Chronic Pseudomonas aeruginosa Lung Infection Is IL-1R Independent, but Relies on MyD88 Signaling.

Cystic fibrosis is associated with chronic Pseudomonas aeruginosa colonization and inflammation. The role of MyD88, the shared adapter protein of the proinflammatory TLR and IL-1R families, in chronic P. aeruginosa biofilm lung infection is unknown. We report that chronic lung infection with the clinical P. aeruginosa RP73 strain is associated with uncontrolled lung infection in complete MyD88-deficient mice with epithelial damage, inflammation, and rapid death. Then, we investigated whether alveolar or myeloid cells contribute to heightened sensitivity to infection. Using cell-specific, MyD88-deficient mice, we uncover that the MyD88 pathway in myeloid or alveolar epithelial cells is dispensable, suggesting that other cell types may control the high sensitivity of MyD88-deficient mice. By contrast, IL-1R1-deficient mice control chronic P. aeruginosa RP73 infection and IL-1ß Ab blockade did not reduce host resistance. Therefore, the IL-1R1/MyD88 pathway is not involved, but other IL-1R or TLR family members need to be investigated. Our data strongly suggest that IL-1 targeted neutralizing therapies used to treat inflammatory diseases in patients unlikely reduce host resistance to chronic P. aeruginosa infection.

IL-36 signaling in keratinocytes controls early IL-23 production in psoriasis-like dermatitis.

IL-36R signaling plays an important role in the pathogenesis of psoriasis. We ought to assess the specific function of IL-36R in keratinocytes for the pathology of Aldara-induced psoriasis-like dermatitis. Il36r ΔK mice presenting deletion of IL-36R in keratinocytes were similarly resistant to Aldara-induced ear inflammation as Il36r -/- mice, but acanthosis was only prevented in Il36r -/- mice. FACS analysis revealed that IL-36R signaling in keratinocytes is mandatory for early neutrophil infiltration in Aldara-treated ears. RNASeq and qRT-PCR experiments demonstrated the crucial role of IL-36R signaling in keratinocytes for induction of IL-23, IL-17, and IL-22 at early time points. Taken together, our results demonstrate that IL-36R signaling in keratinocytes plays a major role in the induction of Aldara-induced psoriasis-like dermatitis by triggering early production of IL-23/IL-17/IL-22 cytokines and neutrophil infiltration.

Intracellular IL-1 Receptor Antagonist Isoform 1 Released from Keratinocytes upon Cell Death Acts as an Inhibitor for the Alarmin IL-1α.

The inflammatory effects of IL-1α/ß are controlled by IL-1R antagonist (IL-1Ra). One IL-1Ra isoform is secreted, whereas three other isoforms (intracellular IL-1Ra [icIL-1Ra] 1, 2, and 3) are supposed to remain intracellular because of the absence of a signal peptide. In contrast to the well-characterized function of the secreted isoform, the biological role of the intracellular isoforms remains largely unclear. icIL-1Ra1 represents the major isoform in keratinocytes. We created icIL-1Ra1-/- mice and investigated the role of icIL-1Ra1 in Aldara (5% imiquimod)-induced psoriasis-like skin inflammation. Naive icIL-1Ra1-/- mice bred habitually and exhibited a normal phenotype. icIL-1Ra1 deficiency aggravated Aldara-induced skin inflammation, as demonstrated by increased ear thickness and increased mRNA levels of key proinflammatory cytokines. No intracellular effect of icIL-1Ra1 could be detected in isolated keratinocytes using RNA-sequencing analysis; however, Aldara treatment led to caspase 1/11-, caspase 8-, and RIPK3-independent keratinocyte cell death accompanied by the release of both icIL-1Ra1 and IL-1α. Furthermore, blocking IL-1α attenuated the clinical severity of Aldara-induced ear thickening in icIL-1Ra1-/- mice. Our data suggest that upon keratinocyte damage icIL-1Ra1 acts extracellularly as an antagonist of the alarmin IL-1α to immediately counteract its inflammatory effects.

