Route of dexamethasone administration influences parasite burden in Strongyloides hyperinfection model

Rodents infected with Strongyloides venezuelensis are experimental models applied to strongyloidiasis research. This study evaluated oral and subcutaneous dexamethasone (DEX) treatments to establish immunosuppression in an experimental model of Strongyloides hyperinfection. Rattus norvegicus Wistar were divided: G I (−): untreated and uninfected animals, G II (+): untreated and infected, G III (o −) orally treated and uninfected, G IV (o +) orally treated and infected, G V (sc −) subcutaneously treated and uninfected, G VI (sc +) subcutaneously treated and infected. For oral administration, DEX was diluted in sterile water (5 µg/ml) and made available to the animals on intervals in experimental days − 5–0, 8–13 and 21–26. For subcutaneous administration, animals received daily injections of DEX disodium phosphate (2 mg/kg). Infection was established by the subcutaneous inoculation of 3000 S. venezuelensis filarioid larvae. Groups were evaluated by egg per gram of feces and parasite females counts and IgG, IgG1 and IgG2a detection. GIV (o +) had egg peaks count on days 13 and 26 and maintained egg elimination until the last experimental day. Parasitic females recovery at day 30 was significantly higher in G IV (o +) when compared to G VI (sc +). Levels of IgG, IgG1 and IgG2a of all groups, except the positive control GII (+), were below the detection threshold. Pharmacological immunosuppression induced by oral administration of DEX produced high parasitic burden, and is a noninvasive method, useful to establish immunosuppression in strongyloidiasis hyperinfection model in rats.


Introduction
Strongyloidiasis is a neglected disease caused by the soiltransmitted helminth Strongyloides stercoralis. Infection resulting from S. stercoralis is the most chronic and deadly of the soil-transmitted helminthiases (Singer and Sarkar 2020). Disease complications lead to morbidity and mortality causing deaths in up to 87% of reported cases in immunocompromised patients with hyperinfection (Genta 1989;Buonfrate et al. 2020).
The global prevalence of the disease in 2017 was estimated to be 8.1% corresponding to 613.9 million people infected . Based on the on the global burden of strongyloidiasis, the World Health Organization (WHO) has included the control of S. stercoralis infection among the targets of the Neglected Tropical Diseases (NTD) Roadmap to 2030 .
Strongyloides venezuelensis is a rodent parasite widely used as an experimental model for human strongyloidiasis studies (Machado et al. 2003;Marques et al. 2016). In rats the experimental infection is established by filarioid larvae subcutaneous injection, and becomes patent 4 to 5 days post infection (dpi) and most of the parasites are expelled about 10-14 dpi (Viney and Kikuchi 2017). A significant difference between S. stercoralis, the human pathogen, and S. venezuelensis, the rodent one, normal life cycles is that hyperinfection and extremely chronic infections are present only in S. stercoralis infection (Breloer and Abraham 2017). Studies have used murine infected with S. venezuelensis and pharmacologically immunosuppressed by glucocorticoid treatments as an experimental model of chronic and complicated forms of strongyloidiasis (Carvalho et al. 2015;Chaves et al. 2015;Couto et al. 2020).
Dexamethasone (DEX) is a very potent anti-inflammatory and immunosuppressant drug of the glucocorticoid (GC) class, having a fluorinated structure and increased affinity for the glucocorticoid receptor (Czock et al. 2005). The GC-glucocorticoid receptor complexes then translocate into the nucleus, influencing downstream gene expression resulting in numerous biologic effects in multiple tissues (Cato et al. 2002). In addition to its extensive action on host immune system, GC is believed to act directly on the parasite Strongyloides sp. (Nutman 2017), and their use could predispose patients to chronic strongyloidiasis (Machado et al. 2011). Direct effects of DEX treatment in humans regarding enhanced fecundity or maintenance of latent forms of S. stercoralis awaits clarification at the transcriptome level (Rodpai et al. 2021). In addition to this aspect, another point to be studied is the form of administration of DEX during strongyloidiasis. There are different routes for administration of DEX, subcutaneous injection offers a reliable absorption, ensuring dosing accuracy (Earp et al. 2008;Li et al. 2017) and oral administration allows bioavailability of 60-100% (Varis et al. 2000).
Given the evident need for studies on strongyloidiasis, mainly due to its social impact on immunocompromised patients or candidates for immunosuppressive/inflammation control treatments and the different routes of administration, this study aimed to evaluate and compare oral and subcutaneous DEX treatment protocols in an experimental model of Strongyloides hyperinfection in rats experimentally infected with S. venezuelensis, assessing immunological and parasitological parameters.

