Macrophage invasion contributes to degeneration of stria vascularis in Pendred syndrome mouse model

Background Pendred syndrome, an autosomal-recessive disorder characterized by deafness and goiter, is caused by a mutation of SLC26A4, which codes for the anion exchanger pendrin. We investigated the relationship between pendrin expression and deafness using mice that have (Slc26a4+/+ or Slc26a4+/-) or lack (Slc26a4-/-) a complete Slc26a4 gene. Previously, we reported that stria vascularis of adult Slc26a4-/- mice is hyperpigmented and that marginal cells appear disorganized. Here we determine the time course of hyperpigmentation and marginal cell disorganization, and test the hypothesis that inflammation contributes to this tissue degeneration. Methods Slc26a4-/- and age-matched control (Slc26a4+/+ or Slc26a4+/-) mice were studied at four postnatal (P) developmental stages: before and after the age that marks the onset of hearing (P10 and P15, respectively), after weaning (P28-41) and adult (P74-170). Degeneration and hyperpigmentation stria vascularis was evaluated by confocal microscopy. Gene expression in stria vascularis was analyzed by microarray and quantitative RT-PCR. In addition, the expression of a select group of genes was quantified in spiral ligament, spleen and liver to evaluate whether expression changes seen in stria vascularis are specific for stria vascularis or systemic in nature. Results Degeneration of stria vascularis defined as hyperpigmentation and marginal cells disorganization was not seen at P10 or P15, but occurred after weaning and was associated with staining for CD68, a marker for macrophages. Marginal cells in Slc26a4-/-, however, had a larger apical surface area at P10 and P15. No difference in the expression of Lyzs, C3 and Cd45 was found in stria vascularis of P15 Slc26a4+/- and Slc26a4-/- mice. However, differences in expression were found after weaning and in adult mice. No difference in the expression of markers for acute inflammation, including Il1a, Il6, Il12a, Nos2 and Nos3 were found at P15, after weaning or in adults. The expression of macrophage markers including Ptprc (= Cd45), Cd68, Cd83, Lyzs, Lgals3 (= Mac2 antigen), Msr2, Cathepsins B, S, and K (Ctsb, Ctss, Ctsk) and complement components C1r, C3 and C4 was significantly increased in stria vascularis of adult Slc26a4-/- mice compared to Slc26a4+/+ mice. Expression of macrophage markers Cd45 and Cd84 and complement components C1r and C3 was increased in stria vascularis but not in spiral ligament, liver or spleen of Slc26a4-/- compared to Slc26a4+/- mice. The expression of Lyzs was increased in stria vascularis and spiral ligament but not in liver or spleen. Conclusion The data demonstrate that hyperpigmentation of stria vascularis and marginal cell reorganization in Slc26a4-/- mice occur after weaning, coinciding with an invasion of macrophages. The data suggest that macrophage invasion contributes to tissue degeneration in stria vascularis, and that macrophage invasion is restricted to stria vascularis and is not systemic in nature. The delayed onset of degeneration of stria vascularis suggests that a window of opportunity exists to restore/preserve hearing in mice and therefore possibly in humans suffering from Pendred syndrome.