The severity of imiquimod-induced mouse skin inflammation is independent of endogenous IL-38 expression.

The IL-1 cytokine family includes eleven members, among which Il-36α, ß and γ, IL-36Ra and IL-38. The IL-36 cytokines are involved in the pathogenesis of psoriasis. IL-38 is also expressed in the skin and was previously proposed to act as an IL-36 antagonist. In this study, we thus examined expression and function of Il-38 in a mouse model of imiquimod (IMQ)-induced skin inflammation. Il-38 mRNA was detected in the epidermis and in primary mouse keratinocytes, but not in dermal fibroblasts. At the peak of IMQ-induced inflammation, skin Il-38 mRNA levels were reduced, whereas Il-36ra mRNA expression increased. The severity of IMQ-induced skin inflammation, as assessed by recording ear thickness and histological changes, was similar in Il-38 KO and WT littermate control mice, while, in contrast, Il-36ra-deficient mice displayed more severe skin pathology than their WT littermates. Il-38-deficiency had no impact on IMQ-induced expression of proinflammatory mediators in the skin in vivo, on the basal expression of various cytokines or chemokines by cultured primary keratinocytes and dermal fibroblasts in vitro, or on the response of these cells to Il-36ß. Finally, after cessation of topical IMQ application, the resolution of skin inflammation was also not altered in Il-38 KO mice. In conclusion, Il-38-deficiency did not impact the development or resolution of IMQ-induced skin inflammation. Our observations further suggest that endogenous Il-38 does not exert Il-36 inhibitory activity in this model, or in cultured skin cells. A potential anti-inflammatory function of Il-38 in mouse skin thus still remains to be demonstrated.

Unopposed IL-18 signaling leads to severe TLR9-induced macrophage activation syndrome in mice.

The term macrophage activation syndrome (MAS) defines a severe, potentially fatal disorder characterized by overwhelming inflammation and multiorgan involvement. Interleukin-18 (IL-18) is a proinflammatory cytokine belonging to the IL-1 family, the activity of which is regulated by its endogenous inhibitor IL-18 binding protein (IL-18BP). Elevated IL-18 levels have been reported in patients with MAS. Herein, we show that on repeated toll-like receptor 9 (TLR9) stimulation with unmethylated cytosine guanine dinucleotide containing single-stranded DNA (CpG), IL-18BP-/- mice display severe MAS manifestations, including increased weight loss, splenomegaly, anemia, thrombocytopenia, hyperferritinemia, and bone marrow hemophagocytosis as compared with wild-type mice. Serum-free IL-18 was detected in CpG-treated IL-18BP-/- mice only. Levels of interferon-γ (IFN-γ) and of IFN-γ signature genes, such as the chemokine Cxcl9 or the transcription factor CIIta, were significantly increased in IL-18BP-/- mice. Blocking IL-18 receptor signaling attenuated the severity of MAS and IFN-γ responses in IL-18BP-/- mice. Blocking IFN-γ had comparable effects to IL-18 inhibition on most MAS manifestations. Our data indicate that endogenous IL-18BP exerts a protective role in CpG-induced MAS and that IL-18, which acts upstream of IFN-γ, is involved in the severity of MAS.

IL-33 receptor ST2 regulates the cognitive impairments associated with experimental cerebral malaria.

Cerebral malaria (CM) is associated with a high mortality rate and long-term neurocognitive impairment in survivors. The murine model of experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA (PbA)-infection reproduces several of these features. We reported recently increased levels of IL-33 protein in brain undergoing ECM and the involvement of IL-33/ST2 pathway in ECM development. Here we show that PbA-infection induced early short term and spatial memory defects, prior to blood brain barrier (BBB) disruption, in wild-type mice, while ST2-deficient mice did not develop cognitive defects. PbA-induced neuroinflammation was reduced in ST2-deficient mice with low Ifng, Tnfa, Il1b, Il6, CXCL9, CXCL10 and Cd8a expression, associated with an absence of neurogenesis defects in hippocampus. PbA-infection triggered a dramatic increase of IL-33 expression by oligodendrocytes, through ST2 pathway. In vitro, IL-33/ST2 pathway induced microglia expression of IL-1ß which in turn stimulated IL-33 expression by oligodendrocytes. These results highlight the IL-33/ST2 pathway ability to orchestrate microglia and oligodendrocytes responses at an early stage of PbA-infection, with an amplification loop between IL-1ß and IL-33, responsible for an exacerbated neuroinflammation context and associated neurological and cognitive defects.

