Investigating neutrophil phenotype and migration mechanisms in the tissue draining lymph node during acute pulmonary infection with Streptococcus pneumoniae

Neutrophils are innate immune cells that form part of the first line of defense against pathogens. These cells are rapidly mobilised in response to infection and inflammation and are equipped with a range of pathogen destruction mechanisms. In recent years it has been demonstrated that neutrophils can also modulate innate and adaptive immune responses. Neutrophils have been found to enter secondary lymphoid organs including lymph nodes, however the phenotype and function of neutrophils in lymph nodes is not well understood. Work in this thesis utilised an acute pulmonary infection model with the lung pathogen Streptococcus pneumoniae, to investigate neutrophil recruitment, phenotype and function in the lung draining (mediastinal) lymph node. It was hypothesised that neutrophil phenotype and behaviours are not cell intrinsic, rather dictated by the localised microenvironment of the cell. To investigate this hypothesis a combination of flow cytometry, immunohistochemistry, quantitative PCR and four dimensional multiphoton explant imaging was used. Neutrophil recruitment to the lung draining lymph node was rapid, peaking at 6 - 12 hours post infection. Neutrophils were localised in the lymphatic sinus, specifically in areas with medullary macrophages. Neutrophil migration in this region was highly dynamic, with swarms of neutrophils observed. When compared to blood, lung and airway neutrophils, lymph node neutrophils showed an intermediate activation profile, similar to lung tissue neutrophils. Interestingly a small population of lymph node neutrophils expressed markers of antigen presenting cells. Neutrophils in the lymph node utilised mainly chemotactic migration mechanisms involving phosphatidylinositde 3-kinase signalling and leukotriene B4, however migration was not completely integrin independent. Thus the results support the hypothesis that neutrophil phenotype and behaviours are environment dependent as opposed to cell-intrinsic, also they demonstrate and support the evolving view of the neutrophil as a complex cell type that has multiple functional profiles and mechanisms of migration

Macrophage transactivation for chemokine production identified as a negative regulator of granulomatous inflammation using agent-based modeling

Cellular activation in trans by interferons, cytokines and chemokines is a commonly recognized mechanism to amplify immune effector function and limit pathogen spread. However, an optimal host response also requires that collateral damage associated with inflammation is limited. This may be particularly so in the case of granulomatous inflammation, where an excessive number and / or excessively florid granulomas can have significant pathological consequences. Here, we have combined transcriptomics, agent-based modeling and in vivo experimental approaches to study constraints on hepatic granuloma formation in a murine model of experimental leishmaniasis. We demonstrate that chemokine production by non-infected Kupffer cells in the Leishmania donovani-infected liver promotes competition with infected KCs for available iNKT cells, ultimately inhibiting the extent of granulomatous inflammation. We propose trans-activation for chemokine production as a novel broadly applicable mechanism that may operate early in infection to limit excessive focal inflammation

Bone marrow-derived and resident liver macrophages display unique transcriptomic signatures but similar biological functions

Abstract: Background and aims: Kupffer cells (KCs), the resident tissue macrophages of the liver, play a crucial role in the clearance of pathogens and other particulate materials that reach the systemic circulation. Recent studies have identified KCs as a yolk sac-derived resident macrophage population that is replenished independently of monocytes in the steady state. Although it is now established that following local tissue injury, bone-marrow derived monocytes may infiltrate the tissue and differentiate into macrophages, the extent to which newly differentiated macrophages functionally resemble the KCs they have replaced has not been extensively studied. Methods and results: Here we show using intravital microscopy, morphometric analysis and gene expression profiling that bone marrow derived “KCs” accumulating as a result of genotoxic injury resemble, but are not identical to their yolk-sac (YS) counterparts. An ion homeostasis gene signature, including genes associated with scavenger receptor function and extracellular matrix deposition, allows discrimination between these two KC populations. Reflecting the differential expression of scavenger receptors, YS-derived KCs were more effective at accumulating Ac-LDL, whereas surprisingly they were poorer than BM-derived KCs when assessed for uptake of a range of bacterial pathogens. The two KC populations were almost indistinguishable in regard to i) response to LPS challenge, ii) phagocytosis of effete RBCs and iii) their ability to contain infection and direct granuloma formation against Leishmania donovani, a KC-tropic intracellular parasite. Conclusions: BM-derived KCs differentiate locally to resemble YS-derived KC in most but not all respects, with implications for models of infectious diseases, liver injury and bone marrow transplantation. In addition, the gene signature we describe adds to the tools available for distinguishing KC subpopulations based on their ontology

