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

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

VZV infects both CD57^– and CD57^bright NK cells and drives CD57 expression.

<p>CD3^–CD56⁺CD57^– NK cells and CD3^–CD56⁺CD57^bright NK cells were isolated from healthy human donor PBMCs by FACS sorting and subsequently mock or VZV infected with or without IL-2 (200 U/ml) for 2 days before analysis by flow cytometry. (A) Diagram describes experimental design of isolating NK cells on CD57 expression, then infecting, and subsequently analysing for infection and phenotype changes. (B) Plots show surface VZV gE:gI expression between subsets from one representative donor. Graph shows frequency of VZV⁺ NK cell subsets when untreated or with IL-2 (shaded) for three donors. Bars indicate mean. (C) Plots show subsequent CD57 expression between mock, bystander and VZV⁺ CD57^– NK cells (left panels) and CD57 versus gE:gI expression for VZV cultured CD57^– NK cells (middle panels), from one representative donor. Graphs show frequency of CD57 expression on mock, bystander and VZV⁺ CD57^– NK cells for three donors. Bars indicate mean. *p < 0.05 (two-tailed paired t test). (D) Histograms show CD16 expression for mock, bystander and VZV⁺ CD57^– NK cells (left panel) and CD57^bright NK cells (right panel) for one representative donor (n = 3).</p

NK cells are productively infected by VZV and support virus transmission.

<p>NK cells (CD3^–CD56⁺) were FACS sorted from healthy human donor CD56⁺-selected lymphocytes following mock or VZV infection for 1 day. (A & B) Staining by IFA of sorted VZV cultured (left panels) or mock cultured (right panels) NK cells for IE63 (A), pORF29 (B) or respective isotype control, with DAPI (n = 3). (C) Sorted VZV cultured NK cells were added to ARPE-19 epithelial cell monolayers. Four days later monolayers were fixed and infectious centres detected with IFA by staining for IE63 and gE:gI, with DAPI. One representative experiment of five is shown.</p

VZV infects human peripheral blood NK cells, CD3⁺CD56⁺ lymphocytes, and T cells.

<p>Healthy human donor PBMCs were inoculated with mock or VZV infected ARPE-19 epithelial cells for 2 days then analysed for infection by flow cytometry. (A) Representative flow cytometry plots of mock or VZV-S infection, examining surface VZV gE:gI expression on live T cells (CD3⁺CD56^–), CD3⁺CD56⁺ lymphocytes, and NK cells (CD3^–CD56⁺). (B) Frequencies of live gE:gI⁺ lymphocytes in total (shaded), compared to specific populations: T cells, CD3⁺CD56⁺ lymphocytes, and NK cells (n = 19). Symbols represent individual donors consistent across lymphocyte populations, and bars indicate mean. Statistical analysis was performed between specific lymphocyte populations. **p < 0.01, ****p < 0.0001 (RM one-way ANOVA with the Greenhouse-Geisser correction and Tukey’s multiple comparisons test). (C) Representative flow cytometry plots of vOka infection, examining surface gE:gI expression on live T cells (CD3⁺CD56^–), CD3⁺CD56⁺ lymphocytes, and NK cells (CD3^–CD56⁺) (n = 3).</p

NK cell markers associated with maturity influence VZV infection of NK cells and are modulated by VZV.

<p>Healthy human donor PBMCs were mock or VZV infected with or without IL-2 (200 U/ml) for 2 days then analysed by flow cytometry. (A) Diagram describes gating strategy and tSNE analysis workflow for samples shown in (B & C). (B & C) tSNE plots show marker expression levels for single parameters on individual cells in the tSNE map for mock and VZV cultured NK cells after 2 days, either untreated (B) or in the presence of IL-2 (C). Arrowheads indicate the CD56^bright NK cell subset, and the outlined population indicates the localisation of VZV⁺ NK cells. One representative experiment of three is shown. (D & E) Plots show CD57 expression between mock and VZV cultured NK cells (D) and between bystander and VZV⁺ NK cells (E), from one representative donor. Graphs show respective frequencies of CD57⁺ NK cells when untreated or with IL-2 (shaded) for four donors. Bars indicate mean. (F) Histograms show CD16 expression for mock, bystander and VZV⁺ NK cells from one representative donor. Graph shows frequency of CD16⁺ NK cells when untreated or with IL-2 (shaded) for six donors. Bars indicate mean. *p < 0.05, **p < 0.01, ***p < 0.001 (Friedman test with Dunn’s multiple comparisons test).</p

IL-2 stimulation of NK cells, CD3⁺CD56⁺ lymphocytes, and T cells enhances VZV infection.

<p>(A) Healthy human donor PBMCs were infected with VZV by cell-associated infection with or without IL-2 (200 U/ml) for 2 days, then analysed by flow cytometry. Plots show surface VZV gE:gI expression from one representative donor and graphs show frequency of live gE:gI⁺ NK cells (CD3^–CD56⁺) (top panels), CD3⁺CD56⁺ lymphocytes (middle panels), and T cells (CD3⁺CD56^–) (bottom panels). Symbols represent individual donors consistent across lymphocyte populations, and bars indicate mean (n = 8). ***p < 0.001, ****p < 0.0001 (two-tailed paired t test). (B & C) Healthy human donor CD56⁺-selected lymphocytes were infected with VZV by cell-associated infection with or without IL-2 (200 U/ml) for 2 days, then analysed by flow cytometry. Plots show surface gE:gI expression from one representative donor and graphs show frequency of live gE:gI⁺ NK cells (B) or CD3⁺CD56⁺ lymphocytes (C). Symbols represent individual donors, consistent across (B & C) (n = 7). *p < 0.05 (two-tailed Wilcoxon matched-pairs signed rank test).</p

Open Access

Targeted blockade in lethal West Nile virus encephalitis indicates a crucial role for very late antigen (VLA)-4-dependent recruitment of nitric oxide-producing macrophage

Targeted blockade in lethal West Nile virus encephalitis indicates a crucial role for very late antigen (VLA)-4-dependent recruitment of nitric oxide-producing macrophages

<p>Abstract</p> <p>Infiltration of Ly6C^hi monocytes from the blood is a hallmark of viral encephalitis. In mice with lethal encephalitis caused by West Nile virus (WNV), an emerging neurotropic flavivirus, inhibition of Ly6C^hi monocyte trafficking into the brain by anti-very late antigen (VLA)-4 integrin antibody blockade at the time of first weight loss and leukocyte influx resulted in long-term survival of up to 60% of infected mice, with subsequent sterilizing immunity. This treatment had no effect on viral titers but appeared to be due to inhibition of Ly6C^hi macrophage immigration. Although macrophages isolated from the infected brain induced WNV-specific CD4⁺ T-cell proliferation, T cells did not directly contribute to pathology, but are likely to be important in viral control, as antibody-mediated T-cell depletion could not reproduce the therapeutic benefit of anti-VLA-4. Instead, 70% of infiltrating inflammatory monocyte-derived macrophages were found to be making nitric oxide (NO). Furthermore, aminoguanidine-mediated inhibition of induced NO synthase activity in infiltrating macrophages significantly prolonged survival, indicating involvement of NO in the immunopathology. These data show for the first time the therapeutic effects of temporally targeting pathogenic NO-producing macrophages during neurotropic viral encephalitis.</p