Deficiency in IL-1 Receptor Type 2 Aggravates K/BxN Serum Transfer-Induced Arthritis in Mice but Has No Impact on Systemic Inflammatory Responses.

The biological activity of IL-1 is tightly regulated by the specific receptor antagonist (IL-1Ra) and the decoy receptor IL-1 receptor type 2 (IL-1R2). The role of IL-1Ra has been well demonstrated in IL-1Ra-deficient mice. In contrast, the role of endogenous IL-1R2 remains widely unknown. To define the functional role of endogenous IL-1R2 in the K/BxN serum transfer arthritis model and in IL-1ß- or LPS-induced systemic inflammation in vivo, IL-1R2-/- mice were created and compared with wild type mice. IL-1R2-/- mice bred habitually and exhibited a normal phenotype. IL-1R2 deficiency aggravated arthritis severity and increased mRNA levels for key cytokines and chemokines such as IL-6, IL-1ß, Cxcl-1, and Cxcl-2 significantly in ankles. There was no effect of IL-1R2 deficiency on the cell-autonomous cytokine response to IL-1ß in the tested cell types, i.e., neutrophils, macrophages, and fibroblasts, but IL-1R2 deficiency on neutrophils increased the IL-1-induced response of fibroblasts in trans. Furthermore, IL-1ß induced shedding of IL-1R2 in vivo. Inflammatory responses to IL-1ß and LPS-induced mortality were not different in IL-1R2-/- compared with wild type mice. Our data demonstrate that the decoy receptor IL-1R2 plays an important inhibitory role in local IL-1- and neutrophil-dependent tissue inflammation as shown in the K/BxN serum transfer arthritis model. In contrast to IL-1Ra, IL-1R2 appears to be less crucial for systemic responses to acute administration of IL-1 or LPS.

IL-36-Induced Toxicity in Neonatal Mice Involves TNF-α Production by Liver Myeloid Cells.

Human and mouse neonates exhibit limited vaccine responses characterized by predominant Th2 and limited Th1 responses. Because IL-36 exerts a synergic adjuvant effect with IL-12, enhancing Th1 polarization in adult (AD) mice, we administered IL-36ß to neonatal (1-wk old) and AD control mice at the time of immunization with tetanus toxoid adsorbed to aluminum hydroxide (TT/Alum). Unexpectedly, the combination of IL-36ß with TT/Alum, which was well tolerated in AD mice, proved toxic and even lethal in neonates. This neonatal toxicity was associated with high Il36r mRNA expression in neonatal liver, resulting in increased cytokine production. Liver Il36r mRNA expression decreased with the termination of fetal liver hematopoiesis, and this decrease correlated with a complete protection from TT/Alum/IL-36ß-induced mortality. The combination of IL-36ß and TT/Alum induced the rapid production of TNF-α and IFN-γ by liver myeloid and lymphoid cells, respectively. These responses were less marked when IL-36ß was used alone, with no adverse effect. The toxicity of IL-36ß + TT/Alum was abrogated by the administration of a neutralizing anti-TNF-α Ab, confirming causality. In conclusion, liver myeloid cells in neonatal mice are an important source of proinflammatory cytokines that may lead to TNF-α-mediated toxicity and even lethality.

IL-1R1-MyD88 axis elicits papain-induced lung inflammation.

