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The ROP16III-dependent early immune response determines the sub-acute CNS immune response and type III Toxoplasma gondii survival

Toxoplasma gondii is an intracellular parasite that persistently infects the CNS and that has genetically distinct strains which provoke different acute immune responses. How differences in the acute immune response affect the CNS immune response is unknown. To address this question, we used two persistent Toxoplasma strains (type II and type III) and examined the CNS immune response at 21 days post infection (dpi). Contrary to acute infection studies, type III-infected mice had higher numbers of total CNS T cells and macrophages/microglia but fewer alternatively activated macrophages (M2s) and regulatory T cells (Tregs) than type II-infected mice. By profiling splenocytes at 5, 10 and 21 dpi, we determined that at 5 dpi type III-infected mice had more M2s while type II-infected mice had more classically activated macrophages (M1s) and these responses flipped over time. To test how these early differences influence the CNS immune response, we engineered the type III strain to lack ROP16 (IIIΔrop16), the polymorphic effector protein that drives the type III-associated M2 response. IIIΔrop16-infected mice showed a type II-like neuroinflammatory response with fewer infiltrating T cells and macrophages/microglia and more M2s and an unexpectedly low CNS parasite burden. At 5 dpi, IIIΔrop16-infected mice showed a mixed inflammatory response with more M1s, M2s, T effector cells, and Tregs, and decreased rates of infection of peritoneal exudative cells (PECs). These data suggested that type III parasites need the early ROP16-associated M2 response to avoid clearance, possibly by the Immunity-Related GTPases (IRGs), IFN-γ dependent proteins essential for murine defenses against Toxoplasma. To test this possibility, we infected IRG-deficient mice and found that IIIΔrop16 parasites now maintained parental levels of PECs infection. Collectively, these studies suggest that, for the type III strain, rop16III plays a key role in parasite persistence and influences the sub-acute CNS immune response. Author Summary Toxoplasma is a ubiquitous intracellular parasite that establishes an asymptomatic brain infection in immunocompetent individuals. However, in the immunocompromised and the developing fetus, Toxoplasma can cause problems ranging from fever to chorioretinitis to severe toxoplasmic encephalitis. Emerging evidence suggests that the genotype of the infecting Toxoplasma strain may influence these outcomes, possibly through the secretion of Toxoplasma strain-specific polymorphic effector proteins that trigger different host cell signaling pathways. While such strain-specific modulation of host cell signaling has been shown to affect acute immune responses, it is unclear how these differences influence the sub-acute or chronic responses in the CNS, the major organ affected in symptomatic disease. This study shows that genetically distinct strains of Toxoplasma provoke strain-specific CNS immune responses and that, for one strain (type III), the acute and sub-acute immune responses and parasite survival are heavily influenced by a polymorphic parasite gene (rop16III).

Introduction 67 68 seen with acute infection in vivo and in vitro (21,22). By examining the peripheral 115 macrophage immune response over time, we determined that, early in infection, type III-116 infected mice have more M2s compared to type II-infected mice and that this response 117 changes over time, leading to fewer M2s in the spleen and brain of type III-infected mice 118 by 21 dpi. To define if the differences in the early macrophage response influenced the 119 subsequent CNS immune response, we engineered the type III strain to lack the rop16 120 gene (IIIΔrop16), which, as noted above, is the driver of the early type III-associated M2 121 response (19,21). Consistent with our hypothesis, compared to the parental type III 122 strain, IIIΔrop16-infected mice showed a more type II-like CNS immune response with 123 fewer macrophages and infiltrating T cells and an increase in M2s in the CNS. 124 Unexpectedly, the IIIΔrop16-infected mice showed a substantial decrease in the CNS 125 parasite burden; a mixed acute inflammatory immune response (i.e. an increase in 126 classically activated macrophages (M1s), M2s, T effector cells, and Tregs); and rapid 127 clearance from the site of inoculation. As the type III strain is sensitive to destruction by 128 Immunity-Related GTPases (IRGs) (25,26), these results suggested that, to persist, the 129 type III strain requires rop16III to dampen the initial immune response, including the IRG 130 response. We tested this possibility by infecting mice that lack the IRG response (27) 131 and found that the IIIΔrop16 strain now maintained parental levels of infection at the site 132 of inoculation. Collectively these data suggest that Toxoplasma strain-specific immune 133 responses persist in the sub-acute phase of disease and that, for the type III strain, 134 rop16III is required for persistence and plays a role in determining acute and sub-acute 135 systemic and CNS immune responses. To determine if genetically divergent Toxoplasma strains cause strain-specific 143 CNS immune responses, we infected mice with either a type II (Prugniaud) or type III 144 (CEP) strain and analyzed the CNS macrophage and T cell immune response at 21 dpi, 145 which we consider a sub-acute time point of CNS infection. We focused on the 146 macrophages/microglial and T cell responses because prior work has established that 147 these cells are essential for controlling acute and chronic toxoplasmosis (1,(28)(29)(30)(31). To 148 quantify macrophages/microglia and T cells in the CNS after Toxoplasma infection, we 149 stained brain sections with antibodies against Iba-1, a pan macrophages/microglial 150 marker, or CD3, a pan-T cell surface marker. Stained sections were then analyzed by 151 light microscopy. We found that type III-infected mice had approximately twice the 152 number of CNS macrophages/microglia compared to type II-infected mice (Fig. 1A,B). 153 Type III-infected mice also had a similar increase in the number of CNS T cells (Fig.  154

