Publications by authors named "Hana Janova"

12 Publications

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Enteric helminth coinfection enhances host susceptibility to neurotropic flaviviruses via a tuft cell-IL-4 receptor signaling axis.

Cell 2021 Mar 25;184(5):1214-1231.e16. Epub 2021 Feb 25.

Department of Medicine, Washington University School of Medicine, St. Louis, St. Louis, MO 63110, USA; Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, St. Louis, MO 63110, USA; Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, St. Louis, MO 63110, USA; The Andrew M. and Jane M. Bursky Center for Human Immunology and Immunotherapy Programs, Washington University School of Medicine, St. Louis, St. Louis, MO 63110, USA. Electronic address:

Although enteric helminth infections modulate immunity to mucosal pathogens, their effects on systemic microbes remain less established. Here, we observe increased mortality in mice coinfected with the enteric helminth Heligmosomoides polygyrus bakeri (Hpb) and West Nile virus (WNV). This enhanced susceptibility is associated with altered gut morphology and transit, translocation of commensal bacteria, impaired WNV-specific T cell responses, and increased virus infection in the gastrointestinal tract and central nervous system. These outcomes were due to type 2 immune skewing, because coinfection in Stat6 mice rescues mortality, treatment of helminth-free WNV-infected mice with interleukin (IL)-4 mirrors coinfection, and IL-4 receptor signaling in intestinal epithelial cells mediates the susceptibility phenotypes. Moreover, tuft cell-deficient mice show improved outcomes with coinfection, whereas treatment of helminth-free mice with tuft cell-derived cytokine IL-25 or ligand succinate worsens WNV disease. Thus, helminth activation of tuft cell-IL-4-receptor circuits in the gut exacerbates infection and disease of a neurotropic flavivirus.
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http://dx.doi.org/10.1016/j.cell.2021.01.051DOI Listing
March 2021

Detection of Synaptic Proteins in Microglia by Flow Cytometry.

Front Mol Neurosci 2020 29;13:149. Epub 2020 Sep 29.

Faculty of Biology, University of Freiburg, Freiburg, Germany.

A growing body of evidence indicates that microglia actively remove synapses , thereby playing a key role in synaptic refinement and modulation of brain connectivity. This phenomenon was mainly investigated in immunofluorescence staining and confocal microscopy. However, a quantification of synaptic material in microglia using these techniques is extremely time-consuming and labor-intensive. To address this issue, we aimed to quantify synaptic proteins in microglia using flow cytometry. With this approach, we first showed that microglia from the healthy adult mouse brain contain a detectable level of VGLUT1 protein. Next, we found more than two-fold increased VGLUT1 immunoreactivity in microglia from the developing brain (P15) as compared to adult microglia. These data indicate that microglia-mediated synaptic pruning mostly occurs during the brain developmental period. We then quantified the VGLUT1 staining in microglia in two transgenic models characterized by pathological microglia-mediated synaptic pruning. In the 5xFAD mouse model of Alzheimer's disease (AD) microglia exhibited a significant increase in VGLUT1 immunoreactivity before the onset of amyloid pathology. Moreover, conditional deletion of TDP-43 in microglia, which causes a hyper-phagocytic phenotype associated with synaptic loss, also resulted in increased VGLUT1 immunoreactivity within microglia. This work provides a quantitative assessment of synaptic proteins in microglia, under homeostasis, and in mouse models of disease.
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http://dx.doi.org/10.3389/fnmol.2020.00149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7550663PMC
September 2020

Long-Term Culture Captures Injury-Repair Cycles of Colonic Stem Cells.

Cell 2019 11 7;179(5):1144-1159.e15. Epub 2019 Nov 7.