Animals and experimental groups
Thirty-six male Rattus norvegicus (Wistar) rats (4-6 weeks old, weighing 100-200 g) were used in the experiments. They were Specific Pathogen Free (SPF) animals and were kept in the facilities of Rede de Biotérios de Roedores of Universidade Federal de Uberlândia (REBIR-UFU), with provision of sterilized feed and water ad libitum. Colony room ambient temperature was 22 ± 2 °C, with artificial lighting 12 h:12 h light-dark cycle.
Animals were divided in 6 groups according to DEX administration route and infection: G I (−): untreated and uninfected animals, G II (+): untreated and infected, G III (o −) orally treated and uninfected, G IV (o +) orally treated and infected, G V (sc −) subcutaneously treated and uninfected, G VI (sc +) subcutaneously treated and infected.

Immunosuppression through oral DEX administration
Orally treated groups: G III (o −) and G IV (o +) received approximately 5 µg/ml of dexamethasone (Brainfarma, Brazil), diluted in water for 5 days prior to infection with S. venezuelensis filarioid larvae, as previously described (Romand et al. 1998), with modifications. The solution prepared was transferred to feeders and made available to animals during administration period. Replacement was performed according to the water consumption. Immunosuppression reinforcement was performed on 8 and 21 dpi, lasting 5 days each, as described.

Immunosuppression through subcutaneous DEX administration
Subcutaneously treated groups: G V (sc −) and G VI (sc +) received daily injections of dexamethasone disodium phosphate (Aché, Brazil) at 2 mg/kg for 30 days. The first administration was done one hour before infection with S. venezuelensis filarioid larvae, as described by Machado et al. (2011).

Parasites and infection
Infective filarioid larvae of S. venezuelensis were obtained from fecal charcoal cultures of infected rats maintained in the REBIR-UFU. Cultures were incubated at 27 °C for 72 h and the larvae were collected by a technique based on larval thermohydrotropism (Rugai et al. 1954). Animals of infected groups: G II (+), G IV (o +) and G VI (sc +) were inoculated subcutaneously with about 3000 filarioid larvae in the abdominal region.

Serum samples collection
Blood samples were collected on days 0 (before infection), 8, 13 and 21 dpi by caudal vein; and at 30 dpi, when the animals were sacrificed by total exsanguination by cardiac puncture. They were previously anesthetized with ketamine hydrochloride (Sespo, Brazil) at 60 mg/kg and xylazine (Sespo, Brazil) at 10 mg/kg and samples were collected in tubes containing EDTA and centrifuged at 1360 g for 10 min. Serum obtained was stored at − 20 °C until use.

Parasite burden: eggs per gram of feces and parasitic females count
Feces from infected animals were collected on 5, 7, 8, 13, 21, 26 and 30 dpi for estimating the number of eggs per gram of feces (EPG) using the Cornell-MacMaster method (Gordon and Whitlock 1939). On the 30 dpi, after euthanasia, the small intestines were removed, sectioned longitudinally and incubated in a Petri dish containing PBS solution, at 37 °C for two hours. The solution was collected and centrifuged at 1000 rpm and the count of parasitic females was performed (Sato and Toma 1990).
Antibody levels: indirect ELISA to detect total IgG, IgG1 and IgG2a levels Saline extract used was produced from 300,000 filarioid larvae (Gonzaga et al. 2011). The follow up of specific S. venezuelensis IgG antibodies and IgG1 and IgG2a detection were done by indirect ELISA, according to (Couto et al. 2020). ELISA protocols were performed using pooled serum samples from each group for each experimental day tested.

