Fig 2: Immunofluorescence staining of the whole mount for immune cells. (A) control cochlea not exposed to noise, (B) Noise exposed cochlea at day 7, (C) Apical turn of the cochlea at day 7 post- noise exposure showing the immune cells (CD45 + ; red) and B cells (B220 + ; green), (D) Basal turn of the cochlea at day 7 post-noise exposure immune cells (CD45 + ; red) and B cells (B220 + ; green), (E) Apical turn of the cochlea at day 7 post-noise exposure showing the myeloid cells (CD11b + ; red) and NK cells (NK1.1 + ; green), (F) Basal turn of the cochlea at day 7 post-noise exposure showing the myeloid cells (CD11b + ; red) and NK cells (NK1.1 + ; green). C’, C”, D’, D”, E’, E”, F’, and F” show the individual images of the respective immune cell. These are the representative figures from 4 mice.

Fig 3: Immunofluorescence staining of the innate immune cells in the noise-exposed cochlea. The cochlea section stained with the marker of various immune cells showed positivity for CD45 (red), CD11b (red), CX3CR1, NK1.1, and neutrophil elastase (green). (A) CD45 + NK1.1 + cells, (B) Cd45 + CD11b + cells (C), CD45 + CX3CR1 cells and (D) CD45 + neutrophil elastase + cells in noise- exposed cochlea at day 7. The arrow shows the presence of immune cells in the cochlea. In rows A, B, C, and D, first panel show the presence of immune cells at × 20 magnification, second panel shows the immune cells at higher magnification (× 40), and the last panels show individual cells for their morphology. These are the represented images (n = 4) for the presence of immune cells in the cochlea.

Fig 4: Auditory Brain Response (ABR) and flow cytometric evaluation of control and noise- exposed mice. The noise-exposed mice showed increased ABR threshold on days 1, 4, 7, and 14 as expected. There was no change in the ABR threshold in control mice (A). ABR threshold shifts at 8 kHz, 16 kHz, and 32 kHz after noise damage. Orange line: 1 day after noise damage, blue line: 4 days after noise damage; red line: 7 days after noise damage; green line: 14 days after noise damage. The numbers in parentheses in the figure indicate the number of mice tested. Noise trauma resulted in significantly increased population of immune cells in mice cochlea (B,C). Live CD45 + cells in the sample were analyzed for B, T, NK, and myeloid cells and myeloid cells were further gated for macrophages and neutrophils depending on their surface- markers as described in methods section. (B) Average percentage population of the B cells (CD45 + B220 +), T cells (CD45 + CD3e +), NK cells (CD45 + NK1.1 +), myeloid cells (CD45 + CD11b +), macrophages (CD45 + CD11b + CX3CR1 +), and neutrophils (CD45 + CD11b + Ly6G + CD11c -) from all mice (male + female together) in the experiment at day 1, day 4, day 7, and day 14 cochlea post noise exposure. B) Percentages of B220 +, CD3e +, CD11b +, CD11b + CX3CR1 +, and CD11b + Ly6G + CD11c- as part of the CD45 + population of immune cells in the control and noise-exposed cochleae. (C) Absolute counts of immune cells in the control and noise-exposed cochleae. Data are presented as mean ± SEM (n = 10, 12, 12, and 12 at day 1, 4, 7, and 14 respectively); *p < 0.05, **p < 0.01, ***p < 0.001; and ****p < 0.0001.

Fig 5: At an early stage of disease, C5a-peptide vaccines reduce the number of activated microglia and amyloid plaque load in the brain. a Brain sections of 15-month-old Tg2576 mice immunized with either AFF1 (n = 23), AFF2 (n = 23), or control (n = 14) vaccines were stained for CD45-positive microglia cells (green). b The average number of CD45high cells in the hippocampal region of all sections. c Amyloid plaques visualized by the 3A5 mAB (green) (d). Percentage of amyloid area of the total brain sections all containing the hippocampal region was calculated by the eDefiniens Software. a, c Cell nuclei were stained with DAPI (blue) and the scale bar refers to 100 µm. b, d Bars represent the group means ± SEM of n animals. The p value was determined using one-way ANOVA test followed by a Dunn’s multiple comparison Test (non-parametric test) and expressed as * p < 0.05 and ** p < 0.01