present invention concerns the identification of pro-pigmenting and depigmenting
agents.
Human skin and hair color depends on different factors, such as seasons, ethnic
origin, sex and age. It is mainly determined by the concentration and distribution, in
keratinocytes, of the melanin produced by melanocytes. Melanin, a natural pigment known
for its anti-radical and sun radiations absorbing properties, is a skin physiological protecting
agent, which exists in two main forms: eumelanin and pheo-melanin.
Melanocytes are specialized cells which synthesize melanin and distribute it to
keratinocytes through particular organelles, the melanosomes.
Mechanisms leading to skin pigmentation are highly regulated and depend on
multiple hormonal and cellular factors. Melanocytes homeostasis enable giving to the skin a
natural, homogenous, more or less intense, pigmentation, conferring an attractive cosmetic
and esthetic aspect.
In most populations, obtaining a brown coloration of the skin and maintaining a
constant coloration of hair are important goals. Keeping a tanned complexion and naturally
pigmented hair are factors highly involved in obtaining an attractive cosmetic aspect.
There is therefore an important need of agents inducing skin and/or hair pigmentation.
Numerous solutions have been proposed in the field of artificial coloration by using
exogenous colorants, such as DHA, supposed to confer to the skin and/or the hair a color as
closed as possible to what it is naturally. Excellent results have been achieved with the
solutions proposed in the prior art, but the compounds used often display important side
effects or are complex mixtures which do not present any specificity.
Finding alternative substances having an effect on skin and/or hair pigmentation
therefore remains a major aim of search.
Inversely, some events, in particular prolonged and repeated exposures to the sun,
as well as hormone variations, can lead to an excessive and anarchical production of
melanin, generating hyperpigmentation, which generally needs an efficient cosmetic
treatment to be diminished or to disappear.
Some cosmetic treatments exist that accelerate disappearance of these
hyperpigmentations. These include for example use of hydroquinone, retinoids, azelaic acid
or glycolic acid. However, most of these treatments are associated with side effects such as
skin irritation, burns, red spots or bleaching.
3
There is therefore a need for new cosmetic treatments, enabling inducing
depigmentation.
NLRP1 is a member of the NLR family of proteins. These proteins derive their name
from the observation that they uniformly possess a nucleotide-binding fold known as NACHT
domain, variable numbers of Leucine-Rich Repeat (LRR) domains and the founder member
of the family, NOD1. NLRs are important components of the innate immune system,
operating as intracellular sensors for pathogen recognition and “danger” signals generated
during tissue injury and cell stress. The LRR domain is the germ line coded pattern
recognition receptor (PRR) that recognizes PAMPS/MAMPs or Pathogen/Micro-organism
Associated Molecular Patterns. NLRs present intracellular analogs of TLRs (Toll like
receptors) (D’Osualdo & Reed, 2011).
Humans have 22 genes encoding proteins that possess the combination of NACHT
and LRR (Martinon and Tschopp, 2004; Ting et al, 2006). Among the 22 human NLR genes,
14 encode proteins that combine the NACHT and LRRs with a protein interation domain
called the PYRIN domain (PYD), while 7 encode proteins that contain a Caspase Associated
Recruitment Domain (CARD). Human NLRP1 has a unique combination of domains
compared to other members of the NLR family. The NLRP1 protein contains both PYRIN and
CARD domains located on opposite ends of the protein, as well as an internal pair ZU5 and
UPA domains that confer intra-proteolytic activity.
NLRP1 gets activated (through LRRs) by appropriate PAMPs (Pathogen Activated
Molecular Patterns) and lead to formation of a multiprotein complex “inflammasome” and
caspase activation. The caspase family members activate IL-1 beta, IL-18 and cause
apoptosis. Bacterial components recognized by NLRP1 include MDP (muramyl dipeptide)
(Faustin B et al, 2007), a component of peptidoglycans produced by Gram-positive and
Gram-negative bacteria. The murine NLRP1 isoform was reported to be crucial for anthrax
lethal toxin-mediated macrophage killing (Boyden ED, 2006), whereas human ortholog is
involved in tissue injury in the context of UV-irradiated keratinocytes (Faustin B et al, 2008).