Chronic Infection Drives Expression of the Inhibitory Receptor CD200R, and Its Ligand CD200, by Mouse and Human CD4 T Cells

Certain parasites have evolved to evade the immune response and establish chronic infections that may persist for many years. T cell responses in these conditions become muted despite ongoing infection. Upregulation of surface receptors with inhibitory properties provides an immune cell-intrinsic mechanism that, under conditions of chronic infection, regulates immune responses and limits cellular activation and associated pathology. The negative regulator, CD200 receptor, and its ligand, CD200, have been shown to regulate macrophage activation and reduce pathology following infection. We show that CD4 T cells also increase expression of inhibitory CD200 receptors (CD200R) in response to chronic infection. CD200R was upregulated on murine effector T cells in response to infection with bacterial, Salmonella enterica, or helminth, Schistosoma mansoni, pathogens that respectively drive predominant Th1- or Th2-responses. In vitro chronic and prolonged stimuli were required for the sustained upregulation of CD200R, and its expression coincided with loss of multifunctional potential in T effector cells during infection. Importantly, we show an association between IL-4 production and CD200R expression on T effector cells from humans infected with Schistosoma haematobium that correlated effectively with egg burden and, thus infection intensity. Our results indicate a role of CD200R:CD200 in T cell responses to helminths which has diagnostic and prognostic relevance as a marker of infection for chronic schistosomiasis in mouse and man

Macrophage Transactivation for chemokine Production identified as a negative regulator of granulomatous inflammation Using agent-Based Modeling

Cellular activation in trans by interferons, cytokines, and chemokines is a commonly recognized mechanism to amplify immune effector function and limit pathogen spread. However, an optimal host response also requires that collateral damage associated with inflammation is limited. This may be particularly so in the case of granulomatous inflammation, where an excessive number and/or excessively florid granulomas can have significant pathological consequences. Here, we have combined transcriptomics, agent-based modeling, and in vivo experimental approaches to study constraints on hepatic granuloma formation in a murine model of experimental leishmaniasis. We demonstrate that chemokine production by non-infected Kupffer cells in the Leishmania donovani-infected liver promotes competition with infected KCs for available iNKT cells, ultimately inhibiting the extent of granulomatous inflammation. We propose trans-activation for chemokine production as a novel broadly applicable mechanism that may operate early in infection to limit excessive focal inflammation

T cells co-express CD200 and CD200R following TCR activation.

<p>A. Naïve C57BL/6 peripheral LN cells were cultured with a titration of anti-CD3 + anti-CD28 (2 µg/ml) for 3d. Contour plots show upregulation of CD200 (upper row) and CD200R (lower row) together with CD44 (R1) in gated CD4 cells. B. DO11.10 LN and spleen cells were cultured with a titration of OVA peptide (ISQAVHAAHAEINEAGR) for 3d. CD200R:CD200 co-expression (R1) is shown in gated CD4 cells. C. Naïve LN cells were stimulated with anti-CD3 (1 µg/ml) + anti-CD28 (2 µg/ml) either transiently on Ab-coated wells for the first 2d then removed to fresh media (blue lines) or chronically with Ab-coated aAPC present throughout the culture (red lines) in Th1 (top row), Th2 (middle row) or non-polarising (IL-7, bottom row) conditions. Histograms show expression of CD200 and CD200R in gated CD4 cells at d3 (left) and d7 (right) compared to naïve levels (grey filled histograms). Data are representative of 6 independent experiments and statistical validation is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035466#pone.0035466.s001" target="_blank">Figure S1A and S3C</a>.</p

Chronic infection induces CD200 and CD200R expression in CD4 T cells.

