Ascaris suum-derived products suppress mucosal allergic inflammation in an interleukin-10-independent manner via interference with dendritic cell function

Abstract

We have previously demonstrated that protection from allergic inflammation by Ascaris suum infection was characterized by a global increase in interleukin-10 (IL-10) and the development of protective CD4(+)/CD25(+) T cells (L. Schopf, S. Luccioli, V. Bundoc, P. Justice, C. C. Chan, B. J. Wetzel, H. H. Norris, J. F. Urban, Jr., and A. Keane-Myers, Investig. Ophthalmol. Vis. Sci. 46:2772-2780, 2005). Here, we used A. suum pseudocoelomic fluid (PCF) in lieu of infection to define molecular mechanisms of allergic protection in a mouse model of allergic inflammation. Mice were sensitized with ragweed (RW) and PCF (RW/PCF), PCF alone, or RW alone and then challenged intratracheally, intranasally, and supraocularly with RW. Histological examination of the eyes and lungs, analysis of the bronchoalveolar lavage fluid (BALF), and characterization of ex vivo cytokine responses were performed to determine allergic inflammatory responses. RW/PCF-treated mice had suppressed allergic immune responses compared to mice given RW alone. To investigate whether IL-10 was involved in PCF-mediated allergic protection, similar experiments were performed using mice genetically deficient for IL-10. Persistent protection from allergic disease was observed in the absence of IL-10, indicating the primary mechanism of PCF protection is IL-10 independent. Ex vivo and in vitro analysis of PCF-treated dendritic cells (DC) demonstrated reduced activation receptor expression and cytokine production in response to either RW or lipopolysaccharide stimulation. These findings extend previous studies that showed infection with A. suum alters expression of allergic disease and suggest that PCF can contribute to this effect by interference with DC function.

FIG. 1.

Allergic conjunctival and pulmonary inflammation following RW sensitization and challenge. Five to eight A/J mice per treatment group were treated with PBS, PCF, RW, or RW and PCF. A. RW challenge in sensitized animals resulted in a significant infiltration of eosinophils into the conjunctiva (***, P < 0.001) compared with all other treatment groups). This allergen-induced eosinophil infiltration was reduced by PCF administration during RW sensitization (RW/PCF). B. Periodic acid-Schiff staining of representative lung tissue sections 72 h after allergen challenge. C. Lung sections were assigned an inflammatory lung score using a scale of 0 to 4, with 4 representing the score for a maximal inflammatory response in the lung. Mice given RW sensitization had a significant increase in lung inflammatory scores compared to those of mice sensitized with PBS or PCF treatment alone post-RW challenge (***, P < 0.001). Mice treated with PCF during RW sensitization had reduced lung inflammatory scores after RW challenge compared to animals given RW alone. D. Total cell numbers were counted from the BALF and demonstrated a significant increase in total cell numbers in the lung lumen in mice given RW sensitization (***, P < 0.001) compared with all other groups of mice. Mice treated with PCF during RW sensitization had significantly reduced cell numbers in the lung lumen after RW compared with animals given RW alone. E. Eosinophils were counted from the BALF as a measure of allergic inflammation. Mice given RW sensitization demonstrated a significant increase in eosinophil infiltrates in the BALF post-RW challenge (***, P < 0.001) compared to all other treatment groups. Mice treated with PCF during RW sensitization had significantly reduced eosinophil numbers in the BALF after RW challenge compared with animals given RW alone.

FIG. 2.

