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Research Article | Open Access
High Triglycerides Are Associated with Low Thrombocyte Counts and High VEGF in Nephropathia Epidemica
Nephropathia epidemica (NE) is a mild form of hemorrhagic fever with renal syndrome. Several reports have demonstrated a severe alteration in lipoprotein metabolism. However, little is known about changes in circulating lipids in NE. The objectives of this study were to evaluate changes in serum total cholesterol, high density cholesterol (HDCL), and triglycerides. In addition to evaluation of serum cytokine activation associations, changes in lipid profile and cytokine activation were determined for gender, thrombocyte counts, and VEGF. Elevated levels of triglycerides and decreased HDCL were observed in NE, while total cholesterol did not differ from controls. High triglycerides were associated with both the lowest thrombocyte counts and high serum VEGF, as well as a high severity score. Additionally, there were higher levels of triglycerides in male than female NE patients. Low triglycerides were associated with upregulation of IFN-γ and IL-12, suggesting activation of Th1 helper cells. Furthermore, levels of IFN-γ and IL-12 were increased in patients with lower severity scores, suggesting that a Th1 type immune response is playing protective role in NE. These combined data advance the understanding of NE pathogenesis and indicate a role for high triglycerides in disease severity.
Nephropathia epidemica (NE) is a mild form of hemorrhagic fever with renal syndrome (HFRS), characterized by kidney insufficiency and hemorrhagic disorders. The causative agent is Puumala virus (PUUV), member of Hantavirus genus, family Bunyaviridae. PUUV targets endothelial cells as viral antigens have been frequently found in endothelial cells, in postmortem tissue [1, 2]. PUUV infection is not cytopathic as cell death attributed to virus replication has not been reported either in vivo or in vitro [1–4]. Therefore the Hantavirus pathogenesis remains largely unknown.
NE is endemic in the Republic of Tatarstan where the highest annual prevalence of 64.4 cases per 100,000 was registered in 1997 . Clinically, NE is characterized by an acute onset of high fever, headache, and abdominal pain. Soon, back pain and decrease urine output are followed indicating the impaired renal function. This disease is characterized by 4 periods, febrile, oliguric, diuresis, and convalescence. The oliguric period is the most critical due to the high likelihood of developing life threatening complications. Recovery begins with the onset of diuresis [1, 2]. At this stage, complications can include disseminated encephalomyelitis and hypopituitarism. NE is characterized by a low fatality rate (0.4%). Postconvalescent sequelae are rare and may involve increased glomerular permeability and moderate hypotension [6, 7].
There are 3 clinical forms of NE: mild, moderate, and severe. The severe form of NE is characterized by prominent hemorrhagic symptoms including petechial and nasal and internal bleeding. In some cases, disturbed blood coagulation presents as disseminated intravascular coagulation (DIC) . Laboratory findings for the severe form include high blood urea and creatinine levels, blood urea nitrogen (BUN) >20 mmol/L, and creatinine up to 600 μmol/L. The moderate form of NE has similar but subtler symptoms, with BUN and creatinine levels over 19 mmol/L and 200–300 mmol/L, respectively. The mild form often remains undiagnosed and characterized by mild headache and fever, with hemorrhagic symptoms restricted to small petechia on mucosa and skin.
Increased vascular permeability is a hallmark of NE pathogenesis. Clinically, this increased vascular leakage manifests as petechia, subconjunctival and gastrointestinal hemorrhaging, and, in severe cases, DIC [9, 10]. Additionally, signs of disturbed hemostasis are evident in laboratory tests including prolonged bleeding, increased prothrombin time, and activated partial thromboplastin times . Furthermore, decreased thrombocyte counts are commonly found in NE patients [11, 12]. Extreme thrombocytopenia has been suggested to be predictive of disease severity. Thrombocytopenia (<60 × 109 platelets/L) was found in patients with serum creatinine >620 μmol/L and was an early prognostic marker for acute renal failure . Thrombocytopenia in NE is associated with platelet consumption, due to endothelial cell activation and repair [11, 13]. Recently, a correlation has been shown between thrombocyte counts and serum VEGF levels. Xu et al. demonstrated that thrombocytopenic disorders, characterized by increased platelet destruction, can exhibit increased levels of plasma VEGF . Although serum VEGF has been shown to be upregulated in HFRS, the severe form of Hantavirus infection [15, 16], little is known about the association between VEGF and thrombocyte counts in NE cases.
