Soluble CD74 Reroutes MIF/CXCR4/AKT-Mediated Survival of Cardiac Myofibroblasts to Necroptosis

Abstract

Background Although macrophage migration inhibitory factor ( MIF ) has been demonstrated to mediate cardioprotection in ischemia/reperfusion injury and antagonize fibrotic effects through its receptor, CD 74, the function of the soluble CD 74 receptor ectodomain ( sCD 74) and its interaction with circulating MIF have not been explored in cardiac disease. Methods and Results Cardiac fibroblasts were isolated from hearts of neonatal mice and differentiated into myofibroblasts. Co-treatment with recombinant MIF and sCD 74 induced cell death ( P<0.001), which was mediated by receptor-interacting serine/threonine-protein kinase ( RIP) 1/ RIP 3-dependent necroptosis ( P=0.0376). This effect was specific for cardiac fibroblasts and did not affect cardiomyocytes. Gene expression analyses using microarray and RT - qPCR technology revealed a 4-fold upregulation of several interferon-induced genes upon co-treatment of myofibroblasts with sCD 74 and MIF (Ifi44: P=0.011; Irg1: P=0.022; Clec4e: P=0.011). Furthermore, Western blot analysis confirmed the role of sCD 74 as a modulator of MIF signaling by diminishing MIF -mediated protein kinase B ( AKT) activation ( P=0.0197) and triggering p38 activation ( P=0.0641). We obtained evidence that sCD 74 inhibits MIF -mediated survival pathway through the C-X-C chemokine receptor 4/ AKT axis, enabling the induction of CD 74-dependent necroptotic processes in cardiac myofibroblasts. Preliminary clinical data revealed a lowered sCD 74/ MIF ratio in heart failure patients (17.47±10.09 versus 1.413±0.6244). Conclusions These findings suggest that treatment of cardiac myofibroblasts with sCD 74 and MIF induces necroptosis, offering new insights into the mechanism of myofibroblast depletion during scar maturation. Preliminary clinical data provided first evidence about a clinical relevance of the sCD 74/ MIF axis in heart failure, suggesting that these proteins may be a promising target to modulate cardiac remodeling and disease progression in heart failure.

Keywords: cell death; heart failure; macrophage migration inhibitory factor; myocardial fibrosis; myofibroblast; necroptosis; soluble CD74.

Figure 1

Co‐treatment with sCD74 and MIF promotes cell death in cardiac myofibroblasts. A, Primary isolated cardiac fibroblasts were co‐stained with the fibroblast marker, vimentin (green), the myofibroblast marker, α‐smooth muscle actin (α‐SMA) (red), and nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI; blue) after 0 (5 hours), 3, and 5 days in culture. Size bar, 200 μm. B, mRNA level of α‐SMA, collagen 1α1 (Col1α1), and fibronectin 1 (FN1) were determined 5 hours (day 0) and 5 days after plating by the RT‐qPCR method. Data represent mean±SEM of 3 independent experiments and were analyzed with a 2‐tailed, unpaired t test. ***P<0.001 vs day 0. C, Cardiac myofibroblasts (MyoFBs) isolated from hearts of wild‐type C57BL/6J (WT) mice were treated with increasing concentrations of sCD74 (0, 0.04, 0.16, 8, 16, and 40 nmol/L) without or with rMIF (8 nmol/L). After 24 hours, cells were stained with trypan blue and cell numbers were assessed. Data were analyzed with a 2‐tailed, unpaired t test and represent means±SEM of at least 7 independent experiments. *P<0.05; **P<0.01; ***P<0.001 without (w/o) MIF vs with MIF respectively; ^§ P<0.05; ^§§ P<0.01 vs untreated control respectively; ^$$ P<0.01; ^$ P<0.001 vs MIF control respectively. D, Murine cardiomyocytes (CMs) were stimulated with 40 nmol/L of sCD74 in the absence or presence of 8 nmol/L of rMIF for 24 hours, followed by trypan blue staining. Percentage of survival of CMs was compared with MyoFBs. Data represent mean±SEM of 6 independent experiments and were analyzed with a 2‐tailed, unpaired t test with multiple correction (n=9). ^$$$ P<0.001 vs control of MyoFBs; **P<0.01 MyoFBs vs CMs. MIF indicates macrophage migration inhibitory factor; RQ indicates relative quantity; rMIF, recombinant MIF; sCD74, soluble CD74.

