Methylprednisolone acetate induces, and Δ7-dafachronic acid suppresses, <em>Strongyloides stercoralis</em> hyperinfection in NSG mice

John Patton

Sandra Bonne-Année

Jessica Deckman

Jessica Hess

Amy Durham+5 authors

Supplementary Material

Supplementary File

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^{Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA, 19107;}

^{Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, 19104;}

^{Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, 75390;}

^{Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, 75390;}

^{Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, 75390;}

^{Division of Pulmonary Medicine, Department of Biochemistry and Molecular Biology, Mayo Clinic, Scottsdale, AZ, 85259;}

^{Division of Parasitic Diseases and Malaria, Centers for Disease Control and Prevention, Atlanta, GA, 30329}

^{To whom correspondence may be addressed. Email:}ude.nretsewhtuostu@ognam.ovad, ude.nnepu.tev@kolj, or ude.nosreffej@maharba.divad.

Contributed by David J. Mangelsdorf, October 30, 2017 (sent for review July 12, 2017; reviewed by Timothy G. Geary and James McKerrow)

Author contributions: J.B.P., S.B.-A., Z.W., S.A.K., D.J.M., J.B.L., and D.A. designed research; J.B.P., S.B.-A., J.D., J.A.H., A.T., T.J.N., Z.W., A.C.D., J.J.L., M.L.E., and J.B.L. performed research; J.B.P., S.B.-A., Z.W., S.A.K., D.J.M., J.B.L., and D.A. analyzed data; and J.B.P., S.B.-A., Z.W., S.A.K., D.J.M., J.B.L., and D.A. wrote the paper.

Reviewers: T.G.G., McGill University; and J.M., University of California, San Diego.

^{J.B.P. and S.B-A. contributed equally to this work.}

Contributed by David J. Mangelsdorf, October 30, 2017 (sent for review July 12, 2017; reviewed by Timothy G. Geary and James McKerrow)

Published under the PNAS license.

Significance

The intestinal parasite Strongyloides stercoralis infects an estimated 100 million people. This nematode’s unique ability to autoinfect its host enables it to persist for decades undetected and to progress to a potentially fatal hyperinfection that often is induced by glucocorticoid treatment. We report a mouse model, involving the NSG strain, that recapitulates all forms of human strongyloidiasis. Even in severely immunocompromised NSG mice, glucocorticoid treatment was required for autoinfection, raising intriguing questions about the mechanism of glucocorticoid action. Notably, administering a nematode-derived steroid, Δ7-dafachronic acid, which acts through a receptor within S. stercoralis to regulate parasite development, significantly diminished autoinfection in glucocorticoid-treated NSG mice. This opens the possibility of new chemotherapy for hyperinfective strongyloidiasis, targeting the parasite’s own steroid hormone mechanisms.

Keywords: Strongyloides stercoralis, hyperinfection, NSG mice, glucocorticoid, dafachronic acid

Significance

Abstract

Strongyloides stercoralis hyperinfection causes high mortality rates in humans, and, while hyperinfection can be induced by immunosuppressive glucocorticoids, the pathogenesis remains unknown. Since immunocompetent mice are resistant to infection with S. stercoralis, we hypothesized that NSG mice, which have a reduced innate immune response and lack adaptive immunity, would be susceptible to the infection and develop hyperinfection. Interestingly, despite the presence of large numbers of adult and first-stage larvae in S. stercoralis-infected NSG mice, no hyperinfection was observed even when the mice were treated with a monoclonal antibody to eliminate residual granulocyte activity. NSG mice were then infected with third-stage larvae and treated for 6 wk with methylprednisolone acetate (MPA), a synthetic glucocorticoid. MPA treatment of infected mice resulted in 50% mortality and caused a significant >10-fold increase in the number of parasitic female worms compared with infected untreated mice. In addition, autoinfective third-stage larvae, which initiate hyperinfection, were found in high numbers in MPA-treated, but not untreated, mice. Remarkably, treatment with Δ7-dafachronic acid, an agonist of the parasite nuclear receptor Ss-DAF-12, significantly reduced the worm burden in MPA-treated mice undergoing hyperinfection with S. stercoralis. Overall, this study provides a useful mouse model for S. stercoralis autoinfection and suggests a therapeutic strategy for treating lethal hyperinfection.

Abstract

The parasitic nematode, Strongyloides stercoralis, infects an estimated 100 million people. Humans acquire infections through skin penetration by infective third-stage larvae (L3i), which undergo development as they migrate to the intestine, where they molt into parthenogenic female worms. Eggs released by female worms hatch in the intestine, and first-stage larvae (L1) may be released in the feces, or they may develop into autoinfective third-stage larvae (L3a). L3a are able to penetrate the bowel and initiate a new infection cycle in the primary host (1). Autoinfection allows S. stercoralis infections to persist in an individual undetected for decades (2, 3). Host symptoms are typically mild and nonspecific and include manifestations such as abdominal pain and diarrhea. However, infected individuals treated with glucocorticoids or coinfected with human T cell lymphotropic virus type 1 (HTLV-1), may develop hyperinfection syndrome. S. stercoralis hyperinfection is characterized by significantly increased parasite burden, the presence of L3a, widespread dissemination of parasites, and translocation of gut bacteria as the L3a penetrate the intestinal walls. Hyperinfection can be life threatening, with mortality reaching 87% when untreated (4 –6).

