IL-13 Is Necessary, Not Simply Sufficient, for Epicutaneously Induced Th2 Responses to Soluble Protein Antigen Herrick et al, The Journal of Immunology, : Grace Chan

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

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

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

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 cells/ul) IL-13 promotes IgE production Pages/T/Th1_Th2.html

IL-4 and IL-13 Produced by Th2 cells Produced by Th2 cells Share similarities in structure and function Share similarities in structure and function May be attributed to sharing the IL-4R  chain in their respective receptor complexes May be attributed to sharing the IL-4R  chain in their respective receptor complexes Also display differences in function Also display differences in function

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

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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-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-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 IL-13 -/- mice  unable to clear N. brasiliensis infection

IL-4-/-WTIL-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)

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? 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? Is IL-13 required or simply sufficient in the cutaneous microenvironment?

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 Animal Model

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

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

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

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

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. 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. Lack of isotype switching in IL-13KOs demonstrates that IL-13 does not suppress Th1 generation, only a decrease in OVA specific Abs.

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

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

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

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 Figure 2 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.

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

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 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 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 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 is not required for Th2 generation when i.p. sensitized IL-13-/- mice can respond to an e.c. applied hapten 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

Discussion The skin, along with the respiratory tract, represents a large barrier to environmental antigens. 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. 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. 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. 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 IL-13 has been found to be necessary in the generation of Th2 responses as opposed to IL-4

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

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. 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. 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. 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. 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 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. 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. 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. 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. This may be the underlying reason for the increased dependence on IL-13 in the skin over IL-4.

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 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. 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 IL-13 is a necessary and critical cytokine in the generation of Th2 responses to e.c. exposure of protein antigens. 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 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 IL-13 may be a possible target for therapy to prevent systemic sensitization

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