1C,D). 155 156
To determine how the influx of these immune cells changed the global CNS 157 cytokine/chemokine environment, we isolated and analyzed protein from brain 158 homogenates of control (saline inoculated) or infected mice using a 25-plex cytokine 159 and chemokine LUMINEX assay. As expected, compared to control mice, type II and 160 type III-infected mice showed a >2-fold increase in most of the pro-inflammatory 161 cytokines and chemokines in the panel (Table S1). A subset of these cytokines and 162 chemokines also showed a >2-fold increase in type III-infected mice compared to type 163 II-infected mice (Fig. 1F). Given the consistent differences we found in the number of 175 macrophages/microglia and T cells ( Fig. 1), we sought to determine if these differences 176 simply reflected disparities in parasite burden. To address this question, we analyzed 177 CNS parasite burden by two methods. First, using DNA isolated from brain 178 homogenates, we performed quantitative PCR (qPCR) for the Toxoplasma-specific 179 gene B1 (32-35). Second, we quantified CNS cyst numbers by performing 180 immunofluorescent assays on brain sections using Dolichos biflorous agglutinin (DBA), 181 a lectin that stains the cyst wall, to identify cysts (36). By both measures, we found that 182 type II and type III-infected mice had equivalent CNS parasite burdens at 21 dpi ( Fig.  183   2A,B). 184

185
To ensure that differences in parasite dissemination to the CNS were not driving 186 the immune response differences, we quantified the parasite burden in the spleen, liver, 187 lungs, and CNS at 5, 10, and 21 dpi using B1 qPCR. At these time points, we found that 188 the parasite burden in these different organs did not differ between type II and type III-189 infected mice ( Fig. 2C-F As these studies represented the first studies to use flow cytometry to compare 208 strain-specific CNS macrophage and T cell responses, we sought to profile major and a prior study that examined CNS macrophages in type II-infected mice showed that 216 macrophages that express MMR also express CXCR3 (41). To validate this staining 217 protocol, we verified that we obtained the same results regardless of whether CD80 and 218 CD86 or CXCR3 and MMR are placed in individual channels or in the same channels 219 (Fig S3). Finally, to further confirm the identity of the M1/M2 populations we isolated 220 CD80 + /CD86 + or MMR + /CXCR3 + splenocytes and used qPCR to determine the 221 expression of IL-12, Arg-1, and IL-4 (21,22,39,41). As expected, the population we 222 defined as M1s (CD80 + /CD86 + ) expressed IL-12 and not Arg-1 and IL-4, while the M2s 223 (MMR + /CXCR3 + ) expressed Arg-1 and IL-4 but not IL-12 ( Fig. S4A-D). In addition, 224 approximately 35% of our CD80 + /CD86 + population was positive for IFN-γ while less 225 than 1% of the MMR + /CXCR3 + population was positive for IFN-γ (Fig. S4E,F). 226 Collectively, these data highly suggest that the macrophage population identified by 227 CD45 + , F4/80 + , CD11b hi , CD11c lo , CD80 + /CD86 + staining is consistent with prior 228 descriptions of M1 macrophages and is pro-inflammatory. Similarly, the macrophage 229 population identified by CD45 + , F4/80 + , CD11b hi , CD11c lo , MMR + /CXCR3 + staining is 230 consistent with prior descriptions of M2 macrophages, which are less inflammatory. 231 From here forward, we will simply denote these populations as M1s and M2s. 232 Based upon these validations, our flow analyses of the CNS immune cells 233 showed that type III-infected mice had approximately half the number and frequency of 234 M2s compared to type II-infected mice (Fig. 3A,B). For M1s, we observed no significant 235 difference in the absolute number or frequency between the groups (Fig. 3C,D). In 236 addition, we found that type III-infected mice had half the number and frequency of 237 Tregs (CD3 + , CD4 + , FoxP3 + ) as compared to type II-infected mice (Fig. 3E,F) and no 238 difference in the number or frequency of Teffs (CD3 + , CD4 + or CD8 + , CD44 + ) ( Table  239 S2). 240 241 Consistent with our findings in the CNS we observed that splenocytes from type 242 III-infected mice had approximately half the absolute number and frequency of M2s as 243 compared to splenocytes from type II-infected mice (Fig. 4A,B). There was no 244 significant difference in the absolute number and frequency of M1s in the spleen (Fig.  245 4C,D). Akin to our CNS data, splenocytes from type III-infected mice had half the 246 number and frequency of Tregs compared to splenocytes from type II-infected mice 247 ( Fig. 4E,F). 248

