A motogenic GABAergic system of mononuclear phagocytes facilitates dissemination of coccidian parasites

Gamma-aminobutyric acid (GABA) serves diverse biological functions in prokaryotes and eukaryotes, including neurotransmission in vertebrates. Yet, the role of GABA in the immune system has remained elusive. Here, a comprehensive characterization of human and murine myeloid mononuclear phagocytes revealed the presence of a conserved and tightly regulated GABAergic machinery with expression of GABA metabolic enzymes and transporters, GABA-A receptors and regulators, and voltage-dependent calcium channels. Infection challenge with the common coccidian parasites Toxoplasma gondii and Neospora caninum activated GABAergic signaling in phagocytes. Using gene silencing and pharmacological modulators in vitro and in vivo in mice, we identify the functional determinants of GABAergic signaling in parasitized phagocytes and demonstrate a link to calcium responses and migratory activation. The findings reveal a regulatory role for a GABAergic signaling machinery in the host-pathogen interplay between phagocytes and invasive coccidian parasites. The co-option of GABA underlies colonization of the host by a Trojan horse mechanism.


Introduction
Gamma-aminobutyric acid (GABA), first identified as a plant and microbe metabolite, is a principal neurotransmitter in the central nervous system (CNS) of vertebrates (Roth et al., 2003). Moreover, recent findings implicate GABAergic signaling in the disease environment of cancer and other inflammatory conditions in humans (Neman et al., 2014;Bhat et al., 2010;Takehara et al., 2007;Li et al., 2017). Neurons and other GABAergic cells synthesize GABA via glutamate decarboxylases (GAD65/67) (Soghomonian and Martin, 1998). GABA is shuttled in and out of cells via GABA transporters (GATs) (Hö glund et al., 2005) and acts via activation of GABA-A receptors (GABA-A Rs) (Olsen and Sieghart, 2008) and GABA-B Rs (Bettler et al., 2004). The GABA-A Rs are pentameric ionotropic chloride channels, normally comprised of three types of subunits: 2 as, 2 bs and a third type of subunit. Nineteen different mammalian GABA-A R subunits (a1-6, b1-3, g1-3, d, e, p, q and r1-3) can combine to form numerous variants of functional heteromeric receptors in neuronal cells. The strength and polarity of GABA signaling is regulated by cation-chloride cotransporters (CCCs) (Kaila et al., 2014). GABA-A R activation by GABA can elicit opening of voltage-dependent calcium (Ca 2+ ) channels (VDCCs) with subsequent Ca 2+ influx into the neuronal cell (Bortone and Polleux, 2009).
Owing to host-pathogen coevolution with reciprocal selection, studies of host-pathogen interactions provide a powerful tool to gain insight into biological processes. The obligate intracellular protozoan Toxoplasma gondii actively invades nucleated cells (Sibley, 2004) and has a broad range of hosts among warm-blooded vertebrates. One third of the global human population is estimated to be chronically infected by T. gondii (Pappas et al., 2009) and severe manifestations may occur upon immunosuppression and during pregnancy (Montoya and Liesenfeld, 2004). Similarly, the related coccidian Neospora caninum represents a pathogen of major importance in veterinary medicine (Dubey et al., 2007). Upon ingestion and after crossing the intestinal epithelium, the tachyzoite stages of these coccidian parasites rapidly disseminate in their intermediate hosts, ultimately establishing latent infection in the CNS (Montoya and Liesenfeld, 2004;Dubey et al., 2007). The mononuclear phagocyte system comprises dendritic cells (DCs), monocytes, macrophages and brain microglia, which mediate multiple immunological functions and are crucial to counteract microbial infection (Guilliams et al., 2014). Early on during infection, tissue-invasive coccidian tachyzoites encounter DCs and other phagocytes, which play a determinant role in mounting a robust host-protective immune response (Liu et al., 2006;Mashayekhi et al., 2011). Paradoxically, T. gondii and N. caninum exploit the inherent migratory ability of DCs and monocytes for dissemination via a Trojan horse mechanism (Sangaré et al., 2019;Lambert et al., 2006;Lambert et al., 2009;Courret et al., 2006;Collantes-Fernandez et al., 2012). Within minutes of active invasion by T. gondii, DCs adopt a hypermigratory phenotype that mediates rapid systemic dissemination in mice (Kanatani et al., 2017;Weidner and Barragan, 2014). GABAergic inhibition of DCs hampers dissemination of T. gondii (Kanatani et al., 2017;Fuks et al., 2012), however the precise mechanisms of action have remained uncharacterized.
