Mitf is required for T cell maturation by regulating dendritic cell homing to the thymus.

Thymic dendritic cells (DCs) promote immune tolerance by regulating negative selection of autoreactive T cells in the thymus. How DC homing to the thymus is transcriptionally regulated is still unclear. Microphthalmia-associated transcription factor (Mitf) is broadly expressed and plays essential roles in the hematopoietic system. Here, we used Mitf-mutated mice (Mitfvit/vit) and found enlargement of the thymus and expansion of CD4/CD8 double-positive T cells. Mitf was highly expressed in a subset of thymic DCs among the hematopoietic system. Genetic mutation or pharmacological inhibition of Mitf in DCs decreased the expression levels of Itga4, which are critical molecules for the homing of DCs to the thymus. Further, inhibition of Mitf decreased thymic DC number. These results suggest a pivotal role of Mitf in the maintenance of T cell differentiation by regulating the homing of DC subsets within the thymus.

Antagonistic regulation by the transcription factors C/EBPα and MITF specifies basophil and mast cell fates.

It remains unclear whether basophils and mast cells are derived from a common progenitor. Furthermore, how basophil versus mast cell fate is specified has not been investigated. Here, we have identified a population of granulocyte-macrophage progenitors (GMPs) that were highly enriched in the capacity to differentiate into basophils and mast cells while retaining a limited capacity to differentiate into myeloid cells. We have designated these progenitor cells "pre-basophil and mast cell progenitors" (pre-BMPs). STAT5 signaling was required for the differentiation of pre-BMPs into both basophils and mast cells and was critical for inducing two downstream molecules: C/EBPα and MITF. We have identified C/EBPα as the critical basophil transcription factor for specifying basophil cell fate and MITF as the crucial transcription factor for specifying mast cell fate. C/EBPα and MITF silenced each other's transcription in a directly antagonistic fashion. Our study reveals how basophil and mast cell fate is specified.

Mutation in KARS: A novel mechanism for severe anaphylaxis.

BACKGROUND: Anaphylaxis is a severe allergic reaction that can be lethal if not treated adequately. The underlying molecular mechanisms responsible for the severity are mostly unknown. OBJECTIVE: This study is based on a clinical case of a patient with extremely severe anaphylaxis to paper wasp venom. This patient has a mutation in the KARS gene, which encodes lysyl-tRNA synthetase (LysRS), a moonlight protein with a canonical function in protein synthesis and a noncanonical function in antigen dependent-FcεRI activation in mast cells. In this study, the objective was to characterize the mutation at the molecular level. METHODS: Analysis of the KARS mutation was carried out using biochemical and functional approaches, cell transfection, Western blot, confocal microscopy, cell degranulation, prostaglandin D2 secretion, and proteases gene transcription. Structural analysis using molecular dynamics simulations and well-tempered metadynamics was also performed. RESULTS: The mutation found, P542R (proline was replaced by arginine at aminoacid 542), affects the location of the protein as we show in biochemical and structural analyses. The mutation resembles active LysRS and causes a constitutive activation of the microphthalmia transcription factor, which is involved in critical mast cell functions such as synthesis of mediators and granule biogenesis. Moreover, the structural analysis provides insights into how LysRS works in mast cell activation. CONCLUSIONS: A link between the aberrant LysRS-P542R function and mast cell-exacerbated activation with increase in proinflammatory mediator release after antigen-IgE-dependent response could be established.

TAF5L functions as transcriptional coactivator of MITF involved in the immune response of the clam Meretrix petechialis.

