Expression of Serum Exosomal miRNA 122 and Lipoprotein Levels in Dogs Naturally Infected by Leishmania infantum: A Preliminary Study.

Current knowledge on the role of exosomal microRNA (miRNA) in canine leishmaniasis (CL), with particular regards to the interaction between miR-122 and lipid alterations, is limited. The aim of this study was to isolate/characterize exosomes in canine serum and evaluate the expression of miR-122 in ten healthy and ten leishmaniotic dogs. Serum exosomes were isolated using a polymer-based kit, ExoQuick® and characterized by flow cytometry and transmission electron microscopy, whereas miR-122-5p expression was evaluated by quantitative reverse-transcriptase polymerase chain reaction. A significant decreased expression of exosomal miR-122-5p, decreased serum levels of high-density lipoproteins, and increased serum levels of low-density lipoproteins were seen in leishmaniotic dogs when compared with healthy dogs. These results suggest that hepatic dysfunctions induced by the parasite interfere with lipoprotein status. The decreased expression of exosomal miR122 represents an additional effect of Leishmania infection in dogs as in people.

1. Introduction

Leishmaniasis is a zoonosis caused by intracellular protozoa of the genus Leishmania transmitted by phlebotomines. During the initial phase of the infection, Leishmania spp. can survive within the Kupffer cells without affecting the hepatic parenchyma [1]. A high tolerability of such cells to Leishmania spp. promotes a parasite survival in the canine liver leading to a perturbation of liver function and, in particular, cholesterol and lipoprotein metabolism [2,3]. In fact, Leishmania parasites are able to modulate the expression of genes associated with cholesterol biosynthesis, uptake, and efflux [2,4]. Cholesterol plays an important role in Leishmania infection since amastigotes are not able to synthesize it de novo [5], however, the mechanistic links between Leishmania infection and lipid changes are complex, multifactorial, and not completely understood. Important differences between promastigotes and amastigotes of Leshmania chagasi have been observed regarding uptake through lipid rafts, subdomains of the plasma membrane that contain high concentrations of cholesterol and glycosphingolipids. A transient disruption of lipid rafts in cell membranes affected promastigote uptake, but not amastigote uptake by macrophages. These findings indicate a difference in the needs of Leishmania parasites regarding both the availability and origin of cholesterol. Leishmania protozoa can alter the metabolism of cholesterol directly or through the effect on lipoproteins; trypanosomatids are able to acquire cholesterol from low-density lipoproteins (LDLs) and high-density lipoproteins (HDLs) by endocytosis [6,7,8].

As in people, Ghosh et al. [9] showed that an inverse association between blood levels of cholesterol and susceptibility to Leishmania donovani infection was present in mice. Contrarily, in leishmaniotic dogs, while hyper/normal cholesterolemia has been detected, high levels of low-density lipoproteins (LDLs) and low levels of high-density lipoproteins (HDLs) have been reported [10,11,12].

Recently, microRNAs (miRNAs) have been used to investigate both lipid metabolism and function in animals [13]. miRNAs are small, 20–22 nucleotides long, posttranscriptional regulators identified in tissues and blood in healthy and diseased people and dogs [14,15]. They act on mRNA primarily as inhibitors (translational repression or degradation) affecting several physiological processes [13]. While in circulation, serum miRNAs are highly degradable, however, when transported in microvesicles (exosomes) these molecules are more stable and can serve as reliable diagnostic biomarkers in diseased patients [16,17,18]. Exosomes being small extracellular mycelial vesicles [19] protect RNA from RNAse degradation [20]. In 2013, Ghosh et al. [21] explored, for the first time, the role played by exosomes in miR-122 expression, the most common miRNA present in the liver tissue, in L. donovani infection in mice. The authors showed that, the glycoprotein gp63, present in Leishmania exosomes, was able to degrade Dicer1 in the hosts’ hepatic cells, reducing the synthesis of miR-122. Considering these premises, the aim of this study was twofold: evaluate the expression of serum exosomal miR-122 and the lipoprotein profile in dogs naturally infected by Leishmania infantum.

