Anti-inflammatory activity of intravenous immunoglobulins protects against West Nile virus encephalitis

A single dose of IVIG is able to protect from lethal WNV infection

To determine the minimum infectious dose of WNV at which the majority of BALB/c mice succumbed to encephalitis, mice were infected intraperitoneally with various doses, and observed for mortality and morbidity. BALB/c mice given doses of 10² or 10³ p.f.u. WNV showed no signs of encephalitis, whereas mice infected with ≥10⁴ p.f.u. WNV succumbed to WNV encephalitis by days 9–11 p.i. (Fig. 1a). To determine if IVIG and WNV-IVIG would protect against a lethal challenge, mice inoculated with 10⁴ p.f.u. WNV were injected intraperitoneally with 4 or 25 mg IVIG or 4 mg WNV-IVIG 24 h p.i. and survival was compared with PBS-treated mice. Whilst all PBS-treated mice succumbed to WNV encephalitis between days 7 and 11 p.i., all IVIG- and WNV-IVIG-treated mice survived (Fig. 1b). Importantly, WNV-infected mice treated with 25 mg IVIG adsorbed to lack WNV E-gp antibodies protected >65 % of mice (Fig. 1c) despite being incapable of neutralizing virus infectivity (Table 1). This result showed that WNV neutralizing activity of IVIG was not critical for protection from WNV encephalitis and that alternative mechanisms could mediate protection (Vogt et al., 2011), which we anticipated based on our demonstration that IVIG protection against HSV1 encephalitis was independent of neutralizing antibodies (Ramakrishna et al., 2011).

 

Fig. 1.

IVIG protects BALB/c mice from WNV encephalitis. (a) Mice were infected intraperitoneally with the indicated doses of WNV and monitored for survival. (b, c) Mice infected with 10⁴ p.f.u. WNV were given PBS, 4 mg IVIG or WNV-IVIG at 24 h p.i. (b) or 25 mg IVIG, desialylated or deglycosylated IVIG, or an IVIG adsorbed to be free of WNV E-gp antibodies and monitored for survival (c). (d) Mice infected with 10⁵ p.f.u. WNV were given 4 mg WNV-IVIG, 25 mg IVIG (1×) at 24 h p.i. and some mice received a second dose (2×) of IVIG at day 9 p.i. n = 6–10 mice per group. Statistically significant values are indicated within each plot.

A recently proposed mechanism for IVIG protection postulated that a minor fraction of IVIG bearing terminal sialic acid residues on Asp297 in the IgG Fc domain, resulting in increased binding affinity for the mouse C-type lectin receptor SIGNR1 (and the human orthologue DC-SIGN) compared with Fc receptors, is crucial for protection from arthritis and immune thrombocytopenia in mouse models (Anthony & Ravetch, 2010; Anthony et al., 2008). According to this anti-inflammatory mechanism, the interaction of 2,6-α-sialylated IgGs (sIgG) with SIGN-R1⁺ macrophages induces IL-33 that drives production of the T_h2 helper cytokines IL-4 and IL-13 by basophils, which ultimately leads to upregulation of the inhibitory FcγRIIB receptor on macrophages (Anthony et al., 2011; Schwab & Nimmerjahn, 2013), thereby increasing the activation threshold for innate monocytes and macrophages. Although this hypothetical mechanism is highly controversial (Sharma et al., 2014; von Gunten et al., 2014), it was nevertheless important to determine if sIgG was involved in protection from WNV encephalitis. We found that there was no significant reduction in protection when WNV-inoculated mice were given 25 mg desialylated IVIG 24 h p.i. as >60 % mice were protected. Moreover, mice treated with either low-dose (4 mg) or high-dose (25 mg) IVIG were protected equally and as sIgG is present in limiting amounts in IVIG, this result further discredits a role for sIgG in protection from WNV encephalitis. Deglycosylation of IVIG abrogated protection against WNV encephalitis. As deglycosylation is known to destabilize the integrity of the IgG Fc domain (Anthony & Ravetch, 2010), this result suggested that IVIG’s protective anti-inflammatory effects were critically dependent on Fc–FcR interactions (Chung et al., 2006).

To determine IVIG protection against WNV encephalitis after high-dose infection, mice inoculated with 10⁵ p.f.u. WNV were treated with either IVIG or WNV-IVIG at 24 h p.i. The generic IVIG used in this study (collected during 2010–2013) had a high WNV antibody neutralizing titre, reflecting the increased exposure of the US population, particularly in the western states, to WNV. A single dose of WNV-IVIG protected all mice inoculated with a high dose of WNV, whereas despite having an equivalent WNV antibody titre, IVIG failed to protect the mice (Fig. 1d, Table 1). The data suggested that WNV-IVIG derived by pooling sera from convalescent patients might contain WNV-specific antibodies of higher affinity or increased in vivo functionality compared with the WNV-specific antibodies in IVIG. It is notable that a single dose of IVIG markedly extended the survival of mice inoculated with high doses of WNV as the mice survived until day 20 p.i. before succumbing to WNV encephalitis (Fig. 1d), whereas the PBS-treated mice all succumbed by day 10 p.i. (Fig. 1a).

