Fig 1.
IFNγ and IL-10 dominate the cytokine profile of T cells isolated from HSV-infected WT mice.
(A) Mononuclear cells isolated from brain stem (BS, top), cervical lymph nodes (CLN, middle) or spleen (bottom) on day 6 or 14 pi from HSV-infected WT mice were probed for IL-10 and IFNγ by intracellular cytokine staining (ICS). Representative FACS plots show cells isolated at day 14 pi. All flow cytometry plots show antigen-stimulated cells (+) except for top left panel “for BS (-)”. Bar plots depict % IL-10- or IFNγ-secreting CD45high, CD4 or CD8 T cells in the (B) BS, (C) CLN or (D) spleen at day 6 and 14 pi. Data representative of 2 (day 14 pi) -3 (day 6 pi) experiments are shown as mean +/- SD (n = 3–4 mice / time point).
Fig 2.
IFNγ is required for survival but not control of HSV replication.
(A) HSV-infected WT, IL-10KO, GKO and Rag-/- mice were monitored for survival from HSE; data are representative of 3–5 experiments (n = WT: 40, IL-10KO: 22, GKO: 36, Rag-/-: 28 mice). ****p<0.0001; *p<0.05. (B) BS was isolated at the indicated time points from HSV-infected WT, IL-10KO, GKO or Rag-/- mice and virus titers determined by plaque assay (n = 8–10 mice per time point), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. p<0.0001 for all time points for Rag-/- mice compared to WT and IL-10KO mice. All Rag-/- mice had died by day 16 pi.
Fig 3.
Neutrophil output from BM and CNS invasion is augmented in the absence of IFNγ.
Representative flow cytometry plots depicting the gating strategy used to distinguish neutrophils (PMN) and inflammatory monocytes (IM) in the (A) BS and (B) blood of HSV-infected GKO (top row) and WT (bottom row) mice: (A) BS mononuclear cells isolated at day 4 pi show CD45high infiltrating leukocytes, CD45high SSChigh CD11b+ CD115- Ly6Ghigh neutrophils (1. PMN) and F480+ CD115+ Ly6Chigh monocytes (2. IMs); and (B) blood mononuclear cells at day 6 pi show SSChigh CD11b+ CD115- Ly6G+ Ly6Cint PMN and CD115+ Ly6G- Ly6Chigh IMs. Bar plots depicting (C) % (left y-axis) and # (right y-axis) of CD45high infiltrating cells in the BS of WT, IL-10KO, GKO and Rag-/- mice at day 6 pi, (D) % (left y-axis) and # (right y-axis) of CD115+ F480+/lo monocytes / macrophages within the BS CD45high infiltrates, (E) % (left y-axis) and # (right y-axis) of Ly6Chigh IMs within the BS CD45high F480+ monocytes / macrophages. Line plots showing (F) % (left y-axis) and # (right y-axis) of Ly6G+ PMN within BS CD45high infiltrates in GKO and WT mice at the indicated time points, (G) % CD11b+ Ly6G+ neutrophils within BM (1 leg = femur + tibia) at the indicated time points, (H) % CD11b+ Ly6G+ PMN or Ly6Chigh IMs in the blood of GKO and WT mice, and (I) 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.
Fig 4.
Regulatory CD4 T cells are impaired in the absence of IFNγ.
FoxP3+ (top) and ICOS+ (bottom) CD4 T cells (left) in (A) CLN and (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). (C) % 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. (D) 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 (E) CLN or (F) 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).
Fig 5.
GKO Neutrophils have a GDSC phenotype and suppress T cell proliferation.
(A-C), Spleen cells isolated at day 6 pi from HSV infected WT or GKO mice were labeled with CFSE and stimulated with HK-HSV to determine effector (e) T cell proliferation at indicated times post culture. GKO or WT T cells that have divided at least one time are depicted as line plots as % proliferating (A) eCD4, (B) eCD8 T or (C) H-2Kb HSV-1 specific gB498 e-tetramer+ (e-Tet+) CD8 T cells. Linear regression analysis showed significant deviation of slope for GKO CD4 (P = 0.041), GKO CD8 (P = 0.0232) and GKO gB498 tet+ CD8 (P = 0.0171) but not WT T cells. (D-E) GKO Innate cells including PMN (CD11b+ Ly6G+) or M/DC (Ly6G- CD11b+ / CD11c+ cells) isolated from blood of GKO mice at day 6 pi cultured with CFSE labeled GKO or WT T cells and CD19+ B cells isolated from spleen cells as in A-C were assessed for suppression of proliferation of (D) eCD4 and eCD8 T cells and (E) memory (m) CD4 and mCD8 T cells and m-H-2Kb HSV-1 gB498-505 tetramer+ (m-Tet+) CD8 T cells. Effector (e) T cells were obtained from spleens of HSV infected WT or GKO mice at day 6 pi while memory (m) T cells were obtained from spleens of immunized mice at day 21 pi. Data is combined from 2 experiments, n = 6 mice per group. PMN: Neutrophils, M/DC: Monocytes/Dendritic cells.
