Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Porphyromonas gingivalis Stimulates TLR2-PI3K Signaling to Escape Immune Clearance and Induce Bone Resorption Independently of MyD88

doi: 10.3389/fcimb.2017.00359

Figure Lengend Snippet: Experimental periodontitis induced by oral challenge with P. gingivalis . Groups of mice were administered P. gingivalis in CMC vs. CMC alone by repeated oral gavage. Six weeks later, the maxillae were harvested and alveolar bone volume was measured by μCT from the cemento-enamel junction to a reference line. The residual bone volume of P. gingivalis infected mice was compared to vehicle-treated mice ( n = 8–10 per group). (A) WT, Tlr2 −/− , Myd88 −/− , and Tlr2/Myd88 double knock-out mice (DKO) infected with P. gingivalis ATCC 381. (B) An independent experiment examined bone loss in WT vs. Myd88 −/− mice infected with P. gingivalis ATCC 381 vs. P. gingivalis ATCC 53977. Ns, non-significant. ** P ≤ 0.01, *** P ≤ 0.005.

Article Snippet: T2.5 monoclonal antibody (mAb) against mouse and human TLR2 was from Hycult Biotech (Uden, Netherlands), mAb 1A6 was a gift from Greg Elson (NovImmune, Geneva, Switzerland), and isotype control mAbs were from BioLegend (San Diego, USA).

Techniques: Infection, Knock-Out

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Porphyromonas gingivalis Stimulates TLR2-PI3K Signaling to Escape Immune Clearance and Induce Bone Resorption Independently of MyD88

doi: 10.3389/fcimb.2017.00359

Figure Lengend Snippet: Detection of P. gingivalis in mouse tissue following the third oral challenge.

Article Snippet: T2.5 monoclonal antibody (mAb) against mouse and human TLR2 was from Hycult Biotech (Uden, Netherlands), mAb 1A6 was a gift from Greg Elson (NovImmune, Geneva, Switzerland), and isotype control mAbs were from BioLegend (San Diego, USA).

Techniques:

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Porphyromonas gingivalis Stimulates TLR2-PI3K Signaling to Escape Immune Clearance and Induce Bone Resorption Independently of MyD88

doi: 10.3389/fcimb.2017.00359

Figure Lengend Snippet: IFN-γ priming enables TLR2-dependent, MYD88-independent signaling in macrophages in response to P. gingivalis challenge. (A) Naïve Myd88 −/− BMM vs. BMM primed with IFN-γ (100 ng/ml) for 2 h were challenged with P. gingivalis (MOI 100) vs. Pam3CSK4 (10 μg/ml). In (B) Myd88 −/− BMM primed with IFN-γ were challenged with increasing MOI of P. gingivalis . (A,B) Supernatants were collected after overnight stimulation and tested for TNF by ELISA. ** P ≤ 0.01, *** P ≤ 0.005.

Article Snippet: T2.5 monoclonal antibody (mAb) against mouse and human TLR2 was from Hycult Biotech (Uden, Netherlands), mAb 1A6 was a gift from Greg Elson (NovImmune, Geneva, Switzerland), and isotype control mAbs were from BioLegend (San Diego, USA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Porphyromonas gingivalis Stimulates TLR2-PI3K Signaling to Escape Immune Clearance and Induce Bone Resorption Independently of MyD88

doi: 10.3389/fcimb.2017.00359

Figure Lengend Snippet: Kinase involvement in the Myd88 −/− response to P. gingivalis . (A) Ly6C+ BM neutrophils or (B–D) BMM were prepared from Myd88 −/− mice and primed with IFN-γ (100 ng/ml for 2 h). (A,B) Inhibitors for PI3K (LY 2940002 100 μM), p38 MAPK (SB 202190 50 μM), mTORC1 (RaPamycin 60 nM) and RAC1 inhibitor (NSC 23766, 50 μM) were added 30 min before challenge with P. gingivalis (MOI 100). DMSO was used as a control at the highest concentration used in the inhibitor wells. Supernatants were collected after overnight stimulation and the percent inhibition of TNF production is shown. (C) Myd88 −/− BMM were similarly primed and Ly294 was added at increasing concentrations 30 min prior to challenge with P. gingivalis . (D) Myd88 −/− BMM were primed with IFN-γ and antibodies (20 μg/ml anti-TLR2 or TLR4 vs. isotype control, I.C.) or LY294 were added prior to challenge with P. gingivalis . * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005.

