Myocardial infarction (MI) occurs following acute thrombotic occlusion of a coronary artery, and triggers a robust inflammatory response. Within hours, neutrophils are recruited to the infarcted myocardium followed by the infiltration of pro-inflammatory Ly6Chi monocytes. Transition from the pro-inflammatory macrophage phenotype (M1) to an anti-inflammatory, pro-resolution phenotype (M2-like) is critical to successful infarct healing. Interventions that polarize macrophages towards an anti-inflammatory 'M2-like' phenotype improve infarct healing in the experimental MI mouse model and reduce subsequent adverse remodelling of the myocardium, but the endogenous mechanisms that regulate repair are not well understood. Furthermore, differences in the resolution of inflammation in C57BL/6 and BALB/c mice, which are two of the commonly used wild-type mouse strains in experimental MI have not been characterised. We previously found that low peripheral blood eosinophil count is associated with increased short-term risk of mortality in low-intermediate risk patients with ischaemic heart disease. This suggests that eosinophils may have a role in the successful remodelling and repair of the heart following myocardial infarction. Eosinophils express a number of immuno-modulating cytokines and lipid mediators implicated in the resolution of inflammation. Increasingly prominent is interleukin-4 (IL-4), a cytokine that has been found to maintain the anti-inflammatory M2-like phenotype in macrophages. We therefore hypothesised that IL-4Rα signalling and recruitment of eosinophils to the myocardium following infarction are key in regulating the subsequent inflammatory response and scar tissue formation during infarct repair and cardiac remodelling. Experimental MI was induced by permanent left anterior descending artery ligation in isofluorane anaesthetized 12-15 week-old male wild-type (WT) BALB/c, WT C57BL/6, IL4Rα-/-, IL-4Rαflox/-, IL-4Rαflox/-LysMCre mice and eosinophil-deficient ΔdblGATA mice. Cardiac function was characterised by high-resolution ultrasound and immune cell infiltration by flow cytometry of single cell infarct and remote zone tissue digests. Blood eosinophil count and 6-month all-cause mortality were assessed in 732 consecutive patients undergoing primary percutaneous coronary intervention for ST-segment elevation myocardial infarction (STEMI). The rate of mortality due to cardiac rupture was significantly higher in C57BL/6 mice in comparison with BALB/c mice at Day 7 post-MI. This was associated with a higher proportion of pro-inflammatory Ly-6Chi macrophages infiltrating the infarct zone tissue of C57BL/6 mice following MI. An accompanying reduction in the number of splenic Ly-6Chi monocytes post-MI, suggestive of splenic monocyte mobilisation, was seen in C57BL/6 mice but not found in BALB/c mice. Furthermore, C57BL/6 mice had a delayed transition in macrophage polarisation towards an anti-inflammatory phenotype. Disruption of IL4Rα signalling, in mice null for the IL4Rα gene, resulted in increased F4/80+ macrophage and pro-inflammatory Ly6Chi macrophage infiltration of the infarct zone and reduced expression of the anti-inflammatory macrophage marker CD206, compared to wild-type controls. Furthermore, expression of GATA3 and ST2, both associated with the immunosuppressive function of (CD4+ Foxp3+) regulatory T cells, was reduced in infarct zone regulatory T cells from IL4Rα-/- mice. These findings were associated with defective wound healing with impaired angiogenesis, increased scar size, disarrayed infarct zone collagen deposition, accompanied by modified expression of plod2 that encodes the collagen cross-linking enzyme lysyl hydroxylase 2. Resulting in greater left ventricular dilatation and loss of cardiac function, as well as a higher 7- day mortality due to cardiac rupture in IL4Rα-/- mice. This indicates that successful infarct repair requires the engagement of IL-4Rα signalling to facilitate the accumulation of anti-inflammatory macrophages and highly immunosuppressive ST2+ regulatory T cells in the heart following MI. Resident cardiac macrophages from naïve hearts of IL-4Rαflox/-LysMCre mice failed to undergo LysMCre-mediated deletion of the IL-4Rα gene, potentially because low or absent expression of Lyz2 (encoding lysozyme M). In both ST-elevation MI (STEMI) patients and mice after acute MI, there was a decline in peripheral blood eosinophil count, with activated eosinophils being recruited to the infarct zone and paracardial adipose tissue of mice. The transcription factors GATA-1 plays a role in the differentiation of eosinophils from eosinophil progenitor cells. Deletion of GATA-1 results in loss of the eosinophil lineage and has been exploited to develop the eosinophil-deficient ΔdblGATA mouse. ΔdblGATA mice were used to address the role of eosinophils in cardiac remodelling following MI. ΔdblGATA mice had increased left ventricular dilatation and reduced ejection fraction after induction of MI, relative to wild-type mice. ΔdblGATA mice had increased scar size with disarrayed infarct zone collagen deposition, accompanied by modified expression of the genes plod2 and lox, which are associated with collagen cross-linking. The proportion of CD206+ anti-inflammatory macrophages was less in the infarct zone of ΔdblGATA mice, but was restored by adoptive transfer of eosinophils from WT mice. Furthermore, adverse cardiac remodelling in eosinophil-deficient ΔdblGATA mice was rescued by provision of IL-4 complex following MI. In conclusion, an enhanced inflammatory response following MI underlies the increased risk of cardiac rupture seen with WT C57BL/6 mice in comparison to WT BALB/c mice. WT BALB/c mice are protected from cardiac rupture, which was associated with an absence of splenic monocyte mobilisation following ischaemic injury. The resolution of inflammation was found to be dependent on IL4Rα signalling which is crucial for cardiac repair and remodelling, through modulating inflammatory cell recruitment and phenotype, as well as scar formation. Eosinophils are recruited to the heart post-MI and are essential for regulating cardiac repair and remodelling, likely through provision of IL-4. Therefore, we were able to show that IL-4Rα signalling and recruitment of eosinophils to the myocardium following infarction are both key in regulating the subsequent inflammatory response and scar tissue formation during infarct healing and cardiac remodelling.

