Through the multiple metabolomics studies performed with clinical samples from leprosy patients it is clear that alterations in the metabolism of lipid mediators derived from ω3 and ω6 PUFA occur with this disease. However, there is a lack of research that directly links these lipid mediators to the breadth of immune responses that occur across the clinical manifestations of leprosy. Detailed investigations to define enzymes and biochemical pathways for lipid mediator synthesis, along with elucidation of lipid mediator receptors and mechanisms by which lipid mediators influence both innate and adaptive immune responses, has nevertheless allowed the development of well supported hypothesis on the function of various lipid mediators in different manifestations of leprosy. A common theme that has emerged from existing studies is that several of the lipid mediators identified in the metabolomics studies of leprosy patients and discussed here (RvD1, LXA4, PGE2, and PGD2) down-regulate the immune-inflammatory responses promoted by Th1 and Th17 cells and facilitate the activity and proliferation Treg cells. This would indicate that *M. leprae* might exploit the pro-resolving activities of lipid meditators to maintain a persistent infection. Nonetheless, some of these lipid mediators such as PGE2 and PGD2, as well as LTB4 can influence the protective response against *M. leprae.* Another emerging theme is that alteration of the balance between pro-inflammatory and pro-resolving lipid mediators has the potential to dramatically skew the Th1/Th17 and Treg responses in leprosy. This same concept also applies to variations in the relative concentration of individual products such as PGE2. Thus, a coordination of the dynamics of the lipid mediator response and that of the adaptive and innate immune systems seems to be a driving factor in the specific presentation of leprosy.

As existing and future data are interpreted to develop models of lipid mediator involvement in the pathology and immunology of leprosy, it is important to consider the complexity of lipid mediator metabolism, and that most lipid mediators can serve as ligands for multiple receptors. Additionally, the spatial and

#### REFERENCES

temporal aspects of lipid mediator metabolism and receptor expression, along with the complementary or opposing activities of multiple lipid mediators must be addressed to fully elucidate the role lipid mediators play in leprosy. Mathematical models, as performed for *M. tuberculosis* infection (150), may be important to elucidate the influence PUFA-derived lipid mediator complexity in disease outcomes that might occur in individuals infected with *M. leprae.* It is also important to highlight that lipid mediators not identified or targeted in previous metabolomics studies on leprosy, may also contribute to immuno-pathogenesis. Thus, further targeted metabolomics investigations supported by orthogonal approaches, such as transcriptomics and proteomics, are needed to elucidate the full complement lipid mediators involved in leprosy and define how systemic alterations in their levels modify the phenotype of innate and adaptive immune cells in different presentations of leprosy. Future research efforts will not only provide an understanding of the contribution of lipid mediators to chronic infectious diseases but also provide the basis for the development of new diagnostic/prognostic and treatment approaches to address leprosy as a public health problem.

## AUTHOR CONTRIBUTIONS

CS and JB contributed to the review of published literature, development of the concepts, and design of the review article, as well as the writing and editing of the manuscript. CS is responsible for the design and concepts of the figures.

### FUNDING

This work was supported by the Heiser Foundation for Leprosy Research of the New York Community Trust, grants P15-000827 and P16-000796 to JB as co-principle investigator (PI) and by the Brazilian Coordination for the Improvement of Higher Education Personnel through the Science without Borders program (10546- 13-8, for the postdoctoral scholarship to CS).

of nerve damage in leprosy. *J Immunol* (2001) 166(10):5883–8. doi:10.4049/ jimmunol.166.10.5883

and prostaglandin E(2). *J Pharmacol Exp Ther* (2008) 326(2):493–501. doi:10.1124/jpet.108.137992

pro-inflammatory and pro-resolving polyunsaturated fatty acid metabolites. *J Chromatogr B Analyt Technol Biomed Life Sci* (2013) 932:123–33. doi:10.1016/ j.jchromb.2013.06.014

control of delayed type hypersensitivity. *Proc Natl Acad Sci U S A* (2006) 103(13):5179–84. doi:10.1073/pnas.0507175103

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer OM and handling editor declared their shared affiliation.

*Copyright © 2018 Silva and Belisle. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

*Marco Alonso Martinez-Guzman1,2, Anabell Alvarado-Navarro2 , Vidal Delgado-Rizo3 , Alejandra Garcia-Orozco1,2, Jorge Arturo Mayorga-Rodríguez 4 , Ana Laura Pereira-Suarez 3 and Mary Fafutis-Morris 2 \**

#### *Edited by:*

*Annemieke Geluk, Leiden University Medical Center, Netherlands*

#### *Reviewed by:*

*Roberta Olmo Pinheiro, Fundação Oswaldo Cruz (Fiocruz), Brazil Mario M. D'Elios, University of Florence, Italy*

> *\*Correspondence: Mary Fafutis-Morris<EMAIL_ADDRESS>
*Specialty section:*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology*

*Received: 24 July 2017 Accepted: 29 January 2018 Published: 13 February 2018*

#### *Citation:*

*Martinez-Guzman MA, Alvarado-Navarro A, Delgado-Rizo V, Garcia-Orozco A, Mayorga-Rodríguez JA, Pereira-Suarez AL and Fafutis-Morris M (2018) Serum Levels of Migration Inhibitory Factor (MIF) and In Situ Expression of MIF and Its Receptor CD74 in Lepromatous Leprosy Patients: A Preliminary Report. Front. Immunol. 9:246. doi: 10.3389/fimmu.2018.00246*

*1Doctorado en Ciencias Biomédicas con Orientación en Inmunología, Departamento de Fisiología, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Guadalajara, Mexico, 2Centro de Investigación en Inmunología y Dermatología, Departamento de Fisiología, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Guadalajara, Mexico, 3 Laboratorio de Inmunología, Departamento de Fisiología, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Guadalajara, Mexico, 4 Laboratorio de Micología, Instituto Dermatológico de Jalisco "Dr. José Barba Rubio", Zapopan, Mexico*

Leprosy is a chronic disease caused by *Mycobacterium leprae* that affects the skin and peripheral nerves. It may present as one of two distinct poles: the self-limiting tuberculoid leprosy and the highly infectious lepromatous leprosy (LL) characterized by *M. leprae*-specific absence of cellular immune response. The pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) enhance the bactericide activities of macrophages after interaction with its receptor, CD74. Importantly, MIF also possesses chemoattractant properties, and it is a key factor *in situ* for the activation of macrophages and in blood to promote leukocytes migration. MIF-mediated activation of macrophages is a key process for the elimination of pathogens such as *Mycobacterium tuberculosis*; however, its participation for the clearance of *M. leprae* is unclear. The aim of this study was to evaluate the serum levels of MIF as well as MIF and CD74 expression in skin lesions of LL and compare it with healthy skin (HSk) taken from subjects attending to dermatological consult. Samples of serum and skin biopsies were taken from 39 LL patients and compared with 36 serum samples of healthy subjects (HS) and 10 biopsies of HSk. Serum samples were analyzed by ELISA and skin biopsies by immunohistochemistry (IHC). IHC smears were observed in 12 100× microscopic fields, in which percentage of stained cells and staining intensity were evaluated. Both variables were used to calculate a semi-quantitative expression score that ranged from 0 to 3+. We found no differences in MIF levels between LL patients and HS in sera. In addition, MIF was observed in over 75% of cells with high intensity in the skin of patients and HSk. Although we found no differences in MIF expression between the groups, a CD74 score statistically higher was found in LL skin than HSk (*p* < 0.001); this was the result of a higher percentage of cells positive for CD74 (*p* < 0.001). As a conclusion, we found that CD74-positive cells are intensely recruited to the skin with LL lesions. In this manner, MIF signaling may be enhanced in the skin of LL patients due to increased expression of its receptor, but further studies are required.

Keywords: leprosy, lepromatous leprosy, skin, serum cytokines, migration inhibitory factor, CD74

### INTRODUCTION

Leprosy is a chronic infectious disease caused by *Mycobacterium leprae*. The disease in patients may develop as one of two poles that differ clinically and immunologically between each other. Few lesions in the body and an active immune response mediated by cells characterize the tuberculoid leprosy (TT) pole; on the other hand, patients who develop lepromatous leprosy (LL) show numerous erythematous macules around the body coupled with *M. leprae*-specific anergy and disrupted immune response. The reasons for developing either pole are still unknown, but the environment and host genetics are probable factors that mediate such polarization (1). As a consequence, the ratio of LL to TT patients varies around the world, and it can be as high as 3:1 in countries like Mexico, where TT cases are scarce (2).

In LL patients, *M. leprae* proliferates in the skin and peripheral nerves (1). Histologically, skin lesions of LL patients are formed by diffuse infiltration of macrophages, lymphocytes, and plasma cells in the dermis, along with a dysfunctional epidermis. Anti-inflammatory cytokines, such as IL-10 and TGF-β, are highly expressed in these lesions when compared with TT lesions (3). As a consequence of the immunological imbalance, macrophages get infested by abundant bacilli and become foamy macrophages (4). These macrophages seem unable to kill *M. leprae* but serve as a reservoir instead and release additional anti-inflammatory mediators and reduce their production of key activator cytokines, namely TNF-α and IL-1β (5).

Macrophage migration inhibition factor (MIF) is a constitutively released cytokine that is able to subvert the antiinflammatory activities of glucocorticoids and also initiates intense immune reactions by stimulating macrophages toward an inflammatory profile that involves productions of ROS, increases in the expression of TLR4, and diminishes cell susceptibility to apoptosis (6, 7). MIF participates not only in skin homeostasis by regulating the differentiation and proliferation of keratinocytes (8) but also in skin disorders by enhancing the activity of inflammatory macrophages (9). To exert its biological activities, MIF needs to interact with its receptor CD74, a molecule with a wide array of described functions in immune cells (10). In addition, CD74 may form a complex with chemokine receptors and lead cells to a process of MIFmediated migration (11).

Only few studies analyze the expression of both MIF and CD74 in infectious diseases, even though MIF has proved to be an important mediator against several infections, especially those caused by *Mycobacterium tuberculosis* (12) and *Leishmania major* through a mechanism dependent on TNF-α and reactive nitrogen intermediates (13). In addition, the early stimulation of CD74 triggers the MAPK and PI3K pathways that lead to proliferation and recruitment of immune cells, thus starting the inflammation in injured tissues (7). However, the specific role of MIF/CD74 interaction in the context of skin diseases has been poorly studied. Due to its relevance in the inflammatory process, it is interesting to know whether the MIF/CD74 axis is implicated in the immunopathology of LL. We previously reported that the susceptibility to develop LL in Western Mexico is associated to the alleles of the STR-794 CATT5–8 polymorphism of the *MIF* gene linked to higher expression of MIF (14). This paradoxical observation relates the inflammatory cytokine MIF with the anti-inflammatory LL pole and is suggestive of an important role for MIF in this spectrum of leprosy. Thus, we are interested to study the levels of MIF in the serum of LL patients as well as the expression of both MIF and CD74 in the skin lesions of LL patients.

#### MATERIALS AND METHODS

#### Subjects and Samples

Patients for this study were diagnosed by Dermatologists working in the Instituto Dermatológico de Jalisco "Dr. José Barba Rubio" according to their clinic, bacilloscopic, and histopathologic characteristics. Due to the low prevalence of TT in Mexico (2), only patients diagnosed as LL and borderline lepromatous in the absence of reactional episodes were included in the study. Patients who presented chronic, inflammatory, and dermatological conditions were excluded. Serum samples from patients were requested upon diagnosis confirmation. Healthy subjects (HS) of similar age and gender characteristics were asked for blood donation to compare MIF serum levels. In total, serum of 39 patients and 36 HS were considered for determination of MIF. The demographic and disease characteristics of participants are shown in **Table 1**.

Tissue samples of 39 patients were obtained for immunohistochemistry (IHC) assays from the biopsies taken for their diagnostics; in addition, 10 tissues of healthy skin (HSk) were obtained from the repertoire of the Institute. Samples were embedded in paraffin for preservation and cut into 3-µm sections for mounting onto precharged slides.

#### Serum Quantification of MIF

Migration inhibitory factor quantification was performed by the ELISA kit for "Human MIF Immunoassay" (Cat. No. DMF00B; R&D, MN, USA) following the manufacturer's indications. Briefly, samples were diluted 10-fold with the kit Calibrator Diluent and 50 µl of the mix were added to the microplate wells, followed by 2-h incubation. Wells were washed and the detection antibody was incubated for 2 h. Wells were washed and the color was developed by 30-min incubation with Substrate Solution in the dark. Finally, stop solution was added and optical density was measured at 450 nm and corrected at 570 nm. The concentration of MIF was calculated interpolating the optical density of samples with a multiparametric curve generated with the MIF standard included in the kit.

