Helminth-induced interleukin-4 abrogates invariant natural killer T cell activation-associated clearance of bacterial infection

Abstract

Helminth infections affect 1 billion people worldwide and render these individuals susceptible to bacterial coinfection through incompletely understood mechanisms. This includes urinary tract coinfection by bacteria and Schistosoma haematobium worms, the etiologic agent of urogenital schistosomiasis. To study the mechanisms of S. haematobium-bacterial urinary tract coinfections, we combined the first tractable model of urogenital schistosomiasis with an established mouse model of bacterial urinary tract infection (UTI). A single bladder exposure to S. haematobium eggs triggers interleukin-4 (IL-4) production and makes BALB/c mice susceptible to bacterial UTI when they are otherwise resistant. Ablation of IL-4 receptor alpha (IL-4Rα) signaling restored the baseline resistance of BALB/c mice to bacterial UTI despite prior exposure to S. haematobium eggs. Interestingly, numbers of NKT cells were decreased in coexposed versus bacterially monoinfected bladders. Given that schistosome-induced, non-natural killer T (NKT) cell leukocyte infiltration may dilute NKT cell numbers in the bladders of coexposed mice without exerting a specific functional effect on these cells, we next examined NKT cell biology on a per-cell basis. Invariant NKT (iNKT) cells from coexposed mice expressed less gamma interferon (IFN-γ) per cell than did those from mice with UTI alone. Moreover, coexposure resulted in lower CD1d expression in bladder antigen-presenting cells (APC) than did bacterial UTI alone in an IL-4Rα-dependent fashion. Finally, coexposed mice were protected from prolonged bacterial infection by administration of α-galactosylceramide, an iNKT cell agonist. Our findings point to a previously unappreciated role for helminth-induced IL-4 in impairment of iNKT cell-mediated clearance of bacterial coexposure.

FIG 1

Bladder wall injection with S. haematobium eggs, with or without subsequent bacterial inoculation, results in mixed leukocyte infiltrates. (A) Microultrasonography of a single representative animal day 6 post-egg injection demonstrates the presence of a bright, echogenic (dense) round granuloma impinging on the left side of the urine-filled (black), otherwise ovoid bladder lumen (lower left quadrant of sonographic view). (B) Flow cytometric analyses of single-cell suspensions isolated from bacterially monoinfected and coexposed bladders demonstrate the presence of macrophages (F40/80⁺ CD11b⁺ SiglecF⁻), B cells (B220⁺), T cells (CD3⁺), eosinophils (F40/80⁺ CD11b⁺ SiglecF⁺), and neutrophils (Gr1^hi CD11b^hi). Data are representative of one of two independent experiments. Coexposed mice are denoted by “Sh+UTI89” (closed circles); monoinfected mice are shown by “UTI89” (open circles). n is 4 to 6 per group. Student t tests were used to compare values between treatment groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0005. Horizontal bars indicate median values. (C) Hematoxylin-and-eosin-stained, paraffin-embedded bladder section from a representative coexposed mouse at 7 days post-UTI89 infection (14 days post-S. haematobium egg injection). Note the presence of a dense egg granuloma in the lamina propria (upper right-hand corner).

FIG 2

S. haematobium-bacterial uropathogen coexposure results in delayed bacterial clearance. Three thousand S. haematobium eggs (Sh+UTI89; closed circles) or control vehicle (UTI89; open circles) were injected into the bladder walls of mice (A to C). Seven days later, all mice were transurethrally inoculated with 10⁸ CFU UTI89. (A and B) Urine was collected for bacterial counts 1, 4, and 7 days post-UTI89 infection. Data shown were compiled from four independent experiments. (C) Urine bacterial titers in coexposed mice were measured weekly (n = 5). Bacterial CFU differences between groups were compared using Mann-Whitney U tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Horizontal bars (A and B) indicate median values.

