Differential Gene Expression of SARS-CoV-2 Positive Bronchoalveolar Lavages: A Case Series.

BACKGROUND: Transcriptomic data on bronchoalveolar lavage (BAL) from COVID-19 patients are currently scarce. OBJECTIVES: This case series seeks to characterize the intra-alveolar immunopathology of COVID-19. METHOD: BALs were performed on 14 patients (5 COVID-19, of which 3 mild and 2 largely asymptomatic, 9 controls). Controls included asthma (n = 1), unremarkable BALs (n = 3), infections with respiratory syncytial virus (n = 1), influenza B (n = 1), and infections with other coronaviruses (n = 3). SARS-CoV-2 RNA load was measured by quantitative nucleic acid testing, while the detection of other pathogens was performed by immunofluorescence or multiplex NAT. RESULTS: Gene expression profiling showed 71 significantly downregulated and 5 upregulated transcripts in SARS-CoV-2-positive lavages versus controls. Downregulated transcripts included genes involved in macrophage development, polarization, and crosstalk (LGALS3, MARCO, ERG2, BTK, RAC1, CD83), and genes involved in chemokine signaling and immunometabolism (NUPR1, CEBPB, CEBPA, PECAM1, CCL18, PPARG, ALOX5, ALOX5AP). Upregulated transcripts featured genes involved in NK-T cell signaling (GZMA, GZMH, GNLY, PRF1, CD3G). Patients with mild COVID-19 showed a significant upregulation of genes involved in blood mononuclear cell/leukocyte function (G0S2, ANXA6, FCGR2B, ADORA3), coagulation (von Willebrand factor [VWF]), interferon response (IFRD1, IL12RB2), and a zinc metalloprotease elevated in asthma (CPA3) compared to asymptomatic cases. In-silico comparison of the 5 COVID-19 BAL cases to a published cohort of lethal COVID-19 showed a significant upregulation of "antigen processing and presentation" and "lysosome" pathways in lethal cases. CONCLUSIONS: These data underscore the heterogeneity of immune response in COVID-19. Further studies with a larger dataset are required to gain a better understanding of the hallmarks of SARS-CoV-2 immunological response.

Von Willebrand factor and the thrombophilia of severe COVID-19: in situ evidence from autopsies.

Background: COVID-19 is accompanied by a hypercoagulable state and characterized by microvascular and macrovascular thrombotic complications. In plasma samples from patients with COVID-19, von Willebrand factor (VWF) levels are highly elevated and predictive of adverse outcomes, especially mortality. Yet, VWF is usually not included in routine coagulation analyses, and histologic evidence of its involvement in thrombus formation is lacking. Objectives: To determine whether VWF, an acute-phase protein, is a bystander, ie, a biomarker of endothelial dysfunction, or a causal factor in the pathogenesis of COVID-19. Methods: We compared autopsy samples from 28 patients with lethal COVID-19 to those from matched controls and systematically assessed for VWF and platelets by immunohistochemistry. The control group comprised 24 lungs, 23 lymph nodes, and 9 hearts and did not differ significantly from the COVID-19 group in age, sex, body mass index (BMI), blood group, or anticoagulant use. Results: In lungs, assessed for platelets by immunohistochemistry for CD42b, microthrombi were more frequent in patients with COVID-19 (10/28 [36%] vs 2/24 [8%]; P = .02). A completely normal pattern of VWF was rare in both groups. Accentuated endothelial staining was found in controls, while VWF-rich thrombi were only found in patients with COVID-19 (11/28 [39%] vs 0/24 [0%], respectively; P < .01), as were NETosis thrombi enriched with VWF (7/28 [25%] vs 0/24 [0%], respectively; P < .01). Forty-six percent of the patients with COVID-19 had VWF-rich thrombi, NETosis thrombi, or both. Trends were also seen in pulmonary draining lymph nodes (7/20 [35%] vs 4/24 [17%]; P = .147), where the overall presence of VWF was very high. Conclusion: We provide in situ evidence of VWF-rich thrombi, likely attributable to COVID-19, and suggest that VWF may be a therapeutic target in severe COVID-19.

Morphologic and molecular analysis of liver injury after SARS-CoV-2 vaccination reveals distinct characteristics.

