Methylprednisolone Acetate Induces, and Δ7-Dafachronic Acid Suppresses, Strongyloides Stercoralis Hyperinfection in NSG Mice

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

John B. Pattona,1, Sandra Bonne-Annéea,1, Jessica Deckmana, Jessica A. Hessa, April Torigiana, Thomas J. Nolanb, Zhu Wangc,d, Steven A. Kliewerc,e, Amy C. Durhamb, James J. Leef, Mark L. Eberhardg, David J. Mangelsdorfc,d,2, James B. Lokb,2, and David Abrahama,2

aDepartment of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA 19107; bDepartment of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104; cDepartment of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390; dHoward Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390; eDepartment of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390; fDivision of Pulmonary Medicine, Department of Biochemistry and Molecular Biology, Mayo Clinic, Scottsdale, AZ 85259; and gDivision of Parasitic Diseases and Malaria, Centers for Disease Control and Prevention, Atlanta, GA 30329

Contributed by David J. Mangelsdorf, October 30, 2017 (sent for review July 12, 2017; reviewed by Timothy G. Geary and James McKerrow) Strongyloides stercoralis hyperinfection causes high mortality rates are treated with the glucocorticoid methylprednisolone acetate in humans, and, while hyperinfection can be induced by immunosup- (MPA) (8). Wild-type (WT) mice are only susceptible to the early pressive glucocorticoids, the pathogenesis remains unknown. Since larval stages of the S. stercoralis life cycle and develop both innate immunocompetent mice are resistant to infection with S. stercoralis, and adaptive responses to control the infection. Multiple com- we hypothesized that NSG mice, which have a reduced innate im- ponents of the murine immune response, including neutrophils, mune response and lack adaptive immunity, would be suscepti- eosinophils, macrophages, and the complement cascade, contrib- ble to the infection and develop hyperinfection. Interestingly, ute to the immune control of S. stercoralis (9). Eosinophils can kill despite the presence of large numbers of adult and first-stage L3i in collaboration with complement factor C3b (10, 11) whereas larvae in S. stercoralis-infected NSG mice, no hyperinfection was ob- neutrophils require C3b and alternatively activated macrophages

