Received: 29 December 2017 DOI: 10.1002/jcb.27048
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Accepted: 23 April 2018
RESEARCH ARTICLE
Cytokines and chemokines differentially regulate innate immune cell trafficking during post kala-azar dermal leishmaniasis Ashish K. Singh1 Vijaya Mahantesh1 Pradeep Das4
| Vidya N. R. Das2 | Ajay Amit1 | Manas R. Dikhit1 | | Shubhankar K. Singh3 | Shyam Naryan3 | Krishna Pandey2 |
| Neena Verma5 | Sanjiva Bimal1
1 Department
of Immunology, Rajendra Memorial Research Institute of Medical Sciences, Patna, India 2 Department
of Clinical Medicine, Rajendra Memorial Research Institute of Medical Sciences, Patna, India 3 Department
of Microbiology, Rajendra Memorial Research Institute of Medical Sciences, Patna, India 4 Department
of Molecular Biology, Rajendra Memorial Research Institute of Medical Sciences, Patna, India 5 Department
of Pathology, Rajendra Memorial Research Institute of Medical Sciences, Patna, India Correspondence Neena Verma, Department of Pathology, Rajendra Memorial Research Institute ofMedical Sciences (ICMR), Agamkuan, Patna 800007, India. Email:
[email protected] Sanjiva Bimal, Department of Immunology, Rajendra Memorial Research Institute of Medical Sciences (ICMR), Agamkuan, Patna 800007, India. Email:
[email protected],
[email protected]
Abstract Post kala-azar dermal leishmaniasis (PKDL) is often considered to be the anthroponotic reservoir of visceral leishmaniasis (VL) in India. A better understanding of the host immune-response in dermal lesions of PKDL patients is therefore of utmost significance to minimize such patients and to restrict VL transmission. Although the innate immune response is known to play an important role in parasite clearance from dermal lesions, the actual contribution of innate cells to the pathogenicity of PKDL is poorly understood. The present study explored the immune-pathogenesis of PKDL patients to understand the expression of CD62L, CD11b, CXCL8/IL-8, and MIP1-α and their contribution in signaling during innate cell trafficking. Twenty-five individuals were enrolled, who comprised eight active and untreated macular cases, seven active and untreated cases with papulo-nodular PKDL manifestations, five successfully treated post PKDL cases and five healthy individuals from a non-endemic region of Bihar, India. The immunological investigation was performed on biopsy specimens prepared with a disaggregation technique and blood samples. We observed that the PMNs in nodular patients displayed decreased L-selectin (CD62L) levels and increased integrin (CD11b) expression compared with those in macular patients. Further analysis showed that lower PMN extravasation in macular patients occurred because of inadequate CXCL8/ IL-8 release. In summary, Leishmania donovani (L. donovani) infection in macular PKDL patients decreased leucocyte rolling (L-selectin shedding) and induced up-regulation of the cellular signaling factors involved in pathogenesis (ERK1/2) as well as down regulated the signaling elements (p38 MAPK) involved in the Th1 response, especially in PMNs. KEYWORDS chemokine, cytokine, innate immunity, macrophages, polymorphonuclear neutrophils, post-kala-azar dermal leishmaniasis
J Cell Biochem. 2018;1–13.
wileyonlinelibrary.com/journal/jcb
© 2018 Wiley Periodicals, Inc.
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1 | INTRODUCTION Approximately, 5-15% of cured visceral leishmaniasis (VL) patients in India and 50-60% in Sudan develop a skin manifestation known as dermal leishmaniasis or postkala-azar dermal leishmaniasis (PKDL), which is clinically represented by the presence of hypo-pigmented macules, erythematous, papules, and/or nodules on the skin.1–3 PKDL is often considered the anthroponotic reservoir of VL in India.4 Comparatively, many of the crucial aspects of the innate immune response remain unresolved in PKDL. Many previous studies emphasized the importance of macrophages (Mφ) in PKDL. However, polymorphonuclear neutrophils (PMNs) can also utilize several strategies to counter Leishmania donovani (L. donovani) by phagocytosis or through the release of the neutrophil extracellular traps (NETs).5 Reports, however, suggested that the survival of Leishmania infantum inside of NETs occurs by a mechanism mediated by parasite-specific nuclease 3′-nucleotidase/ nucleases.6 As the presence of PKDL is a serious concern in VL endemic areas, more in-depth studies on the role of PMNs in the PKDL pathogenesis are greatly needed. In PKDL, infection occurs in dermal lesions of skin; therefore, understanding how circulating leucocytes cross between endothelial cells to reach lymphoid tissues, especially at inflammatory sites at the interface of the external environment, is important. The roles of chemokines, cytokines, and other inflammatory mediators have been described,7–9 but we still lack a proper clarification of this process during PKDL. As is known, leucocyte rolling via cross-linking of L-selectin (CD62L) with its ligand on the endothelium play a crucial role in leucocyte trafficking.7,10 Then, activation events slowly occur with the shedding of CD62L and up-regulation of the β2 integrin, Mac-1 (αMβ2:CD11b/CD18), to help immune cells bind intercellular adhesion molecule 1(ICAM-1) and intercellular adhesion molecule 2 (ICAM-2).11 This process is reported to cause leucocytes to adhere and migrate following support through inflammatory mediators, such as interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF-α) as well as many chemokines, such as macrophage inflammatory protein 1 α (MIP1α) and interleukin 8 (CXCL8/IL8).7–9 The present study aimed to determine alterations in the trafficking abilities of innate immune cells (PMNs and Mφ) and their functions following L.donovani infection through evaluating lesion biopsies as well as circulatory samples from PKDL patients.