#### *In Situ* Characterization of MIF and CD74

We analyzed the expression of MIF and CD74 in skin biopsy samples. Slices of tissue samples were incubated at 60°C for 10 min and deparaffinized on xylene bath for 20 min. Samples were then rehydrated through a series solutions of decreasing concentrations of ethanol. Antigens were retrieved in a bath of sodium citrate solution 10 mM at 95°C for 10 min followed by cooling in a citrate cold solution. Non-specific binding to proteins was blocked by incubation with bovine fetal serum 10% by 30 min; endogenous peroxidase activity of samples was blocked by incubation with H2O2 3% solution by 30 min. Afterward, sections of each biopsy were incubated overnight at 4°C with one of the following primary antibodies: anti-MIF FL-115 (Cat# sc-20121 RRID:AB\_648587) 2 µg/ml or anti-CD74 FL-296 (Cat# sc-20082 RRID:AB\_2075501) from Santa Cruz Biotechnology, Inc., TX, USA. The detection of primary antibodies will be performed using the polymer conjugated to secondary antibodies from Dako EnVision™ + Dual Link System-HRP (Dako Agilent Technologies, Denmark). Finally, sections were counterstained with hematoxylin. The presence of antigens were analyzed in four random 40× fields using the semi-quantitative algorithm of Li et al., which took into account both the intensity of staining and the percentage of stained cells (15). Briefly, percentage of stained cells was transformed into histological index I (HI I) as follows: Neg (0+) ≤ 5% of stained cells; "+" (1+) = 6–25%, "++" (2+) = 26–50%, "+++" (3+) = 51–75%; and "++++" (4+) > 76%. Similarly, a second histological index (HI II) for staining intensity was calculated as: Neg (0+) = no staining, "+" (1+) = low intensity, "++" (2+) = moderate intensity, "+++" (3+) = high intensity. Finally, HI I and HI II were multiplied and ranked as an expression score of Neg if the product of (HI I)\*(HI II) was between 0 and 1; "+" (1+) if the product was between 2 and 4; "++" (2+) if the product was between 5 and 8; and "+++" (3+) if the product was between 9 and 12.

## Statistical Analysis

Qualitative variables of the study were expressed as frequencies ± SD and quantitative variables were expressed as mean values ± SD. Statistical analyses were performed on IBM SPSS Statistics ver. 20. Differences in MIF levels between groups, as well as differences of expression score, percentage of stained cells, and staining intensity for MIF and CD74 were analyzed using Mann–Whitney *U* test. In addition, we studied whether there exists correlation between the bacillary index of LL patients, the soluble levels of MIF as well as the *in situ* expression of MIF and CD74 using the non-parametric one-tailed Rho spearmen correlation coefficient. Statistical analysis resulting with *p* values <0.05 were considered as significant.

#### Ethical Consideration

The study was designed in agreement with the Declaration of Helsinki (16). All participants were informed about the goals of the study and provided written agreement. This study was approved by the ethical committee *Comité de Investigación y Bioseguridad del Centro Universitario de Ciencias de la Salud* of the University of Guadalajara (No. CI-02515).

## RESULTS

#### Serum Levels of MIF

We quantified the serum levels of MIF in 39 LL patients (44.49 ± 20.21 ng/ml) and 36 HS (53.35 ± 34.13 ng/ml). However, we found no significant differences regarding the levels of MIF between groups. Also, we did not find significant correlation between the bacillary index and serum MIF levels (*r* = 0.272, *p* = 0.154).

## MIF-Related Markers in skin

We analyzed the expression of MIF and CD74 in skin biopsies of 39 LL patients as well as 10 samples of HSk.

We found that MIF is highly expressed in the epithelia of both patients and HSk (**Figure 1**). There exist also several MIF+ cells in the dermis in both groups. We observed that MIF is mainly expressed in the cytoplasm but it can also be found in some nuclei. We found no significant differences in the expression score between both groups (**Table 2**). Most of the participants presented 1+ or 2+ of MIF expression score in skin samples. To better understand this index, we analyzed the percentage and intensity index. We found that most of the LL skin samples could be classified as 1+ (37.2%) or 2+ (34.9%); similarly, most of HSk fell into 1+ (30%) and 2+ (40%) categories. Interestingly, the staining of MIF in cells was a little stronger in HSk than in LL samples, but no significant differences were found. In addition, MIF expression score was not correlated to the bacillary index of patients (*r* = 0.190, *p* = 0.153).

CD74 is expressed in the dermis, but its epidermal expression is scarce (**Figure 2**). Although it is expressed in the membrane of some cells, it is mostly expressed in the cytoplasm. Interestingly, we found that its expression score is significantly higher in LL skin than HSk (*p* < 0.001), since 0% of HSk could be classified as 2+ or 3+, whereas 32.6 and 23.3% of LL was classified as 2+ and 3+, respectively (**Table 3**). We analyzed whether this difference

FIGURE 1 | *In situ* expression of migration inhibitory factor (MIF) in healthy skin (HSk) and lepromatous leprosy (LL) skin biopsy. Photographs of MIF-directed immunohistochemistry assays on HSk and skin lesions of LL patients on progressive magnifications are displayed. (A–C) represent the staining of MIF on HSk at 10×, 40×, and 100×, respectively. (D–F) show staining of MIF on LL skin at 10×, 40×, and 100×. The magnified fields are shown within circles. MIF expression is intense on epidermis and annexes of both HSk and LL. In dermis, its expression also seems to be constitutive. MIF is expressed mostly in cytoplasm and can also be found in some nuclei.

TABLE 2 | Ranking of lepromatous leprosy (LL) patients and healthy skin (HSk) according to their score of migration inhibitory factor (MIF) staining.

*Statistical analysis using two-sided Mann–Whitney U test, where p* < *0.05 values were considered as significant.*

was due to an increased infiltrate of CD74+ cells, a higher staining intensity or both. We found that none of the HSk presented an infiltrate of over 1+ (equivalent to 25% of cells), whereas 27.9% LL skin samples presented 2+, 46.5% presented 3+, and 4.7% of LL samples presented 4+. However, the intensity of CD74 staining was not different between groups; notably, CD74 presented high intensity (3+) in up to 40% of HSk and 39.5% of LL. Finally, CD74 expression score was not correlated to the bacillary index of patients (*r* = 0.040, *p* = 0.416).

#### DISCUSSION

The immune response has been well characterized in the advanced forms of leprosy, but there are still several gaps where research is required. One of the main differences between the two poles is the presence of inflammatory cells and markers in TT, meanwhile, anti-inflammatory conditions are rather present in LL patients (3); however, it is not clear what mechanisms lead to such polarization. We previously reported that *MIF* polymorphisms in the promoter region are associated with LL in Western Mexico population (14). Thus, in this work, we investigated the serum levels of MIF in LL patients. Despite the genetic association, we found no differences in the serum levels of MIF between LL patients and HS. Recently, Bansal et al. measured MIF levels in leprosy patients and they also found that the MIF levels in serum are similar between healthy controls and patients presenting LL, borderline lepromatous, or borderline tuberculoid (17).

FIGURE 2 | *In situ* expression of CD74 in healthy skin (HSk) and lepromatous leprosy (LL) skin biopsy. Photographs of CD74-directed immunohistochemistry assays on HSk and skin lesions of LL patients on progressive magnifications are displayed. (A–C) represent the staining of CD74 on HSk at 10×, 40×, and 100×, respectively. (D–F) show staining of CD74 on LL skin at 10×, 40×, and 100×, respectively. The magnified fields are shown within circles. CD74 is scarcely expressed in epidermis and annexes. It is expressed in the dermis, where a major infiltrate can be observed in LL skin than HSk. Paradoxically, it seems to be expressed mostly in cytoplasm rather than the plasma membrane.

TABLE 3 | Ranking of lepromatous leprosy (LL) patients and healthy skin (HSk) according to their score of CD74 staining.

*Statistical analysis using two-sided Mann–Whitney U test, where p* < *0.05 values were considered as significant.*

To obtain more information about MIF role in leprosy, we examined skin biopsies by IHC to inquire about its cellular and tissular localization. As in serum, we found that the expression of MIF is not different between skin lesions of LL patients and HSk, despite the elevated expression of IL-4, IL-10, and TGF-β reported in LL skin lesions (18). Therefore, we may postulate that anti-inflammatory mediators do not reduce the expression of MIF, although they may interfere with MIF capacity to enhance the bactericidal activities of macrophages, which is an intrinsic activity of MIF (6). Since MIF can be stored within the cytoplasm (19), our results most likely reflect the constitutive expression of MIF (20). Noteworthy, MIF presence is crucial for cell homeostasis due to its multiple activities. It may be secreted from the central nervous system to regulate

cell proliferation (6), and it also promotes glucose intake by increasing the expression of GLUT4 (21). Nevertheless, MIF functions may vary according to other microenvironment components and the cell that it is targeting. For example, MIF expression is increased in inflammatory skin diseases such as alopecia areata and skin tumors (22, 23) and MIF orthologs are produced by *Leishmania* spp. to promote the survival on parasite-infected macrophages (24).

Since MIF expression is not different between LL skin and HSk, we measured CD74 expression to determinate if it is involved in leprosy lesions. Paradoxically, we found that its expression is significantly higher in LL skin than HSk. However, the elevated expression of CD74 does not correlate with the antiinflammatory environment of LL lesions. In addition to being MIF receptor, CD74 excerpts several functions that include regulation of vesicular transport, dendritic cells migration, and protection of proteins as a chaperone (11). Noteworthy, we found that CD74 is expressed mostly in the cytoplasm and that few cells actually expressed CD74 on the plasma membrane, suggesting that in LL, CD74 is not acting as a receptor for MIF, whereas its overexpression could contribute to the lack of response toward *M. leprae*. Arguably, the transport of antigens on MHC-II toward the plasma membrane of monocytes could be altered. Indeed, the expression of both MHC-I and MHC-II are reduced in dendritic cells infected with *M. leprae* in a dosedependent manner (25). Moreover, Lee et al. have described that the expression of leukocyte Ig-like receptor A2 (LILRA2) is decreased in TT compared with LL, where it reduced the capacity of monocytes to present leprosy antigens on MHC-II to T cells, although antigen processing was not disrupted (26). Given that CD74 is a chaperone for MHC-II (27) and that it is involved in vesicle transport (11), cytoplasmic CD74 in LL could arrest the movement of antigens-loaded MHC-II toward the cell surface in an LILRA2-mediated mechanism.

Given that 80% of new cases of leprosy Mexico are multibacillary cases (2), our observations of MIF and CD74 are limited to LL patients without treatment. It is important to pursue paucibacillary cases to better detail the role of MIF in leprosy; in particular, the study of MIF in indeterminate leprosy results of particular interest due to the functions of MIF in innate immunity (6, 28). In addition, its participation in reactional episodes has been highlighted by Bansal et al., whose group found increased serum concentration of MIF in erythema nodosum leprosum (17). Further description of MIF and CD74, as well as the possibly involved signaling pathways, namely, PI3K, MAPK could yield valuable insight into leprosy immunopathology.

In summary, we have found that the expression of MIF in LL patients is similar to HS both in serum and in skin. However, the expression of CD74 is significantly increased in the skin lesions of LL patients, although its participation in the physiopathology leprosy remains unclear. Further studies in indeterminate leprosy and paucibacillary leprosy, as well as other infectious and inflammatory diseases of skin, are required to describe the participation of MIF/CD74 in the immune response against leprosy in the skin. In addition, their activities in the systemic response should also be explored. It is important to determine

#### REFERENCES

the molecules to which CD74 is binding to further understand the regulation of antigen presentation in the LL skin. In addition, the characterization of MIF+ and CD74+ cells could provide further insight into the immune microenvironment of skin lesions.

### ETHICS STATEMENT

This study was carried out in accordance with the recommendations of Comité de Investigación y Bioseguridad del Centro Universiario de Ciencias de la Salud of the University of Guadalajara (No. CI-02515) with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Comité de Investigación y Bioseguridad del Centro Universitario de Ciencias de la Salud of the University of Guadalajara (No. CI-02515).

## AUTHOR CONTRIBUTIONS

MM-G aided in ELISA assays and performed IHC identification of MIF and CD74; AA-N directed and designed the IHC assays; VD-R directed the ELISA assays; AG-O performed the ELISA assays; JM-R and AP-S performed the statistical analyses; MF-M is responsible for conception of the work and supervised the progress at all times. All authors participated in drafting, revising, and approval of the manuscript.

## ACKNOWLEDGMENTS

We would like to acknowledge the students in our lab that collaborated in the process, especially Gerardo Saúl Dávila-Espinosa, Karla Lizette Iñiguez-Iñiguez, and Jorge Javier Ricardo Padilla-Arellano, who assisted the different parts of the methods. Also, we recognize the work and help provided by Francisco Arturo Miranda-Gomez and Maria Isabel Medina-Ortega who collected the samples for our study.

## FUNDING

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Martinez-Guzman, Alvarado-Navarro, Delgado-Rizo, Garcia-Orozco, Mayorga-Rodríguez, Pereira-Suarez and Fafutis-Morris. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Vaccines for Leprosy and Tuberculosis: Opportunities for Shared Research, Development, and Application

*Mariateresa Coppola1 \*, Susan J. F. van den Eeden1 , Naoko Robbins2 , Louis Wilson1 , Kees L. M. C. Franken1 , Linda B. Adams2 , Tom P. Gillis 2†, Tom H. M. Ottenhoff 1‡ and Annemieke Geluk1‡*

*1Department of Infectious Diseases, Leiden University Medical Center, Leiden, Netherlands, 2 The National Hansen's Disease Programs, Baton Rouge, LA, United States*

#### *Edited by:*

*Steffen Stenger, Universitätsklinikum Ulm, Germany*

#### *Reviewed by:*

*Maria Florencia Quiroga, Universidad de Buenos Aires, Argentina Elsa Anes, Universidade de Lisboa, Portugal*

> *\*Correspondence: Mariateresa Coppola<EMAIL_ADDRESS>
#### *†Present address:*

*Tom P. Gillis, Research Chair, The Leprosy Mission Canada, Markham, ON, Canada*

*‡ These authors have contributed equally to this work.*

#### *Specialty section:*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology*

*Received: 09 November 2017 Accepted: 05 February 2018 Published: 26 February 2018*

#### *Citation:*

*Coppola M, van den Eeden SJF, Robbins N, Wilson L, Franken KLMC, Adams LB, Gillis TP, Ottenhoff THM and Geluk A (2018) Vaccines for Leprosy and Tuberculosis: Opportunities for Shared Research, Development, and Application. Front. Immunol. 9:308. doi: 10.3389/fimmu.2018.00308*

Tuberculosis (TB) and leprosy still represent significant public health challenges, especially in low- and lower middle-income countries. Both poverty-related mycobacterial diseases require better tools to improve disease control. For leprosy, there has been an increased emphasis on developing tools for improved detection of infection and early diagnosis of disease. For TB, there has been a similar emphasis on such diagnostic tests, while increased research efforts have also focused on the development of new vaccines. Bacille Calmette–Guérin (BCG), the only available TB vaccine, provides insufficient and inconsistent protection to pulmonary TB in adults. The impact of BCG on leprosy, however, is significant, and the introduction of new TB vaccines that might replace BCG could, therefore, have serious impact also on leprosy. Given the similarities in antigenic makeup between the pathogens *Mycobacterium tuberculosis* (*Mtb*) and *M. leprae*, it is well possible, however, that new TB vaccines could cross-protect against leprosy. New TB subunit vaccines currently evaluated in human phase I and II studies indeed often contain antigens with homologs in *M. leprae*. In this review, we discuss pre-clinical studies and clinical trials of subunit or whole mycobacterial vaccines for TB and leprosy and reflect on the development of vaccines that could provide protection against both diseases. Furthermore, we provide the first preclinical evidence of such cross-protection by *Mtb* antigen 85B (Ag85B)-early secretory antigenic target (ESAT6) fusion recombinant proteins in *in vivo* mouse models of *Mtb* and *M. leprae* infection. We propose that preclinical integration and harmonization of TB and leprosy research should be considered and included in global strategies with respect to cross-protective vaccine research and development.

