Neutrophils as Prognostic Factors in Granulomatosis With Polyangiitis (Formerly Named Wegener’s Granulomatosis)

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Brief Title

Neutrophils as Prognostic Factors in Granulomatosis With Polyangiitis (Formerly Named Wegener's Granulomatosis)

Official Title

Neutrophils Function and Identification of Prognostic Factors in Granulomatosis With Polyangiitis (Formerly Named Wegener's Granulomatosis).

Brief Summary

      Anti-neutrophil cytoplasm antibodies (ANCA)-associated vasculitis are rare diseases
      characterized by inflammation of blood vessels. Among the numerous cell types that play a
      role in vasculitis, one of the key actors is the neutrophil. Neutrophils are equipped with
      very powerful molecules that they use to destroy the invading microbes. Therefore, the
      mechanisms controlling neutrophil activation should be tightly controlled. If that is not the
      case, neutrophils may destroy the tissues of the host. This is what happens during chronic
      inflammation in vasculitis. Autoantibodies directed against neutrophils, ANCA, produced thus
      demonstrating that neutrophils are also targets of the immune system in these diseases. In
      addition, molecular studies provided evidence that genes normally silenced in mature
      neutrophils under normal conditions can be re-expressed in neutrophils from patients with
      ANCA-associated vasculitis thus strongly suggesting a profound deregulation of neutrophil
      functions in these conditions. Notably, the investigators have preliminary data showing that
      neutrophils from patients with granulomatosis with polyangiitis (GPA, formerly Wegener's
      granulomatosis), an ANCA-associated vasculitis, interfere with the normal phase of resolution
      of inflammation.

      The objective of the investigators' study is to understand the mechanisms underlying this
      increased activation state and determine if neutrophils could be used to define prognostic
      markers by clinicians to optimize patients' care. Therefore, the investigators plan to study
      the expression of proteins implicated in GPA pathophysiology at the membrane of neutrophils
      when they undergo apoptosis. The investigators will also study the deregulation of protein
      expression in neutrophils. This point will be the molecular translation of neutrophil
      deregulation. This technique is powerful and well adapted to identify by mass spectrometry
      the proteins that will be differentially expressed between the control and the disease state.
      After identification of proteins differentially expressed in patients with GPA, the
      investigators will further investigate whether their expression is modulated during the
      disease course and/or modified by the treatment.

      The investigators believe that understanding these neutrophil perturbations can lead to
      better monitoring of disease activity. Ultimately, the investigators may propose more
      targeted anti-inflammatory therapies which would be better tolerated by patients. the
      investigators also can identify new markers for disease activity which allow clinicians to
      define a better therapeutic strategy.
    

Detailed Description

      A - Specific Aims General aim: This project is designed to study physiopathological
      mechanisms involved in GPA at the cellular and molecular level. the investigators want to
      study neutrophils as prognostic factors and determine the implication of PR3, the major
      autoantigen in the resolution phase of inflammation.

      the investigators' working hypothesis is that structural and functional analysis of PR3 in
      neutrophils will help the investigators to unravel its role in GPA. Hence, our long-term
      objective is to better understand the mechanisms involved in the modulation of PR3 expression
      at the membrane of neutrophils in order to inhibit their activation and propose novel
      anti-inflammatory therapeutic strategies adapted to GPA.

      Present project: The investigators recently demonstrated that in human neutrophils i) PR3 can
      be expressed at the membrane of neutrophils during apoptosis in the absence of degranulation,
      ii) PR3 is a member of a molecular scaffold comprising phospholipidscramblase1 (scramblase)
      and calreticulin externalized during apoptosis, iii) apoptosis-induced PR3 membrane
      expression impairs macrophage reprogramming after phagocytosis of apoptotic neutrophils The
      investigators' hypothesis is that PR3 might have specific molecular partners which can
      facilitate its externalization and/or its persistence at the plasma membrane during
      neutrophil apoptosis thus potentiating its auto-antigenic capacity.

      The specific aims are:

        1. Characterization of apoptosis in neutrophils from GPA patients

        2. Analysis of PR3 partners (CD16, CD11b, Calreticulin, Phospholipidscramblase1) or
           co-expressed protein of interest in inflammation (AnnexinA1,) expression in neutrophils
           from patients with GPA compared to healthy controls

        3. Identification of new partners of PR3 in neutrophils

        4. Analysis of the proteome of neutrophils from GPA patients

      B. Background and Significance 1.1. Introduction GPA is a systemic vasculitis which is mainly
      associated with anti-PR3 ANCA. The role of activated neutrophils in vasculitis lesions is
      suggested by their presence in perivascular infiltrates, especially in kidney glomeruli and
      in the lung.

      PR3 is a serine protease first described within azurophilic granules of neutrophils which are
      NOT supposed to fuse with the plasma membrane but are released within the phagolysosome after
      phagocytosis of the pathogen. We have shown for the first time that the subcellular
      localization of PR3 was not restricted to azurophilic granules, as previously thought, but
      that PR3 was localized in secretory vesicles, a highly mobilizable compartment, thus
      explaining the presence of PR3 at the surface of resting isolated neutrophils. Moreover, a
      high percentage of neutrophils expressing PR3 observed in vasculitis patients could be
      considered as a risk factor for vasculitis. This high expression of membrane PR3 in patients
      with GPA resulted in an increase in ANCA-induced respiratory burst in neutrophils thus
      enhancing the inflammatory process. In vitro the binding of ANCA to PR3 trigger the
      respiratory burst, degranulation and the release of pro-inflammatory cytokines.

