Infection with Francisella tularensis LVS clpB Leads to an Altered yet Protective Immune Response

ABSTRACT Bacterial attenuation is typically thought of as reduced bacterial growth in the presence of constant immune pressure. Infection with Francisella tularensis elicits innate and adaptive immune responses. Several in vivo screens have identified F. tularensis genes necessary for virulence. Many of these mutations render F. tularensis defective for intracellular growth. However, some mutations have no impact on intracellular growth, leading us to hypothesize that these F. tularensis mutants are attenuated because they induce an altered host immune response. We were particularly interested in the F. tularensis LVS (live vaccine strain) clpB (FTL_0094) mutant because this strain was attenuated in pneumonic tularemia yet induced a protective immune response. The attenuation of LVS clpB was not due to an intracellular growth defect, as LVS clpB grew similarly to LVS in primary bone marrow-derived macrophages and a variety of cell lines. We therefore determined whether LVS clpB induced an altered immune response compared to that induced by LVS in vivo . We found that LVS clpB induced proinflammatory cytokine production in the lung early after infection, a process not observed during LVS infection. LVS clpB provoked a robust adaptive immune response similar in magnitude to that provoked by LVS but with increased gamma interferon (IFN-γ) and interleukin-17A (IL-17A) production, as measured by mean fluorescence intensity. Altogether, our results indicate that LVS clpB is attenuated due to altered host immunity and not an intrinsic growth defect. These results also indicate that disruption of a nonessential gene(s) that is involved in bacterial immune evasion, like F. tularensis clpB , can serve as a model for the rational design of attenuated vaccines

Toxoplasma Strain-specific Early Immune Responses Determines Sub-acute CNS Immune Responses

Toxoplasma gondii is an obligate intracellular parasite that infects up to 1/3 of the world’s population. Genetically distinct Toxoplasma strains show differences in acute and chronic virulence in mice, and possibly in humans. In determining the mechanisms that cause these differences, recent in vivo data have revealed that Toxoplasma polymorphic effector proteins can modulate different innate immune responses in a strain-specific manner. But little is known about how these differences play out in the central nervous system (CNS), the major organ affected in human disease. To address this gap, we infected mice with two canonical Toxoplasma strains (type II and type III) and examined the systemic and CNS immune responses. We observed that at 5 days post infection (dpi), type III-infected mice polarized macrophages to alternatively activated (M2) phenotype, whereas, type II-infected mice had more classically activated (M1) phenotype in the spleen. At 21 dpi, we found that despite having similar parasite burden, the CNS of type III-infected mice had a more pro-inflammatory CNS immune response (macrophages and T-cell response, and cytokines/chemokines) and a significantly lower M2s and regulatory T cells as compared to type II-infected mice. Taking these data together, we propose that early immune responses determines the later sub-acute CNS immune response during Toxoplasma infection. To test this idea, we engineered a type III strain that lacks ROP16, the polymorphic effector protein that drives the early M2 response. Type IIIΔROP16-infected mice showed a more type II-like CNS response with lower levels of infiltrating T cells and macrophages/microglia and higher numbers of M2s as compared to type III strain. Collectively these studies suggest that early immune response differences play a key role in determining the later sub-acute immune response

The ROP16III-dependent early immune response determines the subacute 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 pro-inflammatory macrophages and that 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 early 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 pro-inflammatory macrophages, 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), which are 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 subacute CNS immune response.March of Dimes [5-FY15-45]; Arizona Biomedical Research Centre [ADHS14-082991]; National Institute of Neurologic Disorders and Stroke - United States Department of Health & Human Services - National Institutes of Health (NIH) - USANIH National Institute of Neurological Disorders & Stroke (NINDS) [NS095994-02S1]; American Heart Association [16PRE30990019]; United States Department of Health & Human Services - National Institutes of Health (NIH) - USA NIH National Institute of Allergy & Infectious Diseases (NIAID) [F31AI147711]; Howard Hughes Medical Institute - Howard Hughes Medical Institute [52003749]; BIO5 Institute, University of Arizona; National Cancer Institute (UA Flow Cytometry Shared Resource, UA Cancer Center) [P30CA023074]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

In the setting of IFN-γ depletion, <i>Toxoplasma</i> reactivation leads to an increase in parasite burden, GFP⁺ cells, and GFP⁺ astrocytes in the CNS.

