test.(D) The mean luciferase activity (RLU) of the IL-1β probe induced by Pg LPS or OMV in CatB-KD Nuc BV-2 microglia.The data are presented as the mean ± SE of 3 independent experiments.

Figure 5 .
Figure 5. Effects of hBD3 and CA-074Me on nuclear NF-κB p65 translocation following stimulation with Pg LPS and OMVs.(A) Immunofluorescent CLSM images of BV-2 microglia after treatment with Pg LPS or OMVs in the absence or presence of hBD3 (1 µM) or CA-074Me (30 µM).Nuclear NF-κB p65 translocation was visualized by immunohistochemical staining (red).Nuclei were stained blue by Hoechst 33342 (blue).Scale bar = 40 µm.(B) The typical cells were analyzed by line plot profile to show the cytosol and nuclear NF-κB p65 translocation.The fluorescence intensity of NF-κB p65 and Hoechst 33342 in the cells traversed by white lines in (A) was indicated by red and blue lines, respectively.

Figure 6 .
Figure 6.Effects of hBD3 and CA-074Me on the degradation of IκBα after stimulation with Pg LPS and OMVs.(A) The protein level of IκBα in BV-2 microglia after stimulation with Pg LPS (10 µg/mL) for 30 min in the presence or absence of hBD3 (1 µM) or CA-074Me (30 µM).(B) The mean values of the IκBα intensity shown in (A) were measured and normalized against the signal of β-actin.The

Figure 5 . 15 Figure 5 .
Figure 5. Effects of hBD3 and CA-074Me on nuclear NF-κB p65 translocation following stimulation with Pg LPS and OMVs.(A) Immunofluorescent CLSM images of BV-2 microglia after treatment with Pg LPS or OMVs in the absence or presence of hBD3 (1 µM) or CA-074Me (30 µM).Nuclear NF-κB p65 translocation was visualized by immunohistochemical staining (red).Nuclei were stained blue by Hoechst 33342 (blue).Scale bar = 40 µm.(B) The typical cells were analyzed by line plot profile to show the cytosol and nuclear NF-κB p65 translocation.The fluorescence intensity of NF-κB p65 and Hoechst 33342 in the cells traversed by white lines in (A) was indicated by red and blue lines, respectively.

Figure 6 .
Figure 6.Effects of hBD3 and CA-074Me on the degradation of IκBα after stimulation with Pg LPS and OMVs.(A) The protein level of IκBα in BV-2 microglia after stimulation with Pg LPS (10 µg/mL) for 30 min in the presence or absence of hBD3 (1 µM) or CA-074Me (30 µM).(B) The mean values of the IκBα intensity shown in (A) were measured and normalized against the signal of β-actin.The

Figure 6 .
Figure 6.Effects of hBD3 and CA-074Me on the degradation of IκBα after stimulation with Pg LPS and OMVs.(A) The protein level of IκBα in BV-2 microglia after stimulation with Pg LPS (10 µg/mL) for 30 min in the presence or absence of hBD3 (1 µM) or CA-074Me (30 µM).(B) The mean values of the IκBα intensity shown in (A) were measured and normalized against the signal of β-actin.The data relative to the values in untreated cells are presented as the mean ± SE of three-five independent experiments.(C) The protein level of IκBα in BV-2 microglia after stimulation with OMVs (150 µg/mL) for 10 min in the presence or absence of hBD3 (1 µM) or CA-074Me (30 µM).(D) The mean values of the IκBα intensity shown in (C) were measured and normalized against the signal of β-actin.The data relative to the values in untreated cells are presented as the mean ± SE of three independent experiments.

Figure 7 .
Figure 7. Prediction of hBD3 binding to CatB.(A) Structural model of hBD3-bound CatB generated using AlphaFold.The hBD3 model, presented as a ribbon, binds to the molecular surface of CatB.Amino acids are colored based on their pLDDT score.(B) PAE plots of the hBD3 and CatB complex model.(C) Binding of hBD3 to the active formed on the molecular surface of CatB.The surface representation of CatB is shown in gray, except for the S1, S3, and S1' sites, which are shown in light magenta; the S2 and S2' sites, which are shown in magenta; and Cys29, which is shown in yellow.The hBD3 model, presented as a ribbon, is colored cyan.The figures were drawn using the PyMOL software program[33].

Figure 7 .
Figure 7. Prediction of hBD3 binding to CatB.(A) Structural model of hBD3-bound CatB generated using AlphaFold.The hBD3 model, presented as a ribbon, binds to the molecular surface of CatB.Amino acids are colored based on their pLDDT score.(B) PAE plots of the hBD3 and CatB complex model.(C) Binding of hBD3 to the active cleft formed on the molecular surface of CatB.The surface representation of CatB is shown in gray, except for the S1, S3, and S1' sites, which are shown in light magenta; the S2 and S2' sites, which are shown in magenta; and Cys29, which is shown in yellow.The hBD3 model, presented as a ribbon, is colored cyan.The figures were drawn using the PyMOL software program [33].
Amino acids are colored based on their pLDDT score.(B) PAE plots of the hBD3 and CatB complex model.(C) Binding of hBD3 to the active cleft formed on the molecular surface of CatB.The surface representation of CatB is shown in gray, except for the S1, S3, and S1' sites, which are shown in light magenta; the S2 and S2' sites, which are shown in magenta; and Cys29, which is shown in yellow.The hBD3 model, presented as a ribbon, is colored cyan.The figures were drawn using the PyMOL software program[33].

Figure 8 .
Figure 8. Prediction of hBD3 binding to CatL.(A) Structural model of hBD3-bound CatL generated using AlphaFold.The hBD3 model, presented as a ribbon, binds to the molecular surface of CatL.Amino acids are colored based on their pLDDT score.(B) Binding of hBD3 to the active cleft formed on the molecular surface of CatL.The surface representation of CatL is shown in gray, except for S1 and S1'sites, which are shown in light magenta; the S2 and S2' sites, which are shown in magenta; and Cys25, which is shown in yellow.The hBD3 model, presented as a ribbon, is colored cyan.(C) PAE plots of the hBD3 and CatL complex model.The figures were drawn using the PyMOL software program [33].

Table 1 .
Summary of effects of inhibitors on the IL-1β production, NF-κB p65 nuclear translocation and IκBα degradation by BV-2 microglia after stimulation with Pg LPS and OMVs.