Clodronate is not protective in lethal viral encephalitis despite substantially reducing inflammatory monocyte infiltration in the CNS

Bone marrow (BM)-derived monocytes induce inflammation and tissue damage in a range of pathologies. In particular, in a mouse model of West Nile virus (WNV) encephalitis (WNE), nitric oxide-producing, Ly6Chi inflammatory monocytes from the BM are recruited to the central nervous system (CNS) and contribute to lethal immune pathology. Reducing the migration of these cells into the CNS using monoclonal antibody blockade, immune-modifying particles or CSF-1R inhibitors reduces neuroinflammation, improving survival and/or clinical outcomes. Macrophages can also be targeted more broadly by administration of clodronate-encapsulated liposomes, which induce apoptosis in phagocytes. In this study, clodronate reduced the inflammatory infiltrate by 70% in WNE, however, surprisingly, this had no effect on disease outcome. More detailed analysis demonstrated a compensatory increase in neutrophils and enhanced activation status of microglia in the brain. In addition, we observed increased numbers of Ly6Chi BM monocytes with an increased proliferative capacity and expression of SCA-1 and CD16/32, potentially indicating output of immature cells from the BM. Once in the brain, these cells were more phagocytic and had a reduced expression of antigen-presenting molecules. Lastly, we show that clodronate also reduces non-myeloid cells in the spleen and BM, as well as ablating red blood cells and their proliferation. These factors likely impeded the therapeutic potential of clodronate in WNE. Thus, while clodronate provides an excellent system to deplete macrophages in the body, it has larger and broader effects on the phagocytic and non-phagocytic system, which must be considered in the interpretation of data

Circulating effector γδ T cell populations are associated with acute coronavirus disease 19 in unvaccinated individuals

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection causes severe coronavirus disease 2019 (COVID‐19) in a small proportion of infected individuals. The immune system plays an important role in the defense against SARS‐CoV‐2, but our understanding of the cellular immune parameters that contribute to severe COVID‐19 disease is incomplete. Here, we show that populations of effector γδ T cells are associated with COVID‐19 in unvaccinated patients with acute disease. We found that circulating CD27negCD45RA+CX3CR1+ Vδ1effector cells expressing Granzymes (Gzms) were enriched in COVID‐19 patients with acute disease. Moreover, higher frequencies of GzmB+ Vδ2+ T cells were observed in acute COVID‐19 patients. SARS‐CoV‐2 infection did not alter the γδ T cell receptor repertoire of either Vδ1+ or Vδ2+ subsets. Our work demonstrates an association between effector populations of γδ T cells and acute COVID‐19 in unvaccinated individuals

A single-dose intranasal live-attenuated codon deoptimized vaccine provides broad protection against SARS-CoV-2 and its variants

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) continues its significant health and economic impact globally. Despite the success of spike-protein vaccines in preventing severe disease, long-lasting protection against emerging variants and the prevention of breakthrough infections and transmission remain elusive. We generate an intranasal live-attenuated SARS-CoV-2 vaccine, CDO-7N-1, using codon deoptimization. CDO-7N-1 shows highly attenuated replication and minimal or no lung pathology in vivo over multiple passages. It induces robust mucosal and systemic neutralizing antibody and T-cell subset responses, in mice (female K18-hACE2 and male HFH4-hACE2 mice), hamsters, and macaques triggered by a single immunization. Mice and hamsters vaccinated with CDO-7N-1 are protected from challenge with wild-type (WT) SARS-CoV-2 and other variants of concern. Serum from vaccinated animals neutralizes WT SARS-CoV-2, variants of concern (beta and delta), variants of interest (omicron XBB.1.5) and SARS-CoV-1. Antibody responses are sustained and enhanced by repeated immunization or infection with WT SARS-CoV-2. Immunity against all SARS-CoV-2 proteins by CDO-7N-1 should improve efficacy against future SARS-CoV-2 variants

T cell activation markers CD38 and HLA-DR indicative of non-seroconversion in anti-CD20-treated patients with multiple sclerosis following SARS-CoV-2 mRNA vaccination

