Phenotyping the virulence of SARS-CoV-2 variants in hamsters by digital pathology and machine learning.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued to evolve throughout the coronavirus disease-19 (COVID-19) pandemic, giving rise to multiple variants of concern (VOCs) with different biological properties. As the pandemic progresses, it will be essential to test in near real time the potential of any new emerging variant to cause severe disease. BA.1 (Omicron) was shown to be attenuated compared to the previous VOCs like Delta, but it is possible that newly emerging variants may regain a virulent phenotype. Hamsters have been proven to be an exceedingly good model for SARS-CoV-2 pathogenesis. Here, we aimed to develop robust quantitative pipelines to assess the virulence of SARS-CoV-2 variants in hamsters. We used various approaches including RNAseq, RNA in situ hybridization, immunohistochemistry, and digital pathology, including software assisted whole section imaging and downstream automatic analyses enhanced by machine learning, to develop methods to assess and quantify virus-induced pulmonary lesions in an unbiased manner. Initially, we used Delta and Omicron to develop our experimental pipelines. We then assessed the virulence of recent Omicron sub-lineages including BA.5, XBB, BQ.1.18, BA.2, BA.2.75 and EG.5.1. We show that in experimentally infected hamsters, accurate quantification of alveolar epithelial hyperplasia and macrophage infiltrates represent robust markers for assessing the extent of virus-induced pulmonary pathology, and hence virus virulence. In addition, using these pipelines, we could reveal how some Omicron sub-lineages (e.g., BA.2.75 and EG.5.1) have regained virulence compared to the original BA.1. Finally, to maximise the utility of the digital pathology pipelines reported in our study, we developed an online repository containing representative whole organ histopathology sections that can be visualised at variable magnifications (https://covid-atlas.cvr.gla.ac.uk). Overall, this pipeline can provide unbiased and invaluable data for rapidly assessing newly emerging variants and their potential to cause severe disease.

Preclinical models of arthritis for studying immunotherapy and immune tolerance.

Increasingly earlier identification of individuals at high risk of rheumatoid arthritis (RA) (eg, with autoantibodies and mild symptoms) improves the feasibility of preventing or curing disease. The use of antigen-specific immunotherapies to reinstate immunological self-tolerance represent a highly attractive strategy due to their potential to induce disease resolution, in contrast to existing approaches that require long-term treatment of underlying symptoms.Preclinical animal models have been used to understand disease mechanisms and to evaluate novel immunotherapeutic approaches. However, models are required to understand critical processes supporting disease development such as the breach of self-tolerance that triggers autoimmunity and the progression from asymptomatic autoimmunity to joint pain and bone loss. These models would also be useful in evaluating the response to treatment in the pre-RA period.This review proposes that focusing on immune processes contributing to initial disease induction rather than end-stage pathological consequences is essential to allow development and evaluation of novel immunotherapies for early intervention. We will describe and critique existing models in arthritis and the broader field of autoimmunity that may fulfil these criteria. We will also identify key gaps in our ability to study these processes in animal models, to highlight where further research should be targeted.

Intravital microscopy: Imaging host-parasite interactions in lymphoid organs.

Intravital microscopy allows imaging of biological phenomena within living animals, including host-parasite interactions. This has advanced our understanding of both, the function of lymphoid organs during parasitic infections, and the effect of parasites on such organs to allow their survival. In parasitic research, recent developments in this technique have been crucial for the direct study of host-parasite interactions within organs at depths, speeds and resolution previously difficult to achieve. Lymphoid organs have gained more attention as we start to understand their function during parasitic infections and the effect of parasites on them. In this review, we summarise technical and biological findings achieved by intravital microscopy with respect to the interaction of various parasites with host lymphoid organs, namely the bone marrow, thymus, lymph nodes, spleen and the mucosa-associated lymphoid tissue, and present a view into possible future applications.

Intravital imaging of host-parasite interactions in skin and adipose tissues.

Intravital microscopy allows the visualisation of how pathogens interact with host cells and tissues in living animals in real time. This method has enabled key advances in our understanding of host-parasite interactions under physiological conditions. A combination of genetics, microscopy techniques, and image analysis have recently facilitated the understanding of biological phenomena in living animals at cellular and subcellular resolution. In this review, we summarise findings achieved by intravital microscopy of the skin and adipose tissues upon infection with various parasites, and we present a view into possible future applications of this method.