Figure 2

sCD74/MIF‐induced death of myofibroblasts is triggered by RIP1/RIP3‐dependent necroptosis. Cardiac wild‐type myofibroblasts were treated solitarily or simultaneously with MIF and sCD74, and lysates were taken at 10 hours. A and B, Cleaved caspase‐3 levels (Cl Casp 3) as well as (C and D) relative phosphorylation levels of receptor‐interacting serine/threonine‐protein kinase 3 (RIP3) were assessed by Western blotting and immunostaining. Instead of showing the whole blot, only relevant bands were cut out and arranged in the right order. Uncut blots are shown in Figures S3 and S4. Data represent mean±SEM of at least 3 independent experiments. Data were analyzed with a 2‐tailed, unpaired t test. ^$ P<0.05 vs control. E, Cardiac wild‐type fibroblasts were pretreated with dimethyl sulfoxide (DMSO) or a potent necroptosis inhibitor (Nec1s) for 1 hour followed by solitary or co‐treatment with sCD74 and rMIF. After 20 to 24 hours of incubation, cell numbers were quantified by trypan blue staining and automated counting. Data were analyzed with a 2‐tailed, unpaired t test and corrected for multiple comparison (n=9) using Bonferroni's posttest. Data represent mean±SEM of at least 8 independent experiments for inhibition studies. **P<0.01 DMSO vs Nec1s; ^§§§ P<0.001 vs DMSO control; ^$ P<0.05, ^$$$ P<0.001 vs Nec1s control respectively. GAPDH indicates glyceraldehyde 3‐phosphate dehydrogenase; MIF, macrophage migration inhibitory factor; pRIP3, phosphorylated RIP3; rMIF, recombinant MIF; sCD74, soluble CD74.

Figure 3

Treatment with sCD74/MIF significantly upregulates gene expression of type I interferon (IFN)‐induced genes. WT were treated with 40 nmol/L of sCD74 in the absence or presence of 8 nmol/L of rMIF. After 8 hours, mRNA was isolated. A, Microarray analysis was performed, and only genes that were at least 1.5‐fold differentially regulated upon sCD74/rMIF treatment compared with control were depicted. Independent triplicates were performed. Corresponding P values are depicted in Table 1. The 9 genes that were also significantly regulated upon MIF‐treatment were marked as overlap. Type I IFN‐induced genes and genes involved in NF‐κB signaling pathways are indicated as black and dark gray bars, respectively. Genes labeled as gray bars seem not to contribute to specialized function and pathways. B and D, RT‐qPCR was performed with the cDNA and Taqman probes specific for the type I IFN‐induced genes, interferon‐induced protein 44 (Ifi44), immunoresponsive gene 1 (Irg1), and C‐type lectin domain family 4, member e (Clec4e). GAPDH was used as a housekeeping gene. Relative quantity (RQ) values were calculated according to the ΔΔCt method and normalized to control. Data represent mean±SEM of at least 4 independent experiments and were analyzed with a 2‐tailed, unpaired t test with multiple correction (n=5). ^$ P<0.05 vs control; *P<0.05 vs rMIF. GAPDH indicates glyceraldehyde 3‐phosphate dehydrogenase; IFN, interferon; MIF, macrophage migration inhibitory factor; NF‐κB, nuclear factor kappa B; rMIF, recombinant MIF; sCD74, soluble CD74; WT, wild type.

Figure 4

sCD74 changes the kinase activation profile of MIF. Following stimulation of WT myofibroblasts, lysates were taken after 0.5 and 10 hours. Phosphorylation and total protein levels were assessed by Western blotting, band intensities were densitometric analyzed, and relative activation levels were normalized to control. Phosphorylation levels of AKT at (A and C) 0.5 and (B and D) 10 hours as well as the mitogen‐activated protein kinase p38 at (E and G) 0.5 and (F and H) 10 hours were determined. Densitometric analysis of immunostainings as well as representative blots are shown. Instead of showing the whole blot, relevant bands were cut out and arranged in the respective order. Uncut blots are shown in Figures S9 through 12. Data represent mean±SEM of at least 4 independent experiments. Data were analyzed with a 2‐tailed, unpaired t test. ^$ P<0.05, ^$$$ P<0.001 vs control, respectively; *P<0.05 vs rMIF. AKT indicates protein kinase B; MIF, macrophage migration inhibitory factor; pAKT, phosphorylated AKT; pp38, phosphorylated p38; rMIF, recombinant MIF; sCD74, soluble CD74; WT, wild type.

