Lack of Formyl-peptide Receptor 1 Mitigates Atherosclerosis in Hyperlipidemic Mice

Atherosclerosis is a chronic inflammatory disease of the arterial wall with hypercholesterolemia and inflammation representing two major pillars in its pathophysiology. A decade ago, studies unraveled how hypercholesterolemia influences inflammation and revealed that lipids orchestrate leukocyte homeostasis. Hypercholesterolemia-associated monocytosis and neutrophilia directly correlate with lesional leukocyte counts and lesion dimension in mice and with the incidence of cardiovascular events in humans.[1] [2] [3] In mice, for example, counts of circulating Ly6C^hi inflammatory monocytes correlate with atherosclerotic lesion burden.[2] [4] Similarly, circulating neutrophil counts expand under hypercholesterolemia correlating with atherosclerotic plaque size and lesional neutrophil accumulation.[1] [5] [6] Analogous associations were already found in the 1970s, when it was noticed that peripheral leukocyte counts predict future cardiovascular events.[7] Very recently, these observations were extended by providing proof for the causality between high neutrophil counts and the risk for cardiovascular morbidity across several end points.[8] [9]

Formyl-peptide receptor 1 (FPR1) is an ancient receptor for bacterial derived motifs including the needle cap protein of Yersinia pestis [10] as well as of formylated peptides that are released from all bacteria. Yet, given the fact that eukaryote mitochondria are of bacterial origin,[11] it is not surprising that mitochondrial damage in the case of cell necrosis releases host-derived formyl-peptides. Among circulating immune cells, FPR1 is predominantly expressed on neutrophils and hence lack of this receptor impairs neutrophil recruitment in the context of bacterial infections and tissue necrosis.[12] [13] [14] However, the role of FPR1 in vascular inflammation remains thus far unknown.

Hence, we crossed FPR1-deficient (Fpr1^−/− ) and apolipoprotein E-deficient (Apoe^−/− ) animals and fed these mice western diet (WD) or normal chow diet for 4 weeks and performed subsequent flow cytometric and histological sample assessment ([Fig. 1A]). Mice were sacrificed and body weight, plasma cholesterol, and triglyceride levels were determined but remained unaltered between the groups (data not shown). Subsequent analysis of leukocyte counts in blood, however, revealed hypercholesterolemia-associated neutrophilia only in Apoe^−/− but not in Apoe^−/−Fpr1^−/− animals ([Fig. 1B]). Interestingly, this effect could not be recapitulated in bone marrow or spleen of Apoe^−/−Fpr1^−/− mice ([Fig. 1C, D]). The latter indicates a critical role for FPR1 in controlling neutrophil numbers in blood under hyperlipidemic conditions. Given that the frequency of annexin V⁺ neutrophils in blood of FPR1 competent and deficient mice was not different, we concluded that neutrophil apoptosis was likely no contributor to lower neutrophil blood counts in FPR1-deficient mice ([Fig. 1E]). Importantly, the number of colony forming units (CFU) in bone marrow cells increased upon hyperlipidemia but remained unchanged between Apoe^−/− and Apoe^−/− FPR1^−/− mice ([Fig. 1F]). With apoptosis and production being unaltered, we assumed that possibly the mobilization of neutrophils from the bone marrow is impaired. To test this idea, we performed bromodeoxyuridine (BrdU) pulse labeling and quantified the appearance of BrdU-positive neutrophils in blood over time. In agreement with our hypothesis, neutrophils from FPR1-defcient mice exhibited impaired mobilization capacities ([Fig. 1G]).

