Genetic variations in nalp1 mRNA expressions in human vitiligo.

INTRODUCTION: Vitiligo is an acquired autoimmune disease of unknown etiology showing depigmentation of the skin due to the absence of melanocytes. Familial vitiligo suggests a genetic origin to this disease. Chromosome 17 was recently demonstrated to harbor the gene coding for NALP1. PATIENTS AND METHODS: A total of 18 patients of vitiligo were selected on the basis of clinical history. Group 1 (N=8) showing segmental or localized vitiligo with one or two macules on the body. Group 2 (N=10) with generalized or whole body vitiligo. A control group of 10 healthy individuals were selected from our laboratory persons with no history or any infections or skin disease. NALP1 gene expression was studied using RT-PCR assay and the bands quantitated as intensity using volume as measurement and comparison of results was done using SPSS 16 version for statistical analysis. NALP1 gene expression was observed in vitiligo patients with different intensities.
RESULTS: Greater reduction in the intensity was seen in Group I, which was inversely proportional to the volume of the band. The intensity of the NALP1 and the GAPDH gene expression was more in Group 2 patients than that shown by Group 1.
CONCLUSION: This study shows expression of NALP1 gene in patients as well as normals. NALP1 is widely expressed at low levels but is expressed at high levels in immune cells, particularly T cells and Langerhans cells, in which different patterns are seen that are consistent with the particular involvement of NALP1 in skin autoimmunity.

Introduction

Vitiligo has been characterized as an acquired depigmentation of the skin due to the absence of melanocytes. Although the exact cause of the disease or its etiology still remains unknown, several lines of evidence have shown it to be an autoimmune disease.[1] Antibodies to these melanocytes have been detected as well as infiltrating T cells have been observed.[2] This suggests that both humoral and the cellular response may be involved. There is also familial vitiligo observed suggesting a genetic origin to this disease. One third of these cases are associated with one of the autoimmune diseases.[3] The risk of autoimmune and autoinflammatory diseases is thoughtto depend on interactions between environmental factors andspecific variants of specific genes, some of which may confera risk that an individual disease will develop, and others arisk that several different diseases will develop. The genetic aspects in vitiligo research have definitely an intrinsic defect.

The locus for familial vitiligo on chromosome 17 was recently demonstrated to harbor the gene coding for NALP1.[4] NALP1(NACHT-LRR-PYD- containing protein- 1), together with the related protein NALP2, has been identified by Martinon et al.[5] in a databank search for proteins containing a pyrin domain. The protein contains a NACHT domain, an LRR domain (leucine-rich repeat), and a pyrin domain. Proteins with a NACHT domain or an LRR domain are known to be involved in apoptosis or inflammation.[6] NALP1 protein has been shown to be a component of a large, signal-induced multiprotein complex that has been termed as inflammasome.[7] Studies have shown that NALP1 as part of the inflammasome is a direct sensor of bacterial components in host defense against pathogens.

This gene encodes a member of the Ced 4 family of apoptosis proteins. Ced family members contain a caspase recruitment domain (CARD) and are known to be the key mediators of programmed cell death. The encoded protein contains a distinct N-terminal pyrin like motif, which is possibly involved in protein-protein interactions. This protein interacts strongly with caspase 2 and weakly with caspase 9 as well as with Apaf-1. Overexpression of this gene was demonstrated to induce apoptosis in cells. Multiple alternatively spliced transcript variants encoding distinct isoforms have been found for NALP1, but the biological validity of some variants has not been determined.[8]

NALP1 belongs to a family of cytoplasmic proteins that have been implicated in cell responses to apoptotic and inflammatory stimuli.[910] Transient over expression of this gene in cultured cells was sufficient to induce apoptosis.[11] NALP1 mRNA and protein expression levels in myeloid leukemia cells are increased following CREB (cAMP-response element binding protein) activation, suggesting that NALP1 may contribute to modulate the response of these cells to pro-inflammatory stimuli.[12] Multiple alternatively spliced transcript variants encoding distinct isoforms have been found for NALP1.[13] Studies conducted have shown that NALP1 is highly expressed in blood mononuclear cells. NALP1 is mainly expressed in T lymphocytes, granulocytes and monocytes.

