The Role of Type I Interferon in Vitiligo Pathogenesis and Melanoma Immunotherapy

Vitiligo is an autoimmune skin disease in which the pigment producing cells of the epidermis, melanocytes, are targeted for destruction by CD8+ T cells specific for melanocyte/melanoma-shared antigens. Previous work has identified IFNg as the central cytokine driving disease pathogenesis in both human patients and in our mouse model of vitiligo. IFNg signaling induces production of the chemokines CXCL9 and CXCL10, which trigger autoreactive T cell migration into the epidermis where effector T cells can target and destroy melanocytes. However, both IFNg and type I IFN signaling through activation of STAT1 proteins can induce transcription of the chemokines CXCL9 and CXCL10. Therefore, it seems reasonable that type I IFN signaling may also contribute to disease pathogenesis. The role of type I IFN in vitiligo is still unclear. Genome wide association studies identified multiple genes within the type I IFN pathway including TICAM1 and IFIH1 as susceptibility loci in vitiligo. One additional study reported increased epidermal staining of CD123, a marker expressed by pDCs, and the type I IFN induced gene MX1 in vitiligo patient skin. However, this study did not show any functional data to support the role of type I IFN signaling in vitiligo pathogenesis. Since the role of type I IFN in vitiligo is ill-defined, we used two different mouse models of vitiligo to functionally determine the role of type I IFN in disease by inducing vitiligo in hosts which lack the type I IFN receptor (IFNaR). In the first model, we induced vitiligo by adoptive transfer of melanocyte-specific CD8 T cells, which are activated in vivo by infection with recombinant vaccinia virus (VACV) expressing their cognate antigen. Vitiligo induction in IFNaR-deficient mice led to the development of severe disease compared to wild type mice. Acceleration and severity of disease was characterized by increased early recruitment of melanocyte-specific CD8 T cells to the skin, increased production of effector cytokines TNFa and IFNg, and reduced PD-1 expression. Increased production of IFNg by CD8 T cells in the skin of IFNaR-deficient mice led to increased expression of the chemokines CXCL9 and CXCL10 driving disease progression. IFNaR-deficient mice also displayed significantly increased VACV titters compared to wild type hosts. This data reveals a role of type I IFN in the clearance of recombinant VACV. This data also suggests that persistent VACV infection and prolonged antigen exposure in IFNaR deficient hosts is likely driving enhanced activation of melanocyte specific CD8 T cells and the subsequent development of severe vitiligo. Since melanocytes and melanoma cells express shared antigens that can be recognized by CD8 T cells, and because the development of vitiligo after melanoma immunotherapy is a positive prognostic factor for patients, we asked whether VACV vaccine therapy in IFNaR deficient mice would enhance the anti-tumor response to melanoma. B16-F10 inoculated wild type and IFNaR-deficient mice received adoptive transfer of melanocyte-specific CD8 T cells in combination with vaccinia virus expressing their cognate antigen to activate the cells in vivo. Treatment of adoptive T cell transfer and infection with VACV in IFNaR-deficient mice revealed significantly reduced tumor burden compared to wild type mice. Improved tumor regression in IFNaR-deficient hosts was characterized by increased infiltrating cytotoxic T lymphocytes and reduced PD-1 expression. These results further demonstrate that in the absence of type I IFN, hosts mount a robust cytotoxic CD8 T cell response against melanocyte/melanoma antigens and this is likely a result of persistent VACV that leads to prolonged CD8 T cell priming. As a result, IFNaR deficient hosts kill tumor cells more efficiently. To determine whether type I IFN regulates disease pathogenesis in the absence of virus infection, we generated a model of vitiligo in which bone marrow derived dendritic cells (BMDCs) pulsed with the cognate antigen were used to prime melanocyte-specific T cells in place of the viral vector. Induction of vitiligo in IFNaR-deficient hosts using BMDCs revealed no significant differences in disease score compared to wild type hosts. This data clearly demonstrates that type I IFN, in contrast to IFNg, is not required during the effector stage of vitiligo pathogenesis in mice. However, since we intentionally activate transferred melanocyte-specific CD8 T cells with VACV or BMDCs expressing their cognate antigen, our mouse models may circumvent the role of type I IFNs in initiating activation of autoreactive cells and driving autoimmunity. Type I IFN is critical for providing innate immune signals that drive the priming of autoreactive T cells through maturation of DCs by inducing antigen presentation, co-stimulatory molecule expression, and migration to the lymph nodes to encounter naïve T cells. Our mouse models of vitiligo may not capture this process. We have addressed this question by using a TLR ligand to activate BMDCs before transfer into hosts. In fact, activation of BMDCs before transfer leads to significantly enhanced vitiligo in mice and this is partially a result of type I IFN signaling on host cells. Thus, we provide evidence that type I IFNs can enhance the activation of melanocyte-specific CD8 T cells and drive autoimmunity. Collectively, our results show that type I IFN signaling has disparate effects on autoreactive T cell priming in a context dependent manner. We reveal that although type I IFN is not required for the effector phase of vitiligo in mice, maturation of DCs and subsequent type I IFN production can enhance the priming of autoreactive T cells and enhance vitiligo severity. Our studies also reveal that type I IFN is required to clear recombinant attenuated VACV infection and vaccine administration in IFNaR deficient hosts led to a robust autoreactive and anti-tumor response. These insights describing the role of type I IFN in autoimmunity and tumor immunology could have important implications for T cell dependent tumor immunotherapy