Background
Pendred syndrome is an autosomal recessive disorder that is characterized by profound sensorineural deafness, abnormal iodide transport across the thyroid follicular epithelium and an enlarged vestibular aqueduct [1,2]. It is an important condition as it accounts for 1-10% of all cases of hereditary deafness [3]. Pendred syndrome is caused by mutations of the gene SLC26A4, which codes for the protein pendrin [4]. Hearing loss in Pendred syndrome develops in most cases prelingually, which implies that pendrin is not essential for hearing but that a defective pendrin protein causes hearing loss via a secondary mechanism [3,5]. Most Pendred syndrome patients are euthyroid, although the abnormal iodide transport in the thyroid affects the incorporation of iodide into thyroglobulin [6,7]. It is conceivable that the observed thyroid hyperplasia (goiter), which generally develops around puberty, ensures normal levels of thyroid hormone [2].
Pendrin is a Na + -independent exchanger for anions such as Cl -, I -, HCO 3and formate [8][9][10]. Pendrin is expressed in the inner ear, thyroid, kidney, mammary gland, uterus, testes, and placenta [6,[11][12][13][14][15][16][17]. In the thyroid, pendrin is expressed on the apical side of the thyrocytes and mediates Cl -/Iexchange. In the kidney, pendrin is expressed on the apical side of the non-A, non-B intercalated cells, cytoplasmic regions of the type B intercalated cells of cortical collecting tubules, distal convoluted tubules and connecting tubules and mediates Cl -/HCO 3 exchange [14,18]. Loss of pendrin does not effect the arterial pH but results in a lower urinary pH [19]. In the inner ear, pendrin is localized in the outer sulcus epithelial cells, root cells, apical membranes of spiral prominence surface epithelial cells and in apical membranes of spindle-shaped cells of stria vascularis [11,17].
A model for Pendred syndrome, consisting of mice lacking functional expression of pendrin, has recently been developed [20]. Similar to patients suffering from Pendred syndrome, Slc26a4 -/mice are deaf, have an enlarged vestibular aqueduct and appear to be euthyroid. Mice, in contrast to human patients, do not exhibit goiter. Adult Slc26a4 -/mice do not generate an endocochlear potential, which is generated by stria vascularis and is necessary for normal hearing [15,17].
We have shown that adult Slc26a4 -/mice show signs of degeneration of stria vascularis, including hyperpigmentation and marginal disorganization [15,17]. It remains unclear, however, whether hyperpigmentation and marginal cell disorganization occurred before or after the normal onset of hearing (P10 or P15, respectively). Further, it remains unclear whether the disorganized surface epithelial cells were all marginal cells or whether different cells rose to the epithelial surface of stria vascularis, giving rise to the disorganized appearance. In the present study, we determined the time course of hyperpigmentation and marginal disorganization. Further, we tested the hypothesis that hyperpigmentation and marginal cell disorganization is a consequence of tissue inflammation including an invasion of inflammatory cells.

Animals
Breeding pairs of Slc26a4 -/and Slc26a4 +/+ mice were obtained from the colony of Dr Susan Wall (Emory University, Atlanta, GA, USA) to establish a new colony at KSU. Mice used for this study were anaesthetized either with 4% tribromoethanol (0.014 ml/g body weight i.p.) or pentobarbitol (0.1 mg/g body weight, i.p.) and sacrificed by decapitation or by transcardial perfusion. Transcardial perfusion consisted of Clfree solution (6 ml, 1 min) followed by Clfree solution containing 4% paraformaldehyde (24 ml, 4 min Mice that either express (Slc26a4 +/+ or Slc26a4 +/-) or lack (Slc26a4 -/-) a functional pendrin gene were studied at four developmental stages, before and after the age that marks the onset of hearing at postnatal day 10 (P10) and P15, respectively, after weaning (P30-41) and adult (P74-170). Genotypes were determined by PCR as described previously [20]. All procedures involving animals were approved by the Institutional Animal Care and Use Committee of Kansas State University.

Confocal microscopy of whole-mounts
Temporal bones from age matched Slc26a4 -/and Slc26a4 +/mice were rendered blood free by transcardial perfusion with Clfree solution. Stria vascularis was obtained by microdissection and fixed for 2 hrs at 4°C in Clfree solution containing 4% paraformaldehyde, washed twice in Clfree solution and once in PBS-TX and then blocked with 5% BSA in PBS-TX for 45 min at RT and then washed three times in PBS-TX. Stria vascularis was then incubated overnight at 4°C either with Alexa-488 conjugated rat anti-mouse CD68 antibody (1:25, see above) or goat anti-Kcnq1 primary antibody (1:200, C20, Santa Cruz Biotechnology, Santa Cruz, CA, USA) in PBS-TX with 1-3% BSA. Tissues incubated with goat anti-Kcnq1 primary antibody were washed with PBS-TX and incubated for 1 h at 25°C with chicken anti-goat Alexa 594 secondary antibody, 1:1,000 (Molecular Probes, Eugene, OR, USA) in PBS-TX with 1-3% BSA.