Keywords: antigen 85B, early secretory antigenic target, *Mycobacterium leprae*, *Mycobacterium tuberculosis*, tuberculosis, leprosy, hybrid recombinant protein, vaccines

## INTRODUCTION

Tuberculosis (TB) and leprosy are major infectious diseases that are caused by highly related mycobacterial pathogens, *Mycobacterium tuberculosis* (*Mtb*) and *M. leprae.* Although derived from the same mycobacterial ancestor (1), the target organs affected by these highly related mycobacteria (skin and nerves in leprosy; lungs and extrapulmonary lesions in TB) and the resulting

**46**

clinical symptoms, are strikingly different. Notwithstanding these differences, the two poverty-associated diseases also share important characteristics (2–4), including the important role of host cellular immunity in protection. In addition, both diseases display a wide spectrum of (immuno)-pathological features with characteristic granulomatous lesions that often result in chronic disease and require prolonged treatment with multidrug antibiotic therapies (5).

Although rarely lethal, leprosy is enormously feared for causing lifelong handicaps and deformities resulting from irreversible nerve damage. Leprosy is notable for its continued transmission, which results in a stable annual number of approximately 200,000 new cases (6). Moreover, predictions from mathematical modeling indicate that millions linger undetected (7).

Tuberculosis is a major threat due to its high morbidity and mortality, causing an estimated 10.4 million new cases and 1.8 million deaths in 2015 alone (8). This scenario is worsened by HIV co-infection as well as by the emergence of multi-, extensive-, and total-drug resistance (8). Though not as threatening as for TB, anti-microbial resistance also poses a risk for leprosy (9–13), which needs to be considered in post-exposure prophylactic (PEP) treatment strategies in leprosy endemic areas that aim to reduce transmission by administering a single dose of antibiotics to those at high risk of developing leprosy (14).

In order to combat both diseases, global strategies have been endorsed, promoting the implementation of new drugs to shorten lengthy chemotherapeutic regimens, including strategies to avoid occurrence of *de novo* antibiotic resistance (15). In addition, research is focusing on development of improved diagnostics for detection of infection and early stages of disease allowing prophylactic and timely treatment, respectively. In contrast to chemoprophylaxis, vaccines would be expected to give rise to active as well as long-term protection. Therefore, development of novel vaccines is an additional top priority to control TB and leprosy by preventing disease and transmission (6, 16, 17). To explore this further, we here review the current vaccine development pipelines for TB and leprosy focusing on shared features and antigenic components, as well as highlight potential differences and incompatibilities.

#### BACILLE CALMETTE–GUÉRIN (BCG), ONE VACCINE FITS ALL?

*Mycobacterium bovis*, BCG still is the only vaccine used against TB worldwide (18, 19). It is the first live-attenuated bacterial vaccine administered to newborns at or shortly after birth and has been applied in 172 countries (20, 21). In spite of its efficacy against severe TB in children, protection against TB in adolescents and adults is not sufficient to impact on disease and transmission. This urges for new, more efficient vaccines, and alternative strategies to replace or complement BCG (22–24).

Although being introduced and licensed for prevention of TB, BCG was soon recognized to protect partly also from leprosy (25–27). The efficacy of BCG vaccination against TB and leprosy has been evaluated in numerous clinical trials and observational studies. However, these studies also revealed inconsistent and sometimes even contradictory results. BCG's protective effects varied from 2 to 83% and from 58 to 74% in preventing pulmonary and extrapulmonary TB, respectively (28), while its efficacy against leprosy ranged from 26 to 41% in experimental studies to 61% in observational studies, with mild differences between the paucibacillary (62%) and multibacillary (76%) forms (25, 29–31). BCG vaccination does not seem to protect against the third most common mycobacterial disease, Buruli ulcer's disease, although a definite conclusion requires further well-designed prospective studies (32). Apart from its effect on mycobacterial diseases, BCG vaccination has been reported to have significant impact on unrelated diseases, probably through training of the innate immune system to respond more favorably to outer assaults (33, 34).

The remarkable differences in efficacy in various trials for TB and leprosy have been ascribed to several factors, including diversity in the genetic fingerprints of the mycobacterial pathogens in different geographic areas (35, 36), the various BCG strains used in the studies (37, 38), the immune, nutritional, and socioeconomic status of the vaccinees enrolled (39), the presence of helminths or viral coinfections (21, 40, 41), the background exposure to and induction of immunity by environmental mycobacteria, which might mask or block the effects of BCG (42), but the precise reasons for this remain largely unclear.

Our incomplete understanding of which components of the human immune system are responsible for either successful or inefficacious protection following BCG vaccination impedes the rational design of more effective vaccines (43). For instance, the limited efficacy of BCG in preventing local pulmonary TB disease compared to its effects on disseminated forms of TB is well documented, but remains unexplained (19). One hypothesis attributes this finding to its inability to induce durable and effective immune cells that home to the lung (19). Therefore, new routes of BCG administration, such as aerosol or intranasal immunization, are tested to initiate mucosal immunity and promote homing of immune cells to the lung mucosa (44, 45).

Another shortcoming of BCG is that its protective effects against TB as well as leprosy wanes over time, dropping to 14% efficacy after 10–20 years (46), indicating a suboptimal induction of long-term immune memory responses as discussed above (47, 48). Thus, BCG revaccination has been attempted in several countries. As a first attempt, a large trial in Malawi showed that BCG revaccination had limited impact on TB, while reducing the risk of leprosy with 50% (25, 49). Similarly, a large randomized controlled TB trial in Brazil showed that a second dose of BCG in adolescents did not confer better protection than a single dose given at birth (50). In contrast, for leprosy (30, 31), BCG revaccination is officially recommended in Brazil, since the 1970s for household contacts of leprosy patients as a boost to routine neonatal BCG vaccination. More recently, an extensive BCG revaccination trial of household contacts of leprosy patients in Brazil showed that the protection conferred by a booster BCG vaccination was 56% and was independent of previous BCG vaccination (29).

Notwithstanding, this lack of BCG boosting effects in TB and its beneficial effects on leprosy, BCG vaccination can also have less favorable effects, such as increasing the numbers of paucibacillary leprosy cases within the first months after BCG immunization (51). This is thought to be due to excessive boosting of pre-existing *M. leprae*-specific T cells in those already frequently exposed to the bacterium (51, 52), or to hyperinflammatory innate immunity (53, 54). Both mechanisms could lead to pathogenic immunity, such as increased numbers of paucibacillary leprosy and leprosy reactions (55).

Based on the premise that BCG might overcome the phenotypic cellular immunological tolerance against *M. leprae* in multibacillary leprosy, BCG immunotherapy has been trialed in leprosy patients in Venezuela in the 1980s (56). These studies met with limited success, since complications of this therapy were the occasional occurrence of disseminated cutaneous BCG lesions and the induction of leprosy reactional episodes (57). In contrast, a small-sized clinical trial in India studied a combination of MDT and immunotherapy with BCG in newly diagnosed leprosy patients and found a significant reduction in duration of reactions, incidence of type 2 reactions as well as in time to achieve bacterial clearance (58).

In summary, BCG has significant protective efficacy against severe TB in children and against leprosy in adults, while BCG revaccination has added value in leprosy, but not in TB. Future changes in TB vaccination policies might, therefore, also affect leprosy control. To further analyze this issue, we review current vaccine development pipelines and policies for TB and leprosy, focusing on shared target product profiles and antigenic composition.

## VACCINES IN CLINICAL TRIALS: AT THE CROSSROAD BETWEEN LEPROSY AND TB

Although BCG vaccination trials in leprosy were executed decades ago, the current leprosy clinical vaccine pipeline is three times smaller than that of TB (**Figure 1**). This situation is relatively recent considering that in 2001 there were four candidate leprosy vaccines (being) tested in clinical trials vs. none against TB.

#### Vaccine Candidates

The leprosy vaccine pipeline employs both live (26, 60) and killed (26, 56, 61–63) whole cell mycobacterial vaccines as well as adjuvanted recombinant protein vaccines, such as LepVax (64), which have the advantage over BCG and other replicating live vaccines that they can be used safely also in immunocompromised individuals (65). LepVax comprises a hybrid recombinant protein, linking four *M. leprae* antigens: ML2531, ML2380, ML2055, and ML2028 (LEP-F1) (**Table 1**), formulated in a stable emulsion with a synthetic, TLR4 agonist (GLA-SE) as adjuvant which has recently finished pre-clinical testing (66). In line with the extent of the epidemic, the TB vaccine pipeline is much larger. This includes candidates using various delivery platforms, such as virally vectored vaccines (67–70), adjuvanted subunits vaccines (71–74), recombinant BCGs (75), genetically attenuated *Mtbs*, as well as heat killed whole mycobacterial cell-based vaccines (76–79) (**Figure 1**). Evidently, the TB subunit vaccine pipeline has focused on a limited number of candidate *Mtb* antigens, in particular: Ag85A, Ag85B, early secretory antigenic target (ESAT6), TB10.4, Rv1813, Rv2608, Rv3619–3620, Rv1196, and Rv0125 (**Table 1**).

#### Clinical Endpoints

Leprosy and TB vaccines have different target product profiles and clinical endpoints to be considered in efficacy trials, e.g., prevention of infection (POI), prevention of disease (POD), or prevention of recurrence (POR) (80). POD require extensive longitudinal studies due to the long incubation times (years) in TB and leprosy (years-decades), and the limited incidence rates in most populations studied. For these reasons, alternative clinical trial designs have been developed using alternative biologically relevant endpoints, such as prevention of recurrence (POR) in cured TB patients, which evaluate whether relapse rates can be reduced by post-therapy vaccination; or shortening of treatment trials, which evaluate whether treatment length can be reduced by complementary immunotherapy with TB vaccines during the last phase of TB treatment. For leprosy, vaccines could be positioned to help preventing nerve damage in patients, since this clinical endpoint has a much higher frequency in leprosy patients, requires a shorter follow-up period and is a highly relevant endpoint in leprosy. New clinical trial designs with alternative endpoints will be important to accelerate the clinical evaluation of new vaccines for TB and leprosy, and signals detected in such studies can be validated in larger studies against classical endpoints, such as POD and perhaps POI.

## Clinical Trials

In most vaccination trials for leprosy, the protective effects of the tested new vaccine candidates were equivalent to that of BCG (81). Only in one study, vaccination with Indian Cancer Research Centre bacilli (an *M. lep*rae-related cultivable mycobacterium) and BCG plus killed *M. leprae* showed a twofold increased protection against leprosy compared to BCG alone (26). However,

Table 1 | Homology between tuberculosis (TB) vaccine components and *Mycobacterium leprae* proteins.

*a Accession number not known; nnsf, no significant similarity found. bPseudogene.*

*M. indicus pranii* (MIP) (also known as *Mycobacterium w*.) induced protective efficacy below that of BCG. Notwithstanding this result, MIP was evaluated also in a second, large-scale, double-blind trial with a 9-year follow-up (62). In this study, the protective efficacy of MIP in vaccinated household contacts after 3 years was the highest ever reported against leprosy (68%) for a vaccine other than BCG. However, its protective effect dropped considerably after 6 (60%) and 9 (28%) years of follow-up. Despite these conflicting results, MIP is currently being evaluated both as prophylactic and therapeutic vaccine against leprosy in two high endemic districts in India (82) in combination with a single dose of rifampicin (SDR). This design is reminiscent of a previous randomized vaccine field trial in which BCG as well as SDR was provided to leprosy contacts (83).

For TB, several vaccines and vaccine approaches are being pursued, with no new TB vaccine approved, yet for use, since the introduction of BCG in 1921. The results from the recent MVA85A vaccine phase 2b efficacy trial, the first new TB vaccine tested in an efficacy trial, since BCG, showed no improved protection in BCG-vaccinated South African infants (84), despite being highly immunogenic in adults (85). Several trials are ongoing (**Figure 1**), with the first outcomes to become available in 2018.