Statistical analyses
Normality tests were performed. Statistical analyzes of EPG and parasitic females count were performed by the Kruskall Wallis nonparametric test. Mann-Whitney was used for multiple comparisons, first as 2 group test, followed by the Bonferroni-Holm correction. Intergroup evaluation was performed by comparative statistical analysis of parasitological parameters among infected groups [G II (+) vs. G IV (o +); G II (+) vs. G VI (sc +); and G IV (o+) vs. G VI (sc+)].
The cutoff point for each reaction was determined by the mean OD values of serum samples collected before infection (day 0). Levels of antibodies were expressed by ELISA Index (EI), where EI = sample OD / OD cutoff.
Data were presented in median (Md) and interquartile range. P values were considered statistically significant when smaller than 0.05. All tests were performed using GraphPad Prism software version 6.0 (GraphPad Software, San Diego, USA).

Oral administration of DEX leads to the increase in parasite burden
EPG median number for each infected group was performed ( Fig. 1). For G II (+), the higher EPG was reached at day 7 (Md = 16,450), and for G VI (sc +) it was at day 13 (Md = 4350). G IV (o +) presented egg elimination peaks on days 7 (Md = 20,050), 13 (Md = 79,900) and 26 (Md = 26,000). Intergroup statistical analyzes showed on day 7, G II (+) and G IV (o +) EPG medians were higher than those of G VI (sc +). At day 13, G IV (o +) median was higher than that of G II (+) and G VI (sc +). On days 26 and 30, G IV (o +) medians were higher than those of G II (+) and G VI (sc +); and G VI (sc +) medians was higher than that of G II (+).

DEX administration diminishes the detection of total IgG, IgG1 and IgG2a anti-Strongyloides
Detection of anti-S. venezuelensis antibody production in the days after infection was performed by indirect ELISA

