Journal: Frontiers in Microbiology

Article Title: Deciphering macrophage molecular responses to Porphyromonas gingivalis outer membrane vesicles through combined immunoassay and Raman spectroscopic analyses

doi: 10.3389/fmicb.2026.1803640

Figure Lengend Snippet: OMV-induced metabolic adaptations in RAW264.7 cells; OCR (a) and ECAR (b) plots after exposure to increasing concentrations of Pg -OMVs or LPS in combination with IFN- γ for 6 h. In (c,d) , OCR and ECAR plots for cells treated with Pg -OMVs (1.0 μg/mL) or LPS together with IFN-γ as a function of time. Pg -OMVs suppressed mitochondrial respiration and enhanced glycolysis in a dose- and time-dependent manner, indicating macrophage polarization toward a pro-inflammatory phenotype. Data are presented as means ± SD from three independent experiments. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple-comparison test with the control group as a reference. * p < 0.05.

Article Snippet: Murine macrophages RAW264.7 were pre-cultured in DMEM (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum and GlutaMAX TM Supplement (35050061; ThermoFisher Scientific, Waltham, MA, United States).

Techniques: Comparison, Control

Journal: Frontiers in Microbiology

Article Title: Deciphering macrophage molecular responses to Porphyromonas gingivalis outer membrane vesicles through combined immunoassay and Raman spectroscopic analyses

doi: 10.3389/fmicb.2026.1803640

Figure Lengend Snippet: Measurement of key parameters of mitochondrial function in RAW264.7 cells; (a) Assessments of mitochondrial respiration by the seahorse XF Cell Mito Stress Test for cells treated with Pg -OMVs or LPS in combination with IFN-γ for 6 h. The test evaluates multiple components of mitochondrial function, including (b) basal respiration, (c) ATP-linked respiration, (d) maximal respiration, (e) spare respiratory capacity, (f) proton leak, and (g) non-mitochondrial oxygen consumption. OCR was monitored following sequential injections of oligomycin (ATP synthase inhibitor), FCCP (uncoupler), and a mixture of rotenone and antimycin A (complex I and III inhibitors). Data are presented as means ± SD from three independent experiments. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple-comparison test with the control group as a reference. * p < 0.05.

Article Snippet: Murine macrophages RAW264.7 were pre-cultured in DMEM (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum and GlutaMAX TM Supplement (35050061; ThermoFisher Scientific, Waltham, MA, United States).

Techniques: Comparison, Control

Journal: Frontiers in Microbiology

Article Title: Deciphering macrophage molecular responses to Porphyromonas gingivalis outer membrane vesicles through combined immunoassay and Raman spectroscopic analyses

doi: 10.3389/fmicb.2026.1803640

Figure Lengend Snippet: Preserved mitochondrial respiratory activity in OMV-treated cells: (a) OCR results obtained by treating RAW264.7 cells with Pg -OMVs or LPS in combination with IFN-γ for 6 h. Cells were treated with rotenone to inhibit complex I, followed by the addition of duroquinol, which donates electrons directly to complex III. The change in OCR in response to duroquinol was monitored to assess complex III–IV activity. No significant difference in duroquinol-supported oxygen consumption was observed between control and Pg -OMVs-stimulated cells (b) , indicating that complex III–IV–mediated respiration remains intact despite Pg -OMV-induced suppression of basal mitochondrial respiration. Data are presented as means ± SD from three independent experiments. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple-comparison test with the control group as a reference. (c) A gene expression analysis in OMV-activated macrophages is offered; results of the expression levels for selected inflammation-related genes as determined by qPCR. RAW264.7 cells were stimulated with Pg -OMVs or LPS in combination with IFN-γ for 16 h. Data are shown as log 2 fold changes relative to untreated control cells. All analyzed genes (cf. labels in inset) showed statistically significant differences compared with the control in both OMV- and LPS-stimulated groups ( p < 0.05). Finally, (d,e) representative histograms are given, which show CD86 and MHC-II staining intensity, respectively, in non-stimulated, LPS-stimulated, and Pg -OMV-stimulated RAW264.7 cells (cf. labels in inset).

Article Snippet: Murine macrophages RAW264.7 were pre-cultured in DMEM (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum and GlutaMAX TM Supplement (35050061; ThermoFisher Scientific, Waltham, MA, United States).

