Attenuated viral hepatitis in Trem1-/- mice is associated with reduced inflammatory activity of neutrophils

Abstract

TREM1 (Triggering Receptor Expressed on Myeloid Cells 1) is a pro-inflammatory receptor expressed by phagocytes, which can also be released as a soluble molecule (sTREM1). The roles of TREM1 and sTREM1 in liver infection and inflammation are not clear. Here we show that patients with hepatitis B virus (HBV) or hepatitis C virus (HCV) infection manifest elevated serum levels of sTREM1. In mice, experimental viral hepatitis induced by infection with Lymphocytic Choriomeningitis Virus (LCMV)-WE was likewise associated with increased sTREM1 in serum and urine, and with increased TREM1 and its associated adapter molecule DAP12 in the liver. Trem1-/- mice showed accelerated clearance of LCMV-WE and manifested attenuated liver inflammation and injury. TREM1 expression in the liver of wild-type mice was mostly confined to infiltrating neutrophils, which responded to LCMV by secretion of CCL2 and TNF-α, and release of sTREM1. Accordingly, the production of CCL2 and TNF-α was decreased in the livers of LCMV-infected Trem1-/- mice, as compared to LCMV-infected wildtype mice. These findings indicate that TREM1 plays a role in viral hepatitis, in which it seems to aggravate the immunopathology associated with viral clearance, mainly by increasing the inflammatory activity of neutrophils.

Figure 1. Viral hepatitis is associated with increased TREM1 expression and sTREM1 release.

(A) Increased sTREM1 concentrations in blood plasma from patients with hepatitis B (HBV; n = 34) or hepatitis C virus infection (HCV; n = 29), as compared to healthy control subjects (n = 17). (B) Increased sTREM1 concentrations in blood plasma from C57BL/6 mice during acute hepatitis caused by LCMV-WE infection. (C) Increased sTREM1 concentrations in urine from C57BL/6 mice during acute hepatitis caused by LCMV-WE infection. (D) Increased expression of TREM1 RNA in infected livers of C57BL/6 mice during acute hepatitis caused by LCMV-WE infection. (E) Increased expression of DAP12 RNA in infected livers of C57BL/6 mice during acute hepatitis caused by LCMV-WE infection. (F) Increased alanine aminotransferase (ALT) concentrations in blood plasma of C57BL/6 mice during acute hepatitis caused by LCMV-WE infection. *p < 0.05, **p < 0.01.

Figure 2. Accelerated viral clearance and reduced liver damage in LCMV-infected Trem1−/− mice.

(A) At the indicated time-points after LCMV inoculation, livers of Trem1+/+ or Trem1−/− mice were homogenized and the virus titer was determined by the Focus Forming Assay. (B) Reduced viral load of Trem1−/− livers at day 9 and 12 after LCMV inoculation was confirmed by quantitative RT-PCR analysis for the LCMV Z RNA. (C) Frozen liver sections taken at day 9 after LCMV inoculation were stained for the LCMV nucleoprotein with VL4 antibody (green); nuclei were stained with Hoechst 33258 (blue). (D) Protracted ALT elevation in blood plasma of Trem1+/+ mice at day 9 after LCMV inoculation, as compared to Trem1−/− mice. (E) Increased histological activity of Trem1+/+ mice at day 12 after LCMV inoculation, as compared to Trem1−/− mice, determined as the modified histological activity index (mHAI) score. *p < 0.05, **p < 0.01.

Figure 3. TREM1 deficiency does not influence the anti-viral CD8 T cell response.