Some components of GABAergic signaling have been detected in DCs, monocytes, macrophages, T cells and B cells (Bhat et al., 2010;Fuks et al., 2012;Alam et al., 2006;Wheeler et al., 2011;Bhandage et al., 2014). Yet, the precise functions of GABA in immune cells have remained elusive. Here, we have performed a systematic analysis of the GABAergic system of various types of human and murine DCs and monocytes and report a conserved GABAergic machinery implicated in migratory responses. Further, we show that coccidian parasites hijack GABAergic signaling in parasitized phagocytes to promote infection-related dissemination.

Results
Human and murine mononuclear phagocytes exhibit hypermotility and secrete GABA upon challenge with T. gondii and N. caninum To address the impact of coccidian infection on mononuclear phagocyte motility, primary cells from human donors and mice were challenged with freshly egressed tachyzoites of T. gondii and N. caninum. Upon invasion by tachyzoites ( Figure 1A), a rapid increase of migrated distances ( Figure 1B) and elevated velocities ( Figure 1C) were recorded in infected murine bone marrow-derived DCs (mBMDCs), compared with unchallenged cells. Similarly, human monocytes, monocyte-derived DCs (hMoDCs) and primary myeloid DCs (hMDCs) freshly isolated from blood of human donors consistently exhibited hypermotility upon challenge ( Figure 1D,E,F, Figure 1-figure supplement 1A,B, C). Further, phagocytes expressed transcripts of enzymes for GABA synthesis and catabolism ( Figure 1G), which were up-and downregulated, respectively, upon challenge with T. gondii ( Figure 1H). Consistent with this, elevations of GABA concentrations in the supernatants were detected shortly after challenge and increased over time ( Figure 1I). Further, challenge of mononuclear phagocytes with separate strains of the two coccidia resulted in elevated concentrations of GABA in cell supernatants ( Figure 1J). Together, this indicated a putative link between GABA and migratory activation, motivating a further analysis of GABAergic signaling in mononuclear phagocytes.

A conserved repertoire of GABA-A R subunits in human and murine phagocytes
To address GABAergic signaling in phagocytes, human and murine cells were screened for expression of the 19 known GABA-A R subunits (Supplementary file 1), which in neuronal cells combine to form multiple pentameric receptor variants (Olsen and Sieghart, 2008). Interestingly, mBMDCs consistently expressed mRNAs for 10 out of the 19 GABA-A R subunits (a3, a4, a5, b2, b3, g1, g2, d, r1,    Of note, transcriptional expression of the a6, b2 and q subunits, undetectable in unchallenged hMoDCs, was consistently observed in challenged hMoDCs ( Figure 2B,D). Immunocytochemical analyses with available antibodies yielded signal consistent with expression of a3, a5, b3 and r1 subunits ( Figure 2E). We conclude that murine and human phagocytes constitutively transcribed one or more a subunits, one or more b subunits and one or more additional subunits, including r GABA-A R subunits (Supplementary file 2). Additionally, the modulated subunit expression upon challenge with T. gondii motivated an assessment of GABA-A R function upon infection.
Selective pharmacological inhibition of GABA-A Rs abrogates T. gondii-/N. caninum-induced hypermotility in human and murine phagocytes To functionally assess the putative implication of GABA-A Rs in parasite-induced migratory activation of DCs, motility assays were performed in the presence of general and subunit-selective GABA-A R antagonists and modulators. A broad range GABA-A R open-channel blocker (picrotoxin) and a, b and r subunit selective inhibitors (L655 708, SCS and TPMPA, respectively), efficiently inhibited T. gondii-/N. caninum-induced hypermotility in mBMDCs ( Figure  , with a non-significant impact on baseline cell motility. Further, we took advantage of the finding that blockade of GABA transporters (SNAP) inhibited hypermotility in parasitized mBMDCs ( Figure 3E,F) with reduced GABA concentrations in the supernatants ( Figure 5F) to attempt reconstitution of hypermotility by allosteric modulators of GABA-A Rs. Importantly, hypermotility was rescued by an allosteric modulator of b2/b3-containing GABA-A Rs (etomidate), but not by an allosteric modulator of d subunit-containing GABA-A Rs (allopregnanolone) ( Figure 3E,F). Because mBMDCs transcriptionally express b2, b3 and d subunits (Figure 2), the data jointly indicated an implication of b subunits in hypermotility and also differential effects or implication by specific subunits. Together, this indicated that GABA-A Rs are implicated in the motogenic activation of phagocytes upon coccidian challenge and, specifically raised the question of participation of a, b and r GABA-A Rs subunits in hypermotility of phagocytes.