Mid-infrared spectroscopic assessment of nanotoxicity in gram-negative vs. gram-positive bacteria

Nanoparticles appear to induce toxic effects through a variety of mechanisms including generation of reactive oxygen species (ROS), physical contact with the cell membrane and indirect catalysis due to remnants from manufacture. The development and subsequent increasing usage of nanomaterials has highlighted a growing need to characterize and assess the toxicity of nanoparticles, particularly those that may have detrimental health effects such as carbon-based nanomaterials (CBNs). Due to interactions of nanoparticles with some reagents, many traditional toxicity tests are unsuitable for use with CBNs. Infrared (IR) spectroscopy is a non-destructive, high throughput technique, which is unhindered by such problems. We explored the application of IR spectroscopy to investigate the effects of CBNs on Gram-negative (Pseudomonas fluorescens) and Gram-positive (Mycobacterium vanbaalenii PYR-1) bacteria. Two types of IR spectroscopy were compared: attenuated total reflection Fourier-transform infrared (ATR-FTIR) and synchrotron radiation-based FTIR (SR-FTIR) spectroscopy. This showed that Gram-positive and Gram-negative bacteria exhibit differing alterations when exposed to CBNs. Gram-positive bacteria appear more resistant to these agents and this may be due to the protection afforded by their more sturdy cell wall. Markers of exposure also vary according to Gram status; Amide II was consistently altered in Gram-negative bacteria and carbohydrate altered in Gram-positive bacteria. ATR-FTIR and SR-FTIR spectroscopy could both be applied to extract biochemical alterations induced by each CBN that were consistent across the two bacterial species; these may represent potential biomarkers of nanoparticle-induced alterations. Vibrational spectroscopy approaches may provide a novel means of fingerprinting the effects of CBNs in target cells

Classification of agents using Syrian hamster embryo (SHE) cell transformation assay (CTA) with ATR-FTIR spectroscopy and multivariate analysis

The Syrian hamster embryo (SHE) cell transformation assay (pH 6.7) has a reported sensitivity of 87% and specificity of 83%, and an overall concordance of 85% with in vivo rodent bioassay data. To date, the SHE assay is the only in vitro assay that exhibits multistage carcinogenicity. The assay uses morphological transformation, the first stage towards neoplasm, as an endpoint to predict the carcinogenic potential of a test agent. However, scoring of morphologically transformed SHE cells is subjective. We treated SHE cells grown on low-E reflective slides with 2,6-diaminotoluene, N-nitroso-N-ethylnitroguanidine, N-nitroso-N-methylurea, N-nitroso-N-ethylurea, EDTA, dimethyl sulphoxide (DMSO; vehicle control), methyl methanesulfonate, benzoepyrene, mitomycin C, ethyl methanesulfonate, ampicillin or five different concentrations of benzoapyrene. Macroscopically visible SHE colonies were located on the slides and interrogated using attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy acquiring five spectra per colony. The acquired IR data were analysed using Fisher?s linear discriminant analysis (LDA) followed by principal component analysis (PCA)-LDA cluster vectors to extract major and minor discriminating wavenumbers for each treatment class. Each test agent vs. DMSO and treatment-induced transformed cells vs. corresponding non-transformed were classified by a unique combination of major and minor discriminating wavenumbers. Alterations associated with Amide I, Amide II, lipids and nucleic acids appear to be important in segregation of classes. Our findings suggest that a biophysical approach of ATR-FTIR spectroscopy with multivariate analysis could facilitate a more objective interrogation of SHE cells towards scoring for transformation and ultimately employing the assay for risk assessment of test agents