Techniques: Activity Assay, Control, Comparison, Gene Expression, Expressing, Staining

Journal: Frontiers in Microbiology

Article Title: Deciphering macrophage molecular responses to Porphyromonas gingivalis outer membrane vesicles through combined immunoassay and Raman spectroscopic analyses

doi: 10.3389/fmicb.2026.1803640

Figure Lengend Snippet: Confocal imaging of Pg -OMV internalization in RAW264.7 cells; RAW264.7 cells were incubated with fluorescently labeled Pg -OMVs (1 μg/mL) for 3 h. The plasma membrane is shown in green and Pg -OMVs in magenta: (a) Bright-field image, (b) plasma membrane, (c) Pg -OMVs, and (d) merged image of (b,c) . Representative images from three independent experiments are shown. In (e) , high-resolution Raman spectra in the wavenumber interval 910–990 cm −1 are compared for unexposed and for Pg -OMV-stimulated RAW264.7 cells after 3 and 24 h (from top to bottom; cf. labels in inset), in order to clarify the integrity of the OMVs after cell penetration.

Article Snippet: Murine macrophages RAW264.7 were pre-cultured in DMEM (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum and GlutaMAX TM Supplement (35050061; ThermoFisher Scientific, Waltham, MA, United States).

Techniques: Imaging, Incubation, Labeling, Clinical Proteomics, Membrane

Journal: Frontiers in Microbiology

Article Title: Deciphering macrophage molecular responses to Porphyromonas gingivalis outer membrane vesicles through combined immunoassay and Raman spectroscopic analyses

doi: 10.3389/fmicb.2026.1803640

Figure Lengend Snippet: Normalized Raman spectrum of RAW264.7 cells cultured for 6 h without any immunostimulation; the spectrum is divided into zones I–IV for increasing wavenumbers and key vibrational modes and molecules are labeled in inset.

Article Snippet: Murine macrophages RAW264.7 were pre-cultured in DMEM (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum and GlutaMAX TM Supplement (35050061; ThermoFisher Scientific, Waltham, MA, United States).

Techniques: Cell Culture, Labeling

Journal: Frontiers in Microbiology

Article Title: Deciphering macrophage molecular responses to Porphyromonas gingivalis outer membrane vesicles through combined immunoassay and Raman spectroscopic analyses

doi: 10.3389/fmicb.2026.1803640

Figure Lengend Snippet: High spectrally resolved Raman spectra collected at increasing times (3, 6, and 24 h) in Zone I (600–900 cm −1 ) on RAW264.7 cells with no stimulation (a–c) , stimulated with LPS (d–f) , and stimulated with Pg -OMVs (g–k) . Spectra were deconvoluted and all sub-bands numbered (cf. vibrational origins in Table S-III); in inset, the spectral zones of lipid-related Bands 14 and 30 are enlarged and given in inset to each figure. Key signals used in Raman analyses are emphasized with arrows in green color.

Article Snippet: Murine macrophages RAW264.7 were pre-cultured in DMEM (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum and GlutaMAX TM Supplement (35050061; ThermoFisher Scientific, Waltham, MA, United States).

Techniques:

Journal: Frontiers in Microbiology

Article Title: Deciphering macrophage molecular responses to Porphyromonas gingivalis outer membrane vesicles through combined immunoassay and Raman spectroscopic analyses

doi: 10.3389/fmicb.2026.1803640

Figure Lengend Snippet: Plots of (a) viability index, R via , (b) lipid stimulation index, R lip , and cytochrome indexes, (c) R cyt1 , and (d) R cyt2 , as retrieved from areal intensities of selected Raman signals in the spectra of RAW264.7 cells with no stimulation, stimulated with LPS, and stimulated with Pg -OMVs (cf. labels in inset).

Article Snippet: Murine macrophages RAW264.7 were pre-cultured in DMEM (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum and GlutaMAX TM Supplement (35050061; ThermoFisher Scientific, Waltham, MA, United States).

