Passively Administered Pooled Human Immunoglobulins Exert IL-10 Dependent Anti-Inflammatory Effects that Protect against Fatal HSV Encephalitis

HSV-1 is the leading cause of sporadic encephalitis in humans. HSV infection of susceptible 129S6 mice results in fatal encephalitis (HSE) caused by massive inflammatory brainstem lesions comprising monocytes and neutrophils. During infection with pathogenic microorganisms or autoimmune disease, IgGs induce proinflammatory responses and recruit innate effector cells. In contrast, high dose intravenous immunoglobulins (IVIG) are an effective treatment for various autoimmune and inflammatory diseases because of potent anti-inflammatory effects stemming in part from sialylated IgGs (sIgG) present at 1–3% in IVIG. We investigated the ability of IVIG to prevent fatal HSE when given 24 h post infection. We discovered a novel anti-inflammatory pathway mediated by low-dose IVIG that protected 129S6 mice from fatal HSE by modulating CNS inflammation independently of HSV specific antibodies or sIgG. IVIG suppressed CNS infiltration by pathogenic CD11b+ Ly6Chigh monocytes and inhibited their spontaneous degranulation in vitro. FcγRIIb expression was required for IVIG mediated suppression of CNS infiltration by CD45+ Ly6Clow monocytes but not for inhibiting development of Ly6Chigh monocytes. IVIG increased accumulation of T cells in the CNS, and the non-sIgG fraction induced a dramatic expansion of FoxP3+ CD4+ T regulatory cells (Tregs) and FoxP3− ICOS+ CD4+ T cells in peripheral lymphoid organs. Tregs purified from HSV infected IVIG treated, but not control, mice protected adoptively transferred mice from fatal HSE. IL-10, produced by the ICOS+ CD4+ T cells that accumulated in the CNS of IVIG treated, but not control mice, was essential for induction of protective anti-inflammatory responses. Our results significantly enhance understanding of IVIG's anti-inflammatory and immunomodulatory capabilities by revealing a novel sIgG independent anti-inflammatory pathway responsible for induction of regulatory T cells that secrete the immunosuppressive cytokine IL-10 and further reveal the therapeutic potential of IVIG for treating viral induced inflammatory diseases

Bacteroides fragilis polysaccharide A induces IL-10 secreting B and T cells that prevent viral encephalitis

The gut commensal Bacteroides fragilis or its capsular polysaccharide A (PSA) can prevent various peripheral and CNS sterile inflammatory disorders. Fatal herpes simplex encephalitis (HSE) results from immune pathology caused by uncontrolled invasion of the brainstem by inflammatory monocytes and neutrophils. Here we assess the immunomodulatory potential of PSA in HSE by infecting PSA or PBS treated 129S6 mice with HSV1, followed by delayed Acyclovir (ACV) treatment as often occurs in the clinical setting. Only PSA-treated mice survived, with dramatically reduced brainstem inflammation and altered cytokine and chemokine profiles. Importantly, PSA binding by B cells is essential for induction of regulatory CD4+ and CD8+ T cells secreting IL-10 to control innate inflammatory responses, consistent with the lack of PSA mediated protection in Rag−/−, B cell- and IL-10-deficient mice. Our data reveal the translational potential of PSA as an immunomodulatory symbiosis factor to orchestrate robust protective anti-inflammatory responses during viral infections

The Immune Response to Herpes Simplex Virus Type 1 Infection in Susceptible Mice is a Major Cause of CNS Pathology Resulting in Fatal Encephalitis

This study was undertaken to investigate possible immune mechanisms in fatal HSV-1 encephalitis (HSE) after HSV-1 corneal inoculation. Susceptible 129S6 (129) but not resistant C57BL/6 (B6) mice developed intense focal inflammatory brainstem lesions of primarily F4/80+ macrophages and Gr-1+ neutrophils detectable by MRI as early as day 6 post infection (PI). Depletion of macrophages and neutrophils significantly enhanced survival of infected 129 mice. Immunodeficient B6 (IL-7R-/-Kitw41/w41) mice lacking adaptive cells (B6-E mice) transplanted with 129 bone marrow showed significantly accelerated fatal HSE compared to B6-E mice transplanted with B6 marrow or control non-transplanted B6-E mice. In contrast, there was no difference in ocular viral shedding in B6-E mice transplanted with 129 bone marrow or B6 bone marrow. Acyclovir treatment of 129 mice beginning day 4 PI (24 h after HSV-1 first reaches the brain stem) reduced nervous system viral titers to undetectable levels but did not alter brainstem inflammation or mortality. We conclude that fatal HSE in 129 mice results from widespread damage in the brainstem caused by destructive inflammatory responses initiated early in infection by massive infiltration of innate cells

