IL-13%20Is%20Necessary,%20Not%20Simply%20Sufficient,%20for%20Epicutaneously%20Induced%20Th2%20Responses%20to%20Soluble%20Protein%20Antigen

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IL-13 Is Necessary, Not Simply Sufficient, for
Epicutaneously Induced Th2 Responses to Soluble
Protein Antigen

• Herrick et al, The Journal of Immunology, 2003
  170 2488-2495
• Grace Chan

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Agenda

• Background Information
• Question being addressed
• Animal Model
• Results
• Summary
• Discussion and Conclusion

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Atopic disease

• Genetic predisposition to develop local
  anaphylactic reactions to allergens
• Th2 sensitization and responses are known to be
  involved in the pathogenesis of disease

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Th2 Cytokines

• At the site of allergic inflammation
• Th2 cytokines, IL-4, -5, and -13 are abundant
• IL-4, -5, and -13 mediate development of tissue
  eosinophilia, mucus hypersecretion and high IgE
  levels

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Th1 and Th2 cytokines
IL-10 causes the suppression of Th1
responses IL-4 involved in ve feedback to
differentiate CD4 Tcells into Th2 cells IL-5
attracts and activates eosinophils (usually 0-
450 cells/ul) IL-13 promotes IgE production
http//users.rcn.com/jkimball.ma.ultranet/BiologyP
ages/T/Th1_Th2.html
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IL-4 and IL-13

• Produced by Th2 cells
• Share similarities in structure and function
• May be attributed to sharing the IL-4Ra chain in
  their respective receptor complexes
• Also display differences in function

www.sigmaaldrich.com
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Similar functions of IL-4 and IL-13

• Suppression of inflammatory cytokines production
  by macrophages
• Up-regulation of MHCII on monocyte/macrophages
• Induction of endothelial cell VCAM expression

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www.uta.fi/imt/ silvennoinen/work_page.html
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Differing functions of IL-4 and IL-13 (live
pathogen)

• IL-4 deficient mice ? IL-4 independent Th2
  responses observed
• IL-4Ra deficient mice ? more impairment of Th2
  responses than in IL-4-/- mice (impaired
  expulsion of N. brasiliensis worms)
• IL-13-/- mice ? unable to clear N. brasiliensis
  infection

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Differing functions of IL-4 and IL-13 (e.c.
sensitized to OVA)
IL-4-/-
WT
IL-4-/-
IL-13 depleated
STAT6-/-
IL-4-/-
IL-4 independent Th2 response
Lack of IL-4 independent Th2 response
Th2 response
Lack of IL-4 independent Th2 response
IL-13 is responsible for the Th2 response in e.c.
sensitization
IL-4 independent Th2 response is e.c. specific
Taken together IL-13 is involved in IL-4
independent Th2 responses in e.c. sensitization
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Question being Addressed

• Since IL-4 and IL-13 have redundant roles, are
  the IL-4-independent Th2 responses seen in
  IL-4-/- mice a result of IL-13 compensation, or
  does IL-13 play a unique and independent role in
  the generation of Th2 responses?
• Is IL-13 required or simply sufficient in the
  cutaneous microenvironment?

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Animal Model
IL-13 KO
CO2 asphyxiation
Day 14 - i.n. challenge by OVA by i.n. droplet on
days 14,15, 18, 19

• Day 0
• Exposure to OVA /PBS control on shaved back
  (e.c.)
• 4 days

• Day 21
• Sacrifice

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Results

• Comparison of airway inflammatory responses and
  Ab production in e.c. sensitized IL-4-/- and
  STAT6-/- mice
• Previous study In WT mice, Th2 cytokines and Th2
  Ab isotypes are observed. IgG1Th2 and IgG2aTh1
• In IL-4KO lung inflammation comparable to WT, but
  there is a switching of Ab Isotype class from Th2
  to Th1
• In STAT6KO, show reduced inflammatory responses

Figure 1
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Th2 Cytokines

• Mediates high Th2-associated IgG1 and low
  Th1-associated IgG2a
• Th2 mediated airway inflammation