Techniques:

Journal: Frontiers in Microbiology

Article Title: Deciphering macrophage molecular responses to Porphyromonas gingivalis outer membrane vesicles through combined immunoassay and Raman spectroscopic analyses

doi: 10.3389/fmicb.2026.1803640

Figure Lengend Snippet: Key lipid-related bands as extracted from time-resolved and deconvoluted Raman spectra of RAW264.7 cells with no stimulation, stimulated with LPS, and stimulated with Pg -OMVs (cf. labels in inset). In inset, the values of lipid stimulation index, R lip , are given as computed from the respective areal fractions.

Article Snippet: Murine macrophages RAW264.7 were pre-cultured in DMEM (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum and GlutaMAX TM Supplement (35050061; ThermoFisher Scientific, Waltham, MA, United States).

Techniques:

Journal: Frontiers in Microbiology

Article Title: Deciphering macrophage molecular responses to Porphyromonas gingivalis outer membrane vesicles through combined immunoassay and Raman spectroscopic analyses

doi: 10.3389/fmicb.2026.1803640

Figure Lengend Snippet: High spectrally resolved Raman spectra collected at increasing times (3, 6, and 24 h) in Zone IV (1500–1800 cm −1 ) on RAW264.7 cells with no stimulation (a–c) , stimulated with LPS (d–f) , and stimulated with Pg -OMVs (g–k) . Spectra were deconvoluted and all sub-bands numbered (cf. vibrational origins in Table S-IV); Bands 38 and 40, which were analyzed together with Band 18 (in Zone I) to screen cytochrome c oxidation are emphasized with arrows in green color.

Article Snippet: Murine macrophages RAW264.7 were pre-cultured in DMEM (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum and GlutaMAX TM Supplement (35050061; ThermoFisher Scientific, Waltham, MA, United States).

Techniques:

Journal: Frontiers in Microbiology

Article Title: Deciphering macrophage molecular responses to Porphyromonas gingivalis outer membrane vesicles through combined immunoassay and Raman spectroscopic analyses

doi: 10.3389/fmicb.2026.1803640

Figure Lengend Snippet: Key cytochrome c -related bands as extracted from time-resolved and deconvoluted Raman spectra of RAW264.7 cells with no stimulation, stimulated with LPS, and stimulated with Pg -OMVs (cf. labels in inset). In inset, the values of cytochrome indexes, R cyt1 and R cyt2 are given as computed from the respective areal fractions.

Article Snippet: Murine macrophages RAW264.7 were pre-cultured in DMEM (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum and GlutaMAX TM Supplement (35050061; ThermoFisher Scientific, Waltham, MA, United States).

Techniques:

Journal: Frontiers in Microbiology

Article Title: Deciphering macrophage molecular responses to Porphyromonas gingivalis outer membrane vesicles through combined immunoassay and Raman spectroscopic analyses

doi: 10.3389/fmicb.2026.1803640

Figure Lengend Snippet: (a) High spectrally resolved Raman spectra collected at 24 h in the wavenumber interval 950–1,250 cm −1 of Zone II on RAW264.7 cells with no stimulation, stimulated with LPS, and stimulated with Pg -OMVs. Two key-signals at 1060 and 1,125 cm −1 (labeled as Glc) were deconvoluted and assumed as markers for glycolysis enhancements. In (b) , these bands are deconvoluted, extracted, and compared with the phenylalanine bands at 1008 cm −1 , which remained unchanged upon stimulation. The Raman glycosylation index, R gly , was computed from areal intensities and plotted in (c) as a function of time and type of stimulation.

Article Snippet: Murine macrophages RAW264.7 were pre-cultured in DMEM (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum and GlutaMAX TM Supplement (35050061; ThermoFisher Scientific, Waltham, MA, United States).

Techniques: Labeling, Glycoproteomics

Journal: Frontiers in Microbiology

Article Title: Deciphering macrophage molecular responses to Porphyromonas gingivalis outer membrane vesicles through combined immunoassay and Raman spectroscopic analyses

doi: 10.3389/fmicb.2026.1803640

Figure Lengend Snippet: Schematic draft summarizing the effects of Pg -OMVs stimulation on RAW264.7 cells and the Raman markers and related parameters proposed in this study to make a quantification of cells’ metabolic disturbances by Pg -OMVs.

Article Snippet: Murine macrophages RAW264.7 were pre-cultured in DMEM (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum and GlutaMAX TM Supplement (35050061; ThermoFisher Scientific, Waltham, MA, United States).

Techniques: