The Basophil IL-18 Receptor Precisely Regulates the Host Immune Response and Malaria-Induced Intestinal Permeability and Alters Parasite Transmission to Mosquitoes without Effect on Gametocytemia

We have recently demonstrated that basophils are protective against intestinal permeability during malaria and contribute to reduced parasite transmission to mosquitoes. Given that IL-18 is an early cytokine/alarmin in malaria and has been shown to activate basophils, we sought to determine the role of the basophil IL-18R in this protective phenotype. To address this, we infected control [IL18rflox/flox or basoIL-18R (+)] mice and mice with basophils lacking the IL-18R [IL18rflox/flox × Basoph8 or basoIL-18R (−)] with Plasmodium yoelii yoelii 17XNL, a nonlethal strain of mouse malaria. Postinfection (PI), intestinal permeability, ileal mastocytosis, bacteremia, and levels of ileal and plasma cytokines and chemokines were measured through 10 d PI. BasoIL-18R (−) mice exhibited greater intestinal permeability relative to basoIL-18R (+) mice, along with increased plasma levels of proinflammatory cytokines at a single time point PI, day 4 PI, a pattern not observed in basoIL-18R (+) mice. Surprisingly, mosquitoes fed on basoIL-18R (−) mice became infected less frequently than mosquitoes fed on basoIL-18R (+) mice, with no difference in gametocytemia, a pattern that was distinct from that observed previously with basophil-depleted mice. These findings suggest that early basophil-dependent protection of the intestinal barrier in malaria is mediated by IL-18, and that basophil IL-18R–dependent signaling differentially regulates the inflammatory response to infection and parasite transmission.


INTRODUCTION
Malaria results from infection with parasites in the genus Plasmodium transmitted by Anopheles mosquitoes. Despite global malaria eradication efforts, the World Health Organization reported 627,000 deaths and 241 million cases in 2020 (1). Concomitant bacteremia is a complication that contributes to morbidity and mortality associated with malaria across age groups and clinical disease severities (2)(3)(4)(5). Our previous studies with mouse models demonstrated that malaria-induced bacteremia arises from early intestinal mast cell (MC) influx or mastocytosis (6)(7)(8). The appearance of malaria-induced intestinal permeability to enteric bacteria, a phenomenon referred to as "leaky gut," is preceded by a type 2-biased host immune response, specifically by the appearance of IL-4, IL-10, MC protease 1 (Mcpt1), and Mcpt4, as well as increased circulating basophils and eosinophils (8).
We previously showed that basophil-depleted mice exhibited significantly increased intestinal permeability at days 4, 6, and 8 postinfection (PI) with Plasmodium yoelii yoelii 17XNL compared with nondepleted mice (9). Further, basophil-depleted mice exhibited an increase in ileal MC numbers at 8 d PI, suggesting that in the context of malaria, basophils blunt increases in intestinal permeability and mastocytosis. Despite these differences, basophil-depleted and nondepleted mice had similar patterns of bacterial 16S DNA in blood that tracked with rising parasitemia. Network analyses of cytokines and chemokines revealed differences in the immune response to bacteria between genotypes that suggested that basophil-depleted mice were better able to control bacterial translocation at the level of the intestine, while the systemic response was more effective than the intestinal response in controlling bacterial translocation in nondepleted mice (9). Basophil depletion was also associated with increased gametocytemia and increased infection success in the mosquito host Anopheles stephensi, suggesting a previously unknown role for these cells in controlling transmission. Based on these findings, we sought to identify a signal(s) upstream of basophils that could result in these phenotypes.
Basophils have several surface receptors, including TLR2, TLR4, FcεRI, IL-3R, IL-5R, and IL-18R, that contribute to cell activation (reviewed in Refs. [10][11][12]. Given that IL-18 is produced by dendritic cells and macrophages early in malaria (13,reviewed in Ref. 14) and is also released from damaged endothelial cells as an alarmin (reviewed in Ref. 15), the basophil IL-18R was of particular interest. Further, we previously reported that plasma IL-18 was significantly increased at day 4 PI in our mouse model, the same time point at which circulating basophils are significantly increased in P. y. yoelii 17XNL-infected mice (8).
To determine whether basophil-dependent protection of the intestinal barrier is dependent on the basophil IL-18R, we infected IL18r flox/flox × Basoph8 [basoIL-18R (−)] mice and control IL18r flox/flox [basoIL-18R (+)] mice with P. y. yoelii 17XNL to examine malariainduced intestinal permeability and parasite transmission to A. stephensi. Our data revealed that malaria-induced intestinal permeability was strikingly increased on a single day PI, day 4, in basoIL-18R (−) mice relative to basoIL-18R (+) mice. In addition, this time point was marked by significantly increased plasma cytokines and chemokines in basoIL-18R (−) mice relative to uninfected baseline, a pattern that was not evident in infected basoIL-18R (+) mice, suggesting that basophil IL-18R signaling coordinates changes in cytokines and chemokines with intestinal permeability at a precise early time point in malaria. Finally, transmission studies with basoIL-18R (−) and basoIL-18R (+) mice revealed a surprising reversal of the transmission phenotype observed in basophil-depleted versus nondepleted mice (9). In contrast with increased transmission from infected basophil-depleted mice to A. stephensi, we observed decreased transmission from infected basoIL-18R (−) mice relative to basoIL-18R (+) mice, suggesting that basophil-dependent control of parasite transmission likely involves multiple signals from these rare granulocytes.