Th2 cell
IL-4 IL-5 IL-13
Individual Th2 cytokines are responsible for
distinct Th2-associated effector functions
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• Impairment of both lung inflammation and Ab
  responses in e.c. sensitized IL-13-/- mice
• Is IL-13 playing a unique role?
• Other evidence
• IL-13 induces airway eosinophilia
• Lack of eosinophilia seen in STAT6 KO could be
  due to blockage of eosinophil recruitment rather
  than due to a decrease in Th2 priming (proposed
  to be dependent upon IL-13)

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• Experiment after e.c. sensitization, they
  challenged mice with an i.n. OVA.
• Comparing BAL decrease in lymphocytes and total
  BAL cells in IL-13-/- mice
• Comparing Ab decrease in OVA specific IgG1 in
  IL-13-/- mice vs WT suggests defect in priming
  Th2 cells
• Comparing Histology IL-13-/- have no mucus or
  inflammation (lymphocytes decreased) compared to
  STAT6-/- (eosinophils decreased)
• Conclusion IL-13-/- mice have a defect in
  initial sensitization

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Question

• Why is there no isotype class switch seen in
  IL-13KO versus the STAT6KOs?
• The persisting IL-4 in the IL-13KO is suppressing
  the Th1 response maintenance of the Th2 isotype
  is observed.
• Lack of isotype switching in IL-13KOs
  demonstrates that IL-13 does not suppress Th1
  generation, only a decrease in OVA specific Abs.

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• Lack of IL-13 results in defective Th2 cytokine
  production by skin-draining lymph node cells
  after e.c. sensitization
• Confirming that there is a defect in initial
  sensitization after e.c. exposure in IL-13-/-
  mice using Th2 cytokine production as an
  indicator.
• Experiment isolate LN 4 day post e.c. OVA
  exposure and restimulate by in vitro culture with
  OVA.
• Results
• IL-13-/- mice show data consistent with decreased
  Th2 priming
• IL-4-/- show that Th2 responses are preserved
• Conclusion IL-13-/- do, indeed, have a defect
  in early initial sensitization

Figure 3
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• Confirming unique ability of IL-13 to generate
  Th2 responses.
• Other studies
• suggest failure of IL-13-/- to generate Th2
  responses is due to intrinsic defect in IL-13-/-
  to produce IL-4
• In support
• differing outcomes are observed in STAT6-/-
  (deficient in both IL-13 and IL-4 function) vs
  IL-13-/- mice
• Experiment A
• Administered a soluble antagonist against IL-13
  to WT e.c. OVA sensitized mice.
• Results
• IL-13 depleted mice had levels of IL-5 reduced to
  levels comparable to that seen in IL-13-/- mice.
  No difference in IL-4 was seen.

Figure 4-A
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Confirming unique ability of IL-13 to generate
Th2 responses contd Experiment B
Administering IL-13 to IL-13-/- Results
Increased IL-4 (and IL-5) demonstrating the
ability for IL-13-/- to maintain IL-4 secretion.
This effect was dose dependent. Conclusion
IL-13-/- mice are not impaired in their ablity to
produce and secrete IL-4
Figure 4-B, C
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• IL-13 is not required for Th2 responses generated
  by i.p. exposure to OVA
• To ensure that IL-13-/- mice still maintain the
  ability to respond to OVA in the traditional
  Th2-inducing system.
• Experiment i.p. OVA sensitization followed by
  i.n. OVA challenge after 14 days.
• Results decreased inflammatory cells compared to
  WT.
• Contrast with e.c. sensitized mice, i.p.
  sensitized mice showed increased total cells
  compared to sham-immunized mice.

Figure 5

• Since both lymphocytes and eosinophils increased
  compared to sham-immunized while total cells were
  decreased compared to WT, there may be a lack of
  cell recruitment to the airways in i.p.
  sensitized mice.

• However, histological examination does not show
  impairment in inflammation showing no impairment
  in Th2 inflammatory responses.
• Comparing Ab production, IgG1 and IgG2a were
  shown to be high in i.p. sensitized mice vs e.c.
  sensitized mice.
• Conclusion IL-13-/- mice show no inherent defect
  in Ab production or Th2 inflammatory response
  since i.p. sensitization brings out Th2 responses.