Bacteroides fragilis polysaccharide A induces IL-10 secreting B and T cells that prevent viral encephalitis

The gut commensal Bacteroides fragilis or its capsular polysaccharide A (PSA) can prevent various peripheral and CNS sterile inflammatory disorders. Fatal herpes simplex encephalitis (HSE) results from immune pathology caused by uncontrolled invasion of the brainstem by inflammatory monocytes and neutrophils. Here we assess the immunomodulatory potential of PSA in HSE by infecting PSA or PBS treated 129S6 mice with HSV1, followed by delayed Acyclovir (ACV) treatment as often occurs in the clinical setting. Only PSA-treated mice survived, with dramatically reduced brainstem inflammation and altered cytokine and chemokine profiles. Importantly, PSA binding by B cells is essential for induction of regulatory CD4+ and CD8+ T cells secreting IL-10 to control innate inflammatory responses, consistent with the lack of PSA mediated protection in Rag−/−, B cell- and IL-10-deficient mice. Our data reveal the translational potential of PSA as an immunomodulatory symbiosis factor to orchestrate robust protective anti-inflammatory responses during viral infections

Effects of Acyclovir and IVIG on Behavioral Outcomes after HSV1 CNS Infection

Herpes simplex virus 1 (HSV) encephalitis (HSE) has serious neurological complications, involving behavioral and cognitive impairments that cause significant morbidity and a reduced quality of life. We showed that HSE results from dysregulated central nervous system (CNS) inflammatory responses. We hypothesized that CNS inflammation is casually involved in behavioral abnormalities after HSE and that treatment with ACV and pooled human immunoglobulin (IVIG), an immunomodulatory drug, would improve outcomes compared to mice treated with phosphate buffered saline (PBS) or ACV alone. Anxiety levels were high in HSV-infected PBS and ACV-treated mice compared to mice treated with ACV + IVIG, consistent with reports implicating inflammation in anxiety induced by lipopolysaccharide (LPS) or stress. Female, but not male, PBS-treated mice were cognitively impaired, and unexpectedly, ACV was protective, while the inclusion of IVIG surprisingly antagonized ACV’s beneficial effects. Distinct serum proteomic profiles were observed for male and female mice, and the antagonistic effects of ACV and IVIG on behavior were paralleled by similar changes in the serum proteome of ACV- and ACV + IVIG-treated mice. We conclude that inflammation and other factors mediate HSV-induced behavioral impairments and that the effects of ACV and IVIG on behavior involve novel mechanisms

IFNγ inhibits G-CSF induced neutrophil expansion and invasion of the CNS to prevent viral encephalitis

<div><p>Emergency hematopoiesis facilitates the rapid expansion of inflammatory immune cells in response to infections by pathogens, a process that must be carefully regulated to prevent potentially life threatening inflammatory responses. Here, we describe a novel regulatory role for the cytokine IFNγ that is critical for preventing fatal encephalitis after viral infection. HSV1 encephalitis (HSE) is triggered by the invasion of the brainstem by inflammatory monocytes and neutrophils. In mice lacking IFNγ (GKO), we observed unrestrained increases in G-CSF levels but not in GM-CSF or IL-17. This resulted in uncontrolled expansion and infiltration of apoptosis-resistant, degranulating neutrophils into the brainstem, causing fatal HSE in GKO but not WT mice. Excessive G-CSF in GKO mice also induced granulocyte derived suppressor cells, which inhibited T-cell proliferation and function, including production of the anti-inflammatory cytokine IL-10. Unexpectedly, we found that IFNγ suppressed G-CSF signaling by increasing SOCS3 expression in neutrophils, resulting in apoptosis. Depletion of G-CSF, but not GM-CSF, in GKO mice induced neutrophil apoptosis and reinstated IL-10 secretion by T cells, which restored their ability to limit innate inflammatory responses resulting in protection from HSE. Our studies reveals a novel, complex interplay among IFNγ, G-CSF and IL-10, which highlights the opposing roles of G-CSF and IFNγ in regulation of innate inflammatory responses in a murine viral encephalitis model and reveals G-CSF as a potential therapeutic target. Thus, the antagonistic G-CSF-IFNγ interactions emerge as a key regulatory node in control of CNS inflammatory responses to virus infection.</p></div