(A) Equal numbers of infiltrating CD8 T cells in LCMV-infected livers of Trem1+/+ or Trem1−/− mice at day 9 of infection. Each dot represents the absolute number of liver-infiltrating mononuclear cells of one individual mouse. (B) Equal numbers of LCMV-specific CD8 T cells in LCMV-infected of Trem1+/+ or Trem1−/− mice at day 9 of infection, determined by staining with LCMV-gp33 loaded H-2D^b dextramer. Each dot represents the percentage of dextramer+ CD8+ T cells among all CD8+ T cells in the livers of individual mice. (C) Equal numbers of LCMV-responsive CD8 effector T cells in LCMV-infected of Trem1+/+ or Trem1−/− mice at day 9 of infection, determined by intracellular staining of CD8+ T cells for IFN-γ after stimulation with LCMV-gp33 peptide. Each dot represents the percentage of IFN-γ+ CD8+ T cells among all CD8+ T cells in the livers of individual mice. (D) Equal numbers of LCMV-responsive CD8 effector T cells in LCMV-infected of Trem1+/+ or Trem1−/− mice at day 9 of infection, determined by intracellular staining of CD8+ T cells for TNF-α after stimulation with LCMV-gp33 peptide. Each dot represents the percentage of TNF-α+ CD8+ T cells among all CD8+ T cells in the livers of individual mice. (E) Equal degranulation capacity of LCMV-responsive CD8 effector T cells in LCMV-infected of Trem1+/+ or Trem1−/− mice at day 9 of infection, determined by staining for CD107a after stimulation with LCMV-gp33 peptide. Each dot represents the percentage of CD107a+ CD8+ T cells among all CD8+ T cells in the livers of individual mice. (F) Similar LCMV-specific cytotoxic activity of CD8 effector T cells isolated from LCMV-infected Trem1+/+ (white bars) or Trem1−/− mice (black bars) at day 9 of infection, as determined by specific lysis (mean and SEM) of LCMV-infected Hepa1-6 hepatoma cells.

Figure 4. TREM1 expression in LCMV-infected livers of mice by neutrophils.

(A) Frozen liver sections of C57BL/6 mice 9 days after infection with LCMV were stained for TREM1 (green) and Ly6G (red); nuclei were stained with Hoechst 33258 (blue). Co-expression of Ly6G (red) by TREM1-expressing cells (green) in livers of LCMV-infected mice confirmed that TREM1-expressing cells are neutrophils. Scale bar represents 100 μm. (B) Flow cytometric analysis of TREM1 and LY6G expression on neutrophil granulocytes isolated from Trem1+/+ und Trem1−/− C57BL/6 mice 9 days after infection with LCMV. (C) Concentrations of sTREM1 in culture supernatants of primary hepatocytes, liver sinusoidal endothelial cells (LSECs), Kupffer cells or neutrophils 24 hours after isolation from Trem1+/+ C57BL/6 mice and stimulation with LPS (10 ng/ml). Shown are mean values ± SEM. **p < 0.01. (D) Relative TREM1 RNA expression in neutrophils 6 hours after isolation from Trem1+/+ C57BL/6 mice and stimulation with LCMV WE (MOI 5). Shown are mean values ± SEM; **p < 0.01. (E) Release of sTREM1 by neutrophils into cell culture supernatant determined 24 hours after isolation from Trem1+/+ C57BL/6 mice and stimulation with LCMV WE (MOI 5). Shown are mean values ± SEM. **p < 0.01.

Figure 5. Impaired secretion of CCL2 and TNF-α by TREM1−/− neutrophils in response to LCMV.

(A) Neutrophils were isolated from Trem1+/+ C57BL/6 mice, stimulated with LCMV WE (MOI 5) and, after 6 hours, the expression of CCL2, TNF-α, IL-1β, IL-6, MPO, CXCL1, CXCL2, CXCL5, IFN-α and IFN-γ relative to the HPRT house-keeper were determined by qRT-PCR. Shown are mean values ± SEM. *p < 0.05. (B) Relative CCL2 and TNF-α RNA expression in livers from Trem1+/+ and Trem1−/− C57BL/6 mice, sampled 4 days after infection with LCMV. Shown are mean values ± SEM. *p < 0.05.