Sequential polymicrobial infections lead to CNS inflammatory disease: possible involvement of bystander activation in heterologous immunity

Abstract

VV(PLP) is a recombinant vaccinia virus (VV) encoding myelin proteolipid protein (PLP) that has been used to investigate molecular mimicry and autoimmunity. Since virus infections can cause bystander activation, mice were first infected with VV(PLP), and later challenged with wild-type VV, lymphocytic choriomeningitis virus (LCMV), or murine cytomegalovirus (MCMV). Among the VV(PLP)-primed mice, only MCMV challenge induced significant Ki-67(+), CD3(+)T cell infiltration into the central nervous system (CNS) with a mild PLP antibody response. While MCMV alone caused no CNS disease, control VV-infected mice followed with MCMV developed mild CNS inflammation. Thus, heterologous virus infections can induce CNS pathology.

Fig. 1

(a–d) Neuropathology of VV_PLP-primed mice. Five weeks after VV_PLP infection, mice received injection of CFA/BP (a, b) or IL-12 (c, d). VV_PLP-primed mice had meningitis (a) comprised of CD3⁺ T cells (b), 3 weeks after CFA/BP challenge. Two months after IL-12 challenge, VV_PLP-primed mouse had perivascular cuffing (c) in the midbrain with T cell infiltration (d). (a, c) Luxol fast blue staining. (b, d) Immunohistochemistry against CD3. Magnification, (a, b) ×100; (c, d) ×150. (e) Spontaneous proliferation of MNC from VV_PLP-primed mice 3 weeks after immunomodulation. Five weeks after VV_PLP-sensitization, mice were injected with PBS, CFA alone, CFA and BP, IL-12 or VV_SC11. Three weeks after the second challenge, MNCs were isolated from the spleen and inguinal lymph nodes and cultured for 5 days without stimulation. CFA/BP challenged group showed high levels of spontaneous proliferation both in the spleen and the lymph node. Results are means of 2 lymphoproliferative assays using MNCs pooled from 2 to 3 mice.

Fig. 2

Neuropathology of VV_PLP-primed mice, 12 days after challenge with VV (a, b), LCMV (c, d) or MCMV (e, f). (a, b) In VV challenge, the brain parenchyma around the internal capsule (IC) appeared normal and only a few CD3⁺ T cells were seen in the meninges (b, inset). (c) In LCMV challenge, the brain parenchyma appeared normal and mild cell infiltration was seen in the meninges by routine Luxol fast blue staining. (d) However, a consecutive section immunohistostained with CD3 antibody showed substantial T cell infiltration (arrow) in the meninges and parenchyma. (e) In MCMV challenge, perivascular cuffing was obvious around the IC. (f) Infiltrates were predominantly composed of CD3⁺ T cells. (g) A consecutive section immunostained with Ki-67 antibody showed many infiltrating T cells were proliferating. (h) In MCMV challenge, only a few B cells were seen in the meninges (arrow). (a, c, e) Luxol fast blue staining. Immunohistochemistry against CD3 (b, d, f), Ki-67 (g), and B220 (h). Magnification, a–g, ×88; h, ×177; insets b–d, f, g, ×120.

Fig. 3

(a) Mean spleen weight was compared between VV_PLP-primed mice challenged with VV, LCMV or MCMV on day 12. Both MCMV- and LCMV- challenged mice had larger spleens than those of VV-challenged mice (*, P <0.05; **, P < 0.01, by ANOVA). Values are the means ± SEM. Each group consisted of 8 or 9 mice. (b–e) Serum antibody against PLP_139–151 was titrated by ELISA in VV_PLP-primed mice challenged by VV, LCMV MCMV or naïve mice.. (b) MCMV-challenged mice had higher PLP IgG (H+L) titers than VV-challenged mice and naïve mice (*, P < 0.05, ANOVA). LCMV-challenged mice also had higher PLP antibody titers than naïve mice (**, P < 0.01). (c) LCMV-challenged mice had higher IgG1 titer than MCMV-challenged mice (**, P < 0.01). (d) MCMV-challenged mice had higher anti-PLP IgG2c titers than VV and LCMV-challenged mice. (e) MCMV-challenged mice had higher anti-PLP IgG2c/G1 ratio than VV-challenged mice (**, P < 0.01). Values are the means ± SEM of optical density (OD)_{492 nm} at 1:256 (b) or 1: 128 (c–e) dilution of serum, harvested 12 days after VV, LCMV or MCMV challenge. Each group consisted of 5 to 9 mice.