Patients and Methods

A total of 18 patients of vitiligo were selected on the basis of clinical history by a well knowledgeable dermatologist from the OPD clinic of our hospital. The clinical features, their family history and other triggering factors were noted in the immunological proforma. These were further divided into two groups as Group 1 (N=8) showing segmental or localized vitiligo with one or two macules on the body and Group 2 (N=10) with generalized or whole body vitiligo.

A control group of 10 individuals were selected from our laboratory personnel with no history of any infections or skin disease.

Blood for extraction of RNA was collected in clean sterile DEPC treated EDTA tubes. A total of 10 ml of blood was taken after approval by the institute's ethical committee and after the sanction of the project by the Scientific Advisory committee. Informed consent was taken from the patients prior to collection of blood samples.

The study of gene expression is divided into three steps

Extraction of RNA from the blood cells.

Conversion of RNA into cDNA by the reverse transcriptase enzyme and amplification of the product using specific primers.

Analysis of the amplified product using agarose gel electrophoresis.

Cell isolation

Peripheral blood mononuclear cells (PBMNC) were separated from heparinized blood using density gradient separation by overlaying 10 ml of diluted blood in normal physiological saline in equal proportions on histopaque (Sigma density of 1.077) in conical tubes and centrifuging the same at 400 g (1520rpm) for 20 min. Cells separated were then suspended in 11 × 106cells per tube.

Isolation of RNA from PBMNC

11 × 106cells were then homogenized in a homogenizer and to this 1 ml TRI Reagent (Ambion US) was added. This mixture was then incubated for 5 min at room temperature. The cells were then centrifuged at 12,000 rpm for 10 min at 4°C and supernatant was transferred to afresh tube. To this, 100 μl BCP per 1 ml of TRI Reagent solution was added, mixed well, and incubated at room temperature for 15 min. Centrifuged at 12,000 x g for 10-15 min at 4°C and then the aqueous phase was transferred to a fresh tube. 500 μl of isopropanol per 1 ml of TRI Reagent solution was then added and vortexed for 5–10secs and incubated at room temperature for 5-10 min. It was centrifuged at 12,000 x g for 8 min at 4-25°C, and supernatant was discarded.

To this, 1 ml of 75% ethanol per 1 ml of TRI reagent solution was added, centrifuged at 7,500 x g for 5 min, ethanol was then removed and the RNA pellet was air dried. RNA was dissolved in 25 μl DEPC (treated water).1 μl of DNAase I (0.3 μg/μl) was added to the RNA sample. Then it was incubated for 30 min at 37°C. 5 μl of 30 mM EDTA was added and boiled at 75°C for 5 min to inactivate DNAase I and stored at -80°C for longer preservation.

Semiquantitative RT-PCR

The template RNA, primer solutions [Table 1], dNTP Mix, 5x QIAGEN onestep RT-PCR buffer were thawed and all these with RNase-free water were then placed on ice. The solutions needed to be completely dissolved before use to avoid localized differences in salt concentration.

Table 1

Sequence of the primers used in the assay

A master mix was prepared according to Tables 2–3. The master mix typically contains all the components required for RT-PCR except the template RNA. A volume of master mix 10% greater than that required for the total number of reactions to be performed was prepared.

Table 2

Reaction components for one-step RT-PCR of NALP 1

Table 3

Reaction components for one-step RT-PCR of GAPDH

The master mix was mixed thoroughly and dispensed appropriately in 50 μl volumes into PCR tubes. Gently mixed, for example, by pipetting the master mix up and down a few times. Template RNA (2 μg/reaction) was added to the individual PCR tubes. When using a thermal cycler with a heated lid, mineral oil was not used. Otherwise, it is overlayed with approximately 50 μl mineral oil.

The program includes steps for both reverse transcription and PCR. The PCR amplification was started with an initial heating step at 95°C for 15 min to activate HotStarTaq DNA polymerase. For maximum yield and specificity, temperatures and cycling times can be further optimized for each new target and primer pair. However, the protocol gave satisfactory results in most cases. The RT-PCR program was started while PCR tubes were still on ice. Once the thermal cycler reaches 50°C, then the PCR tubes are placed in the thermal cycler [Table 4]. After amplification, samples can be stored overnight at 2-8°C, or at -20°C for longer storage.

