SLC26A4 Targeted to the Endolymphatic Sac Rescues Hearing and Balance in Slc26a4 Mutant Mice

Mutations of SLC26A4 are a common cause of human hearing loss associated with enlargement of the vestibular aqueduct. SLC26A4 encodes pendrin, an anion exchanger expressed in a variety of epithelial cells in the cochlea, the vestibular labyrinth and the endolymphatic sac. Slc26a4 ^Δ/Δ mice are devoid of pendrin and develop a severe enlargement of the membranous labyrinth, fail to acquire hearing and balance, and thereby provide a model for the human phenotype. Here, we generated a transgenic mouse line that expresses human SLC26A4 controlled by the promoter of ATP6V1B1. Crossing this transgene into the Slc26a4 ^Δ/Δ line restored protein expression of pendrin in the endolymphatic sac without inducing detectable expression in the cochlea or the vestibular sensory organs. The transgene prevented abnormal enlargement of the membranous labyrinth, restored a normal endocochlear potential, normal pH gradients between endolymph and perilymph in the cochlea, normal otoconia formation in the vestibular labyrinth and normal sensory functions of hearing and balance. Our study demonstrates that restoration of pendrin to the endolymphatic sac is sufficient to restore normal inner ear function. This finding in conjunction with our previous report that pendrin expression is required for embryonic development but not for the maintenance of hearing opens the prospect that a spatially and temporally limited therapy will restore normal hearing in human patients carrying a variety of mutations of SLC26A4.     

Epithelial cell stretching and luminal acidification lead to a retarded development of stria vascularis and deafness in mice lacking pendrin

Loss-of-function mutations of SLC26A4/pendrin are among the most prevalent causes of deafness. Deafness and vestibular dysfunction in the corresponding mouse model, Slc26a4(-/-), are associated with an enlargement and acidification of the membranous labyrinth. Here we relate the onset of expression of the HCO(3) (-) transporter pendrin to the luminal pH and to enlargement-associated epithelial cell stretching. We determined expression with immunocytochemistry, cell stretching by digital morphometry and pH with double-barreled ion-selective electrodes. Pendrin was first expressed in the endolymphatic sac at embryonic day (E) 11.5, in the cochlear hook-region at E13.5, in the utricle and saccule at E14.5, in ampullae at E16.5, and in the upper turn of the cochlea at E17.5. Epithelial cell stretching in Slc26a4(-/-) mice began at E14.5. pH changes occurred first in the cochlea at E15.5 and in the endolymphatic sac at E17.5. At postnatal day 2, stria vascularis, outer sulcus and Reissner's membrane epithelial cells, and utricular and saccular transitional cells were stretched, whereas sensory cells in the cochlea, utricle and saccule did not differ between Slc26a4(+/-) and Slc26a4(-/-) mice. Structural development of stria vascularis, including vascularization, was retarded in Slc26a4(-/-) mice. In conclusion, the data demonstrate that the enlargement and stretching of non-sensory epithelial cells precedes luminal acidification in the cochlea and the endolymphatic sac. Stretching and luminal acidification may alter cell-to-cell communication and lead to the observed retarded development of stria vascularis, which may be an important step on the path to deafness in Slc26a4(-/-) mice, and possibly in humans, lacking functional pendrin expression.     

Slc26a7 Chloride Channel Activity and Localization in Mouse Reissner’s Membrane Epithelium

