Histamine 1 Receptor Blockade Enhances Eosinophil-Mediated Clearance of Adult Filarial Worms

Abstract

Filariae are tissue-invasive nematodes that cause diseases such as elephantiasis and river blindness. The goal of this study was to characterize the role of histamine during Litomosoides sigmodontis infection of BALB/c mice, a murine model of filariasis. Time course studies demonstrated that while expression of histidine decarboxylase mRNA increases throughout 12 weeks of infection, serum levels of histamine exhibit two peaks-one 30 minutes after primary infection and one 8 weeks later. Interestingly, mice treated with fexofenadine, a histamine receptor 1 inhibitor, demonstrated significantly reduced worm burden in infected mice compared to untreated infected controls. Although fexofenadine-treated mice had decreased antigen-specific IgE levels as well as lower splenocyte IL-5 and IFNγ production, they exhibited a greater than fourfold rise in eosinophil numbers at the tissue site where adult L. sigmodontis worms reside. Fexofenadine-mediated clearance of L. sigmodontis worms was dependent on host eosinophils, as fexofenadine did not decrease worm burdens in eosinophil-deficient dblGATA mice. These findings suggest that histamine release induced by tissue invasive helminths may aid parasite survival by diminishing eosinophilic responses. Further, these results raise the possibility that combining H1 receptor inhibitors with current anthelmintics may improve treatment efficacy for filariae and other tissue-invasive helminths.

Fig 1. Infection of BALB/c mice with L. sigmondontis results in two peaks of histamine release.

Age-matched BALB/c mice were infected with L. sigmondontis and assessed for (A) Circulating histamine in plasma as measured by histamine ELISA, (B) Histidine Decarboxylase (HDC) mRNA from whole blood measured using RT-PCR, fold change relative to uninfected control and (C) Parasite-specific IgE titers as measured by ELISA. Data are combined from two independent experiments of 2–3 animals. *p<0.05, **p<0.01, ***p<0.001, Kruskal-Wallis test followed by Dunn Multiple comparison tests.

Fig 2. Blockade of HR1 but not HR2 results in reduced adult worm burden.

Adult worm burden in BALB/c mice infected with 40 L3s and treated with Fexofenadine (HR1i) or cimetidine (HR2i) for 8 weeks. Data are combined from two independent experiments. **p<0.01, Kruskal-Wallis test followed by Dunn Multiple comparison tests.

Fig 3. Timing of worm clearance in HR1-treated mice.

BALB/c mice were infected with L. sigmodontis by subcutaneous injection of 40 L3 larvae. Fexofenadine (HR1i) was administered in drinking water beginning 3 d prior to infection and continuing until 10, 35, or 56 d after infection. Control mice received no fexofenadine. All mice were euthanized 56 d after infection for enumeration of adult worm burdens. Representative histological sections of A) non-encased and B) granuloma-encased worms stained with hematoxylin and eosin (40x). C) Total numbers of living adult worms (both non-encased and encased) 8 weeks p.i. D) Numbers of encased living worms 8 weeks p.i. Data are combined from two to four independent experiments. *p<0.05, **p<0.01, Kruskal-Wallis test followed by Dunn Multiple comparisons.

Fig 4. Fexofenadine is not directly toxic to L. sigmodontis.

200 L3s were cultured in vitro with daily addition of 200 nM histamine, 1mM fexofenadine, or media. Survival was assessed daily by visual inspection of motility. Data are combined from two independent experiments. Possible differences between groups at each timepoint were assessed by the Kruskal-Wallis test followed by Dunn Multiple comparisons.

Fig 5. Immune modulation by histamine during L. sigmondontis infection.

(A) Total IgE from mice treated with fexofenadine (HR1i) measured by ELISA. (B) Parasite specific (LsAg) IgE in mice treated with fexofenadine (HR1i) measured by ELISA. (C, D, E) IL-4, IL-5 and IFN-γ production by CD4⁺ cells from infected fexofenadine treated mice co-cultured with CD11c⁺ cells from naïve, age-matched mice and activated with anti-CD3/anti-CD28. Cells were cultured 1:10 (CD11c⁺:CD4⁺). Cytokine production was measured by ELISA. Data are from two combined experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, Kruskal-Wallis test followed by Dunn Multiple comparisons.

Fig 6. BALB/c mice treated with fexofenadine have an increase in pleural eosinophils.

(A) Total pleural cells recovered from BALB/c and ΔGATA mice treated with fexofenadine (HR1i). (B) Total pleural eosinophils from BALB/c and ΔGATA mice treated with fexofenadine (HR1i) as determined by flow cytometry. *p<0.05, **p<0.01, Kruskal-Wallis test followed by Dunn Multiple comparisons.

Fig 7. Fexofenadine-mediated worm clearance is dependent on eosinophils.

Wild-type BALB/c and eosinophil deficient ΔdblGATA mice on a BALB/c background were infected with 40 L3-stage L. sigmodontis larvae and treated with fexofedine (HR1i) administered in drinking water for 8 wks. Control wild-type and eosinophil-deficient mice received no fexofenadine. Adult worm burden was determined in all groups at 8 wks. Data are combined from two independent experiments. ** p<0.01 by Kruskal-Wallis test followed by Dunn Multiple comparisons.