Fig. 1 FPR1 deficiency conveys atheroprotection. (A) Experimental setup and main materials applied. Analyses at 4 weeks of western diet (WD) were made in male mice; analyses at 4 months of WD were made in female mice. (B–D) Flow cytometric quantification of CD45⁺Gr1^high CD115⁻ neutrophils in blood (B), bone marrow (C), and spleen (D) from Apoe^−/− or Apoe^−/−Fpr1^−/− mice receiving either normal chow or 4 weeks of WD as indicated. (E) Flow cytometric analysis of annexin V positivity on CD45⁺Gr1^high CD115⁻ neutrophils in blood of Apoe^−/− or Apoe^−/−Fpr1^−/− mice after 4 weeks of WD. (F) Colony-forming units (CFU) per 2 × 10⁴ cells as determined in flushed bone marrow cells from Apoe^−/− or Apoe^−/−Fpr1^−/− mice with and without WD feeding for 4 weeks. (G) Flow cytometric analysis of BrdU⁺ neutrophil counts in blood 0 to 138 hours after BrdU pulse labeling of Apoe^−/− or Apoe^−/−Fpr1^−/− mice (each square represents the mean value of n = 3–7 animals). (H) Re-analysis of a published scRNA-seq dataset (GEO dataset GSE155512). Cells from human carotid plaques were clustered and annotated by established markers. FPR1 expression was selectively detected in cells positive for CD45 and either CSF1R (macrophages) or S100A8 (neutrophils). (I, J) Flow cytometric quantification of CD45⁺Gr1^highCD115⁻ neutrophils and of CD45⁺F4/80⁺ macrophages in aortic cell suspensions from Apoe^−/− or Apoe^−/−Fpr1^−/− mice receiving 4 weeks of WD. (K) Representative images and quantification of Mac2+ cells in aortic root sections of Apoe^−/− or Apoe^−/− Fpr1^−/− mice after 4 months of WD. Scale bar: 250 μm. (L) Pearson correlation plot showing aortic neutrophil numbers plotted against corresponding neutrophil counts in blood of the very same mice. Lines depict linear regression; Pearson r coefficients and p-values are indicated. (M) Representative images and quantification of lesion area after oil-red-O staining in aortic roots of Apoe^−/− or Apoe^−/−Fpr1^−/− mice after 4 weeks of WD. Scale bar: 500 μm. (N) Quantification of lesion area after oil-red-O staining in aortic roots of Apoe^−/− or Apoe^−/−Fpr1^−/− mice after 4 months of WD. (B–G, I–K, M, N) Data depict mean ± SEM, each dot represents a single mouse. Two-sided p-values as depicted and analyzed by unpaired Student's t-test or Mann–Whitney U-test depending on Gaussian distribution determined by the Shapiro–Wilk test. SEM, standard error of the mean.

Preclinical and recent clinical data strongly suggest a correlation between circulating neutrophil counts and cardiovascular disease burden.[1] [8] Here, we analyzed a published human scRNAseq dataset of advanced atherosclerotic lesions (GSE155512,[15]) and an unpublished murine scRNAseq dataset of brachiocephalic arteries from Apoe^−/− mice fed with WD for 16 weeks for cell-specific FPR1 expression. In both datasets, FPR1 expression was restricted to myeloid cells and in particular to neutrophils as evident from clustering of CSF1R and S100A8 together with FPR1 ([Fig. 1H] and data not shown). Further, we studied how lack of FPR1 impacts on early atherogenesis and lesion composition. Therefore, we analyzed the number of neutrophils and macrophages in aortic cell suspensions of WD fed Apoe^−/− and Apoe^−/−Fpr1^−/− mice. Here we revealed a significant reduction of aortic neutrophil and macrophage counts ([Fig. 1I, J]) in Apoe^−/−Fpr1^−/− animals compared with controls. The decrease of lesional macrophages was also noticeable in aortic root cross sections ([Fig. 1K]). These observations are also in line with findings in a murine pulmonary fibrosis model where neutrophil numbers in FPR1-deficient mice were also diminished at sites of inflammation (lung) but not in liver or kidney.[13] To unravel if decreased aortic neutrophil counts can be directly linked to lowered neutrophil numbers in blood, we correlated aortic and blood neutrophil counts and observed a clear correlation in blood versus aorta for WD fed Apoe^−/− and Apoe^−/−Fpr1^−/− mice. Yet, correlations in Apoe^−/−Fpr1^−/− remained at lower absolute neutrophil counts ([Fig. 1L]). To test if these diminished neutrophil counts also abolish atherogenesis, we assessed aortic root lesion sizes in Apoe^−/− and Apoe^−/−Fpr1^−/− mice receiving WD for 4 weeks. Here we revealed a significant reduction of lesion size in Apoe^−/−Fpr1^−/− roots ([Fig. 1M]), an observation that was also made at advanced stages of atherosclerosis after 4 months of WD feeding ([Fig. 1N]).