served even when the mice were treated with a monoclonal antibody PHARMACOLOGY to effectively kill S. stercoralis (9, 11–13). While WT mice are to eliminate residual granulocyte activity. NSG mice were then infected with third-stage larvae and treated for 6 wk with methylpred- resistant to the complete life cycle of S. stercoralis, severely nisolone acetate (MPA), a synthetic glucocorticoid. MPA treatment immune-compromised SCID mice support the development of of infected mice resulted in 50% mortality and caused a significant limited numbers of adult worms and L1 (14). This observation >10-fold increase in the number of parasitic female worms compared suggests that B and/or T cell responses are needed for complete with infected untreated mice. In addition, autoinfective third-stage clearance of the parasites from the tissue of WT mice (14). larvae, which initiate hyperinfection, were found in high numbers A highly immune-compromised strain of mouse, NOD.Cg- scid tm1Wjl in MPA-treated, but not untreated, mice. Remarkably, treatment Prkdc Il2rg /SzJ (NSG), has been developed, with pro- with Δ7-dafachronic acid, an agonist of the parasite nuclear receptor found defects in the adaptive and innate immune responses (15). Ss-DAF-12, significantly reduced the worm burden in MPA-treated mice undergoing hyperinfection with S. stercoralis. Overall, this study Significance provides a useful mouse model for S. stercoralis autoinfection and suggests a therapeutic strategy for treating lethal hyperinfection. The intestinal parasite Strongyloides stercoralis infects an estimated 100 million people. This nematode’s unique ability to Strongyloides stercoralis | hyperinfection | NSG mice | glucocorticoid | autoinfect its host enables it to persist for decades undetected and dafachronic acid to progress to a potentially fatal hyperinfection that often is induced by glucocorticoid treatment. We report a mouse model, he parasitic nematode, Strongyloides stercoralis, infects an involving the NSG strain, that recapitulates all forms of human Testimated 100 million people. Humans acquire infections strongyloidiasis. Even in severely immunocompromised NSG mice, through skin penetration by infective third-stage larvae (L3i), which glucocorticoid treatment was required for autoinfection, raising undergo development as they migrate to the intestine, where they intriguing questions about the mechanism of glucocorticoid ac- molt into parthenogenic female worms. Eggs released by female tion. Notably, administering a nematode-derived steroid, Δ7- wormshatchintheintestine,andfirst-stagelarvae(L1)maybere- dafachronic acid, which acts through a receptor within S. ster- leased in the feces, or they may develop into autoinfective third-stage coralis to regulate parasite development, significantly diminished larvae (L3a). L3a are able to penetrate the bowel and initiate a new autoinfection in glucocorticoid-treated NSG mice. This opens the infection cycle in the primary host (1). Autoinfection allows S. ster- possibility of new chemotherapy for hyperinfective strongyloidi- coralis infections to persist in an individual undetected for decades (2, asis, targeting the parasite’s own steroid hormone mechanisms. 3). Host symptoms are typically mild and nonspecific and include manifestations such as abdominal pain and diarrhea. However, in- 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. per- fected individuals treated with glucocorticoids or coinfected with formed research; J.B.P., S.B.-A., Z.W., S.A.K., D.J.M., J.B.L., and D.A. analyzed data; and human T cell lymphotropic virus type 1 (HTLV-1), may develop J.B.P., S.B.-A., Z.W., S.A.K., D.J.M., J.B.L., and D.A. wrote the paper. hyperinfection syndrome. S. stercoralis hyperinfection is characterized Reviewers: T.G.G., McGill University; and J.M., University of California, San Diego. by significantly increased parasite burden, the presence of L3a, The authors declare no conflict of interest. widespread dissemination of parasites, and translocation of gut bac- Published under the PNAS license. teria as the L3a penetrate the intestinal walls. Hyperinfection can be 1J.B.P. and S.B-A. contributed equally to this work. – life threatening, with mortality reaching 87% when untreated (4 6). 2To whom correspondence may be addressed. Email: [email protected], S. stercoralis naturally infects humans, primates, and canines [email protected], or [email protected]. (7). Gerbils are susceptible to S. stercoralis infection, with all This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. parasitic stages developing. Hyperinfection ensues when gerbils 1073/pnas.1712235114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1712235114 PNAS Early Edition | 1of6 Downloaded by guest on October 5, 2021 NSG mice are derived from the NOD\ShiLtJ background and AB carry multiple alleles reducing the function of the innate immune 80 * 1000 system, including defects in both macrophages and dendritic * 60 * 800 cells, as well as in the complement cascade (16, 17). The SCID 600 40 mutation confers loss of both T and B cell function (18). In * 400 addition, NSG mice have a null mutation of the common gamma 20 200 chain gene, which disrupts signaling of six different interleukins * L1 Recovered Adult Recovered (19). Monocytes and neutrophils are present in NSG mice al- 0 0 though their functional capacity is currently unknown. Both NOD SCID NSG NOD SCID NSG macrophages and dendritic cells are also found in NSG mice but C57BL/6J NOD/SCID C57BL/6J NOD/SCID are functionally compromised due to deficiencies in cytokine C D signaling and other genetic defects of the NOD\ShiLtJ back- 1500 ground (17, 20). Numbers of eosinophils in NSG mice have not * NSG been determined (15). 