Clinical Trial Details
— Status: Not yet recruiting
Administrative data
| NCT number |
NCT06213688 |
| Other study ID # |
RCAPHM23_0328 |
| Secondary ID |
|
| Status |
Not yet recruiting |
| Phase |
N/A
|
| First received |
|
| Last updated |
|
| Start date |
March 2024 |
| Est. completion date |
March 2025 |
Study information
| Verified date |
January 2024 |
| Source |
Assistance Publique Hopitaux De Marseille |
| Contact |
Anaïs Maugard |
| Phone |
0491435186 |
| Email |
anais.maugard[@]ap-hm.fr |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Interventional
|
Clinical Trial Summary
Introduction Pollution is a significant public health issue. Research has shown a positive
correlation between air pollution and chronic inflammatory dermatoses, including psoriasis
and eczema. The incidence of these diseases has been steadily increasing since the beginning
of industrialization. The mechanism behind this association involves the activation of the
aromatic hydrocarbon receptor (AhR). The aryl hydrocarbon receptor (AhR) plays a role in
regulating the balance between T helper 17 (TH17) and regulatory T cells (TREG), as well as
in generating oxidative stress and producing pro-inflammatory cytokines. Studies in cultured
keratinocytes have shown that a non-competitive antagonist that modulates AhR activity can
reduce cutaneous inflammatory processes induced by polycyclic aromatic hydrocarbons (PAHs).
Objectives:
It has been suggested that activation of the AhR by PAHs and dioxins may be related to the
pathogenesis of atopic dermatitis and psoriasis. The main objective is to compare the levels
of AhR pathway activation markers between cases and controls. Secondary objectives include
correlating environmental exposure to AhR ligands with disease severity in patients. Finally,
we will compare the expression of inflammatory and AhR activation markers in cultured
peripheral blood mononuclear cells (PBMCs) after in vitro stimulation with benzo(a)pyrene.
Material and methods:
The study will measure exposure to pollutants by determining blood dioxins and urinary PAH
metabolites. Pro-inflammatory cytokines IL1β, TNFα, IL23, IL17 and IFNγ and Malondialdehyde
(MDA) serum concentrations will be measured by ELISA. The TREG and TH17 lymphocyte population
ratio will be evaluated by flow cytometry on isolated PBMCs. Additionally, the level of
expression of CYP 1A1 and 1B1, pollutant-metabolizing enzymes induced by AhR, will be
assessed on isolated PBMCs. The expression levels of the AhR and NfkB active fractions will
be determined by immunofluorescence. Subsequently, levels of AhR activation markers will be
compared after stimulation of PBMCs with benzo(a)pyrene.
Description:
I. Introduction Pollution is a significant component of the exposome, with established links
to human health. In 2019, pollution was estimated to be responsible for 9 million premature
deaths, making it the fourth leading risk factor for mortality worldwide. Over the last 20
years, pollution-related deaths have increased by 66%. This is due to industrialization,
uncontrolled urbanization, population growth, burning fossil fuels, and a lack of restrictive
policies in some regions.
The cutaneous layer is composed of a complex network of cells that form a mechanical and
biological barrier essential for maintaining the body's integrity and homeostasis. Pollution
can directly impair this function by crossing the barrier or entering the systemic
circulation through inhalation or ingestion.
Atopic dermatitis (AD) and psoriasis are chronic inflammatory skin conditions with
multifactorial origins, including genetic predisposition, immune dysregulation, and
environmental factors. Epidemiological studies have shown a positive correlation between air
pollution and the development of these diseases.
Given their high prevalence and the impact of symptoms on patients' quality of life, a better
understanding of the exposome in these diseases represents a major challenge.
Experimental studies have demonstrated that the aromatic hydrocarbon receptor (AhR) pathway
plays a significant role in the impact of pollutants on psoriasis and AD. AhR is a
transcription factor involved in the response to environmental pollutants, which remains
inactive in a cytoplasmic complex with chaperone proteins. Upon binding with its ligand, AhR
translocates to the nucleus and dimerizes with its nuclear partner, Arnt. The activation of
the complex leads to the expression of numerous genes that contain a consensus sequence known
as the xenobiotic response element (XRE) in their promoters.
The activator ligands of the AhR comprise xenobiotics found in the environment, primarily
polycyclic aromatic hydrocarbons (PAHs), dioxins, and so-called 'dioxin-like' compounds 12.