<p>C57BL/6 (A–E) and 4-get (F) mice were infected with <i>S. mansoni</i>; MesLN (A–D, F) and spleen (E) were analyzed. A. Representative contour plot of CD44 with CD200, and B. CD44 with CD200R is shown for gated CD4 T cells. Upon infection (8 wk), CD44^hiCD200⁺CD4 cells (panel A, R3) increased from 7.71±2.66% (n = 14) to 15.2±3.72% (n = 17, p<0.0001); CD44^hiCD200R⁺CD4 cells (panel B, R3) increased from 4.36±1.69% (n = 14) to 15.3±4.67% (n = 17, p<0.0001). Data were pooled from 4 independent experiments. C. Dot plots show CD200 and CD200R co-expression in CD44^lo and CD44^hi CD4 cells from uninfected and infected mice. D. Graph shows absolute numbers of CD200R⁺ (panel B, R3) and CD200R^– (panel B, R2) CD44^hiCD4 cells from 4 independent experiments. Upon infection CD44hiCD200R+ cells increased from (2.7±0.6)× 10⁵ (n = 14) to (10.8±3.2)×10⁵ (n = 17, ***p = 3.03×10^–10), while CD44^hi CD200R^– cells increased from (7.9±3.3)×10⁵ to (18.6±11.9)×10⁵ (**p = 0.003). E. Contour plots show CD200R expression in proliferating (BrdU⁺) cells, in gated CD4 cells for spleen of uninfected and infected mice. Upon infection BrdU⁺CD200R⁺ CD4 T cells increased from 4.5±0.69 (n = 4) to 46.6±17 (n = 10) p<0.0005. Specificity controls for BrdU staining are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035466#pone.0035466.s007" target="_blank">Figure S7A</a> & B. F. Contour plots (left) show CD200 and eGFP expression; CD200⁺GFP⁺CD4 cells increased from 0.64±0.12% to 13.7±7.24% after infection (p = 0.01, n = 4). Histogram overlays (right) show CD200 and CD200R levels in gated GFP⁺ (green line) and GFP^– (filled histograms) CD4 cells from infected mice. Control stainings for intracellular GFP staining are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035466#pone.0035466.s005" target="_blank">Fig S5B & C</a>. ** = p<0.0001, * = p<0.001. Results are representative of at least 4 (A–D) and 3 (E-F) independent experiments (n = 3 mice/group).</p

Effector cytokine potential is acquired alongside CD200R expression in vivo.

<p>Dot plots show cytokine⁺ gated CD4 cells <i>ex vivo</i> (-P/I) and after recall with pdbU + Ionomycin (+P/I, 5h); gates are based on unstimulated controls (-P/I). A. Infection with <i>S. mansoni</i> (8wk) increased CD200R⁺ IL-4⁺ CD4 cells in MesLN from 0.017±0.019% to 3.39±1.36% and 83±4.14% of IL-4⁺ cells were CD200R⁺, while CD200R^– TNFα⁺ CD4 cells decreased from 47.5±4.66% to 30.0±0.99%. (n = 3, p = 0.01). B. Infection with <i>S. enterica</i> increased IFNγ⁺ CD4 cells in spleen from 1.9±0.65% to 31.1±4.4% and 49.5±9.86% of IFNγ⁺ cells were CD200R⁺, while CD200R^– TNFα⁺ CD4 cells decreased from 19.6±2.31% to 15.1±1.56%. (n = 5, p = 0.01). Results are representative of at least 3 (A, n = 3 mice/group) and 2 (B, n = 5 mice/group) independent experiments.</p

Progressive acquisition of CD200R correlates with effector cytokine secretion.

<p>Peripheral LN cells from BALB/c mice and 4-get mice were analysed ex vivo (Naive) or after culture for 7d in transient TCR or chronic TCR stimulation conditions, as indicated, using polarising culture conditions to promote Th2 (A) or Th1 (B) cytokine differentiation. Intracellular cytokine staining with CD200R expression is shown for TNFα, IL-2, IL-4 and IFNγ. Intracellular staining with anti-GFP was used to enhance eGFP signal in 4-get cells (control stainings for anti-GFP are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035466#pone.0035466.s005" target="_blank">Figure S5B & C</a>). Contour plots show percentage of cytokine⁺ gated CD4 cells; gates were based on unstimulated controls (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035466#pone.0035466.s003" target="_blank">Figure S3A and S3B</a>). Data are representative of 5 independent experiments. Pairwise comparison of multifunctional cytokine loss from replicate cultures is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035466#pone.0035466.s003" target="_blank">Figure S3D-F</a>.</p