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attending the outdoor patient department (OPD) at the Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Patna, India. Informed consent was obtained from each participant and their eligibility for the study was confirmed with two methods: identification of amastigotes (L. donovani) by skin smear examination followed by a nested polymerase chain reaction (nested PCR) test specifically for parasite DNA derived from biopsy samples (Figure 1).12,13 Fifteen active PKDL cases, five post-treated cured PKDL cases and five healthy volunteers recruited from non-endemic areas (negative control) were selected for this study. Among the 15 active cases, 8 were predominantly macular and 7 had papulo-nodular clinical manifestations (Table 1). The five post-treated cases were cured with three, 20-day courses of Amphotericin B separated by 20 day intervals (ie, 60 mg/kg spread over a period of 100 days and 60 infusions).14 All treated PKDL patients had cleared their infection as confirmed through nested PCR.12 Biopsy tissue samples from the healthy subjects were not taken for the study, on ethical consideration. Skin biopsy (4-6 mm) samples were collected from the macular and nodular lesions from the patients categorized as having predominantly macular and papulonodular clinical manifestations of PKDL, respectively, in 1.5 mL sterile vials containing fresh RPMI-1640 medium supplemented with 10% foetal bovine serum (FBS) and antibiotics.15 Whole blood (5 mL) was also collected from the same patients by venipuncture in a lithium heparin (BD, Franklin Lakes, NJ) from each subject for all immunological
2 | MATERIALS AND METHODS 2.1 | Study population and sample collection Skin biopsy samples were collected under proper sterile conditions by a pathologist from suspected PKDL cases
FIGURE 1
A, Flow chart for confirmation of PKDL. Representative figure of (B) Macular and (C) Papulonodular PKDL patient
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TABLE 1 Demographic and clinical features of study group Type of lesion
Macular
Papulonodular
Cured PKDL
Healthy (Control)
Number of PKDL Cases
8
7
5
5
Mean age range (years)
19.13 (12-36)
33.14 (22-48)
29.2 (18-41)
33 (28-42)
Gender (Male/Female)
5/3
6/1
4/1
3/2
Past history of Kala-azar
8
7
5
NA
Microscopic positive for L.d. bodies
8
7
0
ND
PCR positive
8
7
0
ND
Out of fifteen (n = 15) confirmed PKDL patients, eight (n = 8) had predominantly macular lesions and seven (n = 7) had papulonodular lesion, Cured PKDL subjects were cured with three, 20 day course of Amphotericin B separated by 20 days intervals (ie, 60 mg/kg spread over a period of 100 days and 60 infusion) were included in parallel arm to serve as control for tissue biopsy and five healthy volunteers (n = 5) recruited from non-endemic areas to serve as the control group for the cells from circulatory blood. NA, not applicable; ND, not done; PKDL, post kala-azar dermal leishmaniasis.
evaluations. The samples collected in this way were immediately subjected to all the immunological investigations as per the requirements for the different protocols and parameters. The protocols in this study were carried out in accordance with the recommendations outlined in the Helsinki Declaration. Ethical approval was obtained from the Ethical Review Committee of Human Studies, RMRIMS, Patna.
2.4 | Preparation of soluble Leishmania antigen (SLA)
2.2 | Isolation of single cell suspensions from PKDL patient skin biopsies
Peripheral blood mononuclear cells (PBMCs] were isolated following Histopaque 1077 (Sigma-Aldrich, Dorset, UK) density gradient centrifugation at 400 g for 30 min at 20°C from the heparinized peripheral blood of the subjects.20,21 PBMCs were washed twice in RPMI 1640 and resuspended in complete culture medium at a concentration of 1 × 106 cells/mL. Two hundred microliters of cell suspension (1 × 106 cells/mL) that was obtained from the biopsy samples, as mentioned in section 2.2, or PBMCs (1 × 106 cells/mL) were cultured at a concentration of 2 × 105 cells/mL in 24-well tissue culture plates in complete medium for 16 h at 37°C and 5% CO2.22 Cell culture supernatants were harvested and analyzed for cytokines by ELISA techniques according to the manufacturer's instructions. The detection limits were 5.6 pg/mL for IFN-γ (EMD Millipore, Billerica, MA), 2 pg/mL for IL-10 (EMD Millipore) and 3.5 pg/mL for TNF-α (EMD Millipore). All samples were simultaneously run in triplicate wells. Cytokine absorbance was read at a wavelength of 450 nm in an enzyme-linked immunosorbent assay (ELISA) reader (Bio-Rad, iMark, Gurgaon, India).
The PKDL biopsy specimens were prepared for flow cytometry analysis with a disaggregation technique as previously described.16 Biopsy specimens were teased and vortexed vigorously for 10-15 min until the solution turned cloudy. The suspension was filtered through a 70 μm poresize nylon mesh (HIMEDIA, Mumbai, Maharashtra, India) and the cells were labeled for FACS analysis.