1000 * NOD/ The Caenorhabditis elegans nuclear receptor DAF-12, which SCID regulates the switch between dauer and continuous reproductive 500 SCID development (21), is conserved in S. stercoralis (22). The natural L3 Recovered 0 ligands of C. elegans DAF-12, the dafachronic acids (23), have 300 400 500 600 the capacity to signal through homologs of DAF-12 in S. stercoralis NOD SCID NSG μm and Ancylostoma caninum, stimulating resumption of development C57BL/6J NOD/SCID by L3i of these parasites (24, 25) and suppressing morphogenesis of S. stercoralis L3i in both the postparasitic and post–free-living Fig. 1. Susceptibility of immunodeficient mice to infection with Strong- generations (24, 25). The ability of Δ7-dafachronic acid (Δ7-DA) yloides stercoralis. Mice were infected with 5,000 S. stercoralis L3i, and, 6 wk postinfection, mice were necropsied and parasite recoveries in NOD (n = 7), to perturb L3i development in diverse clades of parasitic nema- SCID (n = 10), NOD/SCID (n = 10), and NSG (n = 10) mice were compared with todes bolsters the potential of the DAF-12 signaling pathway as a WT C57BL/6J mice (n = 8). Data presented are means ± SDs (*P ≤ 0.05) of chemotherapeutic target (24). total number of (A) adults, (B) L1, and (C) L3 recovered. (D) L3 recovered Based on the fact that SCID mice are somewhat susceptible to from the tissues of infected SCID, NOD/SCID, and NSG mice were fixed and S. stercoralis infection, it was hypothesized that NSG mice would measured. The experiment was performed twice with the same results. Data be fully susceptible to the infection. Susceptibility of NSG mice to presented are from both experiments. infection with S. stercoralis was compared in the present study with the susceptibility of B6.CB17-Prkdcscid/SzJ (SCID) (18), NOD/ ShiLtJ (NOD) (16, 21), and NOD.CB17-Prkdcscid/J (NOD/SCID) to destroy complement, parasite killing did not occur, indicating (17) mice. A second study hypothesis, based on the current ac- that both sera were sufficient sources of complement for killing cepted dogma that hyperinfection develops in humans due to the of the larvae (Fig. S2). absence of a functional immune response (1, 5, 6, 26, 27), was that Flow cytometry was performed (29) on both blood and spleen NSG mice would spontaneously develop hyperinfection. Finally, cells from uninfected mice comprising each of the five strains we asked whether the naturally occurring C. elegans steroid, Δ7- tested in this study to determine the numbers and percentages of DA, could suppress formation of L3a in NSG mice. innate immune cells. C57BL/6J and SCID mice had equivalent numbers of eosinophils and neutrophils. Neutrophils were also Results unaffected in NOD, NOD/SCID, and NSG mice (Table S1) Susceptibility of Immunodeficient Mice to Infection with S. stercoralis. while eosinophils were absent from NOD mice and the derivative C57BL/6J, NOD, SCID, NOD/SCID, and NSG mice were in- strains NOD/SCID and NSG (Table S1). Based on morpholog- fected s.c. with S. stercoralis L3i. Six weeks postinfection, mice ical differential cell analysis, eosinophils were found in the bone were necropsied, and parasites were collected from all body tis- marrow of NSG mice (Fig. S3) but not in the peripheral blood. sues. All stages of the parasite were absent from C57BL/6J and NOD mice. Similar numbers of adult worms were recovered from Anti-Ly6G Monoclonal Antibody Depletes Granulocytes from NSG SCID (6 ± 6) and NOD/SCID (7 ± 3) mice, and a significantly Mice but Does Not Induce Hyperinfection. We hypothesized that greater number were recovered from NSG mice (50 ± 21) (Fig. L3a and hyperinfection did not develop in NSG mice due to 1A). Parallel to the adult worm recoveries, L1 were recovered in control of the infection by neutrophils. NSG mice were treated low numbers from SCID (2 ± 3) and NOD/SCID (1 ± 2) mice and with an anti-Ly6G monoclonal antibody to eliminate neutrophils. in significantly greater numbers from NSG mice (240 ± 554) (Fig. Anti-Ly6G antibody was effective at near complete elimination 1B). The number of L3 recovered from SCID (158 ± 123) mice of neutrophils from both blood and spleen at all time points was significantly lower than either NOD/SCID (585 ± 480) or NSG tested (Fig. S4). NSG mice, infected with L3i, were treated with (557 ± 543) mice (Fig. 1C). All larvae recovered from the tissues control or anti-Ly6G monoclonal antibodies, and parasites were were within the same size range, indicating that the larvae were all recovered at 2, 4, and 6 wk postinfection. There were no sig- L3i and not L3a (Fig. 1D and Fig. S7A). nificant differences in numbers of adults, L1 or L3, at any time Killing of S. stercoralis larvae by the innate and adaptive re- point regardless of neutrophil depletion. L3a were not observed sponse in mice depends on C3b (28) in collaboration with at any time point in neutrophil-depleted mice (Fig. S5). macrophages, neutrophils, and eosinophils (10–12). As NSG mice have a defect in complement activity (17), we hypothesized NSG Mice Infected with S. stercoralis Treated with MPA Develop that the increased susceptibility of NSG mice to S. stercoralis Hyperinfection. NSG mice were infected with S. stercoralis L3i might be due to a deficiency in formation of complement com- and given 2 mg of MPA weekly. Mortality was observed begin- ponent C3b. C3 levels measured in serum from naive NSG and ning at day 19 in the MPA-treated mice, with a final mortality of C57BL/6J mice were found to be equivalent (Fig. S1). Serum 54%, compared with 11% in controls (Fig. 2A). Animals were recovered from NSG and C57BL/6J mice was used in an in vitro euthanized at day 42 to minimize morbidity and mortality. His- parasite killing assay. Macrophages and neutrophils from topathological analysis of the lungs of infected MPA-treated C57BL/6J mice killed the larvae if serum from NSG or C57BL/6J NSG mice at 6 wk revealed severe pulmonary hemorrhage and mice was added to the cultures. If the sera were heat-inactivated edema, alveolar histiocytosis, and marked interstitial congestion,