Article Snippet: T2.5 monoclonal antibody (mAb) against mouse and human TLR2 was from Hycult Biotech (Uden, Netherlands), mAb 1A6 was a gift from Greg Elson (NovImmune, Geneva, Switzerland), and isotype control mAbs were from BioLegend (San Diego, USA).

Techniques: Concentration Assay, Inhibition

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Porphyromonas gingivalis Stimulates TLR2-PI3K Signaling to Escape Immune Clearance and Induce Bone Resorption Independently of MyD88

doi: 10.3389/fcimb.2017.00359

Figure Lengend Snippet: TLR2-PI3K plays a non-redundant role in the murine and human macrophage response to P. gingivalis . WT murine BMM (A) , RAW264.7 macrophages (B) , and PMA-differentiated human THP-1 cells (C) were primed with IFN-γ. TLR2 and TLR4 were inhibited with blocking antibodies vs. isotype control (I.C.) for 1 h and PI3K was blocked with LY294 prior to challenge with P. gingivalis (MOI 10). Supernatants were collected after overnight incubation and TNF was measured by ELISA. Background (BG) represents IFN-γ primed cells not challenged with P. gingivalis . Cells challenged with P. gingivalis without any blocker are referred to in the graphs as (–). Representative graphs of >3 repeats are shown. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005.

Article Snippet: T2.5 monoclonal antibody (mAb) against mouse and human TLR2 was from Hycult Biotech (Uden, Netherlands), mAb 1A6 was a gift from Greg Elson (NovImmune, Geneva, Switzerland), and isotype control mAbs were from BioLegend (San Diego, USA).

Techniques: Blocking Assay, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Porphyromonas gingivalis Stimulates TLR2-PI3K Signaling to Escape Immune Clearance and Induce Bone Resorption Independently of MyD88

doi: 10.3389/fcimb.2017.00359

Figure Lengend Snippet: TLR2-PI3K signaling suppresses phagocytosis and enhances intracellular survival. (A) RAW264.7 or (B) PMA-differentiated THP-1 cells were treated with blocking antibodies or PI3K inhibitor and then challenged with FITC-labeled P. gingivalis at MOI 10 for 1 h. Cells were then washed, extracellular fluorescence was quenched with trypan blue, and phagocytosis was determined using a fluorescence plate reader (RFU, relative fluorescence units). (C,D) RAW 264.7 cells were treated with TLR blocking antibodies or the PI3K inhibitor prior to challenge with P. gingivalis at MOI 10 for 1 h. Cells were then washed and extracellular bacteria were killed by incubating the cells with Metronidazole and Gentamycin for 1 h. Cells were allowed to recover in fresh media for an additional hour after which they were lysed by DDW for 20 min and lysates were plated on blood agar plates in serial dilution. CFU were enumerated after 7 days of anaerobic growth. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005.

Article Snippet: T2.5 monoclonal antibody (mAb) against mouse and human TLR2 was from Hycult Biotech (Uden, Netherlands), mAb 1A6 was a gift from Greg Elson (NovImmune, Geneva, Switzerland), and isotype control mAbs were from BioLegend (San Diego, USA).

