Central importance of immunoglobulin A in host defense against Giardia spp

Abstract

The protozoan pathogen Giardia is an important cause of parasitic diarrheal disease worldwide. It colonizes the lumen of the small intestine, suggesting that effective host defenses must act luminally. Immunoglobulin A (IgA) antibodies are presumed to be important for controlling Giardia infection, but direct evidence for this function is lacking. B-cell-independent effector mechanisms also exist and may be equally important for antigiardial host defense. To determine the importance of the immunoglobulin isotypes that are transported into the intestinal lumen, IgA and IgM, for antigiardial host defense, we infected gene-targeted mice lacking IgA-expressing B-cells, IgM-secreting B-cells, or all B-cells as controls with Giardia muris or Giardia lamblia GS/M-83-H7. We found that IgA-deficient mice could not eradicate either G. muris or G. lamblia infection, demonstrating that IgA is required for their clearance. Furthermore, although neither B-cell-deficient nor IgA-deficient mice could clear G. muris infections, IgA-deficient mice controlled infection significantly better than B-cell-deficient mice, suggesting the existence of B-cell-dependent but IgA-independent antigiardial defenses. In contrast, mice deficient for secreted IgM antibodies cleared G. muris infection normally, indicating that they have no unique functions in antigiardial host defense. These data, together with the finding that B-cell-deficient mice have some, albeit limited, residual capacity to control G. muris infection, show that IgA-dependent host defenses are central for eradicating Giardia spp. Moreover, B-cell-dependent but IgA-independent and B-cell-independent antigiardial host defenses exist but are less important for controlling infection.

FIG. 1.

Primary G. muris infections of B-cell KO mice. B-cell KO mice (○) and wild-type littermate C57 control mice (•) were infected orally with 10⁴ G. muris cysts. Infection intensity was assessed at the indicated times after infection by determining stool cyst output (top panel) and total trophozoite numbers in the small intestine (bottom panel). All data are means ± SEM from nine or more mice for each data point. The detection limits of the assays are indicated by the gray line in each panel. Asterisks indicate values of B-cell KO mice that are significantly different from controls at the same time point (P < 0.01 by rank sum test).

FIG. 2.

Primary G. muris infections of IgA KO mice. IgA KO mice (○) and wild-type littermate B6129 control mice (•) were infected orally with G. muris cysts, and infection severity was assessed at the indicated times after infection as described for Fig. 1. All data are means ± SEM from 11 or more mice for each data point. The detection limits of the assays are indicated by the gray line in each panel. Asterisks indicate values of IgA KO mice that are significantly different from controls at the same time point (P < 0.05 by rank sum test).

FIG. 3.

Primary G. muris infections of secreted IgM KO mice. Secreted IgM KO mice (○) and wild-type littermate B6129 control mice (•) were infected orally with G. muris cysts, and infection severity was assessed at the indicated times after infection as described for Fig. 1. All data are means ± SEM from 11 or more mice for each data point. The detection limits of the assays are indicated by the gray line in each panel. None of the values in secreted IgM KO mice were significantly different from controls at the same time point, as determined by rank sum tests.

FIG. 4.

Secondary G. muris challenge of immunized B-cell KO and IgA KO mice. C57 control mice (top left), B-cell KO mice (bottom left), B6129 controls (top right), and IgA KO mice (bottom right) were first infected orally with a primary inoculum of 10⁴ G. muris cysts. After 7 weeks, residual infection was cleared by oral metronidazole treatment. Immunized mice were then challenged with a secondary inoculum of 10⁴ G. muris cysts, and infection severity was assessed at the indicated times after secondary challenge by determining total trophozoite numbers in the small intestine. All data are means ± SEM from 10 or more mice for each data point. For comparison, the values from the primary infections of naive mice (from Fig. 1 and 2) are shown in each panel by dashed lines. The detection limits of the assays are indicated by the gray line in each panel. Asterisks indicate values from immunized mice that are significantly different from those in naive mice of the same mouse strain at the same time point (P < 0.01 by rank sum test).

FIG. 5.

G. muris trophozoite distribution along the orad-caudad axis of the small intestine. B-cell KO mice (○) and wild-type littermate C57 control mice (•) were infected orally with 10⁴ G. muris cysts, and the number of trophozoites was determined in each of seven equally spaced segments along the small intestine, with segment 1 being the most orad segment. The numbers were then expressed as a percentage of the total trophozoite load in the small intestine, as shown in a representative example in panel A (top) for two individual mice at 3 weeks after infection. The cumulative percent trophozoite load, i.e., the sum of the percentages of the specific segment and all the segments orad of that segment, was determined for each intestinal segment, as depicted by the symbols in panel A (bottom) for the same mice shown in panel A (top). Subsequently, a sigmoid function was calculated that describes a curve with the best fit to each set of data points, as depicted by the solid and dashed lines in panel A (bottom). Based on this sigmoid function, the midpoint of the infection, i.e., the segment number (or fraction thereof) for which 50% of all trophozoites were on the orad side and 50% on the caudad side of the small intestine, was determined, as shown by the gray lines in panel A (bottom). In the example shown, the midpoint of infection was at segment 2.5 for the C57 control mouse and segment 4.0 for the B-cell KO mouse. Panel B shows a summary of the results from different times after infection. Values are means ± SEM from seven or more mice for each data point. Asterisks indicate values from B-cell KO mice that are significantly different from controls at the same time point (P < 0.05 by Student’s t test).

FIG. 6.

Primary G. lamblia infections of IgA KO mice. IgA KO mice (○) and wild-type littermate B6129 control mice (•) were infected orally with 10⁷ G. lamblia trophozoites. Infection severity was assessed at the indicated times after infection by determining total trophozoite numbers in the small intestine. All data are means ± SEM from six to eight mice for each data point. The detection limits of the assays are indicated by the gray line in each panel. Asterisks indicate values of IgA KO mice that are significantly different from controls at the same time point (P < 0.05 by rank sum test).