Janus-faced Acrolein prevents allergy but accelerates tumor growth by promoting immunoregulatory Foxp3+ cells: Mouse model for passive respiratory exposure

Abstract

Acrolein, a highly reactive unsaturated aldehyde, is generated in large amounts during smoking and is best known for its genotoxic capacity. Here, we aimed to assess whether acrolein at concentrations relevant for smokers may also exert immunomodulatory effects that could be relevant in allergy or cancer. In a BALB/c allergy model repeated nasal exposure to acrolein abrogated allergen-specific antibody and cytokine formation, and led to a relative accumulation of regulatory T cells in the lungs. Only the acrolein-treated mice were protected from bronchial hyperreactivity as well as from anaphylactic reactions upon challenge with the specific allergen. Moreover, grafted D2F2 tumor cells grew faster and intratumoral Foxp3+ cell accumulation was observed in these mice compared to sham-treated controls. Results from reporter cell lines suggested that acrolein acts via the aryl-hydrocarbon receptor which could be inhibited by resveratrol and 3'-methoxy-4'-nitroflavone Acrolein- stimulation of human PBMCs increased Foxp3+ expression by T cells which could be antagonized by resveratrol. Our mouse and human data thus revealed that acrolein exerts systemic immunosuppression by promoting Foxp3+ regulatory cells. This provides a novel explanation why smokers have a lower allergy, but higher cancer risk.

Figure 1. Inhibition of antibody-formation upon exposure to acrolein.

(A) As depicted in the allergic sensitization scheme, mice were nasally sensitized 5 times in biweekly intervals with KLH and/or acrolein or PBS as control. After a nasal challenge, lung resistance was measured by whole body plethysmography (WBP). The following day KLH-specific anaphylactic symptoms, were determined by challenging mice intraperitoneally and recording rectal temperature. (B) KLH-specific antibody-levels in serum of mice nasally treated with PBS (P, n = 4), acrolein (A, n = 8), KLH (K, n = 8) and KLH in combination with acrolein (KA, n = 8). Representative data from two independently performed experiments are shown. Groups were compared by ANOVA following Newman-Keuls Multiple Comparison test. Mean ± SEM; **P < 0.01; ***P < 0.001.

Figure 2. Reduced cytokine-response to KLH in mice exposed to acrolein during sensitization.

Supernatants of splenocytes collected after 72 h stimulation with KLH (25 μg/ml) from mice nasally treated with PBS (P, n = 4), acrolein (A, n = 8), KLH (K, n = 8) and KLH in combination with acrolein (KA, n = 8) were stimulated with KLH for 72 h and analyzed for (A) IFNγ, (B) IL13, (C) IL10, (D) IL5 and (E) BAFF. Representative data from two independently performed experiments are shown. Groups were compared by ANOVA following Newman-Keuls Multiple Comparison test. Mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3. Reduced allergic symptoms in mice exposed to acrolein are associated with an increase in the Foxp3/CD3 ratio in the lung.

(A) Mice nasally treated with KLH (K), acrolein (A), PBS (P) or with KLH in combination with acrolein (KA) were exposed to increasing doses of methacholine and the degree of airway hyperreactivity was measured by whole body plethysmography according to change in area of enhanced pause (Penh). (B) Rectal temperature drop 20 min after ip-challenge with KLH as a parameter to determine systemic anaphylactic reactions (C) Summary of CD4+CD25+Foxp3+ cell numbers counted in the spleens of the different treated mice of two independently performed experiments. (D) Whole lung sections were imaged by TissueFAX and positive stainings were quantified by Histoquest. Summary of immune-histochemical quantification % of CD3+ cells, (D) % of Foxp3+ cells (E) Foxp3/CD3 ratio of lung sections in the different treated groups. (F) Representative regions of lung sections stained for CD3 and Foxp3. 2 stained sections per animal were analysed by TissueFAX. Groups were compared by ANOVA following Newman-Keuls Multiple Comparison test. Mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4. Tumor promotion upon prior exposure to acrolein.

(A) As depicted in the treatment protocol, mice were intranasally pretreated 6 times in biweekly intervals with acrolein or PBS. After one week interval, murine mammary carcinoma cell-line D2F2 derived from BALB/c mice were injected subcutaneously in the right flank and their growth was monitored. (B) Representative pictures of excised tumors of mice previously pre-treated intranasally with PBS (P, n = 8), or acrolein (A, n = 8). Summary of (C) tumor size and (D) body weight of mice 11 days post-tumor engraftment in the different groups. Whole tumor sections were imaged by TissueFAX and positive stainings were quantified by Histoquest. Summary of immune-histochemical quantification of (E) CD3+ cells, (F) Foxp3+ cells and (G) Foxp3/CD3 ratio of tumor sections in the different treated groups. (H) Representative regions of tumor sections stained for CD3, Foxp3 and their respective isotype controls are shown. 2 sections/animal and staining were analyzed by Tissue FAX. Groups were compared by unpaired T test. Mean ± SEM; *P < 0.05; **P < 0.01.

Figure 5. Acrolein promotes Foxp3+ expression via activation of the aryl-hydrocarbon receptor.

(A) NF-κB inhibition was measured with monocytic THP1-XBlue cells, which were treated with LPS and increasing concentrations of acrolein or cinnamaldehyde for 18 hours. Bars represent summary from two independent experiments normalized to cells stimulated with LPS alone. (B) AZ-AhR cells were treated with different concentrations of acrolein or cinnamaldehyde crotonaldehyde, propanal and methacrolein in increasing concentrations (0.018/0.18/1.8/9/18/45/90/180 μM), with (C) acrolein alone or in combination with the antagonists resveratrol or 3′-methoxy-4′-nitroflavone for 18 h, before luciferase-activity was measured in the supernatant. Bars represent data from three independent experiments normalized to medium alone for B and two independent experiments for C. Data presented as mean ± SD, Statistical analyses was made using 2way RM ANOVA following Sidak’s multiple comparisons test, respectively. Significances to controls are depicted. (D) PBMCs were stimulated for 72 h with cinnamaldehyde, acrolein and or resveratrol. Gating Strategy and summary of % of CD4+CD25+Foxp3+ cells as determined by FACS of 5 independent experiments (n = 15). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001.