CD 45 + , CD 68 + and E-cadherin + Expressions in Skin Dogs Naturally Infected by Leishmania infantum

Background: In canine leishmaniasis (CanL), infection occurs through phlebotomine vectors that inoculate the protozoan Leishmania infantum into the skin that infected macrophages and activated dendritic cells (CD). Dogs with CanL present variable clinical manifestations, being common the presence of cutaneous lesions. The aim of this study was to evaluate the expression of CD45, CD68 and E-cadherin associating the skin sentinels cells and to compare the clinical-dermatological manifestations in the skin of dogs naturally infected by L. infantum. Materials, Methods & Results: Dogs infected (n = 22) by L. infantum were divided into asymptomatic group (AD, n = 9), and symptomatic group (SD, n = 13), according criteria based on the presence or absence of skin changes. Dogs non-infected (CD, n = 5) were included as control group. Samples of skin biopsies collected from scapular region were processed by routine histology and labeled by immunohistochemistry with monoclonal antibodies against CD45, CD68 and E-cadherin, and were described as none, mild, moderate and intense. SD presented keratoconjunctivitis, onychogryphose, lichenification, depigmentation, alopecia, hypotrichosis, erythematous dermatitis, exfoliative dermatitis, ulcerative dermatitis and crusted dermatitis, and the frequency these alterations was expressed as percentage. The results of hematological and biochemical parameters were analyzed by Kruskal-Wallis test followed by the Dunn’s test and expressed as mean ± standard deviation, with values P < 0.05. Leukocytosis (not significant), red blood cells, hematocrit and hemoglobin (P < 0.05), total protein serum (P < 0.05), globulins (P < 0.05), albumin and A/G ratio (P < 0.01) were altered in SD in relation to CD. Cutaneous cellular infiltration, composed by macrophages, plasma cells, lymphocytes and neutrophils, was observed in CD. There was an increase of expression of the markers in SD when compared to the other groups, as moderate CD68 expression and L. infantum, and intense CD45 and E-cadherin expressions. Discussion: Cutaneous involvement is very important in CanL, as it corresponds to where is the first interaction between the parasite and the immune system. Dermatological clinical signs, leukocytosis, anemia, globulins levels have been reported for dogs naturally infected by L. infantum. Inflammatory infiltrate was distributed at superficial and deep dermis, which was composed by mononuclear cells as macrophages, plasma cells, lymphocytes and neutrophils. To characterize the immune sentinels cells in the skin it was evaluated CD45, CD68 and E-cadherin expressions. In syntomatic dogs, our results revelead an increase of expression of these markers. CD45 is one of the most abundant molecules expressed on the white blood cell surface in various mammals, while CD68 is a myelomonocytic marker that seems to be retained during monocyte differentiation. In the skin, increased numbers of CD68 are related to dendritic epidermal cells, which can be expressed as CD45/CD1a/HLADR. DCs of the skin, particularly epidermal Langerhans cells (LCs), form networks anchored to neighboring keratinocytes via E-cadherin. Thus, CD45, CD68 and E-cadherin expressions may be related to activation of skin sentinels cells in dogs naturally infected by L. infantum. Our results indicated that CanL modify the CD45, CD68 and E-cadherin expressions, which characterize the immune sentinels cells activation that promove the recruitment the cellular infiltrate, which was composed by macrophages, plasma cells, lymphocytes and neutrophils. Thus, these informations may contribute to the follow-up of CanL progression in skin.


INTRODUCTION
The skin corresponds to the outer covering of body, appearing as a mechanical barrier to environmental and pathogenic insults, consisting of epidermis, dermis and hypodermis.The epidermis and dermis participate in the maintenance of skin homeostasis and have in their composition a variety of pro-inflammatory cells and mediators that initiate and regulate the innate and acquired immune response [11,14].Among the immune cells present in the skin are keratinocytes, T lymphocytes and sentinel cells, as dendritic cells (DCs), macrophages, and mast cells [2,14,23].DCs make a link between the innate and adaptive immune response.They have the ability to capture the pathogen Leishmania spp., and migrate to the lymphoid organs where they present the processed antigens to naive T cells, so that they regulate the cellular response [3,17,23].
Canine leishmaniasis (CanL) is a severe disease, usually subacute or chronic in dogs, transmitted through the bite of female sandfly vectors in the skin, which inoculate in dermis promastigotes forms corresponding to the infective stage of the parasite Leishmania infantum [23].Clinical manifestations of CanL vary widely as a result of numerous pathogenic mechanisms of the immune response and different affected organs [1,23].Cutaneous involvement is very important in CanL, as it corresponds to where is the first interaction between the parasite and the immune system [21].Changes in skin of dogs with CanL are closely related to the progress of infection and present themselves in variable forms such as alopecia, hypotrichosis, hyperkeratosis, lichenification, erythema, exfoliative dermatitis, crusty dermatitis, ulcerative lesion, nodular dermatitis, papular dermatitis or pustular dermatitis [22] .
The aim of this study was to evaluate the CD45 + , CD68 + and E-cadherin + expressions associating the skin immune sentinels cells and to compare the clinical-dermatological manifestations in the skin of dogs naturally infected by Leishmania infantum.

