TNF–alpha, Macrophages, and Virus Spread in the SCN

Abstract

Abstract: : Purpose: To investigate the role of TNF–α and macrophages in the eyes and brain of HSV–1 infected BALB/c mice. Methods: Following uniocular anterior chamber (AC) inoculation of HSV–1, virus spreads to the suprachiasmatic nuclei (SCN) and at day 5 p.i., TNF–α is detected in this area. To determine if macrophages are responsible for production of TNF–α in the SCN and the injected eye, 2×10⁴ of the KOS strain of HSV–1 was injected into the AC of euthymic BALB/c mice. To deplete macrophages, mice in Group 1 (experimental) were injected intravenously with 0.2 ml of dichloromethylene–biphosphate liposomes (Cl₂MBP, clodronate) on day –1, 2, and 5. Mice in Group 2 (control) were injected intravenously with 0.2 ml of liposomes containing PBS on day –1, 2, and 5. On day 0, 2×10⁴ PFU HSV–1 (KOS) in a volume of 2 µl was injected in the AC of each mouse. Mice from both groups were sacrificed at day 2, 3, 4, 5, 6, and 7 p.i. and the eyes, brain, and spleen were removed. Fluorescence immunohistochemistry was used to identify and determine the location of HSV–1, macrophages, and TNF–α in the ipsilateral eye, contralateral eye and SCN of HSV–1 infected mice. The titer of virus in the ipsilateral eye, the ipsilateral SCN, and the contralateral SCN was determined by plaque assay. Depletion of macrophages in HSV–1 infected and non–infected mice was determined by flow cytometry. Results: Double staining for macrophages and for TNF–α revealed that macrophages were the primary source of TNF–α in the injected eye and SCN. Virus staining of the contralateral eye revealed increased virus spread in clodronate–treated mice. The titer of virus in the injected eye and ipsilateral SCN was not significantly different between clodronate–treated and control mice. However, the titer of virus in the contralateral SCN was significantly increased in clodronate treated mice compared with controls on day 5 p.i. (9.09 × 10³ vs. 3.74 × 10² PFU, p<0.05), and day 7 p.i. (1.52 × 10³ vs. 1.41 × 10², p<0.05 ). The titer of virus in the contralateral eye was also significantly increased in clodronate treated mice compared with controls on day 7 p.i. (9.65 × 10⁴ vs. 6.07 × 10³, p<0.05). Flow cytometric analysis of splenocytes revealed significantly lower numbers of systemic macrophages in clodronate treated mice compared with controls. Conclusions: TNF–α, produced by macrophages that infiltrate the contralateral SCN following uniocular AC inoculation of HSV–1, plays a role in limiting virus spread from the SCN to the optic nerve and retina of the injected eye which in turn prevents HSV–1 infection of the retina of the injected eye.

Keywords: herpes simplex virus • immunomodulation/immunoregulation • immunohistochemistry