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1712235114 Patton et al. Downloaded by guest on October 5, 2021 A B Starting at 3 wk postinfection, there were greater numbers of L3 recovered from the MPA-treated infected mice, attaining 100 1500 NSG statistical significance at 5 wk postinfection (Fig. 2D). At 6 wk 1000 NSG : MPA 80 * * postinfection, 248 ± 187 L3 were recovered from infected NSG 500 * ± 60 * mice and 3,459 1,784 from infected mice treated with MPA. 100 Within the L3 population, L3i could be distinguished from L3a 40 80 based on size (Fig. 2E) and shape of the tail (Fig. S7A). The L3i 60 Parasites has a bifid tail whereas the L3a has a bluntly pointed tail (Fig. Percent survival 20 Adults Recovered 40 Parasites + MPA 20 S7B). L3a were not observed in untreated infected NSG mice; 0 0 23456 however, they did develop in infected mice treated with MPA 0 10203040 beginning week 3 and continuing through week 6, with increasing Days Weeks frequency each week within the L3 population (Fig. 2 E and F). CD 16000 * MPA Does Not Alter Switching by Postparasitic S. stercoralis L1 NSG 8000 NSG 12000 NSG : MPA NSG : MPA * Between Homogonic and Heterogonic Developmental Alternatives. 6000 We tested the hypothesis that MPA incites autoinfection by di- 8000 * * * rect action on S. stercoralis by determining whether MPA can 4000 * * 4000 alter the course of development of postparasitic first-stage larvae 100 (PPL1) in culture. PPL1, which are eliminated from the host in L1 Recovered 75 L3 Recovered 2000 the feces, are capable of developing either to free-living males 50 25 and females (heterogonic development) or directly to the L3i 0 0 (homogonic development). When the UPD strain of S. stercoralis 23456 23456 Weeks Weeks is reared at 22 °C, the heterogonic pattern of development predominates; homogonic development by PPL1 can be induced E F 4000 by raising the culture temperature to 37 °C (30). L3i L3i L3a MPA did not elicit a significant departure from the pre- 2 Wk 3000 dominant pattern of heterogonic development seen in controls