Table 4

The thermal cycler was programed as in following table for denaturation, anealing and extension for amplified product

The integrity of purified RNA and RNA from intermediate stage of purification can only be confirmed by agarose gel electrophoresis on denaturing. The ethidium bromide-staining pattern of intact total RNA will have clearly defined 28S and 18S rRNA bands. The ratio of 28S:18S band intensities should be close to 2. Partially degraded RNA appears as smeared rRNA bands with increasing lower molecular weight smear.

RNA yield and purity is spectrophotometrically determined by measuring optical density under ultraviolet (UV) light absorbance (A) of a sample at wavelengths of 230, 260 and 280 nm. Ratios of the absorbances of these wavelengths provide concentration of protein. Significant protein contamination will result in a lower 260 nm/280 nm absorbance ratio (Pure nucleic acid will have 260 nm/280 nm ratio greater than 1.8) showing contamination in RNA samples. cDNA products were visualized by gel electrophoresis using 1% agarose in 10X MOPS buffer (0.2M MOPS+20mM sodium acetate+10mM EDTA+ formaldehyde in DEPC). The formaldehyde loading dye contains 50% glycerol + 10mM sodium acetate + 10 ml EDTA (pH 8.0) + 0.25% xylene cyanol FF in DEPC water and adjusting to a volume of 10 ml. The RNA samples were loaded using RNA sample buffer containing 10 X MOPS + 37% formaldehyde + deionized formaldehyde + 10X formaldehyde loading dye +EtBr. The bands were then observed under UV light after completion of the run as indicated by the dye front.

The bands then analyzed using UVIBAND V 0.99 Software. The intensity of the visual band as seen under UV gel documentation is measured in terms of percent volume where volume corresponds to the sum of intensities included inside the defined area.

Statistical analysis

The data was analyzed using statistical analysis package SPSS version 16. Student's t test was used for all the data.

Results

Table 5 gives the characteristics of the study group comprising 18 patients and 10 healthy controls. These were divided into two groups with segmental and generalized vitiligo and comparative age groups.

Table 5

Characteristics of the study group

Table 6 gives the analysis of NALP1 gene expression by RT-PCR using GAPDH as a reference gene. The intensity of the band is observed and compared with respect to normal control bands. We have observed that this NALP1 gene expression is present in vitilgo patients but the intensity of the expression is different in different patients. Though Group I patients showed greater reduction in the intensity which is inversely proportional to the volume of the band, we observed that the band thickness was smaller than that shown by Group 2 patients. The volume or the intensity of the NALP1 and the GAPDH gene expression was more in Group 2 patients than that shown by Group 1 patients. Thus, this study shows that the expression of NALP1 gene is seen in patients as well as in normals. This is represented in Figure 1.

Table 6

Analysis of gene expression by RT-PCR using relative percent quantification

Figure 1

a) mRNA expression of NALP 1 gene using Reverse transcriptase Polymerised Chain reaction-NALP1 and b) mRNA expression of NALP 1 gene using reverse transcriptase polymerised chain reaction--GAPDH

Discussion

In our studies, we observed reduction in the NALP1 gene expression compared to normals. We have not quantitated the percentage of T cell populations but studies by Gunduz et al[14] have shown that in peripheral blood cells, the highest levels of NALP1 were observed in CD3+ (T-lymphocytes), CD15+ (granulocytes) and CD14+ (monocytes) and langerhans cell populations that are involved in skin autoimmunity. Immunological alterations have been implicated in the etiopathogenesis of vitiligo. T helper/inducer cells, CD4/CD8 ratio and activated (HLA-DR+) T cells are decreased in vitiligo patients, suggesting a role for changes in cellular immunity. In normal conditions, it is seen that NALP1 mRNA expression is maximum and it decreases in disease conditions. Whether this down regulation in the disease condition is the cause of the immune response needs to be determined. But since the vitiligo patients showed decreased NALP 1 gene mRNA expression, there is possibility that NALP1 gene, which is on chromosome 17 located at 17p13, is on a cascade that regulates inflammation and cell death, including myeloid and lymphoid cells, which are white cells that are part of the immune response. The locus for familial vitiligo is found on chromosome 17 and was found to harbor the gene coding for NALP 1. It is seen that specific mutants of NALP1 are possibly associated with vitiligo alone. This may suggest that mutations in NALP1 could lead to deregulation of IL-1 β. This may favor the priming of T cells that ultimately attack the melanocytes.