RNA isolation
Temporal bones were removed and stria vascularis and spiral ligament were obtained by microdissection. Microdissection solutions were changed twice and isolated tissues were washed to minimize contamination between tissue fractions. In addition, liver and spleen were collected and rapidly frozen in liquid nitrogen. Total RNA was isolated and residual DNA contamination was removed by DNase treatment (RNeasy micro, Qiagen, Valencia, CA, USA). Frozen samples of liver and spleen were pulverized and homogenized. Total RNA was isolated and freed from DNA contamination (RNeasy mini, Qiagen). Isolated RNA was either used immediately or diluted and stored at -80°C for later analysis (RNA storage solution, Ambion, Austin, TX, USA).

Gene array
Total RNA was isolated from stria vascularis of adult Slc26a4 +/+ and Slc26a4 -/mice (P148 ± 3 and P153 ± 1, respectively). The minimal amount of blood present within capillaries was deemed insignificant; no cardiac perfusion was performed. Isolated RNA was concentrated and frozen for shipment to the Biotechnology Support Facility at University of Kansas Medical Center. A total of six chips were run; each chip was used to analyze RNA pooled from two animals. Three chips each were used to analyze RNA expression in stria vascularis of Slc26a4 +/+ and Slc26a4 -/mice. RNA was amplified by two rounds of amplification, and cRNA was hybridized to high-density oligonucleotide gene chips (Small sample protocol, version II; mouse 430 2.0 gene chip, Affymetrix, Santa Clara, CA, USA). Gene array data were analyzed using commercial software (GCOS, Affymetrix; Genespring, Silicon Genetics, Redwood City, CA, USA) as well as custom-written macros (Excel, Microsoft, Redmond, WA, USA). Quality metrics conformed with MIAME standards (Table 1). Present/absent calls and averaged signal intensities (average of data obtained from three chips) were used to determine expression and changes in expression levels, respectively.
In the tabulated data summaries, 'Intensity' for Slc26a4 +/+ and for Slc26a4 -/samples represents averages of data from one or more probes. For example, the gene Slc12a2 is represented on the chip by four probes. Present calls (P) were summarized for all three chips, e.g. 12/12 indicates that this gene was called present by all 12 probes (4 × 3 = 12); 9/15 indicates that the gene is represented by 5 probes on the 3 chips (5 × 3 = 15) and that the gene was called present by 9 of the 15 probes.
Ratios of intensity values (Slc26a4 -/to Slc26a4 +/+ ) were calculated for each probe and averaged. Average ratios > 1.000 were reported as Fold with the Direction 'up'. Average ratios < 1.000 were inverted (1/average ratio) and reported as Fold with the Direction 'down'. Fold values are given in the tables only when the gene was called Present (P) in Slc26a4 +/or Slc26a4 -/samples in at least half of the available probes. The direction of the fold change is only given when it exceeded 1.30. Fold changes lower than 1.30 were not considered significant.

Quantitative RT-PCR
Age and sex matched Slc26a4 -/and Slc26a4 +/mice were rendered blood free by transcardiac perfusion with Clfree solution. Total RNA was isolated from microdissected stria vascularis and spiral ligaments, as well as from liver and spleen. In each 96-well plate, the expression of seven different genes as well as 18S rRNA was analyzed. Total Table 1: Quality metrics of gene arrays. Gene array data were deposited at GEO (GSE4749). The required MIAME quality metrics are given as average ± SD.