Nevertheless, to the inventors’ knowledge, no link between NLRP1 expression and
pigmentation has been established so far.
The present invention arises from the unexpected finding by the inventors that, in
response to bacterial PAMPs, NLRP1 forms inflammasome that creates a proinflammatory
environment in skin leading to an activation of keratinocytes, Langerhans cells and dendritic
cells. The microenvironment then leads to an activation of melanocytes, increased melanin
4
formation and/or an abnormal uptake and persistence of melanin in the form of
melanophages, being responsible for hyperpigmentations.
Accordingly, a modulation of NLRP1 expression, in particular a down-modulation of
NLRP1 expression, should restore equilibrium to optimize melanogenesis and clearance of
excessive melanin that may persist as a result of its abnormal uptake by other cell types.
Therefore, identifying active agents that modulate the expression of the NLRP1 gene
is useful to identify new active agents that modulate skin pigmentation.
The present invention thus concerns a method for screening a pigmentation
modulating active agent comprising the steps of:
a) culturing mammalian cells,
b) adding the candidate substance to the mammalian cell culture,
c) after incubation, quantifying the expression level of the NLRP1 gene in the
mammalian cells,
d) comparing the expression level of the NLRP1 gene quantified in step c) with that
quantified starting from a control culture of mammalian cells, obtained under the same
conditions, but in the absence of candidate substance,
e) selecting the candidate substances for which the expression level of the NLRP1
gene is modulated compared to the expression level quantified in the control culture.
It also relates to a method for screening a pro-pigmenting active agent comprising the
steps of:
a) culturing mammalian cells,
b) adding the candidate substance to the mammalian cell culture,
c) after incubation, quantifying the expression level of the NLRP1 gene in the
mammalian cells,
d) comparing the expression level of the NLRP1 gene quantified in step c) with that
quantified starting from a control culture of mammalian cells, obtained under the same
conditions, but in the absence of candidate substance,
e) selecting the candidate substances for which the expression level of the NLRP1
gene is increased compared to the expression level quantified in the control culture.
Another object of the invention concerns a method for screening a depigmenting
active agent comprising the steps of:
a) culturing mammalian cells,
b) adding the candidate substance to the mammalian cell culture,
c) after incubation, quantifying the expression level of the NLRP1 gene in the
mammalian cells,
5
d) comparing the expression level of the NLRP1 gene quantified in step c) with that
quantified starting from a control culture of mammalian cells, obtained under the same
conditions, but in the absence of candidate substance,
e) selecting the candidate substances for which the expression level of the NLRP1
gene is decreased compared to the expression level quantified in the control culture.
The invention also relates to the use of the NLRP1 gene for identifying pigmentation
modulating active agents, in particular for identifying pro-pigmenting active agents or
depigmenting active agents.
In the context of the invention, the term “NLRP1 gene”, also called “CARD7”,
“CLR17.1”, “DEFCAP”, “DEFCAP-L/S”, “NAC”, “NALP1”, “PP1044”, “SLEV1” or “VAMAS1”
refers to the NACHT, LRR and PYD domains-containing protein 1. A cDNA of this gene is
available in the GenBank database under accession number BC051787.1 and is shown as
SEQ ID NO: 1. An amino acid sequence of the protein encoded by this gene is available in
the GenBank database under accession number AAH51787.1 and is shown as SEQ ID NO:
2.
The sequences disclosed by reference to the GenBank database are those available
on April 17, 2012.