Several members of the SLC26 gene family have highly-restricted expression patterns in the auditory and vestibular periphery and mutations in mice of at least two of these (SLC26A4 and SLC26A5) lead to deficits in hearing and/or balance. A previous report pointed to SLC26A7 as a candidate gene important for cochlear function. In the present study, inner ears were assayed by immunostaining for Slc26a7 in neonatal and adult mice. Slc26a7 was detected in the basolateral membrane of Reissner’s membrane epithelial cells but not neighboring cells, with an onset of expression at P5; gene knockout resulted in the absence of protein expression in Reissner’s membrane. Whole-cell patch clamp recordings revealed anion currents and conductances that were elevated for NO₃ ⁻ over Cl⁻ and inhibited by I⁻ and NPPB. Elevated NO₃ ⁻ currents were absent in Slc26a7 knockout mice. There were, however, no major changes to hearing (auditory brainstem response) of knockout mice during early adult life under constitutive and noise exposure conditions. The lack of Slc26a7 protein expression found in the wild-type vestibular labyrinth was consistent with the observation of normal balance. We conclude that SLC26A7 participates in Cl⁻ transport in Reissner’s membrane epithelial cells, but that either other anion pathways, such as ClC-2, possibly substitute satisfactorily under the conditions tested or that Cl⁻ conductance in these cells is not critical to cochlear function. The involvement of SLC26A7 in cellular pH regulation in other epithelial cells leaves open the possibility that SLC26A7 is needed in Reissner’s membrane cells during local perturbations of pH.     

Developmental Changes of ENaC Expression and Function in the Inner Ear of Pendrin Knock-Out Mice as a Perspective on the Development of Endolymphatic Hydrops

Pendrin mutations cause enlarged vestibular aqueducts and various degrees of sensorineural hearing loss. The selective abolition of pendrin causes dilation of the membranous labyrinth known as endolymphatic hydrops, loss of the endocochlear potential, and consequently loss of hearing function. Because Na⁺ transport is one of the most important driving forces for fluid transport, the epithelial Na⁺ channel (ENaC) is believed to play an important role in fluid volume regulation in the inner ear. Therefore, the dysfunction of Na⁺ transport through ENaC by the acidification of endolymph in Pendred syndrome is one of the potential causes of endolymphatic hydrops. We investigated the changes of ENaC expression and function during the development of the pendrin knock-out mouse. In the cochlea, the expression of β and γENaC was significantly increased at P56 in Pds^−/− mice compared with Pds^+/+ mice. In the vestibule, the expression of βENaC was significantly increased at P56, and γENaC expression significantly increased from P6 to P56 in Pds^−/− mice. The ENaC-dependent trans-epithelial current was not significantly different between Pds^+/+ and Pds^−/− mice in Reissner’s membrane or the saccular extramacular roof epithelium at P0, but the current was significantly increased in Pds^−/− mice at P56 compared with Pds^+/+ mice. These findings indicate that the expression and function of ENaC were enhanced in Pds^−/− mice after the development of endolymphatic hydrops as a compensatory mechanism. This result provides insight into the role of Na⁺ transport in the development and regulation of endolymphatic hydrops due to pendrin mutations.     

Expression of the Na+-K+-2Cl- cotransporter in the rat endolymphatic sac

The endolymphatic sac (ES) is a part of the membranous labyrinth that contains the cochlea, vestibular organs, and semicircular canals, and is believed to absorb endolymphatic fluid. Na⁺–K⁺–2Cl⁻ (NKCC) is a cotransporter that occurs as two isoforms (NKCC-1 and NKCC-2). Especially, NKCC-2 is suggested to participate in ES endolymph absorption. In the present study, the expression and cellular localization of NKCC-1 and NKCC-2 in the rat ES were examined by RT-PCR and in situ hybridization, respectively. The findings indicate that both NKCC-1 and NKCC-2 are expressed in the rat ES and suggest that NKCC is involved in ES homeostasis. NKCC-2 may be particularly involved in endolymph absorption. This is the first report confirming NKCC expression in the ES.     