#### Correlates of Protection

Vaccine immunogenicity studies for both leprosy and TB vaccine candidates have mostly focused on their ability to induce type-1 cell-mediated immunity, particularly CD4+ Th cells releasing type 1 helper (Th1) cytokines. Indeed, Th1 immunity is widely considered to be key in controlling mycobacterial infections (86). HIV-induced CD4+ T cell deficiency, and genetic or acquired impairments in type 1 cytokine signaling (IL12-IFN-γ axis), all increase susceptibility to mycobacterial infection and progressive disease in humans and animal models (87–90). In leprosy, the presence of Th1 cytokines in lesions or in lepromin skin reactions has been related to better clinical prognosis and to localized rather than disseminating disease (91, 92). Furthermore, individuals that showed large local reactogenicity after intradermal BCG administration or lepromin injection are reported to have less risk for leprosy onset (93). Observation from a small Dutch cohort of BCG-vaccinated individuals showed that high skin inflammation responders had a larger amount of C-reactive protein in their sera than the low skin inflammation responders. In the same study, at 4, 8, and 12 weeks post-BCG vaccination, PBMCs of individuals with stronger local reactogenicity induced higher IFN-γ production

Figure 2 | IFN-γ secretion after Ag85-ESAT immunization. C57BL/6j and HLA-A2tg mice B6.Cg-Tg (117) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and housed under specific pathogen-free conditions. Recombinant proteins were overexpressed in *E. coli* BL21 (DE3) and purified to remove any traces of endotoxin as described in Ref. (116, 118). For the production of the antigen 85B (Ag85B)-early secretory antigenic target (ESAT6), hybrid recombinant hybrid protein, the Ag85B and ESAT6 genes were fused together by PCR with a linker coding for the amino acids NVA. C57BL/6j mice [(A); 13–14 animals per group] and HLA-A2tg mice [(B); 5 animals per group] were immunized three times subcutaneously with *Mycobacterium tuberculosis* (*Mtb)* Ag85-ESAT or *Mycobacterium leprae* Ag85-ESAT recombinant protein (25 µg) adjuvanted with GLA-SE [glucopyranosyl lipid adjuvant-stable emulsion (23) kindly provided by Infectious Disease Research Institute; Seattle, WA, USA; TLR4 agonist; 20 µg]; or CpG (ODN1826 5′-TCC ATG ACG TTC CTG ACG TT -3′; InvivoGen, San Diego, CA, USA; TLR9 agonist; 50 µg) (119). Splenocytes were harvested 4 weeks after final injections and restimulated *in vitro* with *Mtb* or *M. leprae* Ag85-ESAT hybrid recombinant proteins or the single Ag85B and ESAT6 recombinant proteins (all 10 µg/ml). IFN-γ secretion was analyzed by ELISA after 5 days. All mice were analyzed separately. Data shown indicate the mean and SE value of five mice per group.

after *in vitro* PPD stimulation than the one from the group with less local reaction to BCG (94). This suggests that skin reactogenicity after BCG vaccination causing local inflammation and systemic Th1 responses probably indicate protective immunity to mycobacteria. The failure of MVA85A against TB despite its induction of CD4+ Th1 immunity, the observation that BCGspecific CD4+ and CD8+ T-cell responses did not correlate with protection against TB disease in one study (95) together with the limited results achieved by current leprosy vaccines, clearly underline the need for a better understanding of the host mechanisms that are responsible for protection against both TB and leprosy. Several recent reports in animal models and humans have reported the involvement of other cell subsets in leprosy and TB (96, 97). Discovering these mechanisms may well prove to be a critical step for designing more effective vaccines.

Besides BCG, only MIP and killed *M. vaccae* have been clinically evaluated for both leprosy and TB, although in different trial designs and target populations. MIP has been tested for its putative therapeutic efficacy in tuberculous pericarditis (98) and as mentioned above for its protective efficacy against leprosy (26, 62). Killed *M. vaccae* has been assessed for its ability to prevent TB and leprosy disease in patients or contacts.

However, the administration routes (intramuscular vs. oral vs. intradermal injection of *M. vaccae*) and the eligibility criteria for the recruitment in the two trials (inclusion or not of individuals with BCG scar; HIV-positivity; anti-mycobacterial therapy) were quite diverse, impeding direct comparison of the impact of *M. vaccae* vaccination on both diseases (63, 78).

### ONE SUBUNIT VACCINE FOR BOTH TB AND LEPROSY?

With the exception of *M. habana* (60, 99), the majority of vaccines evaluated for both leprosy and TB were initially designed as TB vaccines, and only evaluated at a later stage for their potential in leprosy. Since *M. leprae* has undergone massive gene reduction (100), not all *Mtb* antigens that are potential targets for TB vaccines have corresponding homologs in *M. leprae*. The first examples are ID83/GLA-SE and ID93/ GLA-SE, two recombinant fusion proteins, formulated with the TLR4L-containing adjuvant GLA-SE, and consisting of three *Mtb* proteins: Rv1813, Rv2608, and Rv3620, with the further addition of Rv3619 in ID93. The amino acid (aa) sequences of Rv3619 and Rv3620 are 58 and 64% identical to the respective *M. leprae* proteins (ML1056 and ML1055, respectively) (**Table 1**). Likely due to these similarities, both *Mtb* hybrid recombinant proteins were also recognized by blood from paucibacillary leprosy patients, although latent *Mtb* infection could have explained these findings as well. Furthermore, when injected subcutaneously these vaccines reduced *M. leprae*induced inflammation and bacterial growth in mouse models of leprosy (65), suggesting that TB subunit vaccines might have efficacy also against leprosy.

In a similar approach, we have investigated another TB subunit vaccine candidate, consisting of two major secreted *Mtb* proteins: *Mtb* ESAT6 and *Mtb* Ag85B, both present in shortterm *Mtb* culture filtrates (101, 102). Ag85B is highly conserved among mycobacterial species, probably due to its critical role in cell wall synthesis as a mycolyltransferase (103). ESAT6 is a secreted virulence protein mainly restricted to the *Mtb* complex organisms (104). Both antigens have been extensively studied in the TB field over the past three decades and proved to be strongly recognized by CD4 Th1-cells of TB patients and latently TB infected (LTBI) individuals (105). Demonstrated to be immunodominant during *Mtb* infection, the two recombinant

Figure 4 | Determination of bacterial burden. C57BL/6j mice were injected with 104 live *Mycobacterium leprae* (121) (viability: 11,000; in 40 µl PBS) in each hind foot pads 4 weeks after the final protein immunization. 7 months after *M. leprae* challenge, mouse footpads were harvested, and *M. leprae* were enumerated by RLEP PCR (122). HLA-A2tg mice were infected with live *Mycobacterium tuberculosis (Mtb)* strain H37Rv 6 weeks after the final protein immunization and 10 weeks after Bacille Calmette–Guérin (BCG) immunization (119). All animals included in the experiments were observed daily in order to ensure ethics requirements and to monitor any adverse effects possibly related to the vaccination or infection. (A) Bacteria were determined by the RLEP PCR from footpads from *M. leprae* infected C57BL/6j mice that had been immunized with GLA-SE adjuvant alone (−), *M. leprae* Ag85-ESAT/GLA-SE, *Mtb* Ag85-ESAT/GLA-SE, or heat killed *M. leprae* (HKML; 2 × 108 in 40 µl; viability: 6,400) as indicated on the *x*-axis. Each symbol represents one mouse. Calculated bacterial loads are expressed as RLEP counts on the *y*-axis. Horizontal lines indicate median values with interquartile range. (B) CFUs were determined in lung homogenates from *Mtb*-infected unimmunized (−) or *Mtb*-infected HLA-A2 tg mice that were immunized with BCG1331 (106 CFU), *M. leprae* Ag85-ESAT or *Mtb* Ag85-ESAT as indicated under the *x*-axis. Each symbol represents one mouse. Bacterial loads are expressed as log10 bacterial counts. Horizontal lines indicate median values with interquartile range. CFU of test and control groups were compared to the controls using the Mann–Whitney test and a *p* < 0.01 was considered significant. \* marks differences that remained significant after multiple test correction using Kruskal–Wallis testing with Dunn's post-test.

proteins were fused into a recombinant hybrid protein, and adjuvanted with the Th1 inducing synthetic adjuvant IC31®. In several animal models, including mice, guinea pigs, and non-human primates, Ag85B-ESAT6/IC31 showed promising protective efficacy against TB disease (106, 107). Based on these results, the vaccine was progressed to human phase 1/2a trials (105, 108, 109). This work demonstrated the vaccine's safety and its remarkable ability to induce long-lasting Th1-type immune reactivity in healthy or HIV-negative, mycobacterially naive individuals, LTBI, and BCG-vaccinated volunteers (109–111) even 3 years after the second vaccination.

In view of several characteristics, Ag85B-ESAT6 is an interesting candidate for leprosy as well. *Mtb* ESAT6 and Ag85B share 68 and 89% aa overlaps (homology according to pre-computed Tuberculist Blastp) with *M. leprae* homologs, ML0049, and ML2028, respectively (**Table 1**). These proteins are widely recognized by antibodies of multibacillary leprosy patients (112, 113), as well as by IFN-γ secreting cells from paucibacillary leprosy patients (64). We previously demonstrated T-cell cross-reactivity between *Mtb* and *M. leprae* ESAT6 in leprosy and TB patients (114). Moreover, a previous study showed that Ag85B overexpression in BCG significantly increased BCG's protective efficacy against *M. leprae* (115). To further explore and compare the efficacy of Ag85B-ESAT6-based vaccines against TB and leprosy, we generated both *Mtb* Ag85B-ESAT6 and *M. leprae* Ag85B-ESAT6 and studied their *in vivo* efficacy in mouse models of *Mtb* and *M. leprae* infection.

### *Mtb* and *M. leprae* Ag85B-ESAT6-Based Vaccines: A Comparative Evaluation

In order to evaluate the immunogenicity of *Mtb*-Ag85B-ESAT6 and *M. leprae*-Ag85B-ESAT6, both hybrid recombinant proteins were produced (116) and injected subcutaneously in wild-type C57BL/6j (BL/6) mice, as well as in C57BL/6j (BL/6) mice expressing an HLA-A\*0201 transgene. The proteins were formulated with GLA-SE (TLR4 agonist) or CpG (TLR9 agonist), respectively, both of which have been reported to drive Th1-type responses. As expected, we detected high levels of IFN-γ released by splenocytes from immunized mice in response to *Mtb* Ag85B-ESAT6, *M. leprae* Ag85B-ESAT6, and their individual components (**Figure 2**). Total IgG, IgA, and IgM levels against *Mtb* Ag85B-ESAT6, *M. leprae* Ag85B-ESAT6, and the individual proteins were increased as well in both mouse strains following immunizations (**Figure 3**). Interestingly, the highest antibody titers were observed against *Mtb* Ag85B-ESAT6, regardless of whether *Mtb* Ag85B-ESAT6 or *M. leprae* Ag85B-ESAT6 had been used to immunize BL/6 mice (**Figure 3A**). Most importantly, both *Mtb* Ag85B-ESAT6/ GLA-SE or *M. leprae* Ag85B-ESAT6/GLA-SE vaccines were capable of inducing host control of *Mtb* and *M. leprae* infection to a significant and comparable extent. Interestingly, *Mtb* Ag85B-ESAT6/GLA-SE controlled *M. leprae* infection significantly better than *M. leprae* Ag85B-ESAT6/GLA-SE (**Figure 4**). In summary, these results suggest that novel subunit vaccines designed for TB, such as *Mtb* Ag85B-ESAT6 could have efficacy against both TB and leprosy.

## CONCLUDING REMARKS

Leprosy and TB are still major poverty-related health concerns. Leprosy is primarily endemic in geographic areas, where TB is also highly prevalent (115). To date, BCG has been used predominantly as a vaccine against TB, but it also contributes to the control of leprosy. However, due to its limited efficacy especially against pulmonary TB in adults, the main and contagious form of TB, novel vaccines are being developed to replace or boost BCG (**Figure 1**). Although these vaccines will likely also impact leprosy incidence, this issue is rarely considered, let alone studied in extensive trials.

There are two leprosy vaccine candidates, MIP in India (82) and LepVax (66), and the TB vaccine pipeline is much more advanced and diverse than the one for leprosy. Even though it is likely that a TB vaccine candidate will emerge, for none of the current TB candidate vaccines, the impact on leprosy is currently being taken into account.

Only two highly similar recombinant subunit TB vaccines, based on the same backbone design, have been tested for their potential use against leprosy (65). Here, we describe original data showing a second TB subunit candidate vaccine platform, based on Ag85B/ESAT6. Collectively, our data suggest that novel TB vaccine candidates can cross-protect against leprosy, providing support for integrating leprosy vaccine research with TB vaccine research (65, 81, 115). At the moment, the most advanced new TB vaccine candidates have been tested in India, Tanzania, China, South Africa, the first two of which have elevated incidences of leprosy. Thus far, none of these recent trials have included evaluation of impact on leprosy, unlike what was done decades ago for BCG(61). We contend that preclinical integration and harmonization of TB/leprosy discovery and development research would well be feasible with respect to the design of subunit vaccines, as we have in fact applied in our recent approach for vaccine antigen discovery (123). With respect to antigen selection algorithms, it is of interest to consider the extensive genomic reduction that *M. leprae* has undergone during evolution (100, 124), causing this *Mycobacterium* to become a highly specialized and obligate intracellular pathogen (125). Studying *M. leprae*'s successful minimalistic approach will reveal genetic and metabolic pathways that pathogenic mycobacteria need to survive in the host, and inspire drug and vaccine efforts to combat both diseases which have put such a heavy toll on humans for millennia.

## ETHICS STATEMENT

The handling of mice was conducted in accordance with the regulations set forward by the animal care committee of the LUMC and in compliance with European Community Directive 86/609 for the care and use of laboratory animals. The experiments at NHDP were performed under a scientific protocol reviewed and approved by the NHDP Institutional Animal Care and Use Committee (Assurance #A3032-01) and were conducted in accordance with all state and federal laws in adherence with PHS policy and as outlined in The Guide for the Care and Use of Laboratory Animals, Eighth Edition.

