Transfected Macrophages in Renal Inflammation

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Transfected Macrophages in Renal Inflammation

• Dr David Kluth
• Senior NKRF Research Fellow
• University of Aberdeen

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Macrophages in Renal Injury

• Glomerular and interstitial macrophages are a
  universal feature of renal inflammation.
• Macrophages products which may propagate renal
  injury
• Proteases
• Reactive oxygen and nitrogen species
• Cytokines IL-1b, TNF-a
• Growth Factors
• Macrophages significant component of transplant
  inflammatory infiltrate

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Macrophage Activation
MHC Class II, CD80/86
LPS/CpG
TLR
NO, IL-1b, TNF-a, IL-12
IFN-g
Proteases
Classically activated- tissue damage
? Mannose Receptor, Arginase ?iNOS, TNF-a
IL-4/IL-13
Alternative activation
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Manipulation of Macrophage Function by disease
? IL-10, TGF-b ? IL-12 ?CCR2
?Angiogeneis ?Antigen presentation
Tumour associated macrophages
Fc-g
LPS
?IL-10 ?IL-12 Favour Th2 response
CD40L
Type II activated macrophage
?TGF-b, PGE2 ?TNF-a, IL-8, MCP-1
Apoptotic cell uptake
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Manipulation of Macrophage function in
transplantation

• Inhibition of iNOS prolonged graft survival (Vos
  et al 2000)
• Transfection of bone marrow cells with allogeneic
  MHC Class II (Sonntag et al 2001) or MHC Class I
  (Fry et al 2002) prolonged grafts
• Transfection of cardiac graft with IL-10 reduced
  leukocyte infiltration (David et al 2000)
• Blockade of NF-kB by decoy oligo improved renal
  graft function (Azuma et al 2003)
• Dendritic cells expressing IL-4 accelerated
  rejection in vascularized heart graft (Kaneko et
  al 2003)

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Macrophages controlling the inflammatory response.
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Methods

• Adenoviral transduction of macrophages
• Efficient 80-90 cells transduced
• Allows assessment in vitro and in vivo
• Inflammatory model of glomerulonephritis
• Nephrotoxic nephritis
• Delivered directly to inflamed glomeruli by renal
  artery injection.

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Ad-IL4 transfected macrophages localise with high
efficiency to inflamed glomeruli.
Day 7 post-injection
Day 1 post-injection
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Ad-IL4 transfected macrophages reduce albuminuria.
Albuminuria (mg/24 hrs)
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Glomerular localisation of IL-4 expressing
macrophages required to reduce albuminuria.
Albuminuria (mg/24hours)
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Ad-IL4 transfected macrophages reduce glomerular
macrophages.
ED1 positive cells/ glomerular area
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IL-10 expressing macrophages in NTN

• Ad-IL10 expressed rat IL-10
• Used primary cultures of rat bone marrow derived
  macrophages (BMDM)
• Injected 6 hours after the onset of disease.
• BMDM labelled with red fluorescent label.

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Glomerular Localisation of Ad-IL10 transfected
Macrophages
Ad-IL10 Transfected BMDM
Ad-null Transfected BMDM
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IL-10 expressing macrophages reduce albuminuria
in NTN.
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IL-10 expressing macrophages reduce glomerular
ED3 positive macrophages
Day 7
ED3 cells/glomerular area
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IL-10 reduces glomerular MHC Class II expression
Control
IL-10 transfected macrophages
Null transfected macrophages
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Histological injury reduced by IL-10 expressing
macrophages
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NF-kB Critical regulator of inflammatory response
TNF, IL-1, LPS, CD40L,
IL1R
CD40
TNFR
TLR4
Ik-B
p65
p50
NF-kB
NF-kB inducible gene
promoter
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Adenoviral vectors

• Ad-IkB mutated at serine 32 and 36 which prevents
  phosphorylation of IkB.
• Ad-p65/RHD contains the rel homology domain so
  can bind DNA but not transactivation domain.

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NF-kB required for macrophage IFN-g responses

• Ad-IkB reduced macrophage IFN-g responses
• Reduced NO and TNF-a
• Inhibited up-regulation of MHC Class II and
  co-stimulatory molecules
• Inhibition of NF-kB reduced IFN-g induced STAT1
  nuclear translocation

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NF-kB dictates the amplitude of the macrophage
inflammatory response
Secondary inflammatory signal
NF-kB
Anti-inflammatory response
Pro-inflammatory response
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Ad-IkB transduced macrophages in vivo

• Ad-IkB transduced macrophages labelled with PKH
  before injection.
• Macrophages injected into renal artery
• Glomeruli isolated and PKH labelled macrophages
  recovered by trypsin /collagenase /DNAase digest.
• Expression of iNOS and MHC Class II determined by
  FACS.

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Glomerular Localisation of Ad-IkB transduced
macrophages
Ad-IkB
Ad-null
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IkB expressing macrophages have reduced MHC Class
II and iNOS expression in vivo
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NF-kB inhibited macrophages reduce glomerular
macrophage infiltration
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NF-kB inhibited macrophages reduce MHC Class II
expression in vivo
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IkB expressing macrophages reduce albuminuria in
NTN
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Summary

• Macrophage can have a spectrum of activities in
  disease, both pro and anti-inflammatory.
• Glomerular localisation of IL-4 or IL-10
  expressing macrophages reduces inflammation
• Macrophages can affect injury locally and at
  distant sites.
• IkB expressing macrophages have anti-inflammatory
  effect in vivo.
• Biasing macrophage function has therapeutic
  potential

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Acknowledgments

• Salah Chettibi
• Heather Wilson
• Andy Rees
• NKRF, BBSRC