Deficiency in astrocyte CCL2 production reduces neuroimmune control of Toxoplasma gondii infection.

Toxoplasma gondii is an obligate intracellular parasite that infects one-third of the world's human population and establishes infection in the brain. Cerebral immune cell infiltration is critical for controlling the parasite, but little is known about the molecular cues guiding immune cells to the brain during infection. Activated astrocytes produce CCL2, a chemokine that mediates inflammatory monocyte recruitment to tissues by binding to the CCR2 receptor. We detected elevated CCL2 production in the brains of C57BL/6J mice by 15 days after T. gondii infection. Utilizing confocal microscopy and intracellular flow cytometry, we identified microglia and brain-infiltrating myeloid cells as the main producers of CCL2 during acute infection, and CCL2 was specifically produced in regions of parasite infection in the brain. In contrast, astrocytes became the dominant CCL2 producer during chronic T. gondii infection. To determine the role of astrocyte-derived CCL2 in mobilizing immune cells to the brain and controlling T. gondii infection, we generated GFAP-Cre x CCL2fl/fl mice, in which astrocytes are deficient in CCL2 production. We observed significantly decreased immune cell recruitment and increased parasite burden in the brain during chronic, but not acute, infection of mice deficient in astrocyte CCL2 production, without an effect on peripheral immune responses. To investigate potential mechanisms explaining the reduced control of T. gondii infection, we analyzed key antimicrobial and immune players in host defense against T. gondii and detected a reduction in iNOS+ myeloid cells, and T. gondii-specific CD4+ T cells in the knockout mice. These data uncover a critical role for astrocyte-derived CCL2 in immune cell recruitment and parasite control in the brain during chronic, but not acute, T. gondii infection.

Meningeal lymphatic drainage promotes T cell responses against Toxoplasma gondii but is dispensable for parasite control in the brain.

The discovery of meningeal lymphatic vessels that drain the CNS has prompted new insights into how immune responses develop in the brain. In this study, we examined how T cell responses against CNS-derived antigen develop in the context of infection. We found that meningeal lymphatic drainage promotes CD4+ and CD8+ T cell responses against the neurotropic parasite Toxoplasma gondii in mice, and we observed changes in the dendritic cell compartment of the dural meninges that may support this process. Indeed, we found that mice chronically, but not acutely, infected with T. gondii exhibited a significant expansion and activation of type 1 and type 2 conventional dendritic cells (cDC) in the dural meninges. cDC1s and cDC2s were both capable of sampling cerebrospinal fluid (CSF)-derived protein and were found to harbor processed CSF-derived protein in the draining deep cervical lymph nodes. Disrupting meningeal lymphatic drainage via ligation surgery led to a reduction in CD103+ cDC1 and cDC2 number in the deep cervical lymph nodes and caused an impairment in cDC1 and cDC2 maturation. Concomitantly, lymphatic vessel ligation impaired CD4+ and CD8+ T cell activation, proliferation, and IFN-γ production at this site. Surprisingly, however, parasite-specific T cell responses in the brain remained intact following ligation, which may be due to concurrent activation of T cells at non-CNS-draining sites during chronic infection. Collectively, our work reveals that CNS lymphatic drainage supports the development of peripheral T cell responses against T. gondii but remains dispensable for immune protection of the brain.

Microglial STAT1-sufficiency is required for resistance to toxoplasmic encephalitis.

