Natural killer cells attenuate cytomegalovirus-induced hearing loss in mice

<div><p>Congenital cytomegalovirus (CMV) infection is the most common non-hereditary cause of sensorineural hearing loss (SNHL) yet the mechanisms of hearing loss remain obscure. Natural Killer (NK) cells play a critical role in regulating murine CMV infection via NK cell recognition of the Ly49H cell surface receptor of the viral-encoded m157 ligand expressed at the infected cell surface. This Ly49H NK receptor/m157 ligand interaction has been found to mediate host resistance to CMV in the spleen, and lung, but is much less effective in the liver, so it is not known if this interaction is important in the context of SNHL. Using a murine model for CMV-induced labyrinthitis, we have demonstrated that the Ly49H/m157 interaction mediates host resistance in the temporal bone. BALB/c mice, which lack functional Ly49H, inoculated with mCMV at post-natal day 3 developed profound hearing loss and significant outer hair cell loss by 28 days of life. In contrast, C57BL/6 mice, competent for the Ly49H/m157 interaction, had minimal hearing loss and attenuated outer hair cell loss with the same mCMV dose. Administration of Ly49H blocking antibody or inoculation with a mCMV viral strain deleted for the m157 gene rendered the previously resistant C57BL/6 mouse strain susceptible to hearing loss to a similar extent as the BALB/c mouse strain indicating a direct role of the Ly49H/m157 interaction in mCMV-dependent hearing loss. Additionally, NK cell recruitment to sites of infection was evident in the temporal bone of inoculated susceptible mouse strains. These results demonstrate participation of NK cells in protection from CMV-induced labyrinthitis and SNHL in mice.</p></div

Ly49H blockade induces co-localization of mCMV-GFP infected cells and NK cells within the temporal bone.

<p>Green fluorescent protein expressed in mCMV-GFP infected cells and red fluorescent protein expressed in NK cells were visualized in cochlear cryosections from NK1.1-tdTomato knock-in mouse temporal bones harvested 3 days post-injection using anti-GFP (green) and anti-RFP antibodies (red). Representative images of 3–4 cochleae examined per group are shown for NK1.1-tdTomato knock-in mice injected with anti-Ly49H antibody only (A), mCMV-GFP only (B), or both (C). Panel D depicts a higher magnification of the auditory nerve region indicated by * in panel B. Panel E depicts a higher magnification of the spiral ganglion region indicated by * in panel C. Scale bars indicate 500 μm in panels A, B, and C and 100 μm in panels D and E. st = scala tympani.</p

Hearing loss in mCMV infected C57BL/6 mice after disruption of NK cell recognition signals.

<p>The effect of Ly49H receptor blockade on hearing loss was evaluated by examining distortion product otoacoustic emission (DPOAE) (A) and auditory brainstem response (ABR) (B) thresholds in post-natal day 28 (4 wks) and post-natal day 42 (6 wks) after intraperitoneal injection of IgG isotype control antibody or Ly49H blocking antibody and/or 200 pfu mCMV-GFP delivered by intracerebral injection on post-natal day 3. Statistically significant threshold differences were seen between the infected mice treated with the IgG isotype control antibody (N = 12 mice) and infected mice treated with the Ly49H blocking antibody 4 weeks post-injection (N = 8 mice) for DPOAE (<i>P</i> < 0.0001) and ABR (<i>P</i> = 0.0001) over the measured tone frequencies. Infected C57BL/6 mice treated with anti-Ly49H antibody and mCMV-GFP showed significant progressive hearing loss at 6 weeks after inoculation compared to thresholds 4 weeks after inoculation (<i>P</i> = 0.001 for DPOAE, <i>P</i> < 0.0001 for ABR). The effect of the mCMV-encoded m157 immunoevasin ligand on hearing loss was tested by comparing DPOAE (C) and ABR (D) in C57BL/6 mice after infection with either a virus deleted for the m157 gene (mCMV Δm157) or its parental wild type virus (mCMV WT1). Statistically significant threshold differences (<i>P</i> < 0.001) were seen between the mCMV WT1 and mCMV Δm157 treated C57BL/6 groups (<i>P</i> < 0.0001 for both DPOAE and ABR). Statistical differences between groups were determined using the Kruskal-Wallis test. ABR and DPOAE thresholds are presented as dB of sound pressure level (dB SPL) as a function of tone frequency in (kHz). Error bars represent standard error of the mean.</p

Hearing loss in mCMV-GFP infected BALB/c and C57BL/6 mice.

<p>Distortion product otoacoustic emission (DPOAE) (A) and auditory brainstem response (ABR) (B) thresholds of uninfected and infected mice were determined on post-natal day 28. Infected mice were treated with 200 pfu mCMV by intracerebral injection on post-natal day 3. Uninfected mice received the same volume of carrier by intracerebral injection on post-natal day 3. Statistically significant differences were seen between uninfected and infected BALB/c mice over the measured tone frequencies for both DPOAE and ABR thresholds (<i>P</i> < 0.0001, N = 6–7 mice per group) and between uninfected (N = 6) and infected (N = 12) C57BL/6 mice for DPOAE (<i>P</i> < 0.005), but not for ABR (<i>P</i> = 0.664). Statistically significant differences were also seen between infected BALB/c and C57BL/6 mice over the measured tone frequencies for both DPOAE and ABR thresholds (<i>P</i> < 0.0001). Statistical comparisons were performed using the Kruskal-Wallis test. ABR and DPOAE thresholds are presented as dB of sound pressure level (dB SPL) as a function of tone frequency in (kHz). Error bars represent standard error of the mean.</p

The mitochondrial metal transporters mitoferrin1 and mitoferrin2 are required for liver regeneration and cell proliferation in mice

International audienceMitochondrial iron import is essential for iron-sulfur cluster formation and heme biosynthesis. Two nuclear-encoded vertebrate mitochondrial high-affinity iron importers, mitoferrin1 (Mfrn1) and Mfrn2, have been identified in mammals. In mice, the gene encoding Mfrn1, solute carrier family 25 member 37 (Slc25a37), is highly expressed in sites of erythropoiesis, and whole-body Slc25a37 deletion leads to lethality. Here, we report that mice with a deletion of Slc25a28 (encoding Mfrn2) are born at expected Mendelian ratios, but show decreased male fertility due to reduced sperm numbers and sperm motility. Mfrn2 -/- mice placed on a low-iron diet exhibited reduced mitochondrial manganese, cobalt, and zinc levels, but not reduced iron. Hepatocyte-specific loss of Slc25a37 (encoding Mfrn1) in Mfrn2 -/- mice did not affect animal viability, but resulted in a 40% reduction in mitochondrial iron and reduced levels of oxidative phosphorylation proteins. Placing animals on a low-iron diet exaggerated the reduction in mitochondrial iron observed in liver-specific Mfrn1/2-knockout animals. Mfrn1 -/-/Mfrn2 -/- bone marrow-derived macrophages or skin fibroblasts in vitro were unable to proliferate, and overexpression of Mfrn1-GFP or Mfrn2-GFP prevented this proliferation defect. Loss of both mitoferrins in hepatocytes dramatically reduced regeneration in the adult mouse liver, further supporting the notion that both mitoferrins transport iron and that their absence limits proliferative capacity of mammalian cells. We conclude that Mfrn1 and Mfrn2 contribute to mitochondrial iron homeostasis and are required for high-affinity iron import during active proliferation of mammalian cells