azar dermal leishmaniasis - Wiley Online Library

DOI: 10.1111/exd.12635

Letter to the Editor

www.wileyonlinelibrary.com/journal/EXD

Decreased presence of Langerhans cells is a critical determinant for Indian Post kala-azar dermal leishmaniasis Shibabrata Mukherjee1, Debanjan Mukhopadhyay1, Claudia Braun2, Joyashree N. Barbhuiya3, Nilay K. Das4, Uttara Chatterjee5, Esther von Stebut2* and Mitali Chatterjee1* 1 Department of Pharmacology, Institute of Postgraduate Medical Education and Research, Kolkata, India; 2Department of Dermatology, University of Medicine, Johannes Gutenberg University, Mainz, Germany; 3Department of Dermatology, School of Tropical Medicine, Kolkata, India; 4 Department of Dermatology, Calcutta Medical College, Kolkata, India; 5Department of Pathology, Institute of Postgraduate Medical Education and Research, Kolkata, India Correspondence: Prof. Mitali Chatterjee, MD PhD, Department of Pharmacology, Institute of PG Medical Education and Research, 244 B, Acharya J C Bose Road, Kolkata 700020, India, Tel.: +91-2204-1428, Fax: +91-33-2223-4135, e-mail: [email protected] and Prof. Esther von Stebut, MD, Department of Dermatology, University of Medicine, Johannes Gutenberg University, Langenbeckstrasse 1, 55131 Mainz, Germany, Tel.: +49-6131-17-5731, Fax: +49-6131-17-5527, e-mail: [email protected] *To be considered as joint corresponding authors.

Abstract: Post kala-azar dermal leishmaniasis (PKDL) is the

dermal sequel of visceral leishmaniasis (VL) and occurs after apparent cure or alongside with VL. It is confined to South Asia (India, Nepal and Bangladesh) and East Africa (mainly Sudan), the incidence being 5-10% and 50-60% respectively. In South Asia, as the transmission of VL is anthroponotic, PKDL patients are the proposed disease reservoir, thus assuming epidemiological significance, its eradication being linked to the control of leishmaniasis. In the absence of an animal model and its low incidence, factors contributing towards the immunopathogenesis of PKDL remain an open-ended, yet pertinent question. This study delineated the lesional immunopathology in terms of granuloma formation, Langerhans cells, tissue macrophages along with mRNA expression of IL-12p40 and IL-10. Our study in Indian PKDL for the first time identified that the number of

Background Leishmaniases, a group of neglected tropical diseases, have variable manifestations depending upon parasite strains, vectors, host genetics and environment. Amongst them, the most intriguing and truly challenging is post kala-azar dermal leishmaniasis (PKDL), the dermal sequel of visceral leishmaniasis (VL). PKDL occurs after apparent cure or alongside with VL and is confined to South Asia (India, Nepal and Bangladesh) and East Africa (mainly Sudan, 1). In South Asia, as the transmission of VL is anthroponotic, PKDL patients are the proposed disease reservoir, thus assuming epidemiological significance, its eradication being linked to the control of leishmaniasis. In the absence of an animal model, factors contributing towards its immunopathogenesis remain unanswered yet pertinent (1).

Question addressed This study delineated the lesional immunopathology in terms of tissue macrophages (MØ), Langerhans cells, granuloma formation along with mRNA expression of IL-12p40 and IL-10.

Experimental design Lesional punch biopsies were serially collected at disease presentation and completion of treatment from 10 patients with PKDL.

232

CD1a+/CD207+ Langerhans cells are decreased and CD68+ macrophages are increased along with the absence of an epitheloid granuloma. Importantly, this decrease in Langerhans cells was associated with decreased mRNA expression of IL-12p40 and increased IL-10. This was reverted with treatment allowing for elimination of parasites and disease resolution along with an increase in Langerhans cells and decrease in macrophages. Thus, in Indian PKDL, absence of a granuloma formation along with a decrease in Langerhans cells collectively caused immune inactivation essential for parasite persistence and disease sustenance. Key words: granuloma – Langerhans cells – Post kala-azar dermal leishmaniasis – tissue macrophages

Accepted for publication 2 January 2015

Immunohistochemical analysis was performed for MØ (CD68), while dermal dendritic cells (dDCs) and Langerhans cells (LCs) were identified using CD1a, CD207 and HLA-DR; mRNA expression for IL-10 and IL-12p40 was measured by RT-PCR (Data S1).

