Toxoplasma gondii commonly causes latent cerebral infection in humans and other vertebrates. Here, we addressed the role of the cerebral vasculature in the passage of T. gondii to the brain parenchyma. Shortly after inoculation in mice, parasites mainly localized to cortical capillaries, in preference over post-capillary venules, cortical arterioles or meningeal and choroidal vessels. Early invasion to the parenchyma (days 1-5) occurred in absence of a measurable increase in blood-brain barrier (BBB) permeability, perivascular leukocyte cuffs or hemorrhage. However, sparse focalized permeability elevations were detected adjacently to replicative parasite foci. Further, T. gondii triggered inflammatory responses in cortical microvessels and endothelium. Pro- and anti-inflammatory treatments of mice with LPS and hydrocortisone, respectively, impacted BBB permeability and parasite loads in the brain parenchyma. Finally, pharmacological inhibition or Cre/loxP conditional knockout of endothelial focal adhesion kinase (FAK), a BBB intercellular junction regulator, facilitated parasite translocation to the brain parenchyma. The data reveal that the initial passage of T. gondii to the central nervous system occurs principally across cortical capillaries. The integrity of the microvascular BBB restricts parasite transit, which conversely is exacerbated by the inflammatory response.The cellular barriers of the central nervous system proficiently protect the brain parenchyma from infectious insults. Yet, the single-celled parasite  The vertebrate central nervous system (CNS) is protected by restrictive cellular barriers that maintain homeostasis and regulate passage of molecules and cells to the brain parenchyma . Three mThe BBB is anatomically localized to the cerebral microvasculature, which is constituted by capillaries with a luminal diameter (Ø) of <10 μm, arterioles and venules (~10–100 μm Ø) . The micInflammation can cause impairment of the blood-CNS barrier functions . Immune Toxoplasma gondii infects a broad range of warm-blooded vertebrates, with an estimated 1/3 of the global human population being chronically infected . Interestingly, immuno-histochemical analyses of brain tissue revealed that T. gondii tachyzoite GFP+ signal preferentially localized to microvessels with an intraluminal diameter less than 10 μm    at a lower relative frequency compared with capillaries, while localization to penetrating arterioles, pre-capillary arterioles and penetrating venules was absent or scarce (nfection  and locanfection . To stud ± 5.97) . Further ± 8.08)  and with ± 8.08) . The accr scarce .i) anatomical localization and (ii) in situ localization in relation to the adjacent vasculature  was related to the vascular marker EB and categorized as intravascular, perivascular or parenchymal localization  infiltration was rapidly observed in the vicinity of parasite foci by days 4–5  and CD45+ cells were retrieved from the brain tissue  and sections with absence of tachyzoites (GFP-) at days 3 or 10  monolayers, of purified brain microvessels and in vivo in mice. First, transcriptional analyses of MBECs challenged with T. gondii demonstrated upregulation of a number of inflammation-associated genes, similar to LPS treatment  reduced inflammation by hydrocortisone treatment before and during infection and (ii) increased inflammation upon low-dose LPS treatment before and during infection. First, we confirmed that hydrocortisone treatment efficiently reduced pro-inflammatory IFN-γ and IL-12 responses upon T. gondii challenge   of the iallenges , with anallenges . Importaallenges . Howeverallenges . Moreoveallenges , indicatallenges , indicatPtk2 in mice. To generate endothelial cell-specific deletion of Ptk2 (ECPTK2 cKO), flox/floxPtk2 mice were crossed with cre+/-Cdh5 mice harboring a tamoxifen-inducible Cre recombinase under the regulation of vascular endothelial cadherin promoter. To confirm FAK deletion in genotyped mice   and with relative lower transmigration frequencies across monolayers with high TCER  , shown tnt (CPS) . First, igh TCER . The TCEigh TCER , in accoigh TCER . In sharigh TCER . Similarigh TCER . In vitrigh TCER , with inigh TCER . Importaigh TCER . In vivoigh TCER . Partly igh TCER . For CPSigh TCER . The lowT. gondii with the brain vasculature in mice.Passage across the cellular blood-CNS barriers is a prerequisite for the establishment of latent cerebral toxoplasmosis in humans and rodents. Here, we studied the crucial interaction of T. gondii from the blood circulation into the brain parenchyma is at the level of cortical capillaries, similarly in three separate mouse strains and for prototypic T. gondii type I and type II strains. The data evidence preferential localization of parasites to capillaries with low α5 laminin/P-glycoprotein expression ratio, a relative less frequent localization to post-capillary venules, and scarce or absence of localization to penetrating arterioles, pre-capillary arterioles and penetrating venules.Our study establishes that the principal point of entry of T. gondii infection of the brain parenchyma. First, from day one post-inoculation, the accumulation of T. gondii tachyzoites in the brain was dramatically inferior to that observed in lung and liver, indicating a relative lower permissiveness of the brain vasculature to adhesion or invasive events by T. gondii. Second, the initial vascular localization with replicating parasites within brain endothelium  interact primarily with the endothelium of cortical capillaries, confirming and extending the earlier reported replication of parasites in brain endothelium at later timepoints (7–9 dpi)  . This lo7–9 dpi) . The cap7–9 dpi) . Hypothehyzoites  and of phyzoites . Among ohyzoites  and T. gmination , future T. gondii can invade the CNS across the choroid plexus (CP) and meningeal vessels, albeit in lower relative numbers compared with total parasite numbers entering across the extensive parenchymal vascular network. However, taking into account the relative smaller endothelial cell surface area and tissue volume of the CP and meningeal vessels related to cortical vasculature, the data indicate that passage is facilitated at the CP. Hypothetically, this may implicate the fenestrated choroidal endothelium, a known locus minoris resistentiae for brain invasion by the causative agent of sleeping sickness Trypanosoma brucei . In contrast, infiltration was abundant by day 10, in line with recent findings (T. gondii passage to the parenchyma (< day 5). However, an inflammatory activation of the BBB endothelium by the infection was evidenced by elevated expression of CAMs but also inflammation-dampening TIMP-1  but, importantly, in absence of leakage of a low-molecular weight permeability tracer  transmigrates across polarized endothelium and epithelium in vitro, with elevated transmigration frequencies upon FAK inhibition. Thus, despite the reduced gliding motility and transmigration abilities of this mutant, the data provide additional evidence that FAK impacts the endothelial process of tachyzoite transmigration, in absence of monolayer disruption and independently of parasite replication in vitro. In mice, surprisingly few CPS parasites were detected in the brain and other peripheral organs shortly after inoculation, consistent with the absence of symptomatology in mice despite the inoculation of doses that are imminently lethal with wildtype ( > 108) and with scarce localization of single CPS tachyzoites intravascularly, and extravascularly in the brain parenchyma. Partly contrasting with in vitro results, elevated total numbers of CPS parasites were not detected in the brain upon FAK inhibition or ablation in vivo. Given the high TCER of the BBB, this may be related to the expected overall reduced invasive and transmigration abilities of this mutant, in combination with a sensitivity limitation of the assay due to scarce total invasion events. Importantly, the low numbers of intravascular CPS parasites compared with wildtype (RH) precluded direct comparisons with wildtype for numbers of translocation events in vivo. Together, the reduced gliding and TCER-related transmigration abilities of this auxotroph mutant upon uracil deprivation indicate that metabolic stress  mice were crossed with C57BL/6-Tg(Cdh5-cre/ERT2)1Rha (cre+/-)Cdh5 mice for endothelial cell-specific deletion of FAK-encoding Ptk2. Experimental mice were flox/floxCdh5cre+/-Ptk2 (ECPTK2 cKO) and flox/floxCdh5cre-/-Ptk2 (ECPTK2 WT) littermates were used as control wild-type mice. wt/wtCdh5cre-/-Ptk2 and wt/wtCdh5cre+/-Ptk2 mice were also used in control experiments. Mice were injected intraperitoneally (i.p.) with 2 mg tamoxifen (Sigma-Aldrich) for 5 consecutive days to induce recombination. Six- to 12-week-old male or female mice were used for experiments. Animals were housed under specific pathogen-free conditions at the Experimental Core Facility, Stockholm University.All utilized mice strains are listed in the Key Resources Table. B6.129P2(FVB)-T. gondii tachyzoites were maintained by serial 2 day passage in human foreskin fibroblast (HFF) monolayers cultured in Dulbecco‘s modified Eagle’s medium  with 10% fetal bovine serum , gentamicin , glutamine , and HEPES . For the CPS line, uracil  was added to the culture medium as indicated (ndicated . Culture6) or i.p. with low doses  of freshly egressed T. gondii tachyzoites. To evaluate the effect of inflammation on parasite loads, T. gondii-infected CD1 mice were inoculated i.p. with LPS  at 0.08 or 0.2 mg/kg/day for seven or 5 days, respectively, starting 1 day before infection or treated subcutaneously with hydrocortisone  at 20 or 40 mg/kg/day for 6 days, starting 3 days before T. gondii infection. For assessment of BBB permeability, control mice were injected i.p. with LPS (2 mg/kg) and sacrificed 6 h post-inoculation. T. gondii-infected mice were inoculated i.p. with the FAK inhibitor VS-6063  at 50 mg/kg twice a day, starting 1 day before T. gondii infection.Mice were inoculated intravenously (i.v.) with high doses  containing 3% Evans blue fluorescent dye (Sigma) and 5% BSA dissolved in phosphate-buffered saline (PBS), prior to organ extraction. Extracted organs were fixed in 4% paraformaldehyde 0,1 M phosphate buffer (PFA) for 2 days and preserved in sucrose solution  for 4 days. Tissues were then frozen on dry ice and 14–50 μm cryosections collected onto gelatin-coated slides. For immunostaining, brains were dissected, snap frozen on dry ice and stored in –80 ° C until sectioned in a cryostat. Brain cryosections of 14 μm thickness on coronal