Elevated serum lipase has been documented in some NE patients , as well as increased serum cholesterol, total phospholipids, and triglycerides , together with upregulated lipid peroxidation . Another study demonstrated low HDCL and total cholesterol, as well as high levels of triglycerides in NE patients . However, little is known about gender differences in lipidemia or any association between serum lipids and cytokine activation in NE.
The objectives of this study were to evaluate changes in serum total cholesterol, HDCL, triglycerides, and cytokine activation in NE patients based on gender, thrombocyte counts, and VEGF and further analyze the association between the severity of the disease and serum lipid, cytokine, VEGF levels, and gender of NE cases. Increased triglycerides were found in NE cases, while total cholesterol levels did not differ significantly between patients and controls. These data indicated that high triglycerides were associated with the lowest thrombocyte counts and high serum VEGF. Furthermore, we found higher triglycerides in male as compared to female NE. Additionally, low triglycerides were associated with upregulation of IFN-γ and IL-12, suggesting activation of Th1 helper cells. Patients with lower severity scores had increased IFN-γ and IL-12 suggesting that a Th1 type immune response plays protective role in NE.
2. Materials and Methods
Two hundred and twenty-eight NE patients (190 male, 38 female; years) admitted to Republican Clinical Hospital for Infectious Disease named after Agafonov, Republic of Tatarstan, were recruited. Serum from 64 NE patients was collected twice (early () and late ()), while a single serum sample was obtained from 164 patients. Diagnosis of NE was established based on clinical presentation and was serologically confirmed by detection of anti-Hantavirus antibodies. In some cases, diagnosis was confirmed using PCR. Serum samples from 56 controls matched for gender, age ( years), and region were collected. The Institutional Review Board of the Kazan Federal University approved this study and informed consent was obtained from each study subject according to the guidelines approved under this protocol (article 20, Federal Law “Protection of Health Rights of Citizens of Russian Federation” N323- FZ, 11.21.2011).
2.2. Multiplex Analysis
Serum cytokine levels were analyzed using Bio-Plex (Bio-Rad, Hercules, CA, USA) multiplex magnetic bead-based antibody detection kits following manufacturer’s instructions. Multiplex kits, Bio-Plex Pro Human Cytokine 27-Plex Panel (IL-1β, IL-1Ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12(p70), IL-13, IL-15, IL-17А, CCL2, CCL3, CCL4, CCL5, CCL11, CXCL10, TNF-α, G-CSF, GM-CSF, IFN-γ, PDGF-BB, and VEGF) and Bio-Plex Human Cytokine 21-Plex Panel (IL-1α, IL-2Ra, IL-3, IL-12(p40), IL-16, IL-18, CCL7, CCL27, CXCL1, CXCL9, CXCL12, HGF, IFN- α2, LIF, M-CSF, MIF, β-NGF, SCF, SCGF-β, TNF-β, and TRAIL), were used for detection of a total of 48 analytes. Serum aliquots analyzed were 50 μL. A minimum of 50 beads per analyte was acquired. Median fluorescence intensities were collected using Luminex 100 or 200 analyzer (Luminex, Austin, TX, USA). Data collected was analyzed using MasterPlex CT control software and MasterPlex QT analysis software (MiraiBio, San Bruno, CA, USA). Standard curves for each analyte were generated using standards provided by manufacturer.
2.3. Serum Lipid Profile
Fasting serum samples were collected early in the morning. Total cholesterol levels were determined using Novochol-200 kit (Vector-Best, Russia) according the manufacturer’s instructions. The optical density (OD 520 nm) of the test serum and calibration sample (provided by the manufacturer) was determined using an Infinite M200 PRO analyzer (Tecan, Port Melbourne, VIC, Australia). Cholesterol levels were calculated using the formula: (mmol/L), where is concentration of cholesterol; is optical density of tested serum; is optical density of calibration sample; and 4.65 mmol/L is concentration of cholesterol in calibration sample.
