Distinct hepatic myeloid and lymphoid cell repertoires are associated with susceptibility and resistance to Ascaris infection

The soil-transmitted helminth Ascaris lumbricoides infects ~800 million people worldwide. Some people are heavily infected, harbouring many worms, whereas others are only lightly infected. The mechanisms behind this difference are unknown. We used a mouse model of hepatic resistance to Ascaris, with C57BL/6J mice as a model for heavy infection and CBA/Ca mice as a model for light infection. The mice were infected with the porcine ascarid, Ascaris suum or the human ascarid, A. lumbricoides and immune cells in their livers and spleens were enumerated using flow cytometry. Compared to uninfected C57BL/6J mice, uninfected CBA/Ca mice had higher splenic CD4+ and γδ T cell counts and lower hepatic eosinophil, Kupffer cell and B cell counts. Infection with A. suum led to expansions of eosinophils, Kupffer cells, monocytes and dendritic cells in the livers of both mouse strains and depletions of hepatic natural killer (NK) cells in CBA/Ca mice only. Infection with A. lumbricoides led to expansions of hepatic eosinophils, monocytes and dendritic cells and depletions of CD8+, αβ, NK and NK T cells in CBA/Ca mice, but not in C57BL/6J mice where only monocytes expanded. Thus, susceptibility and resistance to Ascaris infection are governed, in part, by the hepatic immune system.


Introduction
An estimated 800 million people are infected with the human roundworm Ascaris lumbricoides (Pullan et al., 2014). Despite this high number of infected individuals, ascariasis remains a neglected tropical disease (Deslyper and Holland, 2017;Hotez et al., 2020;World Health Organization, 2020). Intensity of infection is not evenly distributed among the population, where a small subset of the population carries the majority of the worm burden (Bethony et al., 2006). Furthermore, people regain similar worm burdens upon reinfection, even after several rounds of chemotherapy (Seo et al., 1979;Anderson and May, 1982;Croll et al., 1982;Elkins et al., 1986;Holland et al., 1989). This is known as predisposition and appears to be multifactorial in origin (Holland, 2009) with both long-term (host genetics and socio-economic status) and short-term (host-acquired immune system) factors involved (McCallum, 1990).
As heavy worm burden is associated with more severe symptoms (Croll and Ghadirian, 1981;Holland, 2009), it is important to understand the underlying molecular mechanisms associated with the observed predisposition. Because the early life cycle of the parasite includes internal organs of the host, it is necessary to use animal models (Holland et al., 2013). Building on an earlier study (Mitchell et al., 1976), our group ) developed a mouse model for resistance to Ascaris suum infection. We identified two mouse strains, one (C57BL/6J) as a model for susceptibility to heavy infection and another (CBA/Ca) as a model for resistance to Ascaris infection. Using this mouse model, the hepatic stage during larval migration was identified as the most likely time at which the observed differences between the two mouse strains in eventual lung larval burdens were generated (Lewis et al., 2007;Dold et al., 2010). We subsequently used this mouse model to investigate the liver proteomes of the relatively resistant (CBA/Ca) and relatively susceptible (C57BL/6J) mouse strains, infected with A. suum (Deslyper et al., 2016;Deslyper et al., 2019b). We found intrinsic differences between the two mouse strains at the level of oxidative phosphorylation at days 4 and 7 post infection (p.i.) and at the level of the immune response proteins at day 7. The relatively resistant strain had a higher abundance of proteins associated with complement activation, whereas the relatively susceptible strain had a higher abundance of proteins associated with complement inhibition. These mouse models were also found to be suitable for infection with the human ascarid, A. lumbricoides (Deslyper et al., 2020).
The liver has special immunological properties. It receives blood directly from the gut via the hepatic portal vein. This blood carries with it antigens from both gut commensals and dietary products (Doherty, 2016). Because these antigens could cause unwanted chronic inflammatory responses, the hepatic immune system favours tolerance over immunity, which is mediated by specialized liver-resident antigen-presenting cells (Thomson and Knolle, 2010;Crispe, 2011). This feature makes the liver a potentially ideal organ for several parasites, including Ascaris, to incorporate in their migratory path, as a safe refuge and hence for immune evasion (Deslyper et al., 2019a).
To our knowledge, little research has been performed on the immune response in the liver to Ascaris infection. The only evidence of the immune response in the liver is the presence of white spots which have been observed in A. suum-infected pigs (Ronéus, 1966), A. lumbricoides-infected humans (Javid et al., 1999) and A. suum-infected mice (Dold et al., 2010). In the current study, we performed flow cytometry on spleen and liver samples from susceptible and resistant mice before and after infection with A. suum and A. lumbricoides. We selected day 7 p.i., because at this time point we previously found evidence for an altered immune response between the two mouse strains during A. suum infection (Deslyper et al., 2019b).