developing at 22 °C (Fig. 3A). MPA also had no effect on the PHARMACOLOGY 3 Wk 2000 pattern of homogonic development seen in control cultures 4 Wk reared at 37 °C (Fig. 3B). In contrast, treatment with Δ7-DA, a 5 Wk 1000 DAF-12 agonist, eliminated the small proportion of L3i due to

L3i vs L3a Recovered homogonic development seen in control and MPA-treated 6 Wk 0 worms reared at 22 °C (Fig. 3A). Moreover, at 37 °C, Δ7-DA 200 300 400 500 600 23456 treatment completely prevented infective L3i homogonic devel- μm Weeks opment seen in controls and instead induced all of the worms to develop into heterogonic fourth-stage larvae (L4) and free-living Fig. 2. Effect of methylprednisolone acetate (MPA) treatment on NSG mice infected with Strongyloides stercoralis.(A) Survival of NSG mice infected adults (Fig. 3B). with S. stercoralis and treated with MPA. Mice were infected with 5,000 Δ S. stercoralis S. stercoralis L3i and treated with 0.5 mg MPA. Data presented are means ± Orally Administered 7-DA Disrupts Hyperinfection in SDs (*P ≤ 0.05) of the number of (B) adults and (C) L1, and (D) L3 recovered MPA-Treated NSG Mice. Given the ability of Δ7-DA to suppress at 2 through 6 wk postinfection (n = 5 for vehicle and n = 10 for MPA- L3i formation in postparasitic worms, we next tested dafachronic treated mice). (E) Distribution of L3i and L3a within the L3 population re- acid’s effects on hyperinfection in NSG mice. NSG mice were covered 2 to 6 wk postinfection. L3i control worms were isolated from infected with L3i and treated with MPA. Starting at 2 wk post- coprocultures as size reference. (F) Number of L3i and L3a, based on size, infection and continuing for the remainder of the experiment, recovered from MPA-treated NSG mice 2 to 6 wk postinfection. mice were treated with 1 and 10 μM Δ7-DA added to the

with increased numbers of circulating neutrophils. Additionally, intravascular and parenchymal larvae were noted in one mouse in 22 C 37 C AB* Control this group; severe necrosuppurative pleuritis with Gram-negative 100 100 * MPA bacilli was also noted in this mouse. Intestinal lesions in these mice 7 included a mild neutrophilic and histiocytic enteritis with abundant 80 80 larvae and bacterial overgrowth. At the time of death, two mice had larvae invading the gastrointestinal tract, heart, lungs, testis, 60 60 and skeletal muscle (Fig. S6 A and B). Bacteria were also observed in the heart, kidney, and lungs (Fig. S6C and Table S2). 40 40 Beginning at 2 wk postinfection, the number of adult worms 20 20 recovered from the intestines of infected mice treated with MPA Class in Developmental % was significantly higher than seen in untreated infected mice. ND ND The highest number of adults was seen at week 6 (708 ± 684) 0 0 i L3i L3 (Fig. 2B). MPA-treated mice had significantly higher numbers of FLAd L1 in the intestines throughout the infection. The number of L4-FLAd L4- L1 peaked in MPA-treated mice at 5 wk postinfection, with an average of 8,750 ± 7,096 parasites recovered per mouse (Fig. Fig. 3. Proportions of Strongyloides stercoralis L3i and heterogonic fourth- stage larvae (L4) or free-living adults (FLAd) developing at 22 °C (A)or37°C 2C). Between 2 and 6 wk, adult S. stercoralis from MPA-treated (B) in cultures treated with 1 μM methylprednisolone acetate (MPA), 1 μM mice produced a greater number of L1 than adults in untreated Δ7-dafachronic acid (Δ7), or ethanol control. Data are means ± SD of three mice. At 5 wk postinfection, adults in MPA-treated mice pro- biological replicates with total sample sizes for treatment or control groups duced 20 times more L1 per worm than adults in untreated mice. ranging from 111 to 192 parasites (*P ≤ 0.05). ND, not detected.