Background Messenger RNA (mRNA) vaccines provide robust protection against SARS-CoV-2 in healthy individuals. However, immunity after vaccination of patients with multiple sclerosis (MS) treated with ocrelizumab (OCR), a B cell-depleting anti-CD20 monoclonal antibody, is not yet fully understood. Methods In this study, deep immune profiling techniques were employed to investigate the immune response induced by SARS-CoV-2 mRNA vaccines in untreated patients with MS (n=21), OCR-treated patients with MS (n=57) and healthy individuals (n=30). Results Among OCR-treated patients with MS, 63% did not produce detectable levels of antibodies (non-seroconverted), and those who did have lower spike receptor-binding domain-specific IgG responses compared with healthy individuals and untreated patients with MS. Before vaccination, no discernible immunological differences were observed between non-seroconverted and seroconverted OCR-treated patients with MS. However, non-seroconverted patients received overall more OCR infusions, had shorter intervals since their last OCR infusion and displayed higher OCR serum concentrations at the time of their initial vaccination. Following two vaccinations, non-seroconverted patients displayed smaller B cell compartments but instead exhibited more robust activation of general CD4+ and CD8+ T cell compartments, as indicated by upregulation of CD38 and HLA-DR surface expression, when compared with seroconverted patients. Conclusion These findings highlight the importance of optimising treatment regimens when scheduling SARS-CoV-2 vaccination for OCR-treated patients with MS to maximise their humoral and cellular immune responses. This study provides valuable insights for optimising vaccination strategies in OCR-treated patients with MS, including the identification of CD38 and HLA-DR as potential markers to explore vaccine efficacy in non-seroconverting OCR-treated patients with MS

Mobilisation of the murine haematopoietic system in the bone marrow during viral encephalitis

Viral infection of the central nervous system (CNS) with West Nile virus (WNV) or Zika virus (ZIKV) results in a rapid influx of monocyte-derived macrophages, that ultimately induce fatal pathology in the mouse. Whilst these cells are suspected to originate from the bone marrow (BM), little is known about the kinetic and migratory events that might mobilise BM monocytes and their progenitors in response to CNS infection. In this study we conducted comprehensive mapping of the murine central nervous system (CNS) and haematopoietic system in the BM using high-dimensional 29-parameter flow cytometry and 47-parameter mass cytometry (CyTOF). Additionally, we developed and utilized 8-way cell sorting capacities for sorting cells on high-dimensional panels. In order to analyse the resulting datasets, we developed an analysis pipeline for clustering (using the tool ‘FlowSOM’) and dimensionality reduction (using the tool ‘tSNE’) to manage large datasets that we termed ‘Cytometry Analysis Pipeline for large and compleX datasets’ (CAPX, scripts and instructions available at www.sydneycytometry.org.au/capx or www.github.com/sydneycytometry/capx). We used this approach to profile cellular infiltration in virally infected brains, revealing a similar pattern of macrophage-dominated infiltration in WNV and ZIKV (strain MR766) infected brains, but an alternative lymphocyte-dominated infiltration in ZIKV (strain IBH) infected brains. In order to map changes to haematopoiesis during infection, we applied the same approach to the BM during WNV or ZIKV infection. By comprehensively mapping haematopoietic pathways in the BM in steady state and inflammatory conditions, we revealed that viral encephalitis drives a reorganisation of cellular output to favour monocyte production, resulting in activation and expansion of monocytes and monocyte progenitors, with increased proliferation of mature and progenitor populations. In addition, we observed compensatory downregulation of B cell lymphopoiesis, and modification of granulopoiesis in the BM, favouring monocyte expansion. Antibody blockade of various cytokine factors resulted in a reduction of this excessive monocyte proliferation, and in some cases resulted in improved clinical outcomes and reduce cellular infiltration in the brain in WNV-infected mice. In this study we have used high-dimensional cytometry approaches to characterise modifications to the haematopoietic lineage during viral encephalitis that favour production of pathogenic monocytes. Based on these results, we sought to further explore how other models of inflammation might drive alternative changes to different aspects of the haematopoietic system. As such, we examined changes to the haematopoietic system in two models of inflammation. Firstly, we used a mouse model of peripheral infection with lymphocytic choriomeningitis virus (LCMV), resulting in an acute system viral infection. Secondly, we used a mouse model of pulmonary emphysema (PE), which results in chronic, long-term, non-infectious inflammation, with heavy neutrophil and macrophage infiltration into the inflamed lung. In addition to the baseline LCMV model, we were able to utilise a range of genetic knock out mice, which we used to further perturb the haematopoietic system. Whilst monocyte expansion featured prominently in both cases, similar to viral encephalitis, we found notably differences in each model. In the emphysema model, we found differences in monocyte and neutrophil amplification over time, consistent with different phases of disease. Additionally, where B cell lymphopoiesis was severely suppressed in viral encephalitis, B cell proliferation was not suppressed in the emphysema model, despite some modifications to the B cell compartment. Systemic infection with LCMV drove monocyte-dominant changes to the haematopoietic system, as expected. However, mice that had genes related to the IFN system removed exhibit a drastic shift towards granulopoiesis in infected animals only. This dramatic shift also resulted in the appearance of extremely immature granulocytes in infected tissues. Additionally, in stat1 knock out mice, LCMV infection resulted in complete ablation of erythropoiesis in the BM, despite normal numbers of erythrocytes in the blood. In summary, whilst myeloid responses dominated in both scenarios, we found that the re- organisation of haematopoietic priorities was determine both by the severity and acuity of inflammation, as well as on the local signalling environment