2.3 | Innate immune response function in circulatory leucocytes (whole blood assay) Heparinized whole blood (100 µL of blood) collected in polypropylene tubes (Beckton Dickinson [BD], San Jose, CA) was suspended in PBS and incubated with monoclonal antibodies conjugated to anti-CD14 allophycocyanin (APC; BD Pharmingen, San Diego, CA) and anti-CD66b allophycocyanin (APC; BD Pharmingen) in separate tubes with antiCD16 fluorescein isothiocyanate (FITC; BD), anti-CD11b phycoerythrin (PE; BD Pharmingen), and anti-CD62L peridinin-chlorophyll protein (PerCP; BD Pharmingen) in a single staining step for 30 min at room temperature in the dark.17 Erythrocytes were lysed and leucocytes were fixed with FACS lysing solution for 10 min (BD Pharmingen). Cells were washed twice with 1% PBS, 0.5% filtered bovine serum albumin (BSA), and 0.1% NaN3. Next, cells were resuspended in 1% paraformaldehyde and analyzed with a flow cytometer (BD).
Soluble Leishmania antigen was prepared as previously described18,19 and stored at −20°C until use.
2.5 | Determination of total cytokine production
2.6 | Expression of membrane-associated functional molecules on Mφ One hundred microliters cell suspension (1 × 106 cells/mL) that was obtained from biopsy samples as mentioned in section 2.2 or heparinized whole blood, as mentioned in section 2.3 were collected in polypropylene tubes (BD, San Jose, CA) containing RPMI with 10% FBS. These circulatory cells and harvested
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single cell suspensions were incubated with anti-CD14 APC (BD Pharmingen) and anti-CD16 FITC (BD Pharmingen) conjugated antibodies. Staining was performed to analyze forward versus side scatter histograms according to the placement of the R1 gate on CD16 positive (CD16 (pos)) FITC and to analyze data from CD14 positive (CD14 (pos)) Mφ cells to determine the changes in CD62L and CD11b expression. Further, to determine the direct impact of Mφ on CD62L and CD11b expression, cells were further stained with anti-CD11b PE (BD Pharmingen) and anti-CD62L PerCP (BD Pharmingen) conjugated antibodies in a single staining step for 30 min at room temperature in the dark for subsequent flow cytometer analysis. Before the analysis, each stained sample was re-suspended in 500 µL staining buffer. The flow data were evaluated with BD CellQuest Pro software. Negative control samples were incubated with irrelevant isotype-matched antibodies (FITC, PE, PerCP, and APC labeled IgG; BD Pharmingen) in parallel with all experimental samples. The optimal number of events acquired was 10 000 of the gated population and analyzed with BD CellQuest Pro software.
2.7 | Expression of membrane-associated functional molecules on PMNs One hundred microliters cell suspension (1 × 106 cells/mL) obtained from biopsy samples as mentioned in section 2.2 or heparinized whole blood, as mentioned in section 2.3, were collected in polypropylene tubes (BD) containing RPMI with 10% FBS. These circulatory cells and harvested single cell suspensions were incubated with anti-CD66b APC (BD Pharmingen) and anti-CD16 FITC (BD Pharmingen) conjugated antibodies. Staining was performed to analyze forward versus side scatter histograms according to the placement of the R1 gate on CD16(pos) FITC and to analyze data for CD66b positive (CD66b(pos)) PMNs to determine the changes in CD62L and CD11b expression. Further, to determine the direct impact of PMNs on CD62L and CD11b expression, cells were further stained with anti-CD11b PE (BD Pharmingen) and anti-CD62L PerCP (BD Pharmingen) conjugated antibodies in a single staining step for 30 min at room temperature in the dark for subsequent flow cytometer analysis. Before the analysis, each stained sample was re-suspended in 500 µL staining buffer. Flow data were evaluated with BD CellQuest Pro software. Negative control samples were incubated with irrelevant isotype matched antibodies (FITC, PE, PerCP, and APC labeled IgG; Pharmingen) in parallel with all experiment samples. The optimal number of events acquired was 10 000 of the gated population and analyzed with BD CellQuest Pro software.
2.8 | Expression of CD62L on PMNS after stimulation with recombinant TNF-α In a separate experiment, skin biopsies and whole blood sample were collected from five predominantly macular
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PKDL patients in addition to the above mentioned subjects in section 2.1 to evaluate CD62L expression on PMNs after stimulation with recombinant TNF-α (rTNF-α). Cells were collected in polypropylene tubes and suspended in RPMI1640 medium (Gibco) with 10% FCS and incubated at 37°C in a humidified, 5% CO2, 95% air incubator. As such,100 µL heparinized whole blood and 100 µL of dermal lesion suspensions (1 × 106 cells/mL) pulsed with L.donovani were cultured with rTNF-α (BD Pharmingen) at a concentration of 20 ng/mL for 16 h and analyzed for the frequency of PMN shedding.23 After incubation, the PMNs were assessed for rolling alterations by comparing the CD62L expression on their surface with flow cytometry, as mentioned in section 2.7.