Techniques: Blocking Assay, Labeling, Fluorescence, Serial Dilution

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Porphyromonas gingivalis Stimulates TLR2-PI3K Signaling to Escape Immune Clearance and Induce Bone Resorption Independently of MyD88

doi: 10.3389/fcimb.2017.00359

Figure Lengend Snippet: TLR2-PI3K signaling enhances intracellular survival by blocking phago-lysosomal maturation. RAW 264.7 cells were seeded at 3 × 104 cells/ well in Ibidi 8 well m-slides. The cells were untreated (A) or treated with anti-TLR2 (B) , anti-TLR4 (C) , or with the PI3K inhibitor LY 294002 (D) for an hour. Cells were then infected with FITC-labeled P. gingivalis at MOI 10 for 1 h. LysoTracker red was added at 50 nM for the last 10 min of infection. Cells were washed and fixed with 2% formaldehyde and mounted with mounting media. Images were captured using a NIKON confocal microscope at 60X magnification. Yellow color indicates co-localization of P. gingivalis (green) with lysosomes (red). In each field (A–D) the cell in the box is further magnified and shown in the upper right corner. (E) The percent of co-localization was determined by counting cells that demonstrate co-localization as a percentage of all FITC positive cells. (F) Schematic representation of the pathway used by P. gingivalis to evade bactericidal activity without preventing inflammation. *** P ≤ 0.005.

Article Snippet: T2.5 monoclonal antibody (mAb) against mouse and human TLR2 was from Hycult Biotech (Uden, Netherlands), mAb 1A6 was a gift from Greg Elson (NovImmune, Geneva, Switzerland), and isotype control mAbs were from BioLegend (San Diego, USA).

Techniques: Blocking Assay, Infection, Labeling, Microscopy, Activity Assay

Journal: bioRxiv

Article Title: Complement Dysregulation During the Early Phases of Synucleinopathy

doi: 10.64898/2026.04.27.720696

Figure Lengend Snippet: The complement system is an arm of innate immunity, comprising ∼50 proteins present throughout 8ssues and ac8vated by proteoly8c cleavage. a) Complement ac8va8on occurs via one of three pathways: classical, lec8n, and alterna8ve. The classical pathway is ini8ated when C1q binds targets, ac8va8ng C1r and C1s, which cleave C4 and C2 to form the C3 convertase, C4b2a. The lec8n pathway is triggered by mannose-binding lec8n (MBL), collec8ns, or ficolins recognizing microbial- or altered self-carbohydrates, ac8va8ng MASP-1 and MASP-2 to generate the C3 convertase, C4b2a. The alterna8ve pathway is cons8tu8vely ac8ve through spontaneous C3 hydrolysis, forming the free C3 convertase C3(H2O)Bb, and is amplified on surfaces to form the alterna8ve pathways C3 convertase, C3bBb, enhancing complement ac8va8on. b) The central event in the cascade is cleavage of C3 by the C3 convertases to form C3a and C3b, which ini8ate the major effector responses of the system. c) Opsoniza9on/Phagocytosis : C3b and iC3b deposited on target surfaces promote phagocytosis via CR1, CR3, and CR4. Inflammatory Signaling : The anaphylatoxin pep8des, C3a and C5a, mediate inflammatory signaling through C3aR and C5aR, driving chemotaxis, cytokine produc8on, and ac8va8on of immune cells. Membrane ACack Complex (MAC) : C5b ini8ates the terminal pathway by recrui8ng C6–C9 to form the MAC, lysing target cells. Complement ac8vity is 8ghtly regulated at mul8ple levels (shown in pink boxes): C1 inhibitor (C1-INH) and neuronal pentraxins (NPTXs) restrain classical ini8a8on, C4 binding protein (C4BP), Factor H (FH), and FI regulate C3 convertases and alterna8ve pathway amplifica8on; carboxypep8dase-N (CPN) inac8vates anaphylatoxins; the membrane bound regulators CR1, CD46, CD55, and CD59 prevent deposi8on of ac8vated complement on self-cells; clusterin (CLU) and vitronec8n (VTN) prevent MAC assembly and inser8on. Representa8ve complement components (shown in red text) were quan8fied in α-synuclein pre-formed fibril–injected rats and postmortem PD brains.

Article Snippet: Wells were then washed four times with 200μL of PBS-T and incubated in 50μL of a primary antibody specific to a neo-epitope in cleaved (activated) complement C3 (1:500; Hycult HM2257; RRID:AB_1953566) diluted in 2% BSA for 1 hour at RT with gentle shaking.