Figure 2
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IL-13-/- mice are not impaired in generation of
CHS to a hapten To confirm that the general lack
of Th2 responses in the cutaneous
microenvironment is not just due to the inability
of IL-13-/- to respond to antigens. Experiment
Apply DNFB (contact sensitizing agent) onto skin
to determine if there is any contact
hypersensitivity - known to involve CD8 Th1 and
CD8 T effector cells. Th2 believed to
downregulate above response. Results Ear
thickness developed to a greater degree in
IL-13-/- than WT
Figure 6
Conclusion IL-13-/- is able to respond to an
e.c. applied hapten. Swelling may be due to a
lack of Th2 mediated suppression of Th1 responses
suggesting that IL-13 has a unique role to play
in generating Th2 responses in the skin.
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Summary

• Question Is IL-13 required or simply sufficient
  in the cutaneous microenvironment?
• Results
• IL-13 is able to maintain the Th2 cytokine
  profile in IL-4 KO but is unable to compensate
  for the IL-4 suppression of Th1-mediated IgG2a
  production
• IL-13 does not play a role in suppression of Th1
  generation but does have a role in initial
  sensitization
• The lack of IL-13 is not inhibiting the
  production of IL-4, and IL-13-/- mice do not have
  a defect with IL-4 secretion
• IL-13 is not required for Th2 generation when
  i.p. sensitized
• IL-13-/- mice can respond to an e.c. applied
  hapten
• Conclusion IL-13 is necessary, not only
  sufficient, for Th2 sensitization through the
  skin and the requirement for IL-13 is specific to
  the cutaneous microenvironment

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Discussion

• The skin, along with the respiratory tract,
  represents a large barrier to environmental
  antigens.
• The induction of Th2 responses is found to be
  involved in allergic response.
• Since e.c. exposure to protein Ags has been shown
  to generate robust Th2 responses, and many
  children with atopic dermatitis develop allergic
  airway diseases, the skin may be a mode of
  systemic Th2 sensitization in atopic individuals.
• Determination of how responses are generated
  could lead to new therapies to prevent systemic
  sensitization.
• IL-13 has been found to be necessary in the
  generation of Th2 responses as opposed to IL-4

Barrier
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IL-13 function Unique to skin

• IL-13-/- mice showed no inflammation when e.c.
  sensitized as opposed to i.p. sensitized.
• IL-13 has an effect upon sensitization unique to
  the cutaneous microenvironment.
• When there was no subsequent OVA challenge after
  e.c. sensitization, IL-13-/- mice still did not
  produce Th2 cytokines.
• Thus, IL-13 is required early in initial
  sensitization

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IL-13 function Unique to skin

• IL-4 has been shown to be responsible for the
  differentiation of naive T cells to Th2 effector
  cells.
• Generation Th2 responses by i.n. OVA
  sensitization has been shown to be dependent upon
  IL-4 whereas e.c. sensitization is not dependent.
• IL-4-independent Th2 response is probably
  specific to sensitization through the skin.
• Dependence upon IL-13 in the skin (shown in this
  paper) may reflect the relative lack of IL-4 in
  this barrier or the greater receptivity of the
  skin to IL-13 than to IL-4.

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Other findings

• Other groups have found that the IL-7-like
  cytokine, thymic stromal lymphopoietin (TSLP),
  produced by epithelial and keratinocyte is
  greatly expressed in lesions of atopic
  dermatitis.
• DCs responding to TSLP will cause the
  differentiation of naive CD4 T cells to
  differentiate into Th2 cells to produce more
  IL-5, and 13 compared to IL-4.
• Activation of DCs through the skin may be geared
  towards greater IL-13 producing cells.
• This may be the underlying reason for the
  increased dependence on IL-13 in the skin over
  IL-4.

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Other findings

• IL-13 has also been found to be important in the
  clearing of N. brasiliensis worms (an effector
  stage function) over IL-4
• The similar requirement for the clearing of worms
  at the effector stage and for initial
  sensitization by OVA (this paper) may be
  explained by the fact that N. brasiliensis is
  injected through the skin.

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Conclusions

• IL-13 is a necessary and critical cytokine in the
  generation of Th2 responses to e.c. exposure of
  protein antigens.
• Because IL-13 is greatly expressed in the lesions
  of atopic dermatitis, the skin may represent a
  significant site of Th2 sensitization
• IL-13 may be a possible target for therapy to
  prevent systemic sensitization

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Thank You