## AUTHOR CONTRIBUTIONS

Concept of the review: AG and TO. Designed and wrote the review: MC, TO, and AG. Figures and legends: SE, KF, NR, LW, and LA. Final approval of the version to be published: all authors.

#### ACKNOWLEDGMENTS

We want to thank Peter Andersen (SSI, Copenhagen, Denmark) for helpful discussions at the initiation of this project and IDRI (Seattle, WA, USA) for providing GLA-SE. This study was supported by the Q. M. Gastmann-Wichers Foundation, the Netherlands Leprosy

#### REFERENCES

Relief Foundation (ILEP#: 702.02.65), the Order of Malta-Grantsfor-Leprosy-Research (MALTALEP), the American Leprosy Mission, Heiser Program for Research in Leprosy in The New York Community Trust P13-000392, Top Institute Pharma (project D-101-1), ISA Pharmaceuticals, the European Commission [FP7 NEWTBVAC project contract no. LSHP-CT-2003-503367, EC ITN FP7 VACTRAIN project; EC HORIZON2020 TBVAC2020 (Grant Agreement No. 643381)]. The text represents the authors' views and does not necessarily represent a position of the Commission who will not be liable for the use made of such information. Intramural support from the National Hansen's Disease programs was provided for animal studies.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Coppola, van den Eeden, Robbins, Wilson, Franken, Adams, Gillis, Ottenhoff and Geluk. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# mirnome expression analysis reveals new Players on leprosy immune Physiopathology

*Claudio Guedes Salgado1 \*† , Pablo Pinto2,3†, Raquel Carvalho Bouth1 , Angélica Rita Gobbo1 , Ana Caroline Cunha Messias1 , Tatiana Vinasco Sandoval 2 , André Mauricio Ribeiro dos Santos2 , Fabiano Cordeiro Moreira3 , Amanda Ferreira Vidal 2 , Luiz Ricardo Goulart 4 , Josafá Gonçalves Barreto1,5, Moisés Batista da Silva1 , Marco Andrey Cipriani Frade6 , John Stewart Spencer 7 , Sidney Santos 2,3 and Ândrea Ribeiro-dos-Santos 2,3\**

*1 Laboratório de Dermato-Imunologia, Instituto de Ciências Biológicas (ICB), Universidade Federal do Pará (UFPA), Marituba, Brazil, 2 Laboratório de Genética Humana e Médica, ICB, UFPA, Belém, Brazil, 3Núcleo de Pesquisas em Oncologia (NPO), UFPA, Belém, Brazil, 4 Laboratório de Nanobiotecnologia, Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia (UFU), Uberlândia, Brazil, 5 Laboratório de Epidemiologia Espacial (LabEE), Campus Castanhal, UFPA, Belém, Brazil, 6Divisão de Dermatologia, Departamento de Clínica Médica da Faculdade de Medicina de Ribeirão Preto, USP, Ribeirão Preto, Brazil, 7Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO, United States*

#### *Edited by:*

*Vânia Nieto Brito De Souza, Instituto Lauro de Souza Lima, Brazil*

#### *Reviewed by:*

*Rosane M. B. Teles, University of California, Los Angeles, United States Maryam Dadar, Razi Vaccine and Serum Research Institute, Iran*

#### *\*Correspondence:*

*Claudio Guedes Salgado<EMAIL_ADDRESS>Ândrea Ribeiro-dos-Santos<EMAIL_ADDRESS>
*† These authors have contributed equally to this work.*

#### *Specialty section:*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology*

*Received: 28 November 2017 Accepted: 21 February 2018 Published: 09 March 2018*

#### *Citation:*

*Salgado CG, Pinto P, Bouth RC, Gobbo AR, Messias ACC, Sandoval TV, dos Santos AMR, Moreira FC, Vidal AF, Goulart LR, Barreto JG, da Silva MB, Frade MAC, Spencer JS, Santos S and Ribeiro-dos-Santos (2018) miRNome Expression Analysis Reveals New Players on Leprosy Immune Physiopathology. Front. Immunol. 9:463. doi: 10.3389/fimmu.2018.00463*

Leprosy remains as a public health problem and its physiopathology is still not fully understood. MicroRNAs (miRNA) are small RNA non-coding that can interfere with mRNA to regulate gene expression. A few studies using DNA chip microarrays have explored the expression of miRNA in leprosy patients using a predetermined set of genes as targets, providing interesting findings regarding the regulation of immune genes. However, using a predetermined set of genes restricted the possibility of finding new miRNAs that might be involved in different mechanisms of disease. Thus, we examined the miRNome of tuberculoid (TT) and lepromatous (LL) patients using both blood and lesional biopsies from classical leprosy patients (LP) who visited the Dr. Marcello Candia Reference Unit in Sanitary Dermatology in the State of Pará and compared them with healthy subjects. Using a set of tools to correlate significantly differentially expressed miRNAs with their gene targets, we identified possible interactions and networks of miRNAs that might be involved in leprosy immunophysiopathology. Using this approach, we showed that the leprosy miRNA profile in blood is distinct from that in lesional skin as well as that four main groups of genes are the targets of leprosy miRNA: (1) recognition and phagocytosis, with activation of immune effector cells, where the immunosuppressant profile of LL and immunoresponsive profile of TT are clearly affected by miRNA expression; (2) apoptosis, with supportive data for an antiapoptotic leprosy profile based on *BCL2*, *MCL1*, and *CASP8* expression; (3) Schwann cells (SCs), demyelination and epithelial–mesenchymal transition (EMT), supporting a role for different developmental or differentiation gene families, such as Sox, Zeb, and Hox; and (4) loss of sensation and neuropathic pain, revealing that *RHOA*, *ROCK1*, *SIGMAR1*, and aquaporin-1 (*AQP1*) may be involved in the loss of sensation or leprosy pain, indicating possible new therapeutic targets. Additionally, *AQP1* may also be involved in skin dryness and loss of elasticity, which are well known signs of leprosy but with unrecognized physiopathology. In sum, miRNA expression reveals new aspects of leprosy immunophysiopathology, especially on the regulation of the immune system, apoptosis, SC demyelination, EMT, and neuropathic pain.

Keywords: leprosy, immunology, Schwann cells, apoptosis, neuropathic pain, microRNA, miRNome, epigenetics

## INTRODUCTION

Leprosy is an ancient disease caused by *Mycobacterium leprae*, an obligate intracellular pathogen that infects macrophages and Schwann cells (SCs), resulting in nerve and skin lesions with loss of sensation, the hallmark of the disease (1).

After contact with the bacilli, most people control *M. leprae* multiplication and will never develop leprosy (2). If the bacilli survive, the host may develop two stable polar forms of disease, the paucibacillary (PB) tuberculoid (TT) form or the multibacillary (MB) lepromatous (LL) form, besides the three borderline intermediate unstable forms, borderline TT, borderline borderline, and borderline LL. PB patients have a good cellular immune response (CIR) that may restrict bacillus proliferation, resulting in a few lesions that are usually limited to a specific part of the tegument and to a few nerve trunks. MB patients, on the other hand, have a poor CIR with an exacerbated humoral immune response that is not effective for controlling bacillus proliferation. Patients have many lesions disseminated through the body, including the skin and peripheral nerves (3).

The natural history of the disease results in disabilities. Demyelination caused by SC degeneration is one of the main events in leprosy physiopathology, together with exacerbations of the immune response, known as leprosy reactions. While patients evolve a loss of sensation on the skin, they may also have peripheral nerve neuropathic pain that can be exacerbated by the reactions and may last for many years, even after multidrug therapy (MDT) (4).

Genetic studies of portions of the genome that do not encode protein revealed one class of small non-coding RNAs [named microRNAs (miRNAs)] that are involved in posttranscriptional control of gene expression (5). Knowledge about the interaction between miRNA and leprosy is limited (6–12). A recent study demonstrated that a miRNA can influence the mechanism whereby the cell host can prevent bacillus growth and generate natural barriers against infection by *M. leprae* (9). Evidence has shown that miRNAs are able to modulate host antibacterial pathways during the infection process and influence the outcome of disease (9). Analysis of miRNAs that are differentially expressed in distinct poles of the disease could provide a better understanding of targets for an efficient immune response to prevent infection, as well as elucidate novel possible biomarkers for leprosy, for example, subclinical infection and one possible predictor of who will develop leprosy (13, 14).

Upon contact with *M. leprae*, the human immune system must recognize and process the bacteria to activate immune effector cells. During the interaction, the host cells may be induced to undergo apoptosis, hindering bacillus adaptation or maintaining its survival with attenuated microbicide capacity, to shelter the bacilli (15). Demyelination is a key event in leprosy, and SC are critical for myelin production and maintenance on peripheral nerves. Epithelial–mesenchymal transition (EMT) is a biological process in which specialized cells may undergo a phenotypic change to mesenchymal cells, with higher motility, greater resistance to apoptosis, induction of fibrosis, loss of markers for specialized cells, and the acquisition of new proliferation markers (16). Mechanisms responsible for the loss of sensation and neuropathic pain are poorly understood.

Our work presents the first leprosy miRNome from lesions and blood of LP. In addition to describing the miRNAs, we chose those with significant differential expression, searched for their target genes, and constructed possible pathways based on current knowledge of leprosy immune pathophysiology.

## MATERIALS AND METHODS

### Study Design and Participants

A total of 28 biological samples from leprosy patients (LP) before starting MDT treatment who attended the Dr. Marcello Candia Reference Unit in Sanitary Dermatology of the State of Pará (UREMC) on 2014, in Marituba, Pará, Brazil, and individuals without leprosy and with no other diseases [healthy subjects (HS)] were included in the present study, according to the following groups: (a) 17 tissue biopsies samples [11 from LP (6 LL and 5 TT) and 6 skin tissue from HS for controls] and (b) 11 peripheral blood samples [9 from LP (5 LL and 4 TT) and 2 from HS for controls]. **Table 1** describes gender, age, bacterial index, anti-PGL-I optical density, and disability grading of the 11 LP selected for the study.

This study adhered to the Declaration of Helsinki and was approved by the Institute of Health Sciences Research Ethics Committee at Universidade Federal do Pará, certified by CAAE 26765414.0.0000.0018. A written informed consent to publish was obtained from every individual who accepted to participate in this study. The small RNAseq number register is ERP105473 on European Nucleotide Archive database.

#### Total RNA Storage, Extraction, and Quantification

A flowchart (**Figure 1**) presents all the steps performed during miRNA Seq (extraction, library, sequencing data processing and analysis pipeline, target gene identification). The whole peripheral blood samples were collected into a Tempus Blood RNA Tube (Thermo Fisher Scientific, USA) and stored at −20°C until extraction. The skin tissue biopsy samples were collected in a propylene tube with 2 mL RNAlater (Thermo Fisher Scientific, USA) and stored in liquid nitrogen until use. Total RNA was extracted

*a The ID is composed of the clinical form followed by a sequential number. bOptical density of ELISA. NR, not realized.*

Frontiers in Immunology | www.frontiersin.org March 2018 | Volume 9 | Article 463

from the tissue sample using TRIzol reagent (Invitrogen, USA), and samples were eluted in DEPC water and stored in liquid nitrogen. Total RNA was extracted from blood samples using the MagMAX RNA Isolation Kit (Thermo Fisher Scientific, USA). Total RNA quantity and quality assessed were performed with a NanoDrop 1000 (Thermo Scientific, USA) and Agilent 2200 TapeStation (Agilent Technologies, USA).

## Library Preparation and Next-Generation Sequencing (NGS)

The library was prepared using the TruSeq Small RNA Library Preparation Kit (Illumina, Inc., USA) according to the manufacturer's instructions, and all samples used for the library had an initial concentration of 1 μg/5 μL of total RNA. The library was validated and quantified with an Agilent 2200 TapeStation (Agilent Technologies, USA) platform and by real time PCR with the KAPA Library Quantification Kit (KAPA BIOSYSTEM, USA). The libraries were then diluted to a concentration of 4 nM and sequenced using the MiSeq Reagent Kit v3 150 cycle (Illumina, Inc., USA) on a MiSeq System (Illumina, Inc., USA). The tissue and blood samples were sequenced separately.

## Sequencing Data Processing and Analysis-Small RNA-Seq Pipeline

The sequencing data were processed on an Illumina MiSeq reporter and extracted in FASTQ format. A pipeline of pre-processing using the Fastx\_toolkit was applied for a low filter quality, trimers of extreme 3′ reads and contaminant removal. The pipeline was performed according the chronogram: (a) average Phred quality score (*Q*) greater than 30, (b) reads more than 17 nucleotides long, and (c) base calling error probabilities (*P*) greater than 80. Next, read alignment with the human genome (GRCh37) in combination with the miRNA data base (MirBase v.19) was performed using Spliced Transcripts Alignment to a Reference. The miRNA was scored with htseq-count toll, and the results were normalized and analyzed using the Bioconductor-*DESeq2* package with R statistical software. Thus, the following comparisons were conducted: (a) LP vs. HS; (b) TT leprosy vs. HS; (c) LL leprosy vs. HS; (d) TT vs. LL leprosy. Adjusted values of *p* ≤ 0.05 and a log2 fold change >2 were considered statistically significant.

## Target Gene Identification

The genes regulated by the differentially expressed miRNAs detected during the analysis were identified using four tools:(i) TargetCompare (http://lghm.ufpa.br/ferramentas-de-estudos/ targetcompare/) (17); (ii) miRTarBase (http://mirtarbase.mbc. nctu.edu.tw); (iii) DIANA miRPath v.3 (18); and (iv) TargetScan (19). We selected only genes regulated by two or more miRNAs with strong experimental evidences support, such as those confirmed by western blotting, reporter assay or qPCR.

