A new mechanism for inhalational priming: IL-4 bypasses innate immune signals

Abstract

Signaling via innate immune mechanisms is considered pivotal for T cell-mediated responses to inhaled Ags. Furthermore, Th2 cells specific for one inhaled Ag can facilitate priming of naive T cells to unrelated new inhaled Ags, a process we call "Th2 collateral priming". Interestingly, our previous studies showed that collateral priming is independent of signals via the innate immune system but depends on IL-4 secretion by CD4(+) T cells. We thus hypothesized that IL-4 can bypass the need for signals via the innate immune system, considered essential for pulmonary priming. Indeed, we were able to show that IL-4 bypasses the requirement for TLR4- and MyD88-mediated signaling for responses to new allergens. Furthermore, we characterized the mechanisms by which IL-4 primes for new inhaled allergens: "IL-4-dependent pulmonary priming" relies on IL-4 receptor expression on hematopoietic cells and structural cells. Transfer experiments indicate that within the hematopoietic compartment both T cells and dendritic cells need to express the IL-4 receptor. Finally, we were able to show that IL-4 induces recruitment and maturation of myeloid dendritic cells in vivo and increases T cell recruitment to the draining lymph nodes. Our findings bring new mechanistic knowledge to the phenomenon of polysensitization and primary sensitization in asthma.

FIGURE 1

IL-4 bypasses the need for innate immune signals in pulmonary priming. WT (BALB/c) mice received Th2 cells derived from DO11.10 mice with a WT, IL-4 KO, or IL-13 KO background and were challenged intranasally with OVA and KLH (O+K) and rechallenged with OVA (O) or KLH (K) (A), as described previously (9). WT, TLR4d, and MyD88 KO mice were sensitized with OVA plus LPS or with OVA plus IL-4 intranasally. Differential cell counts in BALF (B), serum OVA-specific IgE titers (C), and Ag-specific local cytokine secretion in mLN following in vitro restimulation (D) were measured via microscopic cell differentiation, ELISA, and Multiplex bead assays, respectively, following challenge with OVA. C and D were obtained using TLR4d mice exclusively.

FIGURE 2

IL-4-dependent pulmonary priming depends on signaling via IL-4Rα in structural and hematopoietic compartments. Lethally irradiated WT and IL-4Rα-deficient mice were reconstituted with BM from WT or IL-4Rα-deficient mice to generate mice expressing IL-4Rα in hematopoietic cells only (SC^-/HP⁺), structural cells only (SC⁺/HP^-), or both (SC⁺/HP⁺) or neither (SC^-/HP^-) compartments before sensitization with OVA plus IL-4. Sensitization and challenge were performed as in Fig. 1. However, subimmunogenic doses of OVA (5 μg/mouse) were used, as titrated in previous experiments, to eliminate adjuvant effects through LPS contamination of OVA in mice capable of responding to LPS. Therefore, mice primed with OVA without IL-4 constitute negative controls. Differential cell counts in BALF (A) and OVA-specific IgE titers (B) were measured following challenge with OVA as in Fig. 1. Cytokine production was measured after challenge following in vitro restimulation of mLN cells with OVA via Multiplex bead assays (C).

FIGURE 3

Signaling via IL-4 in CD4⁺ T cells and myeloid DCs partially restores allergic airway inflammation, systemic sensitization, and Th2 polarization. To test which hematopoietic cells are necessary to induce the IL-4-dependent phenotype in pulmonary priming, we performed transfer experiments. BM chimeras lacking IL-4Rα expression in the hematopoietic compartment (SC⁺/HP^-) were reconstituted with BMDCs and/or T cells from WT or IL-4Rα-deficient mice before sensitization with OVA plus IL-4 and compared with BM chimeras sufficient in both compartments (SC⁺/HP⁺). Sensitization and challenge were performed as in Fig. 1, apart from the use of 5 μg of OVA/mouse for sensitization for reasons explained in Fig. 2. Differential cell counts in BALF (A) and OVA-specific IgE titers (B) were measured following challenge with OVA as in Fig. 1. Cytokine production was measured after challenge following in vitro restimulation of mLN cells via Multiplex bead assays (C).

FIGURE 4

IL-4 increases recruitment, costimulatory molecule expression, and Ag presentation by DCs in vivo. WT mice were sensitized with OVA, conjugated to the fluorochromes DQ (lung) or Alexa 647 (mLN) with or without IL-4 to assess the effect of IL-4 on DC Ag uptake, processing, and presentation, as described in Materials and Methods. Twenty-four hours after the last sensitization, lungs were digested and mLNs were mechanically dispersed to obtain single-cell suspensions. Cell counts were assessed microscopically. Total cell numbers were calculated by multiplying total cell counts from single-cell suspensions with respective percentages from flow cytometric analysis. Pulmonary myeloid DCs were identified via flow cytometric staining for nonautofluorescent, CD11c^high-positive, CD11b-positive cells (A). Additionally, the above-referenced costimulatory molecules (B) and uptake of DQ-OVA (C and D) were analyzed on these cells. Lungs were harvested from TLR4d mice treated with OVA with or without IL-4 24 h after last sensitization, and homogenates were obtained and analyzed as described in Materials and Methods (E). Mediastinal lymph node myeloid DCs were identified via flow cytometric staining for CD11c^high-positive cells (F). In addition to CD11c staining, Ag uptake was determined by analysis of Alexa 647⁺ cells (G and H). Filled histograms represent results from animals treated with fluorochrome-labeled OVA alone; unfilled histograms represent animals treated with fluorochrome-labeled OVA plus IL-4.

FIGURE 5

IL-4 treatment increases T cell recruitment to draining LNs. CSFE-labeled CD4⁺ T cells from DO11.10 TCR transgenic mice were transferred to BALB/cJ mice 1 day before priming with OVA (5 μg) with or without IL-4, as described in Materials and Methods. To analyze the effects of IL-4 on T cell priming, T cell proliferation in mLNs was determined via CFSE dilution at the indicated time points (A), while T cell recruitment to draining lymph nodes was assessed by staining for CD4⁺ and CD8⁺ and the TCR-specific Ab KJ1-26⁺ at 72 h (B). Total cell counts were obtained by multiplying respective percentages from flow cytometric analysis with corresponding total cell counts obtained from microscopic cell counts from single-cell suspensions from mLNs.