Department of Pathology and Immunology, Washington University School of Medicine, Saint Louis, MO 63110, USA. Electronic address:

The colonic epithelium can undergo multiple rounds of damage and repair, often in response to excessive inflammation. The responsive stem cell that mediates this process is unclear, in part because of a lack of in vitro models that recapitulate key epithelial changes that occur in vivo during damage and repair. Here, we identify a Hopx colitis-associated regenerative stem cell (CARSC) population that functionally contributes to mucosal repair in mouse models of colitis. Hopx CARSCs, enriched for fetal-like markers, transiently arose from hypertrophic crypts known to facilitate regeneration. Importantly, we established a long-term, self-organizing two-dimensional (2D) epithelial monolayer system to model the regenerative properties and responses of Hopx CARSCs. This system can reenact the "homeostasis-injury-regeneration" cycles of epithelial alterations that occur in vivo. Using this system, we found that hypoxia and endoplasmic reticulum stress, insults commonly present in inflammatory bowel diseases, mediated the cyclic switch of cellular status in this process.
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http://dx.doi.org/10.1016/j.cell.2019.10.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6904908PMC
November 2019

Genetically induced brain inflammation by deletion transiently benefits from microglia depletion.

FASEB J 2019 07 15;33(7):8634-8647. Epub 2019 May 15.

Clinical Neuroscience, Max Planck Institute of Experimental Medicine, Göttingen, Germany.

Reduced expression of 2'-3'-cyclic nucleotide 3'-phosphodiesterase () in humans and mice causes white matter inflammation and catatonic signs. These consequences are experimentally alleviated by microglia ablation colony-stimulating factor 1 receptor (CSF1R) inhibition using PLX5622. Here we address for the first time preclinical topics crucial for translation, most importantly ) the comparison of 2 long-term PLX5622 applications (prevention and treatment) 1 treatment alone, ) the correlation of catatonic signs and executive dysfunction, ) the phenotype of leftover microglia evading depletion, and ) the role of intercellular interactions for efficient CSF1R inhibition. Based on our mouse model and time-lapse imaging, we report the unexpected discovery that microglia surviving under PLX5622 display a highly inflammatory phenotype including aggressive premortal phagocytosis of oligodendrocyte precursor cells. Interestingly, ablating microglia requires mixed glial cultures, whereas cultured pure microglia withstand PLX5622 application. Importantly, 2 extended rounds of CSF1R inhibition are not superior to 1 treatment regarding any readout investigated (magnetic resonance imaging and magnetic resonance spectroscopy, behavior, immunohistochemistry). Catatonia-related executive dysfunction and brain atrophy of mice fail to improve under PLX5622. To conclude, even though microglia depletion is temporarily beneficial and worth pursuing, complementary treatment strategies are needed for full and lasting recovery.-Fernandez Garcia-Agudo, L., Janova, H., Sendler, L. E., Arinrad, S., Steixner, A. A., Hassouna, I., Balmuth, E., Ronnenberg, A., Schopf, N., van der Flier, F. J., Begemann, M., Martens, H., Weber, M. S., Boretius, S., Nave, K.-A., Ehrenreich, H. Genetically induced brain inflammation by deletion transiently benefits from microglia depletion.
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http://dx.doi.org/10.1096/fj.201900337RDOI Listing
July 2019

Astrocytic glutamine synthetase is expressed in the neuronal somatic layers and down-regulated proportionally to neuronal loss in the human epileptic hippocampus.

Glia 2018 05 19;66(5):920-933. Epub 2018 Jan 19.

Institute of Physiology and Pathophysiology, University of Heidelberg, Im Neuenheimer Feld 326, Heidelberg, D-69120, Germany.

Human mesial temporal lobe epilepsy (MTLE) features subregion-specific hippocampal neurodegeneration and reactive astrogliosis, including up-regulation of the glial fibrillary acidic protein (GFAP) and down-regulation of glutamine synthetase (GS). However, the regional astrocytic expression pattern of GFAP and GS upon MTLE-associated neurodegeneration still remains elusive. We assessed GFAP and GS expression in strict correlation with the local neuronal number in cortical and hippocampal surgical specimens from 16 MTLE patients using immunohistochemistry, stereology and high-resolution image analysis for digital pathology and whole-slide imaging. In the cortex, GS-positive (GS+) astrocytes are dominant in all neuronal layers, with a neuron to GS+ cell ratio of 2:1. GFAP-positive (GFAP+) cells are widely spaced, with a GS+ to GFAP+ cell ratio of 3:1-5:1. White matter astrocytes, on the contrary, express mainly GFAP and, to a lesser extent, GS. In the hippocampus, the neuron to GS+ cell ratio is approximately 1:1. Hippocampal degeneration is associated with a reduction of GS+ astrocytes, which is proportional to the degree of neuronal loss and primarily present in the hilus. Up-regulation of GFAP as a classical hallmark of reactive astrogliosis does not follow the GS-pattern and is prominent in the CA1. Reactive alterations were proportional to the neuronal loss in the neuronal somatic layers (stratum pyramidale and hilus), while observed to a lesser extent in the axonal/dendritic layers (stratum radiatum, molecular layer). We conclude that astrocytic GS is expressed in the neuronal somatic layers and, upon neurodegeneration, is down-regulated proportionally to the degree of neuronal loss.
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http://dx.doi.org/10.1002/glia.23292DOI Listing
May 2018