BACKGROUND & AIMS: Liver injury after COVID-19 vaccination is very rare and shows clinical and histomorphological similarities with autoimmune hepatitis (AIH). Little is known about the pathophysiology of COVID-19 vaccine-induced liver injury (VILI) and its relationship to AIH. Therefore, we compared VILI with AIH. METHODS: Formalin-fixed and paraffin-embedded liver biopsy samples from patients with VILI (n = 6) and from patients with an initial diagnosis of AIH (n = 9) were included. Both cohorts were compared by histomorphological evaluation, whole-transcriptome and spatial transcriptome sequencing, multiplex immunofluorescence, and immune repertoire sequencing. RESULTS: Histomorphology was similar in both cohorts but showed more pronounced centrilobular necrosis in VILI. Gene expression profiling showed that mitochondrial metabolism and oxidative stress-related pathways were more and interferon response pathways were less enriched in VILI. Multiplex analysis revealed that inflammation in VILI was dominated by CD8+ effector T cells, similar to drug-induced autoimmune-like hepatitis. In contrast, AIH showed a dominance of CD4+ effector T cells and CD79a+ B and plasma cells. T-cell receptor (TCR) and B-cell receptor sequencing showed that T and B cell clones were more dominant in VILI than in AIH. In addition, many T cell clones detected in the liver were also found in the blood. Interestingly, analysis of TCR beta chain and Ig heavy chain variable-joining gene usage further showed that TRBV6-1, TRBV5-1, TRBV7-6, and IgHV1-24 genes are used differently in VILI than in AIH. CONCLUSIONS: Our analyses support that SARS-CoV-2 VILI is related to AIH but also shows distinct differences from AIH in histomorphology, pathway activation, cellular immune infiltrates, and TCR usage. Therefore, VILI may be a separate entity, which is distinct from AIH and more closely related to drug-induced autoimmune-like hepatitis. IMPACT AND IMPLICATIONS: Little is known about the pathophysiology of COVID-19 vaccine-induced liver injury (VILI). Our analysis shows that COVID-19 VILI shares some similarities with autoimmune hepatitis, but also has distinct differences such as increased activation of metabolic pathways, a more prominent CD8+ T cell infiltrate, and an oligoclonal T and B cell response. Our findings suggest that VILI is a distinct disease entity. Therefore, there is a good chance that many patients with COVID-19 VILI will recover completely and will not develop long-term autoimmune hepatitis.

The pulmonary vasculature in lethal COVID-19 and idiopathic pulmonary fibrosis at single-cell resolution.

AIMS: Severe acute respiratory syndrome coronavirus-2 infection causes COVID-19, which in severe cases evokes life-threatening acute respiratory distress syndrome (ARDS). Transcriptome signatures and the functional relevance of non-vascular cell types (e.g. immune and epithelial cells) in COVID-19 are becoming increasingly evident. However, despite its known contribution to vascular inflammation, recruitment/invasion of immune cells, vascular leakage, and perturbed haemostasis in the lungs of severe COVID-19 patients, an in-depth interrogation of the endothelial cell (EC) compartment in lethal COVID-19 is lacking. Moreover, progressive fibrotic lung disease represents one of the complications of COVID-19 pneumonia and ARDS. Analogous features between idiopathic pulmonary fibrosis (IPF) and COVID-19 suggest partial similarities in their pathophysiology, yet, a head-to-head comparison of pulmonary cell transcriptomes between both conditions has not been implemented to date. METHODS AND RESULTS: We performed single-nucleus RNA-sequencing on frozen lungs from 7 deceased COVID-19 patients, 6 IPF explant lungs, and 12 controls. The vascular fraction, comprising 38 794 nuclei, could be subclustered into 14 distinct EC subtypes. Non-vascular cell types, comprising 137 746 nuclei, were subclustered and used for EC-interactome analyses. Pulmonary ECs of deceased COVID-19 patients showed an enrichment of genes involved in cellular stress, as well as signatures suggestive of dampened immunomodulation and impaired vessel wall integrity. In addition, increased abundance of a population of systemic capillary and venous ECs was identified in COVID-19 and IPF. COVID-19 systemic ECs closely resembled their IPF counterparts, and a set of 30 genes was found congruently enriched in systemic ECs across studies. Receptor-ligand interaction analysis of ECs with non-vascular cell types in the pulmonary micro-environment revealed numerous previously unknown interactions specifically enriched/depleted in COVID-19 and/or IPF. CONCLUSIONS: This study uncovered novel insights into the abundance, expression patterns, and interactomes of EC subtypes in COVID-19 and IPF, relevant for future investigations into the progression and treatment of both lethal conditions.