2.9 | Determination of the MIP-1α and CXCL8/IL8 chemokine pattern One hundred microliters of cells (1 × 106 cells/mL) isolated from biopsy samples, as mentioned in section 2.2, were collected in polypropylene tubes and cultured for 6 h at 37°C and 5% CO2. During the last 4 h, a leucocyte activation cocktail, with Brefeldin A (BD Pharmingen), was added. The leucocyte activation cocktail, BD Golgi plugTM—to-use polyclonal cell activation mixture, contained the phorbol ester, phorbol 12-myristate13-acetate (PMA), a calcium ionophore (ionomycine), and the protein transport inhibitor, Brefeldin A (BD Golgi plug™). After washing, the cells were stained with anti-CD14 APC (BD Pharmingen) and, in a separate tube, anti-CD66b APC(BD). After staining, the cells were washed and permeabilized using Cytofix/Cytoperm buffer (BD Pharmingen) as per the manufacturer's instruction and stained with anti-MIP-1α PE (BD Pharmingen) and antiCXCL8/IL8 PerCP (BD Pharmingen) conjugated antibodies for 30 min at 4°C in the dark. The cells were then washed and re-suspended in 500 µL staining buffer.24,25 Negative control samples were incubated with irrelevant isotype-matched antibodies (FITC, PE, PerCP, and APC labeled IgG; BD Pharmingen) in parallel with all experimental samples. The optimal number of events acquired was 10 000 of the gated population and analyzed with BD CellQuest Pro software.
2.10 | Cell signaling analysis For the expression of extracellular stress-regulated kinase (ERK1/2) and P38 mitogen-activated protein kinase (p38 MAPK) protein in innate immune cells, 100 µL of biopsy single cell suspension (1 × 106 cells/mL), as mentioned in section 2.2, were collected in polypropylene tubes and fixed with paraformaldehyde (1%) at 37°C for 10 min. Cells were washed and permeabilized with cold methanol on ice (30 min). After washing in staining buffer, we stained leucocytes using anti-CD14 APC (BD Pharmingen) and, in
| Cytokine was estimated using ELISA from biopsy tissue and peripheral blood mononuclear cell (PBMC) cell suspension as described in section 2.5 from macular, nodular lesion, cured PKDL, and healthy subjects. Cured PKDL subjects were cured with three, 20 day course of Amphotericin B separated by 20-day intervals (ie, 60 mg/kg spread over a period of 100 days and 60 infusion) were included in parallel arm to serve as control for tissue biopsy and five healthy volunteers (n = 5) recruited from non-endemic areas to serve as the control group for the cells from circulatory blood. Data are mean ± standard error, P ≤ 0.05 were considered as significant. PKDL, post kala-azar dermal leishmaniasis; ND, not done.
12.80 ± 1.16
25.20 ± 2.22 37.40 ± 6.38
15.00 ± 2.93 66.67 ± 7.77
91.29 ± 8.23 64.88 ± 8.07
31.10 ± 2.83 ND
ND 41.20 ± 5.19
22.60 ± 3.53
105.00 ± 11.47 IL-10
147.60 ± 14.49
38.75 ± 3.48 TNF-α
92.33 ± 8.04
26.00 ± 3.56 27.00 ± 4.00 85.00 ± 6.51 60.00 ± 5.92 ND 34.40 ± 2.16 71.50 ± 6.48
151.30 ± 11.61
PKDL cured (n = 5) Nodular (n = 7) Macular (n = 8) Healthy (n = 5) PKDL cured (n = 5)
IFN-γ
The type of cytokines (IFN-γ, TNF-α, and IL-10) produced from the macular and nodular PKDL patients in the circulating cells and dermal lesion are shown in Table 2. There was significantly higher IFN-γ production from the dermal lesions than in the circulating cells in the nodular patients (P = 0.0012). The macular patients had significantly lower yields of IFN- γ from their dermal lesions than the nodular patients (P = 0.0014); however, these levels were higher than the circulatory blood levels of the same macular patient, but they were not statistically significant (P = 0.2691) (Table 2). The negative control samples produced significantly lower yields and they were minimal compared with all experimental groups.
Nodular (n = 7)
3.1 | Macular PKDL patients produce less pro-inflammatory cytokines
Macular (n = 8)
Out of the 25 subjects enrolled in the study, 15 were confirmed PKDL, 5 subjects were treated and cured of PKDL and 5 were healthy controls from a non-endemic area (negative control). In the 15 confirmed PKDL patients, 8 had predominantly macular lesions and 7 had papulo-nodular lesions. Demographic and clinical features of the enrolled subjects are shown in Table 1. To establish the status of the phagocytic cells in the PKDL patients, the cellular profiles of these cells were detected in single cell suspensions from the skin or whole blood using specific markers, includingCD16 (pos)and CD14 (pos)for Mφ andCD16 (pos)and CD66b (pos)for PMNs. The biopsy tissue samples from the healthy subjects were not involved in this study, due to ethical considerations.
Cytokine (pg/mL)
3 | RESULTS
Circulatory blood
We performed non-parametric Mann-Whitney U tests to evaluate differences amongst the groups using GraphPad Prism 5 (USA software). Data are presented as the mean ± standard error of the mean (SE) and the results were regarded as statistically significant at P value ≤ 0.05.
Skin lesion
2.11 | Statistical analysis
Category
a separate tube, anti-CD66b APC (BD Pharmingen), ERK1/ 2 PerCP-Cy™5.5 (BD Pharmingen), and anti-p38 MAPK PE (BD Pharmingen) conjugated antibodies. The cells were then washed and re-suspended in 500 µL staining buffer. Negative control samples were incubated with irrelevant isotype matched antibodies (FITC, PE, PerCP, and APC labeled IgG; BD Pharmingen) in parallel with all experimental samples. The optimal number of events acquired was 10 000 of the gated population and analyzed with BD CellQuest Pro software.
