The isoenzyme of glutaminyl cyclase is an important regulator of monocyte infiltration under inflammatory conditions

Abstract

Acute and chronic inflammatory disorders are characterized by detrimental cytokine and chemokine expression. Frequently, the chemotactic activity of cytokines depends on a modified N-terminus of the polypeptide. Among those, the N-terminus of monocyte chemoattractant protein 1 (CCL2 and MCP-1) is modified to a pyroglutamate (pE-) residue protecting against degradation in vivo. Here, we show that the N-terminal pE-formation depends on glutaminyl cyclase activity. The pE-residue increases stability against N-terminal degradation by aminopeptidases and improves receptor activation and signal transduction in vitro. Genetic ablation of the glutaminyl cyclase iso-enzymes QC (QPCT) or isoQC (QPCTL) revealed a major role of isoQC for pE(1) -CCL2 formation and monocyte infiltration. Consistently, administration of QC-inhibitors in inflammatory models, such as thioglycollate-induced peritonitis reduced monocyte infiltration. The pharmacologic efficacy of QC/isoQC-inhibition was assessed in accelerated atherosclerosis in ApoE3*Leiden mice, showing attenuated atherosclerotic pathology following chronic oral treatment. Current strategies targeting CCL2 are mainly based on antibodies or spiegelmers. The application of small, orally available inhibitors of glutaminyl cyclases represents an alternative therapeutic strategy to treat CCL2-driven disorders such as atherosclerosis/restenosis and fibrosis.

Figure 1. Pyroglutamic acid mediates activity of CCL2

1. Immature forms of human MCPs, e.g. Q¹-CCL2 display cleavage sites for ApP (1) and DP4 (2). CCL2 can also be processed by MMP-1 (3). All human MCPs are putative substrates of QC/isoQC and they contain in their mature state an N-terminal pE-residue and are protected against aminopeptidase cleavage but not against MMP-1 cleavage.

2. N-terminal degradation of human Q¹-CCL2 and pE¹-CCL2 in human plasma monitored using MALDI-TOF mass spectrometry.

3. Internalization of CCR2 from the surface of THP-1 monocytes triggered by CCL2 variants and determined by FACS analysis. (***p < 0.001 and *p < 0.05 vs. control; ^###p < 0.001 and ^##p < 0.01 vs. D³, ⁺⁺p < 0.01 vs. Q¹, one-way ANOVA followed by Tukey post hoc test, n = 3–8, mean ± SEM).

4. Dependence of THP-1 monocyte migration on the concentration of CCL2(Q¹-76) and CCL2(pE¹-76), assessed using a chamber assay and quantification of cells by FACS analysis (***p < 0.001, **p < 0.01, Q¹-CCL2 vs. pE¹-CCL2, two-way ANOVA followed by Bonferroni post-test, n = 3–7, mean ± SEM).

Figure 2. IsoQC is the major pE¹-CCL2 forming enzyme in vivo

1. Analysis of the ratio of total-CCL2 and pE¹-CCL2 secreted from LPS-stimulated primary cells isolated from QC^−/− mice compared to WT littermates (QC^+/+; n = 4–5, mean ± SEM).

2. Analysis of the ratio of total-CCL2 and pE¹-CCL2 secreted from LPS-stimulated primary cells isolated from isoQC^−/− mice compared to WT littermates (isoQC^+/+; ***p < 0.001 vs. isoQC^+/+, Student's t-test, n = 4, mean ± SEM).

3. pE¹-CCL2 formation in serum after peripheral injection of LPS in male QC^−/− mice compared to WT littermates (QC^+/+; n = 6–7, mean ± SEM).

4. pE¹-CCL2 formation in serum after peripheral injection of LPS in male isoQC^−/− mice, compared to WT littermates (QC^+/+; ***p < 0.001 vs. isoQC^+/+ +LPS, Student's t-test, n = 5–8, mean ± SEM).

5. IsoQC-deficiency leads to impaired monocyte recruitment to lungs after intranasal LPS-application in isoQC^−/− compared to WT littermates (isoQC^+/+; *p < 0.05 vs. isoQC^+/+ +LPS, Student's t-test, n = 7–8, mean ± SEM).

6. Analysis of neutrophils in bronchoalveolar fluid after intranasal LPS-application in isoQC^−/− compared to WT littermates (isoQC^+/+; n = 7–8, mean ± SEM).

Figure 3. IsoQC depletion results in impaired monocyte recruitment after thioglycollate challenge

1. Representative FACS analysis showing the infiltrating monocyte population in QC^+/+ and QC^−/− mice dissected by specific staining.

