Michelle Ma

A Protective Role for Type 3 Deiodinase, a Thyroid Hormone-Inactivating Enzyme, in Cochlear Development and Auditory Function

Thyroid hormone is necessary for cochlear development and auditory function, but the factors that control these processes are poorly understood. Previous evidence indicated that in mice, the serum supply of thyroid hormone is augmented within the cochlea itself by type 2 deiodinase, which amplifies the level of T(3), the active form of thyroid hormone, before the onset of hearing. We now report that type 3 deiodinase, a thyroid hormone-inactivating enzyme encoded by Dio3, is expressed in the immature cochlea before type 2 deiodinase. Dio3-/- mice display auditory deficits and accelerated cochlear differentiation, contrasting with the retardation caused by deletion of type 2 deiodinase. The Dio3 mRNA expression pattern in the greater epithelial ridge, stria vascularis, and spiral ganglion partly overlaps with that of thyroid hormone receptor beta (TRbeta), the T(3) receptor that is primarily responsible for auditory development. The proposal that type 3 deiodinase prevents premature stimulation of TRbeta was supported by deleting TRbeta, which converted the Dio3-/- cochlear phenotype from one of accelerated to one of delayed differentiation. The results indicate a protective role for type 3 deiodinase in hearing. The auditory system illustrates the considerable extent to which tissues can autoregulate their developmental response to thyroid hormone through both type 2 and 3 deiodinases.

Type 3 Deiodinase, a Thyroid-Hormone-Inactivating Enzyme, Controls Survival and Maturation of Cone Photoreceptors

Maturation of the mammalian nervous system requires adequate provision of thyroid hormone and mechanisms that enhance tissue responses to the hormone. Here, we report that the development of cones, the photoreceptors for daylight and color vision, requires protection from thyroid hormone by type 3 deiodinase, a thyroid hormone-inactivating enzyme. Type 3 deiodinase, encoded by Dio3, is expressed in the immature mouse retina. In Dio3−/− mice, ∼80% of cones are lost through neonatal cell death. Cones that express opsin photopigments for response to both short (S) and medium-long (M) wavelength light are lost. Rod photoreceptors, which mediate dim light vision, remain essentially intact. Excessive thyroid hormone in wild-type pups also eliminates cones. Cone loss is mediated by cone-specific thyroid hormone receptor β2 (TRβ2) as deletion of TRβ2 rescues cones in Dio3−/− mice. However, rescued cones respond to short but not longer wavelength light because TRβ2 under moderate hormonal stimulation normally induces M opsin and controls the patterning of M and S opsins over the retina. The results suggest that type 3 deiodinase limits hormonal exposure of the cone to levels that safeguard both cone survival and the patterning of opsins that is required for cone function.

Retarded Developmental Expression and Patterning of Retinal Cone Opsins in Hypothyroid Mice

Color vision is mediated by cone photoreceptors that express opsin photopigments with sensitivities to different light wavelengths. Most mammals, including mice, differentially express M and S opsins for response to medium-long and short wavelengths, respectively. Previous studies demonstrated that a thyroid hormone receptor (TRbeta2) is critical for opsin patterning: in TRbeta2-deficient mice, M opsin is lost and all cones instead express S opsin. Here, to investigate the requirement for thyroid hormone in cone development, we studied Tshr(-/-)mice as a model of congenital hypothyroidism. The onset of M opsin expression in Tshr(-/-)mice was severely delayed until after postnatal d 17 (P17), and M opsin expression failed to attain normal levels at older adult ages. S opsin showed a subtler change with an extended distribution pattern over the superior-inferior axis of the retina. Similar opsin abnormalities were detected in wild-type C57BL/6J mice made hypothyroid by methimazole treatment. In Tshr(-/-) mice, T(3) treatment from P8 recovered significant M opsin expression at P17. Tshr(-/-) mice produced normal numbers of cones, indicating that the major requirement for thyroid hormone is in opsin patterning rather than in cone generation. The phenotype is similar to, although milder than, that caused by loss of TRbeta2 and indicates the necessity for thyroid hormone for cone maturation.

Developmental expression of thyroid hormone receptor β2 protein in cone photoreceptors in the mouse

Thyroid hormone receptor &bgr;2 (TR&bgr;2) controls the patterning of cone opsin photopigments that mediate colour vision. We raised an antiserum against TR&bgr;2 to study cone photoreceptor development by western blot and immunostaining analyses. TR&bgr;2-positive cells first appeared between embryonic day 10 (E10) and E12. Numbers increased until near birth, correlating with generation of the cone population. At birth, signals decreased until postnatal day 10, then declined to very low levels in adulthood. TR&bgr;2-positive cells were initially dispersed but became aligned at the edge of the outer neuroblastic layer by E15. Postnatally, these cells migrated inwardly until postnatal day 10, then outwardly to the edge of the outer nuclear layer, the location of mature cones. TR&bgr;2 represents a functionally unique marker for cone development.

Cerebellar Abnormalities in Mice Lacking Type 3 Deiodinase and Partial Reversal of Phenotype by Deletion of Thyroid Hormone Receptor α1

Thyroid hormone serves many functions throughout brain development, but the mechanisms that control the timing of its actions in specific brain regions are poorly understood. In the cerebellum, thyroid hormone controls formation of the transient external germinal layer, which contains proliferative granule cell precursors, subsequent granule cell migration, and cerebellar foliation. We report that the thyroid hormone-inactivating type 3 deiodinase (encoded by Dio3) is expressed in the mouse cerebellum at embryonic and neonatal stages, suggesting a need to protect cerebellar tissues from premature stimulation by thyroid hormone. Dio3(-/-) mice displayed reduced foliation, accelerated disappearance of the external germinal layer, and premature expansion of the molecular layer at juvenile ages. Furthermore, Dio3(-/-) mice exhibited locomotor behavioral abnormalities and impaired ability in descending a vertical pole. To ascertain that these phenotypes resulted from inappropriate exposure to thyroid hormone, thyroid hormone receptor α1 (TRα1) was removed from Dio3(-/-) mice, which substantially corrected the cerebellar and behavioral phenotypes. Deletion of TRα1 did not correct the previously reported small thyroid gland or deafness in Dio3(-/-) mice, indicating that Dio3 controls the activation of specific receptor isoforms in different tissues. These findings suggest that type 3 deiodinase constrains the timing of thyroid hormone action during cerebellar development.

