Overproduction of collagen and diminished SOCS1 expression are causally linked in fibroblasts from i

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Overproduction of collagen and diminished SOCS1
expression are causally linked in fibroblasts
from idiopathic pulmonary fibrosis
Hiroyasu Shoda, Akihito Yokoyama, Ryouhei
Nishinom Taku Nakashima, Nobuhisa Ishikawa,
Yoshinori Haruta, Noboru Hattori, Tetsuji Naka,
Nouoki Kohno

• Biochemical and Biophysical Research
  Communications
• Article Submitted 7 December 2006
• 9 February 2007 R.P.I.
• Michael DAngelo, Elvin Eng,
• Benjamin Klein, Adam Parkinson

353 (2007) 1004-1010
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The Authors

• Department of Molecular and Internal Medicine
• Graduate School of Biomedical Sciences, Hiroshima
  University, Hiroshima, Japan
• Hiroyasu Shoda
• Akihito Yokoyama
• Ryouhei Nishinom
• Taku Nakashima
• Yoshinori Haruta
• Noboru Hattori
• Nouoki Kohno
• Department of Molecular Medicine
• Osaka University Graduate School of Medicine,
  Osaka, Japan
• Tetsuji Naka

yokoyan_at_hiroshima-u.ac.jp
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What is Pulmonary Fibrosis?

• Involves scarring of the interstitium
• Slowly air sacs in lungs are replaced by fibrotic
  tissue
• When the scar forms, the tissue becomes thicker
• Tissues cant transfer oxygen into the
  bloodstream
• Results from a microscopic lung injury
• Usually inflammation ? repair/remodeling
• Believed that PF was a process of chronic repair
• Persistent inflammation ? Matrix production
• and deposition
• PF represents abnormal wound repair
• At some point IPF becomes no longer dependent
• on the inflammatory response for propagation

http//www.pulmonaryfibrosis.org/ipf.htm
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Idiopathic Pulmonary Fibrosis

• IPF has several names
• Cryptogenic Fibrosing Alveolitis (CFA)
• Usual Interstitial Pneumonitis (UIP)
• 50,000 new cases diagnosed annually
• Usually ages (40-60)
• Associations
• May be genetic?
• Autoimmune disorder?
•  Exposure to environmental and occupational
  pollutants (asbestos, silica, organic dusts)
• Cigarette smoking
• Other diseases such (Scleroderma, Rheumatoid
  Arthritis, Lupus, Sarcoidosis)
• Certain medications
• Therapeutic radiation

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Idiopathic Pulmonary Fibrosis

• Symptoms
• Shortness of breath, particularly with exertion
• Chronic dry, hacking cough
• Fatigue and weakness
• Discomfort in the chest
• Loss of appetite
• Rapid weight loss
• Treatments
• Drugs
• Anti-inflammatory
• Exercise therapy

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Idiopathic Pulmonary Fibrosis

• Life expectancy?
• Only 2-4 years

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Why we chose this article?

• 200,000 people in America diagnosed with
  Pulmonary Fibrosis
• No known effective treatment
• No known cure
• Very Recent Article
• Lead to better understanding of disease
• Most of the important things in the world have
  been accomplished by people who have kept on
  trying when there seemed to be no hope at
  all.-Dale Carnegie

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Collagen
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Fibroblasts

• Fibro (fiber), blast (creates)
• Derived from mesenchymal cells
• Are professionals in movement, ECM production,
  and deposition, and shaping other cells.
• Depending on morphology
• Mainly for structural framework and wound healing
  role

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Myofibroblasts

• Form of fibroblast cell that has differentiated
  partially towards a smooth muscle phenotype
• Capable of speeding wound repair by contracting
  the edges of the wound
• After healing is complete, these cells are lost
  through apoptosis

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How Do They Play Together?

• Fibroblasts undergo some type of morphology
• End up migrating through gaps into alveolar space
• Fibroblast attempts to repair the damaged
  alveolus
• Leading to extracellular accumulation
• Normally because of transformation, the
  contraction would close the wound.
• Then, apoptosis mediates the decrease of
  mesenchymal cells.
• Eventually recreating the semi-permeable barrier

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The Problem?

• No evidence for early phase of a IPF condition
• No evidence IPF starts with inflammation
• Begins with injury to alveolar epithelial cells
  (AEC)
• Induces fibroblasts migration/proliferation and
  fibroblast phenotypic change
• Phenotype changes the cytokines and growth
  expressions
• End up damaging AEC even more instead of
  repairing
• Increase AEC apoptosis which cannot compensate
  for the fibroblast/myofibroblast apoptosis

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The Immune Response
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The Immune Response

• The Body has two modes of defense
• The Innate Immune Response
• Comprised of the cells and mechanisms that defend
  the host from infection by other organisms, in a
  non-specific manner.
• The Adaptive Immune Response
• Composed of highly specialized, systemic cells
  and processes that eliminate pathogenic
  challenges