Selected targets were further investigated using the pathway enrichment tool DAVID 6.8 (20) that provides a comprehensive set of functional annotation tools and searches in BioCarta and KEGG pathway maps to help investigators to understand biological meaning behind a large list of genes. The pathway enrichment analysis was performed separately for the following groups: (i) HS vs. LP downregulated miRNAs in tissue; (ii) HS vs. LL downregulated miRNAs in tissue; (iii) HS vs. LL upregulated miRNAs in tissue; and (iv) HS vs. LP downregulated miRNAs in blood.

## RESULTS

This study evaluated two types of leprosy samples, skin biopsies and blood by distinct NGS. For the two sample types, the differential expression profiles of the miRNAs were analyzed to identify possible leprosy biomarkers to assist our understanding of epigenetic control mechanisms of the immune response, apoptosis, SC demyelination, EMT, and neuropathic pain.

## miRNA Sequencing and Differential Expression Profiles of Tissue Samples

This sequencing yielded 4 million reads. After the process pipeline, more than 96% of the reads were aligned with the human genome, and the miRNA count was performed using *htseq-count* (miRNA count ≥ 10), with an average of 36,745 reads per sample and 656 miRNAs expressed in at least one sample.

A heatmap was constructed using the RPKM (Reads per Kilobases per Million) expression for all differentially expression miRNA (**Figure 2**). The analysis identified the RPKM z-score of 67 differentially expressed miRNAs, 43 downregulated and 24 upregulated (**Table 2**; Data Sheet S1 in Supplementary Material) in skin biopsies among HS, LL, and TT. miRNAs (rows) were hierarchically clustered according their expression, and organized according to the three groups, HS, LL, and TT (columns). A hierarchical clustering of the data illustrates how those markers were able to distinguish HS from leprosy patients in general, and LL from TT poles.

**Figure 3** shows the 39 simultaneous differentially expressed miRNAs, for at least two of the three comparisons conducted

as described in **Table 2** (HS vs. LP, HS vs. TT, and HS vs. LL), organized by their fold change showing 24 downregulated and 15 upregulated miRNAs.

three different samples (HS, LL, and TT). The clustering was able to distinguish HS from leprosy patients, as well as LL from TT.

The comparison of extreme poles of leprosy, TT and LL, revealed five differentially expressed miRNAs, of which three were downregulated (*hsa-miR-340-5p, hsa-miR-34a-5p, hsa-miR-362-5p*) and two were upregulated (*hsa-miR-429, hsa-miR-200a-3p*). The *hsa-miR-362-5p* appeared only when TT and LL were compared, but not when LP were compared to HS. The volcano plot shows the only five miRNAs differentially expressed (**Figure 4**).

Table 2 | Number of miRNAs that were differentially expressed in LP (TT and LL) compared with HS in skin biopsy samples.

*LP, leprosy patients; TT, tuberculoid; LL, lepromatous; HS, health subjects.*

#### miRNA Sequencing and Differential Expression Profiles of Whole Blood Samples

Sequencing yielded 6 million reads. After the process pipeline, more than 95% of the reads were aligned with the human genome, and the miRNAs were counted using *htseq-count* (miRNA count ≥ 10), with an average of 371,325 reads per sample and 527 miRNAs expressed in at least one sample.

The differential expression analysis of blood miRNAs was conducted similarly to that applied for the tissue and revealed a total of 10 differentially expressed miRNAs, with nine downregulated (*hsa-let-7f-5p, hsa-miR-126-3p, hsa-miR-126-5p, hsamiR-144-5p, hsa-miR-15a-5p, hsa-miR-20a-5p, hsa-miR-26b-5p,*

Figure 3 | Upregulated or downregulated microRNAs (miRNAs) in at least two comparisons [tuberculoid (TT) vs. healthy subjects (HS), lepromatous (LL) vs. HS, or leprosy patients (LP) vs. HS] on skin biopsies samples. Among the 67 differentially expressed miRNAs (|log2 fold change| > 2 and adjusted *p*-value < 0.05), here we highlight 39 miRNAs differentially expressed between HS versus LP and HS versus LL or TT patients, each indicated by blue, orange, and gray bars, respectively. The barplot represents each miRNA (*y*-axis) absolute log2 fold change (*x*-axis) for each comparison and separated in up- and downregulated miRNA regarding HS.

represents each miRNA differential expression analysis result where the y-axis is −log10 *p*-value of the comparison and the *x*-axis is log2 fold-change (*x*-axis) regarding LL. Differentially expressed miRNAs were considered only if the analysis showed |log2 fold-change| > 2 (indicated by the blue vertical lines) and adjusted *p*-value < 0.05. We found five differentially expressed miRNA (highlighted in red), two upregulated (*hsa-miR-429, hsa-miR-200a-3p*) and three downregulated (*hsa-miR-340-5p, hsa-miR-34a-5p, hsa-miR-362-5p*).

*hsa-miR-106b-5p, hsa-miR-16-5p*) and one upregulated (*hsa-miR-1291*) (**Table 3**; Data Sheet S2 in Supplementary Material). From the differentially expressed miRNAs identified, a heatmap was constructed using RPKM expression and two clusters were observed, with standards of expression able to differentiate LP of HS (**Figure 5**), although comparisons between TT vs. LL showed no differentially expressed miRNAs.

#### Target Gene Identification

Using the differentially expressed miRNAs from either blood or skin lesions, we investigated the genes regulated by them separately for up and down regulated miRNAs. The miRNAs and their targets are described in **Tables 4**–**8**, as follow: (i) HS vs. LP downregulated (**Table 4**) and upregulated (**Table 5**) miRNAs in skin biopsies; (ii) HS vs. LL downregulated (**Table 6**) and upregulated (**Table 7**) miRNAs in skin biopsies; and (iii) HS vs. LP downregulated miRNAs in blood (**Table 8**).

## DISCUSSION

#### Recognition, Engulfment, and Activation of Immune Effector Cells

The metalloproteinase ADAM9 and the integrin ITGA5 are two transmembrane proteins involved in mycobacteria invasion of macrophages (21, 22) and endothelial cells (23). Phagocytosis of *M. leprae* may be stimulated by IGF1R (24), LRP1 (25), and PIK3CA (26). In blood, with the exception of *hsa-miR1291*, all miRNAs were downregulated when LPs were compared with HS, indicating that the phagocytosis of *M. leprae* in LP blood was not Table 3 | Number of miRNAs that were differentially expressed in LP (TT and LL) compared with HS in blood.

*LP, leprosy patients; TT, tuberculoid leprosy; LL, lepromatous leprosy; HS, health subjects.*

inhibited. Furthermore, miRNAs that control *IGF1R* gene were upregulated in lesional tissue of LL patients in comparison to HS (**Figure 6**), which together with the decrease in *IGF1* gene in LL patients (27) may result in inhibition of the microbicidal function of macrophages against *M. leprae* (15) in tissue.

VAC14 is induced to control vacuolation in macrophages (28), while APP produces the oxidative burst (29) and PIK3CA stimulates *M. leprae* phagocytosis (26). Several miRNAs control the expression of *VAC14* and *PIK3CA* genes in lesional tissue and *APP* gene in blood, all of which were downregulated in LP in comparison to HS (**Figure 6**). When LL patients were compared to HS, only miRNAs that control *VAC14* gene were downregulated, indicating that *VAC14* gene expression was necessary for vacuole formation in LL patients.

Protein kinase C epsilon encoded by the *PRKCE* gene was also found to have downregulated miRNAs in LP. PRKCE is coupled to TLR-4, which is responsible for *M. leprae* recognition (30). Upon phosphorylation, two pathways may be activated: *IRF3* gene upregulation may result in the production of IFN-β and IL-10, especially in LL patients (31), or NFκB upregulation, with

RPKM *z*-score of 10 differentially expressed miRNAs. miRNAs (rows) were hierarchically clustered according their expression on HS or LP (columns). The clustering was able to distinguish LP from HS.

the production of proinflammatory cytokines, such as TNF-α and IL-6, by NFκB activation (30). IFN-γ and TNF-α may disrupt the TGF-β pathway by SMAD7 activation followed by TGFBR1 downregulation (32).

Leprosy patients miRNAs control *TGFBR1*, *SMAD7* gene and the zinc finger transcription factor *KLF11* gene, which regulates *SMAD7* expression. LL patients showed a downregulation of miRNAs that control *KLF11* gene expression, which may result in *SMAD7* gene inhibition and an increase in *TGFBR1* gene, with more TGF-β capture contributing to the immunosuppressive profile of LL. SMAD7 also stimulates CHUK, which inhibits the NFKB1 and COX2 inflammatory pathway. Interestingly, the central inflammatory player, NFKB1*,* was also found to be regulated by miRNAs in LL patient lesions (**Figure 6**). It has been previously demonstrated that TGF-β secretion is augmented in LL patients (33) and is secreted by CD4+ CD25+ FOXP3+ T regulatory cells (34), while TGF-β receptors are also increased in lesions of LL patients (35). Additionally, miRNAs that regulated cytoplasmic protein ERBIN were downregulated in LL patient lesions, indicating that ERBIN may also regulate *TGFBR1* gene pathway expression (**Figure 6**).

NOTCH1/2 have different functions in immune regulation, but overall seem to stimulate the immune system participating in the differentiation of naïve T cells (36) and modulating inflammation (37). An important regulator of M1 macrophage differentiation and the Th1 T cell profile in leprosy, the transmembrane protein NOTCH1 (38) was also found to be regulated by LP miRNAs in lesional tissue of LL patients. Additionally, *NOTCH1 gene*, *hsamiR-34a-5p* also control *NOTCH2* and *JAG1* gene in LL lesions (**Figures 6** and **7**). NOCTH1 is known to be activated by JAG1 on endothelial cells, regulating the differentiation of M1 macrophages (38) in PB leprosy. Both NOTCH1 and NOTCH2 are expressed on Th0 cells and are related to Th17 differentiation (39). Furthermore, NOTCH1/2 are expressed on activated Th1 cells and are critical to the protective response against *Leishmania major* infection by the production of IFN-γ (40), which is also important for leprosy protection by JAG1 stimulation (38).

Activation of TLR4, IL15R, IL1R1, and IL1A is important for antimicrobial activity, a key function for infection control. LL patients were found to have upregulated miRNAs for all those genes in lesional skin (**Figures 6** and **7**). TLR4 and IL15R converge to CYP27B1, which converts 25-hydroxyvitamin D (25D) to the Table 4 | List of the genes targeted by two or more differentially expressed miRNAs among the 26 downregulated miRNAs in LP vs. HS skin biopsies.

*a Target gene with strong evidence only.*

*LP, leprosy patients; HS, health subjects.*

active hormone 1,25 dihydroxyvitamin D (1,25D) and links to the vitamin D receptor, resulting in the expression of antimicrobial peptides (9, 41). *IL1R1* and *IL1A*, and *TLR2* gene were found to have upregulated miRNAs for all LP, which are also involved in pathways culminating in antimicrobial activity (9). Although *has-mir-21* seems to be critical for the control of the *TLR4*, *IL15R*, *IL1R1*, and *IL1A* gene expression in a cell culture-based systems, using our human approach, other miRNAs seemed to be more relevant; however, additional studies are necessary for validation.

TNFAIP3 (A20), which is produced by macrophages infected by *M. tuberculosis*, has been recently described as a new NFκB blocker (42). We found that *hsa-miR-125b* is downregulated only in LL, and *hsa-let-7f-5p* is downregulated in LP (**Figure 7**). Both miRNAs inhibit *A20* gene expression, leading to an increase in NFκB production. According to our LP miRNA profile, *hsamiR-125b* and *hsa-let-7f-5p* expression are decreased and therefore do not block *A20* gene, resulting in NFκB abrogation. This phenomenon may drive macrophages toward a M2 profile, with more *TGFB1* (43), *IL6* (44), and *IL10* gene (45) production (all with downregulated miRNAs, **Figure 7**) that may stimulate

Table 5 | List of the genes that were potentially targeted by two or more differentially expressed miRNAs among the 17 upregulated miRNAs in LP vs. HS skin biopsies.

*a Target gene with strong evidence only.*

*LP, leprosy patients; HS, health subjects.*

Th2 cells to produce more IL-4 (44). Upon ligation, the IL-4 receptor activates STAT6, stimulating the transcription of *miR-1301*, *miR-342*, and *miR155*, which supports M2 by activating *BCL2* gene and promotes Th2 activation (46). All three miRNAs were found to be upregulated in LL patients, confirming their importance in driving LP toward a Th2 profile.

In addition, *IL10 gene*, with miRNA downregulated only in LL patients, *IL13* gene miRNAs were downregulated in the blood of all LP. Together with lesional *IL11*, *ETS1* and *CDC42*, *IL10*, and *IL13* gene induce the differentiation of B cells from plasma cells (47–50). The interaction of CD40 with CD40L results in IL-12 production, which is impaired in LL patients (51). In addition to ICAM1, which has been demonstrated to be inhibited in LL (52), *CD40L gene* miRNAs were upregulated in all LP, and the control of *IL2*, *IL1A*, *IL1R*, *TLR4*, and *NFKB1* gene miRNAs were upregulated in LL (**Figure 7**), These phenomena may lead to increased *IL4*, *IL10*, and *TGFB1* production, blocking *IFNG gene* expression (53–56) and resulting in the impaired CIR observed in LL patients.

Table 6 | List of the genes that were potentially targeted by two or more differentially expressed miRNAs among the 34 specific downregulated miRNAs in LL vs. HS skin biopsies.