Perisynaptic Schwann cells phagocytose nerve terminal debris in a mouse model of Guillain-Barré syndrome.

In mouse models of acute motor axonal neuropathy, anti-ganglioside antibodies (AGAbs) bind to motor axons, notably the distal nerve, and activate the complement cascade. While complement activation is well studied in this model, the role of inflammatory cells is unknown. Herein we aimed to investigate the contribution of phagocytic cells including macrophages, neutrophils and perisynaptic Schwann cells (pSCs) to distal nerve pathology. To observe this, we first created a subacute injury model of sufficient duration to allow inflammatory cell recruitment. Mice were injected intraperitoneally with an anti-GD1b monoclonal antibody that binds strongly to mouse motor nerve axons. Subsequently, mice received normal human serum as a source of complement. Dosing was titrated to allow humane survival of mice over a period of 3 days, yet still induce the characteristic neurological impairment. Behaviour and pathology were assessed in vivo using whole-body plethysmography and post-sacrifice by immunofluorescence and flow cytometry. ex vivo nerve-muscle preparations were used to investigate the acute phagocytic role of pSCs following distal nerve injury. Following complement activation at distal intramuscular nerve sites in the diaphragm macrophage localisation or numbers are not altered, nor do they shift to a pro- or anti-inflammatory phenotype. Similarly, neutrophils are not significantly recruited. Instead, ex vivo nerve-muscle preparations exposed to AGAb plus complement reveal that pSCs rapidly become phagocytic and engulf axonal debris. These data suggest that pSCs, rather than inflammatory cells, are the major cellular vehicle for axonal debris clearance following distal nerve injury, in contrast to larger nerve bundles where macrophage-mediated clearance predominates.

Anti-ganglioside antibodies are removed from circulation in mice by neuronal endocytosis.

SEE VAN DOORN AND JACOBS DOI101093/BRAIN/AWW078 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE : In axonal forms of Guillain-Barré syndrome, anti-ganglioside antibodies bind gangliosides on nerve surfaces, thereby causing injury through complement activation and immune cell recruitment. Why some nerve regions are more vulnerable than others is unknown. One reason may be that neuronal membranes with high endocytic activity, including nerve terminals involved in neurotransmitter recycling, are able to endocytose anti-ganglioside antibodies from the cell surface so rapidly that antibody-mediated injury is attenuated. Herein we investigated whether endocytic clearance of anti-ganglioside antibodies by nerve terminals might also be of sufficient magnitude to deplete circulating antibody levels. Remarkably, systemically delivered anti-ganglioside antibody in mice was so avidly cleared from the circulation by endocytosis at ganglioside-expressing plasma membranes that it was rapidly rendered undetectable in serum. A major component of the clearance occurred at motor nerve terminals of neuromuscular junctions, from where anti-ganglioside antibody was retrogradely transported to the motor neuron cell body in the spinal cord, recycled to the plasma membrane, and secreted into the surrounding spinal cord. Uptake at the neuromuscular junction represents a major unexpected pathway by which pathogenic anti-ganglioside antibodies, and potentially other ganglioside binding proteins, are cleared from the systemic circulation and also covertly delivered to the central nervous system.

Neuronal expression of GalNAc transferase is sufficient to prevent the age-related neurodegenerative phenotype of complex ganglioside-deficient mice.