plane were fixed in 4% PFA followed by acetone fixation, permeabilization (0.2% Saponin or 0.3% Triton X-100 in PBS), blocking  and incubation with primary antibodies in IHC buffer  ON at 4 °C or for 3 hr at RT. All sections were then washed in PBS and incubated with corresponding secondary antibodies diluted in IHC buffer for 1 hr at RT, followed by DAPI staining. Microvessels were purified as describe below and seeded onto glass coverslips pre-coated with 1% gelatin, fixed in 4% PFA for 5 min, rinsed in PBS, incubated ON at 4° C with primary antibodies diluted in IHC buffer, and finally stained with secondary antibodies, followed by DAPI staining. All primary and secondary antibodies used are listed in Key Resources Table.Surface rendering tool and replicating tachyzoites were identified as minimum 2 μm spots. The distance from each spot to the border of all surfaces was calculated using the ShortestDistanceToSurfaces tool.Epifluorescence microscopy was performed by using a 20 X objective  or 63 X objective . Z-stack images were captured by confocal microscopy  and processed with ZenBlue software (Zeiss). Two-photon imaging was performed using a Leica SP8 DIVE microscope equipped with 4Tune non-descanned spectral detectors, a HC PL IRAPO 40 x/1.10 water immersion objective (Leica Microsystems) and a tunable INSIGHT DUAL X3 laser . GFP was excited with 924 nm laser pulses, emission was detected at 500–520 nm, Evans blue was excited at 945 nm and detected at 620–750 nm. 3D reconstruction of z-stacks was performed using the 3D Visualization function in the Leica Application Suite X . Hyvolution, a Huygens-powered (Scientific Volume Imaging B.V.) deconvolution function integrated in LAS-X was used for deconvolution. To analyze distances between replicating tachyzoites and vasculature, confocal images were processed with 3D view using Imaris9.5 software. The brain microvasculature was defined by using the T. gondii (GFP+) foci were expressed as number of foci/mm2 of tissue or per section on coronal brain sections from parenchyma, choroid plexus and meninges. A parasite focus was defined as a group of vacuoles with replicating T. gondii (GFP+). Unless differently stated, individual vacuoles with replicating T. gondii tachyzoites (two or more tachyzoites per vacuole) were counted. The size of the foci was determined by counting number of vacuoles and categorized as follows <10, 11–50 or >50 vacuoles/focus. Single tachyzoites were also counted in T. gondii in relation to the cerebral vasculature were performed in the prefrontal cortex area of the brain parenchyma (bregma +2.96 mm to bregma +1.70 mm), using epifluorescence and confocal microscopy (63 X objective). Parasite (GFP+) signal co-localizing with EB signal was defined as intravascular. Parasite signal localized ≤10 μm and >10 μm from the EB signal of the vascular lumen was defined as perivascular and parenchymal, respectively. The vascular lumen and diameter sizes (μm) were determined by a scale-calibrated measurement (ImageJ software) across the EB signal and α5 laminin signal, respectively, in areas with GFP+ foci. To calculate the α5 laminin/P-glycoprotein fluorescence ratio, the mean α5 laminin fluorescence intensity signal was divided by the mean P-glycoprotein fluorescence intensity signal for each vessel. Plaquing assays were performed as described  for 1.5–2 hr at 37 ° C. Digested microvessels were washed and seeded in 12-well plates pre-coated with 1% gelatin (Gibco) in EBM-2/EGM-2 medium (Lonza) with 4 μg/ml puromycin (Santa Cruz). The purity of MBECs was determined by CD31 expression (70%–80%) and ZO-1 expression as detailed in T. gondii tachyzoites  or LPS (100 ng/ml), for six or 12 h, and processed for PCR.Cerebral microvessels from infected and non-infected mice were isolated as described . BrieflyGliding assays were performed as described . Briefly5) were exposed to polarized bEnd.3 and Caco2 monolayers in transwells. Polarization and permeability parameters were determined by measurement of transcellular electrical resistance (TCER) and FITC-dextran (3 kDa), before and after the assay, as detailed in Transmigration assays were performed as described . BrieflyT. gondii was detected in the filter 525/550 channel. Flow cytometry was performed on LSR Fortessa (BD Biosciences) and data were analyzed with FlowJo software v10.To prepare single-cell suspensions, brain tissue was incubated with 1 mg/ml collagenase IV (Gibco) and 10 μg/ml DNase I (Roche) for 1 hr at 37 °C. Brain homogenates were then washed with FACS buffer , centrifuged at 250 g for 10 min and pellets resuspended in Percoll  in PBS at a final concentration of 30%. After centrifugation at 500 g for 30 min, the myelin (top layer) was discarded. Cell pellets were filtered through a 40 μm cell strainer and resuspended in FACS buffer. Spleen and lung cells were isolated by mechanical homogenization on 70 µm cell strainers followed by treatment with red blood cell lysis buffer . Freshly isolated cells from brains, spleens, and lungs were blocked for 30 min in Fc block (BD Biosciences), and incubated with antibodies against CD45  and CD11b . Signal from GFP-expressing -ΔΔCt values were used to calculate fold-change of expression upon challenge in cells and tissues. All primers were designed using Get-prime or Primer-BLAST software (Key Resources Table).Total RNA was extracted from brain, liver or spleen homogenates using TRI Reagent (Sigma), chloroform and isopropanol. Microvessels and MBEC RNA was extracted using the RNeasy Mini kit (Qiagen) according to the manufacturer’s protocols. The amount and purity of isolated RNA was determine using a spectrophotometer  by reading the absorbance at 260 nm and 280 nm. The ratio A260/A280 of RNA from homogenate brain samples was in the range of 1.91–2.06, for microvessels 1.8–2.0 and for MBEC 2.03–2.16. First-strand cDNA was synthesized with Superscript IV Reverse Transcriptase (Invitrogen). Real-time PCR was performed using SYBR green PCR master mix (Kapa Biosystems), forward and reverse primers (200 nM) and cDNA (0.5–1 μg) with a QuantStudio TM 5 Real-Time PCR system (Thermofisher). ΔCt values were calculated with the house-keeping genes HPRT and GAPDH. 2cre+/-Cdh5  mice upon tamoxifen treatment, MBECs were isolated and treated with (Z)–4-Hydroxytamoxifen in vitro  for 24 hr on day 10. Bone-marrow-derived cells were isolated from mice as described .To quantify protein levels of endothelial FAK in escribed . Livers cre+/-Cdh5 mice, and as described by Jackson Laboratory for flox/floxPtk2 mice.Mouse genomic DNA was isolated from ear biopsies using DNeasy Blood & Tissue kit (Qiagen). PCR was performed as described by Taconic Biosciences for Statistical analyses were performed using GraphPad Prism software (v. 8) or TIBCO Statistica software (v. 7). For two-group comparisons, two-tailed Student’s t-test was performed on samples with normal distribution and Mann-Whitney test was used on non-normally distributed data. For multiple comparisons, data with a normal distribution were analyzed by one-way ANOVA, followed by Tukey’s test or Dunnett’s test. ANOVA and ANCOVA- F value and degrees of freedom F  are indicated in the figure legends. For multiple comparisons, Kruskal-Wallis test, followed by Dunn’s post-hoc test was performed when data was not normally distributed. For all statistical tests, values of p ≥ 0.05 were defined as non-significant, and p < 0.05 were defined as significant. There is significant interest in how pathogens like Toxoplasma infect the brain. The present study carefully details early steps in this process by inoculating mice intravenously and monitoring their distribution within brain tissues over the subsequent days. The analysis presented will be a valuable reference for further investigation of this process. The authors perform several perturbations that involve the permeability of the blood-brain barrier as a key determinant of parasite entry into the brain parenchyma. Taken together the study highlights the critical role of the blood-brain barrier in restricting access of Toxoplasma to the brain. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Decision letter after peer review:Toxoplasma gondii from cortical capillaries" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Dominique Soldati-Favre as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Blood-brain barrier-restricted translocation of The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:Detailed comments from reviewers are copied below for your reference. The reviewers were enthusiastic about the potential of the study; however, there was a strong sense that the main hypothesis and title of the study should directly tested as detailed in point 1 below. The reviewers felt that any outcome would be complementary to the present results and should be reported alongside them, either validating the hypothesis or modifying the conclusions of the manuscript.1. Request for additional data. The authors should provide direct evidence for the hypothesis that FAK inhibition or deletion in the vascular endothelium alters parasite translocation by specifically altering barrier function. The reviewers note the various confounding effects of FAK disruption, which extend beyond its role in regulating BBB intercellular junctions. The reviewers were unanimous in requiring experimental evidence using non-replicating strains of Toxoplasma (like CPS) to test this hypothesis since, based on the current model, intravenous injection into WT and BBB FAK KO mice would result in increased translocation of the non-invasive parasites in the latter.Reviewer #1:2. Request for textual modifications or clarification. The itemized reviews list several requests for clarification that can be addressed through changes to the text and the figures. Please respond to all such requests in the resubmission.Toxoplasma gondii, can nevertheless translocate into the brain, so the mechanisms they use to achieve this are of clinical importance. The present study details the early steps of this process by inoculating mice intravenously and monitoring the distribution of parasites within brain tissues over the subsequent days. The result is a highly detailed picture of the early stages of brain infection that recapitulates previous results, and extends their conclusions by examining the role of the blood-brain-barrier (BBB) in this process. Inflammatory stimuli like LPS, which increase BBB permeability, also increased the penetration of Toxoplasma into the parenchyma. Inhibition or knockout of the focal adhesion kinase (FAK), which is important