Immune dysregulation in SHARPIN-deficient mice is dependent on CYLD-mediated cell death.

SHARPIN, together with RNF31/HOIP and RBCK1/HOIL1, form the linear ubiquitin chain assembly complex (LUBAC) E3 ligase that catalyzes M1-linked polyubiquitination. Mutations in RNF31/HOIP and RBCK/HOIL1 in humans and Sharpin in mice lead to autoinflammation and immunodeficiency, but the mechanism underlying the immune dysregulation remains unclear. We now show that the phenotype of the Sharpincpdm/cpdm mice is dependent on CYLD, a deubiquitinase previously shown to mediate removal of K63-linked polyubiquitin chains. Dermatitis, disrupted splenic architecture, and loss of Peyer's patches in the Sharpincpdm/cpdm mice were fully reversed in Sharpincpdm/cpdm Cyld-/- mice. We observed enhanced association of RIPK1 with the death-signaling Complex II following TNF stimulation in Sharpincpdm/cpdm cells, a finding dependent on CYLD since we observed reversal in Sharpincpdm/cpdm Cyld-/- cells. Enhanced RIPK1 recruitment to Complex II in Sharpincpdm/cpdm cells correlated with impaired phosphorylation of CYLD at serine 418, a modification reported to inhibit its enzymatic activity. The dermatitis in the Sharpincpdm/cpdm mice was also ameliorated by the conditional deletion of Cyld using LysM-cre or Cx3cr1-cre indicating that CYLD-dependent death of myeloid cells is inflammatory. Our studies reveal that under physiological conditions, TNF- and RIPK1-dependent cell death is suppressed by the linear ubiquitin-dependent inhibition of CYLD. The Sharpincpdm/cpdm phenotype illustrates the pathological consequences when CYLD inhibition fails.

Bridging mouse and human anatomies; a knowledge-based approach to comparative anatomy for disease model phenotyping.

The laboratory mouse is the foremost mammalian model used for studying human diseases and is closely anatomically related to humans. Whilst knowledge about human anatomy has been collected throughout the history of mankind, the first comprehensive study of the mouse anatomy was published less than 60 years ago. This has been followed by the more recent publication of several books and resources on mouse anatomy. Nevertheless, to date, our understanding and knowledge of mouse anatomy is far from being at the same level as that of humans. In addition, the alignment between current mouse and human anatomy nomenclatures is far from being as developed as those existing between other species, such as domestic animals and humans. To close this gap, more in depth mouse anatomical research is needed and it will be necessary to extent and refine the current vocabulary of mouse anatomical terms.

Microbiome modulates immunotherapy response in cutaneous squamous cell carcinoma.

The gut microbiome is increasingly recognized to alter cancer risk, progression and response to treatments such as immunotherapy, especially in cutaneous melanoma. However, whether the microbiome influences immune checkpoint inhibitor (ICI) immunotherapy response to non-melanoma skin cancer has not yet been defined. As squamous cell carcinomas (SCC) are in closest proximity to the skin microbiome, we hypothesized that the skin microbiome, which regulates cutaneous immunity, might affect SCC-associated anti-PD1 immunotherapy treatment response. We used ultraviolet radiation to induce SCC in SKH1 hairless mice. We then treated the mice with broad-band antibiotics to deplete the microbiome, followed by colonisation by candidate skin and gut bacteria or persistent antibiotic treatment, all in parallel with ICI treatment. We longitudinally monitored skin and gut microbiome dynamics by 16S rRNA gene sequencing and tumour burden by periodic tumour measurements and histologic assessment. Our study revealed that antibiotics-induced abrogation of the microbiome reduced the tumour burden, suggesting a functional role of the microbiome in non-melanoma skin cancer therapy response.

Phenotyping mice with skin, hair, or nail abnormalities: A systematic approach and methodologies from simple to complex.