      Because PR3 membrane expression is an important pathophysiological event in GPA since it is a
      prerequisite for the subsequent activation of neutrophils by ANCA, we sought to determine
      mechanisms involved in PR3 membrane expression. In neutrophils, we demonstrated that
      apoptosis triggered a significantly increased membrane PR3 expression without degranulation.
      In these experiments, we failed to detect MPO at the membrane of neutrophils. Our results
      differ from previously reported data showing that both MPO and PR3 were externalized during
      apoptosis because of the translocation of azurophilic granules to the plasma membrane. This
      phenomenon seems to occur when neutrophils undergo late apoptosis and secondary necrosis,
      leading to expression of all granule proteins on the cell surface.

      Apoptosis-induced PR3 externalization could be of great relevance in the pathophysiology of
      GPA because PR3, which was supposed to be an intracellular antigen, is thus exposed at the
      plasma membrane of apoptotic cells thus amplifying its pro-inflammatory potential, its
      immunogenic properties and interfering with macrophage phagocytosis. We have also
      demonstrated that during apoptosis PR3 expression is associated with the expression of
      calreticulin, a well-known "eat-me signal". We have finally evidenced that the presence of
      PR3 impairs the reprogramming of macrophage to an anti-inflammatory profile after
      phagocytosis of apoptotic cells through the CRT/LRP pathway.

      It can be hypothesized that the expression of the complex PR3/scramblase/CRT and other
      partners (CD11b, CD16) at the surface of apoptotic neutrophils could constitute good
      prognosis markers.. Gene array studies have pointed out a deregulation of neutrophils in GPA.
      We make the hypothesis that the proteome of neutrophils of GPA patients would be very
      informative of its activation state, survival state and that it could be a biological marker
      of clinical severity.

      It is thus pertinent to study the deregulation of neutrophils during their all life cycle:
      their oxidative burst under activation, their physiologic apoptosis, the expression of PR3
      partners. This study will be performed in neutrophils from patients with GPA compared to age
      and sex adjusted healthy controls.

      C. Preliminary studies Analysis of apoptosis in neutrophils from GPA patients In the
      literature, neutrophils from patients with ANCA associated vasculitis (AAV) were found to
      have a disturbed TNFα-induced apoptosis and tends to have a higher rate of apoptosis than
      controls. However, published data on physiologic apoptosis which is the spontaneous apoptosis
      that neutrophils undergo in the absence of exogenous stimuli is contradictory. When calf
      foetal serum is added in medium, neutrophils from GPA patients display a similar apoptosis
      rate than controls. When the serum of patients is used, neutrophils from AAV patients in
      remission have a higher survival rate in vivo than controls, and this difference disappear
      when normal serum is used. These results are in agreement with the discovery that GPA
      patients have a high expression of PCNA which is associated with survival. Therefore, we want
      to study the apoptosis rate of neutrophils in GPA at diagnosis, during relapse and after
      remission on whole blood. These results will be compared to age and sex matched controls.
      Apoptosis will be evaluated by the phosphatidylserine externalization measured by flow
      cytometry after annexin-V labelling and 7-AAD staining on CD15+ cells. PCNA expression will
      be also investigated at the different clinical stage and proteomic profile of apoptotic
      neutrophils will be performed.

      Analysis of scramblase/ Calreticulin expression in neutrophils from GPA patients In a
      cellular model (RBL cell line), we have demonstrated that scramblase extinction by shRNA was
      associated with a decreased expression of phosphatidylserine during apoptosis and that PR3
      apoptosis induced expression was also impaired (unpublished data). We were also able to
      demonstrate that in vitro scramblase and PR3 physically interact. During apoptosis, PR3 is
      also associated with calreticulin at the membrane of neutrophils. These data suggest that the
      association PR3/Scramblase/Calreticulin form a molecular scaffold which is a functional
      platform which could interfere with recognition pathways (like CRT/LRP) between macrophages
      and apoptotic neutrophils. The expression of these partners at the neutrophil membrane during
      apoptosis will be investigated in patients at diagnosis and when remission is obtained.

      Research design and Methods:

        1. Characterization of apoptosis in neutrophils from GPA patients We will determine
           apoptosis rate of neutrophils in whole blood.

           Methodology:

           Research will be performed both on neutrophils isolated from healthy control donors
           obtained in the French Blood Institute (Etablissement Français du Sang) and from
           vasculitis patients from the Department of Internal Medicine of Cochin Hospital headed
           by Professor Loïc Guillevin.

           Apoptosis will be induced "physiologicaly" in whole blood by overnight incubation at
           37°C.

           Phosphatidylserine externalization will be measured by flow cytometry after annexin-V
           labeling associated with 7-AAD staining for CD15+ cells.