2. Materials and Methods

2.1. Animals

Ten mixed breed dogs, naturally infected by L. infantum, and ten mixed breed healthy dogs were recruited in the present study. The diagnosis of CL was based on compatible clinical signs and confirmed by visualization of amastigotes in lymph nodal aspirates and serologically by a positive indirect fluorescent antibody test (IFAT) greater than 1:160 [22,23]. All dogs were also tested for presence of Dirofilaria immitis, Anaplasma phagocytophylum, Borrelia burgdorferi, and Ehrlichia canis antibodies using SNAP® test (Canine SNAP 4Dx, IDEXX laboratories). In order to be enrolled, the dogs with leishmaniasis had to be untreated at the moment of diagnosis and negative to the SNAP test. The healthy dogs had to be clinically healthy, negative to IFAT (<1:40) [22,23] and the SNAP test.

2.2. Samples Collection and Hemato-Biochemical Analysis

Ten mL of peripheral blood were collected from the jugular vein of each dog and put into tubes without anticoagulant (5 mL) and in tubes containing ethylene diamine tetraacetic acid (EDTA) (5 mL). A complete blood cell count was performed within 30 min from the collection using a semi-automatic cell counter (Genius S; SEAC Radom Group, Florence, Italy). Serum was also collected after centrifugation at 327× g for 15 min and it was stored at −20 °C. Serum urea, creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT), bilirubin, alkaline phosphatase (ALP), and total protein (TP) were analyzed using commercially available kits (Reactivos Spinreact S.A. OLOT, Gerona, Spain). Total serum cholesterol, triglycerides, and high-density lipoprotein cholesterol (HDL) were measured using a Dimension EXL analyzer (Siemens Healthcare Diagnostics s.r.l., Milan, Italy); low-density lipoprotein cholesterol (LDL) was calculated using the Friedewald equation [24].

2.3. Exosomes Isolation and Mirna Detection

Exosomes were extracted from the serum using a polymer-based kit, ExoQuick® (System Biosciences Mountain View, Palo Alto, CA, USA) according to a previous study [17]. Exosomes were analyzed by flow cytometry (FC) and characterized by transmission electron microscopy (TEM). Dynamic light scattering and zeta potential determinations were also performed with a Nano ZS 90 (Appendix A).

Isolated exosomes were processed for miRNA isolation using a commercially available kit (exoRNeasy Serum Plasma Kit; Qiagen, Hilden, Germany). Subsequently, the cDNA was amplified by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) following the manufacturer’s instructions (Appendix A).

2.4. Statistical Analysis

The data were tested for normal distribution using the Kolmogorov–Smirnov test (alpha = 0.05). The unpaired two samples Student’s t-test or Mann–Whitney test was performed to evaluate the behavior of each data variable between the two groups (healthy vs. CL). All statistical comparisons were performed using the GraphPad Prism6 Software (GraphPad Software Inc., La Jolla, CA, USA). A p < 0.05 was considered statistically significant.

3. Results

3.1. Clinical Examination and Blood Tests

The median age at the moment of enrollment was four years (range: 1–6) for the healthy group and four years (range: 1–8) for the CL group. The mean body weight was 22.3 ± 5.4 kg and 20.4 ± 4.3 kg for healthy and CL group, respectively. There were four males and six females (three spayed) in the healthy group, whereas five males (one castrated) and five females (two spayed) were present in the CL group. There were no differences in age (Mann–Whitney; p = 0.37), weight (t-test; p = 0.39), or sex (Fisher’s exact; p = 1) between the two groups. The more frequent clinical signs observed in the CL group were lymphadenopathy (80%), weight loss (70%), skin lesions (70%), and splenomegaly (30%). The skin lesions included seborrhea sicca (5) and alopecia (2). The results of hematological and biochemical tests are presented in Table 1.

Table 1

Hematological and biochemical parameters in healthy and affected dogs.