Retrotransposon Insertion in the T-cell Acute Lymphocytic Leukemia 1 (<em>Tal1</em>) Gene Is Associated with Severe Renal Disease and Patchy Alopecia in Hairpatches (<em>Hpt</em>) Mice

<div><p>“Hairpatches” (<em>Hpt</em>) is a naturally occurring, autosomal semi-dominant mouse mutation. <em>Hpt</em>/<em>Hpt</em> homozygotes die in utero, while <em>Hpt</em>/+ heterozygotes exhibit progressive renal failure accompanied by patchy alopecia. This mutation is a model for the rare human disorder “glomerulonephritis with sparse hair and telangiectases" (OMIM 137940). Fine mapping localized the <em>Hpt</em> locus to a 6.7 Mb region of Chromosome 4 containing 62 known genes. Quantitative real time PCR revealed differential expression for only one gene in the interval, T-cell acute lymphocytic leukemia 1 (<em>Tal1</em>), which was highly upregulated in the kidney and skin of <em>Hpt</em>/+ mice. Southern blot analysis of <em>Hpt</em> mutant DNA indicated a new EcoRI site in the <em>Tal1</em> gene. High throughput sequencing identified an endogenous retroviral class II intracisternal A particle insertion in <em>Tal1</em> intron 4. Our data suggests that the IAP insertion in <em>Tal1</em> underlies the histopathological changes in the kidney by three weeks of age, and that glomerulosclerosis is a consequence of an initial developmental defect, progressing in severity over time. The Hairpatches mouse model allows an investigation into the effects of <em>Tal1,</em> a transcription factor characterized by complex regulation patterns, and its effects on renal disease.</p> </div

Antibody blockade of IL-15 signaling has the potential to durably reverse vitiligo

Vitiligo is an autoimmune disease of the skin mediated by CD8(+) T cells that kill melanocytes and create white spots. Skin lesions in vitiligo frequently return after discontinuing conventional treatments, supporting the hypothesis that autoimmune memory is formed at these locations. We found that lesional T cells in mice and humans with vitiligo display a resident memory (TRM) phenotype, similar to those that provide rapid, localized protection against reinfection from skin and mucosal-tropic viruses. Interleukin-15 (IL-15)-deficient mice reportedly have impaired TRM formation, and IL-15 promotes TRM function ex vivo. We found that both human and mouse TRM express the CD122 subunit of the IL-15 receptor and that keratinocytes up-regulate CD215, the subunit required to display the cytokine on their surface to promote activation of T cells. Targeting IL-15 signaling with an anti-CD122 antibody reverses disease in mice with established vitiligo. Short-term treatment with anti-CD122 inhibits TRM production of interferon-gamma (IFNgamma), and long-term treatment depletes TRM from skin lesions. Short-term treatment with anti-CD122 can provide durable repigmentation when administered either systemically or locally in the skin. On the basis of these data, we propose that targeting CD122 may be a highly effective and even durable treatment strategy for vitiligo and other tissue-specific autoimmune diseases involving TRM

Real-time qPCR analysis of <i>Tal1</i> expression using Taqman probes.