Gene silencing of GABA-A R subunits inhibits DC hypermotility
To determine which GABA-A R subunits were implicated in hypermotility, we designed a gene silencing approach in primary DCs. First, based on subunit expression data ( Figure 2) and on the  Figure 4A) and hMoDCs ( Figure 4B). Finally, the impact of gene silencing on hypermotility was assessed. The hypermotility phenotypes remained unaffected in mock-transduced and control shRNA-treated conditions (Ó lafsson et al., 2019; Figure 4C,D,E,F). In contrast, hypermotility was abolished in shb3-and shr1-treated mBMDCs ( Figure 4C,D,G), demonstrating the implication of b3 and r1 subunits. In sha3-treated cells (with two separate constructs), hypermotility was significantly reduced, albeit not abolished, indicating a contribution by the a3 subunit. In hMoDCs, hypermotility was significantly reduced in sha4-treated cells, but not in shr2-treated cells ( Figure 4E,F,G), indicating primarily a dependence on the a4 subunit for hypermotility. Jointly with pharmacological inhibition, these data demonstrate a critical dependency of DC hypermotility on the b3 and r1 subunits for mBMDCs and on the a4 subunit for hMoDCs.  Abolished DC hypermotility by inhibition of GABA synthesis (GAD67) and secretion is rescued by GABA-A R agonism The constitutive elements of a GABA synthesis and transport in human DCs have remained uncharacterized . In addition to GABA synthesis enzymes ( Figure 1G), hMoDCs and mBMDCs expressed GABA transporters ( Figure 5A, C), whose expression was modulated by challenge with T. gondii ( Figure   Sh 2 concentrations ( Figure 5F) in the supernatants of hMoDCs, as previously shown in mBMDCs . Further, exogenous GABA and GABA-A R agonism (muscimol) fully reconstituted hypermotility in hMoDCs in presence of GABA synthesis inhibitor ( Figure 5E, H). This, together with the identification of GAD67 as putative principal GABA synthesis enzyme in hMoDCs ( Figure 1G) motivated gene silencing of GAD67 ( Figure 5I, Figure 4-figure supplement 1B,D, Supplementary file 3). Importantly, the amounts of secreted GABA in the supernatant were strongly reduced in GAD67-silenced infected hMoDCs, approached amounts secreted by unchallenged cells and contrasted with maintained GABA secretion by mock-or shLuc-transduced infected cells ( Figure 5J). Finally, GAD67-silenced infected hMoDCs exhibited abrogated hypermotility ( Figure 5K). Jointly, these data demonstrate a critical dependence of the hypermigratory phenotype on GABA synthesized by GAD67 in hMoDCs.
The GABA signaling regulator NKCC1 impacts hypermotility of phagocytes GABA-A R function in the CNS is regulated by CCCs, which are subdivided in Na-K-Cl cotransporters (NKCCs) and K-Cl cotransporters (KCCs) (Kaila et al., 2014). However, CCCs have remained uncharacterized in phagocytes. A transcriptional expression screen (Supplementary file 1) detected mRNAs of NKCCs and KCCs in mBMDCs ( Figure 6A) and in hMoDCs ( Figure 6B). Notably, upon T. gondii challenge, a strong upregulation of NKCC1 was observed in mBMDCs, which was less accentuated in hMoDCs ( Figure 6C,D). Immunolabeling and western blotting indicated presence of NKCC1/2 proteins in mBMDCs ( Figure 6E,F). Interestingly, antagonism with bumetanide, at concentrations known to inhibit NKCC but not KCC activity in neurons (Orlov et al., 2015), resulted in impaired T. gondii-/N. caninum-induced hypermotility in mBMDCs ( Figure 6G . Importantly, hypermotility was abolished in NKCC1-silenced mBMDCs and hMoDCs ( Figure 6M, N). Next, we addressed if NKCC1 and GABA-A R functions were interconnected. First, we observed that stimulation with GABA or with the GABA-A R agonist muscimol failed to reconstitute hypermotility of mBMDCs in the presence of the NKCC1 antagonist bumetanide ( Figure 6O), contrasting with the reconstitution of hypermotility by GABA and muscimol in cells with abrogated GABA synthesis ( Figure 5G). Second, shNKCC1-or shGAD67-transduced hMoDCs were stimulated with GABA. Upon silencing of GABA synthesis (shGAD67), addition of exogenous GABA reconstituted hypermotility. In contrast, in NKCC1-silenced cells, GABA failed to reconstitute hypermotility ( Figure 6P,Q, Figure 6-figure supplement 1). These data indicate a link between NKCC1 and GABA/GABA-A R function in parasitized phagocytes. Altogether, the data demonstrate a functional implication of the Na-K-Cl cotransporter NKCC1 in T. gondii-/N. caninum-induced hypermotility of phagocytes.
