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  • Atopic disease has a genetic component to it - Genetic predisposition to develop local anaphylactic reactions to allergens - these can be allergens to the airways as well as to the skin Th2 sensitization and responses are known to be involved in the pathogenesis of diseases of this sort
  • The Th2 cytokines, IL-4, 5, and 13 are found in abundance at the sites of allergic inflammation These cytokines mediate the development of eosinophilia (cytotoxic granules for pathogen clearance), mucus hypersecretion, and IgE levels characteristic of allergies
  • Describe pathway: DC  preTh  Th1  cell mediated imm.; DC  preTh  Th2  Ab mediated imm. Looking at the diagram of Th1 vs Th2 cytokines we can see that the Th2 cytokines are involved in suppression of Th1 development, and Th1 cytokines suppress Th2 development (which is also dependent upon IL-4 + feedback) The authors of this study examined the roles of IL-4 and IL-13 and wanted to determine if the function of IL-13 could be unique from IL-4 (next few slides)
  • This diagram shows STAT6 downstream of IL-4. It is also downstream of IL-13. So a double KO of IL-4 and IL-13 is displayed in a STAT6-/- phenotype.
  • Differing functions of IL-4 and IL-13 have been seen in studies with live pathogens. ~IL-4 deficient mice  IL-4 independent Th2 responses observed :. IL-4 is not solely responsible for Th2 responses ~IL-4R  deficient mice  more impairment of Th2 responses than in IL-4 -/- mice (impaired expulsion of N. brasiliensis worms) – remember that IL-4 and IL-13 share the same receptor subunit so the other ligand that may be responsible for the increased impairment of Th2 responses is probably IL-13. ~Indeed, IL-13 -/- mice  unable to clear N. brasiliensis infection
  • ~IL-4-/- mice e.c. sensitized to OVA  Th2 responses equivalent to WT mice (IL-4 Independent response) ~But when WT mice are INTRANASALLY sensitized to OVA  Th2 responses are seen in WT but not in IL-4-/- Therefore: IL-4 independent Th2 responses to OVA are characteristic of e.c. priming as opposed to I.N. priming ~In another study, IL-4-/- depleted of IL-13 by a soluble antagonist and STAT6-/- = DKO of IL4 and IL13,  do not generate Th2 responses after e.c. sensitization Taken together: IL-13 is involved in IL-4 independent Th2 responses in e.c. sensitization
  • General Animal Model used: IL-13 KO, e.c. sensitization on Day 0 by exposure of OVA/PBS control on shaved back with a occluding patch – left on for 4 days Day 14 – I.n. challenge by OVA for six days with administration of the droplet on days 14, 15, 18 and 19. Other points: Day 4; i.n. sens. Day 0; i.p. sens.
  • BAL fluid. 200cells. PREVIOUS STUDIES SHOW THAT In WT mice, both Th2 cytokines and Th2 Ab isotypes are increased upon e.c. sensitization. IgG1=Th2 and IgG2a=Th1 In IL-4KO see the lung inflammation but the switching of Ab Isotype class from Th2 to Th1 :. IL-4 is involved in keeping IgG2a suppressed while the Th2 associated isotype, IgG1 is increased to correlate with the Th2 cytokine profile Looking at Lung inflammation: IL4-/- and WT can generate Th2 cytokines (not shown) and mucus secretion. But STAT6-/- has decreased lung inflammatory responses The difference between IL-4-/- and STAT6-/- is the lack of IL-13 function in the STAT6-/- mouse. So IL-13 is a target
  • Now that IL-13 is a target, 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)
  • Experiment: after e.c. sensitization, they challenged mice with an i.n. OVA on day 14. Results: Comparing BAL: decrease in lymphocytes and total BAL cells in IL-13-/- mice suggest a defect in the initial sensitization to OVA Comparing Ab: decrease in OVA specific IgG1 in IL-13-/- mice vs WT suggests defect in priming Th2 cells IL-13KO had impaired airway inflammation with decreased eosinophils and lymphocytes Comparing Histology: IL-13-/- shows no inflammation or mucus production. STAT6KO has lung inflammation with only a decrease in eosinophils (figure 1) rather than a decrease of lymphocytes as well. Again, this suggests a defect in initial sensitization rather than a defect downstream of sensitization (no inflammation and low lymphs. :.no Ag sensitization).