S. stercoralis naturally infects humans, primates, and canines (7). Gerbils are susceptible to S. stercoralis infection, with all parasitic stages developing. Hyperinfection ensues when gerbils are treated with the glucocorticoid methylprednisolone acetate (MPA) (8). Wild-type (WT) mice are only susceptible to the early larval stages of the S. stercoralis life cycle and develop both innate and adaptive responses to control the infection. Multiple components of the murine immune response, including neutrophils, eosinophils, macrophages, and the complement cascade, contribute to the immune control of S. stercoralis (9). Eosinophils can kill L3i in collaboration with complement factor C3b (10, 11) whereas neutrophils require C3b and alternatively activated macrophages to effectively kill S. stercoralis (9, 11 –13). While WT mice are resistant to the complete life cycle of S. stercoralis, severely immune-compromised SCID mice support the development of limited numbers of adult worms and L1 (14). This observation suggests that B and/or T cell responses are needed for complete clearance of the parasites from the tissue of WT mice (14).

A highly immune-compromised strain of mouse, NOD.Cg-Prkdc^Il2rg^{/SzJ (NSG), has been developed, with profound defects in the adaptive and innate immune responses (}15). NSG mice are derived from the NOD\ShiLtJ background and carry multiple alleles reducing the function of the innate immune system, including defects in both macrophages and dendritic cells, as well as in the complement cascade (16, 17). The SCID mutation confers loss of both T and B cell function (18). In addition, NSG mice have a null mutation of the common gamma chain gene, which disrupts signaling of six different interleukins (19). Monocytes and neutrophils are present in NSG mice although their functional capacity is currently unknown. Both macrophages and dendritic cells are also found in NSG mice but are functionally compromised due to deficiencies in cytokine signaling and other genetic defects of the NOD\ShiLtJ background (17, 20). Numbers of eosinophils in NSG mice have not been determined (15).

The Caenorhabditis elegans nuclear receptor DAF-12, which regulates the switch between dauer and continuous reproductive development (21), is conserved in S. stercoralis (22). The natural ligands of C. elegans DAF-12, the dafachronic acids (23), have the capacity to signal through homologs of DAF-12 in S. stercoralis and Ancylostoma caninum, stimulating resumption of development by L3i of these parasites (24, 25) and suppressing morphogenesis of S. stercoralis L3i in both the postparasitic and post–free-living generations (24, 25). The ability of Δ7-dafachronic acid (Δ7-DA) to perturb L3i development in diverse clades of parasitic nematodes bolsters the potential of the DAF-12 signaling pathway as a chemotherapeutic target (24).

Based on the fact that SCID mice are somewhat susceptible to S. stercoralis infection, it was hypothesized that NSG mice would be fully susceptible to the infection. Susceptibility of NSG mice to infection with S. stercoralis was compared in the present study with the susceptibility of B6.CB17-Prkdc^{/SzJ (SCID) (}18), NOD/ShiLtJ (NOD) (16, 21), and NOD.CB17-Prkdc^{/J (NOD/SCID) (}17) mice. A second study hypothesis, based on the current accepted dogma that hyperinfection develops in humans due to the absence of a functional immune response (1, 5, 6, 26, 27), was that NSG mice would spontaneously develop hyperinfection. Finally, we asked whether the naturally occurring C. elegans steroid, Δ7-DA, could suppress formation of L3a in NSG mice.

Acknowledgments

We thank Hongguang Shao, Grace Zhang, Nicole Bacarella, Erika Klemp, and Lindsay McMenemy for technical assistance. We thank Noelle Williams and the University of Texas Southwestern Preclinical Pharmacology Core for determining the pharmacokinetics of Δ7-DA. We thank Tim Manser for identifying the initial resources and for providing encouragement for these studies. This work was supported by National Institutes of Health Grants AI105856 (to D.A., J.B.L., and D.J.M.), AI22662 (to J.B.L.), OD P40-10939 (to Dr. Charles Vite), and R01DK067158 (to S.A.K. and D.J.M.); by Robert A. Welch Foundation Grants I-1558 (to S.A.K.) and I-1275 (to D.J.M.); and by the Howard Hughes Medical Institute (D.J.M.). The NIH Referral Center Grant OD P40-10939 (to Dr. Charles Vite) provided research materials for the study.

Acknowledgments

Footnotes

The authors declare no conflict of interest.

See Commentary on page 9.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1712235114/-/DCSupplemental.

Footnotes

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