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The Adaptive Immune Response

• The B Cell
• Major cells involved in the creation of
  antibodies that circulate in blood plasma

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The Adaptive Immune Response

• The T-Cells (aka Lymphocytes)
• CD4 helper T-cells
• CD8 T lymphocytes (Killer T Cells)

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The Adaptive Immune Response

• CD4 helper T-cells
• Immune system mediator
• Maximize the capabilities of immune response
• Do not have cytotoxic or phagocytic activity

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The Adaptive Immune Response

• CD8 T lymphocytes (Killer T Cells)
• Induce the death of cells that are infected with
  viruses (and other pathogens) or are otherwise
  damaged or dysfunctional.
• Once activated, undergoes Clonal Expansion to
  mount up a formidable defense

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The Adaptive Immune Response

• CD4 helper T-cells (again)
• Two types of effector CD4 T helper cell
  responses can be induced by a professional APC,
  designated Th1 and Th2. (Only one at a time!!!)
• Which factors differ between a Th1 or Th2 type
  response?
• CYTOKINES!!!!

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The Cytokines

• IFN-?
• What is it?
• Secreted by T-Lymphocytes and Natural Killer
  cells.
• Alters gene transcription
• IFN-? binding to the receptor activates the
  JAK-STAT pathway
• Secreted during a Th1 T-Cell response

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JAK-STAT

• Carries signals from extra cellular proteins
  to the cell nucleus
• STAT Proteins act to Modify gene expression (may
  be activated w/o JAK)
• Plays a role in cell fate.

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The Cytokines

• IL-4
• Secreted by Th2 Cells
• Stimulate activated B-Cell and T-Cell
  Proliferation
• Stimulates the production of more Th2 (IMPORTANT)

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The Complement Cascade
Adaptive Response ? Th2
Innate Response ? Th1
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The Inflammation Process
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The Inflammatory Process

• Includes
• A tissue based startle reaction to trauma
• Go/No-Go decisions based on integration of
  molecular clues for tissue penetration by
  microbes
• The beckoning, instruction and dispatch of cells

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The Inflammatory Process

• Includes (cont)
• Killing of microbes and host cells they infect
• Liquefaction of surrounding tissue to prevent
  microbial metastasis
• The healing of tissues damaged by trauma or by
  the hosts response (clean up)

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The Inflammatory Process
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The Inflammatory Process

• IL-4 and TGF-B stimulate repair
• Activate fibroblasts
• Signaling paths are independent of each other
• Both cytokines secreted by the Th2 Cell
• Fibroblasts produce Type-1 collagen to repair the
  damage done by the immune response

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The Inflammatory Process

• IFN-? inhibits collagen production
• Done two ways
• The TGF-B Pathway
• The IL-4 Pathway
• These Pathways are INDEPENDENT!!

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The Inflammatory Process

• The TGF-B Pathway
• Does not require SOCS1
• Inhibited by IFN-G

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The Inflammatory Process

• IL-4 Pathway
• Regulated by INF-G
• INF-G interaction exclusively mediated by SOCS1

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The Inflammatory Process

• Why is SOCS1 Important?
• This gene encodes a member of the STAT-induced
  STAT inhibitor (SSI)
• The protein encoded by this gene functions
  downstream of cytokine receptors
• Knockout studies in mice suggested the role of
  this gene as a modulator of IFN-gamma action

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The Inflammatory Process

• How does this help you understand the paper?
• If the fibrosis is being causes by an auto-immune
  response, primarily of the Th2 pathway why not
  pump in some INF and calm it down?
• Theoretically it should work, but it doesnt work
  100... Why?

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The Experiment
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Culture Medium and Cytokines

• Fibroblasts were cultured in appropriate media
  supplemented with 10 inactivated FBS.
• Samples maintained at 37C in an atmosphere of
  air with 5 CO2.
• Assays were cultured with IL-4, IFN-?, and
  TGF-ß1, but not FBS.

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Methods ELISA System

• Stands for Enzyme-Linked Immunosorbant Assay
• Used to detect specific protein antigens using
  antibodies
• Antigens will specifically bind to their
  counterpart antibody
• These antibodies are labeled using secondary
  antibodies
• Secondary antibodies bind and cleave a
  colormetric substrate
• Spectrophotometer quantifies color intensity
  given off by the substrate

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Results ELISA System

• Uses monoclonal primary antibodies to bind to
  type 1 collagen.
• Immunoglobin secondary antibodies measured
  primary binding.
• Wild type lung fibroblasts produced much less
  type 1 collagen then SOCS1 deficient mice after
  24 hours.
• Proves that SOCS1 is responsible for type 1
  collagen production.