Parasite eggs
The A. lumbricoides eggs were extracted from adult worms, which were obtained from dewormed children in Ile-Ife, Nigeria, using pyrantel pamoate. The adult worms were transported on ice in 4% formalin. They were dissected upon arrival and the uteri were mechanically broken up and sieved (425 μm). The eggs were placed in 0.05 M H 2 SO 4 (Aldrich) in culture flasks with a ventilated cap for embryonation of the eggs. The flasks were stored at 26°C and oxygenated twice per week.
The A. suum eggs were kindly donated by Dr Johnny Vlaminck (Ghent University). These were shipped in a water solution, stored at 26°C in 0.05 M H 2 SO 4 and oxygenated twice per week.

Infection of mice with Ascaris eggs
Fifteen male mice of both CBA/Ca OlaHsd (Envigo, UK) and C57BL/6J OlaHsd (Comparative Medicine Unit, Trinity College Dublin) were purchased; all mice were 8 weeks old at the time of the experiment. Five mice of each strain received oral gavage (Instech, FTP-20-38-50, USA) with either 1000 eggs of A. suum, 1000 eggs of A. lumbricoides or 100 μL 0.05 M of H 2 SO 4 . The mice were culled at day 7 p.i. and the livers and spleens removed for flow cytometric analysis of immune cells. Additionally, the lungs were removed for larval counts using the modified Baermann method .

Larval recovery and enumeration for the lungs
After 24 h, the resulting samples from the Baermann method were centrifuged at 1389 g for 5 min. The supernatants were removed and 70% ethanol was added (50% v/v). Subsequently, the larvae were counted in 1 mL of sample solution, using a nematode counting chamber (Chalex Corporation, Park City, UT, USA).

Retrieval of spleen and liver immune cells
After dissection, livers and spleens were kept in ice cold phosphatebuffered saline (PBS). Both organs were mechanically minced using sterile scalpels and sieved through a 70 μm-gauge mesh in complete RPMI medium (cRPMI) (RPMI GlutaMAX™ supplemented with 1.25 mM HEPES and 10% foetal bovine serum, pH = 7.4). This resulted in single suspensions of spleen cells, of which the majority are circulating blood cells.
Since the liver contains resident, non-circulating immune cells embedded in a network of sinusoids which traverse the parenchymal tissue (Doherty, 2016), the immune cells must first be enzymatically extracted and separated from the parenchymal cells. The liver cell suspensions were suspended in 50 mL cRPMI and centrifuged for 1 min at 60 g without brake to remove undissociated tissue. The top 45 mL was removed and centrifuged again at 530 g for 10 min at 4°C. The resulting pellet was resuspended in 10 mL of digestion buffer (0.2 g L −1 collagenase from Clostridium histolyticum (Sigma-Aldrich) and 0.02 g L −1 DNase I (Sigma-Aldrich)) and incubated at 37°C for 30 min while shaking. Next, 30 mL PBS was added and left to rest on ice for 5 min before centrifugation at 528 g for 10 min at 4°C. The pellet was resuspended in PBS and layered over Lymphoprep™ (STEMCELL Technologies) and centrifuged at 400 g for 25 min without brake. The buffy coat layer, containing mononuclear cells (MNCs), was removed and kept aside. The pellet, containing erythrocytes and polymorphonuclear cells (PMNs), was incubated for 5 min at room temperature in red cell lysis buffer (0.1 mM EDTA, 155 mM NH 4 Cl, 10 mM KHCO 3 ). Both MNC and PMN were centrifuged for 8 min at 480 g and the pellet resuspended in PBS and counted.