<p>Starting at 4 wpi, isotype control or anti-interferon-γ antibodies were administered every 5 days to Cre reporter mice infected with II-Cre parasites. Mice were sacrificed at 6 wpi after receiving 3 doses of antibody treatment. Brains were sectioned and stained as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005447#ppat.1005447.g001" target="_blank">Fig 1</a>. Confocal microscopy was used to analyze stained brain sections from control and IFN-γ-depleted mice. (a) Representative stitched-grid image of half of a coronal brain section from a control (left) or IFN-γ-depleted (right) mouse. Scale bar, 1 mm. (b) Quantification of cyst number (6 sections/mouse) and GFP⁺ cell number (1 section/mouse) found in control or IFN-γ-depleted mice. N = 4–5 mice/group. *p< 0.05 by independent sample, two-tailed t-test. (c) Quantification of the lineage of GFP⁺ cells by co-localization with antibody stains for neurons, astrocytes, or neither (unidentified). (d) As in (c) but restricting the analysis only to GFP⁺ cells identified as neurons or astrocytes. N = 4–5 mice/group. N = 82–209 GFP⁺ cells/mouse analyzed for cell lineage studies. ***p< 0.001 by independent sample, two-tailed t-test.</p

Neuron-parasite interactions predominate during infection with IRG-resistant <i>Toxoplasma</i> parasites.

<p>Cre reporter mice were infected with either III-Cre or III-Cre parasites that express the type I ROP18 protein (III-Cre-ROP18). At 2 weeks post infection (wpi), brains were harvested, sectioned, stained for astrocytes and neurons, and analyzed by confocal microscopy as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005447#ppat.1005447.g001" target="_blank">Fig 1</a>. (a), (b) Representative merged stitched-grid image of a brain section from a III-Cre (a) or III-Cre-ROP18 (b)-infected mouse. Blue = astrocyte stain (GFAP), Cyan = neuronal stain (neuronal cocktail), Green = GFP expression. Scale bar, 1 mm. (c) Quantification of the lineage of GFP⁺ cells by co-localization with antibody stains for neurons, astrocytes, or neither. N = 100–114 GFP⁺ cells/mouse, N = 5 mice/<i>Toxoplasma</i> strain. (d) As in (c) but restricting the analysis only to GFP⁺ cells identified as neurons or astrocytes. There is no significant difference between the mean percentage of GFP⁺ neurons or astrocytes in III-Cre vs. III-Cre-ROP18 brain sections (p-value = 0.25, independent sample, two-tailed t-test.)</p

<i>Toxoplasma</i> parasites predominantly interact with neurons throughout CNS infection.

<p>Cre-reporter mice were infected with II-Cre or III-Cre <i>Toxoplasma</i> parasites as labeled. Brains were harvested, sectioned, and stained for neurons (anti-neuronal cocktail) and astrocytes (anti-GFAP) at specified time points. Stained sections were analyzed by confocal microscopy to identify if GFP co-localized with stains for neurons, astrocytes, or neither (unidentified). (a) Representative stitched-grid image of a brain section from a III-Cre infected mouse at 3 weeks post infection (wpi). White boxed area in left image is enlarged and separated into the different channels, as labeled (right images). White arrowheads denote GFP⁺ cells that co-localized with anti-neuron staining, red arrowheads denote GFP⁺ cells that did not co-localize with either anti-neuron or anti-astrocyte staining. Left image scale bar, 1 mm. Enlarged image scale bar, 50 μm. (b) Quantification of co-localization for II-Cre infected brain sections at different time points post infection. Bars, mean ±SEM. (c) As in (b) but restricting the analysis only to GFP⁺ cells identified as neurons or astrocytes. (d), (e) As in (b), (c) but for III-Cre infected mice. Bars, mean ±SEM. N = 3–4 mice/time point/<i>Toxoplasma</i> strain. The total number of GFP⁺ cells examined at each time point ranged from 232–372/II-Cre and 368–506/III-Cre. No statistical differences were found in the mean percentage of GFP⁺ neurons across time points in either II-Cre or III-Cre infection (one-way ANOVA, p = 0.13 and p = 0.45 respectively). At 6 wpi, one of the III-Cre infected mice had a substantially lower percentage of GFP⁺ neurons compared to the other mice (67 vs. 91,100,100). Exclusion of this mouse from data analysis changes the mean percentage of GFP⁺ neurons at 6 wpi from 90 ±8 (full data set) to 97 ±3 (1 mouse excluded), which results in a suggestion that in III-Cre infected mice the percentage of GFP⁺ neurons is lower at 1.5 wpi compared to 6 and 12 wpi (one-way ANOVA, p < 0.01). No GFP⁺ cells were found in II-Cre or III-Cre infected brain sections from 0.5 wpi (N = 2 mice/<i>Toxoplasma</i> strain, 9 sections/mouse).</p