Vascular Damage, Thromboinflammation, Plasmablast Activation, T-Cell Dysregulation and Pathological Histiocytic Response in Pulmonary Draining Lymph Nodes of COVID-19.

Although initial immunophenotypical studies on peripheral blood and bronchoalveolar lavage samples have provided a glimpse into the immunopathology of COVID-19, analyses of pulmonary draining lymph nodes are currently scarce. 22 lethal COVID-19 cases and 28 controls were enrolled in this study. Pulmonary draining lymph nodes (mediastinal, tracheal, peribronchial) were collected at autopsy. Control lymph nodes were selected from a range of histomorphological sequelae [unremarkable histology, infectious mononucleosis, follicular hyperplasia, non-SARS related HLH, extrafollicular plasmablast activation, non-SARS related diffuse alveolar damage (DAD), pneumonia]. Samples were mounted on a tissue microarray and underwent immunohistochemical staining for a selection of immunological markers and in-situ hybridization for Epstein Barr Virus (EBV) and SARS-CoV-2. Gene expression profiling was performed using the HTG EdgeSeq Immune Response Panel. Characteristic patterns of a dysregulated immune response were detected in COVID-19: 1. An accumulation of extrafollicular plasmablasts with a relative paucity or depletion of germinal centers. 2. Evidence of T-cell dysregulation demonstrated by immunohistochemical paucity of FOXP3+, Tbet+ and LEF1+ positive T-cells and a downregulation of key genes responsible for T-cell crosstalk, maturation and migration as well as a reactivation of herpes viruses in 6 COVID-19 lymph nodes (EBV, HSV). 3. Macrophage activation by a M2-polarized, CD163+ phenotype and increased incidence of hemophagocytic activity. 4. Microvascular dysfunction, evidenced by an upregulation of hemostatic (CD36, PROCR, VWF) and proangiogenic (FLT1, TEK) genes and an increase of fibrin microthrombi and CD105+ microvessels. Taken together, these findings imply widespread dysregulation of both innate and adoptive pathways with concordant microvascular dysfunction in severe COVID-19.

Determinants of SARS-CoV-2 entry and replication in airway mucosal tissue and susceptibility in smokers.

Understanding viral tropism is an essential step toward reducing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission, decreasing mortality from coronavirus disease 2019 (COVID-19) and limiting opportunities for mutant strains to arise. Currently, little is known about the extent to which distinct tissue sites in the human head and neck region and proximal respiratory tract selectively permit SARS-CoV-2 infection and replication. In this translational study, we discover key variabilities in expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), essential SARS-CoV-2 entry factors, among the mucosal tissues of the human proximal airways. We show that SARS-CoV-2 infection is present in all examined head and neck tissues, with a notable tropism for the nasal cavity and tracheal mucosa. Finally, we uncover an association between smoking and higher SARS-CoV-2 viral infection in the human proximal airway, which may explain the increased susceptibility of smokers to developing severe COVID-19. This is at least partially explained by differences in interferon (IFN)-ß1 levels between smokers and non-smokers.

SARS-CoV-2 infects human pancreatic ß cells and elicits ß cell impairment.

Emerging evidence points toward an intricate relationship between the pandemic of coronavirus disease 2019 (COVID-19) and diabetes. While preexisting diabetes is associated with severe COVID-19, it is unclear whether COVID-19 severity is a cause or consequence of diabetes. To mechanistically link COVID-19 to diabetes, we tested whether insulin-producing pancreatic ß cells can be infected by SARS-CoV-2 and cause ß cell depletion. We found that the SARS-CoV-2 receptor, ACE2, and related entry factors (TMPRSS2, NRP1, and TRFC) are expressed in ß cells, with selectively high expression of NRP1. We discovered that SARS-CoV-2 infects human pancreatic ß cells in patients who succumbed to COVID-19 and selectively infects human islet ß cells in vitro. We demonstrated that SARS-CoV-2 infection attenuates pancreatic insulin levels and secretion and induces ß cell apoptosis, each rescued by NRP1 inhibition. Phosphoproteomic pathway analysis of infected islets indicates apoptotic ß cell signaling, similar to that observed in type 1 diabetes (T1D). In summary, our study shows SARS-CoV-2 can directly induce ß cell killing.