To test the implications of GABAergic signaling on Ca 2+ responses, Ca 2+ indicator dye-loaded mBMDCs were challenged with agonistic and antagonistic stimuli and Ca 2+ responses were measured ( Figure 8A, Video 1). Importantly, perfusion of exogenous GABA consistently generated transient cytosolic Ca 2+ elevations, which were antagonized by the GABA-A R blocker picrotoxin (broad inhibitor) and with maintained responsiveness by purinergic Ca 2+ channels to ATP ( Figure 8B,C,D; Video 1). Further, transients generated by L-type VDCC agonism (Bay K) were effectively antagonized by Ca V 1.3 inhibition (CPCPT), indicative of a prominent role for Ca V 1.3 in the measured Ca 2+ responses ( Figure 8E,F,G, Figure 8-figure supplement 1). Finally, T. gondii-infected cells responded to GABA with transient Ca 2+ influx. In individual cell recordings, Ca 2+ influx was significantly reduced or abolished by application of inhibitors of b-subunit containing GABA-A Rs (SCS) and by picrotoxin, with maintained responses to ATP ( Figure 8H,I,J). Jointly, these data identify a determinant role for GABA-A Rs in the GABA-induced Ca 2+ influx via Ca V 1.3 in DC hypermotility.

Gene silencing and pharmacological antagonism of GABAergic signaling slow DC migration and reduce parasite loads in mice
To address the impact of GABAergic signaling on DC-mediated parasite dissemination, we designed separate approaches that targeted GABA-A R function or the function of the GABA signaling regulator NKCC1 in mice. First, we controlled that inhibitors had non-significant effects on parasite invasion and replication (Figure 9-figure supplement 1A) and a persistent inhibitory effect on hypermotility (Figure 9-figure supplement 1B). Second, inhibitor-pretreated parasitized mBMDCs (CMTMR-labeled) and non-treated parasitized mBMDCs (CMF2HC-labeled) were simultaneously adoptively transferred to mice in a competition assay ( Figure 9A). Fourteen to 18 h post-inoculation, organs were harvested and cells were characterized by flow cytometry ( Figure 9B,C, Figure 9-figure supplement 2A, B,C). Importantly, the ratio of pretreated vs non-treated parasitized mBMDCs was significantly lower in spleen for both GABA-A R inhibitor and NKCC1 inhibitor treatments compared with that for non-treated condition ( Figure 9D). In contrast, non-significant differences were observed in peritoneum ( Figure 9D). This indicated selective reduced migration of mBMDCs pretreated with GABA-A R inhibitors or NKCC1 inhibitor compared with non-treated mBMDCs in individual mice. Third, to asses if the reduced migration of parasitized mBMDCs impacted the infection, parasite loads were assessed by plaquing assays and by qPCR. Importantly, compared with the nontreated condition, treatments significantly decreased the parasite loads in spleen and liver ( Figure 9E, Figure 9-figure supplement 2D, E). Finally, we silenced the GABA-A R subunit r1 or NKCC1, two GABA signaling targets with a significant impact on mBMDC migration in vitro (Figure 4, Figure 6). When gene-silenced mBMDCs challenged with T. gondii were adoptively transferred into mice, significantly reduced parasite loads were quantified in peripheral organs ( Figure 9F), corroborating results of pharmacological treatments ( Figure 9E). Moreover, pharmacological inhibition of GABA-A Rs or NKCC1 yielded a significant reduction of parasites loads in the brain by day seven post-inoculation ( Figure 9G). The data demonstrate a role for the GABA-A R subunit r1 and the GABA signaling regulator NKCC1 in mBMDC-mediated dissemination of T.