for the maintenance of the endothelial barrier, similarly increased the number of parasites found in the parenchyma; however, the interpretation of these observations is severely compromised by the other functions of FAK, including some within immune cells that control parasite function.The critically sensitive nature of the vertebrate brain requires specific adaptations in barrier function and immune response, which ultimately help prevent damage associated with infections. Certain pathogens, like Toxoplasma gondii. The results align well with findings from Konrad et al., (2016) using a similar model. Importantly, the authors examine how entry of parasites into the parenchyma is affected by a variety of perturbations. While many of the perturbations used ultimately affect parasite translocation into the brain, they lack the precision to make specific mechanistic conclusions. Moreover, the authors fail to dissect the contribution of parasite replication within endothelial cells, which would allow them to firmly establish the contribution of the blood-brain barrier in restricting parasite translocation into the brain.The authors have generated a detailed and quantitative picture of brain tissue infection following intravenous inoculation with 1. It seems critical to determine what would be expected from randomly localized vacuoles. E.g. In Figure 3, what would be the average distance of random points within the imaged section from a capillary or a vascular branching point, and what would be the diameter of randomly selected capillaries within a field. Only then can we know whether the observed distribution is unusual.2. Can the authors firmly conclude that at low parasite levels there is no permeability? The authors should consider whether single translocation events would provide sufficient signal to detect vascular leakage.3. The authors need to exclude replication within endothelial cells to firmly establish that the BBB restricts parasite translocation. This could be accomplished by inoculating parasites incable of replication in mice. Without this evidence, it is formally possible that FAK inhibition/knockout or LPS are simply affecting parasite replication within endothelial cells.Reviewer #2:4. Related to point 3, the number of parasite foci trends up in both perturbations of FAK (inhibition and knockout), within the intravascular compartment. Are investigators truly capable of distinguishing between perivascular and intravascular infection, given the lack of a specific marker for endothelial cells? The concern is that in some cases leakage of the endothelium will land parasites in perivascular sites that would look microscopically very similar to vascular sites; this effect could explain the surge in intravascular foci for perturbations that should alter permeability.T. gondii parasites enter the brain, and the factors that limit parasite entrance. Similar to an earlier study  the authors use intravenous inoculation of mice with fluorescent T. gondii tachyzoites, and microscopy to characterize the early events by which parasites enter the brain. As in Konradt et al., the current study provides evidence that parasites are first found in brain capillaries, and then replicate in cerebral endothelial cells prior to infecting the brain parenchyma. Interestingly, parasite replication in cerebral endothelial cells and entry into brain parenchyma observed at days 3-5 post infection does not immediately trigger permeability of the BBB, nor does it lead to substantial leukocyte infiltration. Rather, the increase in BBB permeability (as measured by an increase in fluorescence with vascular tracer Evans Blue) is first observed at day 10 post infection, coinciding with leukocyte infiltration. The authors utilize several imaging strategies to investigate the dynamics of T. gondii entry into the brain. The images produced are compelling and support the conclusions made by the authors, and in general the analysis being performed is clear and compelling. Additionally, the techniques used to measure the presence of inflammation or to manipulate the inflammatory environment are logical and produce compelling results. One area of concern is the potential impact of the non-physiological infection route (high dose intravenous tachyzoites) and the use of a highly virulent, non-cyst forming strain of the parasite (RH) which may lead to altered entry routes to those that occur during natural infection.This paper examines how 1. There is some concern that i.v. injection of large numbers of free tachyzoites might not accurately recapitulate events during normal infection. In addition the majority of experiments used the highly virulent non-cyst forming strain of the parasite (RH), which may use a different route of entry compared to less virulent, cyst forming strains. The authors should acknowledge and address these caveats in the text.2. The conclusion that parasite entry (days 3-5) precedes BBB permeability, leukocyte entry and inflammation (day 10) is drawn from comparison of data from Figures 4, 5 and 6. However, the experiments in these 3 figures uses a variety of different infection routes, doses, parasite and mouse strains, making comparisons challenging. In general, it was not clear why different infection routes, dosages and parasite strains used throughout the manuscript. In addition to describing the logic behind the choices, it would also help the reader if the strains were identified in the text as well as in the figure legend.T. gondii" (line 133) and if and how it differs from "replicating parasite vacuoles" (line 179). Are vacuoles being counted or tachyzoites being counted?3. It would be useful for the authors to define their use of the term "replicating 4. The analysis investigating the distance of the parasites from the nearest capillary in Figure 3 requires some additional clarifying information either in the legend or in the corresponding Methods section. Are the foci being categorized as intravascular/perivascular/parenchymal and the distance for individual parasites within the foci are being measured? The text is unclear. In addition, the authors claim that the extravascular parasites are in close proximity to the capillaries. While the representative images are compelling, the significance of the quantifications being performed is confusing. Why is the comparison between intravascular and extravascular foci relevant ? Perhaps if these data were compared to data from the later D10 time point, the conclusion that the invading parasites are in the vicinity to the capillaries would be easier to substantiate.Reviewer #3:5. The data showing brain parasite load in FAK KO mice  was less compelling than the impact of the FAK inhibitor . How do the authors reconcile a lack of impact on parasite numbers (9b), given the increase in numbers of parasite foci (9e) and number of vacuoles/foci (9f) reported? The authors should provide more information about how they quantified the parameters in Figures 9e, f.There is a lot to like in this submission as it is a focused on characterizing in detail some of the early events in neuro-invasion of the parasite Toxoplasma. There is some overlap in the early figures with the previous Konradt paper . However, it quickly move past that and attempts to focus on whether the BBB has some important protective effects to neuro-invasion. This is an accepted concept that has really not been tested and the authors also use the increased parasite numbers in lungs and liver compared to brain to promote this idea (while the other explanation is that anything particulate injected iv end up in the lungs and liver because of the blood flow and large numbers of phagocytes and capillary beds).Toxoplasma gondii induces FAK-Src-STAT3 signaling to prevents parasite targeting by autophagy – while Loden suggests Toxoplasma targets FAK to disrupt integrin activation and promote hypermotility of infected cells. For the figures 8/9 that address this question the authors need to bolster some of their data sets to make this more compelling (impact on the immune response and recruitment) – rather than leave some obvious gaps that leave a reader unsure whether the data support the conclusions.The authors chose to focus on FAK – which they previously studied in vitro and the most novel aspects of the report are when they start to address the role of this pathway in vivo. Here, there are some concerns. FAK is described as "intercellular junction regulator" but it does much more than that and there is a (somewhat contradictory) literature on its role in Toxoplasm infection – which make this work timely. For example. the PLOS Pathogens 2017 The other major gap is that the authors propose on line 609 that testing which route of entry is relevant is beyond the scope of the current submission. Their 2019 publication proposed FAK is important in vitro for transmigration – and disruption of FAK does a lot of things (detailed below). These authors have a real opportunity to test whether altering the BBB affects parasite entry. Providing the can validate the in vivo interventions they are using to target the BBB, the authors should be able to utilize replication deficient parasite strains (to recapitulate the studies of Konradt) and show whether these free parasites in the blood have an improved ability to cross the BBB when FAK is targeted. It's the whole basis for the model and this is the opportunity to test this directly. This reviewer is agnostic to the outcome, but if these parasites do not access the parenchyma more readily in the KO or inhibitor treated mice then the major conclusion (title) would have to change (which is ok). Its also a way of bypassing all the questions about secondary effects of FAK inhibition.Figure 1 recapitulates the original studies of Konradt that identified infected endothelial cells after iv transfer of parasites and showed preferential localization to small blood vessels. Here the authors also include staining for a5laminin and P-glycoprotein.Figure 2 – confirms the brain is lower than liver/lungs  but do a nice job of characterizing location within 3 compartments of the CNS .It is unclear from panel E how they identified the choroid plexus.Figure 3 – is devoted to rigorously establishing that there is a transition from blood, to perivascular to parenchymal localization and that it's the same for different host-parasite combination. The different host-parasite combinations .Figure 4 is done with hi and low dose ME49 (moderately virulent) top look at kinetics of immune infiltration. Figure 4C shows increased numbers of CD45+ cells associated with the CNS and this is interpreted as increased inflammation at day 4. Sorry – it was not clear if the brains were perfused for this analysis. Did the authors perform an iv labeling experiment to show that these CD45+ cells are in the brain or just in the blood and are expanded as part of the general response to infection.Figure 5 uses the virulent RH – and shows changes in BBB permeability that correlates with parasite numbers (more parasites more marker gets across). Basically, RH parasites injected iv for 5 days – these mice are systemically septic. Figure 5F is not significant but the authors conclude that there are "focal permeability elevations in the vicinity of parasite foci.". The data set quite clearly show no significant difference and if they want to focus on those few data points that are >60, then it would be just as important to highlight those few data points that are <25 . Basically, the authors should consider simplifying their conclusion.T. gondii and conclusion is that there is some upregulation of ICAM. A little hard for me t o know what to do with this data set. Isn't this where they should be looking at FAK dependent integrin expression?Figure 6 looks at some target genes in MBECs from uninfected mice challenged in vitro with Figure 7B – the authors note there were 5-10 mice /group – but some have only 3 or 4 points. It is also unclear if this is a single experiment or pooled data sets? Just looking at the data sets for the LPS treatment  – it does look suspiciously like they may have more parasites (hence the question about repeats) while the micrograph in 7C and data in 7f looks like a lot more parasites. This is interesting but whether they used hydro-corticosteroids or daily low dose LPS they see more parasites in the parenchyma. It is also unclear why the control mice in this experiment do not have any parenchymal parasites when they have them in the earlier figures at an earlier time point. Maybe they just need to clarify this. While there is real value to including LPS as a comparator for BBB permeability – daily low dose LPS does a lot of things and I am (honestly) not clear that this figure helps – especially because they have much more specific interventions in the next figures.Figure 8a – looks like it is underpowered to conclude there is no difference in parasite burden at high dose (a) but there clearly is a difference in all tissues with low dose (b). This suggests a global impact of the inhibitor not an activity restricted to the CNS.Figure 8 – supplemental the concern here is that the analysis of the IFN-g mRNA looks like it is underpowered and its in the brain not the periphery when the authors indicate that there is a not a lot of local inflammation. If the FAK inhibitor has systemic effects on IFN-g then it would result in more parasites and this assay was used in Supp Figure 7 where it surprisingly showed no impact of hydro-corticosteroids. The authors just need to nail down some better assays for impact on the immune response (serum IFN-g protein?). It is also not clear that more parasites and bigger vacuoles are due to early entry. They have more parasites in the periphery which suggests a defect in ability to control them and this would lead to more early entry and increased growth that is independent of any impact on the BBB. This is really a difficult conclusion to make, with the data sets in hand.Figure 9 – key experiment – nicely validate FAK deletion and at day 6 post-infection parasite burden is normal across all tissues (differs from effects of inhibitor) and there is no impact on BBB permeability – but the authors find in panels e and f that there are some more parenchymal parasites at day 6. The main conclusion of this submission really revolves around these (similar) findings in Figure 8 and 9 and this reviewer is concerned that there are other explanations. Early in the manuscript they look at immune infiltration but have not seen if that parameter is altered in Figure 8 or 9. Any alteration in immune recruitment/access/function to the CNS is likely to impact parasite control (as they showed in their earlier figures). Essential revisions:Detailed comments from reviewers are copied below for your reference. The reviewers were enthusiastic about the potential of the study; however, there was a strong sense that the main hypothesis and title of the study should directly tested as detailed in point 1 below. The reviewers felt that any outcome would be complementary to the present results and should be reported alongside them, either validating the hypothesis or modifying the conclusions of the manuscript.1. Request for additional data. The authors should provide direct evidence for the hypothesis that FAK inhibition or deletion in the vascular endothelium alters parasite translocation by specifically altering barrier function. The reviewers note the various confounding effects of FAK disruption, which extend beyond its role in regulating BBB intercellular junctions. The reviewers were unanimous in requiring experimental evidence using non-replicating strains of Toxoplasma (like CPS) to test this hypothesis since, based on the current model, intravenous injection into WT and BBB FAK KO mice would result in increased translocation of the non-invasive parasites in the latter.As requested by the reviewers, we have performed experiments with replication-deficient parasites (CPS). First, because the basic invasive features of the CPS line had not been documented, other than ability to invade fibroblast monolayers over an extended incubation time , we characterized the gliding motility and the transmigration abilities of this mutant in vitro. Further, CPS has been reported to invade brain endothelium and was very rarely retrieved in non-endothelial /brain parenchymal cells in comparison with wildtype (RH) parasites . We undertook a similar approach with the aim of assessing the role of FAK.The data is provided in the new Figure 9 and Figure 9-Supplements 1, 2 and 3. In summary, the in vitro data shows that the CPS mutant has the ability to transmigrate, albeit at a lower frequency across Caco2 cells ) and similar to WT across bEND.3 monolayers (moderate-to-high TCER). Interestingly, both transmigration frequencies and gliding path lengths are rapidly reduced upon uracil deprivation. Note also that the overall decrease in gliding is likely underestimated for CPS as only tachyzoites with gliding trails were assessed and an increasing proportion of CPS tachyzoites exhibited no gliding upon uracil deprivation. Over an extended incubation time, CPS invaded cellular monolayers, consistent with reports in fibroblasts . Importantly, despite the reduced invasive properties of this mutant, treatment of monolayers with FAK inhibitor consistently led to elevated transmigration frequencies, proportionally similar to effects in WT. This indicates that dysregulation of the tight junctions via FAK (but in absence of monolayer disruption or depolarization) facilitates transmigration for this mutant, as previously shown for WT parasites . See Results section (pages 18-19), Figure 9g-j and Figure 9-supplement 2.8 freshly egressed tachyzoites i.v., which can be lethal in < 24 h for WT) few parasites are retrieved in brain and in other organs. This made comparisons difficult with WT for total parasite loads and impossible for reliable quantification of translocation events. Specifically, our aim was to normalize data of invasion of endothelial cells by WT and CPS and relate the endothelial invasion numbers to numbers of parasites in the parenchyma. However, with so few invasion events of endothelial cells by CPS, this was not feasible . As an illustrative example and reference: for any strain inoculated, the parasite loads in brain related to peripheral organs are up to 100 times lower . Similarly, the parasite loads in the brain are up to 100 times lower for CPS compared to RH. Jointly, this makes non-replicating CPS parasite loads in brain minimal , making in situ quantifications very uncertain.In vivo, we noted that very few CPS parasites were retrieved in organs. We therefore opted for a co-infection approach of CPS and WT for more precise comparisons. Similarly, very few invasion events of cells by CPS in different tissues were observed related to WT in peripheral organs and, naturally, even lower numbers in the brain. Thus, despite that dramatically high doses of CPS were inoculated (> 108) which are lethal and not tolerated for wt. Our assumption, based also on the (surprising!) absence of objective symptoms in mice despite extremely high doses, is that, in vivo, very few CPS parasites manage invasion of cells and that high numbers of parasites are very rapidly cleared (in less than 24 h). Jointly, this underscores further the restrictiveness of the BBB for invasion events. We provide, however, data of pharmacological FAK inhibitor treatment and ECFAK KO mice challenged with CPS. Taking into account the low numbers of retrieved parasites, the data show non-significant differences in total parasite counts. Stainings of brain tissue are also provided with intravascular and extravascular localization of CPS in the brain parenchyma. See Results section (p. 18-19), Figure 9k-o, and Figure 9-supplement 3.We compensated with high doses of CPS . Our findings ad that a comparable dramatic reduction of intravascular parasites/infection of brain endothelial cells also takes place upon challenge with CPS, related to WT. In our opinion, this precludes direct comparisons with WT parasites for absolute numbers of translocation events. As indicated above, calculations of the ratios infected endothelial cells/infected parenchymal cells for WT and CPS, respectively, were not feasible based on the overall scarce numbers of CPS parasites in brain. Our interpretation is that, in vitro, this difference in invasion is not easily apparent as CPS invades endothelial cells over time (many hours), while invasion of brain endothelium and across brain endothelium in vivo are dramatically compromised. Given the high TCER of the BBB, this result must also be related to the expected overall reduced invasive and transmigration abilities of this mutant, in combination with a sensitivity limitation of the assay due to scarce total invasion events.T. gondii genotypes have different invasive phenotypes  and rely differently on transportation by leukocytes  during primary infection, these aspects require further study. We agree that a non-replicating mutant with maintained invasive phenotypes would be a very welcome and extremely useful