Allergic asthma is characterized by a strong Th2 response with inflammatory cell recruitment and structural changes in the lung. Papain is a protease allergen disrupting the airway epithelium triggering a rapid inflammation with eosinophilia mediated by innate lymphoid cell activation (ILC2) and leading to a Th2 immune response. In this study, we focused on inflammatory responses to a single exposure to papain and showed that intranasal administration of papain results in the recruitment of inflammatory cells, including neutrophils and eosinophils with a rapid production of IL-1α, IL-1ß, and IL-33. The inflammatory response is abrogated in the absence of IL-1R1 and MyD88. To decipher the cell type(s) involved in MyD88-dependent IL-1R1/MyD88 signaling, we used new cell-specific MyD88-deficient mice and found that the deletion of MyD88 signaling in single cell types such as T cells, epithelial cells, CD11c-positive or myeloid cells leads to only a partial inhibition compared to complete absence of MyD88, suggesting that several cell types contribute to the response. Importantly, the inflammatory response is largely ST2 and IL-36R independent. In conclusion, IL-1R1 signaling via MyD88 is critical for the first step of inflammatory response to papain.

Characterization of seizures induced by acute exposure to an organophosphate herbicide, glufosinate-ammonium.

Glufosinate-ammonium (GLA), the active component of a widely used herbicide, induces convulsions in rodents and humans. In mouse, intraperitoneal treatment with 75 mg/kg GLA generates repetitive tonic-clonic seizures associated with 100% mortality within 72 h after treatment. In this context, we characterized GLA-induced seizures, their histological consequences and the effectiveness of diazepam treatment. Epileptic discharges on electroencephalographic recordings appeared simultaneously in the hippocampus and the cerebral cortex. Diazepam treatment at 6 h immediately stopped the seizures and prevented animal death. However, intermittent seizures were recorded on electroencephalogram from 6 h after diazepam treatment until 24 h, but had disappeared after 15 days. In our model, neuronal activation (c-Fos immunohistochemistry) was observed 6 h after GLA exposure in the dentate gyrus, CA1, CA3, amygdala, piriform and entorhinal cortices, indicating the activation of the limbic system. In these structures, Fluoro-Jade C and Cresyl violet staining did not show neuronal suffering. However, astroglial activation was clearly observed at 24 h and 15 days after GLA treatment in the amygdala, piriform and entorhinal cortices by PCR quantitative, western blot and immunohistochemistry. Concomitantly, glutamine synthetase mRNA expression (PCR quantitative), protein expression (western blot) and enzymatic activity were upregulated. In conclusion, our study suggests that GLA-induced seizures: (a) involved limbic structures and (b) induced astrocytosis without neuronal degeneration as an evidence of a reactive astrocyte beneficial effect for neuronal protection.

The interleukin (IL)-1 cytokine family--Balance between agonists and antagonists in inflammatory diseases.

The interleukin (IL)-1 family of cytokines comprises 11 members, including 7 pro-inflammatory agonists (IL-1α, IL-1ß, IL-18, IL-33, IL-36α, IL-36ß, IL-36γ) and 4 defined or putative antagonists (IL-1R antagonist (IL-1Ra), IL-36Ra, IL-37, and IL-38) exerting anti-inflammatory activities. Except for IL-1Ra, IL-1 cytokines do not possess a leader sequence and are secreted via an unconventional pathway. In addition, IL-1ß and IL-18 are produced as biologically inert pro-peptides that require cleavage by caspase-1 in their N-terminal region to generate active proteins. N-terminal processing is also required for full activity of IL-36 cytokines. The IL-1 receptor (IL-1R) family comprises 10 members and includes cytokine-specific receptors, co-receptors and inhibitory receptors. The signaling IL-1Rs share a common structure with three extracellular immunoglobulin (Ig) domains and an intracellular Toll-like/IL-1R (TIR) domain. IL-1 cytokines bind to their specific receptor, which leads to the recruitment of a co-receptor and intracellular signaling. IL-1 cytokines induce potent inflammatory responses and their activity is tightly controlled at the level of production, protein processing and maturation, receptor binding and post-receptor signaling by naturally occurring inhibitors. Some of these inhibitors are IL-1 family antagonists, while others are IL-1R family members acting as membrane-bound or soluble decoy receptors. An imbalance between agonist and antagonist levels can lead to exaggerated inflammatory responses. Several genetic modifications or mutations associated with dysregulated IL-1 activity and autoinflammatory disorders were identified in mouse models and in patients. These findings paved the road to the successful use of IL-1 inhibitors in diseases that were previously considered as untreatable.