Patton et al. PNAS Early Edition | 3of6 Downloaded by guest on October 5, 2021 drinking water. At necropsy 6 wk postinfection, mice treated with partitioned into L3i (Fig. 4D)andL3a(Fig.4E), it was apparent Δ7-DA in the absence of MPA had parasite numbers equivalent that this decline in total L3 could be attributed to a significant to controls (Fig. 4). In contrast, in mice treated with MPA and decrease in both L3i and L3a in mice treated with MPA and 10 μM Δ7-DA, there was a significant dose-dependent decrease in Δ7-DA (Fig. 4E). These results demonstrate that Δ7-DA has a worms recovered at 6 wk postinfection compared with treatment beneficial therapeutic effect in a mouse model of strongyloidiasis with MPA alone (Fig. 4). This dose-dependent effect of Δ7-DA hyperinfection. was particularly notable in the L3 population. Total L3 recov- Discussion eries trended lower in mice treated with 1 μM Δ7-DA and MPA and were significantly lower in mice treated with 10 μM Δ7-DA In this study, we describe an NSG mouse model that is susceptible to infection by S. stercoralis and importantly can be induced and MPA compared with mice treated with MPA alone (Fig. into a lethal hyperinfection with glucocorticoid treatment. Thus, 4C). When these L3 were analyzed morphologically (Fig. S7) and this model constitutes an inbred murine strain recapitulating the full spectrum of S. stercoralis infection that is observed in humans and therefore should have broad application in characterizing A 40 Adult * 20 Adult this infectious disease. The NSG mouse model also has the po- * tential to be humanized, reconstituted with human hemato- 30 15 poietic stem cells, enabling the study of the human immune response to S. stercoralis infection (17). S. stercoralis infections in 20 10 humans are extremely long-lived (2, 31), through a process of 10 5 autoinfection, whereby L1 develop in the intestine into L3a that penetrate the wall of the lower ileum, colon, or the skin of the

Worm Recovery (X100) 0 0 perianal region, whereby L3a enter the circulation, travel to the lungs, as well as other routes, and then to the small intestine, B 600 L1 600 L1 * thus repeating the life cycle. This maintains the parasite for decades in the human host, with infection levels moderated to 400 400 limit pathogenicity. We conclude that NSG mice are susceptible to infection with S. stercoralis due to an absence of a functional 200 200 immune response and the presence of cues and growth factors required for the parasite to complete its life cycle. When NSG 0 mice were treated with the glucocorticoid MPA, the large

Worm Recovery (X100) 0 number of adult worms was stimulated to hyperproduce L1, of C 800 50 L3 L3 * which a percentage developed into L3a. Some of these L3a 400 40 completed a systemic migration similar to L3i and matured into 30 adults in the intestines, which increased the intestinal worm 90 burden. Unlike L3i, the L3a do not penetrate the skin but mi- 60 20 grate out of the intestine, carrying with them bacteria that can 30 10 potentially induce fatal systemic infections. What properties of the NSG mouse permit its susceptibility to Worm Recovery (X100) 0 0 S. stercoralis infection? NSG mice are characterized by profound D 200 L3i 15 L3i * deficiencies in both innate and adaptive immunity (15). In WT 100 mice, two parasite-killing mechanisms have been identified in the innate immune response, one depending on eosinophils (10, 11) 20 10 and the other on collaboration between neutrophils and macro- 15 5 phages (12). Importantly, the activity of both mechanisms is 10 compromised in NSG mice, contributing to the susceptibility of 5 this strain. The innate and adaptive immune responses also require 0 0 Worm Recovery (X100) complement factor C3b (28). NSG mice lack a functional complement cascade (32) but still maintain serum levels that are E 600 L3a 40 L3a 400 * equivalent to those occurring in WT mice. Furthermore, serum 30 derived from NSG mice was shown to be a sufficient source of C3b 80 in in vitro killing assays. Thus, we conclude that the defect in innate 60 20 immunity in NSG mice that allows S. stercoralis to complete its life 40 10 cycle is not in complement activation but rather in cell activity. 20 Although NSG mice support the complete life cycle of