260
These data strongly suggest that in addition to quantitative differences in the 261 CNS immune response, type II and III-infected mice show differences in the phenotype 262 of immune cells infiltrating into the CNS. Type III infection provokes a more pro-263 inflammatory sub-acute CNS immune response with a relative decrease in the 264 macrophages (M2s) and T cells (Tregs) that suppress the pro-inflammatory response. 265 In addition, as these differences are also seen in splenocytes, these data suggest that, 266 at 21 dpi, the CNS immune response is reflective of the systemic immune response. 267 Finally, the lack of mCherry + and/or GFP + immune cells suggests that our findings are 268 not driven by a small population of immune cells that are actively infected or directly 269 manipulated by parasites.  parasites are polarized to M1s (21,46). As our work was done in vivo at 21 dpi and the 278 prior work was done in vitro or very early in vivo (1-3 dpi), one explanation for these 279 discrepancies is that the in vivo immune response evolves over time. To test this 280 possibility and as we had found that splenocytes were accurate predictors of the CNS 281 immune response, we phenotyped splenocytes from infected mice at 5, 10, and 21 dpi. 282

283
At 5 dpi, we observed that type III-infected mice had an approximately 3-fold 284 higher frequency and 2-fold higher number of splenic M2s as compared to type II-285 infected mice (Fig. 5A). Conversely, at this time point, type II-infected mice showed an 286 increased frequency and 1.5-fold higher number of splenic M1s as compared to type III-287 infected mice (Fig. 5B). By 10 dpi, the macrophage compartment from both type II and 288 type III-infected mice had expanded and no difference in macrophage polarization state 289 was seen ( Fig. 5C-F). By 21 dpi, the splenic macrophage compartment was contracting 290 and now type III-infected mice had fewer splenic M2s both by absolute number and 291 frequency compared to type II-infected mice (Fig. 5C,D). At 5 and 10 dpi, for 292 macrophages from type II or type III-infected mice, we found 1% or less of the 293 macrophage population was infected or injected with parasite protein (i.e. ≤ 1% of the 294 macrophage population was mCherry + and/or GFP + ). We found no strain-specific 295 differences in the Tregs response at 5 or 10 dpi (Fig. 5G,H). 296 297 These data show that early in infection type III-infected mice show a stronger M2 298 macrophage response than type II-infected mice. However, as the infection progresses, 299 the immune response evolves such that by 21 dpi type III-infected mice now have a 300 significant decrease in these anti-inflammatory macrophages compared to type II-301 infected mice, even though parasite dissemination to the CNS is equivalent (Fig. 2). 302 Unlike the macrophage response, we did not observe strain-specific differences in 303 Tregs until 21 dpi. Our data also show that these strain-specific differences are primarily 304 found in uninfected macrophages. 305 306 ROP16III affects the type III CNS immune response and parasite persistence 307 308 Given the evolution of these strain-specific differences from 5 to 21 dpi, we 309 hypothesized that the early macrophage immune response might heavily influence the 310 ensuing sub-acute immune response. We focused on the early macrophage response 311 for several reasons. Tissue resident macrophages are some of the first cells to interface 312 and respond to infecting microbes (47). Consistent with this concept, at 5 dpi, we found 313 strain-specific differences in the macrophage compartment but not the T cell 314 compartment ( Fig. 5A-H). Furthermore, a growing body of literature suggests that this 315 early response influences the ensuing T cell response, possibly through the secretion of 316 cytokines (48). Finally, as noted above, prior in vitro and in vivo work has already 317 established that macrophages infected