Serum triglycerides were determined using the Triglyceride-Novo kit (Vector-Best, Russia) according to manufacturer recommendations. The optic density (520 nm) of serum sample and calibrator (provided by the manufacturer) was determined using Tecan Infinite M200 PRO analyzer (Australia). Triglyceride concentrations were calculated using the formula: (mmole/L), where is concentration of triglycerides; is optical density of serum sample; is optical density of calibration sample; and 2.29 mmol/L is concentration of triglycerides in calibration sample.
Serum levels of HDCL were determined using HDCL-Cholesterol-Novo-A kit (Vector-Best, Russia). Briefly, serum sample (3 μL) or calibrator was mixed with reagent 1 (300 μL), incubated for 5 min at 37°C, and used to determine the optic densities and for the sample and calibrator (provided by the manufacturer), respectively. Then 100 μL of reagent 2 was added and optical densities and were measured for the sample and calibrator, respectively. The optic density (OD 650 nm) was determined using Tecan Infinite M200 PRO analyzer (Australia). The HDCL level was calculated using formula: = , where is concentration of HDCL; ; ; is 1.08 mM/L which is concentration of HDCL in calibration sample.
The HDCL concentrations were calculated using the formula: , where is concentration of HDCL; ; ; is 1.08 mmol/L which is concentration of HDCL in calibration sample.
2.4. RT-PCR Detection of Puumala virus (PUUV) Transcripts
Total RNA from 100 μL of serum was extracted using TRIzol® reagent (Life Technologies, Carlsbad, CA, USA). cDNA was transcribed using Super Script kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Two rounds of PCR were conducted. PCR products were sequenced to confirm Hantavirus strain. Primers used were 5′-GTGAGAA ACACACCACAATACTATG-3′ forward and 5′-CTCTGCGT CGTTGGAGTCGTTC-3′ reverse, which amplifies the S segment RNA giving a first-round product of 328 bp, and 5′-CGGACACACAAAGGACAGGG-3′ forward and 5′-GACGCAGAGAAACACAAGTATAATA-3′ reverse, which amplified a second-round product of 302 bp.
2.5. Statistical Analysis
Statistical analysis was conducted using Minitab software (Minitab, State College, PA, USA); differences between the medians of compared groups were calculated using the Mann-Whitney test for nonparametric data and were considered significant at .
The differential abundance of metabolite concentration was calculated using the Subio Platform (Subio Inc., Kagoshima, Japan) with a differentiation expression threshold of 1.5-fold (-test, ).
3.1. Clinical Presentation
Two hundred and thirty-six NE cases (190 male, 38 female) were recruited for this study (Table 1). The average period of hospitalization was days. NE diagnosis was based on clinical presentation, epidemiological data, and serological confirmation. Additionally, all 228 NE serum samples were analyzed for presence of PUUV RNA. PUUV RNA was detected in the initial serum samples in 49 cases (21.5%). At this time there were decreased platelet counts (), which, by the end of hospitalization, was restored to control levels (). Bleeding and varying degrees of blood coagulation disturbances were detected in 46 patients (20.1%). Six cases developed DIC (2.5%).
Patients were grouped based on the presence of signs of disturbed hemostasis. Symptoms were evaluated to assign severity points: 0, absence of bleeding (100 cases; 42.4%); 1, few skin petechia (102 cases; 43.2%); 2, multiple skin petechia, scleral hemorrhages, and gastrointestinal bleeding (28 cases; 11.9%); and 3, DIC (6 cases; 2.5%).
Patients were also grouped based on disease severity based on clinical presentation, mild, moderate, or severe forms. Each presentation was assigned severity points: 0, mild (100 cases; 42.4%); 2, moderate (102 cases; 43.2%); and 3, severe (31 cases; 14.4%).
3.2. Serum Lipid Profile Analysis
Significant upregulation of triglycerides and HDCL was found in patients at the early stage of the disease (Table 2) compared to controls. In contrast, levels of triglycerides remained significantly upregulated in the late stage of the disease, whereas HDCL were similar to controls. Serum total cholesterol did not change and remained similar to controls.
| differences are between NE 1st and control.|
differences between NE 2nd and control.