The skin and adnexa can be difficult to interpret because they change dramatically with the hair cycle throughout life. However, a variety of methods are commonly available to collect skin and perform assays that can be useful for figuring out morphological and molecular changes. This overview provides information on basic approaches to evaluate skin and its molecular phenotype, with references for more detail, and interpretation of results on the skin and adnexa in the mouse. These approaches range from mouse genetic nomenclature, setting up a cutaneous phenotyping study, skin grafts, hair follicle reconstitution, wax stripping, electron microscopy, and Köbner reaction to very specific approaches such as lipid and protein analyses on a large scale.

The membrane protein ANKH is crucial for bone mechanical performance by mediating cellular export of citrate and ATP.

The membrane protein ANKH was known to prevent pathological mineralization of joints and was thought to export pyrophosphate (PPi) from cells. This did not explain, however, the presence of ANKH in tissues, such as brain, blood vessels and muscle. We now report that in cultured cells ANKH exports ATP, rather than PPi, and, unexpectedly, also citrate as a prominent metabolite. The extracellular ATP is rapidly converted into PPi, explaining the role of ANKH in preventing ankylosis. Mice lacking functional Ank (Ankank/ank mice) had plasma citrate concentrations that were 65% lower than those detected in wild type control animals. Consequently, citrate excretion via the urine was substantially reduced in Ankank/ank mice. Citrate was even undetectable in the urine of a human patient lacking functional ANKH. The hydroxyapatite of Ankank/ank mice contained dramatically reduced levels of both, citrate and PPi and displayed diminished strength. Our results show that ANKH is a critical contributor to extracellular citrate and PPi homeostasis and profoundly affects bone matrix composition and, consequently, bone quality.

Independent DSG4 frameshift variants in cats with hair shaft dystrophy.

Investigations of hereditary phenotypes in spontaneous mutants may help to better understand the physiological functions of the altered genes. We investigated two unrelated domestic shorthair cats with bulbous swellings of the hair shafts. The clinical, histopathological, and ultrastructural features were similar to those in mice with lanceolate hair phenotype caused by loss-of-function variants in Dsg4 encoding desmoglein 4. We sequenced the genomes from both affected cats and compared the data of each affected cat to 61 control genomes. A search for private homozygous variants in the DSG4 candidate gene revealed independent frameshift variants in each case, c.76del or p.Ile26fsLeu*4 in case no. 1 and c.1777del or p.His593Thrfs*23 in case no. 2. DSG4 is a transmembrane glycoprotein located primarily in the extracellular part of desmosomes, a complex of adhesion molecules responsible for connecting the keratin intermediate filaments of neighbouring epithelial cells. Desmosomes are essential for normal hair shaft formation. Both identified DSG4 variants in the affected cats lead to premature stop codons and truncate major parts of the open-reading frame. We assume that this leads to a complete loss of DSG4 function, resulting in an incorrect formation of the desmosomes and causing the development of defective hair shafts. Together with the knowledge on the effects of DSG4 variants in other species, our data suggest that the identified DSG4 variants cause the hair shaft dystrophy. To the best of our knowledge, this study represents the first report of pathogenic DSG4 variants in domestic animals.

Brain and spinal cord lesions in 28 inbred strains of aging mice.

Brain and spinal cord histopathology findings in male and female 20-month-old mice in a large-scale aging study of 28 inbred Jackson Laboratory mouse strains from 7 genetic families are described. Brain sections from selected strains at 12 and 24 months of age or older were also reviewed. Common lesions include axonal dystrophy in the gracile and/or cuneate nucleus in the sensory tract of the dorsal medulla and in the spinal cord in all strains. Hirano-like bodies were seen in 24/28 strains, and mineralization was observed in the thalamus of 9/28 strains. Less common lesions were also seen in the cerebellum, cerebral cortex, and other brain areas. No brain or spinal cord tumors were found. Evidence of an impairment of the ubiquitin-proteasome system (UPS) and/or suspected autophagy was manifested as medullary axonal dystrophy with intra-axonal granular eosinophilic bodies and LC3B immunohistochemistry in most strains. RIIIS/J, the most severely affected strain, showed moderate axonal dystrophy at 12 months, which progressed to severe lesions at 20 months. Comparative pathology in various species is discussed.