Fig 6.
HSV infection of GKO mice elevates neutrophil related cytokines and chemokines.
(A) G-CSF, (B) IL-6, (C) CXCL1 and (D) CXCL2 levels were determined in the sera of WT or GKO mice at the indicated time points using a multiplex ELISA (n = 3–6 mice per time point). G-CSF: ****p<0.0001 for days 4, 6 and 8 pi; IL-6- all time points except day 6 pi and CXCL1: ****p<0.0001; CXCL2- all time points except day 6 pi: *p = 0.02.
Fig 7.
Depletion of G-CSF protects GKO mice by terminating neutrophil responses.
(A) GKO mice were depleted of G-CSF or neutrophils with 3 doses of αG-CSF or αLy6G Ab (250 μg) respectively on days 0, 1 and 4 pi, and observed for survival following HSV infection. ***p<0.0007; ****p<0.0001. Data representative of 2 (for G-CSF)—3 (for Ly6G) experiments (n = 10–20 mice). (B) % Cell subsets isolated from blood mononuclear cells of GKO or αG-CSF-treated GKO mice at day 6 pi, ****p<0.0001, *p<0.05. (C) % (left y-axis) and # (right y-axis) of CD45high cells in the BS of WT, GKO or αG-CSF Ab-treated GKO mice at day 6 pi. Numbers on top of bars indicate % CD45high cells in BS and stacked bars within the CD45high bar depict the proportions of CD3+ and CD11b+ or other cells. (D) % (left y-axis) and # (right y-axis) of CD11b+ cells within BS CD45high infiltrates in WT, GKO and α G-CSF Ab-treated GKO mice. Numbers on top indicate % CD11b+ cells and stacked bars within the CD11b bar depict the proportions of SSClow CD115+ monocytes / macrophages or SSChigh Ly6G+ neutrophils. (E) Percent degranulating (CD107a+b) Ly6G+ neutrophils within CD45high cells isolated from the BS of WT, GKO and αG-CSF Ab-treated GKO mice at day 6 pi, # in parathesis indicate total number of degranulating PMN in the BS. (F) Intracellular staining for G-CSF by splenocytes (left plot) and IL-10 by CD4 T cells in the blood (right plot) of WT, GKO and αG-CSF Ab-treated GKO mice at day 6 pi. # in parenthesis indicate total number of IL-10 secreting CD4 T cells. (G) Intracellular G-CSF (top) and IL-10 (bottom) secretion by BS cells isolated from GKO (left) and αG-CSF Ab-treated GKO mice (right) at day 6 pi.
Fig 8.
IFNγ controls neutrophil responses via apoptosis and SOCS3 expression.
(A) Relative SOCS3 expression from Ly6G+ neutrophils (PMN) isolated from BM and blood (Bld) of WT and GKO mice at day 6 pi, as measured by RT-PCR; levels are relative to PMN isolated from uninfected mice. Blood-derived PMN treated with recombinant IFNγ in vitro for 30 min were compared to untreated neutrophils for SOCS3 expression. Data are representative of 2–3 experiments; *p = 0.013; **p = 0.0035. (B) Bar plots depict Annexin V+ PMN in the blood of WT and GKO mice at days 6 and 8 pi and BS at day 8 pi. Data are representative of 2 experiments with 2–3 mice per group. **p = 0.0011. (C & D) FACS plots show intracellular staining for IL-10 (left plots) and IL-17 (right plots) by γδ T cells isolated from (C) CLN and (D) spleen of GKO mice at day 14 pi. Red dots indicate no antigenic stimulation; blue dots indicate cells stimulated with PMA + ionomycin + HK-HSV.