TAF5L is a component of the P300/CBP-associated factor (PCAF) histone acetylase complex, which serves as a coactivator and takes part in basal transcription such as promoter recognition, complex assembly and transcription initiation. In our study, the full-length sequence of MpTAF5L was identified and characterized in the clam M. petechialis. Sequence analysis showed that the predicted MpTAF5L protein had a N-terminal TAF5-NTD2 domain and a C-terminal WD40-repeats domain. The annotation and evolutionary analysis revealed MpTAF5L had close evolutionary relationship with other invertebrate species. Tissue distribution analysis of TAF5L claimed that it was highly expressed in the mantle, adductor muscle, foot and hepatopancreas. The mRNA expression of MpTAF5L was significantly up-regulated after Vibrio parahaemolyticus challenge, indicating its involvement in the immune response of clam. Yeast two-hybrid assays verified that MpTAF5L can interact with MpMITF (a critical immune-related transcription factor), and our further research clarified this interaction depended upon the N-terminal TAF5-NTD2 domain of MpTAF5L. Moreover, the mRNA expression of MpBcl-2 (a target gene of MITF) was significantly decreased but the mRNA expression of MpMITF was not significantly changed after knockdown of MpTAF5L, which indicated the reduction of MpMITF regulating activity at the same time. These results revealed that MpTAF5L interacted with MpMITF and enhanced the activation of MpMITF, which plays roles in the immune defense against V. parahaemolyticus.

A comparison of imaging software and conventional cell counting in determining melanocyte density in photodamaged control sample and melanoma in situ biopsies.

BACKGROUND: Objective methods for distinguishing melanoma in situ (MIS) from photodamaged skin (PS) are needed to guide treatment in patients with melanocytic proliferations. Melanocyte density (MD) could serve as an objective histopathological criterion in difficult cases. Calculating MD via manual cell counts (MCC) with immunohistochemical (IHC)-stained slides has been previously published. However, the clinical application of this method is questionable, as quantification of MD via MCC on difficult cases is time consuming, especially in high volume practices. METHODS: ImageJ is an image processing software that uses scanned slide images to determine cell count. In this study, we compared MCC to ImageJ calculated MD in microphthalmia transcription factor-IHC stained MIS biopsies and control PS acquired from the same patients. RESULTS: We found a statistically significant difference in MD between PS and MIS as measured by both MCC and ImageJ software (P < 0.01). Additionally, no statistically significant difference was found when comparing MD measurements recorded by ImageJ vs those determined by the MCC method. CONCLUSION: MD as determined by ImageJ strongly correlates with the MD calculated by MCC. We propose the use of ImageJ as a time-efficient, objective, and reproducible tool to assess MD.

Pyruvate dehydrogenase has a major role in mast cell function, and its activity is regulated by mitochondrial microphthalmia transcription factor.

BACKGROUND: We have recently observed that oxidative phosphorylation-mediated ATP production is essential for mast cell function. Pyruvate dehydrogenase (PDH) is the main regulator of the Krebs cycle and is located upstream of the electron transport chain. However, the role of PDH in mast cell function has not been described. Microphthalmia transcription factor (MITF) regulates the development, number, and function of mast cells. Localization of MITF to the mitochondria and its interaction with mitochondrial proteins has not been explored. OBJECTIVE: We sought to explore the role played by PDH in mast cell exocytosis and to determine whether MITF is localized in the mitochondria and involved in regulation of PDH activity. METHODS: Experiments were performed in vitro by using human and mouse mast cells, as well as rat basophil leukemia cells, and in vivo in mice. The effect of PDH inhibition on mast cell function was examined. PDH interaction with MITF was measured before and after immunologic activation. Furthermore, mitochondrial localization of MITF and its effect on PDH activity were determined. RESULTS: PDH is essential for immunologically mediated degranulation of mast cells. After activation, PDH is serine dephosphorylated. In addition, for the first time, we show that MITF is partially located in the mitochondria and interacts with PDH. This interaction is dependent on the phosphorylation state of PDH. Furthermore, mitochondrial MITF regulates PDH activity. CONCLUSION: The association of mitochondrial MITF with PDH emerges as an important regulator of mast cell function. Our findings indicate that PDH could arise as a new target for the manipulation of allergic diseases.

Immune and molecular correlates in melanoma treated with immune checkpoint blockade.