IL-33-mediated protection against experimental cerebral malaria is linked to induction of type 2 innate lymphoid cells, M2 macrophages and regulatory T cells.

Cerebral malaria (CM) is a complex parasitic disease caused by Plasmodium sp. Failure to establish an appropriate balance between pro- and anti-inflammatory immune responses is believed to contribute to the development of cerebral pathology. Using the blood-stage PbA (Plasmodium berghei ANKA) model of infection, we show here that administration of the pro-Th2 cytokine, IL-33, prevents the development of experimental cerebral malaria (ECM) in C57BL/6 mice and reduces the production of inflammatory mediators IFN-γ, IL-12 and TNF-α. IL-33 drives the expansion of type-2 innate lymphoid cells (ILC2) that produce Type-2 cytokines (IL-4, IL-5 and IL-13), leading to the polarization of the anti-inflammatory M2 macrophages, which in turn expand Foxp3 regulatory T cells (Tregs). PbA-infected mice adoptively transferred with ILC2 have elevated frequency of M2 and Tregs and are protected from ECM. Importantly, IL-33-treated mice deleted of Tregs (DEREG mice) are no longer able to resist ECM. Our data therefore provide evidence that IL-33 can prevent the development of ECM by orchestrating a protective immune response via ILC2, M2 macrophages and Tregs.

Critical role of IL-33 receptor ST2 in experimental cerebral malaria development.

Cerebral malaria, a severe complication of Plasmodium falciparum infection, can be modeled in murine Plasmodium berghei ANKA (PbA) infection. PbA-induced experimental cerebral malaria (ECM) is CD8(+) T-cell mediated, and influenced by TH 1/TH 2 balance. Here, we show that IL-33 expression is increased in brain undergoing ECM and we address the role of the IL-33/ST2 pathway in ECM development. ST2-deficient mice were resistant to PbA-induced neuropathology. They survived >20 days with no ECM neurological sign and a preserved cerebral microcirculation, while WT mice succumbed within 10 days with ECM, brain vascular leakage, distinct microvascular pathology obstruction, and hemorrhages. Parasitemia and brain parasite load were similar in ST2-deficient and WT mice. Protection was accompanied by reduced brain sequestration of activated CD4(+) T cells and perforin(+) CD8(+) T cells. While IFN-γ and T-cell-attracting chemokines CXCL9 and CXCL10 were not affected in the absence of functional ST2 pathway, the local expression of ICAM-1, CXCR3, and LT-α, crucial for ECM development, was strongly reduced, and this may explain the diminished pathogenic T-cell recruitment and resistance to ECM. Therefore, IL-33 is induced in PbA sporozoite infection, and the pathogenic T-cell responses with local microvascular pathology are dependent on IL-33/ST2 signaling, identifying IL-33 as a new actor in ECM development.

Role of IL-1ß in experimental cystic fibrosis upon P. aeruginosa infection.

Cystic fibrosis is associated with increased inflammatory responses to pathogen challenge. Here we revisited the role of IL-1ß in lung pathology using the experimental F508del-CFTR murine model on C57BL/6 genetic background (Cftr(tm1eur) or d/d), on double deficient for d/d and type 1 interleukin-1 receptor (d/d X IL-1R1-/-), and antibody neutralization. At steady state, young adult d/d mice did not show any signs of spontaneous lung inflammation. However, IL-1R1 deficiency conferred partial protection to repeated P. aeruginosa endotoxins/LPS lung instillation in d/d mice, as 50% of d/d mice succumbed to inflammation, whereas all d/d x IL-1R1-/- double mutants survived with lower initial weight loss and less pulmonary collagen and mucus production, suggesting that the absence of IL-1R1 signaling is protective in d/d mice in LPS-induced lung damage. Using P. aeruginosa acute lung infection we found heightened neutrophil recruitment in d/d mice with higher epithelial damage, increased bacterial load in BALF, and augmented IL-1ß and TNF-α in parenchyma as compared to WT mice. Thus, F508del-CFTR mice show enhanced IL-1ß signaling in response to P. aeruginosa. IL-1ß antibody neutralization had no effect on lung homeostasis in either d/d or WT mice, however P. aeruginosa induced lung inflammation and bacterial load were diminished by IL-1ß antibody neutralization. In conclusion, enhanced susceptibility to P. aeruginosa in d/d mice correlates with an excessive inflammation and with increased IL-1ß production and reduced bacterial clearance. Further, we show that neutralization of IL-1ß in d/d mice through the double mutation d/d x IL-1R1-/- and in WT via antibody neutralization attenuates inflammation. This supports the notion that intervention in the IL-1R1/IL-1ß pathway may be detrimental in CF patients.