Worm Recovery (X100) 0 0 S. stercoralis, they do not allow formation of significant numbers of MPA ++ ++ L3a or show any common signs of hyperinfection. This is surprising Δ7-DA 1 μM 1 μM 10 μM 10 μM in view of the widely held hypothesis that hyperinfection occurs when there is a deficiency in the immune response (1, 5, 6), which Fig. 4. Effects of orally administered dafachronic acid on the course of may explain the prevalence of hyperinfections that are induced by S. stercoralis infection and hyperinfection in NSG mice treated with MPA. All glucocorticoid and HTLV-1 exposure (33). necropsies were conducted at 6 wk of infection following 4 wk of continual Because neutrophils are one of the few immune effector cells administration of 1 and 10 μM Δ7-dafachronic acid (Δ7) in drinking water, remaining in NSG mice that are known to kill S. stercoralis,we ∼ · −1· −1 which delivers a dose of 0.1 and 1.0 mg kg d , respectively. MPA-treated considered whether these cells might be responsible for blocking the mice received 2 mg i.p. weekly for 6 wk. Data are means ± SDs (*P ≤ 0.05) of the total number of (A) adults, (B)L1,(C) total L3 population, (D) L3i, and (E) development of hyperinfection. However, treatment with an anti- L3a recovered 6 wk postinfection. Left panels represent the 1 μM Δ7-DA Ly6G monoclonal antibody that depleted circulating and splenic treatment group (n = 5 to 8 mice per group from two independent exper- neutrophils from NSG mice did not affect the typical profile of a iments); Right panels represent the 10 μM Δ7-DA treatment group (n = 5to S. stercoralis infection. This observation suggests that neutrophils do 8 mice per group from one experiment). not suppress hyperinfection by S. stercoralis in NSG mice.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1712235114 Patton et al. Downloaded by guest on October 5, 2021 The necessity of glucocorticoid treatment to incite hyper- acid treatment would similarly suppress formation of L3a in NSG infection in the NSG mouse as it does in humans naturally leads to mice with MPA-induced S. stercoralis hyperinfection. Indeed, the the question of what the target of MPA action might be. A plau- most striking effect of the DAF-12 agonist, Δ7-DA, was its sup- sible explanation is that glucocorticoid suppression of the immune pression of L3a populations in S. stercoralis-infected NSG mice system is required. However, ablating residual immune mecha- treated with MPA. The ability of Δ7-DA to markedly decrease the nisms from NSG mice that are known to control S. stercoralis in- worm burden during hyperinfection, particularly of the L3a stage, fection in other strains failed to incite hyperinfection in non–MPA- strongly supports the notion that pharmacologic targeting of the treated animals. These findings prompt two alternative hypotheses. DAF-12 pathway could be an innovative therapeutic strategy. One is that glucocorticoids or their host metabolites promote In conclusion, this study demonstrates the establishment of a hyperinfection through an as yet unknown component of immune mouse model that simulates S. stercoralis infection in humans. function or other physiological mechanism in the host that regu- Importantly, we also provide compelling evidence that targeting lates hyperinfection by the parasite. A potential target of gluco- the nuclear receptor DAF-12 has the capacity to suppress all stages corticoid activity may be the intestine. The intestinal mucosa of parasite development through the autoinfective process. In the contains high levels of the immunosuppressive cytokines TGFβ and aggregate, these findings reinforce the potential of endogenous IL-10 that are produced not only by the regulatory T cells and steroid receptor signaling as a therapeutic target in hyperinfective dendritic cells, but importantly also by epithelial cells (34) that are strongyloidiasis, an often-fatal complication of S. stercoralis in- still present in the NSG mouse. Given the profound effects glu- fection in humans, and possibly in a broad range of other nema- cocorticoids have on the intestine and in protecting the intestinal tode parasitisms of medical and veterinary significance. barrier (35), and the fact that L3a translocation across the intestinal barrier is required for hyperinfection, it is reasonable to speculate Materials and Methods that the intestinal milieu plays an important role in steroid-induced All animal experiments were conducted in compliance with the guidelines set hyperinfection. A second hypothesis is that glucocorticoids incite forth by the Institutional Animal Care and Use Committees (IACUC) at Thomas hyperinfective strongyloidiasis by direct action on the parasite, as Jefferson University, the University of Pennsylvania, and the University of envisioned by Genta (36), independent of host-based mechanisms. Texas Southwestern Medical Center. UPD-strain Strongyloides stercoralis L3i Demonstrating that glucocorticoids can signal through an appro- were obtained from coproculture as previously described (38). Five thousand priate nuclear receptor in the parasite would bolster this alternative L3i were injected s.c. into mice. Necropsy consisted of analysis of all body hypothesis; however, experiments to date have failed to demon- tissues for the presence of parasites. L3i and L3a were differentiated based on lengths of fixed worms. Murine macrophages and polymorphonuclear