Techniques: Binding Assay, Amplification, Chemotaxis Assay, Membrane, Injection

Journal: bioRxiv

Article Title: Complement Dysregulation During the Early Phases of Synucleinopathy

doi: 10.64898/2026.04.27.720696

Figure Lengend Snippet: Rats (n=6-8/sex/group) received intra-striatal injec8ons of α-synuclein (α-syn) preformed fibrils (PFFs) or phosphate buffer saline (PBS) and were sacrificed 2-months post-injec8on. A-b) Representa8ve images of Serine 129 phosphorylated α-syn (pSyn) immunostaining in the substan8a nigra (SN) of PBS ( a ) and α-syn PFF ( b ) injected rats. C-d ) Representa8ve images of MHC-II immunostaining in the SN of PBS ( c ) and α-syn PFF ( d ) injected rats. High magnifica8on images to the right of each panel correspond to the area in the box of respec8ve low magnifica8on images. E-F ) Droplet digital PCR (ddPCR) quan8fica8on of complement component 3 ( C3) expression in the striatum (ST; panel e ) and SN ( f ). Data are C3 normalized to ribosomal potein L13 ( Rpl13 ), analyzed with t-test with Welch’s correc8on. g ) Representa8ve immunoblot of pSyn, α-syn and β-ac8n from the ST. h ) Quan8fica8on of pSyn monomers (∼14-17 kDa), i ) pSyn mul8mers (∼20-50 kDa), j ) total pSyn signal (∼14-50 kDa), and k ) α-syn monomer (∼14-17 kDa) normalized to β-ac8n in the ST. l) Representa8ve immunoblot of pSyn, α-syn and β-ac8n from the SN. m ) Quan8fica8on of pSyn monomers (∼14-17 kDa), n ) pSyn mul8mers (∼20-50 kDa), o ) total pSyn signal (∼14-50 kDa), and p ) α-syn monomer (∼14-17 kDa) normalized to β-ac8n in the SN. q) Representa8ve immunoblot of C3 and β-ac8n from the ipsilateral ST. s ) Representa8ve immunoblot of C3 and β-ac8n from the ipsilateral SN Quan8fica8on of C3 whole molecule (∼190 kDa), C3 α-chain (∼115 kDa), iC3b α-chain (∼75 kDa), and C3c α-chain (∼34kDa) normalized to β-ac8n from the ST ( r ) and SN ( t ), analyzed with t-test with Welch’s correc8on). Scale bars in large images of ( b, d) are 250μm and apply to large images of ( a-d) , while scale bar in the small images of ( b, d) are 50μm and apply to small images of ( a-d) . All data are group means ± standard devia8on expressed a fold change from PBS group.

Article Snippet: Wells were then washed four times with 200μL of PBS-T and incubated in 50μL of a primary antibody specific to a neo-epitope in cleaved (activated) complement C3 (1:500; Hycult HM2257; RRID:AB_1953566) diluted in 2% BSA for 1 hour at RT with gentle shaking.

Techniques: Saline, Immunostaining, Injection, Digital PCR, Expressing, Western Blot

Journal: bioRxiv

Article Title: Complement Dysregulation During the Early Phases of Synucleinopathy

doi: 10.64898/2026.04.27.720696

Figure Lengend Snippet: Male and female rats (n=5-6/group) received intra-striatal injec8ons of α-synuclein (α-syn) preformed fibrils (PFFs) or phosphate buffer saline (PBS) and were sacrificed 2 months post injec8on. a-h ) Representative low magnification immunofluorescent (IF) images of Huc/d (cyan, neuronal marker), serine 129 phosphorylated α-syn (pSyn; green) and complement component 3 (C3; red) and the overlay image in the SNc of PBS ( a-d ) and PFF ( e-h ) injected rats. i-p ) High magnification images corresponding to box in ( d ) and ( h ), for PBS ( i-l ) and PFF injected ( m-p ) rats, respectively. q-r ) Quantification of C3 ( q ) and pSyn ( r ) fluorescence intensity. s ) Regression analysis between pSyn and C3 fluorescence intensity in the SNc of PFF injected rats. t-u ) Quantification of percent area of SNc occupied by C3+ ( t ) and pSyn+ ( u ) staining. v ) Regression analysis between pSyn and C3 percent area staining in the SNc of PFF injected rats. Data expressed as mean fold change (± standard deviation) from PBS controls, analyzed by t-test with Welch’s correction or simple linear regression analyses. w-z ) Representative IF images of pSyn (green; panel w ), the microglial marker, ionized calcium binding adapter molecule 1 (IBA1; cyan; panel x ), C3 (red; panel; y ) and the overlay image ( z ) in the SNc of PFF injected rats. Arrows in ( m-p ) and ( w-z ) indicate areas where C3+ microglia are in direct apposition of neurons containing pSyn+ aggregates. Scale bars in ( h ) is 250μm and applies to ( a-h ), scale bar in ( p ) is 50μm and applies to ( i-p ), scale bar in ( z ) is 50μm and applies to ( w-z )