NALP1 is expressed at high levels in T cells and Langerhans cells, white cells that are involved in skin autoimmunity but in our study we found less expression of NALP1 and hence there may be less T cells and ultimately low immune response and possibility of mutant NALP1 that is associated with vitiligo.

NALP1 encodes NACHT leucine-rich-repeat protein 1, a protein component of the cytoplasmic inflammasome. It is expressed ubiquitously at low levels while higher levels of expression can be seen in cells of the immune system.

Inflammasomes are the cytoplasmic multiprotein complexes that mediate the maturation of the proinflammatory cytokines interleukin -1ß, IL-18 and possibly IL-13 by controlling the activation of the inflammatory caspases-1 and 5.[15] Assembly of inflammasomes depends on NOD-like receptor (NLR) family members such as NALPs, NAIP and IPAF. Various microbial and endogenous stimuli activate different types of inflammasomes.

NALP1, also known as CARD7, DEFCAP, and NAC, is thought to mediate the activation of the innate immune system in response to the so-called pathogen-associated molecular patterns such as bacterial peptides. NALP1 is widely expressed at low levels but is expressed at a high level in immune cells, particularly T cells and Langerhans’ cells, patterns that are consistent with the particular involvement of NALP1 in skin autoimmunity. NALP1 recruits the adapter protein ASC, caspase 1, and caspase 5 to a complex termed as the NALP1 inflammasome, which activates the proinflammatory cytokine interleukin-1β. Serum interleukin-1β levels are elevated in patients with generalized vitiligo, suggesting the involvement of this pathway in the pathogenesis of disease. NALP1 also appears to play a role in cellular apoptosis, its overexpression stimulating caspase-mediated apoptosis in a variety of cell types.[16]

Caspases are cysteinyl-aspartate-specific proteinases known for their role in apoptosis (cell death or apoptotic caspases) and proinflammatory cytokine maturation (inflammatory caspases). They are involved in initiation, execution and regulatory phases of cellular apoptosis. The inflammatory caspases were among the first to be discovered, but only recently the mechanisms leading to their activation and inhibition have begun to be elucidated. We could not study the caspase levels in our patients as we looked into the cellular and immune mechanisms along with gene expression studies.

Cellular apoptosis, a basic form of programmed cell death, is critical for all physiological processes including maintenance of homeostatis in multicellular organisms. Apoptosis plays a causative or a contributing role in various human conditions. Key players in the regulation of cellular apoptosis include death receptors and their ligands, transcriptional regulators, Bcl-2 proteins, caspases and endogenous caspase inhibitors.[17]

If the association between NALP1 and autoimmune and autoinflammatory diseases is confirmed, and if NALP1 variants are found to result in the activation of interleukin-1β, then inhibitors of interleukin-1β and caspase might be effective in the treatment or prevention of NALP1-associated autoimmune and autoinflammatory diseases.[18]

If the action of inflammasome is considered truly then it would help to trigger or enhance the autoimmune part of the disease, which may involve initial contacts between melanocytes or melanocyte fragments and NALP1 positive langerhans rather than being an initial factor in which a systematically dysregulated innate immunity would get rid of epidermal melanocytes.[19]

There is accumulating evidence that T-cell-mediated dominant control of self-reactive T-cells contributes to the maintenance of immunologic self-tolerance and its alteration can cause autoimmune disease. Efforts to delineate such a regulatory T-cell population have revealed that CD25+ cells in the CD4+ population in normal naive animals bear the ability to prevent autoimmune disease in vivo and, upon antigenic stimulation, suppress the activation/proliferation of other T cells in vitro.

The CD25+, CD4+ regulatory T cells, which are naturally anergic and suppressive, appear to be produced by the normal thymus as a functionally distinct subpopulation of T cells. They play critical roles not only in preventing autoimmunity but also in controlling tumor immunity and transplantation tolerance.