tool to address these key questions. See discussion We agree with the reviewers that any outcome of the experiments above is complementary to the results. We provide an in vitro characterization of the transmigratory and motility properties of the CPS line with proof-of-concept that transmigration occurs in absence of replication and that FAK inhibition exacerbates transmigration in vitro. In the revised discussion, we reason that, while lysis on infected endothelial cells -and of epithelial cells in other sites, e.g. the gut - undoubtedly mediates growth across restrictive biological barriers, also other pathways of translocation likely contribute . Further, because different 2. Request for textual modifications or clarification. The itemized reviews list several requests for clarification that can be addressed through changes to the text and the figures. Please respond to all such requests in the resubmission.Please, find below, point-by-point responses to the queries raised by the reviewers.Reviewer #1:The authors have generated a detailed and quantitative picture of brain tissue infection following intravenous inoculation with Toxoplasma gondii. The results align well with findings from Konrad et al., (2016) using a similar model. Importantly, the authors examine how entry of parasites into the parenchyma is affected by a variety of perturbations. While many of the perturbations used ultimately affect parasite translocation into the brain, they lack the precision to make specific mechanistic conclusions. Moreover, the authors fail to dissect the contribution of parasite replication within endothelial cells, which would allow them to firmly establish the contribution of the blood-brain barrier in restricting parasite translocation into the brain.1. It seems critical to determine what would be expected from randomly localized vacuoles. E.g. In Figure 3, what would be the average distance of random points within the imaged section from a capillary or a vascular branching point, and what would be the diameter of randomly selected capillaries within a field. Only then can we know whether the observed distribution is unusual.putative- original capillary of infection/translocation is easily identified for intravascular vacuoles and for the vast majority of perivascular vacuoles (with a clear difference to average distances to any other vessel). However, note that there is more spread of the data among the parenchymal vacuoles , consistent with the purpose of the dissemination process(es) within the parenchymal tissue . However, for most parenchymal vacuoles, the vicinity to other  vacuoles provides the indication of the -putative-original capillary from which translocation was effectuated .This is an interesting question on the relative localization of parasite vacuoles and vasculature. In Figure 1, we show that the intravascular parasites preferentially localize to capillaries and to some extent to post-capillary venules. However, these analyzes were not provided for perivascular/parenchymal or randomly located vacuoles. We have now performed additional analyzes on randomly located parasite vacuoles and show the distribution of distances to surrounding vasculature and their diameter within the same field of view. Overall, the localization of parasite vacuoles in relation to vasculature does not follow a normal or random distribution. Rather, the data show an uneven distribution of distances between vacuoles and vasculature, in most cases with a clear vicinity (<40 micrometers) to one capillary . The -The new compiled data has been added to the new Figure 3f and representative detailed analyses are provided in new Figure 3- Supplement 2, Results . We have added a comment in the discussion .2. Can the authors firmly conclude that at low parasite levels there is no permeability? The authors should consider whether single translocation events would provide sufficient signal to detect vascular leakage.This is a highly relevant question that we´ve given quite some thought past years and we fully agree with the reviewer (absence of evidence is not evidence of absence). The complexity lays in how “permeability” is defined. While a subtle vascular leakage below the detection threshold of the marker Evans Blue cannot be excluded, our data advocate against (1) a generalized BBB dysfunction with enhanced BBB permeability (as in LPS control treatment) or (2) an irreversible BBB damage with hemorrhage . However, because we measured (3) sparse and focalized permeability elevations in the vicinity of parasite foci, this may indicate permeability variations over time and/or reversible permeability elevations, in line with a transient BBB dysregulation.T. gondii challenge compared to LPS challenge, that readily impacted polarization and permeability. Thus, our in vivo observations here are well in line with in vitro data. In summary, we cannot firmly exclude absence of permeability and the data jointly favor a situation encompassing dysregulation (but not irreversible disruption) of the BBB.Thus, it cannot be ruled out that one single parasite translocation event  locally and reversibly destabilizes the BBB in vivo below the detection threshold of Evans Blue. In fact, although in vivo direct evidence is hard to obtain, we consider this as a highly likely possibility based on in vitro data . in vitro, extracellular tachyzoites or parasitized dendritic cells consistently transmigrated without a measurable irreversible disruption ) of the barrier. Importantly, a transient dysregulation of polarized monolayers was picked up by a minor but consistent decrease in TCER but without measurable leakage of the fluorescent tracer . This dose-dependent effect was more subtle for We have further clarified this important aspect and our reasoning in the discussion .3. The authors need to exclude replication within endothelial cells to firmly establish that the BBB restricts parasite translocation. This could be accomplished by inoculating parasites incable of replication in mice. Without this evidence, it is formally possible that FAK inhibition/knockout or LPS are simply affecting parasite replication within endothelial cells.We thank the reviewer for this constructive suggestion. We have addressed our results with replication-deficient parasites at the beginning of this rebuttal and refer to that response to avoid redundance. in vitro, FAK inhibition or knockdown of endothelial FAK by shRNA did not significantly impact parasite replication (WT) within endothelial cells .4. Related to point 3, the number of parasite foci trends up in both perturbations of FAK (inhibition and knockout), within the intravascular compartment. Are investigators truly capable of distinguishing between perivascular and intravascular infection, given the lack of a specific marker for endothelial cells? The concern is that in some cases leakage of the endothelium will land parasites in perivascular sites that would look microscopically very similar to vascular sites; this effect could explain the surge in intravascular foci for perturbations that should alter permeability.The reviewer brings up an interesting thought.The differences between intravascular and perivascular localizations can be best appreciated in the provided 3D reconstructions in the videos and are also illustrated in Figure 3a. For example, S4 video shows examples of intravascular, perivascular  vacuoles in the same field of view. Note also that the perivascular localization can occur in absence of intravascular vacuoles (S10 video).As the reviewer points out, we cannot exclude that, in some rare cases, ambiguous localization might happen but looking at the totality of the data we feel confident that what we define as “perivascular”  or “intravascular”  can be discerned both by 2-photon microscopy in fresh tissue and by confocal in fixed tissue.Note that our description in the paper is related to the luminal signal by EB and that no claim is made is relation to the precise cellular localization. However, it would seem that while intravascular parasites are tightly associated with the endothelial markers alpha4/beta1 laminin and P-glycoprotein, perivascular parasites localize more with GFAP and Iba1 marker . The perivascular localization is, we agree, more difficult to define but also serves the purpose of describing this intermediate localization of parasites in relation to the vascular lumen which is clearly distinct from the more disperse parenchymal localization. We think that this -descriptive categorization- is possibly more faithful to the observations, rather than classifying parasites as strictly parenchymal and strictly intravascular. And, we agree, future investigations, must pin down the precise localization of these parasites and possible host cells (pericytes? Astrocyte end-feet? Microglia? Perivascular macrophages?).The reviewer is also correct that upon counting hundreds of foci, there are examples that are more difficult to determine than others. Our opinion is that invasion is a continuous process, while parasite replication takes place in the different locations and the focus grows successively. The different localizations are not mutually exclusive and co-exist (S4 video).For FAK inhibition, the reviewer correctly points out that the numbers and size of intravascular foci augment compared to the perivascular and parenchymal foci for the high dose/short time (day 3) challenge . However, for the low dose/long time (day 8) challenge the relative increase in vacuole numbers is more accentuated for the parenchymal localization  with a general size increase of the foci . A comparable increase in vascular permeability is observed in both conditions . In Figure 9, the increase in parenchymal localization in proportionally higher compared to intravasc/perivasc. Thus, FAK inhibition or KO does not seem to automatically lead to a relative higher increase in intravascular parasites in relation to the two other localizations  for all conditions. However, we agree that pharmacological FAK inhibition might have other effects that impact parasitemia or even parasite survival in endothelium. These aspects are outlined in the discussion .In case of hemorrhage, the idea that “perivascular sites would look very similar to vascular sites due to enhanced permeability” is appealing but would likely entail a strong focal concentration of the EB marker with parenchymal diffusion (“bleeding”) and increased “diameter” of the affected area (would presumably look as if a larger irregular blood vessel was infected?). We did not observe this type of