IVIG reduces viral titres following WNV infection

To determine if unrestrained viral replication was the cause of death in mice infected with a high dose of WNV (10⁵ p.f.u.), tissues from infected mice treated with PBS or IVIG collected on days 4, 6, 8, 12 and 20 p.i. were used to determine WNV RNA levels. PBS-treated mice had high levels of WNV RNA in peripheral organs such as the spleen at days 4–6 p.i., but replication was controlled by day 8 p.i. (Fig. 2a). In contrast to the high viral titres in spleen at day 4 p.i., virus was not detectable in the brains of PBS-treated mice before day 6 p.i., but thereafter high levels of WNV RNA were detected (Fig. 2b); these mice succumbed to WNV encephalitis by days 8–9 p.i. (Fig. 1a). As expected, hyperimmune WNV-IVIG-treated animals had no viral RNA expression in either the spleen or brain at any time point. Impressively, IVIG reduced virus titres by >10-fold in the spleen on days 4 and 6 p.i. In contrast to the PBS-treated mice, virus was not detected in the brains of IVIG-treated mice until day 8 p.i. (Fig. 2b). Although virus replication in the brains of IVIG- and PBS-treated mice was similar at day 8 p.i., viral titres were reduced in brains from IVIG-treated mice at day 12 p.i. and the majority of these mice had completely controlled virus replication by day 21 p.i. (Fig. 2b). This result suggested that virus replication in the brain was not the major cause of death for these mice.

 

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Fig. 2.

IVIG controls virus replication in the CNS after high-dose WNV infection. BALB/c mice infected with 10⁵ p.f.u. WNV were treated with PBS, IVIG (25 mg) or WNV-IVIG (4 mg) at 24 h p.i. At the indicated time points, (a) spleens and (b) brains were analysed for WNV RNA by TaqMan PCR. All IVIG-treated mice died by day 23 p.i.; PBS-treated mice died by days 9–10 p.i. Data representative of three experiments; n = 3 mice per time point. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

CNS inflammation correlates with mortality following high-dose infection

To determine if CNS inflammation was causally associated with fatal encephalitis in mice inoculated with a high dose of WNV (10⁵ p.f.u.), we analysed cells infiltrating the CNS by flow cytometry. A schematic depicting the analysis of CD45^high and other infiltrating cells is shown in Fig. S1 (available in the online Supplementary Material). Fig. 3(a) shows that CD45^high cells were increased by ~8 % in the brains of PBS-treated WNV-infected mice on days 6 and 8 p.i. compared with IVIG-treated mice. Conversion of percentage CD45^high cells to numbers further highlights the around twofold increase in total CD45^high cells infiltrating the brains of PBS-treated mice compared with IVIG-treated mice (Fig. 3a). F4/80⁺ macrophages and CD11c⁺ DCs were the major cell types present in the CNS infiltrates, and were slightly increased in the brains of IVIG-treated mice compared with PBS-treated mice on days 6 and 8 p.i. (Fig. 3b, c). When presented as numbers, the difference in macrophages and DCs infiltrating the brains of PBS-treated mice compared with IVIG-treated mice on days 6–8 p.i. was shown to be 1.5- to twofold (Fig. 3b, c). Very low numbers of T- and B-cells were present in the brain between days 6 and 8 p.i., suggesting that lymphocytes did not contribute to CNS inflammation at these time points (data not shown).

 

Fig. 3.

Two doses of IVIG inhibit infiltration of Ly6C^high monocytes into the CNS of BALB/c mice infected with a high dose (10⁵ p.f.u.) of WNV. Mononuclear cells isolated from the brains of PBS- and IVIG-treated (25 mg) WNV-infected mice were analysed by flow cytometry for infiltrating cells at the indicated time points. Bar graphs depict percentage (L: left y-axis, depicted as bars) and number (#) (R: right y-axis, depicted as symbols) of (a) CD45^high infiltrating cells in the brain; (b) F4/80⁺ and (c) CD11c⁺ cells within the CD45^high infiltrating cells; and the pathogenic Ly6C^high subset within (d) F4/80⁺ macrophages or (e) CD11c⁺ cells. Data representative of two of three experiments; n = 3 mice per time point. (f) Percentage of the CD45^high infiltrating cells, Ly6C^high subset within F4/80⁺ macrophages or within CD11c⁺ Tip DCs isolated at day 21 p.i. from the brains of WNV-infected mice treated with a single (1×) or two (2×) doses of IVIG, as determined by flow cytometry. Data representative of two experiments; n = 3 mice. *P<0.05.

