TREM2 alters the phagocytic, apoptotic and inflammatory response to Aβ42 in HMC3 cells

Abstract

Alzheimer's disease (AD) is characterized by the accumulation in the brain of extracellular amyloid β (Aβ) plaques as well as intraneuronal inclusions (neurofibrillary tangles) consisting of total tau and phosphorylated tau. Also present are dystrophic neurites, loss of synapses, neuronal death, and gliosis. AD genetic studies have highlighted the importance of inflammation in this disease by identifying several risk associated immune response genes, including TREM2. TREM2 has been strongly implicated in basic microglia function including, phagocytosis, apoptosis, and the inflammatory response to Aβ in mouse brain and primary cells. These studies show that microglia are key players in the response to Aβ and in the accumulation of AD pathology. However, details are still missing about which apoptotic or inflammatory factors rely on TREM2 in their response to Aβ, especially in human cell lines. Given these previous findings our hypothesis is that TREM2 influences the response to Aβ toxicity by enhancing phagocytosis and inhibiting both the BCL-2 family of apoptotic proteins and pro-inflammatory cytokines. Aβ₄₂ treatment of the human microglial cell line, HMC3 cells, was performed and TREM2 was overexpressed or silenced and the phagocytosis, apoptosis and inflammatory response were evaluated. Results indicate that a robust phagocytic response to Aβ after 24 h requires TREM2 in HMC3 cells. Also, TREM2 inhibits Aβ induced apoptosis by activating the Mcl-1/Bim complex. TREM2 is involved in activation of IP-10, MIP-1a, and IL-8, while it inhibits FGF-2, VEGF and GRO. Taken together, TREM2 plays a role in enhancing the microglial functional response to Aβ toxicity in HMC3 cells. This novel information suggests that therapeutic strategies that seek to activate TREM2 may not only enhance phagocytosis and inhibit apoptosis, but may also inhibit beneficial inflammatory factors, emphasizing the need to define TREM2-related inflammatory activity in not only mouse models of AD, but also in human AD.

Keywords: Apoptosis; Aβ; Inflammatory factors; Microglia; Phagocytosis; TREM2.

Fig 1.. HMC3 cells express low to moderate TREM2 levels relative to other cell lines.

TREM2 RNA ΔCT levels (Relative to ACTB) are lower than most other cell lines (A). TREM2 protein (Relative to Actin B) are lower than most other cell lines (B). TREM2 protein relative to TREM2 RNA is lower the HepG2 cell line (C).

Fig 2.. Aβ₄₂ treatment of HMC3 cells increases TREM2.

Treatment of HMC3 cells with 5 μM Aβ for 24 hours increases cell surface TREM2 expression (A). TREM2 full length protein expression is significantly increase after 24 hour 5 μM Aβ₄₂ treatment (25 kDa: p-value = 0.0247) (B). In a separate experiment, full length TREM2 relative to Actin (Western Blot: 25 kDa) is significantly increased, upon 24 hour 5 μM Aβ₄₂ treatment while overexpression of TREM2 (OE) plus Aβ₄₂ treatment does not significantly increase TREM2. Knockdown of TREM2 (siRNA) significantly depletes TREM2 with and without 24 hour 5 μM Aβ₄₂ treatment (C). Fisher’s LSD test without correction for multiple comparisons significant p-value <0.05.

Fig 3.. Lack of TREM2 decreases Aβ₄₂ stimulated phagocytosis in HMC3 cells.

Treatment of HMC3 cells with 5 μM Aβ₄₂ for 24 hours changes cell morphology from resting ramified microglial (A) to activated amoeboid microglia (B). Maximum phagocytosis is observed after 24 hours of 5 μM Aβ₄₂ treatment (lines represent significant p- values <0.01) (C). After 24 hours of 5 μM Aβ₄₂ treatment there is a significant increase in phagocytosis with or without overexpression of TREM2 and upon TREM2 knockdown there is a decrease in phagocytosis with or without 24 hour 5 μM Aβ₄₂ treatment (D).

Fig 4.. Aβ₄₂ treatment alters cell survival, pro-apoptotic and anti-apoptotic factors in HMC3 cells.

HMC3 cell viability significantly declines after 8 hours of 5 μM Aβ₄₂ treatment Bars represent Fisher’s LSD test without correction for multiple comparisons significant p-value <0.05.

Fig 5.. TREM2 modulates apoptotic machinery in HMC3 cells.

(1) Bax, Bad, and pBad are reduced in response to 24 hour Aβ₄₂ treatment. (2) TREM2 overexpression (TREM2 OE) does not influence these apoptotic proteins. (3) TREM2 silencing (TREM2 siRNA) increases Bax. (4) Mcl-1/Bim is increased in response to 24 hour Aβ₄₂ treatment in the context of TREM2 overexpression. (5) Bax, Bcl-xL/Bad are decreased in response to 24 hour Aβ₄₂ treatment in the context of TREM2 silencing. (6) In response to 24 hour Aβ₄₂ treatment TREM2 silencing results in a decrease in Bax compared to TREM2 overexpression. (A) Significance (p-values) are Tukey adjusted for multiple comparisons and mean difference (Mean Diff.) is shown for each apoptotic factor. (B)

Fig 6.. Proinflammatory factors generally increased upon TREM2 overexpression in HMC3 cells.

(1) No change in inflammatory factors after 24 hour Aβ₄₂ treatment. (2) No change in inflammatory factors with TREM2 overexpression. (3) No change in inflammatory factors when TREM2 silenced. (4) No change in inflammatory factors with TREM2 overexpression after 24 hour Aβ₄₂ treatment. (5) FGF-2 is increased and MIP- 1a is decreased when TREM2 silenced after 24 hour Aβ₄₂ treatment. (6) In response to 24 hour Aβ₄₂ treatment TREM2 silencing results in a decrease in IP-10, MIP-1a, IL- 8 and a increase in FGF-2, VEGF, GRO, compared to TREM2 overexpression. (A) Significance (p-values) are Tukey adjusted for multiple comparisons and mean difference (Mean Diff.) is shown for each apoptotic factor. (B)