Neuron-parasite interactions dominate in direct CNS inoculation with <i>Toxoplasma</i>, consistent with the baseline ratio of neurons:astrocytes.

<p>Phosphate Buffer Saline (PBS) or <i>Toxoplasma</i>-Cre parasites (II-Cre) were stereotactically injected into the cerebral cortex of naïve Cre reporter mice. At 3, 6, and 9 days post-injection (dpi) brains were harvested, sectioned, stained for astrocytes and neurons, and analyzed by confocal microscopy for co-localization of GFP with astrocyte or neuronal stains or for baseline numbers of astrocytes and neurons. Brain sections from uninfected mice were also stained for neurons and astrocytes and analyzed for baseline numbers of astrocytes and neurons. (a) Schematic of site of injection. (b) Representative merged stitched-grid images of the injection site at 3 dpi from PBS (left) or <i>Toxoplasma</i>-Cre (right) injected mice. Blue = astrocyte stain (anti-GFAP), Cyan = neuronal stain (anti-neuronal cocktail), Green = GFP expression. Scale bar, 200 μm. (c) Quantification of GFP⁺ cells identified as either astrocytes or neurons by co-localization with staining. N = 3 mice/time point, 100 identified GFP⁺ cells/mouse. No statistical differences were found between the mean percentage of GFP⁺ neurons across time points (one-way ANOVA, p = 0.97). No GFP⁺ cells were seen in PBS injected mice. N = 1 mouse/time point. (d) Schematic of fields of view (FOV) taken to assess neuron and astrocyte numbers across IC infection time points. (e) Quantification of number of astrocytes/FOV. (f) Quantification of number of neurons/FOV. As astrocytes in uninfected mice express little GFAP (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005447#ppat.1005447.s004" target="_blank">S4a Fig</a>), these sections were stained with anti-GFAP, anti-S100β, and anti-ALDH1L1 antibodies. The neuronal cocktail was unchanged. UI, uninfected. N = 3–4 mice/time point, 3 randomly selected FOV/hemisphere/ mouse (6 FOV total/mouse) were counted. * p<0.05, *** p< 0.001, ordinary one-way ANOVA.</p

Neurons are the Primary Target Cell for the Brain-Tropic Intracellular Parasite <i>Toxoplasma gondii</i>

<div><p><i>Toxoplasma gondii</i>, a common brain-tropic parasite, is capable of infecting most nucleated cells, including astrocytes and neurons, <i>in vitro</i>. Yet, <i>in vivo</i>, <i>Toxoplasma</i> is primarily found in neurons. <i>In vitro</i> data showing that interferon-γ-stimulated astrocytes, but not neurons, clear intracellular parasites suggest that neurons alone are persistently infected <i>in vivo</i> because they lack the ability to clear intracellular parasites. Here we test this theory by using a novel <i>Toxoplasma</i>-mouse model capable of marking and tracking host cells that directly interact with parasites, even if the interaction is transient. Remarkably, we find that <i>Toxoplasma</i> shows a strong predilection for interacting with neurons throughout CNS infection. This predilection remains in the setting of IFN-γ depletion; infection with parasites resistant to the major mechanism by which murine astrocytes clear parasites; or when directly injecting parasites into the brain. These findings, in combination with prior work, strongly suggest that neurons are not incidentally infected, but rather they are <i>Toxoplasma</i>’s primary <i>in vivo</i> target.</p></div