Mice
Mice with basophils lacking IL-18R were generated by crossing Basoph8 mice (Jackson Laboratory stock no. 017578, developed by R. Locksley [16]) with IL18r flox/flox mice (provided by R. Flavell). A total of 108 basoIL-18R (−) mice were used as experimental animals, and 91 sex-and age-matched IL18r flox/flox [basoIL-18R (+)] mice were used as controls. Experimental procedures were conducted on both male (n = 87) and female (n = 112) mice at 6-8 wk of age. Mice were housed in ventilated microisolator caging and provided food and water ad libitum. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Idaho (protocol number IACUC-2020-10, approved March 30, 2020).

Mouse infection and monitoring
A total of 108 basoIL-18R (−) mice and 91 basoIL-18R (+) control mice were used across three replicates of time-course studies, six replicates of transmission studies, and one replicate of flow cytometry. Mice were infected as described previously (9,17), with 172 mice injected i.p. with 150 μl of 1 × 10 6 P. y. yoelii 17XNL-infected RBCs and 27 injected i.p. with 150 μl United States Pharmacopeia saline at day 0. Mice were sacrificed at 3 d PI (transmission studies only, when gametocytes are maximally infective to A. stephensi) or at 4, 6, 8, and 10 d PI for blood and/or tissue collection. Starting at 2 d PI, daily parasitemias were calculated from thin blood smears stained with Giemsa as described elsewhere (9). Gametocytemia at 3 d PI was calculated as the number of RBCs infected with gametocytes divided by the total number of RBCs counted in 25 fields viewed at 1000× magnification. Mice were monitored daily for weight loss and reduced activity to determine humane end points (>20% of starting weight, lack of feeding, drinking, and grooming). Blood samples were obtained by cardiac puncture for determination of bacterial 16S ribosomal DNA copies. Plasma samples were prepared from remaining blood and aliquoted for quantification of cytokines, chemokines, Mcpt1, Mcpt4, and IgE. Plasma aliquots were stored at −80° C until analysis. Ileum tissue was collected and snap frozen (~3 cm) for cytokine and chemokine analysis, with the remainder formalin-fixed for histological analysis.

In vivo intestinal permeability
In all three replicates of the time-course studies, intestinal permeability was quantified as described previously (8,9,17). In brief, mice were orally gavaged with 4 kDa FITC dextran solution after a 4-h fast. Plasma was collected 3 h postgavage, and fluorescence was measured using a microplate reader (Molecular Devices LLC, San Jose, CA) at excitation/ emission 490/520 nm.

Ileum histochemistry and MC staining
Formalin-fixed ileum samples were prepared as described previously (8)

Extraction of DNA from blood and bacterial 16S DNA quantitative PCR
DNA was isolated from blood aliquots using the DNeasy Blood & Tissue kit (Qiagen, Germantown, MD) according to the manufacturer's protocol. DNA was diluted to 4 ng/μl, and samples were assayed in triplicate using Maxima SYBR green/ROX qPCR master mix (2×) (Thermo Fisher Scientific, Waltham, MA) and 16S forward and reverse primers (forward 5′-ACTCCTACGGGAGGCAGCAGT-3′, reverse 5′-ATTACCGCGGCTGCTGGC-3′) at final concentrations of 0.4 μM. 16S copies were quantified using a standard curve as described previously (8). Data were analyzed using QuantStudio 6 Flex (Applied Biosystems, Waltham, MA).