Histomorphological patterns of regional lymph nodes in COVID-19 lungs.

BACKGROUND: A dysregulated immune response is considered one of the major factors leading to severe COVID-19. Previously described mechanisms include the development of a cytokine storm, missing immunoglobulin class switch, antibody-mediated enhancement, and aberrant antigen presentation. OBJECTIVES: To understand the heterogeneity of immune response in COVID-19, a thorough investigation of histomorphological patterns in regional lymph nodes was performed. MATERIALS AND METHODS: Lymph nodes from the cervical, mediastinal, and hilar regions were extracted from autopsies of patients with lethal COVID-19 (n = 20). Histomorphological characteristics, SARS-CoV­2 qRT-PCR, and gene expression profiling on common genes involved in immunologic response were analyzed. RESULTS: Lymph nodes displayed moderate to severe capillary stasis and edema, an increased presence of extrafollicular plasmablasts, mild to moderate plasmacytosis, a dominant population of CD8+ T­cells, and CD11c/CD68+ histiocytosis with hemophagocytic activity. Out of 20 cases, 18 presented with hypoplastic or missing germinal centers with a decrease of follicular dendritic cells and follicular T­helper cells. A positive viral load was detected by qRT-PCR in 14 of 20 cases, yet immunohistochemistry for SARS-CoV-2 N-antigen revealed positivity in sinus histiocytes of only one case. Gene expression analysis revealed an increased expression of STAT1, CD163, granzyme B, CD8A, MZB1, and PAK1, as well as CXCL9. CONCLUSIONS: Taken together, our findings imply a dysregulated immune response in lethal COVID-19. The absence/hypoplasia of germinal centers and increased presence of plasmablasts implies a transient B­cell response, implying an impaired development of long-term immunity against SARS-CoV­2 in such occasions.

Characterisation of cardiac pathology in 23 autopsies of lethal COVID-19.

While coronavirus disease 2019 (COVID-19) primarily affects the respiratory tract, pathophysiological changes of the cardiovascular system remain to be elucidated. We performed a retrospective cardiopathological analysis of the heart and vasculature from 23 autopsies of COVID-19 patients, comparing the findings with control tissue. Myocardium from autopsies of COVID-19 patients was categorised into severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positive (n = 14) or negative (n = 9) based on the presence of viral RNA as determined by reverse transcriptase polymerase chain reaction (RT-PCR). Control tissue was selected from autopsies without COVID-19 (n = 10) with similar clinical sequelae. Histological characteristics were scored by ordinal and/or categorical grading. Five RT-PCR-positive cases underwent in situ hybridisation (ISH) for SARS-CoV-2. Patients with lethal COVID-19 infection were mostly male (78%) and had a high incidence of hypertension (91%), coronary artery disease (61%), and diabetes mellitus (48%). Patients with positive myocardial RT-PCR died earlier after hospital admission (5 versus 12 days, p < 0.001) than patients with negative RT-PCR. An increased severity of fibrin deposition, capillary dilatation, and microhaemorrhage was observed in RT-PCR-positive myocardium than in negatives and controls, with a positive correlation amongst these factors All cases with increased cardioinflammatory infiltrate, without myocyte necrosis (n = 4) or with myocarditis (n = 1), were RT-PCR negative. ISH revealed positivity of viral RNA in interstitial cells. Myocardial capillary dilatation, fibrin deposition, and microhaemorrhage may be the histomorphological correlate of COVID-19-associated coagulopathy. Increased cardioinflammation including one case of myocarditis was only detected in RT-PCR-negative hearts with significantly longer hospitalisation time. This may imply a secondary immunological response warranting further characterisation.

[Histomorphological patterns of regional lymph nodes in COVID-19 lungs]. / Reaktionsmuster der lokoregionären Lymphknoten im Abflussgebiet von COVID-19-Lungen.