with type II parasites polarize to M1s while 318 macrophage infected with type III parasites polarize to M2s. Importantly, these studies 319 also determined that specific alleles of Toxoplasma effector proteins (GRA15II for M1s 320 and ROP16III for M2s) drive these macrophage phenotypes (19,21,22), giving us a 321 mechanism for altering these responses. Thus, to determine if early macrophage 322 responses influence the subsequent CNS immune response, we used CRISPR/Cas9 323 (49-53) to engineer a type III strain that lacked rop16 (Fig. S5A). We validated the 324 deletion of rop16 (IIIΔrop16) using locus-specific PCR (Fig. S5B) and a functional assay 325 to show that these parasites no longer induced host cell phosphorylation of STAT6 ( Fig.  326 S5C), the transcription factor linked to the rop16III-associated M2 phenotype (19). Given 327 that this strain should lack the early type III-associated M2 response, we predicted that 328 it would provoke a more type II-like sub-acute CNS immune response. To test this 329 prediction, we infected mice with type II, type III, or III∆rop16 parasites and, at 21 dpi, 330 analyzed the CNS immune response by IHC and flow cytometry. Consistent with our 331 hypothesis, by quantitative IHC, we found that the CNS immune response in III∆rop16-332 infected mice looked akin to type II-infected mice with fewer infiltrating 333 macrophages/microglia and T cells compared to type III-infected mice ( Fig. 6A-E). By 334 flow cytometry, III∆rop16-infected mice again looked similar to type II-infected mice in 335 terms of M2s frequency and absolute number (Fig. 7A,B). The frequency and the 336 absolute number of M1s were not statistically different between type II, type III, or 337 III∆rop16-infected mice (Fig. 7C,D). Unexpectedly, by both Toxoplasma-specific qPCR 338 and cyst count, the III∆rop16-infected mice showed a substantial decrease in the CNS 339 parasite burden compared to type II or type III-infected mice (Fig. 8A,B). 340

341
To verify that the lack of rop16III drove the immune response and parasite burden 342 changes we identified, we generated a IIIΔrop16::ROP16III strain that ectopically 343 expresses rop16III. We validated the expression of rop16III using gene-specific PCR and 344 a functional assay to confirm parasite-induced host cell phosphorylation of STAT6 ( Fig.  345 S5A-C). We then infected mice with type III, IIIΔrop16, or IIIΔrop16::ROP16III parasites 346 and analyzed the CNS immune response by quantitative IHC and flow cytometry. The 347 IIIΔrop16::ROP16III strain produced a CNS immune response akin to the parental type 348 III strain, and distinct from the IIIΔrop16, in terms of macrophage and T cell numbers 349 (Fig. 9A,B), parasite burden (Fig. 9C,D), and M2 number and frequency (Fig. 9E). The 350 M1 number and frequency was not different between the three strains ( Fig. 9F). 351

352
In summary, these data highly suggest that, in the context of a type III infection, 353 rop16III influences the CNS immune response and enables parasite persistence. To confirm that the changes in the CNS were downstream of a change in the 358 acute inflammatory response, at 5 dpi we phenotyped splenocytes from mice infected 359 with type II, type III, or IIIΔrop16 parasites. Unexpectedly, mice infected with the 360 IIIΔrop16 strain showed a mixed immune phenotype with an increase in both M2s and 361 M1s (Fig. 10A,B). In addition, and unlike either type II or type III-infected mice, 362 IIIΔrop16-infected mice showed an increase in the number and frequency of Tregs (Fig.  363   10C) as well as a 3-fold increase in the number of IFN-γ + CD4 and CD8 T cells (Fig.  364 10D,E). Using the mean fluorescence intensity of IFN-γ staining to assess IFN-γ 365 production per cell, we found that T cells from IIIΔrop16 and type II-infected mice 366 produced similar amounts of IFN-γ, which was 1.5-fold higher than T cells from type III-367 infected mice (Fig. 10D,E). 368

