TREM2 deficiency aggravates renal injury by promoting macrophage apoptosis and polarization via the JAK-STAT pathway in mice

Abstract

The triggering receptor expressed on myeloid cells 2 (TREM2) is an immune receptor that affects cellular phenotypes by modulating phagocytosis and metabolism, promoting cell survival, and counteracting inflammation. Its role in renal injury, in particular, unilateral ureteral obstruction (UUO) or ischemia-reperfusion injury (IRI)-induced renal injury remains unclear. In our study, WT and Trem2^-/- mice were employed to evaluate the role of TREM2 in renal macrophage infiltration and tissue injury after UUO. Bone marrow-derived macrophages (BMDM) from both mouse genotypes were cultured and polarized for in vitro experiments. Next, the effects of TREM2 on renal injury and macrophage polarization in IRI mice were also explored. We found that TREM2 expression was upregulated in the obstructed kidneys. TREM2 deficiency exacerbated renal inflammation and fibrosis 3 and 7 days after UUO, in association with reduced macrophage infiltration. Trem2^-/- BMDM exhibited increased apoptosis and poorer survival compared with WT BMDM. Meanwhile, TREM2 deficiency augmented M1 and M2 polarization after UUO. Consistent with the in vivo observations, TREM2 deficiency led to increased polarization of BMDM towards the M1 proinflammatory phenotype. Mechanistically, TREM2 deficiency promoted M1 and M2 polarization via the JAK-STAT pathway in the presence of TGF-β1, thereby affecting cell survival by regulating mTOR signaling. Furthermore, cyclocreatine supplementation alleviated cell death caused by TREM2 deficiency. Additionally, we found that TREM2 deficiency promoted renal injury, fibrosis, and macrophage polarization in IRI mice. The current data suggest that TREM2 deficiency aggravates renal injury by promoting macrophage apoptosis and polarization via the JAK-STAT pathway. These findings have implications for the role of TREM2 in the regulation of renal injury that justify further evaluation.

Fig. 1. Augmented TREM2 expression is observed in UUO kidneys and TREM2 is partially co-expressed with F4/80.

A UUO mouse model was established. TREM2 expression in kidneys was assessed by a qRT-PCR (n = 12) and b IF staining (n = 6). c Co-expression of TREM2 and F4/80 in UUO kidneys of WT mice on POD3 and 7 was detected by IF double-labeling. Green: TREM2; red: F4/80; blue: DAPI. **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. sham group; ^##P < 0.01, ^####P < 0.0001 vs. WT group. POD postoperative day.

Fig. 2. TREM2 deficiency aggravates tubular injury, renal apoptosis, and renal inflammation in UUO mice.

a Left; H&E staining of UUO kidneys from WT and Trem2^−/− mice (n = 7). Black arrows point to damage to kidney tissue (vacuolation of cells, loss of brush border, tubular formation, etc.), right; H&E results were assessed and quantified as the percentage of the tubular damage area. b Kim-1 expression levels in the kidneys of WT and Trem2^−/− mice by IF (n = 6) and c RT-PCR (n = 7). Red: KIM-1; blue: DAPI. Quantitative analysis of KIM-1 IF results by Image Pro Plus 6.0 software. d TUNEL staining of UUO kidney tissues from both groups of mice; TUNEL-positive cells were manually counted, n = 6 per group. Green: TUNEL, blue: DAPI. e TNF-α, IL-1β, and IL-6 mRNA levels were measured by RT-PCR in the UUO kidneys of WT and Trem2^−/− mice, n = 8 per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 3. TREM2 deficiency increases interstitial fibrosis in the kidney after UUO in mice.

a Masson staining of UUO kidney tissues from WT and Trem2^−/− mice; results were quantified by Image Pro Plus 6.0 software to assess the level of renal fibrosis in mice. Immunohistochemical labeling and quantitative analyses for col-I (b) and α-SMA (c) in UUO kidneys showed more renal interstitial fibrosis. n = 5 per group. *P < 0.05, ***P < 0.001, ****P < 0.0001.

Fig. 4. TREM2 deficiency inhibits macrophage survival in vivo and in vitro.