Vestibular Role of KCNQ4 and KCNQ5 K+ Channels Revealed by Mouse Models

The function of sensory hair cells of the cochlea and vestibular organs depends on an influx of K⁺ through apical mechanosensitive ion channels and its subsequent removal over their basolateral membrane. The KCNQ4 (K_v7.4) K⁺ channel, which is mutated in DFNA2 human hearing loss, is expressed in the basal membrane of cochlear outer hair cells where it may mediate K⁺ efflux. Like the related K⁺ channel KCNQ5 (K_v7.5), KCNQ4 is also found at calyx terminals ensheathing type I vestibular hair cells where it may be localized pre- or postsynaptically. Making use of Kcnq4^−/− mice lacking KCNQ4, as well as Kcnq4^dn/dn and Kcnq5^dn/dn mice expressing dominant negative channel mutants, we now show unambiguously that in adult mice both channels reside in postsynaptic calyx-forming neurons, but cannot be detected in the innervated hair cells. Accordingly, whole cell currents of vestibular hair cells did not differ between genotypes. Neither Kcnq4^−/−, Kcnq5^dn/dn nor Kcnq4^−/−/Kcnq5^dn/dn double mutant mice displayed circling behavior found with severe vestibular impairment. However, a milder form of vestibular dysfunction was apparent from altered vestibulo-ocular reflexes in Kcnq4^−/−/Kcnq5^dn/dn and Kcnq4^−/− mice. The larger impact of KCNQ4 may result from its preferential expression in central zones of maculae and cristae, which are innervated by phasic neurons that are more sensitive than the tonic neurons present predominantly in the surrounding peripheral zones where KCNQ5 is found. The impact of postsynaptic KCNQ4 on vestibular function may be related to K⁺ removal and modulation of synaptic transmission.     

Establishment of a knock-in mouse model with the SLC26A4 c.919-2A>G mutation and characterization of its pathology

Recessive mutations in the SLC26A4 gene are a common cause of hereditary hearing impairment worldwide. Previous studies have demonstrated that different SLC26A4 mutations may have different pathogenetic mechanisms. In the present study, we established a knock-in mouse model (i.e., Slc26a4(tm1Dontuh/tm1Dontuh) mice) homozygous for the c.919-2A>G mutation, which is a common mutation in East Asians. Mice were then subjected to audiologic assessment, a battery of vestibular evaluations, and inner ear morphological studies. All Slc26a4(tm1Dontuh/tm1Dontuh) mice revealed profound hearing loss, whereas 46% mice demonstrated pronounced head tilting and circling behaviors. There was a significant difference in the vestibular performance between wild-type and Slc26a4(tm1Dontuh/tm1Dontuh) mice, especially those exhibiting circling behavior. Inner ear morphological examination of Slc26a4(tm1Dontuh/tm1Dontuh) mice revealed an enlarged endolymphatic duct, vestibular aqueduct and sac, atrophy of stria vascularis, deformity of otoconia in the vestibular organs, consistent degeneration of cochlear hair cells, and variable degeneration of vestibular hair cells. Audiologic and inner ear morphological features of Slc26a4(tm1Dontuh/tm1Dontuh) mice were reminiscent of those observed in humans. These features were also similar to those previously reported in both knock-out Slc26a4(-/-) mice and Slc26a4(loop/loop) mice with the Slc26a4 p.S408F mutation, albeit the severity of vestibular hair cell degeneration appeared different among the three mouse strains.     

Differences in the Pathogenicity of the p.H723R Mutation of the Common Deafness-Associated SLC26A4 Gene in Humans and Mice

Mutations in the SLC26A4 gene are a common cause of human hereditary hearing impairment worldwide. Previous studies have demonstrated that different SLC26A4 mutations have different pathogenetic mechanisms. By using a genotype-driven approach, we established a knock-in mouse model (i.e., Slc26a4^{tm2Dontuh/tm2Dontuh} mice) homozygous for the common p.H723R mutation in the East Asian population. To verify the pathogenicity of the p.H723R allele in mice, we further generated mice with compound heterozygous mutations (i.e., Slc26a4^{tm1Dontuh/tm2Dontuh}) by intercrossing Slc26a4^+/tm2Dontuh mice with Slc26a4^{tm1Dontuh/tm1Dontuh} mice, which segregated the c.919-2A>G mutation with an abolished Slc26a4 function. Mice were then subjected to audiologic assessments, a battery of vestibular evaluations, inner ear morphological studies, and noise exposure experiments. The results were unexpected; both Slc26a4^{tm2Dontuh/tm2Dontuh} and Slc26a4^{tm1Dontuh/tm2Dontuh} mice showed normal audiovestibular phenotypes and inner ear morphology, and they did not show significantly higher shifts in hearing thresholds after noise exposure than the wild-type mice. The results indicated not only the p.H723R allele was non-pathogenic in mice, but also a single p.H723R allele was sufficient to maintain normal inner ear physiology in heterozygous compound mice. There might be discrepancies in the pathogenicity of specific SLC26A4 mutations in humans and mice; therefore, precautions should be taken when extrapolating the results of animal studies to humans.     

