Dysregulated interferon-gamma responses during lethal cytomegalovirus brain infection of IL-10-deficient mice

Abstract

Murine cytomegalovirus (MCMV) brain infection induces a transient increase in chemokine production, which precedes the infiltration of CD3(+) lymphocytes. In this study, we hypothesized that an absence of anti-inflammatory cytokines would result in sustained proinflammatory neuroimmune responses. Direct intracerebroventricular injection of MCMV into IL-10 knockout (KO) mice produced an unexpected result: while wild-type animals controlled MCMV, the infection was lethal in IL-10 KO animals. Identical infection of IL-4 KO animals did not produce lethal disease. To further characterize the role of IL-10, infected brain tissue from both wild-type and IL-10 KO animals was assessed for cytokine and chemokine levels, as well as viral gene expression. These data show vastly elevated levels of interferon (IFN)-gamma, and the IFN-gamma-inducible chemokines CXCL9 and CXCL10, as well as IL-6 in brain homogenates obtained from IL-10 KO animals. However, MCMV viral load, glycoprotein B mRNA levels, and titers of infectious virus were similar in both IL-10 KO and wild-type animals. Separation of cells isolated from murine brain tissue into distinct populations using FACS, along with subsequent quantitative RT real-time PCR, showed that brain-infiltrating CD45(hi)/CD11b(-) and CD45(hi)/CD11b(int) were the cellular source of IL-10 in the brain. Taken together, these data demonstrate that MCMV brain infection of IL-10-deficient mice causes lethal disease, which occurs in the presence of a dysregulated IFN-gamma-mediated neuroimmune response.

Fig. 1

Lethal MCMV brain infection in IL-10 KO mice. (A) MCMV RM461 was injected slowly into the right lateral ventricle of IL-10 KO (closed circles, n=15), IL-4 KO (open squares, n=11), and wild-type (Balb/C) (open triangles, n=12) adult mice. Data are expressed as percent of mice in each group surviving at the indicated time point, followed over the 18 d time-course of the experiment. (B) Kinetics of IL-10 expression in the brains of wild-type BALB/c mice (closed circles) in response to MCMV infection. IL-10 mRNA levels were examined over the 15 day time-course of infection using RT real-time PCR. Transcript levels were normalized to HPRT and are presented as mean (± SD) fold increase over animals injected with saline from averaged data obtained using three to ten animals per time point. (C) Levels of IL-10 (pg/mg protein) in brain homogenates obtained from sham-injected (saline) and MCMV-injected wild-type (Balb/C) mice during MCMV infection were measured at 5 days p.i. using ELISA.

Fig. 2

Dysregulated cytokine expression during MCMV brain infection in IL-10 knock out mice. The kinetics of cytokine mRNA expression were measured by quantitative real-time RT-PCR using total RNA extracted from whole brain homogenates obtained from infected wild-type (Balb/C, closed circles) versus IL-10 KO (open circles) mice. Total RNA was extracted, DNAse-treated and reverse transcribed. cDNA obtained was analyzed by qPCR using primers specific to IFN-γ (A), along with ELISA for IFN-γ (B); and IL-6 (C) followed by IL-6 ELISA (D); TNF-α (E) and IL-1β (F). Cytokine mRNA levels were normalized to HPRT and averaged data are expressed as mean ± SD fold increase in relative mRNA induction levels over sham-infected controls from 3–6 animals per time point. Brains from MCMV-infected wild-type and IL-10 KO mice were harvested at 5 days p.i. for ELISA and data are expressed as mean (± SEM) of three to five animals per group.

Fig. 3

Dysregulated chemokine expression during MCMV brain infection in IL-10 knock out mice. Brains from MCMV-infected wild-type (Balb/C) and IL-10 KO mice were harvested at 5 days p.i. Total RNA was extracted, DNAse treated and reverse transcribed. The cDNA obtained was analyzed by quantitative real-time PCR using primers specific to CXCL9 (A) and CXCL9 ELISA (B); CXCL10 (C) and CXCL10 ELISA (D); CCL5 (E) and CCL5 ELISA (F). Chemokine mRNA expression was normalized to HPRT. Averaged data from 3–6 animals per time point are expressed as fold increase in relative mRNA induction over sham-infected controls. Brains from MCMV-infected wild-type and IL-10 KO mice were harvested at 5 days p.i. for ELISA and data are expressed as mean (± SEM) of three to five animals per group.

Fig. 4

Similar viral loads in the brains of IL-10 KO and wild-type animals. (A) Viral load in brain tissue homogenates from both groups of animals was quantified using real-time DNA PCR (5 days p.i.). The amount of MCMV DNA was normalized to β-actin and is presented as mean (± SD) normalized copy number from averaged data using three to five animals per group. (B) Viral titers in brain homogenates of MCMV-infected mice obtained at 5 days p.i. Titers were obtained using TCID₅₀ assay. Data are presented as mean (± SEM) titers from three animals per group. (C) MCMV gB mRNA expression was measured using quantitative real-time RT-PCR. Total RNA was extracted from whole brain homogenates of wild-type (Balb/C) or IL-10 KO animals at 5 days p.i. gB mRNA levels were normalized to HPRT (hypoxanthine guanine phospho-ribosyl transferase) and are presented as mean of normalized expression from averaged data obtained using four to eight animals per group. (D) Viral gB mRNA levels in the brains of infected, immunodeficient (SCID) versus wild-type (Balb/C) mice. Data are presented as mean (± SEM) mRNA expression (normalized to HPRT) obtained using 5 SCID and 3 wild-type animals per group.

Fig. 5

Expression of IL-10 mRNA by brain-infiltrating CD45(hi)/CD11b(−) and CD45(hi)/CD11b(int) inflammatory cells during MCMV infection. (A) Single cell suspensions of brain tissue obtained from saline-injected (sham) and (B) virus infected (MCMV infected) mice (5 animals/group at 5 days p.i) were banded on a 70% Percoll cushion. Brain leukocytes at the 30-70% Percoll interface were collected, labeled with APC-conjugated Abs specific for CD45 and FITC-labeled anti-CD11b Abs, and sorted into four populations (A through D) using FACS. Data presented are representative of three separate experiments using cells from 5 animals each. (B) Total RNA extracted from each of the separated populations (A through D) from virus-infected (infected) or saline-treated (sham) brains (5 days p.i) was analyzed by qRT-PCR for IL-10 expression. Transcript levels were normalized to HPRT expression and are presented as mean (± SEM) using cells isolated from the brain tissues of five animals.