event. We carefully assessed this because it was, in our opinion, crucial to define if hemorrhagic components take place. Alternatively, there could be a milder form of permeability elevation without hemorrhage. We favor this alternative. In Figure 5-Supplement 1, the intensity of the fluorescent marker (EB) between lumen and non-luminal varies significantly. Hypothetically, this situation would be picked up as an intensity elevation with increased “diameter” of the blood vessel, but we did not make that type of observation.We also carefully assessed foci with combined intravascular, perivascular and parenchymal  and did not observe dramatically enhanced signal of blood/permeability tracer in these foci, compared with other foci. Thus, although it cannot be fully excluded, we think that the situation proposed by the reviewer cannot be a common or predominating situation.We have clarified these aspects in the discussion Reviewer #2:1. There is some concern that i.v. injection of large numbers of free tachyzoites might not accurately recapitulate events during normal infection. In addition the majority of experiments used the highly virulent non-cyst forming strain of the parasite (RH), which may use a different route of entry compared to less virulent, cyst forming strains. The authors should acknowledge and address these caveats in the text.The reviewer brings up several important aspects related to the validity of the model and variations among strains. For this reason, we used both ip and iv routes and type I and type II strains. In fact, a higher number of figures/suppl. Figurescontain ME49 data (13) compared to RH data (9).We observed a similar distribution of parasites for the two routes and the two parasite strains. Additionally, permeability analyses were consistent. Jointly, this argues for consistency between type I vs type II and between iv vs ip routes. However, we agree that there are limitations: (1) To be able to consistently obtain quantifiable data, high numbers of parasites had to be injected. This has the drawback of reducing mouse survival/shortening days of infection. (2) For longer mouse survival (day 5-10), lower doses of type II strain were given.In the manuscript, we have clarified further the rationale for using different mouse strains and parasite lines, with a reference . We acknowledge the limitations of the iv/ip infection model and its possible bias for passage in the discussion .2. The conclusion that parasite entry (days 3-5) precedes BBB permeability, leukocyte entry and inflammation (day 10) is drawn from comparison of data from Figures 4, 5 and 6. However, the experiments in these 3 figures uses a variety of different infection routes, doses, parasite and mouse strains, making comparisons challenging. In general, it was not clear why different infection routes, dosages and parasite strains used throughout the manuscript. In addition to describing the logic behind the choices, it would also help the reader if the strains were identified in the text as well as in the figure legend.T. gondii in brain vasculature and brain parenchyma are relatively rare events . Consistent with this, relatively few cysts are formed in the brain in natural infections. Within this biological setting (rare events), technical considerations had to be taken into account in order to repeatedly obtain reproducible and quantifiable data in our study and similarly in other studies with similar approach. Specifically, the caveats of experimental systems  have to be weighed against the benefit of acquisition of quantifiable and reproducible data. If this type of data could reasonably be obtained by natural oral infections, then that would definitively be the way to go.We agree that this required further clarification. It has to be kept in mind that localization of T. gondii crosses the barrier is not an easy task. In this paper, we attempt to document the earliest possible timepoints of CNS penetration, for each condition and mouse/parasite strain. This does not exclude passage at later timepoints. In fact, it is reasonable to consider passage/translocation as a continuous process during primary infection, until parasitemia ceases. However, we admit that we were surprised by the rapid presence of parasites in the parenchyma.We agree that determining the exact timepoint when The reason for using different doses, routes and strains have been brought up above and we have clarified this in the text . For each experiment, the strain, route and dose used are indicated in the figure legends, relevant for the general conclusions.We have also clarified these aspects in the revised Discussion .3. It would be useful for the authors to define their use of the term "replicating T. gondii" (line 133) and if and how it differs from "replicating parasite vacuoles" (line 179). Are vacuoles being counted or tachyzoites being counted?T. gondii tachyzoites (2 or more per vacuole) were counted.Vacuoles with replicating This has been clarified  and precision of the definition of the counting of parasite foci/parasite vacuoles/single tachyzoites has been added to Methods (p. 29).+). Unless differently stated, individual vacuoles with replicating T. gondii tachyzoites (2 or more tachyzoites per vacuole) were counted. The size of the foci was determined by counting number of vacuoles and categorized as follows: <10, 11-50 or >50 vacuoles/focus. Single tachyzoites were also counted in Figure 3e and 3h-k….”“….A parasite focus was defined as a group of vacuoles with replicating T. gondii ? Perhaps if these data were compared to data from the later D10 time point, the conclusion that the invading parasites are in the vicinity to the capillaries would be easier to substantiate.T. gondii tachyzoites (GFP+)” in the figure legend for micrographs is that GFP expression by tachyzoites are used to identify vacuoles with replicating tachyzoites and not a vacuole marker per se (we wanted to avoid this possible confusion). We have provided precision to Methods (p. 29).We agree that these aspects needed additional precision for clarity. We have clarified the definitions of focus/vacuole/single tachyzoite (see above). The reason for using “For Figure 3i-j. We think that, overall, these analyzes are relevant because they show that the blood vessel diameter, distance ranges, frequencies and distribution of parasites in live  tissue analyzed by 2-photon microscopy are comparable to those of fixated tissue , thereby validating and reinforcing the data using fixed tissue in Figure 3e, f. We have further clarified the rationale .T. gondii in relation to a capillary is not a random event .The new Figure 3f provides additional data on the distances of randomly located foci to the vasculature. We think this will clarify further to the reader that the localization of 5. The data showing brain parasite load in FAK KO mice  was less compelling than the impact of the FAK inhibitor . How do the authors reconcile a lack of impact on parasite numbers (9b), given the increase in numbers of parasite foci (9e) and number of vacuoles/foci (9f) reported? The authors should provide more information about how they quantified the parameters in Figures 9e, f.T. gondii compared to B6 mice . Thus, a direct comparison of the two treatments cannot be directly done because mouse strain, doses and days differ. We have indicated the conditions for each experiment in figures legends.The experiments required a number of considerations. First, FAK inhibitor studies allowed the use of CD1 outbred mice , significantly more resistant to For the differences between parasites numbers by plaquing counts (9b), we think the two methods measure partly different things and are complementary. Plaquing assays quantify total parasite numbers from dilutions of homogenized tissues, which may be less precise compared to counting numbers of in situ parasite foci (9e) and vacuoles/foci (9f). Plaquing assays have the advantage though that they are representative of the totality of the tissue, while counts of parasite foci provide insights locally in the parenchyma. We have added precision to the discussion and specifically discuss differences between pharmacological treatment and knockout .For the quantitative parameters and definitions, more precision is now provided under Methods (p. 29).Reviewer #3:The authors chose to focus on FAK – which they previously studied in vitro and the most novel aspects of the report are when they start to address the role of this pathway in vivo. Here, there are some concerns. FAK is described as "intercellular junction regulator" but it does much more than that and there is a (somewhat contradictory) literature on its role in Toxoplasm infection – which make this work timely. For example. the PLOS Pathogens 2017 Toxoplasma gondii induces FAK-Src-STAT3 signaling to prevents parasite targeting by autophagy – while Loden suggests Toxoplasma targets FAK to disrupt integrin activation and promote hypermotility of infected cells. For the figures 8/9 that address this question the authors need to bolster some of their data sets to make this more compelling (impact on the immune response and recruitment) – rather than leave some obvious gaps that leave a reader unsure whether the data support the conclusions.The other major gap is that the authors propose on line 609 that testing which route of entry is relevant is beyond the scope of the current submission. Their 2019 publication proposed FAK is important in vitro for transmigration – and disruption of FAK does a lot of things (detailed below). These authors have a real opportunity to test whether altering the BBB affects parasite entry. Providing the can validate the in vivo interventions they are using to target the BBB, the authors should be able to utilize replication deficient parasite strains (to recapitulate the studies of Konradt) and show whether these free parasites in the blood have an improved ability to cross the BBB when FAK is targeted. It's the whole basis for the model and this is the opportunity to test this directly. This reviewer is agnostic to the outcome, but if these parasites do not access the parenchyma more readily in the KO or inhibitor treated mice then the major conclusion (title) would have to change (which is ok). Its also a way of bypassing all the questions about secondary effects of FAK inhibition.We thank the reviewer for the insightful and constructive comments. We agree with the point of view that FAK is a molecule implicated in many biological processes and have clarified these aspects further in the revised discussion with additional