Healthy (n = 5)
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TABLE 2 Secreted levels of interferon-γ, tumor necrosis factor α and interleukin-10 in macular, nodular clinical manifestation, cured PKDL, and healthy subjects
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Expression of membrane-associated functional molecules on macrophage (Mφ) and polymorphonuclear neutrophils (PMNs) was evaluated using Flow cytometer from biopsy tissue and heparinized whole blood as described in sections 2.6 and 2.7. Skin biopsy samples were collected from macular lesion and nodular lesion from the patients categorized under predominantly macular and papulo-nodular clinical manifestation of PKDL, respectively. Cured PKDL subjects were cured with three, 20 day course of Amphotericin B separated by 20-day intervals (ie, 60 mg/kg spread over a period of 100 days and 60 infusion) were included in parallel arm to serve as control for tissue biopsy and five healthy volunteers (n = 5) recruited from non-endemic areas to serve as the control group for the cells from circulatory blood. Data are mean ± standard error, P ≤ 0.05 were considered as significant. PKDL, post kala-azar dermal leishmaniasis; ND, not done.
89.14 ± 6.391 145 ± 14.73 163.9 ± 13 149.2 ± 10.87 ND 138.9 ± 25.99 292.7 ± 69.78 PMN
507 ± 109.1
109.2 ± 9.296 118.1 ± 12.59 126 ± 10.61 114.6 ± 8.264 ND 155.5 ± 27.31 460.4 ± 95.42 405.1 ± 90.57 Mφ CD11b
2888 ± 300.6
4199 ± 454.8 3905 ± 523.3
2520 ± 308.9 2293 ± 288.2
3241 ± 367.5 3588 ± 369
2356 ± 169.1 ND
ND 2066 ± 312.4
479 ± 64 1551 ± 291.3
919.6 ± 194.5
1556 ± 395 Mφ
PMN
CD62L
1067 ± 208.3
Healthy (n = 5) PKDL cured (n = 5) Nodular (n = 7) Macular (n = 8) PKDL cured (n = 5)
Healthy (n = 5)
Circulatory blood
Nodular (n = 7) Macular (n = 8) Cells Membrane associated surface molecules (MFI)
Diminished L-selectin expression at the surface of resting leucocytes is related to the shedding process during the activation of innate cells. L-selectin is a homing receptor that is cleaved from the surface of leucocytes upon activation and its shedding increases trans-endothelial cell migration out of circulation.28 We evaluated the expression of CD62L on PMNs and Mφ in the macular and the nodular lesions of PKDL patients to evaluate the activated phenotype. CD62L shedding on Mφ in the macular lesions of the PKDL patients was observed, although it remained highly expressed in the circulatory Mφ (P = 0.0047) compared with the cured PKDL patients (P = 0.1709) (Table 3). This decreased CD62L expression in the biopsy skin lesion did not reach statistically significance in the macular lesion. In contrast, there was a significant decrease in CD62L expression on the nodular lesion Mφ compared with the cured PKDL samples (P = 0.0303) (Table 3). Considerable shedding also occurred in the circulatory Mφ of the nodular patients, although more shedding was observed in the dermal lesions of such patients. Additionally, the macular and nodular lesion PMNs of the PKDL patients expressed a significantly high frequency of CD62L and these cells remained in a resting and inactive
Skin lesion
3.2 | L. donovani differentially regulates rolling and adherence of innate cells in the macular and nodular lesions of PKDL patients
Category
The total TNF-α yield was also significantly higher in the dermal lesions of nodular patients than the macular patients (P = 0.0024) and the cured PKDL subjects (P = 0.0043). The macular PKDL patients were somewhat different from the nodular patients in the sense that the amounts of TNF-α produced in the dermal lesions and the circulating blood were almost the same and comparatively low (P = 0.1547), although it was higher than in the cured PKDL subjects (P > 0.05) (Table 2). However, the nodular patients presented with some unusual immunological characteristics that clearly differentiated them from the PKDL patients with macular clinical manifestation. The significance of the IFN-γ, IL-10 paradigm and defined cytokine polarization is linked to resistance and susceptibility and is well established in the pathogenesis of Leishmaniasis.26,27 The quantitative yield of IL-10 produced in the dermal lesions of the nodular patients was always higher than in the dermal lesions of macular PKDL patients, although this level remained very low in the circulating immune cells in such patients (Table 2). The macular patients also produced more IL-10, which was higher in the dermal lesions than in the circulating blood (P = 0.01).However, the overall IL-10 yield remained higher in the nodular patients (Table 2).
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TABLE 3 Sequential changes in trafficking events at innate cells in macular, nodular lesion cured PKDL and healthy subjects
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state in comparison with the cured PKDL samples (P = 0.002 and P = 0.048, respectively). In comparison, the nodular patient PMNs appeared more activated and displayed significantly greater rolling strength in comparison with the macular PKDL patients (P = 0.04). In contrast, the circulatory PMNs in these patients remained in the inactivated state and had higher CD62L expression, which was similar to the cured PKDL patients (P = 0.2677). These findings suggest that the dermal lesions of the nodular patients that were activated against L.donovani contain more Leishmania competent PMNs, whereas the macular patients lack that quality of PMNs in their dermal lesions. These result collectively show that Leishmania parasite differentially regulate the rolling of innate cells to the vascular endothelium in PKDL patients. Additionally, these data suggest that L.donovani impairs the rolling abilities of PMNs, thereby interfering with PMN, but not Mφ, activation during its clinical manifestation in macular and nodular PKDL patients.