369
To verify that these acute peripheral immune response changes were driven by 370 rop16III, we infected mice with the type III, IIIΔrop16, or IIIΔrop16::ROP16III parasites 371 and phenotyped the splenocytes at 5 dpi. As expected the IIIΔrop16::ROP16III strain 372 produced a splenocyte immune response akin to the parental type III strain in terms of 373 M2s, M1s, and Treg frequency and absolute number ( Fig. 11A-C). In the IFN-γ + CD4 374 and CD8 compartment, the IIIΔrop16::ROP16III strain also produced a parental type III 375 response in terms of absolute numbers of CD4 and CD8 IFN-γ + cells and the level of 376 IFN-γ produced per cell (Fig. 11D,E). 377

378
In summary, these data show that type III parasites lacking rop16 produce a 379 mixed inflammatory phenotype at 5 dpi, suggesting that rop16III is an important 380 determinant of the acute immune response to type III parasites. 381 382 Type III parasites depend upon rop16 to avoid early clearance by Immunity-383

Related GTPases (IRGs) 384 385
Given this unanticipated acute inflammatory response including the elevated 386 level of T cell IFN-γ production, and decreased CNS parasite burden at 21 dpi, we 387 hypothesized that the IIIΔrop16 parasites were undergoing an increased level of early 388 clearance. This heightened early inflammatory response and the rapid clearance of 389 parasites would then provoke a strong counterbalancing anti-inflammatory response, 390 causing an increase in both M2s and Tregs early in infection. This hypothesis is 391 particularly appealing because type III strains have a very low expression of the virulent 392 allele of rop18, a parasite virulence gene that disables the interferon-γ-inducible 393 Immunity-Related GTPase system (IRGs) which is a major mechanism by which murine 394 host cells kill intracellular parasites in an IFN-γ dependent manner (25,26,54). To test 395 this possibility, we quantified the frequency of infected peritoneal exudate cells (PECs) 396 at 1, 3, and 5 dpi from mice infected with type II, type III, or III∆rop16 parasites. At 1 and 397 3 dpi, all 3 strains showed the same frequency of infected PECs. But, by 5 dpi, 398 III∆rop16-infected mice showed a 4-6-fold lower rate of infected PECs compared to type 399 II or parental type III-infected mice ( Fig. 12A-C). To confirm that this increase in 400 parasite clearance was secondary to a lack of rop16III, we infected mice with type III, 401 III∆rop16, or IIIΔrop16::ROP16III parasites and assayed the frequency of infected PECs 402 at 3 and 5 dpi. As expected, the IIIΔrop16::ROP16III strain maintained parental levels of 403 infected PECs at both time points (Fig. 12D). 404

