IL-13 Is Necessary, Not IL-13 Is Necessary, Not Simply Sufficient, for Simply Sufficient, for

Epicutaneously Induced Th2 Epicutaneously Induced Th2 Responses to Soluble Protein Responses to Soluble Protein

AntigenAntigenHerrick et al, Herrick et al, The Journal of Immunology, The Journal of Immunology, 2003 2003

170: 2488-2495170: 2488-2495

Grace ChanGrace Chan

AgendaAgenda

Background InformationBackground Information Question being addressedQuestion being addressed Animal ModelAnimal Model ResultsResults Summary Summary Discussion and ConclusionDiscussion and Conclusion

Atopic diseaseAtopic disease

Genetic predisposition to develop local Genetic predisposition to develop local anaphylactic reactions to allergensanaphylactic reactions to allergens

Th2 sensitization and responses are Th2 sensitization and responses are known to be involved in the known to be involved in the pathogenesis of diseasepathogenesis of disease

Th2 CytokinesTh2 Cytokines

At the site of allergic inflammation:At the site of allergic inflammation: Th2 cytokines, IL-4, -5, and -13 are Th2 cytokines, IL-4, -5, and -13 are

abundantabundant IL-4, -5, and -13 mediate development IL-4, -5, and -13 mediate development

of tissue eosinophilia, mucus of tissue eosinophilia, mucus hypersecretion and high IgE levelshypersecretion and high IgE levels

Th1 and Th2 cytokinesTh1 and Th2 cytokinesIL-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

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BiologyPages/T/Th1_Th2.html

IL-4 and IL-13IL-4 and IL-13

Produced by Th2 cellsProduced by Th2 cells Share similarities in Share similarities in

structure and structure and functionfunction May be attributed to May be attributed to

sharing the IL-4Rsharing the IL-4R chain in their chain in their respective receptor respective receptor complexescomplexes

Also display Also display differences in differences in functionfunction

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Similar functions of Similar functions of IL-4 and IL-13IL-4 and IL-13

Suppression of inflammatory cytokines Suppression of inflammatory cytokines production by macrophagesproduction by macrophages

Up-regulation of MHCII on Up-regulation of MHCII on monocyte/macrophagesmonocyte/macrophages

Induction of endothelial cell VCAM Induction of endothelial cell VCAM expression expression

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Differing functions of IL-4 Differing functions of IL-4 and IL-13 (live pathogen)and IL-13 (live pathogen)

IL-4 deficient mice IL-4 deficient mice IL-4 independent Th2 IL-4 independent Th2 responses observed responses observed

IL-4RIL-4R deficient mice deficient mice more impairment more impairment of Th2 responses than in IL-4of Th2 responses than in IL-4-/- -/- mice mice (impaired expulsion of (impaired expulsion of N. brasiliensisN. brasiliensis worms)worms)

IL-13IL-13-/--/- mice mice unable to clear unable to clear N. N. brasiliensisbrasiliensis infection infection

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. Taken together: IL-13 is involved in IL-4 independent Th2 responses in e.c. sensitizationsensitization

Differing functions of IL-4 Differing functions of IL-4 and IL-13 (e.c. sensitized to OVA)and IL-13 (e.c. sensitized to OVA)

Question being AddressedQuestion being Addressed

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

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

ResultsResults

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 CytokinesTh2 Cytokines

Mediates high Mediates high Th2-associated Th2-associated IgG1 and low IgG1 and low Th1-associated Th1-associated IgG2a IgG2a

Th2 mediated Th2 mediated airway airway inflammationinflammation

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

QuestionQuestion

Why is there no isotype class switch Why is there no isotype class switch seen in IL-13KO versus the seen in IL-13KO versus the STAT6KOs?STAT6KOs?

The persisting IL-4 in the IL-13KO is The persisting IL-4 in the IL-13KO is suppressing the Th1 response – suppressing the Th1 response – maintenance of the Th2 isotype is maintenance of the Th2 isotype is observed.observed.

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

SummarySummaryQuestion:Question: Is IL-13 required or simply sufficient in the Is IL-13 required or simply sufficient in the

cutaneous microenvironment?cutaneous microenvironment?

Results:Results: IL-13 is able to maintain the Th2 cytokine profile in IL-4 KO but is 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 unable to compensate for the IL-4 suppression of Th1-mediated IgG2a productionIgG2a production

IL-13 does not play a role in suppression of Th1 generation but does IL-13 does not play a role in suppression of Th1 generation but does have a role in initial sensitizationhave a role in initial sensitization

The lack of IL-13 is not inhibiting the production of IL-4, and IL-13-/- 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 secretionmice do not have a defect with IL-4 secretion

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

Conclusion:Conclusion: IL-13 is necessary, not only sufficient, for Th2 IL-13 is necessary, not only sufficient, for Th2 sensitization through the skin and the requirement for IL-13 sensitization through the skin and the requirement for IL-13 is specific to the cutaneous microenvironmentis specific to the cutaneous microenvironment

DiscussionDiscussion

The skin, along with the respiratory tract, represents a large The skin, along with the respiratory tract, represents a large barrier to environmental antigens.barrier to environmental antigens.