Discussion
Signaling pathways that can drive migration of immune cells, and are alternative to canonical chemokine-mediated migration, have remained poorly understood. Here, we establish that human and murine mononuclear phagocytes possess a conserved GABAergic system that, upon activation, promotes migration in vitro and in vivo. We identified and performed functional tests on the five principal components of GABAergic signaling, namely (i) GABA metabolism, (ii) GABA transportation and secretion, (iii) GABA-A R activation, (iv) GABA signaling regulators CCCs and (v) effector Ca 2+ channel signaling by VDCCs ( Figure 10). The data provide a molecular and cellular framework for assessing the role of the GABAergic system in immune cells.
We demonstrate that a conserved expression of GABAergic molecular components is functionally linked to motility and migratory activation of mononuclear phagocytes upon infection challenge. First, our studies identify GAD67 as the principal GABA synthesizing enzyme in phagocytes. Gene silencing and pharmacological inhibition of GAD67 abrogated secretion of GABA and migratory activation of phagocytes, which was reconstituted by GABA-A R agonism. This supports the notion that GABA is synthesized cytosolically and secreted in vesicle-independent fashion, likely by transport through GATs, for tonic modulations of GABA-A Rs in immune cells, similar to neurons (Kaufman et al., 1991;Feldblum et al., 1993). Second, our data establish that expression of specific GABA-A R subunits determine the motogenic function of GABA-A Rs in phagocytes. The expression of GABA-A R subunit types was diverse, in line with the expression diversity in neurons (Davis et al., 2000;Goetz et al., 2007). Yet, the different phagocyte types consistently expressed a repertoire of GABA-A R subunits sufficient to constitute functional channels, that is: at least one a, one b and one third type of subunit, or homopentamer-forming r subunits. The inhibitory effects by selective pharmacological antagonism on phagocyte hypermotility indicated implication of a, b and r subunits and was confirmed by gene silencing. Importantly, silencing of the a4 subunit (but not r2) or b3 and r1 subunits (but not a3) abolished hypermigration of human and murine DCs, respectively. Jointly, this narrows putative receptor pentamers acting in phagocytes but also highlights a hierarchy among GABA-A R subunits mediating migratory activation or function redundancy among the different subunits. Third, our data identify a determinant role for the CCC NKCC1 in the migratory activation of phagocytes. By pharmacological inhibition and gene silencing in vitro and in vivo, we show that NKCC1 plays a crucial role in the regulation of GABA signaling in phagocytes. Of note, NKCC1 was linked to GABA-A R function and its regulative characteristics together with limited variability -compared with the high diversity in expression of GABA-A R subunits-made NKCC1 a prime target for in vivo experimentation. Finally, we demonstrate that stimulation with GABA elicits Ca 2+ influx transients in the DC cytosol. In line with this, human and murine phagocytes expressed a highly conserved repertoire of VDCC subtypes. Moreover, silencing of the VDCC subtype Ca V 1.3 in human DCs abrogated T. gondii-induced hypermotility, in line with our observations in murine DCs (Kanatani et al., 2017). Jointly, this demonstrates that Ca V 1.3 is determinant to the motogenic action of GABA-A R activation.  Our data establish that T. gondii and N. caninum, two coccidian parasites with a broad range of vertebrate hosts, induce GABAergic signaling in parasitized phagocytes. From a perspective of intracellular parasitism, hijacking a conserved GABAergic system offers the advantage of inducing migratory activation of shuttle phagocytes within minutes after active invasion  to favor systemic dissemination in vertebrate hosts (Lambert et al., 2006;Courret et al., 2006). Activation of GABAergic signaling is relatively fast as GABA binding opens the GABA-A Rs in milliseconds (Farrant and Nusser, 2005) and the elements that synthesize and transport GABA, GADs and GATs, respectively, are constitutionally expressed and upregulated upon infection. Consequently, DCs initiated GABA secretion shortly after T. gondii invasion. Thus, the rapid onset and tight regulation of GABAergic