IL13 Is Necessary Not Simply Sufficient for Epicutaneously

IL-13 Is Necessary, Not Simply Sufficient, for Epicutaneously Induced Th 2 Responses to Soluble Protein Antigen Herrick et al, The Journal of Immunology, 2003 170: 2488 -2495 Grace Chan

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

Atopic disease n n Genetic predisposition to develop local anaphylactic reactions to allergens Th 2 sensitization and responses are known to be involved in the pathogenesis of disease

Th 2 Cytokines n At the site of allergic inflammation: Th 2 cytokines, IL-4, -5, and -13 are abundant n IL-4, -5, and -13 mediate development of tissue eosinophilia, mucus hypersecretion and high Ig. E levels n

Th 1 and Th 2 cytokines IL-10 causes the suppression of Th 1 responses IL-4 involved in +’ve feedback to differentiate CD 4 Tcells into Th 2 cells IL-5 attracts and activates eosinophils (usually 0 - 450 cells/ul) IL-13 promotes Ig. E production http: //users. rcn. com/jkimball. ma. ultranet/Biology. P ages/T/Th 1_Th 2. html

IL-4 and IL-13 n n Produced by Th 2 cells Share similarities in structure and function n n May be attributed to sharing the IL-4 Ra chain in their respective receptor complexes Also display differences in function www. sigmaaldrich. com

Similar functions of IL-4 and IL-13 n n n Suppression of inflammatory cytokines production by macrophages Up-regulation of MHCII on monocyte/macrophages Induction of endothelial cell VCAM expression

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Differing functions of IL-4 and IL-13 (live pathogen) n n n IL-4 deficient mice IL-4 independent Th 2 responses observed IL-4 Ra deficient mice more impairment of Th 2 responses than in IL-4 -/- mice (impaired expulsion of N. brasiliensis worms) IL-13 -/- mice unable to clear N. brasiliensis infection

Differing functions of IL-4 and IL-13 (e. c. sensitized to OVA) IL-4 -/- WT IL-4 -/- IL-13 depleated IL-4 -/- IL-4 independent Th 2 response is e. c. specific Lack of IL-4 independent Th 2 response STAT 6 -/- Lack of IL-4 independent Th 2 response IL-13 is responsible for the Th 2 response in e. c. sensitization Taken together: IL-13 is involved in IL-4 independent Th 2 responses in e. c. sensitization

Question being Addressed n Since IL-4 and IL-13 have redundant roles, are the IL-4 -independent Th 2 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 Th 2 responses? n Is IL-13 required or simply sufficient in the cutaneous microenvironment?

Animal Model IL-13 KO CO 2 asphyxiation Day 0 Day 14 Day 21 -Exposure to OVA /PBS control on shaved back (e. c. ) - i. n. challenge by OVA by i. n. droplet on days 14, 15, 18, 19 -Sacrifice - 4 days

Results Comparison of airway inflammatory responses and Ab production in e. c. sensitized IL-4 -/- and STAT 6 -/- mice • Previous study: In WT mice, Th 2 cytokines and Th 2 Ab isotypes are observed. Ig. G 1=Th 2 and Ig. G 2 a=Th 1 • In IL-4 KO lung inflammation comparable to WT, but there is a switching of Ab Isotype class from Th 2 to Th 1 • In STAT 6 KO, show reduced inflammatory responses Figure 1

Th 2 Cytokines Th 2 cell Mediates high Th 2 associated Ig. G 1 and low Th 1 associated Ig. G 2 a IL-4 IL-5 IL-13 Th 2 mediated airway inflammation Individual Th 2 cytokines are responsible for distinct Th 2 -associated effector functions

Impairment of both lung inflammation and Ab responses in e. c. sensitized IL-13 -/- mice • Is IL-13 playing a unique role? Other evidence: Naïve T cell Th 2 cell • IL-13 induces airway eosinophilia • Lack of eosinophilia seen in STAT 6 KO could be due to blockage of eosinophil recruitment rather than due to a decrease in Th 2 priming (proposed to be dependent upon IL 13) IL-4, -5, -13