Prevention of the Disrupted Enamel Phenotype in Slc4a4-Null Mice Using Explant Organ Culture Maintained in a Living Host Kidney Capsule

Slc4a4-null mice are a model of proximal renal tubular acidosis (pRTA). Slc4a4 encodes the electrogenic sodium base transporter NBCe1 that is involved in transcellular base transport and pH regulation during amelogenesis. Patients with mutations in the SLC4A4 gene and Slc4a4-null mice present with dysplastic enamel, amongst other pathologies. Loss of NBCe1 function leads to local abnormalities in enamel matrix pH regulation. Loss of NBCe1 function also results in systemic acidemic blood pH. Whether local changes in enamel pH and/or a decrease in systemic pH are the cause of the abnormal enamel phenotype is currently unknown. In the present study we addressed this question by explanting fetal wild-type and Slc4a4-null mandibles into healthy host kidney capsules to study enamel formation in the absence of systemic acidemia. Mandibular E11.5 explants from NBCe1^−/− mice, maintained in host kidney capsules for 70 days, resulted in teeth with enamel and dentin with morphological and mineralization properties similar to cultured NBCe1^+/+ mandibles grown under identical conditions. Ameloblasts express a number of proteins involved in dynamic changes in H⁺/base transport during amelogenesis. Despite the capacity of ameloblasts to dynamically modulate the local pH of the enamel matrix, at least in the NBCe1^−/− mice, the systemic pH also appears to contribute to the enamel phenotype. Extrapolating these data to humans, our findings suggest that in patients with NBCe1 mutations, correction of the systemic metabolic acidosis at a sufficiently early time point may lead to amelioration of enamel abnormalities.     

Multiple Roles of the SO42?/Cl?/OH? Exchanger Protein Slc26a2 in Chondrocyte Functions

Mutations in the SO₄²⁻/Cl⁻/OH⁻ exchanger Slc26a2 cause the disease diastrophic dysplasia (DTD), resulting in aberrant bone development and, therefore, skeletal deformities. DTD is commonly attributed to a lack of chondrocyte SO₄²⁻ uptake and proteoglycan sulfation. However, the skeletal phenotype of patients with DTD is typified by reduction in cartilage and osteoporosis of the long bones. Chondrocytes of patients with DTD are irregular in size and have a reduced capacity for proliferation and terminal differentiation. This raises the possibility of additional roles for Slc26a2 in chondrocyte function. Here, we examined the roles of Slc26a2 in chondrocyte biology using two distinct systems: mouse progenitor mesenchymal cells differentiated to chondrocytes and freshly isolated mouse articular chondrocytes differentiated into hypertrophic chondrocytes. Slc26a2 expression was manipulated acutely by delivery of Slc26a2 or shSlc26a2 with lentiviral vectors. We demonstrate that slc26a2 is essential for chondrocyte proliferation and differentiation and for proteoglycan synthesis. Slc26a2 also regulates the terminal stage of chondrocyte cell size expansion. These findings reveal multiple roles for Slc26a2 in chondrocyte biology and emphasize the importance of Slc26a2-mediated protein sulfation in cell signaling, which may account for the complex phenotype of DTD.     