Toxoplasma gondii is a ubiquitous intracellular protozoan parasite that establishes a life-long chronic infection largely restricted to the central nervous system (CNS). Constant immune pressure, notably IFN-γ-STAT1 signaling, is required for preventing fatal pathology during T. gondii infection. Here, we report that abrogation of STAT1 signaling in microglia, the resident immune cells of the CNS, is sufficient to induce a loss of parasite control in the CNS and susceptibility to toxoplasmic encephalitis during the early stages of chronic infection. Using a microglia-specific genetic labeling and targeting system that discriminates microglia from blood-derived myeloid cells that infiltrate the brain during infection, we find that, contrary to previous in vitro reports, microglia do not express inducible nitric-oxide synthase (iNOS) during T. gondii infection in vivo. Instead, transcriptomic analyses of microglia reveal that STAT1 regulates both (i) a transcriptional shift from homeostatic to "disease-associated microglia" (DAM) phenotype conserved across several neuroinflammatory models, including T. gondii infection, and (ii) the expression of anti-parasitic cytosolic molecules that are required for eliminating T. gondii in a cell-intrinsic manner. Further, genetic deletion of Stat1 from microglia during T. gondii challenge leads to fatal pathology despite largely equivalent or enhanced immune effector functions displayed by brain-infiltrating immune populations. Finally, we show that microglial STAT1-deficiency results in the overrepresentation of the highly replicative, lytic tachyzoite form of T. gondii, relative to its quiescent, semi-dormant bradyzoite form typical of chronic CNS infection. Our data suggest an overall protective role of CNS-resident microglia against T. gondii infection, illuminating (i) general mechanisms of CNS-specific immunity to infection (ii) and a clear role for IFN-STAT1 signaling in regulating a microglial activation phenotype observed across diverse neuroinflammatory disease states.

Astrocytes promote a protective immune response to brain Toxoplasma gondii infection via IL-33-ST2 signaling.

It is of great interest to understand how invading pathogens are sensed within the brain, a tissue with unique challenges to mounting an immune response. The eukaryotic parasite Toxoplasma gondii colonizes the brain of its hosts, and initiates robust immune cell recruitment, but little is known about pattern recognition of T. gondii within brain tissue. The host damage signal IL-33 is one protein that has been implicated in control of chronic T. gondii infection, but, like many other pattern recognition pathways, IL-33 can signal peripherally, and the specific impact of IL-33 signaling within the brain is unclear. Here, we show that IL-33 is expressed by oligodendrocytes and astrocytes during T. gondii infection, is released locally into the cerebrospinal fluid of T. gondii-infected animals, and is required for control of infection. IL-33 signaling promotes chemokine expression within brain tissue and is required for the recruitment and/or maintenance of blood-derived anti-parasitic immune cells, including proliferating, IFN-γ-expressing T cells and iNOS-expressing monocytes. Importantly, we find that the beneficial effects of IL-33 during chronic infection are not a result of signaling on infiltrating immune cells, but rather on radio-resistant responders, and specifically, astrocytes. Mice with IL-33 receptor-deficient astrocytes fail to mount an adequate adaptive immune response in the CNS to control parasite burden-demonstrating, genetically, that astrocytes can directly respond to IL-33 in vivo. Together, these results indicate a brain-specific mechanism by which IL-33 is released locally, and sensed locally, to engage the peripheral immune system in controlling a pathogen.

Gasdermin-D-dependent IL-1α release from microglia promotes protective immunity during chronic Toxoplasma gondii infection.

Microglia, resident immune cells of the CNS, are thought to defend against infections. Toxoplasma gondii is an opportunistic infection that can cause severe neurological disease. Here we report that during T. gondii infection a strong NF-κB and inflammatory cytokine transcriptional signature is overrepresented in blood-derived macrophages versus microglia. Interestingly, IL-1α is enriched in microglia and IL-1ß in macrophages. We find that mice lacking IL-1R1 or IL-1α, but not IL-1ß, have impaired parasite control and immune cell infiltration within the brain. Further, we show that microglia, not peripheral myeloid cells, release IL-1α ex vivo. Finally, we show that ex vivo IL-1α release is gasdermin-D dependent, and that gasdermin-D and caspase-1/11 deficient mice show deficits in brain inflammation and parasite control. These results demonstrate that microglia and macrophages are differently equipped to propagate inflammation, and that in chronic T. gondii infection, microglia can release the alarmin IL-1α, promoting neuroinflammation and parasite control.