Article Snippet: Wells were then washed four times with 200μL of PBS-T and incubated in 50μL of a primary antibody specific to a neo-epitope in cleaved (activated) complement C3 (1:500; Hycult HM2257; RRID:AB_1953566) diluted in 2% BSA for 1 hour at RT with gentle shaking.

Techniques: Saline, Marker, Injection, Fluorescence, Staining, Standard Deviation, Binding Assay

Journal: bioRxiv

Article Title: Complement Dysregulation During the Early Phases of Synucleinopathy

doi: 10.64898/2026.04.27.720696

Figure Lengend Snippet: Male and female rats (n=5-6/group) received intra-striatal injec8ons of α-synuclein (α-syn) preformed fibrils (PFFs) or phosphate buffer saline (PBS) and sacrificed 2-months post-injec8on. a-h ) Representative low magnification images of DAPI (blue, nuclear marker), pSyn (green, phospho-Ser129 α-syn) and C3 (red, complement 3) and the overlay in the ipsilateral cortex (Cx) of PBS ( a-d ) and α-syn PFF ( e-h ) injected rats. i-p) High magnification images corresponding to box in ( d ) and ( h ), for PBS ( i-l ) and PFF ( m-p ) injected rats, respectively. q-r ) Quantification of C3 ( q ) and pSyn ( r ) fluorescence intensity in the Cx. s ) Linear regression between pSyn and C3 fluorescence intensity in the Cx of PFF injected rats. t-u) Quantification of percent area of Cx occupied by C3+ ( t ) and pSyn+ ( u ) staining. v ) Linear regression between pSyn and C3 percent area staining in the Cx of PFF injected rats. w-dd ) Representative low magnification images of DAPI (blue), pSyn (green) and C3 (red) and overlay image in the ipsilateral striatum (ST) of PBS ( w-z ) and PFF ( aa-dd ) injected rats. ee-ll) High magnification images corresponding to box in ( z ) and ( dd ), for PBS ( ee-hh ) and PFF ( ii-ll ) injected rats, respectively. mm-nn ) Quantification of C3 ( mm ) and pSyn ( nn ) fluorescence intensity in the ST. oo ) Linear regression between pSyn and C3 fluorescence intensity in the ST of PFF injected rats. pp-qq) Quantification of percent area of ST occupied by C3+ ( pp ) and pSyn+ ( qq ) staining. rr ) Linear regression between pSyn and C3 percent area staining in the ST of PFF injected rats. Data expressed as mean fold change (± standard deviation) from PBS controls (analyzed with unpaired t-test with Welch’s correction or simple linear regression analyses). Scale bars in ( h; dd) are 250μm and apply to ( a-h; w-dd) , respectively. Scale bars in ( p; ll) are 50μm and apply to ( i-p; ee-ll) , respectively.

Article Snippet: Wells were then washed four times with 200μL of PBS-T and incubated in 50μL of a primary antibody specific to a neo-epitope in cleaved (activated) complement C3 (1:500; Hycult HM2257; RRID:AB_1953566) diluted in 2% BSA for 1 hour at RT with gentle shaking.

Techniques: Saline, Marker, Injection, Fluorescence, Staining, Standard Deviation