Gangliosides are widely expressed sialylated glycosphingolipids with multifunctional properties in different cell types and organs. In the nervous system, they are highly enriched in both glial and neuronal membranes. Mice lacking complex gangliosides attributable to targeted ablation of the B4galnt1 gene that encodes ß-1,4-N-acetylegalactosaminyltransferase 1 (GalNAc-transferase; GalNAcT(-/-)) develop normally before exhibiting an age-dependent neurodegenerative phenotype characterized by marked behavioral abnormalities, central and peripheral axonal degeneration, reduced myelin volume, and loss of axo-glial junction integrity. The cell biological substrates underlying this neurodegeneration and the relative contribution of either glial or neuronal gangliosides to the process are unknown. To address this, we generated neuron-specific and glial-specific GalNAcT rescue mice crossed on the global GalNAcT(-/-) background [GalNAcT(-/-)-Tg(neuronal) and GalNAcT(-/-)-Tg(glial)] and analyzed their behavioral, morphological, and electrophysiological phenotype. Complex gangliosides, as assessed by thin-layer chromatography, mass spectrometry, GalNAcT enzyme activity, and anti-ganglioside antibody (AgAb) immunohistology, were restored in both neuronal and glial GalNAcT rescue mice. Behaviorally, GalNAcT(-/-)-Tg(neuronal) retained a normal "wild-type" (WT) phenotype throughout life, whereas GalNAcT(-/-)-Tg(glial) resembled GalNAcT(-/-) mice, exhibiting progressive tremor, weakness, and ataxia with aging. Quantitative electron microscopy demonstrated that GalNAcT(-/-) and GalNAcT(-/-)-Tg(glial) nerves had significantly increased rates of axon degeneration and reduced myelin volume, whereas GalNAcT(-/-)-Tg(neuronal) and WT appeared normal. The increased invasion of the paranode with juxtaparanodal Kv1.1, characteristically seen in GalNAcT(-/-) and attributed to a breakdown of the axo-glial junction, was normalized in GalNAcT(-/-)-Tg(neuronal) but remained present in GalNAcT(-/-)-Tg(glial) mice. These results indicate that neuronal rather than glial gangliosides are critical to the age-related maintenance of nervous system integrity.

Orally administered antigen can reduce or exacerbate pathology in an animal model of inflammatory arthritis dependent upon the timing of administration.

Currently, treatments for rheumatoid arthritis (RA) are focussed on management of disease symptoms rather than addressing the cause of disease, which could lead to remission and cure. Central to disease development is the induction of autoimmunity through a breach of self-tolerance. Developing approaches to re-establish antigen specific tolerance is therefore an important emerging area of RA research. A crucial step in this research is to employ appropriate animal models to test prospective antigen specific immunotherapies. In this short communication, we evaluate our previously developed model of antigen specific inflammatory arthritis in which ovalbumin-specific T cell receptor transgenic T cells drive breach of tolerance to endogenous antigens to determine the impact that the timing of therapy administration has upon disease progression. Using antigen feeding to induce tolerance we demonstrate that administration prior to articular challenge results in a reduced disease score as evidenced by pathology and serum antibody responses. By contrast, feeding antigen after initiation of disease had the opposite effect and resulted in the exacerbation of pathology. These preliminary data suggest that the timing of antigen administration may be key to the success of tolerogenic immunotherapies. This has important implications for the timing of potential tolerogenic therapies in patients.

Developing a xenograft model of human vasculature in the mouse ear pinna.

Humanised xenograft models allow for the analysis of human tissue within a physiological environment in vivo. However, current models often rely on the angiogenesis and ingrowth of recipient vasculature to perfuse tissues, preventing analysis of biological processes and diseases involving human blood vessels. This limits the effectiveness of xenografts in replicating human physiology and may lead to issues with translating findings into human research. We have designed a xenograft model of human vasculature to address this issue. Human subcutaneous fat was cultured in vitro to promote blood vessel outgrowth prior to implantation into immunocompromised mice. We demonstrate that implants survived, retained human vasculature and anastomosed with the circulatory system of the recipient mouse. Significantly, by performing transplants into the ear pinna, this system enabled intravital observation of xenografts by multiphoton microscopy, allowing us to visualise the steps leading to vascular cytoadherence of erythrocytes infected with the human parasite Plasmodium falciparum. This model represents a useful tool for imaging the interactions that occur within human tissues in vivo and permits visualization of blood flow and cellular recruitment in a system which is amenable to intervention for various studies in basic biology together with drug evaluation and mechanism of action studies.

Spatiotemporal Modeling of the Key Migratory Events During the Initiation of Adaptive Immunity.