references . Further, we have characterized the transmigration of replication-deficient parasites (CPS) in vitro and in vivo.Figure 1 recapitulates the original studies of Konradt that identified infected endothelial cells after iv transfer of parasites and showed preferential localization to small blood vessels. Here the authors also include staining for a5laminin and P-glycoprotein.T. gondii preferentially localizes to vessels with a lumen < 10 micrometers. We felt it was crucial to firmly establish this preferential localization at the earliest possible timepoint – permitting reliable quantifications- because parasites infecting endothelium by day 7-9 could either have replicated locally earlier days or have replicated in other peripheral organs and later been transported with the cerebral circulation. Later in the paper, we also confirm this result using outbred mice (CD1) and another parasite strain  . This needed more precision to the reader and we have clarified these aspects in the text .Yes, the data in Figure 1 (3 days post inoculation (dpi), RH strain, B6 mice) is consistent with the data in Konradt et al.,  showing that Figure 2 – confirms the brain is lower than liver/lungs  but do a nice job of characterizing location within 3 compartments of the CNS .It is unclear from panel E how they identified the choroid plexus.T. gondii. Precision has been added to the text .For parasite loads, we reason as above because it was important to determine if the dramatic differences in parasite loads between brain and other peripheral organs with parenchyma (≈10-100 fold depending on timepoint and dose) occur very early (even after iv injection with rapid systemic spread of parasites) and depend on mouse strain, or if they would depend primarily on differences in parasite replication and dissemination (or combinations). In a sense, because the differences in parasite loads are present from day 1 (before significant parasite expansion takes place), similarly in both mouse strains, we think these data indirectly illustrate the restrictiveness of the BBB compared to other endothelial barriers in peripheral organs and motivate a further exploration of the BBB. This may indicate that the brain vasculature is less permissive to adhesion or invasive events by The choroid plexus was identified by its anatomical localization and its unique morphological structure upon staining and perfusion of Evans Blue dye. A Suppl. figure has been added to the manuscript .Figure 3 – is devoted to rigorously establishing that there is a transition from blood, to perivascular to parenchymal localization and that it's the same for different host-parasite combination. The different host-parasite combinations .Figure 3 is complemented with a supplementary Figure that contains the different host-parasite combinations .Figure 4 is done with hi and low dose ME49 (moderately virulent) top look at kinetics of immune infiltration. Figure 4C shows increased numbers of CD45+ cells associated with the CNS and this is interpreted as increased inflammation at day 4. Sorry – it was not clear if the brains were perfused for this analysis. Did the authors perform an iv labeling experiment to show that these CD45+ cells are in the brain or just in the blood and are expanded as part of the general response to infection.T. gondii . Brains were therefore not perfused and localization of leukocytes in the brain parenchyma is instead shown by immunofluorescence stainings . The reviewer is correct that part of the abundant leukocyte infiltration could emanate from expansion  however the main point here was that some parasites enter the CNS before a significant leukocyte infiltration is observed. We wanted to provide the reader with the information that measurable passage occurs in absence of an important infiltrative process and before day 10. We have clarified this in the text The purpose of these experiments was to determine the total leukocyte infiltration, specifically at the early timepoints and how leukocyte numbers (intravascular and extravascular) relate to the early passage of Figure 5 uses the virulent RH – and shows changes in BBB permeability that correlates with parasite numbers (more parasites more marker gets across). Basically, RH parasites injected iv for 5 days – these mice are systemically septic. Figure 5F is not significant but the authors conclude that there are "focal permeability elevations in the vicinity of parasite foci.". The data set quite clearly show no significant difference and if they want to focus on those few data points that are >60, then it would be just as important to highlight those few data points that are <25 . Basically, the authors should consider simplifying their conclusion.+) and sections with absence of tachyzoites (GFP-) at days 3 or 10 . However, a subset of GFP+ vessels exhibited elevated EB signal, .We write “Overall, non-significant differences in permeability were observed when comparing brain sections containing tachyzoites . Possibly a larger material  could provide a more definitive answer. We have moved the more speculative part under results (above) to the discussion .Figure 6 looks at some target genes in MBECs from uninfected mice challenged in vitro with T. gondii and conclusion is that there is some upregulation of ICAM. A little hard for me t o know what to do with this data set. Isn't this where they should be looking at FAK dependent integrin expression?We agree and have expanded on this. At the time of our submission, another manuscript where we study the roles of integrins and CAMs in vitro was under revision and is now published. By citing these data, we now better link these aspects to possible mechanisms of passage in infected leukocytes with reference to  and also passage/invasion of endothelium and epithelium by extracellular tachyzoites . The putative connection to FAK dysregulation awaits further investigation. Ongoing work indicates a role for CAMs in this process . We have provided precision to these aspects in the text and additional references have been added .Figure 7B – the authors note there were 5-10 mice /group – but some have only 3 or 4 points. It is also unclear if this is a single experiment or pooled data sets? Just looking at the data sets for the LPS treatment  – it does look suspiciously like they may have more parasites (hence the question about repeats) while the micrograph in 7C and data in 7f looks like a lot more parasites. This is interesting but whether they used hydro-corticosteroids or daily low dose LPS they see more parasites in the parenchyma. It is also unclear why the control mice in this experiment do not have any parenchymal parasites when they have them in the earlier figures at an earlier time point. Maybe they just need to clarify this. While there is real value to including LPS as a comparator for BBB permeability – daily low dose LPS does a lot of things and I am (honestly) not clear that this figure helps – especially because they have much more specific interventions in the next figures.This point is well taken and we agree that the effects of anti- and pro-inflammatory treatments are probably as complex as the infection processes themselves. We included these treatments because they impact/modulate the infection process in different ways. Yet, as pointed out by the reviewer, both treatments result in elevated parasite numbers. We do not draw any major conclusion from this, other than pro-inflammatory LPS treatment – elevating BBB permeability – impacts parasite numbers and so does anti-inflammatory treatment, likely by dampening immune responses. The total parasite loads are provided as a guideline to the reader for comparisons. We have toned down the argumentation in the discussion and also provided additional nuance Related to parasites in parenchyma: When possible, outbred CD1 mice are used (this figure 7 for example) because of their superior resistance to T. gondii compared with B6 mice . Further, dose and inoculation route naturally impacted parasite loads and mouse survival time. Thus, the conditions for different figures are not necessarily directly comparable and address different aspects.Specifically in Figure 7, CD1 mice are inoculated with low dose i.p.. Parenchymal parasites can be quantified by day 3 in B6 mice inoculated with high dose i.v. , however low dose i.p. does not allow reliable quantification by IF by day 6 and therefore only day 10 is shown . These adjustments (strain/dose/administration route) were done to assure survival of the mice and in order to follow strict guidelines for minimization of severe symptoms, following ethical permit protocols. The rationale for use of different mouse strains, parasite strains and administration routes has been further clarified (results p. 7) and each figure legend indicates mouse strain, dose and administration route.Related to replicates: The reason for the fewer points in some graphics were technical . These experiments have been repeated and additional datapoints added. The compiled results reinforce the trend previously observed and show significant differences between the conditions .Figure 8a – looks like it is underpowered to conclude there is no difference in parasite burden at high dose (a) but there clearly is a difference in all tissues with low dose (b). This suggests a global impact of the inhibitor not an activity restricted to the CNS.This is very possible or, also that the relative inhibition for high dose  is inferior to the cumulative impact that FAK inhibition has over time . Regardless, we do not make major conclusions on this, total parasite loads are given as reference for loads in the brain and we think it does not change the overall conclusions of the paper. We have expanded on the possible “side effects” of pharmacological FAK inhibition in the revised discussion and advantages/disadvantages compared with conditional knockout Figure 8 – supplemental the concern here is that the analysis of the IFN-g mRNA looks like it is underpowered and its in the brain not the periphery when the authors indicate that there is a not a lot of local inflammation. If the FAK inhibitor has systemic effects on IFN-g then it would result in more parasites and this assay was used in Supp Figure 7 where it surprisingly showed no impact of hydro-corticosteroids. The authors just need to nail down some better assays for impact on the immune response (serum IFN-g protein?). It is also not clear that more parasites and bigger vacuoles are due to early entry. They have more parasites in the periphery which suggests a defect