Microglia ablation alleviates myelin-associated catatonic signs in mice.

J Clin Invest 2018 02 18;128(2):734-745. Epub 2017 Dec 18.

DFG Research Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Göttingen, Germany.

The underlying cellular mechanisms of catatonia, an executive "psychomotor" syndrome that is observed across neuropsychiatric diseases, have remained obscure. In humans and mice, reduced expression of the structural myelin protein CNP is associated with catatonic signs in an age-dependent manner, pointing to the involvement of myelin-producing oligodendrocytes. Here, we showed that the underlying cause of catatonic signs is the low-grade inflammation of white matter tracts, which marks a final common pathway in Cnp-deficient and other mutant mice with minor myelin abnormalities. The inhibitor of CSF1 receptor kinase signaling PLX5622 depleted microglia and alleviated the catatonic symptoms of Cnp mutants. Thus, microglia and low-grade inflammation of myelinated tracts emerged as the trigger of a previously unexplained mental condition. We observed a very high (25%) prevalence of individuals with catatonic signs in a deeply phenotyped schizophrenia sample (n = 1095). Additionally, we found the loss-of-function allele of a myelin-specific gene (CNP rs2070106-AA) associated with catatonia in 2 independent schizophrenia cohorts and also associated with white matter hyperintensities in a general population sample. Since the catatonic syndrome is likely a surrogate marker for other executive function defects, we suggest that microglia-directed therapies may be considered in psychiatric disorders associated with myelin abnormalities.
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http://dx.doi.org/10.1172/JCI97032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5785265PMC
February 2018

A presumed antagonistic LPS identifies distinct functional organization of TLR4 in mouse microglia.

Glia 2017 07 4;65(7):1176-1185. Epub 2017 May 4.

Institute of Neuropathology, University Medical Center Göttingen, Göttingen, 37075, Germany.

Microglia as principle innate immune cells of the central nervous system (CNS) are the first line of defense against invading pathogens. They are capable of sensing infections through diverse receptors, such as Toll-like receptor 4 (TLR4). This receptor is best known for its ability to recognize bacterial lipopolysaccharide (LPS), a causative agent of gram-negative sepsis and septic shock. A putative, naturally occurring antagonist of TLR4 derives from the photosynthetic bacterium Rhodobacter sphaeroides. However, the antagonistic potential of R. sphaeroides LPS (Rs-LPS) is no universal feature, since several studies suggested agonistic rather than antagonistic actions of this molecule depending on the investigated mammalian species. Here we show the agonistic versus antagonistic potential of Rs-LPS in primary mouse microglia. We demonstrate that Rs-LPS efficiently induces the release of cytokines and chemokines, which depends on TLR4, MyD88, and TRIF, but not CD14. Furthermore, Rs-LPS is able to regulate the phagocytic capacity of microglia as agonist, while it antagonizes Re-LPS-induced MHC I expression. Finally, to our knowledge, we are the first to provide in vivo evidence for an agonistic potential of Rs-LPS, as it efficiently triggers the recruitment of peripheral immune cells to the endotoxin-challenged CNS. Together, our results argue for a versatile and complex organization of the microglial TLR4 system, which specifically translates exogenous signals into cellular functions. Importantly, as demonstrated here for microglia, the antagonistic potential of Rs-LPS needs to be considered with caution, as reactions to Rs-LPS not only differ by cell type, but even by function within one cell type.
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http://dx.doi.org/10.1002/glia.23151DOI Listing
July 2017

CD14 is a key organizer of microglial responses to CNS infection and injury.

