N UCLEOTIDES RELEASED FROM INFECTED CELLS TRIGGER MICROGLIA RECRUITMENT AND

4.4.1. Neurotropic virus infection induces the production of inflammatory mediators

Next, we aimed to investigate the production of inflammatory mediators induced by neurotropic virus infection, therefore we measured several inflammatory cytokines and chemokines that are commonly upregulated in response to virus infection in neuronal and astroglial cell cultures (Lokensgard, Cheeran, Hu, Gekker, & Peterson, 2002). As a positive control, we used bacterial lipopolysaccharide (LPS), which is a widely used pro-inflammatory stimulus. LPS treatment induced a robust increase of TNFα, IL-6, CXCL1, CCL5 (RANTES), G-CSF and MCP-1 in astrocytes and CCL5, MCP-1 and CXCL1 increase in neurons.

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However, we noticed that PRV infection increased only CCL5 levels in both cell types at mRNA and peptide levels 24 hours after infection (Fig.10.).

Figure 10. In response to viral infection inflammatory cytokines and chemokines are produced by astroglia, microglia and neuronal cell cultures in vitro. a-i, Cytokine levels were measured 24 hours after PRV infection from conditioned medium of astroglial and neuronal cultures by cytometric bead array (CBA). As a positive control, LPS treatment was induced parallel with virus infection. For qPCR measurement cell homogenates were collected as well. a-f, one-way ANOVA, followed by Tukey’s post hoc test. a,****p<0.0001 Cont vs LPS; b, *p<0.05 Cont vs LPS; c, ***p<0.001 Cont vs LPS; d, ****p<0.0001 Cont vs LPS,

***P<0.001 Cont vs LPS, Cont vs PRV, *p<0.05 Cont vs LPS; e, ****p<0.0001 Cont vs LPS,

**p<0.01 Cont vs LPS; f, ****p<0.0001 Cont vs LPS; g-i, two-way ANOVA followed by Sidak’s multiple comparisons test; h, ****p<0.0001 BDG vs LPS; i, ****p<0.0001 BDG vs LPS, ***p<0.001 BDG vs LPS (Fekete et al. 2018).

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4.4.2. Release of purinergic nucleotides triggers rapid microglia activation

Since synthesis and release of chemokines could last for several hours and our in vivo data suggested rapid microglia recruitment to sites of virus infection, we checked whether purine nucleotides such as ATP that are chemotactic for microglia could be released from infected cells at a short time scale (Davalos et al., 2005a). We found that cultured neurons released ATP after virus infection, which was associated with reduced ATP, ADP, AMP and adenosine levels in cell lysates within hours (Fig.11.a,b,) upon the expression of the immediate-early marker GFP, which precedes the expression of viral structural proteins required for productive infection (Dénes et al., 2006). The changes in purinergic metabolites were associated with increased ecto-ATPase levels in infected cells (Fig.11.c,) but were not due to apoptosis or necrosis, since at the early stages of infection neurons expressing high levels of GFP showed no uptake of propidium iodide (PI) (Fig.11.g,h,). In addition, increased ecto-ATPase levels and NTDPase1 expression were found in microglia at sites of virus infection in the brain, indicating that microglia respond to changes in the levels of purine nucleotides (Fig.11,d,e,) (Sperlágh & Illes, 2007).