Prior reports implicated Ly6C^high monocytes as major determinants of the pathology of WNV encephalitis (Getts et al., 2008; Terry et al., 2012). Interestingly, we found that Ly6C^high monocytes were also the major inflammatory cell subset causally associated with HSV1 encephalitis and we demonstrated that IVIG treatment impeded CNS infiltration by these pathogenic cells (Ramakrishna et al., 2011). To determine if these macrophages and DCs were inflammatory, we stained the cells for several surface markers. We found that 50 % of F4/80⁺ macrophages and 66 % of CD11c⁺ DCs expressed high levels of Ly6C in the brains of PBS-treated mice (Fig. 3d, e). In contrast, only 11 % of F4/80⁺ macrophages and 13 % of CD11c⁺ DCs in the brains of IVIG-treated mice were Ly6C^high. Similarly, at day 8 p.i., 70 % of F4/80⁺ macrophages and 40 % of CD11c⁺ DCs in the brains of PBS-treated mice expressed high levels of Ly6C (Fig. 3d, e). However, Ly6C expression was reduced to 40 % on F4/80⁺ macrophages and 7 % on CD11c⁺ DCs in the brains of IVIG-treated mice (Fig. 3d, e). These data confirmed that IVIG suppressed the generation of inflammatory Ly6C^high macrophages and Tip DCs during WNV infection, similar to our observations in HSV1-infected mice (Ramakrishna et al., 2011). Unexpectedly, CD45^high infiltrates, specifically Ly6C^high F4/80⁺ macrophages (95 %) and CD11c⁺ DCs (90 %), were increased in the brains of IVIG-treated mice at day 12 p.i. (Fig. 3d, e), suggesting infiltration of inflammatory cells into the CNS had resumed. This renewed inflammation correlated with increased mortality in high-dose infected mice treated with a single dose of IVIG. Thus, for mice inoculated with a high dose of WNV, IVIG affords transient protection up to day 20 p.i. and thereafter the mice succumb to encephalitis as a consequence of renewed CNS infiltration by pathogenic Ly6C^high macrophages from around day 12 p.i.

Two doses of IVIG suppresses inflammation in mice infected with 10⁵ p.f.u. WNV

As resurgent CNS infiltration by inflammatory Ly6C^high macrophages and DCs resulted in death of all mice inoculated with a high dose of WNV that received a single IVIG dose (Fig. 3a–e), we investigated whether a second dose of IVIG would improve survival. Treatment with two doses of IVIG significantly augmented protection, resulting in survival of ~70 % of mice infected with a high dose of WNV (Fig. 1c). To determine if the second IVIG dose enhanced survival by suppressing inflammation, mononuclear cells were analysed by flow cytometry for CD45^high cells infiltrating the CNS of mice treated with either a single dose of IVIG or two doses of IVIG. As shown in Fig. 3(f), at day 21 p.i., CD45^high cells, particularly F4/80⁺ macrophages and CD11c⁺ DCs, were markedly increased in the brains of WNV-infected mice given a single dose of IVIG compared with mice given a second dose of IVIG on day 9 p.i. (48 versus 16 %). Flow cytometry analysis of F4/80⁺ macrophages and CD11c⁺ cells within the CNS revealed that >70 % of F4/80⁺ macrophages and >75 % of CD11c⁺ DCs in the CNS of mice given a single dose of IVIG expressed high levels of Ly6C (Fig. 3f). In contrast, only 10 % of F4/80⁺ macrophages and 15 % of CD11c⁺ DCs were Ly6C^high in the brains of high-dose infected mice given two doses of IVIG (Fig. 3f). Intriguingly, mice that were protected by a second dose of IVIG had increased levels of CD8 T- and B-cells (~60 %) infiltrating the brain at day 21 p.i., indicating that these cells might play an important role in protection (not shown). These results are consistent with two doses of IVIG enhancing survival of mice inoculated with a high dose of WNV by exerting a greater moderating effect on the massive inflammatory response arising in the brains of these mice (Fig. 1d).

We thank Don Forthal (Director, BSL3 Biosafety Training Program; Chief, UCI Division of Infectious Diseases) for access to the ABSL3 facility; Gary Landucci (Director, BSL3 Training and Development Manager, BSL3 Laboratory) for training and assistance throughout the project and Tran B. Phan for technical assistance in the ABSL3 laboratory; and Pamela Foster and Shelley Scanlon for financial management and administrative assistance, respectively. Funded by National Institutes of Health (U54 A1065359: Principal Investigator, Alan Barbour; subaward 2011-2716: Principal Investigator, E. Cantin).