in ability to control them and this would lead to more early entry and increased growth that is independent of any impact on the BBB. This is really a difficult conclusion to make, with the data sets in hand.We agree and have clarified this figure to better show the variability between individual mice. As discussed above, the effects of treatments are likely multiple and these inflammatory parameters are provided as guidance that responses are not out of range (very high or very low) but rather in a comparable range. This does not exclude local or systemic differences that would require detailed analysis and cannot be made with the data sets in hand.We have modified the text under results  and bring up the possible impact of FAK inhibition on immune responses in the discussion Figure 9 – key experiment – nicely validate FAK and at day 6 post-infection parasite burden is normal across all tissues (differs from effects of inhibitor) and there is no impact on BBB permeability – but the authors find in panels e and f that there are some more parenchymal parasites at day 6. The main conclusion of this submission really revolves around these (similar) findings in Figure 8 and 9 and this reviewer is concerned that there are other explanations. Early in the manuscript they look at immune infiltration but have not seen if that parameter is altered in Figure 8 or 9. Any alteration in immune recruitment/access/function to the CNS is likely to impact parasite control (as they showed in their earlier figures).The reviewer brings up an important point. In Figure 4 we show that leukocyte infiltration days 1-5 is subtle and becomes accentuated by day 10. Thus, by day 6 it is still not prominent but could be affected by endothelial FAK deletion.We have performed an additional validation testing expression of FAK in bone-marrow derived cells from mice. By western blotting, we show that while FAK is reduced in endothelial cells, FAK expression is maintained in bone marrow-derived cells .We address the caveats of each system utilized in Figure 8 and 9 in the revised discussion


There was no clear evidence whether the initial dose of enzalutamide affects the incidence of adverse events (AEs), and oncological outcome in patients with castration-resistant prostate cancer (CRPC).The clinical charts of 233 patients with CRPC treated with enzalutamide were reviewed retrospectively. After 1:3 propensity score matching (PSM), 124 patients were divided into a reduced dose group and a standard dose group, and the prostate specific antigen (PSA) response and the incidence of AEs were compared.190 patients with CRPC initiated with standard dose enzalutamide were younger and better performance status compared with 43 patients beginning with reduced dose. After PSM, the baseline characteristics were not different between the standard and the reduced dose group. In the PSM cohort, the PSA response rate was significantly lower in the reduced dose group than in the standard dose group . The incidence rates of AEs were not statistically different between the groups .Initiating treatment with a reduced dose of enzalutamide did not significantly decrease the incidence rate of AEs, and it showed poorer PSA response rate. There is no clear rationale for treating with a reduced initial dose of enzalutamide to reduce the incidence of AEs. Prostate cancer (PC) is the most common type of cancer among elderly men worldwide . OverallEnzalutamide, a next-generation androgen receptor (AR)-targeted agent, was approved for metastatic CRPC based on the results of phase 3 double-blind, randomised trials (PREVAIL and AFFIRM) , 5. In tPatients sometimes require dose reduction or discontinuation of enzalutamide because of severe AEs . Thus, tThis study was approved by the Ethics Committee of both The Jikei University School of Medicine ((30-390(9411)) and University of the Ryukyus, Graduate School of Medicine. The requirement for informed patient consent was waived due to the study’s retrospective design.We retrospectively reviewed the clinical records of patients with CRPC treated with enzalutamide from June 2014 to December 2018 at The Jikei University Hospitals and University of the Ryukyus, Graduate School of Medicine and its affiliated institution. Patients were excluded from this study based on the following criteria: 1) lack of clinical information, 2) short follow-up periods (<1 month), and 3) prior treatment with abiraterone acetate. Ultimately, 233 patients were enrolled in this study. The initial dose of enzalutamide was decided based on an individual patient’s condition and the institutional policies, i.e., one institution (JU) used the standard dose and the other (RU) used a reduced dose as the starting dose in general. Patients were classified based on initial use of the standard dose or a reduced dose of enzalutamide, and the efficacy and safety of each dose were compared. The treatment sequence for CRPC was essentially decided according to the guidelines . CRPC waThe patients’ characteristics in the standard and reduced dose groups shown in Tables The baseline characteristics of the 233 patients are listed in The patients’ median age was 77 years , and median PSA level before enzalutamide treatment was 14.4 ng/mL . Among the 233 patients, 42% had undergone local therapies, such as prostatectomy or radiotherapy. Approximately 70% of the patients were initially treated with ADT plus bicalutamide, and the median time to CRPC was 18.5 months. Regarding the allocation of patients based on the initial dose of enzalutamide, 190 (81.6%), 25 (10.7%), 15 (6.4%), and 3 (1.3%) patients were administered 160, 120, 80, and 40 mg of enzalutamide, respectively. When stratifying the patients to the standard or reduced dose group, the patients in the reduced dose group were significantly older and had poorer PS than those in the standard dose group . AlthougThe patients’ baseline characteristics after PSM are shown in After 1:3 PSM, 31 and 93 patients were stratified to the reduced and the standard dose groups, respectively. The baseline characteristics were not significantly different between the groups.In the PSM cohort, the PSA response after enzalutamide treatment is shown in The PSA response rate after enzalutamide treatment was significantly higher in the standard dose group than in the reduced dose group . The proportion of patients with a PSA decline of >90% was significantly higher in the standard dose group than in the reduced dose group . The median follow-up period was 17 months. Overall, 84 patients (67.7%) had PSA progression and 24 patients (19.4%) died due to any cause. The Kaplan-Meier curves for PSA-PFS according to the initial dose of enzalutamide are presented in The initial dose of enzalutamide was not a significant predictive factor for PSA-PFS. In the multivariate analyses, time to CRPC within 12 months was the independent predictive factor for PSA progression .During the follow-up period, the proportion of patients requiring a dose reduction due to AEs tended to be higher in the standard dose group than in the reduced dose group, even though the difference was not statistically significant . Moreover, the discontinuation rate of enzalutamide due to AEs was not significantly different between the groups . The AE profile of the patients is presented in Tables The incidence rates of AEs of any grade and grades 3–5 were 30.2% and 8.9%, respectively, in the entire cohort. Fatigue and appetite loss were frequent AEs observed in this cohort . The incIn the present study, we assessed the influence of the initial dose of enzalutamide on the oncological outcome and incidence of AEs in patients with CRPC. The results of the PSM cohort indicated that the PSA response after enzalutamide treatment was lower in the reduced dose group than in the standard dose group even though the PSA-PFS was not statistically different. On the other hand, the incidence and severity of AEs were not significantly different between the groups.A dose escalation study of enzalutamide (30–360 mg per day) was conducted in previous preliminary and phase 1–2 trials, which found a dose-dependent increase of enzalutamide in the plasma , 16. In However, there are a number of patients with severe AEs after administration of the standard dose of enzalutamide in real-world practice. Actually, 8.9% of patients had grade 3–5 AEs in the present study, even though the incidence was not higher than that of the previous phase 3 study Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The authors have explored the impact of the initial dose of enzalutamide on the treatment response and incidence of AEs in patients with CRPC. They report that initial dose modification of enzalutamide did not decrease the incidence of AEs and might impair the efficacy. The topic is clinically significant as the patients are getting older, as written in the manuscript. The manuscript is concise and well-written. Some points to be clarified are as below.1) In the univariate and multivariate analysis, 'Time to CRPC <12m' is independent risk factor for the prediction of PSA progression-free survival (Table 4). Then, I suppose some patients who had long, durable response with previous ADT (Time to CRPC >12m) might be treated with initial dose reduction. In contrast, some with resistant to ADT might be treated without dose modification. So , Could you check and provide subgroup analysis between these two group according to Time to CRPC?2) It would be good to provide relative dose intensity if you can calculate from the subjects.3) In Table 2, the Median time to CRPC (IQR) of the two groups seems different (median time 10.5 vs. 20.5). Although, P-value is not statistically significant. I recommend checking this value and provide other values, such as mean time or distribution if possible.4) Please add abbreviations to each table.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  6 Sep 2021Thank you for reviewing our manuscript and offering us the opportunity to submit the revised one. We would also like to thank PLOS ONE Editorial Board that provides an opportunity to improve the manuscript with their helpful comments. We have revised the manuscript according to the reviewers’ suggestions.AttachmentResponse to Reviewer.docSubmitted filename: Click here for additional data file. 20 Sep 2021Initial dose reduction of enzalutamide does not decrease the incidence of adverse events in castration-resistant prostate cancerPONE-D-21-17359R1Dear Dr. Tsuzuki,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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