Mouse genome-wide association and systems genetics identifies Lhfp as a regulator of bone mass.

Bone mineral density (BMD) is a strong predictor of osteoporotic fracture. It is also one of the most heritable disease-associated quantitative traits. As a result, there has been considerable effort focused on dissecting its genetic basis. Here, we performed a genome-wide association study (GWAS) in a panel of inbred strains to identify associations influencing BMD. This analysis identified a significant (P = 3.1 x 10-12) BMD locus on Chromosome 3@52.5 Mbp that replicated in two separate inbred strain panels and overlapped a BMD quantitative trait locus (QTL) previously identified in a F2 intercross. The association mapped to a 300 Kbp region containing four genes; Gm2447, Gm20750, Cog6, and Lhfp. Further analysis found that Lipoma HMGIC Fusion Partner (Lhfp) was highly expressed in bone and osteoblasts. Furthermore, its expression was regulated by a local expression QTL (eQTL), which overlapped the BMD association. A co-expression network analysis revealed that Lhfp was strongly connected to genes involved in osteoblast differentiation. To directly evaluate its role in bone, Lhfp deficient mice (Lhfp-/-) were created using CRISPR/Cas9. Consistent with genetic and network predictions, bone marrow stromal cells (BMSCs) from Lhfp-/- mice displayed increased osteogenic differentiation. Lhfp-/- mice also had elevated BMD due to increased cortical bone mass. Lastly, we identified SNPs in human LHFP that were associated (P = 1.2 x 10-5) with heel BMD. In conclusion, we used GWAS and systems genetics to identify Lhfp as a regulator of osteoblast activity and bone mass.

SHARPIN regulates collagen architecture and ductal outgrowth in the developing mouse mammary gland.

SHARPIN is a widely expressed multifunctional protein implicated in cancer, inflammation, linear ubiquitination and integrin activity inhibition; however, its contribution to epithelial homeostasis remains poorly understood. Here, we examined the role of SHARPIN in mammary gland development, a process strongly regulated by epithelial-stromal interactions. Mice lacking SHARPIN expression in all cells (Sharpincpdm), and mice with a stromal (S100a4-Cre) deletion of Sharpin, have reduced mammary ductal outgrowth during puberty. In contrast, Sharpincpdm mammary epithelial cells transplanted in vivo into wild-type stroma, fully repopulate the mammary gland fat pad, undergo unperturbed ductal outgrowth and terminal differentiation. Thus, SHARPIN is required in mammary gland stroma during development. Accordingly, stroma adjacent to invading mammary ducts of Sharpincpdm mice displayed reduced collagen arrangement and extracellular matrix (ECM) stiffness. Moreover, Sharpincpdm mammary gland stromal fibroblasts demonstrated defects in collagen fibre assembly, collagen contraction and degradation in vitro Together, these data imply that SHARPIN regulates the normal invasive mammary gland branching morphogenesis in an epithelial cell extrinsic manner by controlling the organisation of the stromal ECM.

Hair follicle dystrophy in a litter of domestic cats resembling lanceolate hair mutant mice.

BACKGROUND: A new congenital hair-shaft abnormality resembling the lanceolate hair phenotype of rodents is described in a litter of four domestic short hair (DSH) cats. Data relating to hair shaft and follicle disorders remain scarce in veterinary medicine. OBJECTIVES: To describe and compare structural abnormalities in these cats with other hair dystrophies in cats and other mammals. ANIMALS: A DSH cat litter with progressive noninflammatory alopecia. METHODS AND MATERIALS: Histopathological evaluation, scanning and transmission electron microscopy, and X-ray based element analysis defined the hair and skin changes in cats born with alopecia. Findings were compared to archival data from normal cats and lanceolate hair (Dsg4lahJ ) and Keratin 75 (Krt75tm1Der ) mutant mice. RESULTS: Light and scanning electron microscopy of the hairs revealed lance- or spear-head shaped defects of the hair tip. Histological findings were swollen hair shafts, initially above the hair bulb matrix and later found in the distal parts of the telogen hair follicles, similar to those observed in Dsg4lahJ Krt75tm1Der mutant mice. Transmission electron microscopy of the hair shaft and hair follicles showed a loss in the normal structure of the guard hairs in the alopecic cats. There was a statistically significant decrease in sulfur content just below the defects in the hair shafts (trichothiodystrophy). CONCLUSION AND CLINICAL IMPORTANCE: A rare form of congenital alopecia resulting in follicular dystrophy is described in cats which is similar to hair follicle and hair-shaft changes reported in several mutant mouse strains with single gene mutations in adhesion molecules or keratin genes.

