Clodronate is not protective in lethal viral encephalitis despite substantially reducing inflammatory monocyte infiltration in the CNS

Abstract

Bone marrow (BM)-derived monocytes induce inflammation and tissue damage in a range of pathologies. In particular, in a mouse model of West Nile virus (WNV) encephalitis (WNE), nitric oxide-producing, Ly6C^hi inflammatory monocytes from the BM are recruited to the central nervous system (CNS) and contribute to lethal immune pathology. Reducing the migration of these cells into the CNS using monoclonal antibody blockade, immune-modifying particles or CSF-1R inhibitors reduces neuroinflammation, improving survival and/or clinical outcomes. Macrophages can also be targeted more broadly by administration of clodronate-encapsulated liposomes, which induce apoptosis in phagocytes. In this study, clodronate reduced the inflammatory infiltrate by 70% in WNE, however, surprisingly, this had no effect on disease outcome. More detailed analysis demonstrated a compensatory increase in neutrophils and enhanced activation status of microglia in the brain. In addition, we observed increased numbers of Ly6C^hi BM monocytes with an increased proliferative capacity and expression of SCA-1 and CD16/32, potentially indicating output of immature cells from the BM. Once in the brain, these cells were more phagocytic and had a reduced expression of antigen-presenting molecules. Lastly, we show that clodronate also reduces non-myeloid cells in the spleen and BM, as well as ablating red blood cells and their proliferation. These factors likely impeded the therapeutic potential of clodronate in WNE. Thus, while clodronate provides an excellent system to deplete macrophages in the body, it has larger and broader effects on the phagocytic and non-phagocytic system, which must be considered in the interpretation of data.

Keywords: CNS infection; bone marrow monopoiesis; clodronate; macrophage depletion; monocyte-derived cells; monocyte-mediated inflammation; neuroinflammation; virus-induced encephalitis.

Figure 1

Clodronate reduces inflammatory monocyte infiltration without improving clinical outcomes in lethal viral infection. (A) Schematic of experimental design. Mice were infected with WNV, injected intravenously with clodronate-encapsulated liposomes (CLO) at 5 dpi and culled at 7 dpi. (B) Flow cytometry dot plots showing the frequency of neutrophils, infiltrating MCs and microglia in the brains of PBS- (blue outline) and CLO-treated (green outline) mice at 7 dpi. Numbers on dot plots represent the frequency of each subset out of the total leukocyte population in the brain. (C, D) Number (C) and tSNE plot (D) of CD45⁺ leukocyte subsets in the brain of PBS- and CLO-treated mice at 7 dpi. (E-I) Weight (E), percent of weight lost at 6 and 7 dpi (F), clinical score at 7 dpi (G) and survival of mice infected with a lethal dose (lethal dose 100%, LD₁₀₀) (H) or sublethal dose (lethal dose 50%, LD₅₀) of WNV (I) and treated with PBS or CLO. (J, K) Viral load in the brain of 7 dpi mice treated with PBS or CLO, as determined by the number of plaque-forming units (PFU) using a virus plaque assay (J) and Wnv expression with qPCR (K). (L) Expression of select chemokines, pro-inflammatory and anti-inflammatory genes at 7 dpi in the brain of mice treated with PBS or CLO. Data is presented as mean ± SEM from 1-4 independent experiments with at least four mice per group, except the survival study which used a minimum of 25 mice per group ns, not statistically significant.

Figure 2

Clodronate abrogates splenic monocyte populations which are potentially pathogenic in WNV infection. (A) tSNE plot showing CD45⁺ splenocytes isolated from PBS- and clodronate (CLO)-treated mice at 7 dpi. (B) Number of myeloid subsets in the spleen of PBS- and CLO-treated mice at 7 dpi. (C) Number of Ly6C^hi monocytes in the spleen over the course of WNV infection. (D) Percent change in BrdU incorporation (proliferation) of splenic monocytes at 3-7 dpi, relative to 0 dpi (i.e., 100%). (E) Number of cells in the brain of splenectomized and WNV-infected mice at 7 dpi. Mice were splenectomized 14 days prior to infection and culled at 7 dpi. (F-H) Percent of weight lost (F) and survival of non-splenectomized and splenectomized mice infected with a lethal dose (lethal dose 100%, LD₁₀₀) (G) or sublethal dose (lethal dose 50%, LD₅₀) of WNV (H). Data is presented as mean ± SEM from 2-4 independent experiments with at least 4 mice per group, except the survival study which used a minimum of 18 mice per group.

Figure 3

Clodronate-encapsulated liposomes promote a “left-shift” in the bone marrow. (A) tSNE plot showing BM cells isolated from PBS- and clodronate (CLO)-treated, WNV-infected mice at 7 dpi. (B) Number of cell subsets in the BM of PBS- and CLO-treated mice at 7 dpi. (C) Geometric mean of select markers on Ly6C^hi mature BM monocytes from PBS- and CLO-treated mice at 7 dpi. (D) Frequency of BrdU⁺ (proliferating) cell subsets in the bone marrow of PBS- and CLO-treated mice at 7 dpi. Data is presented as mean ± SEM from two-three independent experiments with at least three mice per group.

Figure 4

Clodronate alters effector functions of MCs infiltrating the virus-infected CNS. (A) Schematic of experimental design. Mice were infected with WNV, injected intravenously with clodronate (CLO)-encapsulated liposomes at 4 dpi, 4 and 5 dpi, 5 dpi or 4, 5 and 6 dpi and euthanized at 5, 6 and 7, respectively. (B-D) Number of Ly6C^hi inflammatory MCs in the brain of mice injected with 1, 2 or 3 doses of CLO and sacrificed on 5 (B), 6 (C) and 7 (D), respectively. (E) Frequency of DAF-FM⁺ neutrophils, Ly6C^hi MCs, microglia (MG)-like MCs and microglia in the brain of mice treated intravenously with CLO at 5 dpi and culled at 7 dpi. (F, H) Percent difference in the geometric mean of select markers upregulated or downregulated by Ly6C^hi MCs and microglia in CLO-treated mice compared to PBS-treated mice at 7 dpi. A Mann-Whitney test comparing geometric means for each marker from both groups was used to generate p values. (G, I) Histograms showing the expression of select markers on Ly6C^hi MCs (G) and microglia (I) in mice treated with PBS or CLO and culled at 7 dpi. (J) Flow cytometry dot plot showing the frequency of IMP⁺ leukocytes in the brain of WNV-infected mice at 7 dpi treated with IMP or IMP and CLO. (K) Number of IMP⁺ cells in the brain of WNV-infected mice treated with IMP or IMP and CLO. (L) Survival of mice infected with a sublethal dose (LD₅₀) of WNV and treated with IMP and/or CLO or PBS. Data is presented as mean ± SEM from two-three independent experiments with at least six mice per group, except the survival study which used a minimum of 10 mice per group.