Developmental changes in the cochlear hair cell mechanotransducer channel and their regulation by transmembrane channel–like proteins

Vibration of the stereociliary bundles activates calcium-permeable mechanotransducer (MT) channels to initiate sound detection in cochlear hair cells. Different regions of the cochlea respond preferentially to different acoustic frequencies, with variation in the unitary conductance of the MT channels contributing to this tonotopic organization. Although the molecular identity of the MT channel remains uncertain, two members of the transmembrane channel–like family, Tmc1 and Tmc2, are crucial to hair cell mechanotransduction. We measured MT channel current amplitude and Ca²⁺ permeability along the cochlea’s longitudinal (tonotopic) axis during postnatal development of wild-type mice and mice lacking Tmc1 (Tmc1−/−) or Tmc2 (Tmc2−/−). In wild-type mice older than postnatal day (P) 4, MT current amplitude increased ∼1.5-fold from cochlear apex to base in outer hair cells (OHCs) but showed little change in inner hair cells (IHCs), a pattern apparent in mutant mice during the first postnatal week. After P7, the OHC MT current in Tmc1−/− (dn) mice declined to zero, consistent with their deafness phenotype. In wild-type mice before P6, the relative Ca²⁺ permeability, P_Ca, of the OHC MT channel decreased from cochlear apex to base. This gradient in P_Ca was not apparent in IHCs and disappeared after P7 in OHCs. In Tmc1−/− mice, P_Ca in basal OHCs was larger than that in wild-type mice (to equal that of apical OHCs), whereas in Tmc2−/−, P_Ca in apical and basal OHCs and IHCs was decreased compared with that in wild-type mice. We postulate that differences in Ca²⁺ permeability reflect different subunit compositions of the MT channel determined by expression of Tmc1 and Tmc2, with the latter conferring higher P_Ca in IHCs and immature apical OHCs. Changes in P_Ca with maturation are consistent with a developmental decrease in abundance of Tmc2 in OHCs but not in IHCs.     

Functional significance of glutamate–cysteine ligase modifier for erythrocyte survival in vitro and in vivo

Erythrocytes endure constant exposure to oxidative stress. The major oxidative stress scavenger in erythrocytes is glutathione. The rate-limiting enzyme for glutathione synthesis is glutamate–cysteine ligase, which consists of a catalytic subunit (GCLC) and a modifier subunit (GCLM). Here, we examined erythrocyte survival in GCLM-deficient (gclm^−/−) mice. Erythrocytes from gclm^−/− mice showed greatly reduced intracellular glutathione. Prolonged incubation resulted in complete lysis of gclm^−/− erythrocytes, which could be reversed by exogenous delivery of the antioxidant Trolox. To test the importance of GCLM in vivo, mice were treated with phenylhydrazine (PHZ; 0.07 mg/g b.w.) to induce oxidative stress. Gclm^−/− mice showed dramatically increased hemolysis compared with gclm^+/+ controls. In addition, PHZ-treated gclm^−/− mice displayed markedly larger accumulations of injured erythrocytes in the spleen than gclm^+/+ mice within 24 h of treatment. Iron staining indicated precipitations of the erythrocyte-derived pigment hemosiderin in kidney tubules of gclm^−/− mice and none in gclm^+/+ controls. In fact, 24 h after treatment, kidney function began to diminish in gclm^−/− mice as evident from increased serum creatinine and urea. Consequently, while all PHZ-treated gclm^+/+ mice survived, 90% of PHZ-treated gclm^−/− mice died within 5 days of treatment. In vitro, upon incubation in the absence or presence of additional oxidative stress, gclm^−/− erythrocytes exposed significantly more phosphatidylserine, a cell death marker, than gclm^+/+ erythrocytes, an effect at least partially due to increased cytosolic Ca²⁺ concentration. Under resting conditions, gclm^−/− mice exhibited reticulocytosis, indicating that the enhanced erythrocyte death was offset by accelerated erythrocyte generation. GCLM is thus indispensable for erythrocyte survival, in vitro and in vivo, during oxidative stress.     