Transmission studies
Transmission studies were carried out as described previously (9,17). In brief, A. Stephensi (Indian strain, reared as described in Ref. 18), were fed on 6-to 8-wk-old male or female basoIL-18R (−) mice (n = 23) or basoIL-18R (+) mice (n = 25) at 3 d PI. Mice were anesthetized with ketamine (50 mg/kg) and xylazine (5 mg/kg) and placed individually on top of a carton containing ~60 3-to 5-d-old female mosquitoes. Mosquitoes were allowed to feed for 15 min. After feeding was complete, mice were euthanized via CO 2 asphyxiation followed by cervical dislocation. Non-blood-fed mosquitoes were removed from the cartons. At 10 d postfeeding, 25-35 mosquitoes per carton were dissected and stained with mercurochrome to quantify numbers of oocysts per midgut (infection intensity) and number of mosquitoes with at least one midgut oocyst out of the total fed mosquitoes (infection prevalence).

Statistical analyses
Parasitemia, intestinal permeability, bacterial 16S DNA copies per microliter of blood and Mcpt1, Mcpt4, IgE, MCs per high-power field, and cytokine/chemokine concentrations were analyzed by Robust Regression and Outlier Removal method (maximum false discovery rate Q = 1%) to exclude outliers. Nonnormal data were compared among time points using the Kruskal-Wallis test followed by Dunn's multiple-comparison test of each time point between genotypes. Normally distributed data were analyzed using Brown-Forsythe & Welch ANOVA Mean oocysts per midgut and gametocytemia were compared using a Mann-Whitney U test. Infection prevalence was analyzed by Fisher's exact test. The p values <0.05 for all analyses were considered significant.

Ethics statement
All experiments were performed with the approval of the Institutional Animal Care and Use Committee of the University of Idaho (protocol number IACUC-2020-10, approved March 30, 2020).

Basophil IL-18R deficiency had no effect on P. y. yoelii 17XNL parasitemia
Parasitemia in basoIL-18R (−) mice was not different at any time point relative to that in basoIL-18R (+) mice (Fig. 1). This pattern was consistent with that observed in basophildepleted and nondepleted mice (9), suggesting that phenotypic differences between the two genotypes were not confounded by differences in parasite burden.

Basophil IL-18R deficiency was associated with increased intestinal permeability at day 4 PI and ileal MC influx at day 10 PI
Given our prior observations of MC-dependent intestinal permeability after P. yoelii infection (6,7) and that basophil depletion was associated with increased intestinal permeability at 4, 6, and 8 d PI and increased ileal MC numbers at 8 d PI (9), we sought to assess the role of basophil IL-18R in these phenotypes. BasoIL-18R (−) mice exhibited a notable increase in intestinal permeability to FITC-dextran compared with basoIL-18R (+) mice at day 4 PI (Fig. 2), followed by a gradual return to baseline by 10 d PI. Interestingly, this peak in intestinal permeability coincides with a previously observed peak in circulating basophils in our model at 4 d PI (8), suggesting that basophil IL-18R contributes to protection against intestinal permeability at this time point. NASDCE staining of ileum sections revealed significantly increased MCs per field in basoIL-18R (−) mice relative to basoIL-18R (+) mice at 10 d PI (Fig. 3A-C). This observation was similar to findings in basophil-depleted mice, which showed significantly increased ileal MCs at day 8 PI relative to nondepleted mice (9). Patterns in both mutant mouse lines, therefore, were notably delayed relative to the infection-associated increase in ileal MCs in wild type mice at 4 d PI (8), suggesting that early malaria-induced MC influx into the ileum is at least partly dependent on basophils and signaling through IL-18R.  (26), were significantly increased compared with uninfected mice in both genotypes at days 6, 8, and 10 PI, with no significant differences between genotypes at any time point (Fig. 3D). This pattern was also observed in basophildepleted mice (9), suggesting that plasma Mcpt1 levels are not basophil dependent. Plasma levels of Mcpt4, largely a product of connective tissue MCs (21), were not significantly different from uninfected levels in either genotype at any time PI, despite a trend toward increased levels in basoIL-18R (−) mice at 10 d PI (Fig. 3E). We previously observed that P. y. yoelii 17XNL-infected wild type mice exhibited significantly increased plasma Mcpt4 levels at days 4 and 8 PI relative to uninfected controls (8), but this pattern was absent in infected, basophil-depleted mice relative to nondepleted control mice (9), suggesting that Mcpt4 synthesis is dependent on basophils and signaling through IL-18R Circulating IgE levels were significantly different between the two genotypes at day 8 PI, with levels in basoIL-18R (−) mice higher than uninfected baseline at this time point (Fig. 3F), potentially reflecting the observation that accumulation of IL-18 triggers IgE (27).