Chips
Scale RNA from stria vascularis and spiral ligaments or from spleen and liver of one Slc26a4 -/and one matched Slc26a4 +/mouse was analyzed in duplicate reactions in parallel to allow paired comparisons (paired t-test). qRT-PCR was performed in the presence of 0.5× SYBR green I on total RNA isolated from individual animals using gene specific primers (One step RT-PCR kit, Qiagen; iCycler, BioRad, Hercules, CA, USA; SYBR green I, Molecular Probes; Table 2). RT was performed for 30 min at 50°C and 15 min at 95°C. PCR consisted of 40 cycles of 1 min at 60°C, 1 min at 72°C, 20 s hot measurement, and 1 min at 94°C. Specificity of primers was verified by sequencing. The generation of a single product of the appropriate size was verified by agarose gel electrophoresis.
Template molecules were quantified according to T = 10^log (P Ct )/(E avg^Ct ), where P Ct is product molecules at C t , E avg is the average efficiency and C t is cycle at which the fluorescence of the product molecules reached a set threshold. Efficiencies for individual reactions was obtained from the slope of the log-linear phase of the growth curve using an Excel-based program (LinRegPCR) [21].
The number of product molecules at C t (P Ct ) was calculated by amplifying known numbers of 18S rRNA (T 18S ) molecules according to P Ct = T 18S × E 18S^Ct , where E 18S is the average efficiency of all the 18S rRNA reactions. X00525], respectively. The mass of 18S rRNA per 1 μg of total RNA was estimated to be 0.284 μg, equivalent to 4.7 × 10 -13 mol or 2.8 × 10 11 molecules of 18S rRNA (molecular weight of 18S rRNA estimated to be 598,080).

Western blotting
Temporal bones from age matched Slc26a4 -/and Slc26a4 +/mice were rendered blood free by transcardial perfusion with Clfree solution. Stria vascularis and spiral ligament were isolated by microdissection. Proteins in stria vascularis and spiral ligament from one animal were isolated by heating (10 min, 95°C) in 20 μl of a diluent (Compound B, NanoOrange, Invitrogen). After cooling (20 min, RT), the isolated protein was quantified (NanoOrange, Invitrogen) and either used immediately in Western blots or stored at -80°C.

Statistics
Numeric data are presented at average ± sem, unless specified otherwise. The number (n) of animals, blots or cells is given. Differences were determined by paired t-tests. Significance was assumed at p < 0.05.
Marginal cells can be identified by there expression of the K + channel Kcnq1 in their apical membrane [17]. To gain information on the identity of surface epithelial cells, expression of Kcnq1 was visualized by immunocytochemistry and F-actin, a marker for tight junctions, was visualized by phalloidin staining. Marginal cells at all ages of Slc26a4 +/mice and at P10 and P15 of Slc26a4 -/mice expressed the K + channel Kcnq1 evenly. Little variation was observed in cell surface areas at all ages of Slc26a4 +/mice and at P10 and P15 of Slc26a4 -/mice, and variations appeared normally distributed (Figs 2 and 3). The average cell surface area of marginal cells in Slc26a4 +/mice was 104 ± 2 μm 2 (n = 120). No significant differences were detected in Slc26a4 +/mice of different ages. In contrast, the average cell surface areas of marginal cells in P10 and P15 Slc26a4 -/mice were significantly larger, 236 ± 12 μm 2 (n = 30) and 232 ± 12 μm 2 (n = 30), respectively. This Kcnq1-expressing marginal cells are likely to remain functional, given that Kcnq1 is essential for K + secretion and the endolymphatic K + concentration was found to be normal (~ 140 mM) in adult Slc26a4 -/mice [17,22,23].

Expression analysis by gene array
Gene expression in stria vascularis was analyzed by gene array to gain data on the cause of marginal cells reorganization. The quality of the gene array was determined by quality metrics (Table 1), and the quality of the expression analysis was determined by an evaluation of genes that are known to be expressed in stria vascularis (Table 3) and by an evaluation of genes that are known to be expressed in neighboring tissues that could have served as sources of contamination (Table 4). As expected, genes known to be expressed in marginal, intermediate basal and spindle cells were present in Slc26a4 +/+ and Slc26a4 -/mice (Table  3). Interestingly, genes known to be expressed in marginal cells were downregulated in Slc26a4 -/mice (Table 3A). In contrast, no change in expression was observed for genes known to be expressed in intermediate and/or basal cells (Table 3B). These data are consistent with a partial degeneration of marginal cells.
Genes known to be expressed in neighboring tissues were not found in stria vascularis, with the exception of otospiralin (Otos). Otos in known to be expressed in spiral ligament [24], which is the tissue adjacent to stria vascularis (Table 4). Taken together, these data demonstrate that gene array analysis provided reliable data of expression in stria vascularis.