FIG 3

The activation status of bladder NKT cells in coexposed mice affects bacteriuria. (A) Flow cytometric analyses of single-cell suspensions isolated from bacterially monoinfected and coexposed bladders demonstrated the presence of NKT cells (DX5⁺ CD3⁺). Data from two independent experiments are shown and separated by a vertical dashed line. Coexposed mice are denoted by “Sh+UTI89” (closed circles); monoinfected mice are shown by “UTI89” (open circles). (B) Intracellular cytokine staining of bladder iNKT cells from monoinfected and coexposed mice (n = 6/group). Data from two independent experiments are shown and separated by a vertical dashed line. (C) Effect of α-GalCer on UTI89 clearance in coexposed mice. Coexposed mice were given intraperitoneal α-GalCer (2 μg, “Sh+UTI89+α-GalCer” [diamonds]) on days 0, 2, and 5 (or no α-GalCer [squares]) after UTI89 infection. Urine bacterial titers were examined at 1 and 7 days post-UTI89 infection. Data were compiled from two to four independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Horizontal bars indicate median values. MFI, median fluorescence intensity. Mann-Whitney U tests (B) and Student t tests (A and C) were used to compare values between treatment groups.

FIG 4

Baseline bacterial clearance kinetics are restored in coexposed mice in the absence of IL-4R signaling. (A) Twenty-six bladder cytokines were measured by Luminex assays after 2 days of UTI89 infection. Granulocyte colony-stimulating factor (G-CSF), IL-23, and eotaxin were below or above the range of detection (data not shown). Coexposed mice are denoted by “Sh+UTI89” (closed circles); monoinfected mice are shown by “UTI89” (open circles) (n = 4/group). (B) After 7 days of UTI89 infection, pelvic lymph nodes were collected from monoinfected (vehicle bladder wall injection followed by transurethral administration of UTI89) and coexposed (bladder wall injection with S. haematobium eggs followed by transurethral administration of UTI89) mice, and cytokine gene transcription was measured by quantitative RT-PCR (n = 4/group). (C) Urine bacterial titers were determined in coexposed wild-type (“WT”), IL-4Rα^−/−, or 11B11 antibody-treated wild-type mice 1 and 7 days post-UTI89 infection. Data were compiled from two to four independent experiments. (D) Cytokines in bladder homogenates from IL-4Rα^−/− (open circles) or wild-type (“WT,” closed circles) coexposed mice were determined by Luminex assays (n = 4). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0005. Horizontal bars indicate median values. Comparisons between treatment groups were performed using Student t tests (A, B, and D) as well as analysis of variance followed by post hoc pairwise comparisons (C).

FIG 5

Voiding patterns and granuloma sizes are similar in coexposed wild-type and IL-4Rα^−/− mice. (A) Similar voiding frequencies and total voided volumes were observed in coexposed wild-type (“WT”) and IL-4Rα^−/− mice. Data were compiled from two independent experiments. (B) At 7 and 13 days post-S. haematobium egg injection, the sizes of bladder granulomata of coexposed mice treated with phosphate-buffered saline (PBS) or 11B11 antibody were measured sonographically using the VisualSonics Vevo 2100 image system (n = 8/group). Horizontal bars indicate median values.

FIG 6

Coexposed mice downregulate expression of CD1d and MHC class II on bladder APC. CD1d (A) and MHC class II (B) expression on CD11c⁺ CD3⁻ B220⁻ DC and F4/80⁺ CD11b⁺ SiglecF⁻ macrophages were analyzed in monoinfected (“WT UTI89,” open circles) and coexposed (“WT Sh+UTI89,” closed circles) wild-type and coexposed IL-4Rα^−/− (“IL4Rα^−/− Sh+UTI89,” open triangles) mice. Data are representative of one of two independent experiments. MFI, median fluorescence intensity. Treatment groups were compared using analysis of variance followed by post hoc pairwise comparisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0005. Horizontal bars indicate median values.