An updated guide for the perplexed : cytometry in the high-dimensional era

High-dimensional cytometry experiments measuring 20-50 cellular markers have become routine in many laboratories. The increased complexity of these datasets requires added rigor during the experimental planning and the subsequent manual and computational data analysis to avoid artefacts and misinterpretation of results. Here we discuss pitfalls frequently encountered during high-dimensional cytometry data analysis and aim to provide a basic framework and recommendations for reporting and analyzing these datasets

Varicella zoster virus productively infects human natural killer cells and manipulates phenotype.

Varicella zoster virus (VZV) is a ubiquitous human alphaherpesvirus, responsible for varicella upon primary infection and herpes zoster following reactivation from latency. To establish lifelong infection, VZV employs strategies to evade and manipulate the immune system to its advantage in disseminating virus. As innate lymphocytes, natural killer (NK) cells are part of the early immune response to infection, and have been implicated in controlling VZV infection in patients. Understanding of how VZV directly interacts with NK cells, however, has not been investigated in detail. In this study, we provide the first evidence that VZV is capable of infecting human NK cells from peripheral blood in vitro. VZV infection of NK cells is productive, supporting the full kinetic cascade of viral gene expression and producing new infectious virus which was transmitted to epithelial cells in culture. We determined by flow cytometry that NK cell infection with VZV was not only preferential for the mature CD56dim NK cell subset, but also drove acquisition of the terminally-differentiated maturity marker CD57. Interpretation of high dimensional flow cytometry data with tSNE analysis revealed that culture of NK cells with VZV also induced a potent loss of expression of the low-affinity IgG Fc receptor CD16 on the cell surface. Notably, VZV infection of NK cells upregulated surface expression of chemokine receptors associated with trafficking to the skin -a crucial site in VZV disease where highly infectious lesions develop. We demonstrate that VZV actively manipulates the NK cell phenotype through productive infection, and propose a potential role for NK cells in VZV pathogenesis

Image_2_Clodronate is not protective in lethal viral encephalitis despite substantially reducing inflammatory monocyte infiltration in the CNS.tiff

Bone marrow (BM)-derived monocytes induce inflammation and tissue damage in a range of pathologies. In particular, in a mouse model of West Nile virus (WNV) encephalitis (WNE), nitric oxide-producing, Ly6Chi inflammatory monocytes from the BM are recruited to the central nervous system (CNS) and contribute to lethal immune pathology. Reducing the migration of these cells into the CNS using monoclonal antibody blockade, immune-modifying particles or CSF-1R inhibitors reduces neuroinflammation, improving survival and/or clinical outcomes. Macrophages can also be targeted more broadly by administration of clodronate-encapsulated liposomes, which induce apoptosis in phagocytes. In this study, clodronate reduced the inflammatory infiltrate by 70% in WNE, however, surprisingly, this had no effect on disease outcome. More detailed analysis demonstrated a compensatory increase in neutrophils and enhanced activation status of microglia in the brain. In addition, we observed increased numbers of Ly6Chi BM monocytes with an increased proliferative capacity and expression of SCA-1 and CD16/32, potentially indicating output of immature cells from the BM. Once in the brain, these cells were more phagocytic and had a reduced expression of antigen-presenting molecules. Lastly, we show that clodronate also reduces non-myeloid cells in the spleen and BM, as well as ablating red blood cells and their proliferation. These factors likely impeded the therapeutic potential of clodronate in WNE. Thus, while clodronate provides an excellent system to deplete macrophages in the body, it has larger and broader effects on the phagocytic and non-phagocytic system, which must be considered in the interpretation of data.</p