Assessment of the proportion of infiltrating macrophages (CD11b⁺ F4/80⁺) in UUO kidneys of two groups of mice (gated on CD45⁺ cells) by a Flow cytometry (n = 7) and b IF staining (n = 6). c WT, Trem2^−/− BMDM for Annexin V-PI apoptosis detection by Flow Cytometry, n = 3 per group. Annexin V⁺ PI⁻ represents early apoptosis, while Annexin V⁺ PI⁺ represents late apoptosis. (d) WT, Trem2^−/− BMDM in vitro culture day 0-7 for CCK-8 results (WT BMDM as control), n = 9 per group. e Western blotting assay of pro-caspase 3 (n = 8), cleaved-caspase 3 (n = 8), and Bcl2 (n = 4) proteins in WT, Trem2^−/− BMDM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 5. TREM2 regulates macrophage survival through the Akt/mTOR pathway.

a Volcano plot for WT, Trem2^−/− BMDM difference comparison. The horizontal coordinates indicate the logarithmic value of the difference in ploidy between the two subgroups, and the vertical coordinates indicate the negative Log10 value of the FDR of the difference between the two subgroups. Red (Trem2^−/− upregulated relative to WT expression) and blue (expression downregulated) points indicate a significant difference in gene expression (judged by FDR < 0.05, and more than two-fold difference), and black points indicate a lack of difference. b WT, Trem2^−/− KEGG enrichment bar graph of differential genes in BMDM. The vertical coordinate represents each signaling pathway, the horizontal coordinate represents the number of that signaling pathway as a percentage of the number of all differential genes, the darker the color the smaller the Q value, and the value on the bar represents the number of that signaling pathway and the Q value. c Heatmap of WT, Trem2^−/− BMDM for differential comparative clustering analysis. Each column in the graph represents a sample, each row represents a gene, and the expression of genes in different samples is indicated by different colors, with red colors indicating higher expression and blue colors indicating lower expression. d The differences in expression levels of mTOR, p-mTOR (ser2448), Akt, p-Akt (ser473), and p70S6K, key proteins of the mTOR signaling pathway, were detected by western blotting, and the bands analyzed in grayscale and statistically by using ImageJ software, n = 7–8. e Comparison of the CCK-8 results of WT and Trem2^−/− BMDM in vitro cultures after the addition of cyclocreatine days 3–5, n = 8. **P < 0.01, ****P < 0.0001.

Fig. 6. TREM2 deficiency promotes macrophage polarization.

a Fluorescent representative images of iNOS⁺ cells in UUO kidneys of WT and Trem2^−/− mice. Quantification of the percentage of area occupied by iNOS⁺ cells in the two groups of mice was performed using Image Pro Plus 6.0 software n = 6–7. Red: iNOS, blue: DAPI. b Fluorescent representative images of CD206⁺ cells in the UUO kidneys of WT and Trem2^−/− mice. The percentage of area occupied by red fluorescence of CD206⁺ cells in both groups of mice was quantified using Image Pro Plus 6.0 software, n = 6–7. Red: CD206, blue: DAPI. Flow cytometry representative images of (c) CD86⁺ (n = 6) and (e) CD206⁺ (n = 9–10) macrophages in the UUO kidneys of two groups of mice (gated on CD11b⁺F4/80⁺ cells). Absolute counts of (d) CD86⁺ (n = 6–9) and (f) CD206⁺ (n = 9–10) macrophages in the two groups of mice. *P < 0.05, **P < 0.01.

Fig. 7. TREM2 deficiency promotes BMDM polarization via the JAK2-STAT1/3 pathway.

a Flow representative images of BMDM polarized to M1 and M2 following LPS or IL-4 stimulation (gated on CD11b⁺F4/80⁺ cells), superimposed images of two sets of peak and statistical analysis of BMDM polarization, n = 6. b Flow representative images of BMDM polarized to M1 and M2 following TGF-β1 stimulation (gated on CD11b⁺F4/80⁺ cells) respectively, superimposed images of two sets of peak and statistical analysis of BMDM polarization, n = 4. Heatmap of mRNA levels of BMDM (c) M1- and (d) M2-related markers. e Protein expression levels of JAK2, p-JAK2 (Tyr1007/1008), STAT1, p-STAT1 (Tyr701), STAT3, and p-STAT3 (Tyr705) were detected by western blotting in WT and Trem2^−/− BMDM in control and TGF-β1 groups, respectively. f Western blotting of the key proteins associated with the JAK-STAT pathway was analyzed in the grayscale, n = 4–8. g The measurement of mRNA and protein levels of SOCS1 and SOCS3, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 8. TREM2 deficiency aggravated IRI-induced renal injury and fibrosis and promoted macrophage polarization in mice.

a Left; H&E staining of IRI kidneys from WT and Trem2^−/− mice. right; H&E results were assessed and quantified as the percentage of the tubular damage area. b Masson staining of IRI kidney tissues from WT and Trem2^−/− mice; results were quantified by Image Pro Plus 6.0 software to assess the level of renal fibrosis in mice. c Flow cytometry representative images of CD86⁺ and CD206⁺ macrophages in the IRI kidneys of two groups of mice (gated on CD11b⁺F4/80⁺ cells). n = 5 per group. *P < 0.05, ****P < 0.0001.