405
Finally, to directly test if the IRGs were the mechanism by which the IIIΔrop16 406 parasites were being cleared, we assayed the frequency of infected PECs at 3 and 5 407 dpi in mice that lack both Irgm1 and Irgm3, key mediators of IRG pathway (27,55-58). 408 We reasoned that if the IRGs mediated the rop16III-dependent increase in clearance, 409 then in Irgm1/3 KO mice, the IIIΔrop16 strain should now maintain parental levels of 410 PECs infection at 5dpi, which is what we found (Fig. 12E) responses (5 dpi), with high levels of IL-12 and IFN-γ production (Fig S4). This early 440 pro-inflammatory response then provokes a counter-balancing anti-inflammatory 441 response that leads to a rise in M2s, which continues as parasites disseminate and 442 enter the brain. This systemic response is then recapitulated in the CNS as parasites 443 establish a chronic CNS infection (Fig. 13A). Conversely, for type III parasites, during 444 acute infection, type III-infected macrophages are polarized to M2s via rop16III leading 445 to increased IL-4 production (Fig S4), which promotes a mixed systemic inflammatory 446 response with more early M2s, resulting in decreased levels of IFN-γ and IL-12 and 447 increased levels of IL-4 (Fig S4) compared to type II-infected mice. This less pro-448 inflammatory early response enables type III parasites to evade early clearance and 449 avoids provoking the compensatory anti-inflammatory response, so as type III parasites 450 disseminate to the CNS, the immune response that occurs is now strongly pro-451 inflammatory (Fig. 13B). parasite burden in distal organs while also increasing IL-12 production by PECs. These 473 data suggest that the increase in IL-12, which should result in higher IFN-γ production, 474 does not adversely affect the IΔrop16 parasites (19). Conversely, we used a type III 475 strain, which is genetically similar to the type I strain but avirulent in mice because of its' 476 low expression of rop18, a key protein for blocking murine IFN-γ-dependent cell intrinsic 477 defenses (25,26,54). In the context of low rop18 expression and therefore high 478 susceptibility to the IRGs, the ability of rop16I/III to decrease the early IFN-γ response 479 (Fig. 10E) by decreasing IL-12 and increasing the M2 response, appears crucial for 480 type III strains to avoid rapid clearance during the very earliest part of infection. This 481 proposed mechanism is supported by the data in the Irgm1/3 knockout mice (Fig. 12) 482 as well as prior work showing that IRG-mediated clearance of intracellular parasites is a 483 major murine IFN-γ-dependent mechanism for controlling Toxoplasma (25,26,54). 484 Collectively, these data suggest that, in vivo, rop16I/III is dispensable for type I strains 485 but essential for type III strains, a discrepancy potentially explained by differences in 486 rop18 expression. 487

488
We have also shown that the strain-specific polarizations previously identified in 489 infected macrophages (21) also extends to uninfected macrophages found in the 490 spleen. Based upon the work of others showing that type II-infected macrophages 491 secrete more IL-12 while type I/III-infected macrophages secrete less IL-12 and more 492 IL-4 (19,21,22,60), we speculate that the strain-specific differences in secreted 493 cytokines from infected immune cells propagates the immune cell polarization 494 differences to uninfected immune cells (Fig. 13). 495 496 Finally, we have shown that the IIIΔrop16 strain is able to elicit a strong brain immune 497 response, including infiltration of T cells and likely monocytes, despite having a very low 498 CNS parasite burden (Fig. 6-8). The finding of a much stronger CNS immune response 499 than parasite burden is consistent with prior work showing that immune cells can and do 500 infiltrate into the CNS in the setting of a strong systemic immune response without brain 501 infection or pathology (61). We suggest that our data add to the growing body of 502 literature that the "immune privileged" status of the CNS is less absolute than previously and xylazine (4.8 mg/ml) cocktail and transcardially perfused with ice cold phosphate 544 buffered saline. As previously described, after harvesting organs, the left half of the 545 mouse brain was fixed in 4% paraformaldehyde in phosphate buffer, kept at 4°C 546 overnight, rinsed in PBS, and then was embedded in 30% sucrose (42,45). Post fixation 547 and sucrose embedding, the brain was sectioned into 40 µm thick sagittal sections 548 using a freezing sliding microtome (Microm HM 430). Sections were stored as free-549 floating sections in cryoprotective media (0.05 M sodium phosphate buffer containing 550 30% glycerol and 30% ethylene glycol) until stained and mounted on slides. The right 551 half of the brain was sectioned coronally into 2 halves and stored in separate tubes. 552 These tubes were flash frozen and stored at -80°C until used for protein or DNA Iba-1 + cells/FOV was quantified by manually counting cells with FIJI software (63). 577 These analyses was performed on 3 brain sections per mouse, after which the resulting 578 numbers were then averaged to obtain the average number of Iba1 + or CD3 + immune 579 cells/brain section/mouse. Investigators quantifying CD3 + and Iba-1 + cells were blinded 580 to the infection status of the mouse until after the data were collected. 581 582