Discussion
This is the first study to evaluate and compare oral and subcutaneous DEX treatments in the induction of pharmacological immunosuppression in rats experimentally infected with S. venezuelensis. Immunosuppressive GC therapies may increase susceptibility and facilitate the establishment of parasites in their hosts, so they are applied to several models of experimental infection, such as S. stercoralis infected NGS mice (Patton et al. 2018); monkeys (Callithrix penicillata) infected by S. stercoralis (Mati et al. 2014); Balb/c mice infected by S. venezuelensis (Machado et al. 2011); Wistar rats infected with Echinococcus multilocularis (Joekel and Deplazes 2017); gerbils infected by Haemonchus contortus (Gressler et al. 2019); and mice infected with Toxoplama gondii (Zhang et al. 2017).
Oral treatment with DEX is practical, safe, and noninvasive. It has been described by Romand et al. (1998) to establish progressive, non-lethal and constant immunosuppression in Neospora caninum infected mice. The protocol has been applied to the immunosuppression of rats infected with S. venezuelensis (Chaves et al. 2015;Carvalho et al. 2015;Gonçalves et al. 2016;Marques et al. 2016). DEX is diluted in the water consumed by the animals and it is not possible to control the amount ingested by each animal to establish the concentration in mg/kg administered of the drug. In the present study, regular reinforcements of DEX oral treatment (8 and 21 dpi) were inserted in order to prolong immunosuppression up to 30 dpi.
DEX subcutaneous treatment was first applied by Machado et al. (2011) in mice infected experimentally by S. venezuelensis. The administration of daily subcutaneous injections is advantageous since it is possible to determine the concentration of administered drug in mg/kg. The exposure is performed on a daily basis, ensuring the immunosuppressive effect on every day of the experiment. The mechanical stress to which the animals are subjected in this administration scheme constitutes a disadvantage and may generate physical weakness and increase susceptibility to secondary infections.
In this study, G IV (o +) presented the highest number of EPG, mainly on days 13 and 26, soon after the end of the oral immunosuppressive reinforcements. G IV (o +) and GVI (sc +) maintained high egg elimination until the last experimental day (day 30), while egg elimination of the positive control G II (+) was drastically reduced after day 21. In primates treated with DEX stool analyzes did not exhibit parasitological negativity during the patent infection period, and a larger number of S. stercoralis larvae were observed in the feces in relation to immunocompetent animals (Mati et al. 2014), similar to the observed in the present study. In relation to females recovered, Machado et al. (2011) reported an increased parasite burden after DEX subcutaneous administration in S. venezuelensis infected mice. Other study reported significantly higher numbers of S. stercoralis parasite females recovered in subcutaneous (2.5 mg/kg, 5 consecutive days) treated primates than in immunocompetent primates (Mati et al. 2014). Our data differs from both studies, since there was no statistical difference in the number of females recovered between the control group G II (+) and the immunosuppressed ones G IV (o +) and G VI (sc +). In G IV (o +) a higher number of females was recovered if compared to G VI (sc +).
In addition to the route of administration, the differences in results may also be due to oral DEX administration "pulses". The pharmacokinetics is beyond the scope of the work, but even so, the set of DEX oral route administration is more effective to generate bigger parasitic load, and this may be due to the better interaction of DEX with the parasite, since its absorption occurs in the intestinal wall, where parasitic females reside.
DEX not only suppress immune response, but also act directly on the parasite. It increases the fertility of adult female worms and consequently a high number of eggs and subsequent rhabditiform larvae is produced, contributing to the occurrence of high parasitic burden (Keiser and Nutman 2004;Machado et al. 2011;Genta 1992) hypothesized that the interaction of host-derived and parasite-expressed ecdysteroid-like receptors with GC could accelerate the transformation process of rabditoid larvae in filarioid larvae of S. stercoralis. Mansfield et al. (1996) presented evidence that GC can act in the rejuvenation of reproductively latent parasitic females, which return to produce viable eggs, and consequently larvae. In this sense, occurrence of higher parasitic burden in group treated orally (G IV o +) could also be explained by the better interaction of DEX with the parasite, since its absorption occurs in the intestinal wall, where parasitic females reside.
Reduction in the detection of specific antibodies was also observed in other studies that applied DEX treatment protocols. Machado et al. (2011) detected the inhibition of DEX-induced IgG1, IgG2a and IgE antibody levels in mice infected experimentally by S. venezuelensis. DEX treatment also reduced levels of IgG, IgG1 and IgE in the experimental infection of C57BL/6 mice by Echinococcus granulosus (Zhang et al. 2016). The production of antibodies is easily inhibited in species sensitive to GC. The primary mechanism consists of the inhibition or lysis of B lymphocytes, but also increases the catabolism of immunoglobulins and partial compromise of their synthesis (McEwen et al. 1997). Both treatments led to the suppression of aspects related to humoral response with reduced detection levels of rat IgG, IgG1 and IgG2a anti-Strongyloides.

Conclusion
These findings are interesting and suggest that further studies on the influence of administration route of DEX and GC in human strongyloidiasis high risk areas and specific populations will assist in treatment decisions. Studies in experimental models allow us to better understand aspects of the infection. The route of DEX administration influences on parasite burden, and oral treatment of R. norvegicus Wistar has proved to be a useful, safe and non-invasive route of administration of DEX to establish pharmacological immunosuppression in an experimental model for strongyloidiasis hyperinfection in rats, causing increased parasitic burden and decreased humoral immune response.
Author contributions LQC: designed and performed the experiments, analyzed and interpreted the data, and wrote the draft of the manuscript. BPC: assisted in carrying out the experiments, analysis and interpretation of data. EFGC: assisted in carrying out the experiments, analysis and interpretation of data. JENS: assisted in carrying out the experiments, analysis and interpretation of data. VSR: wrote, reviewed, and edited the manuscript. HTG: assisted in analysis and interpretation of data and wrote and reviewed the manuscript. JMCC: designed the experiments, acquired funding, supervised and reviewed the manuscript. All authors approved the final version of the manuscript.
Data availability All relevant data generated during this study are included in the article.

Conflict of interest The authors declare no competing interests.
Ethics approval Experimental procedures were performed in accordance with the guidelines of Brazilian College of Animal Experimentation (COBEA) and received prior approval by the Comissão de Ética na Utilização de Animais (CEUA) -Universidade Federal de Uberlândia (UFU), (protocol number 103/16).