The induction of Th2 responses is found to be involved in The induction of Th2 responses is found to be involved in allergic response.allergic response.

Since e.c. exposure to protein Ags has been shown to Since e.c. exposure to protein Ags has been shown to generate robust Th2 responses, and many children with generate robust Th2 responses, and many children with atopic dermatitis develop allergic airway diseases, the skin atopic dermatitis develop allergic airway diseases, the skin may be a mode of systemic Th2 sensitization in atopic may be a mode of systemic Th2 sensitization in atopic individuals.individuals.

Determination of how responses are generated could lead to Determination of how responses are generated could lead to new therapies to prevent systemic sensitization.new therapies to prevent systemic sensitization.

IL-13 has been found to be necessary in the generation of IL-13 has been found to be necessary in the generation of Th2 responses as opposed to IL-4Th2 responses as opposed to IL-4

IL-13 function – Unique to IL-13 function – Unique to skinskin

IL-13-/- mice showed no inflammation when e.c. IL-13-/- mice showed no inflammation when e.c. sensitized as opposed to i.p. sensitized.sensitized as opposed to i.p. sensitized.

IL-13 has an effect upon sensitization IL-13 has an effect upon sensitization unique to the unique to the cutaneous microenvironment.cutaneous microenvironment.

When there was no subsequent OVA challenge When there was no subsequent OVA challenge after e.c. sensitization, IL-13-/- mice still did not after e.c. sensitization, IL-13-/- mice still did not produce Th2 cytokines.produce Th2 cytokines.

Thus, IL-13 is required Thus, IL-13 is required early early in initial sensitizationin initial sensitization

IL-13 function – Unique to skinIL-13 function – Unique to skin

IL-4 has been shown to be responsible for the IL-4 has been shown to be responsible for the differentiation of naive T cells to Th2 effector cells.differentiation of naive T cells to Th2 effector cells.

Generation Th2 responses by i.n. OVA sensitization Generation Th2 responses by i.n. OVA sensitization has been shown to be dependent upon IL-4 has been shown to be dependent upon IL-4 whereas e.c. sensitization is not dependent.whereas e.c. sensitization is not dependent.

IL-4-IL-4-inindependent Th2 response is probably specific to dependent Th2 response is probably specific to sensitization through the skin.sensitization through the skin.

Dependence upon IL-13 in the skin (shown in this Dependence upon IL-13 in the skin (shown in this paper) may reflect the relative lack of IL-4 in this paper) may reflect the relative lack of IL-4 in this barrier or the greater receptivity of the skin to IL-barrier or the greater receptivity of the skin to IL-13 than to IL-4.13 than to IL-4.

Other findingsOther findings

Other groups have found that the IL-7-like cytokine, thymic Other groups have found that the IL-7-like cytokine, thymic stromal lymphopoietin (TSLP), produced by epithelial and stromal lymphopoietin (TSLP), produced by epithelial and keratinocyte is greatly expressed in lesions of atopic keratinocyte is greatly expressed in lesions of atopic dermatitis.dermatitis.

DCs responding to TSLP will cause the differentiation of naive DCs responding to TSLP will cause the differentiation of naive CD4CD4+ + T cells to differentiate into Th2 cells to produce more IL-T cells to differentiate into Th2 cells to produce more IL-5, and –13 compared to IL-4.5, and –13 compared to IL-4.

Activation of DCs through the skin may be geared towards Activation of DCs through the skin may be geared towards greater IL-13 producing cells.greater IL-13 producing cells.

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

Other findingsOther findings

IL-13 has also been found to be important in the IL-13 has also been found to be important in the clearing of clearing of N. brasiliensisN. brasiliensis worms (an effector worms (an effector stage function) over IL-4 stage function) over IL-4

The similar requirement for the clearing of worms The similar requirement for the clearing of worms at the effector stage and for initial sensitization at the effector stage and for initial sensitization by OVA (this paper) may be explained by the fact by OVA (this paper) may be explained by the fact that that N. brasiliensisN. brasiliensis is injected through the skin. is injected through the skin.

ConclusionsConclusions

IL-13 is a necessary and critical cytokine in IL-13 is a necessary and critical cytokine in the generation of Th2 responses to e.c. the generation of Th2 responses to e.c. exposure of protein antigens.exposure of protein antigens.

Because IL-13 is greatly expressed in the Because IL-13 is greatly expressed in the lesions of atopic dermatitis, the skin may lesions of atopic dermatitis, the skin may represent a significant site of Th2 represent a significant site of Th2 sensitizationsensitization

IL-13 may be a possible target for therapy IL-13 may be a possible target for therapy to prevent systemic sensitizationto prevent systemic sensitization

Thank YouThank You