2. Quantification of infiltrating monocytes and granulocytes in QC^+/+ mice (black bars) and QC^−/− mice (open bars; n = 8–13, mean ± SEM).

3. Lavage fluid from (B) was analyzed for total-CCL2 (black bars) and pE¹-CCL2 (open bars; n.s., not significant, Student's t-test, mean ± SD).

4. Representative FACS analysis showing the infiltrating monocyte population in isoQC^+/+ and isoQC^−/− mice.

5. Quantification of infiltrating monocytes and granulocytes in isoQC^+/+ mice (black bars) and isoQC^−/− mice (open bars; ***p < 0.001 vs. isoQC^+/+ Thio, Student's t-test, n = 10–14, mean ± SEM).

6. Lavage fluid from (E) was analyzed for total-CCL2 (black bars) and pE¹-CCL2 (open bars; ***p < 0.001 vs. pE¹-CCL2 from isoQC^+/+ mice, Student's t-test, mean ± SD).

Figure 4. Pharmacological inhibition of QC/isoQC activity in vitro and in vivo reduces pE-CCL2 activity

1. Analysis of total-CCL2 (black bars) and pE¹-CCL2 (open bars) after application of varying doses PQ529 to LPS-stimulated primary murine glia cells isolated from C57BL/6J WT mice compared to unstimulated controls (***p < 0.001 vs. pE¹-CCL2 (0 µM PQ529), ANOVA followed by Tukey post hoc test, n = 3–4, mean ± SEM).

2. Analysis of CCL2 gene expression in LPS-stimulated primary glia cells derived from Fig 4A(*p < 0.05, **p < 0.01 vs. PQ529 0 µM, ANOVA followed by Tukey post hoc test, n = 3–4, mean ± SEM).

3. Representative FACS image showing the reduction of infiltrating monocytes after application of PQ529 (30 mg/kg, i.p.).

4. Dose-dependent reduction of infiltrating monocytes in absence (black bars) or presence (red bars) of intraperitoneal PQ529 treatment (**p < 0.01 vs. Thio (+), ANOVA followed by Tukey post hoc test, n = 5–6, mean ± SEM, female mice).

5. Inhibition of monocyte infiltration after oral application of PQ50 (red bars) and PQ529 (white bar; **p < 0.01 vs. Thio (+), ANOVA followed by Tukey post hoc test, n = 5–6, mean ± SEM, female mice).

Figure 5. IsoQC is ubiquitously expressed in human tissue

RT-PCR analysis of various human tissues in comparison to isoQC expression in atherosclerotic vessel segments. The inset shows a Western blot analysis of six cases (5 male, 1 female, mean age 53.8 years) revealing the expression of isoQC in atherosclerotic segments of the Arteria femoralis superficialis compared to expression in livers of isoQC^+/+ and isoQC^−/− mice as internal standard.

Figure 6. QC/isoQC-inhibition alleviates atherosclerotic pathology in a model of cuff-induced accelerated atherosclerosis

1. Monocyte adhesion and total adhering cells 2 days after cuff placement in absence (black bars) or presence (open bars) of PQ50 (QCI) treatment (*p < 0.05 vs. control, Student's t-test, n = 5, mean ± SD).

2. In addition, the CCL2-positive area was calculated in cross-sections within the media and neointima in absence (black bars) and presence (open bars) of PQ50 (QCI) treatment (**p < 0.01, ***p < 0.001 vs. control. Student's t-test, n = 5, mean ± SD).

3. Morphometric analysis of cuffed vessel segments shows a reduction in the degree of lumen stenosis (*p < 0.05 vs. control, Student's t-test, n = 10, mean ± SD). Inset: Example for lumen stenosis after 2 weeks (black arrows).

4. Neointima formation and media thickness (inset) of the cuffed vessel segments of mice sacrificed after 2 weeks, treated in absence (black bars) and presence (open bars) of QC-inhibitor PQ50 (QCI; *p < 0.05 vs. control, Student's t-test, n = 10, mean ± SD).

Figure 7. The role of isoQC for regulating monocyte recruitment in CCL2-driven disorders

IsoQC is the major enzyme for maturation of CCL2 in vivo ensuring stability and receptor activation leading to monocyte migration. Application of small molecules, which inhibit isoQC provokes the secretion of immature forms (Q¹-CCL2) prone to aminopeptidase cleavage generating truncated forms (}-CCL2), devoid of bioactivity under physiological conditions.