Figure 5

sCD74 redirects the MIF/CXCR4‐profibrotic signal into a CD74‐mediated antifibrotic signal. First, cardiac WT fibroblasts were treated with CXCR2‐inhibitor SB225002, CXCR4‐inhibitor AMD3100, or appropriate vehicle controls for 1 hour. Subsequently, (A) Cd74 ^−/− cells, (B) wild‐type (WT) cells, (D) SB225002‐, and (E) DMSO‐pretreated myofibroblasts as well as (G) AMD3100‐ and (H) ddH₂O‐pretreated WT myofibroblasts were subjected to 40 nmol/L of sCD74 either alone or together with 8 nmol/L of MIF. Cell numbers were quantified by trypan blue staining and automated counting and normalized to untreated control. (C, F, and I) For comparison, data from Cd74 ^−/−, CXCR2, and CXCR4 inhibition studies were overlaid with their appropriate vehicle control. Data represent mean±SEM of at least (A) 14, (B) 12, (D) 6, (E) 6, (G) 6, and (H) 6 independent experiments. Data were analyzed with a 2‐tailed, unpaired t test and corrected for multiple comparison (A, B, D, E, G, and H: n=5; C, F, and I: n=4) using Bonferroni's posttest. ^$$ P<0.01, ^$$$ P<0.001 vs control within group respectively; *P<0.05, **P<0.01, ***P<0.001 vs MIF or sCD74 respectively; ^§§ P<0.01, ^§§§ P<0.001 comparison between WT and deficient or inhibited myofibroblasts respectively. CXCR, C‐X‐C chemokine receptor; DMSO, dimethyl sulfoxide; MIF, macrophage migration inhibitory factor; rMIF, recombinant MIF; sCD74, soluble CD74; WT, wild type.

Figure 6

Circulating CD74 and MIF concentrations in healthy, CHD, and HF patients. Using an ELISA technique, we analyzed (A) MIF and (B) sCD74 concentrations in plasma samples of healthy volunteers (n=4), patients with coronary heart disease (CHD; n=5), and patients with advanced heart failure (HF; n=4). C, Ratio of CD74/MIF was calculated by dividing the molar serum concentration of circulating CD74 (19.4 kDa) by MIF (12.5 kDa). Data were analyzed with a 2‐tailed, unpaired t test without multiple correction and represent mean±SEM. *P<0.05 vs CHD cohort; ^$ P<0.05, ^$$ P<0.01 vs healthy cohort, respectively. MIF indicates macrophage migration inhibitory factor; sCD74, soluble CD74.

Figure 7

Proposed model of molecular switch between the profibrotic MIF/CXCR4 signal and antifibrotic MIF/CD74 signal. Recombinant MIF triggers CXCR4 internalization, which requires the presence of CD74. Subsequently, MIF/CXCR4 axis mediates survival by AKT activation. Although sCD74/MIF still induces CXCR4 (and CXCR2) internalization, AKT signaling is disturbed. However, the CXCR4/AKT axis seems to be important to suppress cell death. As soon as MIF‐mediated CXCR4 activation is inhibited, signaling by CD74 predominates resulting in RIP1 and RIP3 phosphorylation and, finally, necroptosis. Furthermore, sCD74/MIF seems to be recognized in a DAMP‐like manner to activate components typical for the antimicrobial defense system, such as type 1 interferon (IFN)‐induced genes. AKT indicates protein kinase B; CXCR, C‐X‐C chemokine receptor; DAMP, danger‐associated molecular pattern; MIF, macrophage migration inhibitory factor; RIP1/3, receptor‐interacting serine/threonine‐protein kinases 1 and 3; rMIF, recombinant MIF; sCD74, soluble CD74.