Basophil IL-18R deficiency had no effect on bacterial 16S DNA levels in blood during P. y. yoelii 17XNL infection
There were no differences in bacterial 16S DNA copies in blood between genotypes at any time point PI (Fig. 4). Similar to the findings in Céspedes et al. (8), blood 16S DNA copies in both basoIL-18R (−) mice and basoIL-18R (+) mice increased over the course of infection, becoming significantly elevated at days 8 and 10 PI (Fig. 4). These observations were similar to those for basophil-depleted and nondepleted mice, which exhibited rising blood 16S copies over the course of infection that did not differ between the genotypes at any time point (9).

Transmission studies
We have observed that mice depleted of basophils developed more gametocytes in circulation, and the mosquitoes that fed on these mice developed greater numbers of parasites relative to infected, nondepleted mice (9). Therefore, we sought to test whether the effects of basophils on gametocytemia and transmission could be attributed to basophil IL-18R To the contrary, however, the percentage of infected mosquitoes was reduced in mosquitoes that fed on basoIL-18R (−) mice relative to those that fed on basoIL-18R (+) mice (Fig. 7A), but there was no difference in infection intensity (oocysts per midgut) in mosquitoes that fed on mice with these genotypes (Fig. 7B). Gametocyte numbers trended lower in basoIL-18R (−) mice but were not significantly different between genotypes (Fig.  7C). These observations suggest that the basophil IL-18R regulates transmission biology, but further investigation is needed to identify the mechanisms by which basophils control gametocytemia and parasite transmission.