Taken together, we here identify FPR1 as a key regulator of blood neutrophil counts under hypercholesterolemia hence fostering atherosclerosis lesion formation.

Publication History

Received: 16 April 2024

Accepted: 06 May 2024

Article published online:
23 May 2024

• References

• 1 Drechsler M, Megens RT, van Zandvoort M, Weber C, Soehnlein O. Hyperlipidemia-triggered neutrophilia promotes early atherosclerosis. Circulation 2010; 122 (18) 1837-1845
• 2 Swirski FK, Libby P, Aikawa E. et al. Ly-6Chi monocytes dominate hypercholesterolemia-associated monocytosis and give rise to macrophages in atheromata. J Clin Invest 2007; 117 (01) 195-205
• 3 Yvan-Charvet L, Pagler T, Gautier EL. et al. ATP-binding cassette transporters and HDL suppress hematopoietic stem cell proliferation. Science 2010; 328 (5986) 1689-1693
• 4 Soehnlein O, Drechsler M, Döring Y. et al. Distinct functions of chemokine receptor axes in the atherogenic mobilization and recruitment of classical monocytes. EMBO Mol Med 2013; 5 (03) 471-481
• 5 Döring Y, Drechsler M, Wantha S. et al. Lack of neutrophil-derived CRAMP reduces atherosclerosis in mice. Circ Res 2012; 110 (08) 1052-1056
• 6 Zernecke A, Bot I, Djalali-Talab Y. et al. Protective role of CXC receptor 4/CXC ligand 12 unveils the importance of neutrophils in atherosclerosis. Circ Res 2008; 102 (02) 209-217
• 7 Friedman GD, Klatsky AL, Siegelaub AB. The leukocyte count as a predictor of myocardial infarction. N Engl J Med 1974; 290 (23) 1275-1278
• 8 Luo J, Thomassen JQ, Nordestgaard BG, Tybjærg-Hansen A, Frikke-Schmidt R. Neutrophil counts and cardiovascular disease. Eur Heart J 2023; 44 (47) 4953-4964
• 9 Soehnlein O, Döring Y. Beyond association: high neutrophil counts are a causal risk factor for atherosclerotic cardiovascular disease. Eur Heart J 2023; 44 (47) 4965-4967
• 10 Osei-Owusu P, Charlton TM, Kim HK, Missiakas D, Schneewind O. FPR1 is the plague receptor on host immune cells. Nature 2019; 574 (7776) 57-62
• 11 Martijn J, Vosseberg J, Guy L, Offre P, Ettema TJG. Deep mitochondrial origin outside the sampled alphaproteobacteria. Nature 2018; 557 (7703) 101-105
• 12 Grommes J, Drechsler M, Soehnlein O. CCR5 and FPR1 mediate neutrophil recruitment in endotoxin-induced lung injury. J Innate Immun 2014; 6 (01) 111-116
• 13 Leslie J, Millar BJ, Del Carpio Pons A. et al. FPR-1 is an important regulator of neutrophil recruitment and a tissue-specific driver of pulmonary fibrosis. JCI Insight 2020; 5 (04) e125937
• 14 McDonald B, Pittman K, Menezes GB. et al. Intravascular danger signals guide neutrophils to sites of sterile inflammation. Science 2010; 330 (6002) 362-366
• 15 Pan H, Xue C, Auerbach BJ. et al. Single-cell genomics reveals a novel cell state during smooth muscle cell phenotypic switching and potential therapeutic targets for atherosclerosis in mouse and human. Circulation 2020; 142 (21) 2060-2075