Genomic instability in newborn with short telomeres.

UNLABELLED: Telomere length is considered to be a risk factor in adults due to its proved association with cancer incidence and mortality. Since newborn present a wide interindividual variation in mean telomere length, it is relevant to demonstrate if these differences in length can act also as an early risk indicator. To answer this question, we have measured the mean telomere length of 74 samples of cord blood from newborns and studied its association with the basal genetic damage, measured as the frequency of binucleated cells carrying micronuclei. In addition, we have challenged the cells of a subgroup of individuals (N = 35) against mitomycin-C (MMC) to establish their sensitivity to induced genomic instability. Results indicate that newborn with shorter telomeres present significantly higher levels of genetic damage when compared to those with longer telomeres. In addition, the cellular response to MMC was also significantly higher among those samples from subjects with shorter telomeres. Independently of the causal mechanisms involved, our results show for the first time that telomere length at delivery influence both the basal and induced genetic damage of the individual. IMPACT: Individuals born with shorter telomeres may be at increased risk, especially for those biological processes triggered by genomic instability as is the case of cancer and other age-related diseases.

Type I interferons contribute to experimental cerebral malaria development in response to sporozoite or blood-stage Plasmodium berghei ANKA.

Cerebral malaria is a severe complication of Plasmodium falciparum infection. Although T-cell activation and type II IFN-γ are required for Plasmodium berghei ANKA (PbA)-induced murine experimental cerebral malaria (ECM), the role of type I IFN-α/ß in ECM development remains unclear. Here, we address the role of the IFN-α/ß pathway in ECM devel-opment in response to hepatic or blood-stage PbA infection, using mice deficient for types I or II IFN receptors. While IFN-γR1⁻/⁻ mice were fully resistant, IFNAR1⁻/⁻ mice showed delayed and partial protection to ECM after PbA infection. ECM resistance in IFN-γR1⁻/⁻ mice correlated with unaltered cerebral microcirculation and absence of ischemia, while WT and IFNAR1⁻/⁻ mice developed distinct microvascular pathologies. ECM resistance appeared to be independent of parasitemia. Instead, key mediators of ECM were attenuated in the absence of IFNAR1, including PbA-induced brain sequestration of CXCR3⁺-activated CD8⁺ T cells. This was associated with reduced expression of Granzyme B, IFN-γ, IL-12Rß2, and T-cell-attracting chemokines CXCL9 and CXCL10 in IFNAR1⁻/⁻ mice, more so in the absence of IFN-γR1. Therefore, the type I IFN-α/ß receptor pathway contributes to brain T-cell responses and microvascular pathology, although it is not as essential as IFN-γ for the development of cerebral malaria upon hepatic or blood-stage PbA infection.

IL-12Rß2 is essential for the development of experimental cerebral malaria.