CD4+ and CD8+ T Cells Can Act Separately in Tumour Rejection after Immunization with Murine Pneumotropic Virus Chimeric Her2/neu Virus-Like Particles

Background

Immunization with murine pneumotropic virus virus-like particles carrying Her2/neu (Her2MPtVLPs) prevents tumour outgrowth in mice when given prophylactically, and therapeutically if combined with the adjuvant CpG. We investigated which components of the immune system are involved in tumour rejection, and whether long-term immunological memory can be obtained.

Methodology and Results

During the effector phase in BALB/c mice, only depletion of CD4⁺ and CD8⁺ in combination, with or without NK cells, completely abrogated tumour protection. Depletion of single CD4⁺, CD8⁺ or NK cell populations only had minor effects. During the immunization/induction phase, combined depletion of CD4⁺ and CD8⁺ cells abolished protection, while depletion of each individual subset had no or negligible effect. When tumour rejection was studied in knock-out mice with a C57Bl/6 background, protection was lost in CD4^−/−CD8^−/− and CD4^−/−, but not in CD8^−/− mice. In contrast, when normal C57Bl/6 mice were depleted of different cell types, protection was lost irrespective of whether only CD4⁺, only CD8⁺, or CD4⁺ and CD8⁺ cells in combination were eradicated. No anti-Her2/neu antibodies were detected but a Her2/neu-specific IFNγ response was seen. Studies of long-term memory showed that BALB/c mice could be protected against tumour development when immunized together with CpG as long as ten weeks before challenge.

Conclusion

Her2MPtVLP immunization is efficient in stimulating several compartments of the immune system, and induces an efficient immune response including long-term memory. In addition, when depleting mice of isolated cellular compartments, tumour protection is not as efficiently abolished as when depleting several immune compartments together.     

Mutations of KCNJ10 Together with Mutations of SLC26A4 Cause Digenic Nonsyndromic Hearing Loss Associated with Enlarged Vestibular Aqueduct Syndrome

Mutations in SLC26A4 cause nonsyndromic hearing loss associated with an enlarged vestibular aqueduct (EVA, also known as DFNB4) and Pendred syndrome (PS), the most common type of autosomal-recessive syndromic deafness. In many patients with an EVA/PS phenotype, mutation screening of SLC26A4 fails to identify two disease-causing allele variants. That a sizable fraction of patients carry only one SLC26A4 mutation suggests that EVA/PS is a complex disease involving other genetic factors. Here, we show that mutations in the inwardly rectifying K⁺ channel gene KCNJ10 are associated with nonsyndromic hearing loss in carriers of SLC26A4 mutations with an EVA/PS phenotype. In probands from two families, we identified double heterozygosity in affected individuals. These persons carried single mutations in both SLC26A4 and KCNJ10. The identified SLC26A4 mutations have been previously implicated in EVA/PS, and the KCNJ10 mutations reduce K⁺ conductance activity, which is critical for generating and maintaining the endocochlear potential. In addition, we show that haploinsufficiency of Slc26a4 in the Slc26a4^+/− mouse mutant results in reduced protein expression of Kcnj10 in the stria vascularis of the inner ear. Our results link KCNJ10 mutations with EVA/PS and provide further support for the model of EVA/PS as a multigenic complex disease.     