Protein Extraction, Quantification and Multiplex LUMINEX Assay 583 584
The caudal quarter of the flash frozen brain tissue was homogenized in 585 radioimmunoprecipitation assay buffer (1% Triton-X, 0.1% SDS, 1X PBS) and 1% 586 phosphatase inhibitor cocktail (P5726-1ML, Sigma-Aldrich) and 1% protease inhibitor 587 cocktail (P8340-1ML, Sigma-Aldrich). As previously described, the samples were 588 sonicated on ice in 3s bursts until homogenized and then were centrifuged at 4°C (62). 589 The protein concentration of each sample was measured using Direct Detect ® Infrared 590 Spectrometer. Each sample was stored at -80°C until the LUMINEX ® assay was 591 Eppendorf Mastercycler ep realplex 2.2 system using primers listed in Table S3. 606 GAPDH was used as house-keeping gene to normalize parasite DNA levels. Results 607 were calculated as previously described (35,62). For quantification of Arg-1, IL-4, and 608 IL-12, total RNA from sorted M1 and M2 cells was extracted with TRIzol™ (Life 609 Technologies, Grand Island, NY) and according to the manufacturer's instructions. First 610 strand cDNA synthesis was performed using a High-Capacity cDNA Reverse 611 Transcription kit (4368814, ThermoFisher) and following the manufacturer's instructions. 612 Arg-1, IL-4, and IL-12 were amplified using SYBR Green fluorescence detection with the 613 Eppendorf Mastercycler ep realplex 2.2 system using the primers are listed in Table S3  614 (21,22,41). GAPDH was used as the house-keeping gene for normalization. The 615 reaction condition were as follows: 2 min at 50°C, 2 min at 90°C, followed by 40 cycles 616 of 15 sec at 95°C, 15 sec at 58°C, and 1 min at 72°C, followed by a melting curve 617 analysis. All the plasmids and primers used to make and validate the IIIΔrop16 strain are listed in 687   Table S3. The rop16-targeting CRISPR plasmids (sg rop16Up and sg rop16Down) were 688 constructed from sgUPRT using a previously described Q5 mutagenesis protocol (51-689 53). To generate a plasmid for inserting hxgprt into the rop16 locus, upstream (500-bp) 690 and downstream (500-bp) regions directly adjacent to the sgROP16Up and sg 691 rop16Down target sequence were used to flank hxgprt via sequential restriction cloning. To disrupt rop16 in type IIIΔhpt, we transfected parasites with 3 plasmids: the sg 696 rop16Up CRISPR and sg rop16Down CRISPR plasmids and the pTKO (69) plasmid 697 containing rop16 homology regions surrounding a selectable marker (hxgprt) or with the 698 toxofilin-Cre cassette (44) (Fig. S5). Selection by growth for 4 to 8 days in 25 mg/ml 699 mycophenolic acid and 50 mg/ml xanthine (70) was used to obtain stably resistant 700 clones with hxgprt integration. These clones were subsequently screened by PCR to 701 confirm disruption of the rop16 locus (Fig. S5) supplemented with 50 μg/mL of Zeocin (InvivoGEN, 11006-33-0) for 4 hour and then 711 added to HFF monolayers supplemented with 5μg/mL Zeocin to select for integrants. 712 This process was repeated 3 times prior to plating by limiting dilution to isolate single 713 clones. Single clones were subsequently screened by PCR for rop16 integration (Fig.  714   S5) were collected by peritoneal lavage with 10 ml of cold 1 x PBS. PECs were incubated in 722 FcBlock for 10 min as described above. PECs were subsequently stained with CD45 723 marker, followed by with live/dead staining as described above, and then analyzed 724 Polymorphic  cytokines or chemokines from a 25-plex assay that showed a >2-fold difference 978 between protein levels in brain homogenates from type II and type III-infected mice. 979 Table S1 has full list of cytokines/chemokines. N=8 mice/infected group. p-values 980 determined by one-way ANOVA with Bonferroni post-hoc test. type II or type III parasites, and brains were harvested at noted time points. A. 986 Quantification of CNS Toxoplasma burden at 21 dpi using quantitative PCR (qPCR) for 987 the Toxoplasma-specific B1 gene and host GAPDH gene (housekeeping gene). 988 Toxoplasma and mouse genomic DNA were isolated from brain homogenates. B. 989 Quantification of Toxoplasma cyst burden at 21 dpi in brain sections stained with 990 Dolichos biflorous agglutinin (DBA), which stains the cyst wall. Stained sections were 991 then analyzed by epifluorescent microscopy to quantify DBA + mCherry + cysts. For A,B. 992 Bars, mean + SEM. N= 8 mice/infected group. ns= not significant; two-way ANOVA with 993  Table S3. List of Primers used throughout the paper. 1202