Study population
Twenty-seven mixed-breed adult dogs of both genders (ages ranging from 2 to 6 years) were selected.They were provided by the Zoonosis Control Center, in Fortaleza (Ceará, Brazil), a region with a high prevalence of CanL.The experimental animals were selected based on the results of DPP rapid test Canine Visceral Leishmaniasis 1 , serologic ELISA test 1 and bone marrow parasitological test.Negative animals in serological and parasitological tests were considered not infected and used as control animals (CD, n = 5).Dogs infected (n = 22) by L. infantum were divided into asymptomatic group (AD, n = 9), and symptomatic group (SD, n = 13), according criteria based on the presence or absence of skin changes.SD presents the characteristic cutaneous manifestations of CanL, including skin lesions, onychogryphosis and keratoconjunctivitis. Blood samples were collected by jugular venipuncture for determination of hematological (Mindray BC-200 Vet ® ; Mindray BA 88A ® ) 2 and biochemical (Labtest ® ) 3 parameters.

Skin sample
The collection of skin specimens was carried out after euthanasia (Potassium chloride 10%) of dogs with a barbituric anesthesia (Thiopentax ® ) 4 .Skin sections of 5 mm were collected with the help of a punch from the scapular region.

Histological analysis
Skin samples were fixed in 10% buffered neutral formalin and were embedded in paraffin for routine histological processing.Five-micrometer sections were cut and stained with hematoxylin and eosin (HE).The analysis of histological sections was performed under a light microscope (Nikon -Eclipse E200 ® ) 5 at a 200x magnification.We assessed the degree (none, mild, moderate and intense) of cellular inflammatory infiltrate in dermis, according to the average subjective perception of two observers.

CD45 + , CD68 + and E-cadherin + immunohistochemistry
Sections (5 μm) were mounted on silanized glass slides and subsequently subjected to an incubator at 36ºC.Antigen retrieval was performed using citrate buffer pH 9.0 for 30 min at 97ºC.The activity of endogenous peroxidase was inhibited by Hydrogen Peroxide 3% 6 for 10 min and the slides were subjected to monoclonal antibody mouse anti-human CD45 + (clone 2B11 + PD7/26) 6 , CD68 + (Clone KP1) 6 monoclonal antibody and mouse anti-human E-cadherin + (UMR-38 clone) 6 Flex incubated for 1 h at room temperature.The slides were then washed twice in PBS and then incubated with En-Vision polymer reagent (EnVision TMþDual connection System/HRP) 6 for 30 min at room temperature.Finally, diamine benzidine (DAB) 6 was applied for 10 min.Sections were counterstained with Mayer's hematoxylin 6 for 5 min.The intensity of the staining was analyzed by light microscopy (Leica DM2000®) 7 at 200x magnification.The expression of the markers was classified as none (-), mild (+), moderate (++) and intense (+++), according to the average subjective perception of two observers [16].

L. infantum immunohistochemistry
Cutaneous sections prepared from paraffinembedded biopsies were cut into 5 μm sections and mounted on silanized slides.Slides were deparaffinised in xylene, and the tissue was rehydrated using graded alcohols.The slides were incubated in a 4% hydrogen peroxide solution and PBS (0.01 M, pH 7.2), followed by incubation in normal goat serum (1:100 dilution).Antibody from rabbits experimentally infected with L. infantum (1:200 dilution in 0.01 M PBS), donated by the Institute of Tropical Medicine of São Paulo, was used as the primary antibody.The slides were then incubated for 22 h at 4°C in a humid chamber.Then, the PBS lavage was performed and the slides were incubated again in a biotin solution, and then washed again with PBS and incubated with the streptavidin-peroxidase complex for 20 min at room temperature.The reaction was carried out with the addition of 0.024% diamino-benzidine 8 and 0.16% hydrogen peroxide 8 .Analysis of amastigote forms of L. infantum was performed by light microscopy 7 at 200x magnification.The intensity of staining was classified as none, mild, moderate and intense, according to the average subjective perception of two observers [8].