Results The majority of patients had polymorphic lesions (n = 9, 90%), while one had macular lesions (Fig. 1a). Diffuse, heavy lymphohistiocytic infiltrate confined to the dermal compartment was observed in 8 of 10 patients (Fig. 1b), and none had an epithelioid granuloma (Fig. 1b). Leishmania, an intra-cellular pathogen ensures its survival by deviously manipulating MØ, emphasizing the importance of studying the dermal distribution of CD68+MØ/histiocytes. In Sudanese PKDL, macrophages reside within granulomas, whereas in Indian PKDL, even in the absence of a mature granuloma, there was heavy infiltration of CD68+ MØ vis-a-vis healthy controls, 118.8 (70.2–150.4) vs 15.2 (11.2–18.4) cells/mm2, P < 0.05, which decreased with treatment 18.6 (11.9–39.4 cells/mm2, P < 0.001, Fig. 1c,d) which corroborated when individually analysed (Fig. 1e). At presentation, ovoid Leishmania amastigotes (Leishman Donovan bodies, LD bodies) were detected within

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 215–239


(a)

(a)

(b)

(b)

(c)

(d)

(e)

(c)

(f) (d)

(e) (g)

Figure 1. Dermal infiltration in patients with post kala-azar dermal leishmaniasis (PKDL). (a) Representative clinical features of PKDL (polymorphic and macular variant) at disease presentation and post-treatment. (b) Representative profile of H&E-stained sections of a healthy control along with a patient with PKDL at disease presentation and post-treatment (magnification, 109 objective). (c) Representative IHC profile of CD68+ cells in the dermal biopsy of a healthy control, a patient with PKDL at disease presentation and post-treatment (magnification, 109 objective). (d) Comparison of CD68+ cells of healthy controls (■), patients with PKDL at disease presentation (▲) and post-treatment (▼), *P < 0.05. Each horizontal bar represents the median value. (e) Before (●) and after (○) plots of CD68+ cells in patients with PKDL.

histiocytes by Giemsa staining (Figure S1) and were absent following treatment. Dendritic cells (DC), the major immune sentinels of host defense, are critical for antigen presentation. DCs in the epidermis are called Langerhans cells, CD1a positive, (LCs) and were significantly decreased in PKDL when compared with healthy donors, 11.7 (2.9–12.4) vs 45.2 (39.2–52.1) cells/mm2, P < 0.05; this was substantiated by CD207 staining (Fig. 2a,b,c). Importantly, LCs were morphologically altered, as demonstrated by loss of elongated cellular protrusions (Fig. 2b). With treatment, their morphology was reverted, and increased significantly to 26.0 (16.9–36.8) cells/ mm2, P < 0.05), yet remained significantly lower than healthy


(h)

(i)

(j)

Figure 2. Dermal distribution of CD1a+ cells in patients with post kala-azar dermal leishmaniasis (PKDL). (a) Representative IHC profile showing distribution of CD1a+ cells in the dermal biopsy of a healthy control, patient with PKDL at disease presentation and post-treatment (magnification, 109 and 409 objectives). (b) Representative IHC profile showing distribution of CD207+ cells in the dermal biopsy of a healthy control, patient with PKDL at disease presentation and posttreatment (magnification, 409 objective). (c) and (e) Distribution of CD1a+ cells in the epidermal and dermal compartment, respectively, of healthy controls (■), patients with PKDL at disease presentation (▲) and post-treatment (▼), *P < 0.05. Each horizontal bar represents the median value. (d) Before (●) and after (○) plots of CD1a+ cells in the epidermis of patients with PKDL. (f) A representative profile of IL-10 and IL-12p40 mRNA expression in lesional tissue from patients with PKDL before (Pre 1–3) and after treatment (Post 1–3) along with skin from three healthy individuals (N 1–3). (g) mRNA expression of IL-10 in dermal biopsies of healthy controls (■), patients with PKDL at disease presentation (▲) and post-treatment (▼). *P < 0.05, **P < 0.01. Each horizontal bar represents the median value. (h) Before (●) and after (○) plots of mRNA expression of IL-10 in patients with PKDL. (i) mRNA expression of IL-12p40in dermal biopsies of healthy controls (■), patients with PKDL at disease presentation (▲) and post-treatment (▼), **P < 0.01. Each horizontal bar represents the median value. (j) Before (●) and after (○) plots of mRNA expression of IL-12p40 in patients with PKDL.

controls, P < 0.05 (Fig. 2a–c); this was corroborated on an individual basis (Fig. 2d). These LCs did not migrate to the dermis as six of nine patients showed a near total absence of dDCs (Fig. 2a, e), which remained unchanged with treatment (Fig. 2a,e). The lesional mRNA expression of IL-10 during active disease was significantly upregulated as compared to healthy individuals,

233


39.80 (14.10–89.05) vs 0.00, P < 0.001, its cellular source yet to be defined. However, IL-12p40 mRNA expression remained undetected during active disease and in healthy controls. Treatment translated into a significant reduction in mRNA expression of IL-10, 9.50 (4.10–31.30, P < 0.05) and increment of IL-12p40, 3.8 (2.17–4.77, P < 0.01, Fig. 2f,g,i); this was corroborated on a paired basis (Fig. 2h,j).

