Tim-3 is dispensable for allergic inflammation and respiratory tolerance in experimental asthma

Abstract

T cell immunoglobulin and mucin domain-containing molecule-3 (Tim-3) has been described as a transmembrane protein, expressed on the surface of various T cells as well as different cells of innate immunity. It has since been associated with Th1 mediated autoimmune diseases and transplantation tolerance studies, thereby indicating a possible role of this receptor in counter-regulation of Th2 immune responses. In the present study we therefore directly examined the role of Tim-3 in allergic inflammation and respiratory tolerance. First, Tim-3-/- mice and wild type controls were immunized and challenged with the model allergen ovalbumin (OVA) to induce an asthma-like phenotype. Analysis of cell numbers and distribution in the bronchoalveolar lavage (BAL) fluid as well as lung histology in H&E stained lung sections demonstrated a comparable degree of eosinophilic inflammation in both mouse strains. Th2 cytokine production in restimulated cell culture supernatants and serum IgE and IgG levels were equally increased in both genotypes. In addition, cell proliferation and the distribution of different T cell subsets were comparable. Moreover, analysis of both mouse strains in our respiratory tolerance model, where mucosal application of the model allergen before immunization, prevents the development of an asthma-like phenotype, revealed no differences in any of the parameters mentioned above. The current study demonstrates that Tim-3 is dispensable not only for the development of allergic inflammation but also for induction of respiratory tolerance in mice in an OVA-based model.

Fig 1. Immunization protocols.

(A) Induction of allergic phenotype. WT and Tim-3^-/- mice were sensitized at days 0 and 14 by -i.p. application of OVA in Alum, resulting in an allergic phenotype; whereas negative control mice were i.p. immunized with NaCl 0.9% in Alum. Sensitization was followed by i.n. challenges at consecutive days 14-16 and 20-22 with either OVA or saline, respectively. Section of all mice at day 23. (B) Induction of tolerant phenotype. In order to induce a tolerant phenotype, mice received i.n. tolerization with OVA in saline at days -6 and -3 (square), followed by i.p. immunization with OVA in Alum at days 0 and 14 and i.n. challenges with OVA at consecutive days 14-16 and 20-22. Section of all mice at day 23.

Fig 2. BALF analysis and OVA-specific immunoglobulin E.

(A, B) Bronchoalveolar lavage fluid was obtained from each individual mouse (WT, Tim-3^-/-) at section day to determine total cell count (A) and number of eosinophils (B). No significant differences were detected between WT and Tim-3^-/- OVA and TOL mice, resulting in comparable allergic and tolerant phenotypes. (Data of three independent experiments, n = 12-15 animals per group.) (C) Significant and comparable decrease in IgE production for WT and Tim-3^-/- TOL mice compared to OVA controls. (One representative out of three independent experiments, n = 5-6 animals for each group.) Mann-Whitney-U-test. * p ≤ 0.05; ** p ≤ 0.01. All data are presented as mean ± SEM.

Fig 3. OVA-specific cytokine production and proliferation of splenic lymphocytes.

(A-C) Production of IL-5 (A), IL-10 (B) and IL-13 (C) in cell culture supernatants of spleens showed a significant decrease after tolerization compared to allergic (OVA) animals in both, WT and Tim-3^-/- mice. (D) Tolerant mice showed a significant increase of IFN-γ in Tim-3^-/- mice. (E) Proliferation of splenic lymphocytes by cell titer glow assay after OVA restimulation showed decreased proliferation in TOL animals of WT and Tim-3^-/- mice. Increased levels of luminescence were detected in allergic mice, both WT and Tim-3^-/- animals, no significant differences between WT and Tim-3^-/- mice. (One representative out of three independent experiments; n = 5-6 animals per group.) Student`s unpaired t-test.* p ≤ 0.05, ** p ≤0.01, n.s. p > 0.05. All data are presented as mean ± SEM.

Fig 4. Lung histology analysis.

(A-D) Lungs were stained for eosinophilic lung inflammation (H&E, A) or mucus secretion (PAS, C), followed by computer-based quantification using an image analyzing program (B and D). Significantly decreased inflammation and mucus production was found in TOL mice compared to allergic controls (OVA). In contrast, WT and Tim-3^-/- mice were not significantly altered. (Three independent experiments; n = 5-6 animals per group.) Mann-Whitney-U-test. * p ≤ 0.05, ** p ≤0.01, n.s. p > 0.05. All data are presented as mean ± SEM.

Fig 5. Antigen-specific Th cells.

(A, B) WT or Tim-3^-/- mice were immunized with OVA/Alum i.p. (OVA), PBS/Alum i.p. (Alum), or received OVA i.n. (TOL) before challenge with OVA i.n. as described in methods. 23 days later mice were sacrificed and single cell suspensions prepared from spleens were analyzed flow cytometrically. The frequency (A) and absolute numbers (B) of CD4⁺ Th cells (black bars) and CD4⁺CD154⁺ cells (grey bars) were determined as described in methods. Number of animals per group was two. Error bars represent the standard deviation.