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 Naïve T cell Th 2 cell Comparing Ab: decrease in OVA specific Ig. G 1 in IL-13 -/- mice vs WT suggests defect in priming Th 2 cells Comparing Histology: IL-13 -/- have no mucus or inflammation (lymphocytes decreased) compared to STAT 6 -/- (eosinophils decreased) IL-4, -5, -13 Conclusion: IL-13 -/- mice have a defect in initial sensitization Figure 2

Question Why is there no isotype class switch seen in IL-13 KO versus the STAT 6 KOs? n n The persisting IL-4 in the IL-13 KO is suppressing the Th 1 response – maintenance of the Th 2 isotype is observed. Lack of isotype switching in IL-13 KOs demonstrates that IL-13 does not suppress Th 1 generation, only a decrease in OVA specific Abs.

Lack of IL-13 results in defective Th 2 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 Th 2 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 Th 2 priming • IL-4 -/- show that Th 2 responses are preserved Conclusion: IL-13 -/- do, indeed, have a defect in early initial sensitization Figure 3

Confirming unique ability of IL-13 to generate Th 2 responses. Other studies: • suggest failure of IL-13 -/- to generate Th 2 responses is due to intrinsic defect in IL-13 -/- to produce IL-4 In support: • differing outcomes are observed in STAT 6 -/- (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

Confirming unique ability of IL-13 to generate Th 2 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 Figure 4 -B, C

IL-13 is not required for Th 2 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 Th 2 -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 shamimmunized mice. • Since both lymphocytes and eosinophils increased compared to shamimmunized while total cells were decreased compared to WT, there may be a lack of cell recruitment to the airways in i. p. sensitized mice. Figure 5 • However, histological examination does not show impairment in inflammation showing no impairment in Th 2 inflammatory responses. • Comparing Ab production, Ig. G 1 and Ig. G 2 a 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 Th 2 inflammatory response since i. p. sensitization brings out Th 2 responses. Figure 2

IL-13 -/- mice are not impaired in generation of CHS to a hapten To confirm that the general lack of Th 2 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 CD 8 Th 1 and CD 8 T effector cells. Th 2 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 Th 2 mediated suppression of Th 1 responses suggesting that IL-13 has a unique role to play in generating Th 2 responses in the skin.

Summary Question: Is IL-13 required or simply sufficient in the cutaneous microenvironment? Results: n n n IL-13 is able to maintain the Th 2 cytokine profile in IL-4 KO but is unable to compensate for the IL-4 suppression of Th 1 -mediated Ig. G 2 a production IL-13 does not play a role in suppression of Th 1 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 Th 2 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 Th 2 sensitization through the skin and the requirement for IL-13 is specific to the cutaneous microenvironment

Discussion n n The skin, along with the respiratory tract, represents a large barrier to environmental antigens. The induction of Th 2 responses is found to be involved in allergic response. Since e. c. exposure to protein Ags has been shown to generate robust Th 2 responses, and many children with atopic dermatitis develop allergic airway diseases, the skin may be a mode of systemic Th 2 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 Th 2 responses as opposed to IL-4

IL-13 function – Unique to skin n IL-13 -/- mice showed no inflammation when e. c. sensitized as opposed to i. p. sensitized. n n 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 Th 2 cytokines. n Thus, IL-13 is required early in initial sensitization

IL-13 function – Unique to skin n n IL-4 has been shown to be responsible for the differentiation of naive T cells to Th 2 effector cells. Generation Th 2 responses by i. n. OVA sensitization has been shown to be dependent upon IL-4 whereas e. c. sensitization is not dependent. n n IL-4 -independent Th 2 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.

Other findings n n 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 CD 4 + T cells to differentiate into Th 2 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.

Other findings n n 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.

Conclusions n n n IL-13 is a necessary and critical cytokine in the generation of Th 2 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 Th 2 sensitization IL-13 may be a possible target for therapy to prevent systemic sensitization

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