3.3 | Role of inflammatory mediators (TNF-α and IL-8) during rolling 3.3.1 | Increased tendency of PMN’S to diminish expression of L-selectin (CD62L) following stimulation with rTNF-α As shown earlier, macular PKDL patients produced more IL-10 and their PMNs also appeared to be inactivated, as they expressed higher CD62L levels, which is an indication that the initial trafficking abilities of the PMNs was defective. Continuous recruitment is a pre-requisite to co-operate with pre-existing cells to mount a strong counter attack against parasites at inflammatory sites. Reports suggest that many inflammatory mediators (eg, histamine, IL-1, and TNF-α) regulate this process.29–31 We evaluated for rolling alterations by comparing their surface CD62L expression after stimulation with rTNF-α by flow cytometry (Figure 2C-E). The major PMN pool displayed higher CD62-L shedding levels (an activation indicator) compared with a lesion suspension cultured in the absence of rTNF-α (P < 0.05) (Figure 2).
3.3.2 | Not only TNF-α but also minimal IL-8 support contributed to significantly lower PMN extravasation in macular PKDL patients Given the role of chemokines in influencing sequential proteolytic cleavage of L-selectin (CD62L) followed by activation of leucocyte-expressed integrin (CD11b) and its interaction with the immunoglobulin super family members, ICAM-1, and VCAM-1, which are required for extravasation, we determined the intracellular production of the CXC class of chemokines, such as human CXCL8/ IL8 and MIP-1α.
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As illustrated in Figures 3C and 3D both Mφ and PMNs in the macular and nodular lesion of PKDL patients expressed significantly increased MIP-1α levels compared with the cured PKDL patients(P < 0.05). Like the above results, we observed significantly increased CXCL8/IL8 levels, except in the macular lesion PMNs. The macular lesion PMNs exhibited a non-significant difference with the cured PKDL PMNs (P > 0.05); however, they had significantly decreased CXCL8/IL8 expression compared with the nodular lesions (P ≤ 0.05) (Figures 3A and 3B). Thus, it seems that CXCL8/ IL8 may be a possible reason for the lower PMN extravasation observed in the macular PKDL patients.
3.4 | Adherence and extravasation (CD11b upregulation) of innate cells remains intact during macular and nodular clinical manifestations Additionally, we compared the expression levels of another activation marker, the β2-integrin CD11b, in PKDL patient lesions and circulating leucocytes under different clinical manifestations (PMNs and Mφ). The activity pattern between the Mφ and PMNs looked almost similar in the macular and nodular patient circulatory pools, which indicated that CD11b expression remained high during infection and was similar to the cured PKDL patient levels (P > 0.05). Interestingly, we observed a striking difference in the abilities of these cells to expressCD11b in the skin lesions. Although, the Mφ in both the macular and nodular participants presented with increased CD11b expression, the expression of this activation marker was significantly depressed, especially on macular patient PMNs (P = 0.0427) (Table 3).
3.5 | Role of ERK1/2 and P38 MAPK during trafficking of innate cells in PKDL To validate that the inadequate release of TNF-α and IL-8 could be a possible reason for the lower PMN extravasation level in the PKDL patient macular lesions, we examined for alterations in signaling events. The MAPK kinases, including ERK JNK and p38, were reported to affect inflammation, apoptosis, and migration.32 As such, the roles of ERK1/2 and p38 MAPK in Mφ and PMN chemotaxis in PKDL patients were elucidated. While the Mφ in both the macular and nodular patients expressed p38 MAPK at a lower fluorescence intensity, most of the PMNs in the nodular PKDL patients produced p38 MAPK (MFI, 467.71 ± 125.5) at a significantly higher intensity compared with the cured PKDL patients (MFI,177.0 ± 14.55) (P = 0.048) (Figures 4A and 4B). The p38 MAPK levels in the PMNs of the macular PKDL patients were significantly decreased compared with the nodular PKDL patients (MFI, 155.4 ± 33.73, 467.7 ± 125.5, respectively; P = 0.0289 (Figures 4A, 4B, 4E, and 4F). Moreover, the p38
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FIGURE 2 Increased tendency of PMN's to diminish expression of L-selectin (CD62L) following stimulation with rTNF-α. CD62L on PMNs (CD16 (pos) CD66b (pos), represented as mean fluorescence intensity (MFI). A, shows CD62L expression on PMN's of Dermal lesion macular PKDL (n = 5), B, shows CD62L expression on PMN's of circulatory cells of macular PKDL (n = 5) pulsed with L.donovani were cultured with rTNF-α (BD) at a concentration of 20 ng/mL for 16 h and analyzed for the frequency shedding as described in sections 2 and 2.7. Representative figure of (C) gating strategy for CD16(pos) cells (D) CD62L expression on Unstimulated (UNS) PMNs (E) CD62L expression on PMNs after stimulation with rTNF-α. Data are mean ± standard error, P ≤ 0.05 were considered as significant. *P ≤ 0.05, **P ≤ 0.01, ns (nonsignificant), Mann-Whitney U test
MAPK fluorescence intensity was even lower than in the cured PKDL patients (MFI, 177.0 ± 14.55). In contrast to p38 MAPK, the Mφ and PMNs in the macular patient lesions were highly positive for ERK1/2 compared with the nodular patients (Figures 4C and 4D). However, the increased ERK1/2 intensity in the Mφ was always higher than in the PMNs. Thus, downregulation of p38 MAPK interfered with extravasation, especially for the dermal lesion PMNs of the macular patients. On the other hand, p38 MAPK up regulation in the nodular patients increased the chances of directional migration of innate cells into the dermis.