Macrophage invasion of stria vascularis
Gene array analysis revealed that stria vascularis of adult Slc26a4 -/mice expresses markers specific and/or consistent with the presence of macrophages (Table 5). Expres- Reorganization of marginal cells  Genes that are expected to be expressed in the organ of Corti, the modiolus or the vestibular labyrinth but not in stria vascularis (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16), and genes that are expected to be expressed in spiral ligament but not in stria vascularis (17)(18)(19) are listed. For parameters 'P', 'Fold' and 'Direction', see the Gene array section in Methods. Fold changes are given when a gene was called Present (P) in Slc26a4 +/or Slc26a4 -/samples in at least half of the available probes. The direction of the fold change is only given when it exceeded 1.30. Fold changes lower than 1.30 were not considered significant. sion of Mac2 antigen, Itgax, Cd45, Cd83 and Cd68 is limited to leucocytes and hematopoietic cells including macrophages and dendritic cells [25]. Expression of major histocompatibility complex II (MHCII) proteins is limited to antigen-presenting cells including macrophages [25]. The expression of Lysz, a lysosomal enzyme, has been shown to be a marker for monocytes and macrophages [26]. Further, the increased expression of major histocompatibility complex I, of complement components and of cathepsins is consistent with the presence of macrophages although the expression of the gene is not limited to macrophages [27].

Macrophage invasion is restricted to stria vascularis
The observation that stria vascularis of adult mice is invaded by macrophages raises the question whether macrophage invasion is systemic and hence found in other organs, such as spleen and liver, or whether macrophage invasion is restricted to stria vascularis and hence not seen in the adjacent tissue, spiral ligament. This is an important issue, as the inner ear is immunologically responsive to systemic infections [30].
Total RNA was isolated from stria vascularis, spiral ligament, liver and spleen from age and sex matched Slc26a4 -/and Slc26a4 +/mice. The expression of a select group of transcripts was evaluated by qRT-PCR. Possible contamination between stria vascularis and the adjacent spiral ligament was evaluated by quantifying the expression of Otos and tyrosinase (Tyr) under the assumption that Tyr is expressed in stria vascularis and not in spiral ligament, and Otos in spiral ligament and not in stria vascularis. The Tissue specificity of macrophage invasion Figure 4 Tissue specificity of macrophage invasion. Transcripts of markers specific for or consistent with the presence of macrophages in stria vascularis, spiral ligament, spleen and liver of Slc26a4 +/and Slc26a4 -/mice at P34 and/or P86 were quantified by qRT-PCR. Significant changes between Slc26a4 +/and Slc26a4 -/mice are marked with an asterisk (*). Numbers between bars represent the number of animal pairs analyzed. Note that significant increases were mainly seen in stria vascularis, to a lesser degree in spiral ligament, but not in spleen or liver. These data suggest that macrophage invasion is specific to stria vascularis.
Time course of macrophage invasion Figure 5 Time course of macrophage invasion. Transcripts of markers specific for or consistent with the presence of macrophages in stria vascularis and spiral ligament of Slc26a4 +/and Slc26a4 -/mice at different ages were quantified by qRT-PCR to determine the time course of macrophage invasion during development. Significant changes between Slc26a4 +/and Slc26a4 -/mice are marked with an asterisk (*). Numbers between symbols represent the number of age-matched animal pairs analyzed. Note that significant differences were not seen before P34, suggesting that macrophage invasion occurred after weaning (P22).
Expression of complement components and macrophage markers including C1r, C3, Ptprc (= Cd45), Cd83 and Lyzs was increased in stria vascularis of Slc26a4 -/mice compared to Slc26a4 +/mice (Fig. 4). Expression of these genes was not upregulated in spiral ligament with the exception of Lyzs and Cd45. Further, the expression of these genes was not upregulated in liver or spleen (Fig. 4). Taken together, these data suggest that macrophage invasion in Slc26a4 -/mice is restricted to stria vascularis.