Glia 2016 Apr 18;64(4):635-49. Epub 2015 Dec 18.

Institute of Neuropathology, University of Göttingen, Göttingen, 37075, Germany.

Microglia, innate immune cells of the CNS, sense infection and damage through overlapping receptor sets. Toll-like receptor (TLR) 4 recognizes bacterial lipopolysaccharide (LPS) and multiple injury-associated factors. We show that its co-receptor CD14 serves three non-redundant functions in microglia. First, it confers an up to 100-fold higher LPS sensitivity compared to peripheral macrophages to enable efficient proinflammatory cytokine induction. Second, CD14 prevents excessive responses to massive LPS challenges via an interferon β-mediated feedback. Third, CD14 is mandatory for microglial reactions to tissue damage-associated signals. In mice, these functions are essential for balanced CNS responses to bacterial infection, traumatic and ischemic injuries, since CD14 deficiency causes either hypo- or hyperinflammation, insufficient or exaggerated immune cell recruitment or worsened stroke outcomes. While CD14 orchestrates functions of TLR4 and related immune receptors, it is itself regulated by TLR and non-TLR systems to thereby fine-tune microglial damage-sensing capacity upon infectious and non-infectious CNS challenges.
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http://dx.doi.org/10.1002/glia.22955DOI Listing
April 2016

Tyrphostin AG126 exerts neuroprotection in CNS inflammation by a dual mechanism.

Glia 2015 Jun 2;63(6):1083-99. Epub 2015 Mar 2.

Institute of Neuropathology, University of Göttingen, Germany.

The putative protein tyrosine kinase (PTK) inhibitor tyrphostin AG126 has proven beneficial in various models of inflammatory disease. Yet molecular targets and cellular mechanisms remained enigmatic. We demonstrate here that AG126 treatment has beneficial effects in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. AG126 alleviates the clinical symptoms, diminishes encephalitogenic Th17 differentiation, reduces inflammatory CNS infiltration as well as microglia activation and attenuates myelin damage. We show that AG126 directly inhibits Bruton's tyrosine kinase (BTK), a PTK associated with B cell receptor and Toll-like receptor (TLR) signaling. However, BTK inhibition cannot account for the entire activity spectrum. Effects on TLR-induced proinflammatory cytokine expression in microglia involve AG126 hydrolysis and conversion of its dinitrile side chain to malononitrile (MN). Notably, while liberated MN can subsequently mediate critical AG126 features, full protection in EAE still requires delivery of intact AG126. Its anti-inflammatory potential and especially interference with TLR signaling thus rely on a dual mechanism encompassing BTK and a novel MN-sensitive target. Both principles bear great potential for the therapeutic management of disturbed innate and adaptive immune functions.
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http://dx.doi.org/10.1002/glia.22803DOI Listing
June 2015

Bordetella adenylate cyclase toxin differentially modulates toll-like receptor-stimulated activation, migration and T cell stimulatory capacity of dendritic cells.

PLoS One 2014 1;9(8):e104064. Epub 2014 Aug 1.

Laboratory of Molecular Biology of Bacterial Pathogens, Institute of Microbiology of the Academy of Sciences of the Czech Republic, Prague, Czech Republic.