BACKGROUND: A dysregulated immune response is considered one of the major factors leading to severe COVID-19. Previously described mechanisms include the development of a cytokine storm, missing immunoglobulin class switch, antibody-mediated enhancement, and aberrant antigen presentation. OBJECTIVES: To understand the heterogeneity of immune response in COVID-19, a thorough investigation of histomorphological patterns in regional lymph nodes was performed. MATERIALS AND METHODS: Lymph nodes from the cervical, mediastinal, and hilar regions were extracted from autopsies of patients with lethal COVID-19 (n = 20). Histomorphological characteristics, SARS-CoV­2 qRT-PCR, and gene expression profiling on common genes involved in immunologic response were analyzed. RESULTS: Lymph nodes displayed moderate to severe capillary stasis and edema, an increased presence of extrafollicular plasmablasts, mild to moderate plasmacytosis, a dominant population of CD8+ T­cells, and CD11c/CD68+ histiocytosis with hemophagocytic activity. Out of 20 cases, 18 presented with hypoplastic or missing germinal centers with a decrease of follicular dendritic cells and follicular T­helper cells. A positive viral load was detected by qRT-PCR in 14 of 20 cases, yet immunohistochemistry for SARS-CoV-2 N-antigen revealed positivity in sinus histiocytes of only one case. Gene expression analysis revealed an increased expression of STAT1, CD163, granzyme B, CD8A, MZB1, and PAK1, as well as CXCL9. CONCLUSIONS: Taken together, our findings imply a dysregulated immune response in lethal COVID-19. The absence/hypoplasia of germinal centers and increased presence of plasmablasts implies a transient B­cell response, implying an impaired development of long-term immunity against SARS-CoV­2 in such occasions.

Orbit Image Analysis: An open-source whole slide image analysis tool.

We describe Orbit Image Analysis, an open-source whole slide image analysis tool. The tool consists of a generic tile-processing engine which allows the execution of various image analysis algorithms provided by either Orbit itself or from other open-source platforms using a tile-based map-reduce execution framework. Orbit Image Analysis is capable of sophisticated whole slide imaging analyses due to several key features. First, Orbit has machine-learning capabilities. This deep learning segmentation can be integrated with complex object detection for analysis of intricate tissues. In addition, Orbit can run locally as standalone or connect to the open-source image server OMERO. Another important characteristic is its scale-out functionality, using the Apache Spark framework for distributed computing. In this paper, we describe the use of Orbit in three different real-world applications: quantification of idiopathic lung fibrosis, nerve fibre density quantification, and glomeruli detection in the kidney.

Eosinophilic oesophagitis: relevance of mast cell infiltration.

AIMS: Eosinophilic oesophagitis (EoE) is a chronic inflammatory disease characterised clinically by symptoms of oesophageal dysfunction and histopathologically by a prominent eosinophilic inflammation. Despite eosinophils having a histologically predominant position, their role in the immunopathogenesis of the disease is still questionable. Several other inflammatory cells are involved and may also play a critical role. The purpose of this study was to characterise the mast cell infiltration, and to correlate it with the clinical state of EoE. METHODS AND RESULTS: Using immunohistochemistry and quantitative morphometry, we investigated eosinophils and mast cells extensively in oesophageal biopsies from patients with active EoE and from patients with EoE in remission, and compared the findings with healthy individuals. In EoE, epithelium and lamina propria were similarly infiltrated with eosinophils. In contrast, mast cells infiltration was limited to the epithelium, displaying a localised immune response. Interestingly, whereas epithelial mast cells and eosinophils were high in active EoE, some patients in remission, e.g. normalised epithelial eosinophils, showed remaining high numbers of mast cells. Patient clustering supported two groups of patients in clinical remission, differentiating based on presence or absence of epithelial mast cells. CONCLUSIONS: Active EoE is characterised in addition to the well-known tissue eosinophilia by a marked epithelium-restricted mast cell infiltration. Of interest, in a subgroup of patients, mast cell infiltration persisted despite clinical remission. To elucidate the clinical consequence of persistent epithelial mast cells infiltration further studies are required following patients in clinical remission longitudinally.

A fully automated image analysis method to quantify lung fibrosis in the bleomycin-induced rat model.