It has been previously demonstrated that severe Hantavirus cases are characterized by a “lipid paradox,” with serum cholesterol lower but triglycerides higher in HFRS cases than controls . Therefore, we sought to determine whether similar serum lipid profiles are found in severe NE. A total of 6 NE patients (5 male, 1 female; age years) were identified as having severe NE based on the diagnosis of DIC (Table 3). Serum total cholesterol and HDCL levels did not differ from controls; however, triglycerides were significantly higher in severe NE cases than controls. These data support Clement et al. observation of the high triglycerides in severe NE cases .
3.3. Gender Differences in Serum Lipid and Cytokine Activation Profile in NE Patients
When serum lipid profiles were analyzed based on gender, only male cases had significantly higher triglyceride levels than male controls (Table 4). Although upregulated in female NE serum, triglycerides did not differ significantly when compared to female controls. There were no differences between serum level of triglycerides and total cholesterol between male and female NE patients. HDCL levels were significantly lower in female NE compared to female controls, while HDCL was not significantly different in all male patients. Total cholesterol levels did not differ between male and female NE patients as well as between corresponding gender controls.
|P value between female control and female NE; P value between male control and male NE; P value between male and female NE.|
Serum triglyceride levels were significantly increased in NE patients as compared to controls (Figure 1). Also, triglyceride levels differed in NE males and females when compared to controls. It should be noted that once they increased in early stage of the disease, triglycerides remained steady through the convalescent stage in male patients, while female triglycerides continued increasing through early and late stages of the disease (Figure 1(a)). When gender differences in HDCL levels were analyzed, only a slight increase in HDCL level was detected in male NE patients compared to controls, while a steep increase of the HDCL was found in female patients (Figure 1(b)).
Further analysis revealed that more cytokines were significantly upregulated in females than male NE cases when compared to their corresponding controls (33 versus 9) (Table 4). Interestingly, IFN-γ and IL-12(p40) were both upregulated in female NE cases, while only IL-12(p40) was significantly higher in male NE cases. Additionally, female cases had significantly lower scores for disease severity ( versus ; ) and hemorrhagic disturbances ( versus ; ) (Table 5). Although not significant, thrombocyte counts in female patients were higher than in males. IFN-γ and IL-12 are cytokines that play a key role in the activation of Th1 lymphocytes [22, 23]. Upregulation of these cytokines in female NE suggests that there is activation of Th1 lymphocytes. Previously, we have shown an association between the mild form of NE and elevated serum IFN-γ and IL-12 . Therefore, these data suggest that lower severity scores in female cases are associated with activation of Th1 type immune response.
Compared to males, female NE cases had significantly higher levels of TRAIL, CXCL1, and CCL2. These cytokines stimulate mononuclear leukocyte chemotaxis, apoptosis, and angiogenesis. This indicates that differences in clinical presentation between NE in males and females are associated with gender-dependent activation of these cytokines.
3.4. Serum Lipid and Cytokine Activation Analysis Based on Thrombocyte Count
Next, changes in serum lipid and cytokines were analyzed based on thrombocyte counts. Thrombocyte counts below 50,000 cells/μL are considered to be a risk factor for bleeding [25–27]. Recently, a correlation was demonstrated between thrombocyte counts and low density lipids in patients with the severe form of dengue infection , suggesting a role for lipid metabolism in the pathogenesis of thrombocytopenia. Therefore, we sought to determine whether low thrombocyte counts were associated with changes in lipid and cytokine profiles of NE cases. NE patients were separated into two groups: low (<50,000 cells/μL) and high (>50,000 cells/μL) thrombocyte counts. Disease severity scores did not differ between patients with high and low thrombocyte counts (Table 6). Neither serum total cholesterol nor triglyceride levels differed between these two groups of patients, although serum levels for each lipid were higher than control (Table 7). However, HDCL was significantly increased in NE cases with low thrombocyte counts, while in NE patients with high thrombocyte counts HDCL was significantly decreased.