Parameter	Healthy	Leishmaniotic	Reference Range [25,26,27]	p-Value
RBC (×106/dL)	6.5 ± 0.6	5.5 ± 1.6	5.5–8.5	0.01
Hb (g/dL)	14.9 ± 1.5	10.7 ± 3.1	12–18	0.0001
Hct (%)	44.3 ± 3.2	34.4 ± 9.6	37–55	0.0009
MCV (fL)	69.2 ± 3.4	63.4 ± 4.8	60–77	0.008
MCH (pg)	21.1 ± 1.4	19.6 ± 1.4	20.5–24.2	<0.0001
MCHC (%)	33.8 ± 1.1	30.9 ± 0.9	32–36	<0.0001
PLT (×103/mm3)	295.4 ± 89.5	279.3 ± 74.4	200–500	0.7
WBC (×103/mm3)	10.3 ± 1.9	10.9 ± 1.5	6–17	0.5
Azotemia (mg/dL)	42.2 ± 6.0	48.0 ± 17.6	21–59	0.7
Crea (mg/dL)	0.8 ± 0.2	1.4 ± 1.0	0.5–1.5	0.7
ALT (U/L)	41.9 ± 11.8	35.1 ± 8.3	21–102	0.1
AST (U/L)	34.6 ± 10.2	34.3 ± 12.9	23–66	0.9
ALP (U/L)	111.8 ± 28.2	110.3 ± 32.1	20–156	0.9
BIL (mg/dL)	0.3 ± 0.1	0.3 ± 0.1	0.1–0.5	1
GGT (U/L)	3.0 ± 1.4	2.4 ± 1.2	1.2–6.4	0.4
GLU (mg/dL)	73.8 ± 12.0	74.1 ± 13.5	65–118	0.9
TRIG (mg/dL)	80.0 ± 23.0	86.1 ± 22.3	20–112	0.5
CHOL (mg/dL)	193.6 ± 49.3	223.7 ± 63.8	135–270	0.3
HDL (mg/dL)	89.7 ± 19.1	47.4 ± 26	49–165	<0.0001
LDL (mg/dL)	87.9 ± 49.5	159.1 ± 51.1	5–86	0.01
TP (g/dL)	6.2 ± 0.5	9.1 ± 1.7	5.4–7.1	0.0003
Alb	3.4 ± 0.4	2.9 ± 0.5	2.6–3.3 g/dL	0.02

RBC: red blood cells; Hb: hemoglobin; Hct: hematocrit; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration; Plt: platelet; WBC: white blood cells; Crea: creatinine; ALT: alanine aminotransferase; AST: aspartate aminotransferase; ALP: alkaline phosphatase; BIL: total bilirubin; GGT: gamma glutamil transferase; GLU: glucose; TRIG: triglycerides; CHOL: total cholesterol; HDL: high-density lipoprotein cholesterol; LDL: low density lipoprotein cholesterol; TP: total protein; Alb: albumin.

In particular, 70% of affected dogs had a non-regenerative normocytic normochromic anemia. In addition, CL dogs had a significant reduction in total red blood cells (p = 0.01), hematocrit (p = 0.0009), hemoglobin (p = 0.0001), mean corpuscular volume (MCV, p = 0.008), mean corpuscular hemoglobin (MCH, p < 0.0001), and mean corpuscular hemoglobin concentration (MCHC, p < 0.0001). The biochemical parameters were also altered in the CL group compared to the healthy dogs. In particular, levels of TPs (p = 0.0003) and LDLs (p = 0.01) were significantly increased, whereas the level of HDLs was significantly decreased (p < 0.0001).

3.2. Exosomes Isolation and miRNA Detection

Serum exosomes were detected as round vesicles of heterogeneous sizes via negative stain observed by TEM (Figure 1).

Figure 1

Transmission electron micrograph of exosomes isolated from serum. The morphology is observed by negative staining.