SiglecF+Gr1hi eosinophils are a distinct subpopulation within the lungs of allergen-challenged mice

Although eosinophils as a group are readily identified by their unique morphology and staining properties, flow cytometry provides an important means for identification of subgroups based on differential expression of distinct surface Ags. Here, we characterize an eosinophil subpopulation defined by high levels of expression of the neutrophil Ag Gr1 (CD45+CD11c−SiglecF+Gr1hi). SiglecF+Gr1hi eosinophils, distinct from the canonical SiglecF+Gr1− eosinophil population, were detected in allergen‐challenged wild‐type and granule protein‐deficient (EPX−/− and MBP‐1−/−) mice, but not in the eosinophil‐deficient ΔdblGATA strain. In contrast to Gr1+ neutrophils, which express both cross‐reacting Ags Ly6C and Ly6G, SiglecF+Gr1hi eosinophils from allergen‐challenged lung tissue are uniquely Ly6G+. Although indistinguishable from the more‐numerous SiglecF+Gr1− eosinophils under light microscopy, FACS‐isolated populations revealed prominent differences in cytokine contents. The lymphocyte‐targeting cytokines CXCL13 and IL‐27 were identified only in the SiglecF+Gr1hi eosinophil population (at 3.9 and 4.8 pg/106 cells, respectively), as was the prominent proinflammatory mediator IL‐13 (72 pg/106 cells). Interestingly, bone marrow‐derived (SiglecF+), cultured eosinophils include a more substantial Gr1+ subpopulation (∼50%); Gr1+ bmEos includes primarily a single Ly6C+ and a smaller, double‐positive (Ly6C+Ly6G+) population. Taken together, our findings characterize a distinct SiglecF+Gr1hi eosinophil subset in lungs of allergen‐challenged, wild‐type and granule protein‐deficient mice. SiglecF+Gr1hi eosinophils from wild‐type mice maintain a distinct subset of cytokines, including those active on B and T lymphocytes. These cytokines may facilitate eosinophil‐mediated immunomodulatory responses in the allergen‐challenged lung as well as in other distinct microenvironments.

The timecourse of apoptotic cell death during postnatal remodeling of the mouse cochlea and its premature onset by triiodothyronine (T3)

Apoptosis underlies various forms of tissue remodeling during development. Prior to the onset of hearing, thyroid hormone (T3) promotes cochlear remodeling, which involves regression of the greater epithelial ridge (GER), a transient structure of columnar cells adjacent to the mechanosensory hair cells. We investigated the timecourse of apoptosis in the GER and the influence of ectopic T3 on apoptosis. In saline-treated mice, activated caspase 3-positive cells were detected in the GER between postnatal days 7 and 13 and appeared progressively along the cochlear duct from base to apex over developmental time. T3 given on P0 and P1 advanced the overall program of apoptosis and remodeling by ~4 days. Thyroid hormone receptor β was required for these actions, suggesting a receptor-mediated process of initiation of apoptosis. Finally, T3 given only at P0 or P1 resulted in deafness in adult mice, thus revealing a transient period of susceptibility to long-term damage in the neonatal auditory system.

Silkworm larvae plasma (SLP) assay for detection of bacteria: False positives secondary to inflammation in vivo.

The silkworm larvae plasma (SLP) assay has been developed as a means to detect bacterial peptidoglycan as a surrogate for live bacteria. Here, we present results that indicate that generation of melanin by this assay is not fully reliable as a surrogate marker for bacterial count.

Alternaria alternata challenge at the nasal mucosa results in eosinophilic inflammation and increased susceptibility to influenza virus infection

Eosinophils in the nasal mucosa are an elemental feature of allergic rhinitis.

Administration of immunobiotic Lactobacillus plantarum delays but does not prevent lethal pneumovirus infection in Rag1−/− mice

Administration of immunobiotic Lactobacillus plantarum (Lp) directly to the respiratory mucosa promotes cross‐protection against lethal pneumovirus infection via B‐cell‐independent mechanisms. In this study, we examined Lp‐mediated cross protection in Rag1−/− mice which cannot clear virus from lung tissue. Although Lp was initially protective, Rag1−/− mice ultimately succumbed to a delayed lethal outcome associated with local production of the proinflammatory cytokines CCL1, ‐2, and ‐7, granulocyte recruitment, and ongoing virus replication. By contrast, CD8null mice, which are fully capable of clearing virus, are protected by Lp with no delayed lethal outcome, granulocyte recruitment to the airways, or induction of CCL7. Repeated administration of Lp to virus‐infected Rag1−/− mice had no impact on delayed mortality. Moreover, administration of Lp to the respiratory mucosa resulted in no induction of IFN‐α or ‐β in Rag1−/− or wild‐type mice, and IFN‐abR gene deletion had no impact on Lp‐mediated protection. Overall, our findings indicate that although Lp administered to the respiratory tract has substantial impact on lethal virus‐induced inflammation in situ, endogenous virus clearance mechanisms are needed to promote sustained protection. Our results suggest that a larger understanding of the mechanisms and mediators that limit acute virus‐induced inflammation may yield new and useful therapeutic modalities.