Statistical analysis
Analyses were performed using the statistical software 9 .Results were expressed as mean ± standard deviation (SD).Prior, data were submitted to Grubbs and Kolmogorov test to verify the presence of outliers and to determine data homoscedasticity, respectively.Data of clinical changes observed were expressed as a percentage.For hematological and biochemical analyzes it was used Kruskal-Wallis test followed by the Dunn's test.In all cases, significance was defined at P < 0.05.
Hematological and serum biochemical parameters are shown in Table 1.Regarding the white blood cell (WBC) count, AD and SD differed significantly (P < 0.05).It's worth highlighting the presence of leukocytosis in AD (16.89 ± 3.74) when compared to the CD (12.60 ± 2.29), although it is not statistically significant.Red blood cells (RBC), hematocrit (HCT) and hemoglobin (HGB) were reduced in SD when compared to CD (P < 0.05).There was no significant difference between the groups regarding to platelets (PLT) count.Total protein serum (TPS) levels differ (P < 0.05) between AD and SD.Globulins levels were elevated (P < 0.05) but the albumin and A/G ratio were decreased (P < 0.01) in SD in relation to CD.The skin histopathological findings were showed in Figure 3. Cutaneous cellular infiltration was identified in 54.0% (12/22) of the naturally infected dogs, being 83.3% (10/12) in SD and 16.7% (2/12) in AD.Inflammatory infiltrate was present in AD and SD as a mild (Figure 3B) and mild to moderate (Figure 3C), respectivelly, which was composed by mononuclear cells as macrophages, plasma cells, lymphocytes and neutrophils.The cellular infiltrate shown a distribution multifocal in the superficial and deep dermis.
The main immunohistochemical findings were showed in Table 2 and Figure 3.There was a greater expression of the markers in SD when compared to the other groups, with moderate CD68 + expression and L. infantum, and intense CD45 + and E-cadherin + expressions.It is worth noting that in AD there was a greater expression of CD45 + , while in CD it was highlighted CD68 + and E-cadherin + .

DISCUSSION
Cutaneous involvement is very important in CanL, as it corresponds to where is the first interaction between the parasite and the immune system [21].All dermatological clinical signs observed for the SD have been reported for infected by Leishmania infantum [21,22].The presence of leukocytosis in AD was described by Reis et al. [20].RBC, HCT and HGB reduced in SD were also reported in previous studies [5,15,20].Regarding PLT count, previously described data, also did not observe significant difference between the groups [15].High levels of TPS and globulins, and decreased albumin and A/G ratio in SD were reported by other authors [7].
Inflammatory infiltrate present as a mild to moderate, composed by mononuclear cells as macrophages, plasma cells, lymphocytes and neutrophils, is a characteristic of CanL [6].In addition, the cellular infiltrate shown in the superficial and deep dermis with multifocal distribution, consists of findings described by Reis et al. [21].
A greater expression of the markers CD45+, CD68+ and E-cadherin+ in SD when compared to the other groups, may be related to activation of skin sentinels cells in dogs naturally infected by L. infantum.CD45 + is one of the most abundant molecules expressed on the white blood cell surface in various mammals, while CD68 + is a myelomonocytic marker that seems to be retained during monocyte differentiation.The differential expression of CD45 + isoforms in canine leukocytes were shown to vary among leukocyte phenotype and by the activation status [9].In dogs, it was demonstrate that leukocytes and CD45 + epithelial cells was characteristic of an immature stage [4].In the skin, increased numbers of CD68 + are related to dendritic epidermal cells, which can be expressed as CD45 + /CD1a -/HLA -DR + [13].DCs of the skin, particularly epidermal Langerhans cells (LCs), form networks anchored to neighboring keratinocytes via Ecadherin, a component of epithelial cell junctions that is also expressed by LCs.It has been demonstrated that E-Cadherin-mediated contacts induce DC maturation [10].On the other hand, the expression of cadherins have been observed during tumor progression [18].E-cadherin + is expressed in not only canine cutaneous histiocytomas and mastocytomas, but also multiple other canine tumor [12,19].

CONCLUSION
Our results indicated that CanL modify the CD45 + , CD68 + and E-cadherin + expressions, which characterize the immune sentinels cells activation that promove the recruitment of cellular infiltrate composed by macrophages, plasma cells, lymphocytes and neutrophils.Thus, these informations may contribute to the follow-up of CanL progression in skin.

Declaration of interest.
The authors report no conflicts of interest.The authors alone are responsible for the content and writing of the paper.

Figure 1 .
Figure 1.Skin macroscopic lesions of dogs diagnosed with CanL.A, dermatitis ulcerative in ear; B and D, dry exfoliative dermatitis areas diffused by the animal body; C, dermatitis crusted nasal region; D, diffuse alopecia; E, keratoconjunctivitis and hypotrichosis periocular; F, diffuse hypotrichosis; G, onychogryphosis; H, lichenification and hyperpigmentation diffuse the animal's body.

Figure 2 .
Figure 2. Dermatological clinical findings of dogs naturally infected by Leishmania infantum.

Funding.
We would like to thank the CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico -"National Counsel of Technological and Scientific Development") and FINEP (Empresa Fiannciadora de Estudos e Projetos) for financial support.Ethical approval.All procedures, treatments and animal care were approved by the Ethical Committee in Animal Use of the State University of Ceará, Brazil (CEUA, Protocol no.6459434/2015).

Table 1 .
Hematological and biochemical analyses parameters in asymptomatic and symptomatic dogs naturally infected by Leishmania infantum.