Immunotherapy for metastatic melanoma has a decades-long history, and the relatively recent use of checkpoint inhibitors has revolutionized treatment. Durable and sometimes complete remission of metastatic melanoma is now achievable in some patients who receive checkpoint-blocking therapy. However, it is unclear why some patients fare better than others. This review highlights several molecular indicators of response to checkpoint inhibition in metastatic melanoma, focusing on tumor programmed death ligand 1 expression, major histocompatibility complex class I expression, mutational load in the tumor, and T-cell infiltration into the tumor. In addition, clinical correlates of response, notably vitiligo and other immune-related adverse events, can potentially shed light on the mechanisms by which checkpoint blockade may achieve such great success, particularly in melanoma. The authors propose that microphthalmia-associated transcription factor-a key regulator of melanocyte survival, melanin production, and melanoma transformation-produces a molecular landscape in melanocytes and melanoma cells that can make melanomas particularly susceptible to checkpoint blockade and also can result in immune attack on normal melanocytes. Cancer 2017;123:2143-53. © 2017 American Cancer Society.

The enigma of the role of protein inhibitor of activated STAT3 (PIAS3) in the immune response.

Protein inhibitor of activated STAT3 (PIAS3), the main cellular inhibitor of signal transducers and activator of transcription 3 (STAT3), has been described as a modulator of DNA binding transcription factors. The exploration of the emerging roles of PIAS3 in immune regulation is a growing and fascinating field. Recent discoveries have shed new light on the key role of PIAS3 in the regulation of transcriptional activity, and on the molecular mechanism involved. These findings suggest that the known functions of this signalling molecule are merely the "tip of the iceberg". This article reviews the challenging questions regarding the link between PIAS3 and the intracellular signalling in immune cells. Some of the known functions of PIAS3 that potentially modulate key proteins in the immune system will also be discussed.

CD4+ T-cell-mediated anti-tumor immunity can be uncoupled from autoimmunity via the STAT4/STAT6 signaling axis.

Previous reports have suggested that autoimmune sequelae may be an unavoidable consequence of successful immunization against tumor-associated antigens, which are typically non-mutated self-antigens. Using a melanoma model, we demonstrated that CD4(+) T-cell-mediated anti-tumor immunity and autoimmunity could be separated by modulating the STAT4/STAT6 signaling axis. Our results have revealed an unexpected dichotomy in the effector phase following cancer vaccination where anti-tumor immunity is mediated via a STAT6 and IL-4-dependent pathway, whereas autoimmune pathology is mediated via STAT4 through a mechanism that relies partially on IFN-gamma. Our results offer a possibility to elicit specific anti-tumor responses without triggering unwanted tissue autoimmune diseases.

MITF Regulates Downstream Genes in Response to Vibrio parahaemolyticus Infection in the Clam Meretrix Petechialis.

The microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix-leucine zipper protein that plays a key role in cell proliferation, survival and immune defense through the direct transcriptional control of downstream genes. We have found that MITF participates in the immune response to Vibrio parahaemolyticus infection in the clam Meretrix petechialis. In this study, we focused on how MITF functions in immunity. First, PO, CTSK, and BCL-2 were identified as the target genes of MpMITF in the clam by RNAi. EMSAs showed direct binding between the MpMITF protein and the E-box of the MpPO, MpCTSK, and MpBCL-2 promoters. Yeast one-hybrid assays also suggested that MpMITF could activate the expression of these three downstream genes. These results demonstrated that the transcriptional expression of MpPO, MpCTSK, and MpBCL-2 is directly regulated by MpMITF. Second, we analyzed the roles of MpPO, MpCTSK, and MpBCL-2 in clam immunity. The mRNA expression of MpPO, MpCTSK, and MpBCL-2 increased significantly after V. parahaemolyticus challenge, which implied that these genes might take part in the immune defense against V. parahaemolyticus challenge in clams. The purified recombinant proteins, MpPO and MpCTSK, inhibited the growth of V. parahaemolyticus. Additionally, the apoptosis rate of clam haemocytes rose significantly when the activity of MpBCL-2 was suppressed. These results revealed that MpPO, MpCTSK, and MpBCL-2 are involved in the immune defense against V. parahaemolyticus. This study supports the idea that the MpMITF pathway plays a key role in immune defense through the direct regulation of the downstream genes MpPO, MpCTSK, and MpBCL-2 in the clam, M. petechialis.

