Associations between serum L-arginine and ficolins in the early phase of acute ischemic stroke – A pilot study

Introduction: Activation of both the L-arginine and the lectin pathway contributes to the pathophysiology and the outcome of acute ischemic stroke (AIS). However, the interplay between the two systems has not yet been examined. Methods: A total of 44 patients with AIS were recruited into this study. Serial measurement of serum L-arginine, asymmetric and symmetric dimethylarginine (ADMA, SDMA), and hsCRP, ﬁ colin-2, ﬁ colin-3, MAP-1, MASP-3 and mannose-binding lectin (MBL) were analyzed within 6 h after onset of stroke and 72 h later. Outcomes were assessed as National Institutes of Health Stroke Scale (NIHSS) worsening by 24 h, poststroke infection, and death by 1 month. Results: In the hyperacute stage of AIS, ﬁ colin-3, MAP-1 and MBL were positively correlated with L-arginine within 6 h after onset of symptoms ( p < 0.05 respectively). Signi ﬁ cantly lower ﬁ colin-3 and MASP-3 levels were found at 72 h in patients, who developed post-stroke infection after day 4, when compared to patients without post-stroke infections ( p =0.03 and p =0.009). At 72 hours, ﬁ colin-3 levels negatively correlated with S100B ( p =0.01). Ficolin-3 at 72 post-stroke hours remained an independent predictor of post-stroke infection, while only hsCRP was an independent predictor of 30-day mortality. Conclusion: Early consumption of ﬁ colin-3 is associated with complications such as post-stroke infections. In the hyperacute phase of AIS, the positive correlation between ﬁ colins and the NO donor L-arginine may re ﬂ ect the protective role of L-arginine presumably by improving the cerebral microcirculation in a prothrombotic environment induced by complement activation. C-reactive protein and L-arginine and lectin pathway


Introduction
Post-stroke inflammation and immunodepression are mediated by systemic immune cells, endothelial cells, microglia, and neurons. 1À3 Both infection and inflammation can influence the outcome of ischemic stroke. 4À6 Markers for immunodepression, inflammation and infection are being intensively investigated. 7À9 Activation of the complement system is a key element in the ischemic cascade resulting in an unfavorable outcome due to secondary brain injury. 10 Several studies have explored the temporal profile of the lectin pathway molecules in the sera of patients with acute ischemic stroke (AIS) showing: (i) a decrease in concentration of ficolins in the very early phase of stroke; (ii) unchanged concentration during the subacute phase; (iii) an inverse correlation between ficolin-3 and astrocyte-derived S100B in the follow-up samples suggesting that greater size of the infarct results in higher consumption of ficolin-3. 11,12 Besides, the low levels of ficolins were found to be associated with an unfavorable prognosis in AIS. 11,13 The mannose-binding lectin (MBL) pathway also contributes to the pathological processes. A deficiency of MBL is associated with smaller infarction size and a more favorable outcome. 14 In focal cerebral ischemia model, MBL was a key player in the pathogenesis of ischemic injury suggesting that MBL inhibition may be a relevant therapeutic target in humans with a wide therapeutic window of application. 15,16 Besides complement activation, other inflammatory processes are also known to contribute to the pathogenesis and outcome of the ischemic stroke. 1 Elevated serum levels of L-arginine pathway molecules were observed in the very acute phase of ischemic stroke indicating a more pronounced endothelial dysfunction compared with asymptomatic significant carotid stenosis. 17 Moreover, an increased level of the NO donor L-arginine was found in patients with AIS on admission. 17 A transient elevation of the L-arginine/asymmetric dimethylarginine (ADMA) ratio was observed at the critical 24 post-stroke hours suggesting the protective role of L-arginine, and changes in the L-arginine pathway were predictive of post-stroke infections. 17 In addition, the concentration of L-arginine and ADMA/ symmetric dimethylarginine (SDMA) differentially correlated with thrombo-inflammation in the hyperacute phase of ischemic stroke and such correlations were independently associated with post-stroke infection. 18 Besides, CRP is an independent predictor of cerebrovascular events and prognosis after stroke. CRP is an important molecule in the progression of cerebral tissue injury. 19 Complement activation and elevated CRP levels were independently associated with the clinical severity and different outcome measures of ischemic stroke, indicating their additive effect. 6 An early elevation of CRP inversely correlated with the concentration of L-arginine suggesting that L-arginine may play a protective role and the low L-arginine levels are associated with a higher concentration of CRP, a risk factor for ischemic stroke. 17 In contrast, further elevation of CRP by 72 hours was associated with high concentration of ADMA, 17 contributing to brain injury by reducing cerebral blood flow and facilitating excitotoxic neuronal death. 20 Early elevation of CRP was a strong predictor of mortality, while late elevation predicted post-stroke infection as an early subclinical sign of infection. 21 Here, we aimed to explore the interplay between the lectin and L-arginine pathway and their impact on poststroke infection and outcome.