Regulatory CD4 T cells are impaired in the absence of IFNγ.

<p>FoxP3⁺ (top) and ICOS⁺ (bottom) CD4 T cells (left) in <b>(A)</b> CLN and <b>(B)</b> spleen of WT mice at day 8 pi. FoxP3⁺ (top row) and FoxP3^- (bottom row) CD4 T cells probed for CD62L and CD25 expression (middle) and ICOS expression (right). <b>(C)</b> % FoxP3 Tregs within splenic CD4 T cells isolated from WT or GKO mice at the indicated time points; data representative of 2–3 experiments are shown as mean ± SD. <b>(D)</b> FoxP3⁺ CD25⁺ CD4 T cells (Tregs, 5x10⁶) or ICOS⁺ (10⁷) CD4 T cells isolated from naïve WT (nWT), infected WT (iWT) or GKO (iGKO) mice at day 8 pi were adoptively transferred to naïve WT recipients, which were then challenged with HSV but did not receive IVIG, and monitored for survival (n = 6–8 mice). **p = 0.002. CD4 (top row) or γδ (bottom row) T cells isolated from <b>(E)</b> CLN or <b>(F)</b> spleen of GKO mice at day 6 pi were probed for IL-10 and IL-17 by ICS. Blue dots indicate no antigenic stimulation; red dots indicate cells stimulated with PMA + ionomycin + heat-killed HSV (HK-HSV).</p

Neutrophil output from BM and CNS invasion is augmented in the absence of IFNγ.

<p>Representative flow cytometry plots depicting the gating strategy used to distinguish neutrophils (PMN) and inflammatory monocytes (IM) in the <b>(A)</b> BS and <b>(B)</b> blood of HSV-infected GKO (top row) and WT (bottom row) mice: <b>(A)</b> BS mononuclear cells isolated at day 4 pi show CD45^high infiltrating leukocytes, CD45^high SSC^high CD11b⁺ CD115^- Ly6G^high neutrophils (1. PMN) and F480⁺ CD115⁺ Ly6C^high monocytes (2. IMs); and <b>(B)</b> blood mononuclear cells at day 6 pi show SSC^high CD11b⁺ CD115^- Ly6G⁺ Ly6C^int PMN and CD115⁺ Ly6G^- Ly6C^high IMs. Bar plots depicting <b>(C)</b> % (left y-axis) and # (right y-axis) of CD45^high infiltrating cells in the BS of WT, IL-10KO, GKO and Rag^-/- mice at day 6 pi, <b>(D)</b> % (left y-axis) and # (right y-axis) of CD115⁺ F480^+/lo monocytes / macrophages within the BS CD45^high infiltrates, <b>(E)</b> % (left y-axis) and # (right y-axis) of Ly6C^high IMs within the BS CD45^high F480⁺ monocytes / macrophages. Line plots showing <b>(F)</b> % (left y-axis) and # (right y-axis) of Ly6G⁺ PMN within BS CD45^high infiltrates in GKO and WT mice at the indicated time points, <b>(G)</b> % CD11b⁺ Ly6G⁺ neutrophils within BM (1 leg = femur + tibia) at the indicated time points, <b>(H)</b> % CD11b⁺ Ly6G⁺ PMN or Ly6C^high IMs in the blood of GKO and WT mice, and <b>(I)</b> the ratio of PMN to IM in the blood of GKO and WT mice at the indicated time points. Data representative of 2–3 experiments are shown as mean +/- SD; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.</p