Time course of macrophage invasion
The finding that macrophage invasion was restricted to stria vascularis raises the question of when in development macrophage invasion occurs. Total RNA was isolated from stria vascularis and spiral ligament from sex and age matched Slc26a4 -/and Slc26a4 +/mice and a select group of transcripts was quantified by qRT-PCR. At P15, no difference in the expression of Lyzs, C3 and Cd45 was found in stria vascularis or in spiral ligament between Slc26a4 -/and Slc26a4 +/mice (Fig. 5). After weaning, and persistent into adulthood, however, the expression of Lyzs, C3 and Cd45 was higher in stria vascularis of Slc26a4 -/mice compared to Slc26a4 +/mice. A similar trend was observed with spiral ligament.
The expression of Lyzs was determined based not only on the transcript level but also on the protein level, which may be less prone to contamination between neighboring tissues. Western blotting revealed that Lyzs protein is upregulated in stria vascularis but not in spiral ligament of post-weaning Slc26a4 -/mice (Fig. 6).
Macrophage invasion was not only determined by gene expression analysis but also by immunohistochemistry, using CD68 as a marker. The specificity of the anti-CD68 antibody was verified by using bone marrow cells as a positive control and heavily pigmented cells of the vestibular labyrinth as a negative control (Fig. 7). Staining for CD68 was clearly associated with hyperpigmentation in stria vascularis in post-weaning and adult Slc26a4 -/mice (Fig.  7). Figure 7 Controls for immunohistochemistry. Controls for CD68 immunohistochemistry were obtained from cryosections of the cochlea. Top: bone marrow cells congregated in cavities of the bony cochlear wall served as a positive control. Bottom: heavily pigmented cells in the connective tissue underneath vestibular dark cells (VDC) served as a negative control. Left: confocal immunohistochemistry of CD68. Right: corresponding bright field images. Scale bars represent 10 μm.

Controls for immunohistochemistry
Lysozyme protein expression Figure 6 Lysozyme protein expression. Protein expression of lysozyme (Lyzs) in stria vascularis and spiral ligament of postweaning Slc26a4 +/and Slc26a4 -/mice was determined by Western blotting. Actin expression served as a normalization control. Significant changes between Slc26a4 +/and Slc26a4 -/mice are marked with an asterisk (*). The number of animal pairs (n) is given. Note that protein expression of Lyzs was significantly increased in stria vascularis, but not in spiral ligament.

Conclusion
The data demonstrate that hyperpigmentation of stria vascularis and marginal cell reorganization in Slc26a4 -/mice occur after weaning, coinciding with an invasion of macrophages. The data suggest that macrophage invasion contributes to tissue degeneration in stria vascularis, and that macrophage invasion is restricted to stria vascularis and is not systemic in nature. The delayed onset of degeneration of stria vascularis suggests that a window of opportunity exists to restore/preserve hearing in mice and humans suffering from Pendred syndrome.

Competing interests
The author(s) declare that they have no competing interests.

Authors' contributions
SVJ and AO drafted the text and PW finalized the manuscript. SVJ carried out confocal immunocytochemistry and morphometry. SVJ and PW isolated tissue fractions by microdissection. AO designed primers, isolated RNA and performed quantitative RT-PCR. RS prepared gene array data for submission under MIAME standards. RJM carried Figure 8 Macrophage invasion in stria vascularis. Macrophages were visualized by CD68 immunohistochemistry in whole mounts of stria vascularis and cryosections of the cochlear lateral wall. Top row: (a-d), CD68 staining in whole mounts of stria vascularis from P33 Slc26a4 +/and Slc26a4 -/mice. Immunostaining of CD68 (a and d) and corresponding bright field images (b and c) are shown. Second and third row: (e-p), CD68 staining in cryosections of the cochlear lateral wall from P33 and P80 Slc26a4 +/and Slc26a4 -/mice. Immunostaining of CD68 (e, h, k, and n), corresponding bright field images (f, i, l, and o) and merged images (g, j, m, and p) are shown. Note that CD68 expression is restricted to hyperpigmented areas of stria vascularis in Slc26a4 -/mice and that no expression of CD68 was observed in Slc26a4 +/mice. Scale bars represent 10 μm.

Macrophage invasion in stria vascularis
out confocal immunocytochemistry and Western blotting, SF advised on immunology. PW mined gene array data. SMW, LAE and EDG provided mice prior to the establishment of a colony at KSU. PW conceived the study. All authors have read and approved the final manuscript.