           Western blot analysis using a specific antibody against PCNA will also be used to
           visualize its expression.

        2. Analysis of scramblase, PR3, calreticulin, AnnexinA1, expression in apoptotic
           neutrophils from GPA patients We will determine the membrane expression of PR3, CRT,
           PLSCR, AxA1, in apoptotic neutrophils in the different subsets of neutrophils: non
           apoptotic ones (AnnexinV-, 7AAD-), early apoptotic neutrophils (AnnexinV+, 7AAD-) and
           late apoptotic neutrophils (AnnexinV+, 7AAD+). This determination will be done for
           patients at the time of diagnosis (in active state; BVAS>3) and at remission.

           Methodology

             -  Neutrophils will be isolated from blood using Dextran gradient and Ficoll.

             -  Investigation of PR3, CRT, PLSCR, AxA1, membrane expression will be measured on
                isolated neutrophils by flow cytometry after overnight incubation at 37°C with
                assessment of apoptosis by annexin-V labeling and 7AAD staining.

        3. Study of the respiratory burst in neutrophils of GPA patients One of the main functions
           of neutrophils is the production of reactive oxygen species, we will determine the
           oxidative metabolism of neutrophils due to different stimuli (f-MLP, opsonized zymosan
           beads by complement, opsonized zymosan beads by IgG, PMA) with or without a previous
           priming by TNFα.

           Methodology:

           Exploration of the respiratory burst will be done in whole blood with a chemoluminescent
           (luminol) technique. Neutrophils will be primed by TNFα or stimulated without priming.
           The burst response will be analysed during 90 minutes after stimulation.

        4. Deregulation of neutrophil Cytokinome Neutrophils are capable to synthetize a large
           number of cytokines (IL-1, TNFα, IL-6, IL-12), growth factors like GM-CSF and
           chimiokines like IL-8. More recently it has been shown that neutrophils are also great
           producers of BAFF (B-cell activating factor) and that production is superior to those by
           monocytes or dendritic cells when stimulated by INF-γ. In GPA serum BAFF levels have
           been shown to be higher than controls and recently targeting B-lymphocytes therapeutics
           have confirmed in large controlled trials their importance in AAV.

           Methodology:

           Serum of patients will be collected at the same time than neutrophils. They will be
           analyzed using BD™ Cytometric Bead Array (CBA) with detection of IL-8, IL-1β, IL-6,
           IL-10, TNF, and IL-12p70. Neutrophils which will be isolated as in 1) will be stimulated
           by INF-γ, LPS and G-CSF to analyze their production and secretion of BAFF/BlyS
           determined by ELISA.

        5. Deregulation of neutrophil Proteome and functional study of new PR3 partners Gene array
           studies have shown a probable deregulation of neutrophils in AAV, in this study we will
           study this deregulation at the protein level.

      Methodology:

      To study the proteome of GPA patient when active and in remission compared to healthy
      controls we will use a differential in gel electrophoresis of cytosolic samples of
      neutrophils obtained by sonication. Identification of proteins of interest will be done by
      mass spectrometry MALDI-TOF-TOF. Functional validation of these proteins by over-expression
      or siRNA extinction will be performed.
    


Study Type

Observational


Primary Outcome

level of neutrophils


Condition

Granulomatosis With Polyangiitis

Intervention

Blood samples and clinical data

Study Arms / Comparison Groups

 GPA patients
Description:  GPA patients with an active disease at inclusion

Publications

* Includes publications given by the data provider as well as publications identified by National Clinical Trials Identifier (NCT ID) in Medline.

Recruitment Information


Recruitment Status

Other

Estimated Enrollment

93

Start Date

May 25, 2012

Completion Date

May 18, 2018

Primary Completion Date

May 18, 2018

Eligibility Criteria

        Inclusion Criteria:

        For patients with GPA

          -  Systemic or localized GPA with ACR (American College of Rheumatology) criteria.

          -  BVAS > 3.

          -  ANCA anti-PR3 or anti-MPO

          -  Consent form signed

        For patients with MPA

          -  Systemic MPA with Chapel Hill criteria.

          -  BVAS > 3.

          -  ANCA anti-MPO

          -  Consent form signed

        For patients with EGPA

          -  Systemic EGPA with ACR (American College of Rheumatology) criteria.

          -  BVAS > 3.

          -  ANCA anti-PR3 or anti-MPO

          -  Consent form signed

        For atherosclerotic patients

          -  Ischemic stroke

          -  Coronary heart attack

          -  Peripheral vascular disease responsible for intermittent claudication

          -  Carotid Stenosis

        Exclusion Criteria:

          -  Pregnancy

          -  <18 yr

          -  Malignancy

          -  Infectious diseases: HIV, HBV, HCV
      

Gender

All

Ages

18 Years - N/A

Accepts Healthy Volunteers

Accepts Healthy Volunteers

Contacts

, , 

Location Countries

France

Location Countries

France

Administrative Informations


NCT ID

NCT01862068

Organization ID

NI 10017


Responsible Party

Sponsor

Study Sponsor

Assistance Publique - Hôpitaux de Paris


Study Sponsor

, , 


Verification Date

April 2021