Adenylate cyclase toxin (CyaA) is a key virulence factor of the whooping cough agent Bordetella pertussis. The toxin targets CD11b-expressing phagocytes and delivers into their cytosol an adenylyl cyclase (AC) enzyme that subverts cellular signaling by increasing cAMP levels. In the present study, we analyzed the modulatory effects of CyaA on adhesive, migratory and antigen presenting properties of Toll-like receptor (TLR)-activated murine and human dendritic cells (DCs). cAMP signaling of CyaA enhanced TLR-induced dissolution of cell adhesive contacts and migration of DCs towards the lymph node-homing chemokines CCL19 and CCL21 in vitro. Moreover, we examined in detail the capacity of toxin-treated DCs to induce CD4(+) and CD8(+) T cell responses. Exposure to CyaA decreased the capacity of LPS-stimulated DCs to present soluble protein antigen to CD4+ T cells independently of modulation of co-stimulatory molecules and cytokine production, and enhanced their capacity to promote CD4(+)CD25(+)Foxp3(+) T regulatory cells in vitro. In addition, CyaA decreased the capacity of LPS-stimulated DCs to induce CD8(+) T cell proliferation and limited the induction of IFN-γ producing CD8(+) T cells while enhancing IL-10 and IL-17-production. These results indicate that through activation of cAMP signaling, the CyaA may be mobilizing DCs impaired in T cell stimulatory capacity and arrival of such DCs into draining lymph nodes may than contribute to delay and subversion of host immune responses during B. pertussis infection.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0104064PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4118975PMC
November 2015

Intraperitoneal prophylaxis with CpG oligodeoxynucleotides protects neutropenic mice against intracerebral Escherichia coli K1 infection.

J Neuroinflammation 2014 Jan 23;11:14. Epub 2014 Jan 23.

Institute of Neuropathology, University Medical Center Göttingen, Göttingen, Germany.

Background: Prophylaxis with unmethylated cytosine phosphate guanidine (CpG) oligodeoxynucleotides (ODN) protects against several systemic experimental infections. Escherichia coli is a major cause of Gram-negative neonatal bacterial meningitis and also causes meningitis and meningoencephalitis in older and immunocompromised patients.

Methods: Wild-type (wt) and Toll-like receptor 9 (TLR9)-deficient mice were rendered neutropenic by intraperitoneal administration of the anti-Ly-6G monoclonal antibody. Immunocompetent and neutropenic mice received intraperitoneal CpG ODN or vehicle 72 h prior to induction of E. coli K1 meningoencephalitis.

Results: Pre-treatment with CpG ODN significantly increased survival of neutropenic wt mice from 33% to 75% (P = 0.0003) but did not protect neutropenic TLR9-/- mice. The protective effect of CpG ODN was associated with an enhanced production of interleukin (IL)-12/IL-23p40 with sustained increased levels in serum and spleen at least for 17 days after conditioning compared to buffer-treated animals. CpG-treated neutropenic wt mice showed reduced bacterial concentrations and increased recruitment of Ly6ChighCCR2+ monocytes in brain and spleen 42 h after infection. The levels of macrophage inflammatory protein 1α (MIP-1α) and interferon gamma (IFN-γ) in spleen were higher 42 h after infection in CpG-treated compared to buffer-treated neutropenic animals. In immunocompetent mice, prophylaxis with CpG ODN did not significantly increase survival compared to the buffer group (60% vs. 45%, P = 0.2).

Conclusions: These findings suggest that systemic administration of CpG ODN may help to prevent bacterial CNS infections in immunocompromised individuals.
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http://dx.doi.org/10.1186/1742-2094-11-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3906862PMC
January 2014

The nucleotide-binding oligomerization domain-containing-2 ligand muramyl dipeptide enhances phagocytosis and intracellular killing of Escherichia coli K1 by Toll-like receptor agonists in microglial cells.

J Neuroimmunol 2012 Nov 11;252(1-2):16-23. Epub 2012 Aug 11.

Institute of Neuropathology, University Medical Center Göttingen, Göttingen, Germany.

Increasing the phagocytic activity of microglia could improve the resistance of immunocompromised patients to CNS infections. We studied the microglial responses upon stimulation with the Nod2 ligand muramyl dipeptide (MDP) alone or in combination with a TLR1/2, 3 or 4 agonist. MDP caused a mild release of NO, but induced neither a significant release of pro-inflammatory cytokines nor an expression of molecules associated with professional antigen presentation. Using the Escherichia coli K1 model, microglial pre-stimulation with MDP enhanced bacterial phagocytosis which was strengthened on TLR-pre-stimulated cells. Dual pre-stimulation of Nod2 and TLR1/2 or 4 caused maximal phagocytosis and intracellular killing.
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http://dx.doi.org/10.1016/j.jneuroim.2012.07.012DOI Listing
November 2012