signaling by-passes the need for, presumably slower, transcriptional regulation. Additionally, the GABA signaling regulator NKCC1 plays an important role in the hypermigration of parasitized phagocytes, presumably by impacting chloride concentration and thus, GABA-A R function. Consequently, inhibition of GABA-A Rs or NKCC1 in adoptively transferred pharmacologically pretreated or gene silenced DCs, significantly reduced the migration of parasitized DCs and parasite loads in mice. Importantly, hampered dissemination to peripheral vital organs was evident early during infection and, later, resulted in reduced parasite loads in the CNS. Jointly with present data, different approaches show that targeting (i) GABA synthesis/transportation , (ii) GABA-A R signaling, (iii) GABA-A R regulation/NKCC1 hampers systemic dissemination and parasite loads in the brain. However, inhibition of VDCC signaling inhibited systemic dissemination but non-significantly impacted parasite loads in the brain (Kanatani et al., 2017), indicating that specific GABAergic signaling components contribute differently or indicating redundancy in VDCC signaling. Because invasive coccidian parasites need to reconcile their obligate intracellular existence with the need for dissemination, the hijacking of migratory leukocytes represents a secluded replication niche that facilitates dissemination. The identified GABAergic determinants provide a molecular framework for assessing if other protozoa, intracellular bacteria (Kim et al., 2018) or viruses (Zhu et al., 2017) utilize the GABAergic signaling of phagocytes for dissemination. Because hypermigratory parasitized DCs gondii-challenged mBMDCs prelabeled with CMF2HC or CMTMR dye. Cells were analyzed by flow cytometry at 14-18 h post-inoculation (gating Figure 9 continued on next page exhibit chemotaxis (Weidner and Barragan, 2014) and GABA impacts the secretion of pro-inflammatory cytokines (Bhandage et al., 2018), the impact of GABAergic signaling in the inflammatory microenvironment of infection needs to be further investigated, also in the setting of acute and chronic infection and neuroinflammatory responses in the CNS . Thus, hypermigration and chemotaxis are not antithetical and may, in fact, cooperatively potentiate the migratory potential of parasitized phagocytes and therefore also the dissemination of coccidia Weidner et al., 2013;García-Sánchez et al., 2019).

A C
Our study provides the first exhaustive characterization of a GABAergic machinery in myeloid mononuclear phagocytes. Recent findings also indicate GABAergic responses by macrophages (Januzi et al., 2018), microglia , lymphocytes (Bhandage et al., 2018) and bovine immune cells (García-Sánchez et al., 2019). Altogether, this highlights that GABAergic signaling by immune cells may be more the rule than the exception. Along these lines, GABA has a motogenic role in embryonic interneuron migration in the developing fetus (Bortone and Polleux, 2009). Furthermore, GABAergic signaling has newly been linked to the metastasis of multiple cancer types (Sizemore et al., 2014;Wu et al., 2014), including gliomas, pancreatic cancer and breast cancer (Neman et al., 2014;Takehara et al., 2007;Smits et al., 2012). The peripheral GABAergic system also appears implicated in various autoimmune diseases, such as multiple sclerosis (Bhat et al., 2010), type I diabetes (Li et al., 2017;Bhandage et al., 2018) and rheumatoid arthritis (Tian et al., 2011), where GABAergic inhibition dampens the inflammatory response. It will be important to assess if the motogenic molecular components identified here are also implicated in the inflammatory responses and in cancer cell metastasis. Moreover, the patho-physiological cellular microenvironments result in expression of specific subtypes of GABA receptors (Bhandage et al., 2014;Smits et al., 2012) and thus, selective compounds have been identified in neuropsychiatric drug design (Korpi and Sinkkonen, 2006;Krall et al., 2015). Additionally, anesthetics targeting GABA-A Rs have been implicated in the impairment of immune cell function during human surgery (Wheeler et al., 2011). Thus, receptor subtypes or other GABAergic components may be targeted to modulate migration of GABAergic cells (Miao et al., 2010) and our data provide a molecular framework for therapeutic targets of clinical relevance.