A Th1 response is required for the development of Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (ECM). The role of pro-Th1 IL-12 in malaria is complex and controversial. In this study, we addressed the role of IL-12Rß2 in ECM development. C57BL/6 mice deficient for IL-12Rß2, IL-12p40, or IL-12p35 were analyzed for ECM development after blood-stage PbA infection in terms of ischemia and blood flow by noninvasive magnetic resonance imaging and angiography, T cell recruitment, and gene expression. Without IL-12Rß2, no neurologic sign of ECM developed upon PbA infection. Although wild-type mice developed distinct brain microvascular pathology, ECM-resistant, IL-12Rß2-deficient mice showed unaltered cerebral microcirculation and the absence of ischemia after PbA infection. In contrast, mice deficient for IL-12p40 or IL-12p35 were sensitive to ECM development. The resistance of IL-12Rß2-deficient mice to ECM correlated with reduced recruitment of activated T cells and impaired overexpression of lymphotoxin-α, TNF-α, and IFN-γ in the brain after PbA infection. Therefore, IL-12Rß2 signaling is essential for ECM development but independent from IL-12p40 and IL-12p35. We document a novel link between IL-12Rß2 and lymphotoxin-α, TNF-α, and IFN-γ expression, key cytokines for ECM pathogenesis.

Protein kinase C-theta is required for development of experimental cerebral malaria.

Cerebral malaria is the most severe neurologic complication in children and young adults infected with Plasmodium falciparum. T-cell activation is required for development of Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (CM). To characterize the T-cell activation pathway involved, the role of protein kinase C-theta (PKC-θ) in experimental CM development was examined. PKC-θ-deficient mice are resistant to CM development. In the absence of PKC-θ, no neurologic sign of CM developed after blood stage PbA infection. Resistance of PKC-θ-deficient mice correlated with unaltered cerebral microcirculation and absence of ischemia, as documented by magnetic resonance imaging and magnetic resonance angiography, whereas wild-type mice developed distinct microvascular pathology. Recruitment and activation of CD8(+) T cells, and ICAM-1 and CD69 expression were reduced in the brain of resistant mice; however, the pulmonary inflammation and edema associated with PbA infection were still present in the absence of functional PKC-θ. Resistant PKC-θ-deficient mice developed high parasitemia, and died at 3 weeks with severe anemia. Therefore, PKC-θ signaling is crucial for recruitment of CD8(+) T cells and development of brain microvascular pathology resulting in fatal experimental CM, and may represent a novel therapeutic target of CM.

Automated image analysis of cytokinesis-blocked micronuclei: an adapted protocol and a validated scoring procedure for biomonitoring.

Micronuclei (MN) frequencies in peripheral blood lymphocytes have been used worldwide as a biomarker of chromosomal damage for genotoxicity testing and biomonitoring studies. Automation of MN analysis would provide faster and more reliable results with minimizing subjective MN identification. We developed an automated facility for the scoring of the in vitro MN cytokinesis-block assay for biomonitoring on Giemsa-stained slides, fulfilling the following criteria: applicable to the cytokinesis-block micronucleus methodology, discriminating between mono-, bi- and polynucleated cells, MN scoring according to HUMN scoring criteria, false-negative MN rate <10% and false-positive (FP) MN rate <1%. We first adapted the slide preparation protocol to obtain an optimal cell density and dispersion, which is important for image analysis. We developed specific algorithms starting from the cell as a detection unit. The whole detection and scoring process was separated into two distinct steps: in the first step, the cells and nuclei are detected; then, in the second step, the MN are searched for in the detected cells. Since the rate of FP MN obtained by the automatic analysis was in the range of 0.5-1.5%, an interactive visual validation step was introduced, which is not time consuming and allows quality control. Validation of the automated scoring procedure was undertaken by comparing the results of visual and automated scoring of micronucleated mono- and binucleated cells in human lymphocytes induced by two clastogens (ionizing radiation and methyl methane-sulphonate), two aneugens (nocodazole and carbendazim) and one apoptogen (staurosporine). Although the absolute MN frequencies obtained with automated scoring were lower as compared to those detected by visual scoring, a clear dose response for MNBN frequencies was observed with the automated scoring system, indicating that it is able to produce biologically relevant and reliable results. These observations, together with its ability to detect cells, nuclei and MN in accordance with the HUMN scoring criteria, confirm the usability of the automated MN analysis system for biomonitoring.