Probiotic Bifidobacterium breve Induces IL-10-Producing Tr1 Cells in the Colon

Specific intestinal microbiota has been shown to induce Foxp3⁺ regulatory T cell development. However, it remains unclear how development of another regulatory T cell subset, Tr1 cells, is regulated in the intestine. Here, we analyzed the role of two probiotic strains of intestinal bacteria, Lactobacillus casei and Bifidobacterium breve in T cell development in the intestine. B. breve, but not L. casei, induced development of IL-10-producing Tr1 cells that express cMaf, IL-21, and Ahr in the large intestine. Intestinal CD103⁺ dendritic cells (DCs) mediated B. breve-induced development of IL-10-producing T cells. CD103⁺ DCs from Il10 ^−/−, Tlr2 ^−/−, and Myd88 ^−/− mice showed defective B. breve-induced Tr1 cell development. B. breve-treated CD103⁺ DCs failed to induce IL-10 production from co-cultured Il27ra ^−/− T cells. B. breve treatment of Tlr2 ^−/− mice did not increase IL-10-producing T cells in the colonic lamina propria. Thus, B. breve activates intestinal CD103⁺ DCs to produce IL-10 and IL-27 via the TLR2/MyD88 pathway thereby inducing IL-10-producing Tr1 cells in the large intestine. Oral B. breve administration ameliorated colitis in immunocompromised mice given naïve CD4⁺ T cells from wild-type mice, but not Il10 ^−/− mice. These findings demonstrate that B. breve prevents intestinal inflammation through the induction of intestinal IL-10-producing Tr1 cells.     

Role of JNK in a Trp53-Dependent Mouse Model of Breast Cancer

The cJun NH₂-terminal kinase (JNK) signal transduction pathway has been implicated in mammary carcinogenesis. To test the role of JNK, we examined the effect of ablation of the Jnk1 and Jnk2 genes in a Trp53-dependent model of breast cancer using BALB/c mice. We detected no defects in mammary gland development in virgin mice or during lactation and involution in control studies of Jnk1^−/− and Jnk2^−/− mice. In a Trp53^−/+ genetic background, mammary carcinomas were detected in 43% of control mice, 70% of Jnk1^−/− mice, and 53% of Jnk2^−/− mice. These data indicate that JNK1 and JNK2 are not essential for mammary carcinoma development in the Trp53^−/+ BALB/c model of breast cancer. In contrast, this analysis suggests that JNK may partially contribute to tumor suppression. This conclusion is consistent with the finding that tumor-free survival of JNK-deficient Trp53^−/+ mice was significantly reduced compared with control Trp53^−/+ mice. We conclude that JNK1 and JNK2 can act as suppressors of mammary tumor development.     

Absence of Macrophage Inflammatory Protein-1α Prevents the Development of Blinding Herpes Stromal Keratitis

Prior studies in our laboratory have suggested that the CC chemokine macrophage inflammatory protein-1α (MIP-1α) may be an important mediator in the blinding ocular inflammation which develops following herpes simplex virus type 1 (HSV-1) infection of the murine cornea. To directly test this hypothesis, MIP-1α-deficient (−/−) mice and their wild-type (+/+) counterparts were infected topically on the scarified cornea with 2.5 × 10⁵ PFU of HSV-1 strain RE and subsequently graded for corneal opacity. Four weeks postinfection (p.i.), the mean corneal opacity score of −/− mice was 1.1 ± 0.3 while that of the +/+ mice was 3.7 ± 0.5. No detectable infiltrating CD4⁺ T cells were seen histologically at 14 or 21 days p.i. in −/− animals, whereas the mean CD4⁺ T-cell count per field (36 fields counted) in +/+ hosts was 26 ± 2 (P < 0.001). In addition, neutrophil counts in the −/− mouse corneas were reduced by >80% in comparison to the wild-type controls. At 2 weeks p.i., no interleukin-2 or gamma interferon could be detected in six of seven −/− mice, whereas both T-cell cytokines were readily demonstrable in +/+ mouse corneas. Also, MIP-2 and monocyte chemoattractant protein-1 protein levels were significantly lower in MIP-1α −/− mouse corneas than in +/+ host corneas, suggesting that MIP-1α directly, or more likely indirectly, influences the expression of other chemokines. Interestingly, despite the paucity of infiltrating cells, HSV-1 clearance from the eyes of −/− mice was not significantly different from that observed in +/+ hosts. We conclude that MIP-1α is not needed to control virus growth in the cornea but is essential for the development of severe stromal keratitis.     

