Interferongamma and Interleukin 6 Modulate the Susceptibility of Macrophages to Human Immunodeficien

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• Interferon-gamma and Interleukin 6 Modulate the
  Susceptibility of Macrophages to Human
  Immunodeficiency Virus Type 1 Infection
• Zaitseva, M. Lee, S. Lapham, C. Taffs, R.
  King, L. Romantseva, T. Manischewitz, J. and
  Golding, H. 2000. Blood 96(9)3109-3117.
• Presented by Alice Cioara

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Cytokines

• Important effector molecules
• May exert multiple effects on viral entry and
  reverse transcription as well as reactivation
• Proinflammatory cytokines
• Interferon-gamma
• Interleukin 6

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Interferon ?

• Maps to chromosome 12q24.1
• Is a dimeric protein
• Interferon ? (IFN- ?) and receptor complexes are
  rapidly internalized by endocytosis
• Has antiviral and antiparastic activities
• Inhibits the proliferation of a number of normal
  and transformed cells

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Interferon ?

• A modulator of T-cell growth and functional
  differentiation
• Regulates the expression of MHC class 2 genes
• In macrophages stimulates release of reactive
  oxygen species
• Involved in processes of bone growth and inhibits
  bone resorption
• Mediates host defense

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Interferon ?

• Is a potent activator of mononuclear phagocytes
• Produced by T and B cells, natural killer cells,
  and lymphocytes
• Activated by antigens
• Has both suppressive and enhancing effects

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Interleukin 6

• Maps to 7p21-p14
• Involved in T cell proliferation
• Influences antigen-specific immune responses
• Physiological stimuli for synthesis
• Bacterial endotoxins

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Interleukin 6

• Produced by
• Monocytes, fibroblasts and endothelial cells
• After stimluation also produced by macrophages
  T-cells and B-lymphocytes
• Limited to leukocyte subpopulations
• Promotes inflammatory events
• Serves a protective role

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Chemokines

• Superfamily composed of 20 different leukocyte
  chemoattractants
• Small proinflammatory proteins that recruit and
  activate leukocytes and inhibit the viral
  co-receptor function
• Are chemotactic cytokines
• 2 categories
• CC (beta)
• CXC (alpha)

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Chemokines

• CC (beta)
• Non-syncytium-inducing
• Marcophagetropic variants use R5 viruses
• Slow to replicate
• Do not infect CD4 T-cell lines
• CXC (alpha)
• Syncytium-inducing
• T-cell line adapted use CXC chemokine receptor 4
  (CXCR4)

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HIV

• CC chemokine are fusion coreceptors for IV entry
  into macrophages
• CCR5 major determinant of entry
• Macrophages are the main target and serve as a
  reservoir of HIV
• Macrophages more efficiently infected by M-tropic
  HIV strains
• Activation of Macrophages

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HIV

• The cytokine action created during the onset of
  an opportunistic infection or as a result of
  local inflammation may affect HIV infection of
  macrophages by providing a more favorable
  environment for emerging T-tropic viruses

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IFN ? AND Il-6

• To determine whether proinflammatory cytokines
  affect the ability of the macrophages to fuse
  with X4 envelope-expressing cells
• Procedure
• Macrophages were generated
• Treated with cytokines (IFN ?, TNF?, IL-6, and
  IL10)
• Combined with cells expressing a T-tropic IIIB
  envelope

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IFN ? AND Il-6

• Only IFN ? IL-6 exhibited enhancement in the
  numbers of syncytia
• Monocyte-derived macrophages (MDM) obtained
• Mixed (ratio 11) with X4 envelope-expressing
  target cells.
• Quantified of syncytia and analyzed

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IFN ? AND Il-6

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Infection of MDMs with X4

• To see if there is a correlation between
  enhancement of X4 HIV-1 viral entry and an
  increase in fusion of cytokine-treated MDMs
• Analysis of viral DNA at 48 hours using PCR was
  performed
• MDM incubated with INF ? or IL6
• Washed to remove cytokines
• Infected with HIV-1(NL4-3)
• DNA extracted after infection

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Infection of MDMs with X4

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Viral DNA in cytokine-treated MDMs

• To determine if an increase in the primary
  production of treated viral DNA would increase
  viral reproduction
• 6-day MDMs incubated with cytokines
• Washed and then infected with HIV(LAI)

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Viral DNA in cytokine-treated MDMs
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Viral DNA in cytokine-treated MDMs

• Conclusion of Data
• IL-6 INF ? increase efficiency of X4-tropic
  viral entry, thus resulting in an increased
  production of infection of MDMs with T-tropic
  HIV-1