Statistical analysis
The flow cytometry data were overdispersed, therefore a negative binomial distribution was found to be most appropriate. The MASS package (Venables and Ripley, 2002) was used for the negative binomial (log link) on each cell type. The most parsimonious model, a negative binomial without interaction between mouse strain and infection status of the mouse, was considered the default model. This model was compared to an interaction model, with an interaction between the mouse strain and infection status of the mouse. If the difference in absolute values of the Akaike information criterion (AIC) of the models (Δ i = AIC i − AIC min ) was greater than 2, then the interaction model was used (Burnham and Anderson, 2004). Post-hoc tests with a multivariate testing adjustment were performed using contrasts in the emmeans package (Lenth, 2019). Post-hoc tests were performed between mouse strains for each species.

Effect of A. suum infection on spleen and liver cell numbers and phenotypes
Spleen lymphoid cells Infection with A. suum did not elicit a statistically significant change in the numbers of any of the investigated spleen cell populations, CD8 + T cells, CD4 + T cells, αβ T cells, B cells, natural killer (NK) cells, γδ T cells, NKT cells or activated cells (Fig. 1). However, an intrinsic difference between the two mouse strains was observed for CD4 + T cells (z ratio: 2.505, P < 0.05) and γδ T cells (z ratio: 5.644, P < 0.01). For these cell populations, there were significantly higher numbers in the CBA/Ca mouse strain, compared to the C57BL/6J mouse strain, both with and without infection.
Differences in liver lymphoid cell numbers are mainly between mouse strains For the liver lymphocytes (Fig. 2), the numbers of only one cell type, NK cells, was found to be statistically significantly different (z ratio: −2.766, P < 0.05) when comparing infected samples to their uninfected controls, and that occurred only for the CBA/ Ca strain. Here, the control samples were found to have more NK cells than the infected samples. An intrinsic difference between the two mouse strains, was found for hepatic B cell numbers (z ratio: −7.054, P < 0.01), with the C57BL/6J strain having a higher number of B cells compared to the CBA/Ca strain. Although the numbers of the other lymphoid cell populations tested were similar in both mouse strains, after infection with A. suum, the numbers of several cell types were found to be present in higher numbers in the livers of C57BL/6J mice compared with those of CBA/Ca mice. These are: CD8 + T cells (z ratio: −2.783, P < 0.05), B cells (z ratio: −7.054, P < 0.01), NK cells (z ratio: −4.003, P < 0.01), γδ T cells (z ratio: −2.761, P < 0.05) and NKT cells (z ratio: −3.293, P < 0.01).

Differences between hepatic myeloid cells are mainly between control and infection
Analysis of liver myeloid cells, revealed higher numbers of eosinophils (z ratio: −5.070, P < 0.01), and KCs (z ratio: −4.143, P < 0.01) in uninfected C57BL/6J compared to uninfected CBA/ Ca livers (Fig. 3). For the eosinophils, this difference disappeared under A. suum infection. However, for the KCs this difference remained with the C57BL/6J mouse strain having a higher cell count (z ratio: −4.143, P < 0.01) than the CBA/Ca strain.

Splenic lymphocytes
When the immune cell composition in spleens of C57BL/6J and CBA/Ca mice infected with A. lumbricoides were examined, the frequencies of CD4 + T

cells, CD8 + T cells, αβ T cells, B cells, NK cells, γδ T cells, NKT cells and activated T cells were found
to be similar to those in uninfected mice. The only statistically significant differences (Fig. 4) found were higher numbers of CD4 + cells (z ratio: 2.505, P < 0.05) and γδ T cells (z ratio: 5.644, P < 0.01) in the CBA/Ca strain compared to the C57BL/ 6J strain. These higher numbers of CD4 + and γδ T cells were found in both uninfected and infected CBA/Ca mice.

Differences between liver myeloid cells
Analysis of hepatic myeloid cell numbers in control and A. lumbricoides-infected mice revealed that eosinophils (z ratio: 5.978, P < 0.01), DC (z ratio: 3.969, P < 0.01) and monocytes (z ratio: 2.483, P < 0.05) were expanded in infected CBA/Ca mice, whereas only monocytes (z ratio: 2.483, P < 0.05) were expanded in infected C57BL/6J mice. KCs were the only cell type whose numbers differed significantly in the two mouse strains after infection with A. lumbricoides (z ratio: −4.143, P < 0.01), with the C57BL/6J-infected samples having a higher number of cells compared to CBA/Ca-infected samples (Fig. 6).