The above cDNA sequence is given as a reference, without any limitative feature. In
the context of the present invention, the term “expression level of a gene” denotes the
expression level of the gene having, as a coding sequence, the specified reference cDNA,
but also any variant or polymorphic form of this human gene and which could have a coding
sequence differing from this cDNA by substitution, addition, deletion of one or more
nucleotide(s). In particular, the variants or polymorphic forms of a given gene will have a
coding sequence displaying at least 90%, preferably 95%, more preferably 97% sequence
identity with the reference coding sequence of this gene. In particular, the variants or
polymorphic forms of a gene display the same biological function as the gene having as a
coding sequence the specified reference cDNA.
In the context of the invention, the percentage of identity between two nucleotide
sequences may be determined by global alignment of the two sequences using the
Needleman-Wunsch algorithm, for example with the Needle software using the DNAfull
matrix and using 10 as gap-open parameter, and 0.5 as gap-extend parameter.
“Express” or “expression” means enabling the information contained in a gene or in a
DNA sequence to manifest, for example by producing a protein by activation of cell functions
involved in transcription and translation of the corresponding genetic or DNA sequence.
6
The expression level of a gene may be measured:
- by detecting the mRNA level of said gene by suitable technique of molecular
biology, such as the one described notably in Sambrook, Fritsch & Maniatis, Molecular
Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, New York (‘Sambrook et al., 1989”) or in DNA Cloning: A Practical
Approach, Volumes I and II (D.N. Glover ed. 1985) F.M. Ausubel et al. (eds.), Current
Protocols in Molecular Biology, John Wiley & Sons, Inc (1994); or
- by detecting the level of the protein encoded by said gene by any suitable
biochemical or immunological technique.
For detecting the NLRP1mRNA level, cells may be treated to extract cellular RNAs,
and NLRP1-specific mRNAs are then detected and quantified for example by semiquantitative
RT-PCR, with labeling of the amplified products with ethidium bromide and
separation by electrophoresis, or by real-time/quantitative RT-PCR, using primers and
optionally one or more probe(s) specific of the mRNA to be detected.
For detecting the level of the protein encoded by the NLRP1 gene, cells may be
treated to extract proteins and the NLRP1 proteins can then be detected for example by
immunoassay, in particular by ELISA or RIA, or by Western Blot, or any suitable technique
known from the skilled person, using NLRP1-specific antibodies.
Primers, probes or antibodies constitute detection means of the level of expression of
the NLRP1 gene.
Primers are oligonucleotides for amplifying a target sequence, in the present case
NLRP1 mRNA, by extension of the oligonucleotide after hybridization to the target sequence.
Probes are oligonucleotides, which are generally labeled with a detectable moiety, for
detecting an amplified target sequence by hybridization to the target sequence.
Primers and probes have the capacity to hybridize to the target sequence in suitable
conditions of temperature and ionic strength (stringency conditions). High stringency
conditions correspond for example to the use of an hybridization solution of 50% formamide,
5X or 6X SSC, where SSC is a solution of 0.15 M NaCl, 0.015 M Na-citrate, and are usable
for primers or probes having the highest melting temperatures (Tm). Preferably, primers and
probes have a sequence complementary to the target mRNA to be detected, even if,
according to the stringency conditions used, some mismatches between bases are possible.
The term “antibody” denotes conventional, monoclonal or polyclonal antibodies, or
fragments thereof, as well as single domain antibodies or fragments thereof, in particular a
variable heavy chain of single domain antibody. For carrying out the methods of the invention,
one or more antibody(ies) directed against the protein encoded by the NLRP1 gene can be
used.
7
Advantageously, the probes or antibodies can be used in a form bound to a solid
support, preferably in a format compatible with a high yield analysis such as a microarray or
a microtitration plaque.
Preferably, the mammalian cell culture is from a skin sample. Still preferably, the
mammalian cell culture is a culture of keratinocytes, melanocytes, fibroblasts, Langerhans
cells and/or skin dendritic cells.
A “candidate substance” may be any molecule or agent, for example organic, mineral
or particulate, as well as any microorganism.