4 | DISCUSSION PKDL infection is a dermatosis that usually occurs after VL caused by L. donovani. It is characterized by hypo-pigmented
macules, erythematous, papules, and/or nodule skin lesions on entire body surface.33 PKDL patients in India exist with VL in same endemic communities. PKDL cases are of considerable epidemiological importance because these patients act as parasite reservoirs.33 There are ample reports to suggest that L. donovani parasite transmission from PKDL cases to other individuals by Phlebotomus sand flies massively contributed to the various kala-azar epidemics in Bihar in the 1970s.34 Because the disease is a dermatosis in nature, it was of interest to analyze the course of events, especially the immune cell trafficking, which can help recruit such cells to the dermal lesion inflammatory site. Unfortunately, reports on the relationship between innate immune responses and pathogenicity in PKDL patient dermal lesions have thus far remained unsatisfactory. Hence, the present study explored the immuno-pathogenesis of PKDL patients to understand the expression levels of CD62L,
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FIGURE 3 Inadequate production of CXCL8/IL8 from macular PKDL patients may be possible reason for lower extravasation. MIP1α and CXCL8/IL8 production from Mφ and PMNs, represented as percentage gated (% gated). A, shows CXCL8/IL8 production from Mφ, (B) shows CXCL8/IL8 production from PMNs, (C) shows MIP1α production from Mφ, (D) shows MIP1α production from PMNs of macular, nodular, and cured PKDL. Cured PKDL (Control) subjects were cured with three, 20 day course of Amphotericin B separated by 20 days intervals (ie, 60 mg/ kg spread over a period of 100 days and 60 infusion) were included in parallel arm to serve as control for tissue biopsy. Data are mean ± standard error, P ≤ 0.05 were considered as significant. *P ≤ 0.05, **P ≤ 0.01, ns (non-significant), Mann-Whitney U test
CD11b, CXCL8/IL8, and MIP1α and their contribution in signaling during innate cell trafficking (Mφ & PMNs). We observed a trend that L. Donovani infection in macular PKDL patients decreases leucocyte rolling (L-selectin shedding) and induces the up-regulation of cellular signaling factors involved in pathogenesis (ERK1/2) while down-regulating signaling elements (p38 MAPK) involved in the Th1 response, especially in PMNs. PMNs and Mφ both have a vital role in pathophysiology and clearance of Leishmania infection and are particularly important in PKDL where infection occurs in the dermal lesion. However, the purification of such cells, particularly the dermal lesion PMNs using flow cytometry, has thus far remained unsatisfactory, as various phenotypic markers, including CD11b, CD14, CD15, and CD62L produced in-consistent results.35,36 We selected CD16 and CD66b as neutrophil markers based on a report that these two surface markers are consistently expressed on PMNs independent of their location, expression level, and
disease state37 (Figure 2C-E). To evaluate the Mφ function during the innate immune response, we used CD16 (pos) andCD14 (pos)cells. The gating strategy for Mφ was to gate the entire CD16(pos) population (R1) and then subsequently evaluate the CD14(pos) Mφ population. As previously shown, nonclassical CD14 (pos)&CD16 (pos)cells usually resemble tissue macrophages.38–40 The cytokine behavior of immune cells is still not very clear in PKDL patient skin lesions when presented with macular and nodular clinical manifestation. We presented some important evidence in this context, which can exploit to understand the immune response caused by Leishmania, and thus dermal leishmaniasis, in PKDL patients. Decreased concentrations of the inflammatory cytokines, IFN-γ, and TNF-α, were mostly seen in the macular patients. The low level of these cytokines suggests their significance, as Leishmania interrupted rolling activities, especially of PMNs, in PKDL patients with macular manifestation. The
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FIGURE 4 Role of ERK1/2 and p38 MAPK during trafficking of innate cells in PKDL. Expression of p38 MAPK and ERK1/2 on Mφ and PMNs and represented as mean fluorescence intensity (MFI). A and B, show p38 MAPK expression on Mφ and PMN's of macular lesion (A) and nodular lesion of PKDL (B). C and D, show ERK1/2 expression on Mφ and PMN's of macular lesion (C) and nodular lesion of PKDL (D). Cured PKDL (Control) subjects were cured with three, 20 day course of Amphotericin B separated by 20 days intervals (ie, 60 mg/kg spread over a period of 100 days and 60 infusion) were included in parallel arm to serve as control for tissue biopsy. Representative figure of, (E) p38 MAPK expression on PMNs of Macular PKDL, (F) p38 MAPK expression on PMNs of Nodular PKDL. Data are mean ± standard error, P ≤ 0.05 were considered as significant. *P ≤ 0.05, **P ≤ 0.01, ns (non-significant), Mann-Whitney U test
integrin Mac-1/CR-3 (CD11b/CD18) and chemokine (CXCL8/IL8) analyses also showed that PMNs, especially in the macular cases, were comparatively lesser prepared for tissue extravasation.