Intratracheal administration of bleomycin induces fibrosis in the lung, which is mainly assessed by histopathological grading that is subjective. Current literature highlights the need of reproducible and quantitative pulmonary fibrosis analysis. If some quantitative studies looked at fibrosis parameters separately, none of them quantitatively assessed both aspects: lung tissue remodeling and collagenization. To ensure reliable quantification, support vector machine learning was used on digitalized images to design a fully automated method that analyzes two important aspects of lung fibrosis: (i) areas having substantial tissue remodeling with appearance of dense fibrotic masses and (ii) collagen deposition. Fibrotic masses were identified on low magnification images and collagen detection was performed at high magnification. To insure a fully automated application the tissue classifier was trained on several independent studies that were performed over a period of four years. The detection method generates two different values that can be used to quantify lung fibrosis development: (i) percent area of fibrotic masses and (ii) percent of alveolar collagen. These two parameters were validated using independent studies from bleomycin- and saline-treated animals. A significant change of these lung fibrosis quantification parameters- increased amount of fibrotic masses and increased collagen deposition- were observed upon intratracheal administration of bleomycin and subsequent significant beneficial treatments effects were observed with BIBF-1120 and pirfenidone.

Biomarker-guided clinical development of the first-in-class anti-inflammatory FPR2/ALX agonist ACT-389949.

AIMS: The main objectives of these two phase I studies were to investigate safety and tolerability as well as the pharmacokinetic/pharmacodynamic profile of the novel potent and selective formyl peptide receptor type 2 (FPR2)/Lipoxin A4 receptor (ALX) agonist ACT-389949. A challenge model was used to assess the drug's anti-inflammatory potential, with the aim of selecting a dosing regimen for future patient studies. METHODS: Two double-blind, randomized phase I studies investigated the safety, tolerability, pharmacokinetics and pharmacodynamics of ACT-389949 at different doses and dosing regimens. Drug exposure was correlated with target engagement markers such as receptor internalization and cytokine measurements. The effect of FPR2/ALX agonism on neutrophil migration was studied in a lipopolysaccharide (LPS) inhalation model. RESULTS: ACT-389949 was well tolerated. Maximum concentrations were reached around 2 h after dosing, with a mean terminal half-life of 29.3 h [95% confidence interval (CI) 25.5, 33.7]. After multiple-dose administration, exposure increased by 111% (95% CI 89, 136), indicating drug accumulation. Administration of ACT-389949 resulted in a dose-dependent, long-lasting internalization of FPR2/ALX into leukocytes. Pro- and anti-inflammatory cytokines were dose-dependently but transiently upregulated only after the first dose. No pharmacological effect on neutrophil count was observed in the LPS challenge test performed at steady state. CONCLUSIONS: FPR2/ALX agonism with ACT-389949 was shown to be safe and well tolerated in healthy subjects. Receptor internalization and downstream mediators pointed towards a desensitization of the system, which may explain the lack of effect on neutrophil recruitment in the LPS challenge model.

A semi-automated method to assess intraepidermal nerve fibre density in human skin biopsies.

AIMS: Evaluation of intraepidermal nerve fibres (IENFs) in skin biopsies is used in the diagnosis of small-fibre neuropathies. The number of IENFs is assessed manually under a microscope, with an inter-rater variability of ~25%. Unless the images are digitized, there is no documentation. Our aim was to develop a method for standardized semi-automated quantification (SAQ) and documentation of IENF density. METHODS AND RESULTS: We analysed samples from four different university centres that were immunostained according to local protocols. Images were acquired through the Z-plane with a whole slide scanner. orbit image analysis software was used to create an analysable image and develop a reliable algorithm for IENF detection. Rebuilt images revealed well-contrasted nerves, allowing detection of IENFs (automated). The software presented the detected nerves for confirmation by the operator (manual). As compared with the conventional microscopy count, the SAQ achieved correlation coefficients of 0.99 and 0.96 and interfacility variabilities of 19% and 23%, respectively. We found better reproducibility with fluorescence-stained specimens than with bright-field images. CONCLUSIONS: The new semi-automated method has high experimenter-independent reproducibility when based on nerve detection by fluorescence and is easy to perform, even by untrained users. The IENF counting is electronically well documented.

Dual induction of TREM2 and tolerance-related transcript, Tmem176b, in amyloid transgenic mice: implications for vaccine-based therapies for Alzheimer's disease.