DISCUSSION
Basophils are short-lived (~2.5 d) in circulation (33), have roles beyond allergy in finetuning host immune responses (34,35), and are activated in both mouse and human malaria (8,36). The studies herein affirm our observations (9) that basophils contribute to the maintenance of intestinal barrier integrity during malaria and extend these observations to include a role for basophil IL-18R. Although the mechanism by which this occurs remains to be elucidated, basophil-depleted mice and basoIL-18R (−) mice showed increased intestinal permeability from days 4-8 PI and at day 4 PI, respectively (9) (Fig. 2). Collectively, our data suggest for the first time, to our knowledge, that basophil IL-18R protects against early Coinciding with the peak in intestinal permeability at day 4 PI, basoIL-18R (−) mice showed increases in 14 plasma cytokines and chemokines ( Fig. 6A-G, J-P) compared with baseline, a pattern not observed in the basoIL-18R (+) mice. Six of these, IL-12p40, IFN-γ, TNF-α, IL-6, IL-4, and IL-9, have previously been demonstrated to cause degradation of tight junctions between epithelial cells and increased intestinal permeability in other disease models (reviewed in Refs. [37][38][39][40]. IL-10, largely considered to be protective in maintaining intestinal barrier integrity (41,42), was also increased in the plasma at days 4 and 10 PI compared with baseline in basoIL-18R (−) mice, but not in basoIL-18R (+) mice. Interestingly, IL-10 has also been shown to enhance MC responses mediated by IgE, and sensitized IL-10 −/− mice were protected from MC influx into the jejunum after OVA challenge (43). Increased levels of IL-4 in conjunction with IL-3, which can induce MC proliferation (44,45,reviewed in Ref. 46), and IL-9, an important regulator of intestinal immunity and mucosal MCs (47) (54). Other studies have highlighted dichotomous roles for MIP-1α and MIP-1β in regulating systemic/type 1 and mucosal/type 2 host immune responses, respectively (55). These studies reported that MIP-1α treatment promoted robust production of IgG and IgM, while MIP-1β treatment promoted IgA and IgE production, in addition to lower levels of IgG and IgM (55). Interestingly, basoIL-18R (−) mice, which showed increases in circulating levels of MIP-1β beginning at day 4 PI and continuing through day 10 PI (Fig. 6P), also showed increased circulating IgE at 8 d PI (Fig. 3E), while basoIL-18R (+) mice, in which MIP-1β never increased above baseline levels, did not. Given that MCs express the FcεRI (56) and that binding of IgE to this receptor promotes their survival (57), this increase in circulating IgE at day 8 PI could contribute to the influx of MCs seen at day 10 PI in the baSOIL-I8R (−) mice ( Fig. 3A-C). In addition, in parasitic infections, the proinflammatory cytokine and alarmin IL-1α is a strong coactivating stimulus for MCs, resulting in MC synthesis of IL-3, IL-5, IL-6, and IL-9, which can have autocrine and paracrine effects (IL-3, IL-9) on MCs (58). Synthesis of G-CSF can be induced by IL-1α (reviewed in Ref. 59) and is a master regulator of neutrophilic homeostasis, mobilization, and the release of neutrophil extracellular traps (60). MC reconstitution of C57BL/6 Kit W-sh/W-sh mice has been associated with reduced G-CSF levels and restoration of neutrophils to wild type levels in these mutant mice (61), suggesting that elevated MCs (Fig. 3A) and reduced G-CSF levels at 10 d PI may be associated with enhanced neutrophilic responsiveness in basoIL-18R (−) mice and the downward trend in bacterial 16S DNA copies in these mice at 10 d PI relative to basoIL-18R (+) mice (Fig. 4). Together, these data suggest that basophil IL-18R protects against increases in intestinal permeability PI in part by regulating early production of inflammatory cytokines and chemokines, which may in turn protect against MC influx in the ileum.
Overall, basoIL-18R (−) mice showed increases from baseline in numerous plasma cytokines and chemokines, including IL-2 (Fig. 6A), INF-γ (Fig. 6C), TNF-α (Fig. 6D), IL-3 (Fig. 6E), IL-6 (Fig. 6F), G-CSF (Fig. 6G), IL-10 ( Fig. 6J), IL-4 (Fig. 6K), KC (Fig.  6M), and MIP-1α (Fig. 6O) at day 4 PI that were absent in both basoIL-18R (+) mice and basophil-depleted mice (9). These unique increases in the plasma cytokines, many of which have been previously shown to regulate intestinal permeability, co-oc-cur with the peak of intestinal permeability at 4 d PI in our model. This suggests that basophil IL-18 signaling may be important for controlling the early inflammatory response to infection and promoting increased MC influx into the intestine later during infection. Many groups have noted instances where organisms that are genetic knockouts display no obvious phenotype, while knockdown organisms exhibit marked phenotypic changes (reviewed in Ref. 62). This observation has been attributed to multiple mechanisms of genetic compensation and/or transcriptional adaptation (reviewed in Ref. 62). Thus, although this phenomenon has not been studied in the context of basophil depletion, it is an important consideration when comparing findings across different strains of mutant mice. In addition, because the IL-18R on basophils is absent from birth, there is the possibility that there could be differential immune responses to infection. Nevertheless, given the consistencies in the major phenotypes of increased intestinal permeability and MC influx in both basoIL-18R (−) mice and basophil-depleted mice compared with their respective controls, we maintain that basophils protect against, rather than contribute to, malaria-induced damage to the intestinal barrier and play an important role in controlling the inflammation that underlies this increased permeability.
Our data have shown that increased intestinal permeability and ileal MC influx observed in basophil-depleted mice after P. y. yoelii 17XNL infection (9) were recapitulated in basoIL-18R (−) mice with minor temporal shifts, but the effect of basophil depletion on parasite transmission to A. stephensi was reversed in basoIL-18R (−) mice. That is, mosquitoes that fed on basoIL-18R (−) mice became infected less frequently than mosquitoes fed on infected basoIL-18R (+) mice, with no difference in intensity of infection (Fig. 7A, 7B). Plasma TNF-α and IFN-γ, both shown to have gametocidal properties (63)(64)(65), were significantly increased in basoIL-18R (−) mice close to the peak of transmission to mosquitoes (Fig. 6C, 6D). This may explain the trend toward decreased gametocytemia in basoIL-18R (−) mice relative to basoIL-18R (+) mice (Fig. 7C). However, more work is necessary to elucidate the mechanism(s) by which basophil-mediated control of transmission occurs, because these could have major implications for malaria-control efforts.   In vivo intestinal permeability quantified by plasma FITC-dextran concentration after oral gavage of P. y. yoelii 17XNL-infected and uninfected control mice of each genotype.
Each dot represents a single mouse. Data were analyzed with Kruskal-Wallis test followed by Dunn's multiple comparison between genotypes at each time point and between the respective uninfected controls. The p values <0.05 were considered significant. *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001.