Adaptive immunity alters distinct host feeding pathways during nematode induced inflammation, a novel mechanism in parasite expulsion

Abstract

Gastrointestinal infection is often associated with hypophagia and weight loss; however, the precise mechanisms governing these responses remain poorly defined. Furthermore, the possibility that alterations in feeding during infection may be beneficial to the host requires further study. We used the nematode Trichinella spiralis, which transiently inhabits the small intestine before migrating to skeletal muscle, as a biphasic model of infection to determine the cellular and molecular pathways controlling feeding during enteric and peripheral inflammation. Through the infection of genetically modified mice lacking cholecystokinin, Tumor necrosis factor α receptors and T and B-cells, we observed a biphasic hypophagic response to infection resulting from two separate immune-driven mechanisms. The enteroendocrine I-cell derived hormone cholecystokinin is an essential mediator of initial hypophagia and is induced by CD4+ T-cells during enteritis. In contrast, the second hypophagic response is extra-intestinal and due to the anorectic effects of TNFα during peripheral infection of the muscle. Moreover, via maintaining naive levels of the adipose secreted hormone leptin throughout infection we demonstrate a novel feedback loop in the immunoendocrine axis. Immune driven I-cell hyperplasia and resultant weight loss leads to a reduction in the inflammatory adipokine leptin, which in turn heightens protective immunity during infection. These results characterize specific immune mediated mechanisms which reduce feeding during intestinal or peripheral inflammation. Importantly, the molecular mediators of each phase are entirely separate. The data also introduce the first evidence that I-cell hyperplasia is an adaptively driven immune response that directly impinges on the outcome to infection.

Figure 1. T. spiralis induced enteritis and peripheral myositis produces a CCK dependent and independent bi-phasic hypophagia.

Food intake of naïve and infected wild-type (A) and CCK^lacZ (B) mice, derived via weighing chow daily. Representative CCK (C) and lacZ (D) I-cell staining from day 9 p.i. wild-type and CCK^lacZ duodenum respectively. Black arrows indicate I-cells. Black bar = 100 µm. (E) Number of CCK/LacZ positive I-cells/20 VCU in wild-type and CCK^lacZ mice as determined from immunohistochemistry or immunofluorescence respectively. Data (n = 8–10 mice/group) from two independent experiments. *, P<0.05; **, P<0.01; or ***, P<0.005 between naïve and infected groups, error bars represent SE of means.

Figure 2. Adoptive transfer of CD4+ T-cells to SCID mice restores hypophagia during enteritis.

Food intake of naïve and infected wild-type (A), SCID (B) and adoptively transferred SCID mice (E), derived via weighing chow daily. Number of CCK positive I-cells in wild-type, SCID and adoptively transferred SCID mice (C) and number of goblet cells in wild-type SCID and adoptively transferred SCID mice (D); cells/20 VCU accessed via immunohistochemistry and periodic acid-Schiff's histology staining respectively. Data (n = 4 mice per group) from 2 independent experiments. *, P<0.05; **, P<0.01; or ***, P<0.005 between naïve and infected groups, error bars represent SE of means.

Figure 3. Secondary hypophagia during T. spiralis induced peripheral inflammation is absent in p55/p75 −/− mice.

(A) TNFα serum levels during infection in wild-type mice determined via cytometric bead array (B) Number of CCK positive cells/20 VCU, as determined from immunohistochemistry in wild-type and p55/p75 −/− mice. Food intake of naïve and infected wild-type (C) and p55/p75 −/− (D) mice, derived via weighing chow daily. Data (n = 7–16 mice per group) are from three independent experiments. *, P<0.05; **, P<0.01; or ***, P<0.005 between naïve and infected groups, error bars represent SE of means.

Figure 4. Hypophagia and weight loss during T. spiralis induced enteritis reduces the pro-inflammatory adipokine leptin.

(A) Change in basal weight during infection. (B) Serum leptin levels during infection, determined via ELISA. (C) Representative CD4+ intracellular IFN-y and IL-4 flow cytometry plots during Th2 polarization in control and leptin treated mLN cells. Data (n = 4–8 mice per group) are from two independent experiments. *, P<0.05; **, P<0.01; or ***, P<0.005 between naïve and infected groups or for indicated comparisons, error bars represent SE of means.

Figure 5. The maintenance of basal leptin levels during T. spiralis infection results in delayed parasite expulsion.

(A) Serum leptin levels in PBS and leptin treated mice, determined via ELISA. (B) Adult and larval worm burdens in infected mice. (C) IFN-y, IL-4 and IL-13 cytokine levels from Ag-specific re-stimulation of day 8 p.i. mLN cells from PBS and leptin treated mice, grey lines represent naïve levels; determined via cytometric bead array. (D) Mast cells/20 VCU in PBS and leptin treated mice; accessed via toludine blue staining. Data (n = 9 mice per group) are from two independent experiments. *, P<0.05; **, P<0.01; or ***, P<0.005 between naïve and infected groups or for indicated comparisons, error bars represent SE of means.