  • Class Question: Why is there no isotype class switch seen in IL-13KO versus the STAT6KOs? A suprression of Th1 response is observed by the maintenance of Th2 isotype and this may be a function of persisting IL-4 in the IL-13 KO. Comparing Ab: decrease in OVA-specific IgG1 in IL-13-/- mice vs WT suggests defect in priming Th2 cells BUT comparing IgG1 levels to IgG2a levels IgG1 remains higher than levels of IgG2a…:. Therefore, there was no isotype switching in IL-13KO mice – only a decrease in overall OVA-specific Abs which could be consistent with IL-4 persistence. :.IL-13 does not play a role in suppression of Th1 generation, since we didn’t see an increase in IgG2a in IL-13KO. Conclusion: IL-13-/- mice have a defect in initial sensitization
  • Confirming defect in initial sensitization after e.c. exposure in IL-13-/- mice - U sing Th2 cytokine production as an indicator Experiment: isolate skin-draining LN 4 day post e.c. OVA exposure and restimulate by in vitro culture with OVA. Results: IL-13 KO mice showed that, even before an OVA challenge there was decreased IL-5 and IL-13 (KO) compared to WT in supernatants. IFN-gamma was comparable. Data are consistent with decreased Th2-priming. Comparing with IL-4-/- (which still have IL-13), see that after e.c. OVA exposure, IL-5 and IL-13 are increase but IFN-gamma was not detected. Th2 responses are preserved in IL-4-/- Conclusion: IL-13-/- do, indeed, have a defect in early initial sensitization
  • The lack of IL-13 is not inhibiting production of IL-4, and IL-13KOs do not have a problem with IL-4 secretion
  • To ensure that IL-13-/- mice still maintain the ability to respond to OVA in the traditional Th2-inducing system and do not have an inherent defect for this response due to the KO… Because can see Th2 responses in IL-13ko, it means that IL-13 is not required for i.p. sens. Th2 response
  • Wanted to confirm that the general lack of Th2 responses in the cutaneous microenvironment wasn’t just due to the inability of IL-13-/- to respond to antigens. Dinitrofluorobenzene which is known to involve CD8 Th1 and T effector cells. Results: Ear thickness developed to a greater degree in IL-13-/- than WT
  • ~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. ~Although IL-13 does not play a role in suppression of Th1 generation, it 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 (the traditional Th2 inducing system) ~IL-13 mice can respond to an e.c. applied hapten
  • The skin represents a large barrier to environmental antigens along with the respiratory tract. The induction of Th2 responses is found to be involved in the allergic response.
  • When i.n. sens. Then need IL-4 But when e.c. sens, then can be IL-4 INdependent
  • Along these lines…
  • Transcript

    • 1. 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
    • 2. Agenda
      • Background Information
      • Question being addressed
      • Animal Model
      • Results
      • Summary
      • Discussion and Conclusion
    • 3. 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
    • 4. 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
    • 5. 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/BiologyPages/T/Th1_Th2.html
    • 6. IL-4 and IL-13
      • Produced by Th2 cells
      • Share similarities in structure and function
        • May be attributed to sharing the IL-4R  chain in their respective receptor complexes
      • Also display differences in function
      www.sigmaaldrich.com
    • 7. 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
    • 8. www. uta . fi / imt / silvennoinen /work_page.html
    • 9. Differing functions of IL-4 and IL-13 (live pathogen)
      • IL-4 deficient mice  IL-4 independent Th2 responses observed
      • IL-4R  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
    • 10. IL-4-/- WT IL-4-/- IL-4 independent Th2 response Th2 response Lack of IL-4 independent Th2 response IL-4 independent Th2 response is e.c. specific IL-4-/- STAT6-/- IL-13 depleated Lack of IL-4 independent Th2 response IL-13 is responsible for the Th2 response in e.c. sensitization Taken together: IL-13 is involved in IL-4 independent Th2 responses in e.c. sensitization Differing functions of IL-4 and IL-13 (e.c. sensitized to OVA)
    • 11. 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?
    • 12. Animal Model
      • Day 0
      • Exposure to OVA /PBS control on shaved back (e.c.)
      • 4 days
      Day 14 - i.n. challenge by OVA by i.n. droplet on days 14,15, 18, 19
      • Day 21
      • Sacrifice
      IL-13 KO CO2 asphyxiation
    • 13. Results Figure 1
      • 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. IgG1=Th2 and IgG2a=Th1
      • 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
    • 14. 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
    • 15.
      • 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)
      Th2 cell IL-4, -5, -13 Naïve T cell Barrier
    • 16.
      • 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
      Figure 2 Th2 cell IL-4, -5, -13 Naïve T cell Barrier
    • 17. 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.
    • 18.
      • 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
    • 19.
      • 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
    • 20. Figure 4-B, C Confirming unique ability of IL-13 to generate Th2 responses – cont’d 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
    • 21.
      • 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.
      • C ontrast with e.c. sensitized mice, i.p. sensitized mice showed increased total cells compared to sham-immunized mice.
      Figure 5 Figure 2
      • S ince 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.
    • 22. 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 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. Figure 6
    • 23. 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
    • 24. 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
    • 25. 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
    • 26. 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- in dependent 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.
    • 27. 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.
    • 28. 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.
    • 29. 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
    • 30. Thank You