PATHBIO: an international training program for precision mouse phenotyping.

Design and production of genetically engineered mouse strains by individual research laboratories, research teams, large-scale consortia, and the biopharmaceutical industry have magnified the need for qualified personnel to identify, annotate, and validate (phenotype) these potentially new mouse models of human disease. The PATHBIO project has been recently established and funded by the European Union's ERASMUS+ Knowledge Alliance program to address the current shortfall in formally trained personnel. A series of teaching workshops will be given by experts on anatomy, histology, embryology, imaging, and comparative pathology to increase the availability of individuals with formal training to contribute to this important niche of Europe's biomedical research enterprise. These didactic and hands-on workshops are organized into three modules: (1) embryology, anatomy, histology, and the anatomical basis of imaging, (2) image-based phenotyping, and (3) pathology. The workshops are open to all levels of participants from recent graduates to Ph.D., M.D., and veterinary scientists. Participation is available on a competitive basis at no cost for attending. The first series of Workshop Modules was held in 2019 and these will continue for the next 2 years.

2-deoxy D-glucose treatment does not elicit a hair growth response in alopecia areata.

2-deoxy D-glucose (2DG) was tested for efficacy in treating alopecia areata using the C3H/HeJ skin graft model. 2DG has proven to be efficacious in treatment of various mouse models of autoimmunity with minimal serious side effects noted. This agent has been shown to normalize abnormally activated T-cell populations while also preventing cell surface expression of NKG2D; key factors defining alopecia areata disease progression. Daily oral ingestion of 2DG via drinking water to mice with patchy or diffuse alopecia areata for 16 weeks failed to prevent expansion of alopecia or cause regrowth of hair in treated mice. Histologically, there were no differences between treated and control groups. These results indicate that, while 2DG is effective for some autoimmune diseases, it was not efficacious for the cell-mediated autoimmune mouse disease, alopecia areata.

Nail abnormalities identified in an ageing study of 30 inbred mouse strains.

In a large-scale ageing study, 30 inbred mouse strains were systematically screened for histologic evidence of lesions in all organ systems. Ten strains were diagnosed with similar nail abnormalities. The highest frequency was noted in NON/ShiLtJ mice. Lesions identified fell into two main categories: acute to chronic penetration of the third phalangeal bone through the hyponychium with associated inflammation and bone remodelling or metaplasia of the nail matrix and nail bed associated with severe orthokeratotic hyperkeratosis replacing the nail plate. Penetration of the distal phalanx through the hyponychium appeared to be the initiating feature resulting in nail abnormalities. The accompanying acute to subacute inflammatory response was associated with osteolysis of the distal phalanx. Evaluation of young NON/ShiLtJ mice revealed that these lesions were not often found, or affected only one digit. The only other nail unit abnormality identified was sporadic subungual epidermoid inclusion cysts which closely resembled similar lesions in human patients. These abnormalities, being age-related developments, may have contributed to weight loss due to impacts upon feeding and should be a consideration for future research due to the potential to interact with other experimental factors in ageing studies using the affected strains of mice.

Gain of function p.E138A alteration in Card14 leads to psoriasiform skin inflammation and implicates genetic modifiers in disease severity.