Discussion
In this study, we investigated the immune response in the liver during Ascaris infection, using a model of hepatic resistance, where one mouse strain (C57BL/6J) is relatively susceptible and another mouse strain (CBA/Ca) is relatively resistant to Ascaris infection (Lewis, 2006;Deslyper et al., 2020). The larval burdens in the lungs at day 7 p.i. support this model, with means of 7 and 31 A. suum larvae observed in the relatively resistant and relatively susceptible mouse strains, respectively, and 1 and 3 A. lumbricoides larvae observed in the resistant and susceptible strains.
The results of the current study indicate that the differences in susceptibility to Ascaris infection between the two mouse strains
Our data indicate that infection with Ascaris is mainly associated with expansions or influxes of populations of myeloid cells in the livers. The relatively susceptible mouse strain had more eosinophils than the relatively resistant strain. However, under infection with either A. suum or A. lumbricoides, both mice showed expansions of eosinophils in their livers. Given the importance of eosinophils in parasite immunity in the lung and the gut, this is an expected finding (Enobe et al., 2006;Masure et al., 2013;Nogueira et al., 2016;Vlaminck et al., 2016;Weatherhead et al., 2018;Gazzinelli-Guimaraes et al., 2019). Eosinophils have been reported to expand in murine lungs during A. suum infection (Enobe et al., 2006;Nogueira et al., 2016;Weatherhead et al., 2018;Gazzinelli-Guimaraes et al., 2019) and are involved in reducing larval numbers (Gazzinelli-Guimaraes et al., 2019). In pigs, the presence of eosinophils has been linked to elimination of Ascaris in the gut (Masure et al., 2013;Vlaminck et al., 2016). Eosinophils also play roles in human ascarid infections, with expansions of these cells and elevated production of eosinophil cationic protein in putatively immune Nigerian children (McSharry et al., 1999) and chronically-infected Ecuadorian children (Reina Ortiz et al., 2011). Eosinophils can also paralyse Schistosoma mansoni (McLaren et al., 1984) and kill Trypanosoma cruzi and Brugia malayi in vitro (Hamann et al., 1990). A recent study found that immune serum-activated human macrophages coordinate with eosinophils to immobilize A. suum larvae (Coakley et al., 2020). The results of the current study further implicate hepatic eosinophils in the immune response to A. suum and A. lumbricoides in both mouse strains.
Monocytes and macrophages are also thought to contribute to immunity against Ascaris, being present in lung samples from A. suum-infected mice (Gazzinelli-Guimaraes et al., 2019) and capable of recognizing and responding to A. suum in vitro Coakley et al., 2020). We found that uninfected C57BL/6J and CBA/Ca had similar numbers of monocytes in their livers, and these cells were found in significantly higher numbers after infection with either A. suum or A. lumbricoides. However, the liver-resident macrophages, Kupffer cells, were present in higher numbers in the relatively susceptible C57BL/6J mice compared to the resistant CBA/Ca mice, both without and with infection by either Ascaris type. Under A. suum infection, however, there was a significant increase in Kupffer cell numbers for both mouse strains, but the relatively susceptible strain still had more of this cell type than the relatively resistant strain.
DCs are of interest in parasite infections because these cells are required for the induction of Th2 responses. Previous studies have found that A. suum has immunomodulatory effects on human DCs in vitro and that A. suum can upregulate a negative regulator of reactive oxygen species production (Favoretto et al., 2014;Midttun et al., 2018;Summan et al., 2018;Arora et al., 2020). We found that both myeloid and plasmacytoid DC were found in similar numbers in the livers of the two mouse strains, and that their numbers were significantly higher in A. suum-infected livers but only slightly higher in A. lumbricoides-infected livers.
Immune responses against parasites are controlled by a number of lymphoid cell types, in particular those that produce Th2 cytokines, such as interleukin (IL)-4, IL-5 and IL-13, which promote immunoglobulin E production and recruit and activate eosinophils and mast cells. We enumerated CD4 + and CD8 + T cells, T cells expressing αβ and γδ T cell receptors, NKT cells, B cells and NK cells in the spleens and livers of A. suum and A. lumbricoides-infected C57BL/6J and CBA/Ca mice. We found that the relatively resistant mouse strain had significantly more CD4 + and γδ T cells in their spleens than the relatively susceptible strain before infection and after infection with either ascarid species. While both CD4 + and γδ T cells can release Th2 cytokines, future studies are required to determine if the increased numbers of these cells in CBA/Ca mice compared to C57BL/6 mice reflect expansions of Th2 cells. A previous study, however, observed a reduction in CD4 + IL-4 + T cells in the spleen of A. suum-infected BALB/c mice (Gazzinelli-Guimarães et al., 2013), suggesting that the parasite may inhibit Th2 cell differentiation.
Analysis of liver lymphocytes from the relatively susceptible C57BL/6 mice revealed a pattern whereby the numbers of every investigated cell population increased slightly or significantly with infection by either Ascaris species. The opposite was true for the relatively resistant CBA/Ca mice, where all hepatic lymphoid cell numbers decreased with infection. This observation suggests, surprisingly, a more pronounced immune response to Ascaris by lymphoid cells in the susceptible C57BL/6 mice. It applies to B cells and conventional αβ T cells and their CD4 + and CD8 + T cell subsets, which can mediate pathogen-specific adaptive immunity via selective Th1/Th2/Th17 cytokine secretion and antibody production. It also applies to the innate lymphocytes, including γδ T cells, NK and NKT cells, which are uniquely abundant in the liver (Doherty, 2016).
NK cells, γδ T cells and NKT cells recognize conserved structures on pathogens and stressed host cells and respond rapidly by killing target cells and rapidly releasing cytokines. They account for the majority of lymphocytes in the liver and are thought to play roles in the initiation of immune responses against pathogens and tumours in an environment where immune tolerance is favoured over active immunity (Doherty, 2016). Little is known about the roles of these innate lymphocytes in Ascaris infection, but they are implicated in immunity against other parasites. γδ T cells are found in elevated numbers in the blood of patients with cutaneous leishmaniasis (Darabi et al., 2002), Toxoplasma gondii (Prigione et al., 2006) and Schistosoma infections (Schwartz et al., 2014) and in the mesenteric lymph nodes of Schistosoma-infected mice (Yu et al., 2014). γδ T cells numbers also increase after the acute phase of Plasmodium infection (Mamedov et al., 2018) and are required for the induction of immunity against Plasmodium in vaccination studies in mice (Zaidi et al., 2017). Subsets of NKT cells produce IFN-γ and IL-4 during egg deposition in the liver in mice infected with S. mansoni (Mallevaey et al., 2006;Mallevaey et al., 2007). Although these innate lymphocyte populations can selectively release Th2 cytokines and can polarize immune responses towards Th2, future studies are required to determine if any of these innate lymphocyte populations exhibit skewed Th2