strate such signaling and so fail to support the hypothesis of direct PHARMACOLOGY effects of glucocorticoids on S. stercoralis. Notably, immunosup- leukocytes (PMNs) were derived as previously described and used in in vitro killing assays (12). Serum from C57BL/6J and NSG mice was used as a source pressive steroids fail to activate the DAF-12 nuclear receptor that is of complement. Then, 0.5 mg of anti-Ly6G antibody, RB6-8C5, was injected i.p. found in both C. elegans and S. stercoralis (22). In contrast, we Δ twice per week for 6 wk to eliminate neutrophils. Mice were treated with found that 7-dafachronic acid, which is a known agonist for DAF- 2 mg of methylprednisolone acetate (MPA) once per week for 6 wk by i.p. 12, suppressed glucocorticoid-induced hyperinfection (see next injection. Δ7-DA was synthesized as described (39). Mice were inoculated s.c. paragraph). This finding is significant because Ss-DAF-12 is ho- with L3i and then given weekly i.p. injections of MPA for 6 wk. Beginning in mologous to the nuclear receptor that governs switching between week 3 and continuing through week 6, Δ7-DA was administered to mice dauer and continuous developmental fates in C. elegans L3 (23). continuously at concentrations of 1 μMand10μM in their drinking water Furthermore, results of the present in vitro developmental along with the weekly MPA injections. Mice consumed 5 to 7 mL/d of water, −1 −1 switching experiment indicated that MPA cannot promote direct which results in a delivery dose of ∼0.1 and ∼1.0 mg·kg ·d of Δ7-DA, respectively. L1, isolated from the intestines of gerbils inoculated with L3i in the development by postparasitic S. stercoralis L1 to free living L3i and Δ so failed to support the alternate hypothesis. However, this result absence of MPA treatment, were transferred to DMEM with either MPA or 7- DA and cultured at 37° or 22 °C. Differential counts, of L3i and worms having does not discount that MPA promotes the development of L1 to developed to heterogonic fourth-stage larvae or free-living adults, were done the parasitic L3a (24). There presently is no method that promotes 24 h after culture inoculation for steroid-treated worms. Statistics and mo- development of S. stercoralis L3a in vitro. lecular, biological, and pharmacokinetic analyses were performed as described The most consistent effect of dafachronic acid on Strongyloides in SI Materials and Methods. development, and the one most relevant to the present study, is its capacity to suppress formation of L3i, which are similar in ACKNOWLEDGMENTS. We thank Hongguang Shao, Grace Zhang, Nicole form to L3a, in the progeny of both free-living adults of Bacarella, Erika Klemp, and Lindsay McMenemy for technical assistance. We Strongyloides papillosus (37) and S. stercoralis (23, 24) and in the thank Noelle Williams and the University of Texas Southwestern Preclinical Pharmacology Core for determining the pharmacokinetics of Δ7-DA. We progeny of parasitic female S. stercoralis (24) that are voided thank Tim Manser for identifying the initial resources and for providing from the host in feces. In the latter instance, development of encouragement for these studies. This work was supported by National In- L1 harvested from the intestines of infected gerbils and reared at stitutes of Health Grants AI105856 (to D.A., J.B.L., and D.J.M.), AI22662 (to 37 °C was shifted from its predominating pattern of direct de- 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 velopment to L3i, and instead the majority of these L1 developed (to D.J.M.); and by the Howard Hughes Medical Institute (D.J.M.). The NIH into free-living female worms. In view of these suppressive ef- Referral Center Grant OD P40-10939 (to Dr. Charles Vite) provided research fects on L3i morphogenesis, we hypothesized that dafachronic materials for the study.

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