Psoriasis (PS) is a common inflammatory and incurable skin disease affecting 2-3% of the human population. Although genome-wide association studies implicate more than 60 loci, the full complement of genetic factors leading to disease is not known. Rare, highly penetrant, gain-of-function, dominantly acting mutations within the human caspase recruitment domain family, member 14 (CARD14) gene lead to the development of PS and psoriatic arthritis (PSA) (a familial p.G117S and de-novo p.E138A alteration). These residues are conserved in mouse and orthologous Knock-In (KI) mutations within Card14 were created. The Card14tm.1.1Sun allele (G117S) resulted in no clinically or histologically evident phenotype of the skin or joints in young adult or old mice. However, mice carrying the Card14tm2.1Sun mutant allele (E138A) were runted and developed thick, white, scaly skin soon after birth, dying within two weeks or less. The skin hyperplasia and inflammation was remarkable similarity to human PS at the clinical, histological, and transcriptomic levels. For example, the skin was markedly acanthotic and exhibited orthokeratotic hyperkeratosis with minimal inflammation and no pustules and transcripts affecting critical pathways of epidermal differentiation and components of the IL17 axis (IL23, IL17A, IL17C, TNF and IL22) were altered. Similar changes were seen in a set of orthologous microRNAs previously associated with PS suggesting conservation across species. Crossing the Card14tm2.1Sun/WT mice to C57BL/6NJ, FVB/NJ, CBA/J, C3H/HeJ, and 129S1/SvImJ generated progeny with epidermal acanthosis and marked orthokeratotic hyperkeratosis regardless of the hybrid strain. Of these hybrid lines, only the FVB;B6N(129S4) mice survived to 250 days of age or older and has led to recombinant inbred lines homozygous for Card14E138A that are fecund and have scaly skin disease. This implicates that modifiers of PS severity exist in mice, as in the familial forms of the disease in humans.

Hyaline Arteriolosclerosis in 30 Strains of Aged Inbred Mice.

During a screen for vascular phenotypes in aged laboratory mice, a unique discrete phenotype of hyaline arteriolosclerosis of the intertubular arteries and arterioles of the testes was identified in several inbred strains. Lesions were limited to the testes and did not occur as part of any renal, systemic, or pulmonary arteriopathy or vasculitis phenotype. There was no evidence of systemic or pulmonary hypertension, and lesions did not occur in ovaries of females. Frequency was highest in males of the SM/J (27/30, 90%) and WSB/EiJ (19/26, 73%) strains, aged 383 to 847 days. Lesions were sporadically present in males from several other inbred strains at a much lower (<20%) frequency. The risk of testicular hyaline arteriolosclerosis is at least partially underpinned by a genetic predisposition that is not associated with other vascular lesions (including vasculitis), separating out the etiology of this form and site of arteriolosclerosis from other related conditions that often co-occur in other strains of mice and in humans. Because of their genetic uniformity and controlled dietary and environmental conditions, mice are an excellent model to dissect the pathogenesis of human disease conditions. In this study, a discrete genetically driven phenotype of testicular hyaline arteriolosclerosis in aging mice was identified. These observations open the possibility of identifying the underlying genetic variant(s) associated with the predisposition and therefore allowing future interrogation of the pathogenesis of this condition.

Intraflagellar transport 27 is essential for hedgehog signaling but dispensable for ciliogenesis during hair follicle morphogenesis.