Distribution of mast cells within the mouse heart and its dependency on Mitf.

Although mast cell distribution has been described in both human and canine hearts, cardiac mast cells in mice have yet to be categorically localized. We therefore sought to describe mast cell distribution within the mouse heart and characterize their dependence on the Microphthalmia-associated transcription factor (Mitf). Cardiac mast cells were visualized using Toluidine Blue and avidin staining, and their distribution within the heart described. Cardiac mast cells were most prevalent in the epicardium (50%) or myocardium (45%). Less frequently, mast cells were noted in the endocardium (5%). Within the myocardium, 31% of the mast cells had perivascular location. By studying two different Mitf mutant strains, Mitfmi-vga9 and MitfMi-wh, we demonstrated that these mutations led to near-complete deficiency of cardiac mast cells. Accordingly, expression of the mMCP-4 and mMCP-5 genes was lost and chymase enzyme activity was severely reduced. Additionally, hearts from mice heterozygous for these Mitf mutations contained significantly fewer mast cells compared to wild-type mice. Our results demonstrated that the distribution of cardiac mast cells in mice is different from humans and dogs. Cardiac mast cells are dependent on Mitf expression, with loss-of-function mutation in the Mitf gene leading to near-complete lack of cardiac mast cells. Loss of a single Mitf allele is sufficient for relative mast cell deficiency.

Canine leishmaniasis: Genome-wide analysis and antibody response to Lutzomyia longipalpis saliva.

The anti-inflammatory properties of sand fly saliva favor the establishment of the Leishmania infantum infection. In contrast, an antibody response against Lutzomyia longipalpis saliva is often associated with a protective cell-mediated response against canine visceral leishmaniasis. Genetic studies may demonstrate to what extent the ability to secrete anti-saliva antibodies depends on genetic or environmental factors. However, the genetic basis of canine antibody response against sand fly saliva has not been assessed. The aim of this study was to identify chromosomal regions associated with the anti-Lu. longipalpis salivary IgG response in 189 dogs resident in endemic areas in order to provide information for prophylactic strategies. Dogs were classified into five groups based on serological and parasitological diagnosis and clinical evaluation. Anti-salivary gland homogenate (SGH) IgG levels were assessed by Enzyme-Linked Immunosorbent Assay (ELISA). Genomic DNA was isolated from blood samples and genotyped using a SNP chip with 173,662 single nucleotide polymorphism (SNP) markers. The following linear regression model was fitted: IgG level = mean + origin + sex + age + use of a repellent collar, and the residuals were assumed as pseudo-phenotypes for the association test between phenotypes and genotypes (GWA). A component of variance model that takes into account polygenic and sample structure effects (EMMAX) was employed for GWA. Phenotypic findings indicated that anti-SGH IgG levels remained higher in exposed and subclinically infected dogs than in severely diseased dogs even in regression model residuals. Five associated markers were identified on chromosomes 2, 20 and 31. The mapped genes included CD180 (RP105) and MITF related to the rapid activation of B lymphocytes and differentiation into antibody-secreting plasma cells. The findings pointed to chromosomal segments useful for functional confirmation studies and a search for adjuvant molecules of the anti-saliva response.

Target identification and validation in systemic autoimmunity.