Methods
This research has been complied with all the relevant national regulations, institutional policies, and in accordance the tenets of the Helsinki Declaration. The study was approved by the local ethics committees. Written informed consent was obtained from all individuals included in this study.

Subjects
Patients with AIS were prospectively examined ( Table 1). Part of this cohort overlapped with a previously published cohort. 11 Patients were enrolled upon the first occurrence of acute ischemic stroke only; all patients had neuroimaging (most of them brain MRI, but at least cranial CT). All patients with definite acute clinical symptoms were enrolled regardless of etiology i.e. lacunar or territorial infarct caused by thrombosis or emboli. Exclusion criteria were infectious diseases, fever < 4 weeks before stroke, an elevated white blood cell (WBC), erythrocyte sedimentation rate (ESR), high- sensitivity CRP (hsCRP, cut-off value < 5 mg/L), procalcitonin on admission (cut-off value < 0.05 ng/mL), positive chest X-ray, hemorrhagic stroke defined by an acute cranial CT scan, and those who declined to participate in the study. 11,21 An evidence-based guideline 22 was followed to detect post-stroke infectious complications (in short, physical and laboratory measures including WBC, ESR, hsCRP, PCT, fever, abnormal urine, chest X-ray or positive cultures). Such complications, predominantly pneumonia and urinary tract infection occurred on the 5th day as an average. The severity of stroke was assessed each day by National Institutes of Health Stroke Scale (NIHSS). 23 Outcome measures were the worsening of NIHSS by 24 h, post-stroke infections, and 30-day mortality.

Biomarkers
Serial measurement of serum L-arginine, ADMA and SDMA, and serum hsCRP, ficolin-2, ficolin-3, MAP-1, MASP-3 and MBL were analyzed within 6 h after onset of stroke and 72 hours later. Ficolin-2 and ficolin-3, mannose-binding lectin (MBL), MBL/Ficolin associated Protein-1 (MAP-1), and MBL associated serine protease-3 (MASP-3) were determined by ELISA-based methods at the Laboratory of Molecular Medicine, Department of Clinical Immunology, Rigshospitalet, Copenhagen, Denmark as described earlier. 24 Serum S100B concentrations were measured by an ELISA method (BioVendor, Modrice, Czech Republic). Serum concentrations of hsCRP were measured by particle-enhanced immunturbidimetric assay, using an automated laboratory analyzer (Roche Cobas Integra 400, Basel, Switzerland). The serum concentration of L-arginine, ADMA and SDMA were determined by High Pressure Liquid Chromatography (HPLC), at the Department of Applied Chemistry, University of Debrecen, Hungary. The determination of L-arginine and its derivatives was described earlier. 17,18 Statistical analysis Statistical analyses were performed using SPSS 20.0 software (IBM Corp., Armonk, NY). Comparison of categorical variables between the groups was performed by chi-square test. Normal distribution of continuous variables was checked by Kolmogorov-Smirnov test. MannÀWhitney U test was used to compare not normally distributed parameters. Correlation analysis was performed calculating Spearman's correlation coefficient (r). Binary logistic regression including baseline variables (age, gender, BMI, hsCRP, S100B, L-arginine and lectin pathway molecules) was used to explore independent predictors and ROC analysis was performed to calculate the cut-off value. Results were considered significant, if p < 0.05.

Results
A total of 44 patients (mean age: 67, SD: 10 years, male: 21) (AIS) were recruited to this study. The demography and clinical parameters are shown in Table 1.