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Methods Western Blotting

• Proteins in a sample are sorted by size using a
  gel electrophoresis.
• Place a membrane on the gel. The positive charge
  on the membrane and negative charge on the gel
  pushes the proteins across and generates an
  imprint.
• Incubate the membrane after adding the primary
  antibodies.
• Creates primary antibody-protein linking
• Incubate the membrane after adding secondary
  antibodies.
• Secondary antibodies bind to primary antibodies
  on protein in question.
• Secondary antibodies have conjugated enzymes
  attached for florescence.
• Incubate mix in a serum specific to your
  composition
• Add x-ray film to gel
• Film will capture light emitted from conjugated
  enzymes
• Shows where protein of interest is located.

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Results Western Blotting

• Graph a
• More type 1 collagen is produced by
  SOCS1-deficient fibroblasts.
• Graph c

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Methods RNeasy

• Used for RNA purification.
• Membrane binds to RNA molecules longer than 200
  nucleotides.
• Everything else is washed away using a spin
  column.

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Methods Nunc Maxisorp

• Coating for well plates.
• Uses polystyrene 96 which has a high protein
  binding capability.
• Gives better results for assays.

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MethodsReal Time RT-PCR

• Real Time PCR amplifies primer and measures DNA
  or RNA content each cycle.
• Uses fluorescent tags to determine quantity of
  target
• Used with a standard curve to determine initial
  primer quantity

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Methods Real Time RT-PCR

• More accurate than Northern Blot (RNA) or
  Southern Blot (DNA)
• Fluorescent tags are activated by cleavage from a
  quench molecule by DNA polymerase

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Results and Discussion
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Overproduction of Collagen

• Human IPF fibroblasts produced significantly more
  type-I collagen than normal fibroblasts.
• Tested using ELISA
• IPF fibroblasts had more pro-a1-(I) collagen mRNA

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SOCS1 deficiency

• Using RT-PCR SOCS1 mRNA expression tested
• SOCS1 mRNA expression lower in IPF fibroblasts

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MEF collagen expression
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Causation

• SOCS1 deficient and normal wild-type MEF were
  given IFN-?
• Normal MEF were stimulated to produce SOCS1 when
  exposed to IFN-?
• SOCS1 deficient MEF did not produce SOCS1 mRNA
  when exposed
• SOCS1 deficient MEF produced significantly more
  type-I collagen mRNA than normal MEF
• SOCS1 deficiency increases type-I collagen
  production

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Over-expression of SOCS1

• Wild type MEF expressing myc-tagged SOCS1 protein
  were created
• Inserted using a retroviral factor
• Myc-SOCS1 was tested and found to be functional
  active by its inhibition of pSTAT6 via IL-4
• IL-4 is active when pSTAT6 is present.
• Absence of pSTAT6 and presence of IL-4 and
  SOCS1-myc means SOCS1 is active.

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Over expression of SOCS1 via SOCS1-myc

• Myc-SOCS1 found to inhibit collagen production in
  normal and SOCS1 deficient MEF when compared to
  controls with no myc-SOCS1 gene.
• Suggests that overproduction of SOCS1 can inhibit
  fibroblast collagen production.
• Type-I collagen production in fibroblasts is
  causally linked to SOCS1 expression.

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Role of IFN-?, Normal MEF

• Measured amount of type-I collagen produced in
  response to the addition of IFN-? TGF-ß1 and IL-4
• IFN-? reduced collagen production proportional to
  dose

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Role of IFN-?, Normal MEF

• TGF-ß1 and IL-4 increased collagen production
• IFN-? inhibited effects of TGF-ß1 and IL-4

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Role of IFN-?, SOCS1 deficient MEF

• IFN-? inhibited collagen production based on
  dose. Greater inhibition compared to normal MEF

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Role of IFN-gamma, SOCS1 deficient MEF

• TGF-ß1 and IL-4 increased collagen production
• IFN-? inhibits TGF-ß1 induced collagen production
  but not IL-4 induced collagen production
• Inhibitory effects of IFN-? on TGF-ß1 is
  independent of SOCS1 expression
• Effects of IFN-? on IL-4 is mediated by SOCS1.
• Suppression of collagen production by IFN-? is
  both dependent and independent of SOCS1

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IFN-? and IPF fibroblasts

• IFN-? inhibited collagen production more in IPF
  fibroblasts than normal fibroblasts.

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IFN-? and IPF fibroblasts

• IFN-? was more effective in increasing SOCS1 mRNA
  in IPF fibroblasts than in healthy lung
  fibroblasts

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Discussion

• SOCS1 and IFN-? were both shown to decrease the
  spontaneous production of collagen type-1 in IPF
  and MEF
• SOCS1 regulates IFN-? through negative feedback
• IFN-gamma inhibits TGF-ß1 induced collagen
  independent of SOCS1
• IFN-? inhibits IL-4 induced collagen through a
  mechanism dependent on SOCS1

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Discussion

• IFN-? was shown not to significantly improve
  quality of life in a study of IPF patients over
  placebo.
• Is this the expected result?
• What could be the cause for the limited
  effectiveness of treatment?

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