In systemic, humoral autoimmune diseases such as systemic lupus erythematosus, a pathogenic hyperactivation of T- and B-lymphocytes results in the elaboration of high-titer, high-affinity autoantibodies that mediate end organ damage. Although several T-dependent and -independent cell-surface molecules and cytokines, such as CD154 and the interferons, have been proposed to play key roles in disease, they have remained insufficient to explain fully the pathophysiology of these syndromes and have so far failed to yield premium therapeutic targets. Recent genome-based approaches to autoimmunity, however, have revealed novel pathogenic targets, including genes that negatively regulate T- and/or B-cell effector differentiation, as well as genes that specifically regulate T-cell-B-cell collaboration. It is hoped that continued investigation of such pathogenic targets will yield novel and specific therapeutic agents for the treatment of human autoimmune conditions.

MITF and c-Jun antagonism interconnects melanoma dedifferentiation with pro-inflammatory cytokine responsiveness and myeloid cell recruitment.

Inflammation promotes phenotypic plasticity in melanoma, a source of non-genetic heterogeneity, but the molecular framework is poorly understood. Here we use functional genomic approaches and identify a reciprocal antagonism between the melanocyte lineage transcription factor MITF and c-Jun, which interconnects inflammation-induced dedifferentiation with pro-inflammatory cytokine responsiveness of melanoma cells favouring myeloid cell recruitment. We show that pro-inflammatory cytokines such as TNF-α instigate gradual suppression of MITF expression through c-Jun. MITF itself binds to the c-Jun regulatory genomic region and its reduction increases c-Jun expression that in turn amplifies TNF-stimulated cytokine expression with further MITF suppression. This feed-forward mechanism turns poor peak-like transcriptional responses to TNF-α into progressive and persistent cytokine and chemokine induction. Consistently, inflammatory MITF(low)/c-Jun(high) syngeneic mouse melanomas recruit myeloid immune cells into the tumour microenvironment as recapitulated by their human counterparts. Our study suggests myeloid cell-directed therapies may be useful for MITF(low)/c-Jun(high) melanomas to counteract their growth-promoting and immunosuppressive functions.

Value of melanocytic-associated immunohistochemical markers in the diagnosis of malignant melanoma: a review and update.

Since the identification of S100 protein as an immunohistochemical marker that could be useful in the diagnosis of melanoma in the early 1980s, a large number of other melanocytic-associated markers that could potentially be used to assist in the differential diagnosis of these tumors have also been investigated. A great variation exists, however, among these markers, not only in their expression in some subtypes of melanoma, particularly desmoplastic melanoma, but also in their specificity because some of them can also be expressed in nonmelanocytic neoplasms, including various types of soft tissue tumors and carcinomas. This article reviews the information that is currently available on the practical value of some of the markers that have more often been recommended for assisting in the diagnosis of melanomas, including those that have only recently become available.

Partial rescue of B cells in microphthalmic osteopetrotic marrow by loss of response to type I IFNs.

The microphthalmic (mi) mouse exhibits deficiencies in the development of osteoclasts, melanocytes, mast cells and marrow B cells. Previously, we demonstrated that the marrow of such mice over-express receptor activator of nuclear factor kappaB (RANK) ligand (RANKL). RANKL has been shown to induce the production of IFN-beta, a type I IFN. Additionally, maturing B cells have been shown to undergo apoptosis in response to type I IFNs including IFN-beta during differentiation. We hypothesized that the loss of B cells in the marrow of mi mice was due to the over-expression of IFN-beta as a result of heightened RANK-RANKL signaling. Creating a mouse with the mi genotype that was non-responsive to IFN-beta (lacking the type I IFNR) allowed us to test this hypothesis. These mice demonstrated an elevated number of marrow B cells and marrow precursor cells compared with mi animals possessing the type I IFNR. Intriguingly, type I IFNR-deficient wild-type animals also demonstrated an increased number of precursor cells in the marrow, but not an expansion of B220-positive pre-B cells, compared with wild type, suggesting that modulation of type I IFN responses directly controls the development of marrow constituents.