Cerebrospinal Fluid Biomarkers of Simian Immunodeficiency Virus Encephalitis : CSF Biomarkers of SIV Encephalitis

Abstract

Antiretroviral therapy has led to increased survival of HIV-infected patients but also increased prevalence of HIV-associated neurocognitive disorders. We previously identified YKL40 as a potential cerebrospinal fluid (CSF) biomarker of lentiviral central nervous system (CNS) disease in HIV-infected patients and in the macaque model of HIV encephalitis. The aim of this study was to define the specificity and sensitivity along with the predictive value of YKL40 as a biomarker of encephalitis and to assess its relationship to CSF viral load. CSF YKL40 and SIV RNA concentrations were analyzed over the course of infection in 19 SIV-infected pigtailed macaques and statistical analyses were performed to evaluate the relationship to encephalitis. Using these relationships, CSF alterations of 31 neuroimmune markers were studied pre-infection, during acute and asymptomatic infection, at the onset of encephalitis, and at necropsy. YKL40 CSF concentrations above 1122 ng/ml were found to be a specific and sensitive biomarker for the presence of encephalitis and were highly correlated with CSF viral load. Macaques that developed encephalitis had evidence of chronic CNS immune activation during early, asymptomatic, and end stages of infection. At the onset of encephalitis, CSF demonstrated a rise of neuroimmune markers associated with macrophage recruitment, activation and interferon response. CSF YKL40 concentration and viral load are valuable biomarkers to define the onset of encephalitis. Chronic CNS immune activation precedes the development of encephalitis while some responses suggest protection from CNS lentiviral disease.

Keywords: Biomarker; Cerebrospinal fluid; Encephalitis; Human immunodeficiency virus; Simian immunodeficiency virus; YKL40.

Fig. 1. Macaques with SIV encephalitis show SIV-infected microglial nodules and increased SIV viral load and YKL40 expression in frontal cortical tissue

Macaques were defined as having SIV encephalitis (SIVE) by histological examination of brain sections. Hematoxylin and eosin (H&E) stains show the presence of perivascular cuffing, a microglial nodule and multinuclear giant cells (a). Many cells in microglial nodules stain with an antibody against the SIV envelope protein (b). Microglial nodules and macrophage infiltrates can be observed with CD68 immunostains (c). SIV-infected macaques without encephalitis show no infiltrate (H&E) (d). In situ hybridization for YKL40 shows cells expressing YKL40 RNA surrounding a microglial nodule (e). YKL40 concentrations in frontal cortical tissue extracts of macaques with SIVE (red) are significantly elevated compared to SIV-infected macaques without encephalitis (blue), P = 0.002 (f). SIV RNA viral loads in frontal cortical tissue from macaques with SIVE (red) are higher than SIV-infected macaques without encephalitis (blue), P = 0.0007 (g). Correlation between frontal cortical SIV RNA viral load and frontal cortical YKL40 levels (h).

Fig. 2. Macaques that develop encephalitis have increased CSF YKL40 and SIV RNA concentrations

Preserved CSF samples obtained from 19 macaques from prior studies were analyzed for YKL40 concentrations (a) and SIV viral load (b). Macaques that were determined to have presence of SIVE based on post-mortem histological findings are shown in red while SIV-infected non-encephalitic macaques are depicted in blue. The dashed lines (a and b) represent the concentration determined statistically likely to be associated with encephalitis, 1122 ng/ml for CSF YKL40 concentrations and 1.65 × 10⁵ copies/ml CSF for SIV CSF viral load. Necropsy analyses of terminally ill SIV-infected non-encephalitic macaques (blue) show a significantly lower CSF YKL40, P = 0.0006 (c) and CSF SIV viral load, P = 0.0479 (d) than macaques with SIVE. The black lines represent the median.

Fig. 3. CSF YKL40 and SIV RNA concentrations are tightly correlated in macaques that develop encephalitis

Spearman rank correlation of CSF YKL40 concentrations and CSF SIV viral load in PTM that develop encephalitis (a) (red) and SIVE-infected non-encephalitic PTM (b) (blue).

Fig. 4. CSF YKL40 concentrations increase concomitantly with CSF viral load in macaques that developed SIV encephalitis

CSF SIV RNA concentrations (blue, left axis) are graphed separately for each macaque to show the relationship with CSF YKL40 concentrations (purple, right axis) over the course of infection. 10 macaques that developed encephalitis (a) showed YKL40 increases concomitant with elevated CSF viral load while SIV-infected terminally ill non-encephalitic macaques (b) generally maintain similar CSF YKL40 levels throughout infection.

Fig. 5. Some neuroimmune markers indicate chronic immune activation at all stages of infection in macaques that develop encephalitis

Multiplex quantitation of 31 cytokines present in the CSF was performed on samples from baseline (d0), acute infection (d10 and d14), asymptomatic infection (d28 and d42), development of encephalitis (rise of YKL40), and at necropsy (nec). Compared to SIV-infected non-encephalitic macaques (blue), PAI-1 (a), IL-6 (b), VEGF (c), and CCL2 (d) were elevated in macaques that developed SIVE (red) during acute infection (except VEGF), and when encephalitis developed. Bars represent median concentrations of the indicated cytokine.

Fig. 6. The majority of elevated neuroimmune markers became elevated as encephalitis developed and were associated with macrophage recruitment and activation

Multiplex quantitation of 31 cytokines present in the CSF was performed on samples from baseline (d0), acute infection (d10 and d14), asymptomatic infection (d28 and d42), development of encephalitis (rise of YKL40), and at necropsy (nec). IL-1β (a), MIF (b), IL-8 (c), IFN-γ (d), CXCL9 (e), CXCL11 (f), CCL4 (g), TGF-β (h) were elevated when encephalitis (red) developed or shortly before. SIV-infected non-encephalitic macaques (blue) show little elevation of these markers. Bars represent median concentrations of the indicated cytokine.

Fig. 7. Non-encephalitic SIV-infected macaques have detectable CSF IL-10 suggesting a protective response

Multiplex quantitation of 31 cytokines present in the CSF was performed on samples from baseline (d0), acute infection (d10 and d14), asymptomatic infection (d28 and d42), development of encephalitis (rise of YKL40), and at necropsy (nec). Bars represent median concentrations of IL-10.

Fig. 8. Macaques with elevated CSF YKL40 concentrations have elevated CSF neurofilament light chain concentrations

CSF neurofilament light chain concentrations (NFL) were measured by ELISA at baseline (d0), day 14 post-infection (d14), day 42 post-infection (d42) and necropsy. Results were compared to CSF YKL40 concentrations in the same samples.

Fig. 9. Neuroimmune markers in frontal cortical tissue protein extracts

Multiplex quantitation of 10 cytokines in frontal cortical tissue protein extracts was performed. Compared to SIV-infected macaques without encephalitis (blue), IL-6 (a), IFN-γ (b), VEGF (c), CXCL9 (d), CCL2 (e), CXCL11 (f), IL-1β (g), CCL4 (h), and IL-8 (i) were elevated in macaques that developed SIVE (red). IL-10 was similar in SIV-infected macaques with and without encephalitis (j). Bars represent median concentration of indicated cytokine.