This signalling pathway is considered an 'adaptive' response to detoxify the body from
various exogenous compounds. However, prolonged activation of the AhR by such agents may lead
to deregulation of the inflammatory process, contributing to the development of psoriasis and
AD.
The aryl hydrocarbon receptor (AhR) regulates the transcription of cytochromes 1A1 (CYP1A1)
and 1B1 (CYP1B1), which are members of a multigene family of enzymes involved in xenobiotic
metabolism. These enzymes catalyse the bioactivation of HAPs into electrophilic metabolites
leading to activation of NFκB, a key player in the inflammatory response. During their
catalytic cycle, these CYP1 enzymes produce reactive oxygen species (ROS), which generate
oxidative stress. In addition, the AhR is responsible for regulating the expression of
pro-inflammatory cytokines, including IL1β, TNFα, IL23, IL17 and IFNγ. It can directly affect
the balance between Th17 lymphocytes and regulatory T cells (Tregs), potentially contributing
to cutaneous inflammatory processes.
However, further research is needed to better define the role of AhR in the pathogenesis of
chronic inflammatory dermatoses. This will help in the management and prevention of these
diseases, especially when environmental risk factors are associated with predisposition.
II. Objective:
It is hypothesized that the activation of AhR with exogenous ligands may play a role in the
development of atopic dermatitis and psoriasis.
The main objective is to compare the levels of AhR pathway activation markers between cases
and controls.
Secondary objectives include correlating environmental exposure to AhR ligands with disease
severity in patients.
Finally, we will compare the expression of inflammatory and AhR activation markers in
cultured peripheral blood mononuclear cells (PBMCs) after in vitro stimulation with
benzo(a)pyrene.
III. Material and methods:
We will be carrying out a case-control study:
- Case: Patients with chronic inflammatory dermatosis corresponding to psoriasis or atopic
dermatitis.
- Controls: People consulting the dermatology department who do not have these
pathologies.
All participants, including patients and controls, will undergo consultation/hospitalisation
and biological testing as part of their usual medical care. Procedures performed as part of
the protocol will not interfere with their care.
A sample of 10 ml of blood will be taken for toxicological analysis. 20 ml of blood will be
taken for evaluation of inflammatory and AhR activation markers.
A. Pollutant measurement The blood sample will be centrifuged, the serum will then be
collected and stored at -20 C until the blood dioxins are analysed.
2,3,7,8-tétrachlorodibenzo-p-dioxin (TCDD) and polychlorobiphényle 118 (PCB118) will be
measured in blood using gas chromatography coupled with high-resolution mass spectrometry
(GC-HR-MS).
For biological monitoring of exposure to PAHs,1-Hydroxypyrene (1-OHP) will be measured in
urine using high-performance liquid chromatography-mass spectrometry (LC-MS-MS)
B. Evaluation of inflammatory and AhR activation markers
- PBMCs will be isolated from a 20mL blood sample using a standard Ficoll® gradient
protocol. The isolated cells and sera will be aliquoted and frozen for subsequent
experiments on the AhR signalling pathway.
- Enzyme-Linked Immunosorbent Assay (ELISA) will be used to measure pro-inflammatory
cytokines IL1β, TNFα, IL23, IL17 and IFNγ.
- Malondialdehyde (MDA) concentration will be determined using the ELISA method as a
biomarker for lipid peroxidation, which indicates the level of oxidative stress caused
by reactive oxygen species (ROS).
- Fluorochrome-conjugated monoclonal antibodies (CD4, CD25, CD127, FOXP3, Il17) will be
used in fluorescence activated cell sorter (FACS) to assess the numbers and ratios of
different lymphocyte populations in PBMCs. The investigation will focus on Th17 and Treg
levels.
- FACS will be used to assess the expression of CYP1A1 and CYP1B1, which are prototypes of
AhR target genes, in PBMCs after specific targeting.
- The levels of expression of the active fractions of AhR and NfkB will be determined
using direct immunofluorescence technique.
- PBMCs will be cultured with anti-CD3+/-BaP (50 nM) in a cell incubator (37°C, 5% CO2+
controlled humidity) for 48-72 hours. The parameters of interest, including oxidative
stress, pro-inflammatory cytokines, and assessment of the AhR pathway, will be
reassessed after lymphocyte reactivation by anti-CD3 in the presence or absence of BaP.