<p>The Taqman-65 and -33 probes span exons 3 and 4, and exons 4 and 5, respectively (A). While the increased expression of <i>Tal1</i> in <i>Hpt</i>/+ mice versus +/+ controls is significant with both probe sets in all organs tested, the more 3′ Taqman-33 shows greater up-regulation than the Taqman-65 probe located more 5′(B).</p

A) Sybr Green qPCR expression analysis of genes in the 6.7 Mb candidate region of Chromosome 4.

<p>Relative gene expression levels of 62 genes from the adult kidneys of <i>Hpt</i>/+ mice (1.5–15 months of age) within the candidate interval are shown (n = 3). Only <i>Tal1</i> showed a 18-fold increase, whereas no greater than 3-fold change was observed among other genes in the interval. The SybrGreen-<i>TAL1</i> Probe is located in the non-coding cDNA region of exon 5 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053426#pone-0053426-g007" target="_blank">Fig.7A</a>). <b>B)</b><b>Tissue-specific expression of </b><b><i>Tal1.</i></b> Relative gene expression levels of <i>Tal1</i> in <i>Hpt</i>/+ versus +/+ animals using Sybr Green qPCR (n = 3). <i>Tal1</i> is overexpressed in the kidney, skin, thymus and brain of <i>Hpt</i>/+ mice, while there is no significant expression difference in liver or spleen. As there were no statistically significant differences in the expression values of 4 day, 14 day, 6 month, or 10–15 month <i>Hpt</i>/+ mice tested, expression values for each genotype were pooled from all age groups.</p

Renal function.

<p>A (male), B (female). Blood urea nitrogen (BUN) levels in individual <i>Hpt</i>/+ and +/+ mice. C (male), D (female). Urine albumin:creatinine ratio (ACR) in individual <i>Hpt</i>/+ and +/+ mice. Renal function progressively declines in the <i>Hpt</i>/+ animals. BUN values are significantly elevated (p<0.05) in <i>Hpt</i>/+ male (A) and female (B) mice compared with sex-matched +/+ controls at all time points (unpaired two-tailed T-test with Welsh’s correction). Urinary ACR is significantly elevated (P<0.05) as early as one month of age in males (C) and by three months of age in females (D) (unpaired two-tailed T-test with Welsh’s correction).</p

Renal histopathology of<b>Hpt</b>/+ and +/+ female mice at 1, 4, and 52 weeks of age.

<p>Representative glomeruli from <i>Hpt/</i>+ and +/+mice, at 1 week, 4 weeks, and 52 weeks of age. There are no marked histopathological changes between <i>Hpt</i>/+ and +/+ glomeruli at 1 week of age. At 4 weeks of age, <i>Hpt</i>/+ glomeruli show enlargement, diffuse mesangial matrix expansion (D) and a few dilated glomerular capillaries (arrowheads). At 12 months of age, <i>Hpt</i>/+ glomeruli show diffuse (D) and nodular (N) mesangial matrix expansion with mesangiolysis (L), capillary aneurysms (arrowheads), and thickening of glomerular (long arrows) and capsular (short arrows) basement membranes. Hematoxylin and Eosin (H&E), Periodic Acid Schiff (PAS), Jones Methenamine Silver (JMS) stains, and type IV collagen immunohistochemistry are shown; x600.</p

High resolution mapping of the <i>Hpt</i> mutation.

<p>Fine mapping of the <i>Hpt</i> mutation by segregation analysis of the HPT/LeJ-<i>Hpt</i>/+ x CAST/EiJ) F1 hybrid x C3HeB/FeJ cross. All of the 182 N2 offspring typed for the Chromosome 4 markers had one allele derived from C3HeB/FeJ (A) and the other allele derived from either C57BL/6J (B) or CAST/EiJ (C). Shown are the AB (gray) and AC (white) genotypes of 25 markers in the six most informative mutant N2 mice (<i>Hpt</i>/+, genotype designation AB) recombinant between <i>D4Mit176</i> and <i>D4Mit202</i>. The markers <i>D4Mit 352</i>, <i>hptssr22, hptssr23,</i> and <i>hptssr25</i> are non-recombinant with the <i>Hpt</i> mutation in all six mice, and the new flanking markers <i>D4Mit 199</i> and <i>hptssr50</i> refine the candidate gene interval to a 6.7 Mb region.</p