Cytokines from the BALF (A to C) and antigen-specific recall assays (D and E) in response to RW challenge. The levels of the cytokines IL-5 (A), IL-13 (B), IL-4 (C), and IFN-γ (data not shown) were measured in the BALF. The level of the proallergic cytokines IL-5 (A), IL-13 (B), and IL-4 (C) were significantly higher (P < 0.05) in the BALF of RW-sensitized and -challenged mice than in control mice given PBS or PCF alone. Mice given PCF during RW sensitization had a significant reduction (P < 0.05) in the amount of IL-5 (A) and IL-13 (B) in the BALF compared with animals given RW sensitization alone. ND, not detected. (C) The level of IL-4 in the BALF from animals given PCF during RW sensitization was also reduced compared to that of animals sensitized with RW alone, but this reduction did not reach the level of significance. The level of the cytokines IL-5 (D) and IL-13 (E) were also assessed in antigen-specific recall assays in response to RW challenge and in response to RW challenge in vitro in splenocytes from RW-sensitized and -challenged mice compared with control mice given PBS or PCF alone (***, P < 0.001). Mice given PCF treatment prior to challenge had a significant reduction in the amount of both IL-5 (D) and IL-13 (E) in in vitro antigen-specific recall assays.

FIG. 3.

Allergic pulmonary inflammation following OVA sensitization and challenge. Five to eight BALB/c mice per treatment group were sensitized with OVA to determine alterations of the allergic inflammatory response to a nonpollen allergen (OVA) post-PCF treatment. A. Periodic acid-Schiff staining of representative lung tissue sections 72 h after allergen challenge. B. OVA challenge in sensitized mice resulted in increased pulmonary inflammatory scores (**, P < 0.01) compared with all other treatment groups. Mice given PCF during OVA sensitization had a significant reduction of pulmonary inflammation compared to animals given OVA sensitization alone.

FIG. 4.

Allergic conjunctival and pulmonary inflammation following RW sensitization and challenge in IL-10-deficient mice. Five to eight IL-10-deficient mice were treated with PBS, RW, PCF, or RW and PCF. A. Five conjunctival sections per mouse per treatment group were counted to determine eosinophil infiltration 72 h postallergen challenge. RW sensitization and challenge in IL-10-deficient animals led to increased eosinophil infiltrates in the conjunctiva compared with all other groups (*, P < 0.05). Mice treated with PCF during RW sensitization had a reduced eosinophilic ocular infiltration after RW challenge compared with animals given RW alone. B. IL-10-deficient mice given RW sensitization had a significant increase in lung inflammatory scores compared to mice sensitized with PBS or PCF treatment alone post-RW challenge (***, P < 0.001). Mice treated with PCF during RW sensitization had a reduced lung inflammatory score after RW challenge compared with animals given RW alone.

FIG. 5.

Ex vivo analysis of peritoneal lavage cells. A. Peritoneal lavage of mice 6 days after i.p. injection from PBS, RW, PCF, RW/PCF, OVA, or OVA/PCF treatment demonstrated a significant decrease in total numbers of peritoneal lavage cells than what was observed in naïve animals or from mice 24 h postperitoneal injection (P < 0.05). B. FACS analysis of lavage cells revealed significant increases in the percentage of CD11c⁺ DC cells in all groups of animals 24 h after i.p. injection compared with naïve control animals (P < 0.001) that was further increased in all groups at the 6-day post-i.p. time point (P < 0.001).

FIG. 6.

Ex vivo and in vitro analysis of DC activation. CD11c⁺ cells removed ex vivo from the peritoneal cavity, spleens, and mediastinal LN after either i.p. sensitization with PCF, RW, or RW/PCF (A) or i.p. sensitization with PCF, OVA, or OVA/PCF (B). i.p. injection of either RW or OVA (A and B, respectively; green) led to increased expression of the costimulatory marker CD86 compared to intraperitoneal DC from mice that received sham sensitization with PBS (red) or PCF alone (blue). (A) Intraperitoneal DC derived from RW/PCF-treated mice (brown) showed reduced levels of CD86 compared to DC from mice in the RW alone group (green). (B) Similar reductions were seen in mice given OVA/PCF treatment (brown) compared with injection of OVA alone (green). Expression of LPS-induced CD40 (C) (green) and CD86 (D) (green) was dramatically reduced in BMDC in vitro after preexposure to PCF (D) (blue). (E) LPS-induced production of IL-12 was significantly inhibited in a dose-dependent manner when the BMDC were preexposed with increasing doses of PCF.