A distinct brain pathway links viral RNA exposure to sickness behavior

Sickness behaviors and metabolic responses to invading pathogens are common to nearly all types of infection. These responses evolved to provide short-term benefit to the host to ward off infection, but impact on quality of life, and when prolonged lead to neurodegeneration, depression, and cachexia. Among the major infectious agents, viruses most frequently enter the brain, resulting in profound neuroinflammation. We sought to define the unique features of the inflammatory response in the brain to these infections. We demonstrate that the molecular pathway defining the central response to dsRNA is distinct from that found in the periphery. The behavioral and physical response to the dsRNA mimetic poly I:C is dependent on signaling via MyD88 when it is delivered centrally, whereas this response is mediated via the TRIF pathway when delivered peripherally. We also define the likely cellular candidates for this MyD88-dependent step. These findings suggest that symptom management is possible without ameliorating protective antiviral immune responses.


Mice
Knockout mice of TLR2, TLR3, TLR4, MDA5, IL-1R, MyD88, TRIF, IPS-1 and age-sex-matched C57BL/6J wild type (WT) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). TLR9 KO mice were provided by Dr. Mary Stenzel-Poore. Double knockout (DKO) mice of TRIF/IPS-1 were generated by intercrossing TRIFKO and IPS-1KO mice as described previously (Kumar et al., 2008;Menasria et al., 2013). KO and DKO mice were genotyped using standard protocols from The Jackson Laboratory. All mice were housed in rooms with controlled temperature (25 ± 2 0 C) and illumination (12 h light/12 h dark) and provided ad libitum access to water and food (Purina rodent diet 5001; Purina Mills, St. Louis, MO, USA) unless otherwise stated, and were allowed to acclimate for at least 7 d before procedures. Adult mice between 8-12 wk of age were used for in vivo experiments, and newborn mice (postnatal day 1 to 3) generated from our own breeding colonies were used for in vitro experiments. All studies were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Oregon Health & Science University.
For in vivo experiments, one day before each experiment commenced, the animals were weighed and divided into treatment groups such that the mean body weights of each group were similar. During the experiments, food intake and body weight were measured at the same time each day, unless otherwise noted.

Intracerebroventricular and Intraperitoneal Injections
Under isofluorane anesthesia, 26-gauge lateral ventricle cannulas (PlasticsOne, Roanoke, VA, USA) were placed in mice using a stereotactic alignment instrument (Kopf, Tujunga, CA, USA) at the following coordinates relative to bregma: 1.0 mm X, -0.5 mm Y and -2.25 mm Z. Mice were then individually housed and allowed to recover from surgery for at least 7 days. Poly I:C (Sigma, St. Louis, MO, USA) was dissolved in 0.9% saline according to the manufacturer's instruction. 0.9% saline or 20 µg of poly I:C (per mouse) was given in 2 µl total volume through intracerebroventricular (icv) injection. For peripheral treatment, 0.9% saline or poly I:C at 10 mg/kg body weight was injected intraperitoneally (ip).

Body Temperature and Locomotor Activity Measurement
Body temperature (BT) and voluntary home cage locomotor activity (LMA) were measured using a MiniMitter system (MiniMitter, Bend, OR, USA) as described previously Grossberg et al., 2011). Briefly, under isofluorane anesthesia, transponders for sensing BT and LMA were implanted adjacent to the abdominal aorta in the retroperitoneal space. Mice were allowed to acclimate for at least 7 days before BT and movement in x-, y-and z-axes were recorded in 5-minute intervals (Vital View, MiniMitter). Δ BT was calculated in B and D using formula Δ BT = BT post-injection -BT pre-injection at a same ZT point during the 48h period of before and after treatment.

Nocturnal Feeding Studies
All feeding studies were performed at night, as described previously . Mice were individually housed for conditioning for at least 7 days. 2 days before treatment, mice were placed in clean individual cages with measured amounts of food. Baseline body weight change and 24 h food intake were obtained on two consecutive days. On the experimental day, 3h before lights off, mice were administered poly I:C at 20 µg /mouse (icv) or 10 mg/kg body weight (ip), respectively, and pre-weighed food pellets were placed into each cage at 5:00 PM. Food was weighed at 4 time points (2 h, 4 h, 16 h and 24 h). Body weights were measured at 2 time points (16 h and 24 h). Care was taken to minimize stress and light exposure to the animals during the nighttime of food measurements.

Tissue Collection
For RT-PCR analysis in tissues, 6 h after icv injection, mice were deeply anesthetized using a ketamine cocktail and sacrificed by trans-cardiac perfusion (≥50 ml) with PBS to remove intravascular blood. Brains and inter-scapular brown adipose tissue (BAT) were immediately removed. Hypothalamic and cortical blocks were dissected. Hypothalami, cortices and BAT were snap frozen and stored in -80 0 C until analysis. For immunohistochemistry (IHC) analysis of lymphocytes in brain tissue, after two icv injections (two doses/mouse within 24 h), mice were perfused by PBS