*a Target gene with strong evidence only.*

*LL, lepromatous leprosy; HS, health subjects.*

MicroRNAs targeting the CD8+ T cell differentiation gene *ZEB2* gene and activation genes *CADM1* and *CRTAM* gene were found to be downregulated in LP patients (**Figure 7**). The adhesion molecule CADM1, which is expressed on dendritic cells (DCs), induces a CD8+ cytotoxic profile upon ligation to CRTAM Table 7 | List of genes potentially targeted by two or more differentially expressed miRNAs among the 14 specific upregulated miRNAs LL vs. HS skin biopsies.

*Target gene with strong evidence only.*

*a*

*LL, lepromatous leprosy; HS, health subjects.*

T-cells, with the release of IL2 and IFNG (57, 58). Although *IFNG* gene miRNAs were downregulated, *IL2 gene* miRNAs were upregulated in LL patients, suggesting a posttranscriptional blocking of *IL2* released from CD8+ T cells in LP.

Considering Th1, Th2, and Th17, we detected miRNAs controlling all three axes in LP. On the Th1 axis, although *IL12A* and *IFNG* gene miRNAs were downregulated in blood, *IL2 gene*, a key cytokine for Th1 proliferation (59), together with *IL1A*, *IL1R1*, *IL15R*, *TLR4*, and *NFKB gene*, were all found to be regulated by miRNAs in the lesional skin of LL patients. For Th2, *IL6*, and *IL10 gene*, together with the miRNAs *hsamiR-125b*, *miR-1301*, *miR-342*, and *miR-155*, were all regulated by miRNAs, maintaining a suppressive profile in LL patients. Th17 differentiation is influenced by different factors. We found that *SHC1 gene*, which activates *STAT3 gene*, and *IL6* gene that can induce Th17 differentiation in association with *TGFB1gene* (60), both had downregulated miRNAs in LL patients, together with upregulation of the miRNA *hsa-miR-326*, which was described as a Th17 inductor. Although Th17 is known to produce IL8 (61), we found miRNAs controlling the expression of this chemokine in lesions of LL patients (**Figure 7**), corroborating the absence of this chemokine in polymorphonuclear cells of LL patient unstimulated blood (62).

*a Target gene with strong evidence only.*

*LP, leprosy patients; HS, health subjects.*

#### Apoptosis

The role of apoptosis during *M. leprae* infection is not clear, and different research settings have demonstrated both anti- (24, 63) and proapoptotic (64, 65) features, in addition to possible differences depending on the clinical form of leprosy (66). BCL2 has been shown to be highly expressed in LL patients (66), and *BCL2* and *MCL1 gene* are induced by *M. leprae* on monocytes (63), while CASP8 activity in LL is decreased (67). We found several miRNAs controlling apoptosis pathways in LP. In addition to the downregulation of all miRNAs acting directly on the antiapoptotic gene *BCL2* and its family member *MCL1*, especially in LL lesions, the proapoptotic *gene CASP8* inducers *MYC*, *FAS,* and *FADD gene* were found to have upregulated miRNAs in LP, while the *FASLG* inhibitor *GRB2* (68) presented downregulated miRNAs in LL patients (**Figure 8**).

AKT1 and PIK3CA kinases appeared to be central molecules involved in the apoptosis pathway related to *M. leprae*. In contrast, *PIK3CA* miRNAs were downregulated in all forms of leprosy in comparison to HS, and *AKT1* miRNAs was more prominent in LL patient lesions (**Figure 8**). Interestingly, PHLPP1, a serine-threonine family member that has never described in leprosy and that controls AKT1 (69), was found to have downregulated miRNAs in LL lesions, indicating that *PHLPP1 gene* may have a role in apoptosis control in LP. Furthermore, AKT1 blocks the expression of the proapoptotic *genes YAP1* and *FOX03*, while stimulate the antiapoptotic *gene MDM4*. YAP1 has a proapoptotic function after DNA damage of tumor cells (70), and FOXO3 blocks BCL2 (71). In addition to being inhibited by AKT1, *FOXO3 gene* was found to have upregulated miRNAs in all LP cases (**Figure 8**). Upon *FOXO3* blockade, *RBL1/2* are transcribed and indirectly block *TP53* expression by blocking *E2F1* (72). MDM4, P53 regulator is also stimulated by AKT1. miRNAs found in our miRNome regulate *FOXO3* and *E2F1* in all forms of leprosy, while miRNAs for *YAP1*, *MDM4*, and *TP53* were found exclusively in LL. Taken together, these findings show that the influence of LP miRNAs of those pathways in leprosy may result in an antiapoptotic profile.

Considering the miRNA profile controlling cell receptors, in addition to *FAS gene*, we found five other genes related to apoptosis control by miRNA in *M. leprae* infection: *TGFBR1*, involved in TGF-β signaling, a cytokine known to induce tolerance (73, 74) with a suppressive potential of Tregs in LL patients (75); *ITGA5*, an α-integrin linked to *M. tuberculosis* infection of macrophages (22); *VEGFR2*, known to participate in *M. tuberculosis* dissemination by triggering angiogenesis (76), while its ligand, *VEGFA*, has been demonstrated to be expressed in leprosy lesions (77); *PDGFRA*, which was shown to be upregulated in SC 27 days after *M. leprae* infection (78), and its ligand *PDGF,* a potential marker for erythema nodosum leprosum (79); and *IGF1R*, the receptor of *IGF1*, which inhibits macrophage and SC apoptosis upon *M. leprae* infection, in turn stimulating the production and secretion of *IGF1* (15).

In contrast to downregulated miRNAs controlling *PIK3CA*, *AKT1*, *BCL2*, and *MCL1*, miRNAs controlling the expression of the proapoptotic genes *MYC*, *E2F1*, and *FOXO3* were all found to be upregulated in lesional tissue. Additionally, miRNAs for *FAS* and *FADD*, members of the *CASP8* proapoptotic pathway, were also found to be upregulated (**Figure 8**). Taken together, concerning miRNA regulation, our data suggest an antiapoptotic profile for leprosy in general, driven by *BCL2*, *MCL1*, and *CASP8*.

#### SCs, Demyelination, and EMT

Demyelination is a pathologic process that destroys the myelin sheath and involves multiple factors, including inflammatory responses or infections (80). LP demyelination is the ultimate consequence of leprosy neuritis, and LL patients exhibit myelinated and non-myelinated SC infected by *M. leprae* (81).

Upon invasion, *M. leprae* stimulates *ERBB2* independently of *ERBB3* (82), resulting in *ERK1/2* activation, which leads to peripheral nerves demyelination (83). *ERBB2* miRNAs were downregulated in LL patients (**Figure 9**), indicating a possible role for the *SOX2* and *JUN* pathway in demyelination and EMT (78). However, *SOX2* miRNAs were found to be upregulated in all LP, while *ZEB1/2* miRNAs were downregulated in LL patients (**Figure 9**), indicating that ZEB1/2 may regulate SOX2 (84)

expression to inhibit demyelination and EMT. Although *ZEB1* expression increases after *M. leprae* infection or after TGF-β stimulation (85), while ZEB2 is essential after nerve injury by allowing remyelination and functional recovery (86), they may be regulated by ERBIN, which blocks the TGFBR1 pathway (87), and the downregulated miRNAs were observed only in LL patients (**Figure 9**).

Schwann cell lesions induce the antiapoptotic molecule *AKT1* and the mitogen *MET*, both of which are regulated by miRNAs in LL patients. AKT1 signaling, one of the most important pathways involved in myelination (88), may be activated in injured peripheral nerves (89). We found that miRNAs for *AKT1* were downregulated in LL patients, indicating a possible role for *AKT1* in LP myelination (**Figure 9**). In parallel, MET, an important inducer of neural injury (90) and the *KRAS*, *RAF1*, and *MAPK14 genes* involved in EMT pathways (91) were found to have miRNAs in LL patient lesions. In contrast, *MAPK14* and *RAF1 genes* had downregulated miRNAs, and *MET* and *KRAS genes* had miRNAs upregulated, indicating a strict control of the first steps of the pathway. *MET* and *KRAS* also regulate cell motility through RAC1 and CDC42, which are important for actin rearrangement and cellular polarity (92).

Very few works have described the effects of *HOX* and *SNAI genes* on leprosy pathophysiology. It has been demonstrated that upon *M. leprae* infection, SC may switch off differentiation genes, such as *SOX10* and *p75ntr*, while switching on EMT genes, especially the *HOX* family (78) and *CD44*, which are considered a marker for EMT in SC (78), with downregulated miRNAs in our LP skin lesion samples. Our study did not find miRNAs regulating *p75ntr*, which is compatible with the switching off demonstrated in the previous work; however, we detected upregulated miRNAs for *SOX10* only in LL, downregulated miRNAs for *SNAI2* and *HOXB5*, with the latter only in LL patient lesions, and upregulated miRNAs for *HOXB7*, *HOXB8*, and *HOXC8* in all LP (**Figure 9**). Interestingly, the only genes that were detected in the earlier *in vitro* work and that for which we also detected miRNAs were *SNAI2* and *HOXB8*. miRNAs for *HOXB5*, *HOXB7*, and *HOXC8* were found in our work, but their expression was not detected previously.

*HOXB5 gene* is a marker for long-term hematopoietic stem cells (93) and it affects the differentiation of the vascular endothelium development from precursor cells (94). It is known that endothelial cells are important for the entry and maintenance of *M. leprae* in nerves (95) and that vasculitis may be observed in leprosy reactions, with endothelial proliferation in Lucio's phenomena (1). *HOXB7* is associated with EMT in breast cancer cells *via* the canonical TGF-β pathway (96), while *HOXC8* mutant mice present motoneuron abnormalities with analog molecular defects compared with mutant mice for retinaldehyde dehydrogenase 2 synthesizing enzyme (97), which is responsible for retinoic acid synthesis, with atrophy of the distal projections of the ramus profundus of the radialis nerve that supply the extensor muscles of the forepaw, resulting in forepaw neuromuscular defects. Vitamin A levels in LP have been shown

to be low in comparison to healthy controls, and much lower in LL patients (98).

*SNAI2* and *HOXB8* have been demonstrated to be upregulated in SC infected with *M. leprae in vitro* (78). SNAI2 is activated by the canonical TGF-β pathway (99), inhibiting CDH1 (E-cadherin) and resulting in cellular EMT (100). We found that upregulated miRNAs controlled *SMAD2* and *SMAD3* expression in LP. *SMAD3* miRNAs were significantly upregulated only in LL patients, possibly participating in the control of SC-EMT (**Figure 9**). *HOXB8* null mutants show altered sensory responses in mice, probably due to a smaller number of neurons and neural disorganization (101), indicating that *HOXB8* expression levels may be related to sensory alterations in LP.

## Loss of Sensation and Neuropathic Pain in Leprosy

ROCK1 is a Rho-associated protein kinase that is present in different signaling pathways in neurons (102), is known to regulate SC myelination (103), and may be activated by *RHOA gene* (104), for which we found downregulated miRNAs in LL lesions. *ROCK1 gene* stimulated by TGFBR1 induced EMT of SC *via* the MAPK14 pathway, which leads to SOX9 activation and SC EMT associated with the blockade of SOX10 (**Figure 9**), a recognized inducer of cell differentiation (105). We observed upregulated miRNAs for *SOX10 gene* in LL lesions, indicating that it may be expressed in LL patients. Myelin-associated inhibitors upon ligation with p75ntr activate *RHOA*, resulting in demyelination through *ROCK1 genes* (106), while *ROCK1* stimulated by *BDNF* through p75ntr and *RHOA* may lead to inflammation and pain (107).

LPAR1 signaling is required to initiate neuropathic pain after nerve injury. Mice lacking *LPAR1 gene* do not present signs of neuropathic pain, and inhibition of *RHOA* and *ROCK1* also prevent neuropathic pain (108). We found that miRNAs for *LPAR1* were downregulated in LL patients in both blood and lesions, indicating that the receptor may be available for ligation in LP. *RHOA* miRNAs were also downregulated in LL patients, while *ROCK1* was upregulated in all LP, indicating an attempt to control EMT, demyelination and pain.

The miRNA *hsa-miR-1291* was the only differentially expressed miRNA in both skin tissue and blood samples. It was predicted to regulate the aquaporin-1 (*AQP1*) gene (109), which influences the hydration, elasticity and glycerol permeability of skin (110). In LL lesions, frequent overexpression of lipid metabolism genes (111) indicates that *M. leprae* uses host lipids for growth and virulence. Therefore, downregulation of *hsa-miR-1291* in skin lesions could modulate *AQP1* expression and increase glycerol permeability to promote fatty acid metabolism. Altered *AQP1*

expression may improve our comprehension of some well-known clinical issues related to leprosy, such as the dryness found in skin lesions. Moreover, *MEF2C*, a transcription factor for *AQP1* (112), was found to have upregulated miRNAs in the skin lesions of LL patients, which could result in an absence of *AQP1* transcription in LL patients, contributing to loss of sensation.

Aquaporins may be key molecules in leprosy pathophysiology. *AQP1* knockout mice have impaired pain sensation (113), and human trigeminal neurons that mediate head nociception and innervate the oral mucosa express *AQP1*, indicating an involvement in sensory transduction (114). Peripheral nerve system expression of *AQP1* has been seldom investigated, but it has been shown in the sciatic nerve (115) and in Ruffini mechanoreceptors (116). *AQP4* is expressed in the olfactory epithelium (117) and in retinal glia (118), and it is the target of anti-AQP4 antibodies in autoimmune neuromyelitis optica (119). Loss of sensation is the hallmark of leprosy, but there is no definite mechanism explaining this phenomenon.