Probiotics are defined as live microorganisms which when administered in adequate
amounts confer a health benefit to the consumer. The most common formulation of probiotics
is a fresh fermentation product or dried bacteria supplements and their consumption has
been associated with a variety of health benefits for the consumer. The genomic era has
provided novel opportunities for the discovery and characterization of bacterial probiotic
effector molecules that elicit specific responses in the intestinal system. The cytoplasmic
membrane of Gram positive bacteria, which constitute the vast majority of commercially
applied probiotics, is covered by a thick cell wall consisting of multiple layers of peptidogycan,
capsular polysaccharide, lipoprotein and teichoic acids that contain MAMPs (Microorganism
Associated Molecular Patterns).
NLRP1 can detect muramyl dipeptide that is present in peptidoglycan of most Gram
positive and Gram negative bacteria, including probiotic strains such as Escherichia coli str.
Nissle 1917 or Lactobacillus plantarum (Wells JM et al, 2011). These studies show an
extremely beneficial role of signaling through NLR to maintain homeostasis and recognition
of microorganisms by intestinal epithelial cells.
Because of this interaction with NLRP1, probiotics can include microorganisms of
interest able to modulate NLRP1 expression in skin cells. Accordingly, in the methods of
screening of the invention, the active agent may be a probiotic microorganism.
The present invention will be further illustrated by the following examples.
Examples
Example 1
This example demonstrates the association between an irregular expression of the
NLPR1 gene in skin and the presence of pigmentation disorders.
8
Skin biopsy samples from individuals suffering from inflammatory pigmentation
disorders, including hyper and hypopigmented samples, are studied and compared with skin
biopsy samples from healthy individuals.
The level of expression of the NLRP1 gene is quantified in each skin sample and
compared with a control sample.
Example 2
This example demonstrates that active agents that modulate the level of expression
of the NLRP1 gene are able to modulate skin pigmentation.
Dead and alive probiotics are applied to skin samples. The level of expression of the
NLRP1 gene is then quantified and the level of pigmentation of the skin is observed.
The same experiments are performed on different cell types, including keratinocytes,
melanocytes, fibroblasts, Langerhans cells and skin dendritic cells.

I/We claim:
1. A method for screening a pigmentation modulating active agent comprising the steps of:
a) culturing mammalian cells,
b) adding the candidate substance to the mammalian cell culture,
c) after incubation, quantifying the expression level of the NLRP1 gene in the
mammalian cells,
d) comparing the expression level of the NLRP1 gene quantified in step c) with that
quantified starting from a control culture of mammalian cells, obtained under the same
conditions, but in the absence of candidate substance,
e) selecting the candidate substances for which the expression level of the NLRP1
gene is modulated compared to the expression level quantified in the control culture.
2. A method for screening a pro-pigmenting active agent comprising the steps of:
a) culturing mammalian cells,
b) adding the candidate substance to the mammalian cell culture,
c) after incubation, quantifying the expression level of the NLRP1 gene in the
mammalian cells,
d) comparing the expression level of the NLRP1 gene quantified in step c) with that
quantified starting from a control culture of mammalian cells, obtained under the same
conditions, but in the absence of candidate substance,
e) selecting the candidate substances for which the expression level of the NLRP1
gene is increased compared to the expression level quantified in the control culture.
3. A method for screening a depigmenting active agent comprising the steps of:
a) culturing mammalian cells,
b) adding the candidate substance to the mammalian cell culture,
c) after incubation, quantifying the expression level of the NLRP1 gene in the
mammalian cells,
d) comparing the expression level of the NLRP1 gene quantified in step c) with that
quantified starting from a control culture of mammalian cells, obtained under the same
conditions, but in the absence of candidate substance,
e) selecting the candidate substances for which the expression level of the NLRP1
gene is decreased compared to the expression level quantified in the control culture.
10
4. The method according to any one of claims 1 to 3, wherein said active agent is a probiotic
microorganism.
5. Use of the NLRP1 gene for identifying pigmentation modulating active agents.
6. The use according to claim 5, for identifying pro-pigmenting active agents.
7. The use according to claim 5, for identifying depigmenting active agents.