Discussion Intra-cellular pathogens such as Leishmania have evolved survival strategies to combat the hostile environment of macrophages, which via their ability to present antigens to T helper cells, tilt differentiation of T helper (Th) cells towards a Th2 anti-inflammatory phenotype. Additionally, formation of granulomas enhanced generation of an immune privileged site, as evidenced in cutaneous and experimental VL, wherein the robust granuloma microenvironment showed a strong association with antileishmanial responses (2). Although cellular infiltration is common to South Asian and Sudanese PKDL, the occurrence of granulomas was absent in the former (Fig. 1a), suggesting that granuloma formation in the latter accounted for its self-resolving nature (3). In an animal model of tuberculosis, an increased presence of IL-10 inhibited granuloma formation and facilitated disease progression (4). Similarly, in PKDL, the raised lesional IL-10 (Fig. 2e) limited granuloma formation and parasite survival ensued. The unique capacity of DCs to sample, sites of pathogen entry, respond to microbial signals and subsequently activate na€ıve Tcells, contribute to their critical role in initiating antimicrobial immunity (5). As pathogens causative for plague, typhoid, influenza, etc. eliminate DCs so as to evade the immune system, the reduction in PKDL of LCs (Fig. 2a–c) and dDCs (Fig. 2a,d) similarly attenuated immune-activating properties and facilitated parasite persistence. The decrease in CD1a was not due to its selective downmodulation on activated LC/dDC, as MHC-class II staining

References

1 Mukhopadhyay D, Dalton J E, Kaye P M et al. Trends Parasitol 2014: 30: 65–74. 2 Tuon F F, Fernandes E R, Pagliari C et al. Parasite Immunol 2010: 32: 769–772. 3 Ismail A, Gadir A F, Theander T G et al. J Cutan Pathol 2006: 33: 778–787. 4 Cyktor J C, Carruthers B, Kominsky R A et al. J Immunol 2013: 190: 2778–2790. 5 von Stebut E, Belkaid Y, Jakob T et al. J Exp Med 1998: 188: 1547–1552. 6 Ganguly S, Mukhopadhyay D, Das N K et al. J Invest Dermatol 2010: 130: 1013–1022.

234

also decreased (Figure S2) indicating that in PKDL, LC/dDCs were attenuated. Except for human immunodeficiency virus and human papilloma virus (6), a decrease in LCs has not been reported and importantly, distinguishes PKDL from Lepromatous leprosy, a disease with overlapping clinical features. In Leishmaniasis, the disease-promoting activity of IL-10 has been attributed to its ability to render macrophages unresponsive to activation signals and thereby nullify its microbicidal mechanisms (7,8). This was endorsed in PKDL wherein the increased lesional IL-10 (Fig. 2) generated a pro-parasitic milieu, via deactivation or alternative activation of macrophages (9). Accordingly, despite the increased presence of lesional MØ (Fig. 1c–e), disease resolution did not occur (Figure S1) indicating their inactivation. Furthermore, as treatment caused an increase in IL12p40 and LCs, concomitant with a decrease in IL-10 and absence of LD bodies, it strengthened our proposition that in PKDL, this decrease in LCs along with increased IL-10 caused immune inactivation that allowed for disease sustenance, emphasizing the importance of immunomodulation as a chemotherapeutic strategy against Leishmaniasis.

Acknowledgements Financial assistance was provided by Indian Council for Medical Research (ICMR), Council for Scientific & Industrial Research (CSIR) and Department of Science & Technology (DST), Govt. of India (MC) and Bundesministeriumf€ urBildung und Forschung (BMBF) and Deutsche Forschungsgemeinschaft (DFG; Ste 833/6-2), Germany (EvS). SM and DM received fellowships from DST-INSPIRE and ICMR, respectively.

Authors contribution MC, EvS, SM, DM: experimental design; SM, CB, UC, NKD: experiments and analysed data. MC, EvS, SM, DM, NKD, JNB: drafted and critically reviewed the manuscript.

Conflict of interest The authors have declared no conflicting interests.

7 Mukhopadhyay D, Das N K, Roy S et al. J Infect Dis 2011: 204: 1427–1436. ~a L et al. Histol 8 Tapia F J, Caceres-Dittmar G, Acun Histopathol 1989: 4: 499–508. 9 Gordon S, Taylor P R. Nat Rev Immunol 2005: 5: 953–964.

Supporting Information

Figure S1. Representative profile of giemsa stained sections of a patient with PKDL at disease presentation and post treatment (magnification, 1009 objective). Figure S2. Representative IHC profile showing distribution of HLA-DR+ cells in the dermal biopsy of a healthy control, patient with PKDL at disease presentation and post treatment under (magnification, 109 and 409 objectives).

Additional supporting data may be found in the supplementary information of this article. Data S1. Materials and methods.