Hair follicle morphogenesis requires precisely controlled reciprocal communications, including hedgehog (Hh) signaling. Activation of the Hh signaling pathway relies on the primary cilium. Disrupting ciliogenesis results in hair follicle morphogenesis defects due to attenuated Hh signaling; however, the loss of cilia makes it impossible to determine whether hair follicle phenotypes in these cilia mutants are caused by the loss of cilia, disruption of Hh signaling, or a combination of these events. In this study, we characterized the function of Ift27, which encodes a subunit of intraflagellar transport (IFT) complex B. Hair follicle morphogenesis of Ift27-null mice was severely impaired, reminiscent of phenotypes observed in cilia and Hh mutants. Furthermore, the Hh signaling pathway was attenuated in Ift27 mutants, which was in association with abnormal ciliary trafficking of SMO and GLI2, and impaired processing of Gli transcription factors; however, formation of the ciliary axoneme was unaffected. The ciliary localization of IFT25 (HSPB11), the binding partner of IFT27, was disrupted in Ift27 mutant cells, and Ift25-null mice displayed hair follicle phenotypes similar to those of Ift27 mutants. These data suggest that Ift27 and Ift25 operate in a genetically and functionally dependent manner during hair follicle morphogenesis. This study suggests that the molecular trafficking machineries underlying ciliogenesis and Hh signaling can be segregated, thereby providing important insights into new avenues of inhibiting Hh signaling, which might be adopted in the development of targeted therapies for Hh-dependent cancers, such as basal cell carcinoma.

Corneocyte proteomics: Applications to skin biology and dermatology.

Advances in mass spectrometry-based proteomics now permit analysis of complex cellular structures. Application to epidermis and its appendages (nail plate, hair shaft) has revealed a wealth of information about their protein profiles. The results confirm known site-specific differences in levels of certain keratins and add great depth to our knowledge of site specificity of scores of other proteins, thereby connecting anatomy and pathology. An example is the evident overlap in protein profiles of hair shaft and nail plate, helping rationalize their sharing of certain dystrophic syndromes distinct from epidermis. In addition, interindividual differences in protein level are manifest as would be expected. This approach permits characterization of altered profiles as a result of disease, where the magnitude of perturbation can be quantified and monitored during treatment. Proteomic analysis has also clarified the nature of the isopeptide cross-linked residual insoluble material after vigorous extraction with protein denaturants, nearly intractable to analysis without fragmentation. These structures, including the cross-linked envelope of epidermal corneocytes, are comprised of hundreds of protein constituents, evidence for strengthening the terminal structure complementary to disulphide bonding. Along with other developing technologies, proteomic analysis is anticipated to find use in disease risk stratification, detection, diagnosis and prognosis after the discovery phase and clinical validation.

Cicatricial Alopecia Research Foundation meeting, May 2016: Progress towards the diagnosis, treatment and cure of primary cicatricial alopecias.

Primary cicatricial alopecias (PCAs) are a group of skin diseases in which there is progressive and permanent destruction of hair follicles followed by replacement with fibrous tissue. Unfortunately, by the time patients seek clinical evaluation of their hair loss, the skin is already inflamed and/or scarred, so there is little hope for a return to their normal hair growth pattern. Clinical and basic science investigations are now focusing on three forms of human PCA: lichen planopilaris (LPP), frontal fibrosing alopecia (FFA) and central centrifugal cicatricial alopecia (CCCA). Transcriptome, lipidome and other new technologies are providing new insight into the pathogenesis of some of these diseases that are being validated and further investigated using spontaneous and genetically engineered mouse models.

Screening Gene Knockout Mice for Variation in Bone Mass: Analysis by µCT and Histomorphometry.

PURPOSE OF REVIEW: The international mouse phenotyping consortium (IMPC) is producing defined gene knockout mouse lines. Here, a phenotyping program is presented that is based on micro-computed tomography (µCT) assessment of distal femur and vertebra. Lines with significant variation undergo a computer-based bone histomorphometric analysis. RECENT FINDINGS: Of the 220 lines examined to date, approximately 15% have a significant variation (high or low) by µCT, most of which are not identified by the IMPC screen. Significant dimorphism between the sexes and bone compartments adds to the complexity of the skeletal findings. The µCT information that is posted at www.bonebase.org can group KOMP lines with similar morphological features. The histological data is presented in a graphic form that associates the cellular features with a specific anatomic group. The web portal presents a bone-centric view appropriate for the skeletal biologist/clinician to organize and understand the large number of genes that can influence skeletal health. Cataloging the relative severity of each variant is the first step towards compiling the dataset necessary to appreciate the full polygenic basis of degenerative bone disease.