Rolling on the wall of a blood vessel by a new pool of innate cells helps them transmigrate into dermal tissue to support pre-existing cells to counter proliferating parasites and prevent the spread of infection.8 Tethering and rolling on
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E-selectin upon the endothelium wall by leucocytes requires L-selectin (CD62L), which is a carbohydrate domain common to both sialyl Le and sialyl-Lewis X.8,41 The activation process is rapid and includes the shedding of the rolling element (L-selectin), firm adherence and diapedesis, which are mediated by heterodimer β2-integrins, which consists of α- and β- subunits (CD11a/CD18).8,42 Reports on neutrophil rolling mediated by P-selectin on blood vessel walls are regulated by the activities of several inflammatory mediators, such as histamines,30 TNF-α, and IL-8.31 As preceding experiments indicated that lower TNF-α expression was observed in macular patients and that Leishmania interrupted PMN rolling in macular patients, we closely scrutinized the CD62L shedding trends after costimulation of leucocytes with rTNF-α. The results presented in this study demonstrate that lower TNF-α amounts diminished the tethering and rolling activities of PMNs in PKDL patients. Despite a report that TNF-α promotes neutrophil apoptosis,43 TNF-α based treatments were reported to be effective in many inflammatory situations.44,45 Reduced responses to anti-leishmanial treatments has been a feature observed in most PKDL patients thus far. Hence, evidence for a role of TNF-α in PMN trafficking to Indian PKDL patient dermal lesions is worth pursuing. This study further shows that although the percentages of MIP-1α positive CD14 (pos), CD16 (pos) (Mφ), and CD66b (pos) , CD16 (pos) population were elevated, the percentage of CXCL8/IL-8 positive, CD66b(pos)(PMNs) was significantly reduced. The significance of this finding is that CXCL8/IL8 expression has a strong impact on CD66b (pos) PMNs, which have conserved cysteines separated by amino acids and works by activating PMNs that then produce integrins (CD11b) to render strong adhesiveness of PMNs to the endothelial wall and helps in their generation of microbial oxygen radicals. Therefore, inadequate CXCL8/IL8 production might contribute to lower PMN extravasation in macular PKDL patients. These results suggest the existence of signaling mechanisms that regulate the retention and recruitment of innate immune cells within inflammatory sites in nodular patients. Additionally, this begged the question as to why PMNs are pre-dominantly affected by L. donovani parasite infection more predominantly in macular patients. This led us to examine the signaling trends that occur in the two PKDL patient groups. Previous reports suggest that Leishmania species either resist or become susceptible to clearance by their influence on immune responses, which are polarized to either high or low IL-12 to IL-10 response ratios.46 There are reports these ratios are controlled by the reciprocal ERK1/2 and P38 MAPK signaling.47,48 Here, we showed that most PMNs from macular patients had up-regulated ERK1/2 levels. This effect of Leishmania together with the mobility and p38 MAPK induction of macular patient PMNs provides a strong
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argument for the need to test whether the inadequate initial support of TNF-α and IL-8, induced mainly by L. donovani, is a critical factor in the functioning of innate cells in PKDL patients, especially with macular clinical manifestations. These results are a strong indication that the production of anti-inflammatory cytokines (TNF-α and IFN-γ) in dermal lesions, in which parasites make first contact with innate cells, is crucial to enhance the adhesive forces and further optimize cell trafficking and chemotaxis under the directional control of p38 MAPK and chemokines. PKDL patients with macular clinical manifestations are usually considered to be difficult to treat. According to our results, it is possible that ERK1/2 activation in macular patients promotes IL-10 production, leading to diminished pro-inflammatory cytokine levels and interference of chemokine trafficking in the potential second pool of innate cells. The findings obtained from this study signify the importance of developing new control strategies that boost immune cell trafficking in PKDL. These new approaches might help to minimize PKDL occurrences and hence to restrict VL transmission in endemic areas. ACKNOWLEDGMENTS The authors acknowledge Rajendra Memorial Research Institute of Medical Sciences, Indian Council of Medical Research, Patna 800 007, India for providing fellowship support to one of the investigators (Ashish Kumar Singh) in this study. The technical services rendered by lab technical staffs Santosh Sinha, Rakhi Kumari, and Tathagat Gupta are highly acknowledged and appreciated. CONFLICTS OF INTEREST The authors declare no conflict of interest in any regards. AUTHORS' CONTRIBUTION AKS, NV, PD, SB, and SKS conceived and designed the experiments, AKS, V, AA, and SN performed the experiments, AKS, SB, KP, and VNRD analyzed the data, SB PD, and NV contributed reagents/materials/analysis tools, and AKS, SB, NV, and MRD wrote the paper. REFERENCES 1. Ramesh V, Mukherjee A. Post-kala-azar dermal leishmaniasis. Int J Dermatol. 1995;34:85–91. 2. Zijlstra EE, Musa AM, Khalil EA, El Hassan IM, El-Hassan AM. Post-kala-azar dermal leishmaniasis. Lancet Infect Dis. 2003;3: 87–98. 3. WHO. 2012. Post-kala-azar dermal leishmaniasis: a manual for case management and control: report of a WHO consultative meeting, Kolkata, India, 2-3 July 2012.
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How to cite this article: Singh AK, Das VNR, Amit A, et al. Cytokines and chemokines differentially regulate innate immune cell trafficking during post kala-azar dermal leishmaniasis. J Cell Biochem. 2018;1–13. https://doi.org/10.1002/jcb.27048