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Role of CXCR4 in IFN ? and IL-6

• To determine the fusion of X4-tropic envelope
  with cytokine-treated MDMs was CXCR4 dependent

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Role of CXCR4 in IFN ? and IL-6

• IL-6- and IFN- -induced increase in the fusion of
  macrophages with T-tropic envelope-expressing
  cells is CXCR4 dependent
• Experiment Cytokine Inhibitor of syncytia with
  IIIB envelope
• 1 None None 51
• IFN ? RANTES 458
• 2 None None 171
• IFN
  ? RANTES 729
• 3 None None 61
• 
  IL-6 RANTES 874
• 4 None None 11
• IL-6 None 5712

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Role of CXCR4 in IFN ? and IL-6

• The previous data exhibits that CXCR4 does in
  fact participate in the fusion between X4-tropic
  envelopes with cytokine-treated macrophages

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Enhanced MDMs, X4-tropic and CXCR4

• Flow cytometry was done on the MDMs which were
  used in the fusion experiments to determine if
  there was a correlation between X4-tropic
  envelopes with enhanced MDMs and CXCR4 surface
  expression
• 12 different experiments were conducted

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Enhanced MDMs, X4-tropic and CXCR4
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Enhanced MDMs, X4-tropic and CXCR4

• The previous data shows that the fusion between
  X4-tropic envelopes with the cytokine-treated
  MDMs does depend on CXCR4.
• This fusion, however, is not due to an increase
  in the levels of surface CXCR4 expression.

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SDF-1-induced Ca flux

• To determine if IFN ? or IL-6 caused
  conformational changes in CXCR4 (in macrophages)
  which resulted in the improvement of ligand
  binding

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SDF-1-induced Ca flux

• The previous data suggests that the fusion of
  X4-tropic envelope with the cytokine-treated MDMs
  does depend on CXCR4.
• However, it is not ascribed to an increase in the
  level of CXCR4 expression

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Effect of Cx and H7 on IFN ? and IL-6

• To determine if X4-tropic envelopes fusion with
  enhanced MDMs is linked with Stat1/3 activation
• MDMs pretreated with an inhibitor of Stat1/3 H7
• These cells then mixed with IIIB
  envelope-expressing cells

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Effect of Cx and H7 on IFN ? and IL-6

• H7 and cycloheximide inhibit IL-6- and IFN-
  -induced fusion of macrophages with T-tropic
  envelope-expressing cells
• Cytokine Inhibitor
  No. of syncytia
• None None 7  4
• IL-6 None 72  9
• IL-6 Cx 6  2
• IL-6 H7 9  1
• None Cx 8  4
• None H7 3  0
• None None 14  1
• IFN None 61  13
• IFN Cx 13  1
• IFN H7 12  2
• None Cx 11  1
• None H7 9  1

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IFN ? and CCR5

• The reduction in the R5-tropic viral entry and
  reproduction detected in the presence of IFN ?
  could be mediated by down-modulation of CD48
  and/or the expression of CCR5
• Fusion assays
• Surface staining
• Performed 11 experiments

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IFN ? and CCR5
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IFN ? and CCR5

• Treatment of MDM with IFN ?
• Slight reduction of surface CCR5
• Inhibition of MDMs with M-tropic envelopes
  significant

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Effect of IL-6 and Macrophages

• To determine if cytokines have the ability to
  change the susceptibility of macrophages to
  primary viral isolates

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Effect of IL-6 and Macrophages

• The previous data shows that proinflammatory
  cytokines do have the ability to increase the
  susceptibility of macrophages to the infection
  with multiple primary X4 viruses

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Cytokine-treated MDMs secrete ß -chemokine

• To determine the mechanism liable for the effects
  of the fusion of MDMs with X4 and R5
  envelope-expressing cells along with the cytokine
  treatment

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Cytokine-treated MDMs secrete ß -Chemokines

• IFN- and IL-6 induce chemokine production in
  macrophages
• Treatment MIP-1 (pg/mL) MIP-1
  (pg/mL)
• None 143  35 66  3
• IFN- 909  37 1425  12
• IL-6 647  5 2176  44

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Discussion

• Exhibited
• IFN ? and IL-6 exert a positive effect on the
  susceptibility of MDMs to infection with X4 HIV-1
• Induced an increase in X4 HIV-1 viral entry
• Infection of MDMs
• Promoted fusion of X4 and MDMs

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Discussion

• Inhibitory effect
• IFN ? on R5 viral entry and the R5
• reproduction of MDMs fusion with target cells
• ß chemokines were detected from IFN ? IL-6
  treated MDMs.

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