Finally, the non-protein amino acid GABA has developed into an essential neurotransmitter of the evolved vertebrate CNS. However, GABA precedes the development of the CNS as a metabolismand stress-related signaling molecule in prokaryotes, invertebrates and plants (Hudec et al., 2015;MacRae et al., 2012;Pinan-Lucarré et al., 2014). Here, we add that mononuclear phagocytes express a conserved GABAergic system linked to their migratory functions, which coccidian parasites can hijack for dissemination. Thus, GABA also acts as an interspecies signaling molecule in hostmicrobe interactions.  Figure 9- figure supplement 2A, B,C). Plots in upper, center and lower rows show, respectively, non-treated cells, cells pretreated with GABA-A R inhibitors (GABA-A R i) and NKCC1 inhibitor (NKCC1 i). (D) Bar graph shows the ratio of treated cells (CD11c + GFP + CMTMR + ) to non-treated cells (CD11c + GFP + CMF2HC + ) in peritoneal lavage and spleen after adoptive transfer of T. gondii-challenged mBMDCs (n = 7-10 mice per group). (E) Parasite loads in spleen and liver of C57BL/6 mice at day 4 post-inoculation of pharmacologically treated cells (GABA-A R i, NKCC1 i) related to nontreated cells and measured by plaquing assays (n = 5-6 mice per group). (F) Parasite loads in spleen and liver of C57BL/6 mice at day 5 post-inoculation of gene-silenced cells (shr1, shNKCC1) related to mock-and control shLuc-transduced cells and measured by plaquing assays (n = 6 mice per group). (G) Parasite loads in spleen and brain of CD1 mice at day 7 post-inoculation of pharmacologically treated cells (GABA-A R i, NKCC1 i) related to nontreated cells and measured by plaquing assays (n = 9-10 mice per group). Bar graphs show mean + SEM. Statistical significance was assessed by ordinary one-way ANOVA with Tukey's multiple comparison test, *p<0.05, **p<0.01, ***p<0.001, ns p!0.05. The online version of this article includes the following figure supplement(s) for figure 9:    (Zeiss Observer Z.1). Motility tracks for 50-60 cells per treatment were analyzed using ImageJ software for each experiment.

Lentiviral vector production and transduction
Self-inactivating Lentiviral particles were produced from Lenti-X 293 T cells (Takara Bio, Gothenburg, Sweden) by co-transfecting (i) a lentiviral vector, pLL3.7 or pLKO.1, containing self-complementary hairpin DNA oligos targeting specific mRNA (Supplementary file 3), (ii) psPAX2 packaging vector and (iii) pCMV-VSVg envelope vector, as described previously (Kanatani et al., 2017;Ó lafsson et al., 2019). The lentiviral supernatants were used for transduction. The lentivirus transduction efficiency was examined in murine NE4Cs and human SH-Sy5y cell lines and further, knockdown efficiency was determined in NE4Cs (Figure 4-figure supplement 1). Murine BMDCs were transduced on day 3 of culturing, whereas human MoDCs were transduced day 3 and day 5. Transduction efficiency was examined for eGFP expression by epifluorescence microscopy, followed by expression analysis by qPCR for knock-down of targeted mRNA. All the target conditions were compared to mock-condition and a positive control, non-related sequence (luciferase, Luc). Transduced cells were further used for experiments.

Plaquing assays
Plaquing assays were performed as described . Briefly, organs were extracted and homogenized on 70 mm cell strainers. The numbers of viable parasites per g of tissue were determined by plaque formation on HFF-1 monolayers.
Replication assay mBMDCs were challenged with freshly egressed tachyzoites (ME49/PTG-GFP, 1 h, MOI 2). Cells were washed by centrifugation to remove extracellular parasites and seeded in 96-well plates. Cells were imaged by epifluorescence microscopy to detect number of parasites per vacuoles at 6 h and 24 h post-infection in presence of pharmacological treatments.

Data mining and statistical analyses
Motility plots were compiled using ImageJ with manual tracking and chemotaxis plugin. X-and y-axes in the plots show distances in mm. For motility assays, box-whisker and scattered dot plots represents median velocities (mm/min) with boxes marking 25 th to 75 th percentile and whiskers marking 10 th and 90 th percentiles of the datasets. Gray circles represent velocities from individual cells. Accumulated distances travelled by the cells (% cells tracked are binned at a range of 2 mm distance) are represented as histograms. Bar graphs show mean + SEM. Heat maps represent transcriptional changes in mRNA expression (2 -DCt ) upon challenge with T. gondii. Red and blue color scales indicate percentage increase and decrease in expression, respectively, normalized to expression in unchallenged cells at the same time point, respectively. Data mining and statistical analyses were performed using GraphPad Prism 7.0 (La Jolla, CA, USA). The statistical significance is represented as p<0.05 (*), p<0.01 (**), p<0.001 (***) or non-significant p!0.05 (ns).