Post-stroke infection
Patients were dichotomized into two subgroups: with (n=7) and without post-stroke infection (n=37). The stroke severity based on NIHSS at 24 h was significantly higher in patients with post-stroke infection compared to those without such complication (19.7 §5.0 vs. 6.5 §6.0, p=0.001). Post-stroke infection was developed in 7 patients on the average 5th post-admission day. Five of them died due to severe infection (predominantly pulmonary complication).
Significantly lower ficolin-3 and MASP-3 levels were found at 72 h in patients, who developed post-stroke infection later, when compared to patients without poststroke infections (p=0.03 and p=0.009) (Figure 2

Discussion
Here, we examined association between the L-arginine and lectin pathway molecules in the sera of patients with AIS within the first 72 post-stroke hours.
In the hyperacute phase of AIS, we observed a positive correlation between ficolin-3, MAP-1, MBL and the NO donor L-arginine reflecting the protective role of L-arginine presumably by improving the cerebral microcirculation in a prothrombotic environment induced by complement activation. 25 Beside initiating activation of complement via the lectin pathway, ficolins may trigger activation of the immune system by production of nitric oxide (NO) by macrophages, thus limiting the infection and concurrently orchestrating the subsequent adaptive immune response. 26 In acute coronary syndrome, the kinetics of oxidized lowdensity lipoprotein receptor-1 (LOX-1), a lectin like molecule, was described with an acute increase up to 12 postevent hour and a subsequent decrease, while NOx derived from the L-arginine-nitric oxide pathway, decreased promptly suggesting an impaired NO metabolism during acute ischemic coronary event. 27 Based on these observations, we assume that an increased availability of the L-arginine might be a counteracting response in the very early phase of ischemic stroke providing the following beneficial effects via restoration of NO production; (i) regulation of the vascular tone; (ii) playing a role as a cytotoxic effector in the immune system; (iii) acting as an intercellular neurotransmitter in the nervous system.
In the subacute phase of ischemic stroke, significant differences were found in the serum concentration of both,  ficolin-3 and L-arginine by 72 h showing that clinically more severe AIS (NIHSS16) was associated with lower ficolin-3 11,17 and higher L-arginine serum levels. It seems reasonable, that the ficolin-3 induced NO generation is exhausted in patients with worsening stroke despite a higher L-arginine supply in the subacute phase. A higher NIHSS score at 24 h reflects an early progression of AIS, thus the opposite change in serum ficolin-3 (decrease between 6 and 72 h) and L-arginine levels (increase between 6 and 72 h) may also suggest that higher infarct size is accompanied by further consumption of ficolin-3, which is presumably antagonized by a further L-arginine availability. Similarly, MBL, another component of the lectin pathway, showed negative correlation with ADMA an indicator of endothelium dysfunction, and also S100B, an indicator of infarct size suggesting an association between extended brain tissue injury and increased consumption of MBL by post-stroke 72 h. In accordance with our previous data, the S100B protein reaches its peak serum concentration at 72 h after the onset of stroke. 21 In addition, high plasma concentration of ADMA, an endogenous inhibitor of NO synthase, was observed in patients with silent brain infarcts on MRI. 28 Consumption of ficolin-3 and MASP-3 by 72 h was also associated with complications such as post-stroke infections. Recently MASP-2 was reported playing a pivotal role in driving tissue injury and unfavorable outcomes in mouse models of ischemic brain injury. 29 In our earlier studies, low ficolin-3 and elevated hsCRP concentrations were independently predictive for worse outcome. 10,11 Our finding, that ficolin-3 at 72 post-stroke hours was found to be an independent predictor of post-stroke infection, is in accordance with those observations that ficolin-3 has the highest post-stroke concentration and the greatest complement-activating capacity among the lectin pathway initiators. 30 The fact, that the independent predictor of 30-day mortality was only CRP, but not ficolin-3, suggests that they reflect two different pathways of inflammation contributing to the outcome of stroke. 11,18,31 Although the sample size is small, our findings may suggest that both the lectin and L-arginine pathway plays a crucial role in post-ischemic stroke pathology including immunodepression leading to a poor outcome. Limitations of this study should be also mentioned: (i) the size and localization of the infarct could have been more accurately determined by follow-up MR imaging instead of S100B; (ii) the functional outcome based on the modified Rankin Scale was not assessed here; and (iii) it would have been interesting to clarify the recovery time of the lectin pathway by a follow-up study. However, the detailed mechanisms remain largely unknown, therefore larger studies will be required to establish the clinical implications of our finding.

Declaration of Competing Interest
None declared.