Initiation of adaptive immunity involves distinct migratory cell populations coming together in a highly dynamic and spatially organized process. However, we lack a detailed spatiotemporal map of these events due to our inability to track the fate of cells between anatomically distinct locations or functionally identify cell populations as migratory. We used photo-convertible transgenic mice (Kaede) to spatiotemporally track the fate and composition of the cell populations that leave the site of priming and enter the draining lymph node to initiate immunity. We show that following skin priming, the lymph node migratory population is principally composed of cells recruited to the site of priming, with a minor contribution from tissue resident cells. In combination with the YAe/Eα system, we also show that the majority of cells presenting antigen are CD103+CD11b+ dendritic cells that were recruited to the site of priming during the inflammatory response. This population has previously only been described in relation to mucosal tissues. Comprehensive phenotypic profiling of the cells migrating from the skin to the draining lymph node by mass cytometry revealed that in addition to dendritic cells, the migratory population also included CD4+ and CD8+ T cells, B cells, and neutrophils. Taking our complex spatiotemporal data set, we then generated a model of cell migration that quantifies and describes the dynamics of arrival, departure, and residence times of cells at the site of priming and in the draining lymph node throughout the time-course of the initiation of adaptive immunity. In addition, we have identified the mean migration time of migratory dendritic cells as they travel from the site of priming to the draining lymph node. These findings represent an unprecedented, detailed and quantitative map of cell dynamics and phenotypes during immunization, identifying where, when and which cells to target for immunomodulation in autoimmunity and vaccination strategies.

Differential binding patterns of anti-sulfatide antibodies to glial membranes.

Sulfatide is a major glycosphingolipid in myelin and a target for autoantibodies in autoimmune neuropathies. However neuropathy disease models have not been widely established, in part because currently available monoclonal antibodies to sulfatide may not represent the diversity of anti-sulfatide antibody binding patterns found in neuropathy patients. We sought to address this issue by generating and characterising a panel of new anti-sulfatide monoclonal antibodies. These antibodies have sulfatide reactivity distinct from existing antibodies in assays and in binding to peripheral nerve tissues and can be used to provide insights into the pathophysiological roles of anti-sulfatide antibodies in demyelinating neuropathies.

A Novel Cellular Pathway of Antigen Presentation and CD4 T Cell Activation in vivo.

Dendritic cell activation of CD4 T cells in the lymph node draining a site of infection or vaccination is widely considered the central event in initiating adaptive immunity. The accepted dogma is that this occurs by stimulating local activation and antigen acquisition by dendritic cells, with subsequent lymph node migration, however the generalizability of this mechanism is unclear. Here we show that in some circumstances antigen can bypass the injection site inflammatory response, draining freely and rapidly to the lymph nodes where it interacts with subcapsular sinus (SCS) macrophages resulting in their death. Debris from these dying SCS macrophages is internalized by monocytes recruited from the circulation. This coordinated response leads to antigen presentation by monocytes and interactions with naïve CD4 T cells that can drive the initiation of T cell and B cell responses. These studies demonstrate an entirely novel pathway leading to initiation of adaptive immune responses in vivo.

In vivo multiplex molecular imaging of vascular inflammation using surface-enhanced Raman spectroscopy.

Vascular immune-inflammatory responses play a crucial role in the progression and outcome of atherosclerosis. The ability to assess localized inflammation through detection of specific vascular inflammatory biomarkers would significantly improve cardiovascular risk assessment and management; however, no multi-parameter molecular imaging technologies have been established to date. Here, we report the targeted in vivo imaging of multiple vascular biomarkers using antibody-functionalized nanoparticles and surface-enhanced Raman scattering (SERS). Methods: A series of antibody-functionalized gold nanoprobes (BFNP) were designed containing unique Raman signals in order to detect intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and P-selectin using SERS. Results: SERS and BFNP were utilized to detect, discriminate and quantify ICAM-1, VCAM-1 and P-selectin in vitro on human endothelial cells and ex vivo in human coronary arteries. Ultimately, non-invasive multiplex imaging of adhesion molecules in a humanized mouse model was demonstrated in vivo following intravenous injection of the nanoprobes. Conclusion: This study demonstrates that multiplexed SERS-based molecular imaging can indicate the status of vascular inflammation in vivo and gives promise for SERS as a clinical imaging technique for cardiovascular disease in the future.