Neuroimmune semaphorin 4A downregulates the severity of allergic response

Abstract

To define the role of semaphorin 4A (Sema4A) in allergic response, we employed Sema4A⁻/⁻ and wild-type (WT) mice in the experimental model of ovalbumin (OVA)-induced allergic airway inflammation. We observed a selective increase in eosinophilic airway infiltration accompanied by bronchial epithelial cell hyperplasia in allergen-treated Sema4A⁻/⁻ mice relative to WT mice. This enhanced inflammatory response was associated with a selective increase in bronchoalveolar lavage (BAL) interleukin 13 (IL-13) content, augmented airway hyperreactivity, and lower regulatory T cell (Treg) numbers. In vivo allergen-primed Sema4A⁻/⁻ CD4+ T cells were more effective in transferring T helper type 2 (Th2) response to naive mice as compared with WT CD4+ T cells. T-cell proliferation and IL-13 productions in OVA₃₂₃₋₃₃₉-restimulated Sema4A⁻/⁻ cell cultures were upregulated. Generated bone marrow chimeras showed an equal importance of both lung-resident cell and inflammatory cell Sema4A expression in optimal disease regulation. These data provide a new insight into Sema4A biology and define Sema4A as an important regulator of Th2-driven lung pathophysiology.

Figure 1

Experimental protocols of the allergen treatment (a) and the in vivo adoptive cell transfer followed by the antigen nebulizations (b). Models are detailed in the Materials and Methods section.

Figure 2

Sema4A deficiency increases the severity of allergic airway response in mice. (A) WT and Sema4A^−/− mice were immunized with OVA as shown in Supplemental Figure 1a. Control mice were challenge with PBS. (a) The average numbers (n=3–5) of BAL total cells (white bars) macrophages (black bars), eosinophils (forward hatched bars), lymphocytes (reverse hatched bars) and neutrophils (gray bars) ± SEM in one of three representative experiments are shown. *, # p<0.05, differences in absolute numbers of total cells and eosinophils in BAL between OVA-challenged WT and Sema4A^−/− mice. (b) Lung tissue sections were stained with H&E for inflammation and (c) Periodic acid stain (PAS) for mucus cell hyperplasia evaluation. Magnification used for pictures is 40x and for inserts is 100x. (d) Anti-OVA specific IgG Abs in serum samples (n=4 mice/group) were measured using corresponding ELISA kits as described in Material and Methods. *p<0.05, OVA-treated WT versus Sema4A^−/− mice. (e) The levels of cytokines in BAL fluids of OVA-challenged WT (open bars) and Sema4A^−/− (black bars) mice measured by either ELISA or protein array were combined. Data are shown as mean ± SEM (n = 3–6 mice). *p<0.05, OVA-challenged WT versus Sema4A^−/− mice. (f) For cytokine CBA in lung lysates and 5x concentrated BALF data were acquired by BD FACS Calibur and shown are FlowJo generated dot plots for individual proteins for an individual mouse in one of 2 representative experiments (n=2/group). For a numeric representation of cytokine concentration, CBA data were calculated using the BD CBA software. *p<0.02 and *p<0.004, MCP-1 and IL-17A in WT vs Sema4A−/− mice, correspondingly. (g) Airway reactivity is shown as the percent increase in PenH over a baseline (n = 6 – 8 mice/group in the separate 3 experiments). *p< 0.05, OVA-treated WT mice versus Sema4A^−/− mice for the corresponding doses of methacholine. Forced oscillation measurements were done in anesthetized mice employing FlexiVent (SCIREQ) and airway resistance is graphed as cm H₂O/ml/s (n = 2 mice per group).

Figure 3

Sema4A expression on BM-derived cells and lung resident cells is critical for the downregulation of allergic airway response. The indicated bone marrow chimeras were generated as described in Materials and Method. Non-irradiated WT mice and the chimeras were immunized with Alum or OVA/Alum and challenged as indicated. (a) Lungs were lavaged and the cells in the BAL were analyzed by the differential counting. The average numbers of BAL total cells (white bars) macrophages (black bars), eosinophils (hatched bars), lymphocytes (dotted bars) and neutrophils (grid bars) ± SEM are shown (n=3–4 mice/group). *, †, #p<0.05, differences in absolute numbers of total cells, macrophages and eosinophils, respectively, in BAL of Sema4A^−/− (d) – Sema4A^−/− (r) versus WT (d) – Sema4A^−/− (r) or Sema4A^−/− (d) – WT (r). WT (d) – WT (r) group was assessed in a separate experiment. (b) Representative lung histology (H&E, 40x; inserts, 100x) from PBS- and OVA-challenged mice is shown. (c) Representative periodic acid stain (PAS) for mucus cell hyperplasia. Magnification used for pictures is 40x. (d) Cytokine contents in concentrated BAL measured using a CBA Th1/Th2/Th17 kit. Data are shown as FlowJo generated dot plots and CBA software calculated cytokine concentrations for polled samples from 2 mice in one of 2 representative experiments.

Figure 4

CD4+ T cells but not DC from OVA-challenged Sema4A^−/− mice transfer allergic eosinophilic response to naïve mice followed by allergen challenges. Mice which received either lung DC (CD11c+CD11b+ cells) or spleen CD4+ T cells obtained from either OVA-challenged WT or Sema4A^−/− mice were subjected to three consecutive allergen nebulizations and assessed for lung inflammatory response 24h later (n=3–5). Allergen challenged mice which did not receive cell transfer served as controls. Note the inflammatory cell accumulations in the lung tissues of WT and Sema4A^−/− mice after Sema4A^−/− CD4+ T cell transfer followed by allergen nebulizations.

Figure 5

Increased T cell and decreased Treg numbers are associated with a heightened allergic response in Sema4A^−/− mice. WT and Sema4A^−/− mice were immunized and challenged with OVA as depicted in Supplemental Figure 1a. Lungs were digested with collagenase-DNAse and spleens were sterily processed omitting a digestion step. Single cell suspensions were analyzed by FACS for the expression of CD4 and CD8 T cell markers (a, b). Gated CD4+ T cells were further analyzed for the expression of CD25 and Foxp3 and the relative number of CD4+Foxp3+ cells was ascertained in comparison to a control rat IgG protein stain (a). Spleen T and B cell relative numbers in one out of three representative independent experiments are shown (b).

Figure 6

Sema4A deficiency does not affect the in vitro recall T cell response to a whole allergen but upregulates the response to the OVA_323–339 peptide challenge. (a–c) Spleen MNC were obtained from allergen-treated WT (open circles or bars) and Sema4A^−/− (black circles or bars) mice as described in the Materials and Methods. Cells were stimulated with either increasing doses of OVA (A) or 200 μg/ml of OVA_323–339 peptide (b–c) for 72h. (a–b) Proliferation was measured in triplicate wells. [H] was added to the cultures for the last 24h before cell harvest. Data are presented as mean cpm ± SEM of [H] incorporation in 72h cultures. (b) Means and SEM are shown for the measured O.D. of cells incubated with WST-1 solution for the colorimetric proliferation visualization. Means and SEM of [H] incorporation in 72h cultures. (c) Supernatants were taken at 72h from the in vitro cell cultures restimulated with OVA_323–339 peptide. IL-5 and IL-13 contents are expressed as the average of corresponding cytokine concentration for 2 representative samples for each point.