Vaccine-based autoimmune (anti-amyloid) treatments are currently being examined for their therapeutic potential in Alzheimer's disease. In the present study we examined, in a transgenic model of amyloid pathology, the expression of two molecules previously implicated in decreasing the severity of autoimmune responses: TREM2 (triggering receptor expressed on myeloid cells 2) and the intracellular tolerance-associated transcript, Tmem176b (transmembrane domain protein 176b). In situ hybridization analysis revealed that both molecules were highly expressed in plaque-associated microglia, but their expression defined two different zones of plaque-associated activation. Tmem176b expression was highest in the inner zone of amyloid plaques, whereas TREM2 expression was highest in the outer zone. Induced expression of TREM2 occurred co-incident with detection of thioflavine-S-positive amyloid deposits. Transfection studies revealed that expression of TREM2 correlated negatively with motility, but correlated positively with the ability of microglia to stimulate CD4(+) T-cell proliferation, TNF (tumour necrosis factor) and CCL2 (chemokine ligand 2) production, but not IFNgamma (interferon gamma) production. TREM2 expression also showed a positive correlation with amyloid phagocytosis in unactivated cells. However, activating cells with LPS (lipopolysaccharide), but not IFNgamma, reduced the correlation between TREM2 expression and phagocytosis. Transfection of Tmem176b into both microglial and macrophage cell lines increased apoptosis. Taken together, these data suggest that, in vivo, Tmem176b(+) cells in closest apposition to amyloid may be the least able to clear amyloid. Conversely, the phagocytic TREM2(+) microglia on the plaque outer zones are positioned to capture and present self-antigens to CNS (central nervous system)-infiltrating lymphocytes without promoting pro-inflammatory lymphocyte responses. Instead, plaque-associated TREM2(+) microglia have the potential to evoke neuroprotective immune responses that may serve to support CNS function during pro-inflammatory anti-amyloid immune therapies.

Transmission and spreading of tauopathy in transgenic mouse brain.

Hyperphosphorylated tau makes up the filamentous intracellular inclusions of several neurodegenerative diseases, including Alzheimer's disease. In the disease process, neuronal tau inclusions first appear in the transentorhinal cortex from where they seem to spread to the hippocampal formation and neocortex. Cognitive impairment becomes manifest when inclusions reach the hippocampus, with abundant neocortical tau inclusions and extracellular beta-amyloid deposits being the defining pathological hallmarks of Alzheimer's disease. An abundance of tau inclusions, in the absence of beta-amyloid deposits, defines Pick's disease, progressive supranuclear palsy, corticobasal degeneration and other diseases. Tau mutations cause familial forms of frontotemporal dementia, establishing that tau protein dysfunction is sufficient to cause neurodegeneration and dementia. Thus, transgenic mice expressing mutant (for example, P301S) human tau in nerve cells show the essential features of tauopathies, including neurodegeneration and abundant filaments made of hyperphosphorylated tau protein. By contrast, mouse lines expressing single isoforms of wild-type human tau do not produce tau filaments or show neurodegeneration. Here we have used tau-expressing lines to investigate whether experimental tauopathy can be transmitted. We show that injection of brain extract from mutant P301S tau-expressing mice into the brain of transgenic wild-type tau-expressing animals induces assembly of wild-type human tau into filaments and spreading of pathology from the site of injection to neighbouring brain regions.

Immunoglobulin M deposition in cutaneous nerves of anti-myelin-associated glycoprotein polyneuropathy patients correlates with axonal degeneration.

Anti-myelin-associated glycoprotein (MAG) neuropathy is an antibody-mediated polyneuropathy. We correlated clinical features, immunoglobulin (Ig) M blood levels, IgM deposition and axonal degeneration in skin biopsies of anti-MAG neuropathy patients. By confocal microscopy, IgM deposits were found exclusively within perineurium-enclosed nerves; they were not found on single, non-perineurium-ensheathed myelinated axons. There was a linear correlation between IgM accumulation in nerve fascicles with IgM blood levels but not with anti-MAG antibody titer or disease duration. Axons with specific IgM deposits had signs of axonal damage, including neurofilament disintegration. Nodal structures were intact even at sites where the axons showed pathologic changes. Ultrastructural analysis revealed degeneration of myelinating Schwann cells. Taken together, these findings suggest that in anti-MAG neuropathy patients, IgM deposits are entrapped within cutaneous perineurium-ensheathed nerve bundles where they accumulate in the endoneurial space. High local IgM levels in the endoneurium may be required for IgM deposition on myelin and subsequent axonal injury and degeneration. This study underlines the importance of early, effective anti-B-cell treatments for preventing progression of this neuropathy.