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Gwendoline Deslyper et al. phenotypes in response to Ascaris infection. Future studies are also required to explain why innate T cells exhibit opposite dynamics in the relatively susceptible and relatively resistant mouse strains in response to Ascaris infection. In summary, our data demonstrate that infection with A. suum or A. lumbricoides results in increases in the numbers of myeloid cells, including monocytes, dendritic cells, Kupffer cells and eosinophils in the livers of both C57BL/6J and CBA/Ca mice, suggesting that these cells are likely to contribute to parasite elimination. Unexpectedly, the numbers of all hepatic lymphocyte subsets examined increased in the susceptible C57BL/6J mice but decreased in the relatively resistant CBA/Ca mice after infection with both parasite species, suggesting that the susceptible mice mounted more robust immune responses to the worms. This might be explained by the responses being more tolerogenic in C57BL/6J mice, leading to higher subsequent worm burdens in the lungs. Liver dendritic cells, Kupffer cells and monocytes are well-documented to preferentially induce tolerance over immunity to antigens encountered in the liver (Thomson and Knolle, 2010;Crispe, 2011;Doherty, 2016). Alternatively, the livers of the susceptible C57BL/6J mice may receive higher numbers of worms producing stimulatory antigens, requiring a more robust hepatic immune response compared with that of CBA/Ca mice. Consistent with the latter hypothesis, intestinal/rectal worm burdens are reported to be higher in C57BL/6J mice compared to CBA/Ca mice 6 days p.i. (Lewis et al., 2007), suggesting that predisposition may take place before the ascarid reaches the liver. Uninfected and Ascaris-infected CBA/Ca mice had higher numbers of CD4 + T cells and γδ T cells in their spleens which might arise from mesenteric lymph node activation by parasite antigens. Future functional studies are needed to elucidate the immunogenic vs tolerogenic roles of hepatic leucocytes in Ascaris infection.