Expression of the muscle-associated gene MYF6 in hairy cell leukemia.

Hairy cell leukemia (HCL) is a purine analog-responsive B-cell malignancy containing the BRAF V600E mutation, expressing CD22, CD11c, CD103, tartrate resistant acid phosphatase (TRAP) CD25, CD123, and annexin 1A. BRAF V600E and the latter 4 markers are usually absent in the more aggressive and chemoresistant variant HCLv. To evaluate differences between HCL and HCLv, expression microarrays comparing HCL with HCLv were performed for 24694 genes using 47323 probes. Microarray data from 35 HCL and 27 HCLv purified samples showed the greatest HCL-HCLv difference in the muscle-associated gene MYF6, expressed by its 2 probes 18.5- and 10.8-fold higher in HCL than HCLv (p<0.0001). By real-time quantitative PCR (RQ-PCR), 100% of 152 classic HCL samples were MYF6-positive, vs 5 (6%) of 90 blood donors. MYF6-expression was also detected in 18 (35%) of 51 with HCLv, 11 (92%) of 12 with HCL expressing unmutated IGHV4-34, 35 (73%) of 48 with chronic lymphocytic leukemia (CLL), and 1 (8%) of 12 with mantle cell lymphoma. Hypomethylation status of MYF6 supported expression in HCL more than HCLv. Posttreatment blood samples becoming negative by flow cytometry remained MYF6+ by RQ-PCR in 42 (48%) of 87 HCL patients, and MYF6 RQ-PCR could detect 1 HCL in 105 normal cells. MYF6, universally expressed in HCL and in most CLL samples, may be a useful biomarker for these leukemias. Further studies are underway to determine the role of MYF6 in HCL.

The Notch signaling pathway promotes basophil responses during helminth-induced type 2 inflammation.

Type 2 inflammation drives the clearance of gastrointestinal helminth parasites, which infect over two billion people worldwide. Basophils are innate immune cells that support host-protective type 2 inflammation during murine infection with the helminth Trichuris muris However, the mechanisms required for basophil function and gene expression regulation in this context remain unclear. We show that during T. muris infection, basophils localized to the intestine and up-regulated Notch receptor expression, rendering them sensitive to Notch signals that rapidly regulate gene expression programs. In vitro, Notch inhibition limited basophil cytokine production in response to cytokine stimulation. Basophil-intrinsic Notch signaling was required for T. muris-elicited changes in genome-wide basophil transcriptional programs. Mice lacking basophil-intrinsic functional Notch signaling had impaired worm clearance, decreased intestinal type 2 inflammation, altered basophil localization in the intestine, and decreased CD4+ T helper 2 cell responses following infection. These findings demonstrate that Notch is required for basophil gene expression and effector function associated with helminth expulsion during type 2 inflammation.

IL-10 and ICOS Differentially Regulate T Cell Responses in the Brain during Chronic Toxoplasma gondii Infection.

Control of chronic CNS infection with the parasite Toxoplasma gondii requires ongoing T cell responses in the brain. Immunosuppressive cytokines are also important for preventing lethal immunopathology during chronic infection. To explore the loss of suppressive cytokines exclusively during the chronic phase of infection, we blocked IL-10R in chronically infected mice. Consistent with previous reports, IL-10R blockade led to severe, fatal tissue destruction associated with widespread changes in the inflammatory response, including increased APC activation, expansion of CD4+ T cells, and neutrophil recruitment to the brain. We then sought to identify regulatory mechanisms contributing to IL-10 production, focusing on ICOS, a molecule implicated in IL-10 production. Unexpectedly, ICOS ligand (ICOSL) blockade led to a local expansion of effector T cells in the brain without affecting IL-10 production or APC activation. Instead, we found that ICOSL blockade led to changes in T cells associated with their proliferation and survival. We observed increased expression of IL-2-associated signaling molecules CD25, STAT5 phosphorylation, Ki67, and Bcl-2 in T cells in the brain, along with decreased apoptosis. Interestingly, increases in CD25 and Bcl-2 were not observed following IL-10R blockade. Also, unlike IL-10R blockade, ICOSL blockade led to an expansion of both CD8+ and CD4+ T cells in the brain, with no expansion of peripheral T cells or neutrophil recruitment to the brain and no severe tissue destruction. Overall, these results suggest that IL-10 and ICOS differentially regulate T cell responses in the brain during chronic T. gondii infection.