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Figure 11. Microglia react to nucleotides released upon viral infection. a, and b, graphs show that ATP, ADP, AMP and adenosine levels were measured with HPLC analysis in the supernatant (a) and in cellular fractions (b) of control and virus-infected neuronal cell cultures . c, Enzyme histochemistry and densitometric analysis of ecto-ATPase show that the enzyme activity increased markedly upon infection. d, Electron microscopic image shows NTDPase1 expression of microglia (red) in PRV-infected brain. NTDPase1⁺ microglia is in contact with a disintegrating PRV-immunopositive neuron (blue). e, NTDPase1⁺ microglia in control and infected brains. f, Densitometric analysis of NTDPase1 enzyme, measured from control and infected mouse brains. g, h, Propidium iodide (PI, red), was used as viability marker to show that neuronal death in cell cultures was not increased 16 hours after infection. All data are expressed as mean ± s.e.m a-b, n=4 cell culture per group, unpaired t-test, *p<0.05; **p<0.001 c, n=6 cell culture per group, unpaired t-test, ***p≤0.001 f, n=7 brain slice per group, unpaired t-test, **p<0.01. Scale bars: c, 25 µm; d, 50 µm; e, 10 µm; g, 50 µm (Fekete et al. 2018).

4.4.3. Microglia recruitment to infected neurons is mediated by P2Y12 receptors in vitro

To further investigate the mechanisms mediating microglial responses to purinergic nucleotides we established co-cultures of P2X7^-/- or P2Y12^-/- microglia and wild type astrocytes. Similar to that seen in wild type microglia, motility of P2X7 deficient cells decreased when exposed to infected cells (Fig.8.a,b,) and trajectories showed characteristic localized pattern due to regular scanning activity, suggesting that P2X7 deficiency does not impede recognition of virus-infected cells by microglia (Fig.12.e,). In contrast, virus exposed P2Y12^-/- deficient microglia showed marked increase in their motility (Fig.12.f-j,) with trajectories characteristic of random walk behaviour and lacking the localized pattern.

(Fig.12.k,). This suggests that these cells are unable to display targeted recruitment in response to viral infection. Moreover P2Y12-deficient microglia showed very low phagocytic activity in virus exposed environment, compared to wild type and P2X7^-/- microglia that showed marked increase in phagocytosis (Fig.12.l-n,). Thus, P2Y12 is a key contributor in recognition and phagocytosis of compromised cells by microglia in vitro.

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Figure 12. Microglial recruitment and phagocytosis are mediated by purinergic signalling upon virus infection. a, and f, Time-dependent average velocities of P2X7 (nc=67, ni=74) and P2Y12 (nc=51, ni=59) deficient microglial cells in control and infected astroglia cultures. Decreased velocity of P2X7^-/- and the lack of such decrease of P2Y12^-/- microglia cells can be detected in infected cultures. b, and g, Average velocities of P2X7^-/- and P2Y12

-/- microglia over 24 hours in control and infected astrocyte cultures. c, and h, Frequency distribution of time-dependent average velocities of P2X7^-/- and P2Y12^-/- microglial cells in control and infected astroglial cultures. d, and j, Average displacement of P2X7 and P2Y12 deficient microglia in different time intervals of migration in control and infected astroglial cell cultures. Error stripes correspond to s.e.m. e, and k, Individual trajectories of equal number (n=50) randomly chosen P2X7^-/- and P2Y12^-/- microglial cells over 24 hours in control and infected astrocyte cultures. For better comparison of migration directionality individual cell trajectories were centered to start from the origo. P2X7^-/- microglia showed more localised migration pattern and scanning activity in infected cultures compared to the random walk behaviour under control condition. P2Y12^-/- microglial cells showed the absence of this localised scanning behaviour in their trajectories in infected cultures. l, Phagocytic activity of wild type (WT), P2X7 and P2Y12 deficient microglia. Expression of immediate-early GFP marker can be seen in infected astrocytes. Red arrowheads point to phagocytosed, infected cells. m-n, Percentage of phagocytic events by wild type, P2X7^-/- and PY12^-/- microglia. Note that P2Y12 deficient microglial cells are unable to phagocytose infected cells. b and g, n=121 cell per group, unpaired t-test, ****p<0.0001. m and n, n=9 cells per group, one-way ANOVA, **p<0.001; *** p<0.0001 **** p<0.0001, ns = not significant (Fekete et al. 2018).

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4.5. Microglial P2Y12 receptors mediate recruitment of microglia and elimination of