The size determination was further investigated using a Zetasizer Nano resolved in an average size of 131 ± 4 nm with a Z potential of −27 ± 0.5 mV. Fluorescein isothiocyanate (FITC) positive singlets were 99.5%, 100%, and 94.4% for CD63, CD9, and CD81, respectively (Figure 2).

Figure 2

Identification of canine exosomes. Exosomes, obtained from canine serum, were labeled with Exo-FITC and incubated respectively with anti-CD63, anti-CD9 and anti-CD81 Exo-Flow FACS magnetic beads. The data percentage of captured exosomes is shown.

A total of 12 ng/μL of miRNA was isolated from serum exosomes. Using qRT-PCR, both miR-122 and RNU6-2, with Ct values of 35.3 ± 0.4 and 32.5 ± 0.6 respectively, were detected. When exosomal miR122 levels were compared between healthy and leishmaniotic dogs, a significantly lower (p = 0.004) expression was seen in the latter group (Figure 3).

Figure 3

Expression of miR-122 gene expression in canine serum exosomes from healthy (n = 10) and leishmaniotic (n = 10) dogs. Boxes and whiskers graph. The boxes indicate the quartiles and the mean. **: p < 0.01.

4. Discussion

Although in the present study, a significant modification of the level of serum cholesterol was not present, a significant alteration of serum LDL and HDL levels were seen in the CL group, in agreement with previous studies [10,11,12,21]. Such alterations may suggest a lipid perturbation associated with Leishmania infection. These data are also in agreement with Carvalho et al. [28] showing that people with clinical manifestation of visceral leishmaniasis have high triacylglycerol and very-low-density lipoprotein (VLDL) levels, but low HDL levels. Different mechanisms may be implicated in the reduction of HDL levels during Leishmania infection; these may include decreased hepatic synthesis and secretion of apolipoproteins [29], increased endothelial lipase activity [30], and displacement of apoA-I by serum amyloid A [31]. In addition to their primary role in lipid transport, HDLs have also been associated with anti-inflammatory and anti-oxidant activity, vascular endothelial cell activation, nitric oxide (NO) production, expression of inflammatory mediators, and endothelial progenitor cell proliferation [32,33,34,35]. A reduction of HDL levels could represent a mechanism of defense that the protozoa uses to contrast the leishmanicidal activity of NO in infected macrophages [36]. In a recent study, Rodrigues Santos et al. [37] showed that human monocytes, experimentally infected by L. infantum, had two times higher parasitism in the presence of VLDL and HDL than when these lipoproteins were absent.

This is the first study in leishmaniotic dogs showing higher levels of serum exosomal miR-122, a microRNA recently indicated as a good candidate marker for liver diseases in the absence of liver-specific biochemical markers [15]. In leishmaniotic dogs, liver damage can be present with or without specific clinical signs as well as with a low or high parasitic burden. Indeed, liver granulomas (effector T cells, macrophage/dendritic cell) have been described in asymptomatic dogs with low parasite burdens while not organized granulomas were detected in the liver of symptomatic dogs with high parasite burdens [38]. In this study, liver biopsies were not performed (absence of increased cytopathic markers of liver toxicity, e.g., ALT), however, a lower level of albumin and exosomal miR-122 in the absence of renal and enteric signs, suggests liver dysfunction rather than liver damage. This dysfunction associated with a reduction of circulating miR-122 in leishmaniotic dogs may lead to the hypothesis that, like in mice [21], Leishmania parasites may play a potential role in the regulation of specific miRNA through the gp63 in dogs.

Future research should consider enrolling a significantly higher number of dogs naturally infected by L. infantum in order to study the connection of exosomal miR-122 obtained both from serum and from liver biopsies with the levels of circulant gp63.

5. Conclusions

In summary, the results of the present study suggest that alterations of the lipid metabolism, low HDL and high LDL serum levels along with a lower miR-122 expression may indirectly mirror hepatic alterations induced by L. infantum in dogs. However, because of the low number of animals enrolled, further studies are warranted to better define the role of miR-122 as a potential biomarker of hepatic damage/disfunction during canine leishmaniasis.