Cytokiner eller interleukiner är signalpeptider med låg molekylär vikt somreglerar många viktiga funktioner. De kan delas in i två grupper beroende på deraseffekt på celler. Interleukin 4 (IL-4) till exempel kan tillhöra gruppentillväxtfaktorer medan interleukin 6 (IL-6) kan tillhöra gruppen aktiverings- ellerdifferentieringsfaktorer. IgM-receptorn eller B-cellsreceptorn, BCR, finns på Bcelleroch är membranbundna immunoglobuliner (mIg) som har tvåhuvuduppgifter; att förmedla signaler som styr B-cellens utveckling samt att bindain antigen som sedan ska presenteras för T-celler. I studien aktiverades B-cellermed antikropp mot IgM (anti-IgM) samt rekombinant IL-4. Efter stimuleringanalyserades IL-6 produktionen med enzyme-linked immunosorbent assay(ELISA). Syftet med studien var att karakterisera uttrycket av IL-4 receptorn pålymfom- och leukemiceller med flödescytometri och polymeras chain reaction(PCR) samt produktion av IL-6 med ELISA. ELISA-analysen visade att tvåcellinjer, stimulerade Sp53 och stimulerade samt kontroller av WaC3CD5+, gaven ökning av IL-6 produktionen. Vid jämförelse med ELISA-resultaten ochflödescytometri-analyserna ser man att WaC3CD5+ som producerat storamängder IL-6, har en liten andel IL-4 receptorer på cellytan. Resultatet av PCRanalysenvisar att det var framförallt Sp53 som fick höga mängder IL-4R mRNA,men även I83, U2932 och WaC3CD5+ fick positiva resultat. Resultaten i dennastudie är preliminära och för att få mer säkerställda resultat krävs att alla analysergörs om ett antal gånger för att få ett mer tillförlitligt resultat. / Cytokines or interleukins are signal peptides of low molecular weight, whichregulates many important functions. They can roughly be divided into two groupsor divisions due to their effect on cells. Interleukin 4 (IL-4), for example, belongsto the group growth factors while interleukin-6 (IL-6) belongs to the groupactivation or differentiation factors. IgM receptors or B cell receptors, BCR, areexpressed on B cells and are membrane-bound immunoglobulins (mIg) and havetwo main functions: to convey signals that control B cell activation and to bindantigen which will then be presented to T cells. In the study B cells were activatedwith antibodies against IgM (anti-IgM) and recombinant IL-4. After stimulationthe IL-6 production was analysed by enzyme-linked immunosorbent assay(ELISA). The purpose of this study was to characterize the expression of IL-4receptor in lymphoma and leukemia cells by flow cytometry and polymerasechain reaction (PCR) and furthermore the production of IL-6 by ELISA. ELISAanalysis showed that two cell lines, stimulated Sp53 and stimulated and control ofWaC3CD5+, resulted in an increased IL-6 production. When comparing ELISAresults and flow cytometry assays, it can be seen that WaC3CD5+, whichproduced large amounts of IL-6, has a small percentage of IL-4 receptors on thecell surface. The results of the PCR analysis shows that particularly Sp53displayed high amounts of IL-4 mRNA, but also I83, U2932 and WaC3CD5+were positive for IL-4 mRNA. The results of this study are preliminary, and to getmore trustworthy results, all analyses have to be repeated to get more reliableresults.