Fig. 4

(a, b) Clinical course of MCMV challenged mice following PBS (○), VV_SC11 (●) and VV_PLP (▲) infection. Mice were first injected i.p. with PBS, VV_SC11, or VV_PLP on day –25. On day 0, mice were challenged with MCMV i.p. VV_PLP-primed mice showed significantly less weight increase and higher righting reflex score than PBS-injected mice (*, P < 0.05; **, P <0.01). Shown are means of 5 to 10 mice per group. (c) MCMV challenge of PBS-injected mice did not cause lesions in the white matter (WM) or the granular layer (gr) of the cerebellum. (d) MCMV challenge induced perivascular cuffing (arrow) in the cerebellar white matter of mice primed with VV_PLP. (e) One week after challenge, a CD3⁺ T cell was seen around the vessel in a PBS-injected mouse challenged with MCMV, (f) while MCMV challenge induced T cell infiltration (arrow) in the thalamus of VV_PLP-primed mice. (g) Two weeks after infection, PBS-injected mice challenged with MCMV had minor T cell infiltration (arrow) in the cerebellar white matter (WM), where a consecutive section, Fig. 4c, appeared normal. (h) A consecutive section of Fig. 4d showed substantial T cell infiltration in the parenchyma and perivascular area. (c, d) Luxol fast blue staining. (e–h) Immunohistochemistry against CD3. Magnification, (c, d) ×200; (e–h), ×100. (i) Serum PLP antibody responses. One (open column) and 2 weeks (closed column) after MCMV challenge, serum PLP_139–151 antibody titers were compared between mice injected with PBS, VV_SC11 or VV_PLP, 25 days before MCMV challenge. At 2 weeks after MCMV challenge, VV_SC11- and VV_PLP-primed mice had higher PLP antibody titers, compared with that of naïve mice (shown by dotted line) (**, P < 0.01). Shown are mean OD_{492 nm} at 1:256 dilution of sera ± SEM. Each group consisted of 5 to 10 mice.

Fig. 5

Adjuvant effect of TMEV challenge in VV_PLP-primed mice. (a) TMEV challenge of VV_SC11-primed mice caused no parenchymal lesions in the cerebellar white matter (WM). Gr: granular cell layers of the cerebellum. (b–d) TMEV challenge of VV_PLP-primed mice resulted in inflammation (b) comprised of CD3⁺ T cells (d) in the cerebellum, but viral antigen (c) was negative. (a, b) Luxol fast blue staining. (c, d) Immunohistochemistry against TMEV antigen (c) and CD3 (d). (a–d) Magnification, ×300.

Fig. 6

Weight change and neuropathology of mice infected i.c. with VV_PLP. (a) Mean body weight ± SEM of mice infected i.c. with VV_PLP. After infection, all mice showed significant weight loss during the first week of infection. Mice that survived this acute stage recovered completely with no obvious clinical signs during the 4-month observation period. (b) One week after i.c. VV_PLP infection, MNC and PMN infiltrates with amorphous material and fragmented nuclei (arrow) were seen in the subarachnoid space (sub), but no lesions were seen in the CNS parenchyma (pr). (c) Hydrocephalus was seen at 4 months after VV_PLP infection. Note severe dilatation of lateral ventricles (*), compared with normal mouse brain (inset). (b) Hematoxylin and eosin staining. (c) Luxol fast blue staining. Magnification: b, ×300; c × 10; c, inset × 4.