Impaired Innate Immunity in Tlr4 ?/? Mice but Preserved CD8+ T Cell Responses against Trypanosoma cruzi in Tlr4-, Tlr2-, Tlr9- or Myd88-Deficient Mice

The murine model of T. cruzi infection has provided compelling evidence that development of host resistance against intracellular protozoans critically depends on the activation of members of the Toll-like receptor (TLR) family via the MyD88 adaptor molecule. However, the possibility that TLR/MyD88 signaling pathways also control the induction of immunoprotective CD8⁺ T cell-mediated effector functions has not been investigated to date. We addressed this question by measuring the frequencies of IFN-γ secreting CD8⁺ T cells specific for H-2K^b-restricted immunodominant peptides as well as the in vivo Ag-specific cytotoxic response in infected animals that are deficient either in TLR2, TLR4, TLR9 or MyD88 signaling pathways. Strikingly, we found that T. cruzi-infected Tlr2^−/−, Tlr4^−/−, Tlr9^{−/ −} or Myd88^−/− mice generated both specific cytotoxic responses and IFN-γ secreting CD8⁺ T cells at levels comparable to WT mice, although the frequency of IFN-γ⁺CD4⁺ cells was diminished in infected Myd88^−/− mice. We also analyzed the efficiency of TLR4-driven immune responses against T. cruzi using TLR4-deficient mice on the C57BL genetic background (B6 and B10). Our studies demonstrated that TLR4 signaling is required for optimal production of IFN-γ, TNF-α and nitric oxide (NO) in the spleen of infected animals and, as a consequence, Tlr4^−/− mice display higher parasitemia levels. Collectively, our results indicate that TLR4, as well as previously shown for TLR2, TLR9 and MyD88, contributes to the innate immune response and, consequently, resistance in the acute phase of infection, although each of these pathways is not individually essential for the generation of class I-restricted responses against T. cruzi.     

Attenuation of Reactive Gliosis Does Not Affect Infarct Volume in Neonatal Hypoxic-Ischemic Brain Injury in Mice

Background

Astroglial cells are activated following injury and up-regulate the expression of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. Adult mice lacking the intermediate filament proteins GFAP and vimentin (GFAP^−/−Vim^−/−) show attenuated reactive gliosis, reduced glial scar formation and improved regeneration of neuronal synapses after neurotrauma. GFAP^−/−Vim^−/− mice exhibit larger brain infarcts after middle cerebral artery occlusion suggesting protective role of reactive gliosis after adult focal brain ischemia. However, the role of astrocyte activation and reactive gliosis in the injured developing brain is unknown.

Methodology/Principal Findings

We subjected GFAP^−/−Vim^−/− and wild-type mice to unilateral hypoxia-ischemia (HI) at postnatal day 9 (P9). Bromodeoxyuridine (BrdU; 25 mg/kg) was injected intraperitoneally twice daily from P9 to P12. On P12 and P31, the animals were perfused intracardially. Immunohistochemistry with MAP-2, BrdU, NeuN, and S100 antibodies was performed on coronal sections. We found no difference in the hemisphere or infarct volume between GFAP^−/−Vim^−/− and wild-type mice at P12 and P31, i.e. 3 and 22 days after HI. At P31, the number of NeuN⁺ neurons in the ischemic and contralateral hemisphere was comparable between GFAP^−/−Vim^−/− and wild-type mice. In wild-type mice, the number of S100⁺ astrocytes was lower in the ipsilateral compared to contralateral hemisphere (65.0±50.1 vs. 85.6±34.0, p<0.05). In the GFAP^−/−Vim^−/− mice, the number of S100⁺ astrocytes did not differ between the ischemic and contralateral hemisphere at P31. At P31, GFAP^−/−Vim^−/− mice showed an increase in NeuN⁺BrdU⁺ (surviving newly born) neurons in the ischemic cortex compared to wild-type mice (6.7±7.7; n = 29 versus 2.9±3.6; n = 28, respectively, p<0.05), but a comparable number of S100⁺BrdU⁺ (surviving newly born) astrocytes.

Conclusions/Significance

Our results suggest that attenuation of reactive gliosis in the developing brain does not affect the hemisphere or infarct volume after HI, but increases the number of surviving newborn neurons.