Detection of prosecretory mitogen lacritin in nonprimate tears primarily as a C-terminal-like fragment.

PURPOSE: Lacritin is a human tear glycoprotein that promotes basal tear protein secretion in cultured rat lacrimal acinar cells and proliferation of subconfluent human corneal epithelial cells. When topically added to rabbit eyes, lacritin promotes basal tearing. Despite these activities on several species, lacritin's presence in nonprimate tears or other tissues has not been explored. Here we probed for lacritin in normal horse tears. METHODS: Sequences were collected from the Ensembl genomic alignment of human LACRT gene with high-quality draft horse genome (EquCab2.0) and analyzed. Normal horse tears were collected and assayed by Western blotting, ELISA, and mass spectrometry. Newly generated rabbit antibodies, respectively, against N- and C-terminal regions of human lacritin were employed. RESULTS: Identity was 75% and 45%, respectively, at nucleotide and protein levels. Structural features were conserved, including a C-terminal amphipathic α-helix. Anti-C-terminal antibodies strongly detected a ∼13 kDa band in horse tears that was validated by mass spectrometry. In human tears, the same antibody detected uncleaved lacritin (∼24 kDa) strongly and C-terminal fragments of ∼13 and ∼11 kDa weakly. Anti-N-terminal antibodies were slightly reactive with a ∼24 kDa horse antigen and showed no reaction with the anti-C-terminal-reactive ∼13 kDa species. Similar respective levels of horse C-terminal versus N-terminal immunoreactivity were apparent by ELISA. CONCLUSIONS: Lacritin is present in horse tears, largely as a C-terminal fragment homologous to the mitogenic and bactericidal region in human lacritin, suggesting potential benefit in corneal wound repair.

Tear lacritin levels by age, sex, and time of day in healthy adults.

PURPOSE: Several small proteomic studies suggest that the prosecretory tear protein lacritin may be selectively downregulated in dry eye syndrome and in blepharitis, yet little information is available about normal baseline levels. This study assessed lacritin levels in tears from healthy individuals and addressed whether they differ according to sex, age, or time of day. METHODS: Rabbit antibodies against lacritin N-terminal peptide EDASSDSTGADPAQEAGTS (Pep Lac N-Term) were generated and characterized against human recombinant lacritin and N-65 truncation mutant. Basal tears were collected from 66 healthy individuals ranging in age from 18 to 52 years, and at four times during one 24-hour period from 34 other individuals. Lacritin levels were then analyzed by ELISA and Western blotting. RESULTS: Anti-Pep Lac N-Term bound lacritin, but not truncation mutant N-65 that lacks the N-terminal antigenic site. Tear lacritin levels followed a normal distribution with a mean of 4.2 ± 1.17 ng/100 ng total tear protein. Levels differed little by age or sex, and decreased slightly between 4 and 8 hours in a 24-hour cycle. Tear-blocking effects were minimal, as suggested by spiking of tears with recombinant lacritin. CONCLUSIONS: Anti-Pep Lac N-Term-detectable lacritin comprises ~4.2 ng/100 ng total tear protein in healthy individuals, with no significant differences between males and females or among individuals between 18 and 52 years old. Levels decrease slightly in the late afternoon. These findings provide a baseline for future immunodiagnostic studies of lacritin in dry eye and other ocular diseases.