Development of Acanthocheilonema viteae in Meriones shawi: Absence of microfilariae and production of active ES‐62

Abstract Aims ES‐62 is a well‐studied anti‐inflammatory molecule secreted by L4‐adult stage Acanthocheilonema viteae. We maintain the life cycle of A viteae using Meriones libycus as the definitive host. Here, we investigated whether the full life cycle could be maintained, and functional ES‐62 produced, in a related jird species—Meriones shawi. Methods and Results Adult worms were produced in comparable numbers in the two species, but very few microfilariae (MF) were observed in the M shawi bloodstream. M shawi ES‐62 produced ex vivo was functional and protective in a mouse model of arthritis. Myeloid‐derived cells from naïve and infected jirds of both species were compared with respect to ROS production and osteoclast generation, and some differences between the two species in both the absence and presence of infection were observed. Conclusions The life cycle of A viteae cannot be successfully completed in M shawi jirds but L3 stage worms develop to adulthood and produce functional ES‐62. Preliminary investigation into jird immune responses suggests that infection can differentially modulate myeloid responses in the two species. However, species‐specific reagents are required to understand the complex interplay between A viteae and its host and to explain the lack of circulating MF in infected M shawi jirds.


| INTRODUC TI ON
; allergic diseases such as asthma 7 and, more recently, obesity-accelerated ageing, 8 in mouse models.
We routinely maintain the full life cycle of A viteae in the jird, M libycus, and adult worms can be retrieved and cultured ex vivo to isolate and purify ES-62. Despite being an excellent host for A viteae, our M libycus colony is difficult to maintain due to the absence of a commercial source for providing new members (the colony was transferred to Strathclyde from the National Institute for Medical Research, London, in 1991) resulting in a high degree of inbreeding that makes it challenging to produce healthy offspring. A viteae can also be developed in the more readily available closely related Meriones unguiculatus, 9 but our previous use of this species resulted in production of female worms notably smaller than those developed in M libycus (results not shown). We therefore explored whether we could establish the life cycle in a further closely related readily available species of jird, Meriones shawi, and whether nematodes from these jirds could produce functional ES-62. We were also interested in investigating the effect of ES-62 on the jird immune response. Despite having extensively studied the effect of ES-62 in mouse models of disease, we know very little about how the molecule interacts with the immune system of a receptive host. Work in this area has been hampered by the lack of jird-specific reagents required to investigate the effect of ES-62 on specific immune cell populations; however, we were able to study the effects of the nematode product on certain functional responses of host cells.

| Acanthocheilonema viteae life cycle
The Acanthocheilonema viteae life cycle was maintained in Meriones libycus and in soft ticks (Ornithodoros moubata), as described previously. 10 Briefly, adult male jirds were infected subcutaneously with 120 arthropod-derived larvae (L3) which matured into adult worms over the course of 3 months. The number of MF was then determined in a small blood sample (5 µL), and jirds with an appropriate level of MF (approximately 120 MF in 5 µL blood) were used to infect ticks to complete the life cycle. Adult worms were obtained from jirds by dissection from under the skin of jird pelts following exsanguination using CO 2 and counted and sexed-female worms were identified as larger nematodes while males are shorter and have a 'corkscrewed' phenotype. The worms were cultured ex vivo in 'complete' RPMI 1640 medium (containing 50 U/mL penicillin and 50 µg/mL streptomycin, supplemented with 10% glucose [Life Technologies]) for generation of ES-62 as described previously. [5][6][7][8] The same procedures were applied to Meriones shawi.

| Reactive oxygen species (ROS) flow cytometry assay
The presence of ROS in BM monocytes and BMMs was measured using 2′,7′-dichlorofluorescein diacetate (DCF-DA, Sigma). Briefly, ROS in BM monocytes was measured following red cell lysis, while ROS in BMMs was measured after stimulation with pathogen-asso-

| In vitro osteoclast differentiation
Osteoclasts (OCs) were differentiated from naïve or infected M libycus or M shawi BM as previously described using mouse M-CSF and RANKL reagents. 10 BM was assessed for OC differentiation by TRAP staining (Leukocyte Acid Phosphatase Kit, Sigma) on day 6, and cells that stained positive for TRAP with ≥3 nuclei were counted as OCs.
Images were obtained on an EVOS FL Auto Cell Imaging System at 20× magnification with scale bars set at 200 µm.

| Collagen-induced arthritis model
Male DBA/1 mice (8-10 weeks; Harlan Olac), for the collagen-induced arthritis (CIA) model, 5,11 were maintained at the Central Research Mice were treated subcutaneously with PBS or M shawi purified, endotoxin-free ES-62 (2 µg/dose) on days −2, 0 and 21. Joint damage (articular score) was scored as previously described. 5,11 Draining lymph nodes were dissected at cull and processed and analysed by flow cytometry based on size and granularity. Lymphocytes were categorized as either resting (small size and nongranular) or activated (small size and granular), and the percentage of activated cells within the lymphocyte gate was calculated.

| Statistics
All data were analysed using GraphPad Prism 6 software, employing statistical analysis by Student's t test, one-way ANOVA with Fisher's LSD post-test, two-way ANOVA with Tukey's multiple comparisons and linear regressions where appropriate. In all cases, *P < .05, **P < .01 and ***P < .001.

| Acanthocheilonema viteae fecundity is host sub-species specific
Acanthocheilonema viteae is considered to only fully mature in a limited range of hosts of the rodent family Gerbillae, for example M libycus and Gerbillus hirtipes. 12 Figure 1E).
Additionally, treatment with M shawi ES-62 was able to significantly suppress lymphocyte activation following ex vivo stimulation compared to control mice, confirming that M shawi ES-62 was immunomodulatory in the model ( Figure 1F).

| Acanthocheilonema viteae trains host myeloid cell responses in Meriones libycus
Meriones shawi adult worms were found to produce live MF when cultured ex vivo, and examination of these by light microscopy revealed no obvious difference from those produced by females de-

ROS Production (RFU)
Naive Infected Naive Infected arthritis. As mentioned earlier, 11 ES-62 can inhibit osteoclastogenesis in protecting against joint disease in the CIA model of RA, but the differential effect observed in the two jird species suggests it may not be active in this way in the present study, at least in M shawi.

CO N FLI C T O F I NTE R E S T
The authors have no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
JD and FL contributed equally to this manuscript, maintained the

D I SCLOS U R E S
None.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.