*AQP1* participates in the mechanism of thermic and chemical pain, likely controlling neuronal ionic nociceptive homeostasis (113). Membrane depolarization activates *ATP1A1*, which regulates sodium potassium channels, and *TRPV1*, which is responsible for calcium influx into the cell. Calcium acts on calcitonin gene-related peptide (CGRP), which together with substance P (SP) results in pain (113). In addition to CGRP, calcium also stimulates *SIGMAR1* (120), for which we detected downregulated miRNAs in LP lesional skin (**Figure 9**). *SIGMAR1* acts as a chaperone for *IP3R* to maintain calcium signaling from the endoplasmic reticulum to mitochondria, and it has been implicated in pain (121). In contrast, *SIGMAR1* agonists potentiate pain, antagonists potentiate analgesia (122), and neuropathic pain was strongly attenuated in *SIGMAR1* knockout mice (123). Interestingly, *AQP1* silencing in tumor cells abrogates the expression of *RHOA* and *TGFB1/2* (124), indicating a possible mechanism to maintain EMT, demyelination, inflammation and pain through *AQP1*, *SIGMAR1*, *RHOA*, and *ROCK1* in leprosy.

Taken together, our data suggest an important role for miRNA expression in leprosy immunophysiopathology, especially the regulation of different parameters of the immune system, apoptosis, SC demyelination, EMT, and neuropathic pain. The epigenetic control of the genes expressed in leprosy lesions and blood by miRNAs may provide new insights into the different facets of leprosy, from *M. leprae*–host cell interactions to new therapeutic targets.

Figure 9 | MicroRNAs (miRNAs) related to the epigenetic control of genes involved in demyelination, epithelial–mesenchymal transition (EMT), inflammation, pain, and loss of sensation were differentially expressed in leprosy patient (LP). Different pathways, including canonical and non-canonical pathways of TGF-β, may drive cell toward EMT, with a Schwann cell (SC) undifferentiated phenotype consisting of the absence of p75ntr, in gray, *SOX10*, with upregulated miRNAs in lepromatous (LL) and blockade by the *SOX9* pathway, and the presence of *CD44*, which had downregulated miRNAs in LL. *RHOA* miRNAs were downregulated in LL, stimulating *ROCK1* to drive EMT, inflammation and pain, which was controlled by upregulated miRNAs in all LP. *AQP1*, the only gene with upregulated miRNAs in blood in LP and downregulation in lesional skin, is one of the genes responsible for osmotic regulation. In lesions, downregulated miRNA for *AQP1* indicated that *AQP1* might not be expressed in the nerve, which was consistent with the upregulation of miRNAs for the *MEF2C AQP1* transcription factor, possibly resulting in depolarization and calcium influx stimulating *SIGMAR1*, which also had downregulated miRNAs. The low expression level of *SIGMAR1* may result in loss of sensation, but its overexpression may result in pain. Depolarization may stimulate *RHOA* to maintain a cycle of demyelination, inflammation, loss of sensation, and pain.

#### ETHICS STATEMENT

This study was carried out in accordance with the recommendations of Brazilian National Ethics Committee (CONEP) guidelines, approved by Pará Federal University Ethics Committee number CAAE 26765414.0.0000.0018, with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Pará Federal University Ethics Committee.

#### AUTHOR CONTRIBUTIONS

CS, PP, JB, MS, SS, and AR designed research; CS, RB, AG, AM, and MF enrolled patients, performed, and registered clinical diagnosis; CS, PP, TS, AS, FM, AV, SS, and AS performed research; CS, PP, RB, AG, AM, TS, AS, FM, AV, LG, JS, SS, and AS analyzed the data; CS, PP, RB, AG, AM, JS, SS, and AS wrote the article; CS, PP, RB, AG, AM, TS, AS, FM, AV, LG,

#### REFERENCES

1. Scollard DM, Adams LB, Gillis TP, Krahenbuhl JL, Truman RW, Williams DL. The continuing challenges of leprosy. *Clin Microbiol Rev* (2006) 19:338–81. doi:10.1128/CMR.19.2.338-381.2006

JB, MS, MF, JS, SS, and AS agree with manuscript results and conclusions.

### FUNDING

We acknowledge the support of CNPq (308980/2015-8; 428964/2016-8, 304413/2015-1, and 305258/2013-3), CAPES PROAMAZONIA (3288/2013), CAPES Biocomputacional (3381/ 2013), PROPESP/UFPA, SESPA, FAPESPA (ICAAF 155/2014), UFPA, and MS/FAEPAFMRP-USP (749145/2010 and 767202/ 2011), MALTALEP-2012, J William Fulbright Scholar to Brazil award 2015–16, and The Heiser Fund of the New York Community Trust. The funders had no role on the manuscript data or concept.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at http://www.frontiersin.org/articles/10.3389/fimmu.2018.00463/ full#supplementary-material.

a possible role for tumour necrosis factor-alpha. *Immunology* (2003) 109:156–64. doi:10.1046/J.1365-2567.2003.01630.X

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Salgado, Pinto, Bouth, Gobbo, Messias, Sandoval, dos Santos, Moreira, Vidal, Goulart, Barreto, da Silva, Frade, Spencer, Santos and Ribeirodos-Santos. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

*Roberta Olmo Pinheiro1 \*, Veronica Schmitz1 , Bruno Jorge de Andrade Silva1 , André Alves Dias2 , Beatriz Junqueira de Souza1 , Mayara Garcia de Mattos Barbosa1 , Danuza de Almeida Esquenazi1 , Maria Cristina Vidal Pessolani2 and Euzenir Nunes Sarno1*

*1 Leprosy Laboratory, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil, 2Cellular Microbiology Laboratory, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil*

Leprosy is an infectious disease that may present different clinical forms depending on host immune response to *Mycobacterium leprae*. Several studies have clarified the role of various T cell populations in leprosy; however, recent evidences suggest that local innate immune mechanisms are key determinants in driving the disease to its different clinical manifestations. Leprosy is an ideal model to study the immunoregulatory role of innate immune molecules and its interaction with nervous system, which can affect homeostasis and contribute to the development of inflammatory episodes during the course of the disease. Macrophages, dendritic cells, neutrophils, and keratinocytes are the major cell populations studied and the comprehension of the complex networking created by cytokine release, lipid and iron metabolism, as well as antimicrobial effector pathways might provide data that will help in the development of new strategies for leprosy management.

Keywords: leprosy, innate immune responses, skin, *Mycobacterium leprae*, autophagy, toll-like receptors, inflammasomes

## TRANSMISSION OF LEPROSY

Leprosy is a chronic granulomatous disease, which affects dermis and peripheral nerves and also can involve the eye, the mucosa of the upper respiratory tract, muscle, bone, and testes, caused by the intracellular pathogen *Mycobacterium leprae* (1, 2).

Early diagnosis of leprosy is a prerequisite for effective therapy and rehabilitation. According to Ridley (3) the earliest lesion in leprosy is an intraepidermal lymphocytic infiltration. Although the transmission pathways of *M. leprae* are not fully understood (4), there are several results that suggest that *M. leprae* is mainly dispersed by the nose, not the skin. The protective mucosal innate mechanism in the respiratory tract may contribute to low infectivity of *M. leprae* after exposition. The release of bacilli by multibacillary patients supports a respiratory transmission (5). Viable bacilli have been found for at least 2 days in discharged nasal secretion (6). The hypothesis of respiratory transmission is validated by studies that demonstrated that adhesins present in *M. leprae* surface, like heparin-binding hemagglutinin and histone-like protein may attach in alveolar and nasal epithelial cells and both cell types are capable of sustaining bacterial survival (7, 8). In addition, a previous study demonstrated that mce1a gene is found in *M. leprae* genome and that mce1a product is associated with *M. leprae* entry into respiratory epithelial cells (9).

## HISTOPATHOLOGICAL FEATURES IN LEPROSY

The association of the histopathologic aspects and the immune state of the patient has made it the basis of the all leprosy classification and has helped to understanding the immunologic background of this disease and its transmission.

#### *Edited by:*

*Vânia Nieto Brito De Souza, Instituto Lauro de Souza Lima, Brazil*

#### *Reviewed by:*

*António Gil Castro, University of Minho, Portugal Antonio C. R. Vallinoto, Institute of Biological Sciences (ICB) of Federal University of Pará, Brazil Francisco Javier Sánchez-García, Instituto Politécnico Nacional, Mexico*

#### *\*Correspondence:*

*Roberta Olmo Pinheiro<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology*

*Received: 25 September 2017 Accepted: 27 February 2018 Published: 28 March 2018*

#### *Citation:*

*Pinheiro RO, Schmitz V, Silva BJA, Dias AA, de Souza BJ, de Mattos Barbosa MG, de Almeida Esquenazi D, Pessolani MCV and Sarno EN (2018) Innate Immune Responses in Leprosy. Front. Immunol. 9:518. doi: 10.3389/fimmu.2018.00518*

**75**

The histopathology of the nose demonstrates that the majority of all bacilli are present mainly in macrophages, as observed in lepromatous skin and other tissues. Bacilli were also seen inside monocytes, Schwann cells, polymorphs and columnar and goblet cells of the pseudostratified epithelium, secretory gland, and ducts (10).

Ridley and Jopling (11) classification establishes that the disease may present different clinical forms that may vary accordingly to histopathological findings and the immune status of the host. Tuberculoid or paucibacillary leprosy is characterized by cell-mediated immune responses to mycobacterial antigens and low infection whereas lepromatous or multibacillary leprosy is characterized by humoral immune response and high bacillary load. The different degree of cellular immune response to *M. leprae* is responsible for different types of granulomatous reaction. Analysis of skin lesion cells demonstrated that epithelioid cells are usually seen in paucibacillary patients [tuberculoid (TT) and borderline tuberculoid], whereas foamy macrophages are found in multibacillary cases [borderline lepromatous (BL) and lepromatous lepromatous (LL)]. Macrophages may present a granular eosinophilic cytoplasm with large numbers of bacilli in early and active lesions. In older lesions, on the other hand, cells are highly vacuolated and the cytoplasm has a foamy appearance (1). Recent studies have demonstrated that the macrophages in lepromatous skin cells are positive for ADRP, suggesting that their foamy aspect may be derived from lipid bodies accumulation induced by *M. leprae* (12, 13).

Two types of leprosy reactions may occur in leprosy patients. Reversal reaction is an acute inflammatory episode in skin and nerves that occurs because of an increase or emergence of cellular immunity against *M. leprae* antigens in lower or previously non-responder patients and may occur in patients of the whole clinical spectrum, except the tuberculoid, TT form (14). In addition, neuritis is frequently associated with reversal reaction episodes. Erythema nodosum leprosum (ENL) occurs in approximately 50% of patients from lepromatous pole due to a complex interaction between innate and cellular immunity poorly understood. Reversal reaction lesions show activated epithelioid macrophages, organized or not as granuloma (15, 16). The hallmark of ENL is an infiltrate of neutrophils in the profound dermis and hypodermis, frequently accompanied by macrophages (17–20). However, neutrophils are not always present (21–23) and skin fragments collected after 72 h demonstrate the presence of lymphocytes, plasma cells, and mast cells (24).

The pathogenesis of nerve destruction varies accordingly the clinical form of the disease (25); although the understanding of mechanisms associated with nerve damage and regeneration in leprosy-associated neuropathy are not fully understood (26). In the pure neural leprosy, bacilli are rarely detected despite the clinical neurological impairment. In multibacillary cases, which show macrophages in considerable numbers within the nerve, bacilli are in greater numbers, often as large bundles or globi. Ultrastructural analyses demonstrate that BL and LL foamy macrophages and vacuolated Schwann cells contain numerous electrondense structures considered as deteriorated and fragmented *M. leprae*. The dense materials are also found in the cytoplasm of vascular endothelial cells. In lepromatous cells both Schwann and endothelial cells frequently harbor *M. leprae* (25). The nerves are progressively destroyed and replaced by fibrous tissue, in both paucibacillary and multibacillary cases (27).

The peripheral nerve damage in leprosy often results in sensory and motor dysfunctions that lead to permanent deformities and/ or disabilities (28). Innate immune and inflammatory genes were modulated by *M. leprae* during early infection (29). Therefore, the understanding of the innate immune pathways in the local of infection is crucial for the development of new strategies to control leprosy and its reactional episodes (**Table 1**).

#### INNATE IMMUNE CELLS IN LEPROSY

The use of monoclonal antibodies to label specific membrane antigens is one of the most used tools to identify the presence and the frequency of different cell populations in tissue. Several studies demonstrated an enormous diversity in cell phenotypes present in different tissues. The proportions of each cell type amongst the total population of non-lymphoid mononuclear cells are different in the various leprosy infiltrates (58). In addition, the characterization of different cell phenotypes in dermis and epidermis has been shown by many studies (16, 59–61).

Despite the existence of predominant macrophage phenotypes well described in literature, between the polar forms of leprosy, it is widely recognized that some terminologies are simplistic and cells like macrophages may present a broad spectrum of differentiation states, continuously regulated by a myriad of signals from the microenvironment (62, 63). In conjunction of Th1–Th2 dichotomy, macrophages have been classified in M1 and M2. Stimulation with proinflammatory cytokines as interferon (IFN)-γ activate M1 macrophages, characterized by enhanced antimicrobial, inflammatory, and antigen-presenting properties, whereas cytokines like interleukin (IL)-4 and IL-13 activate M2 macrophages, which portray anti-inflammatory actions, being associated with tissue repair and fibrosis (62, 63).

Table 1 | Innate immunity-modulating strategies and possible therapeutic targets.

Our previous study has demonstrated that in skin cells from lepromatous patients that developed reversal reaction there is a coexistence of M1 and M2 populations in the midst of the inflammatory environment, together with a wide diversity of DC subsets (15, 64). The hallmark of the reversal reaction has been broadly accepted as the appearance of immature and loose epithelioid granulomas, which differ from the typical mature epithelioid granuloma seen in the TT forms. The epithelioid cell is described as a non-phagocyte of unknown ontogeny with high secretory capacity that could be a differentiation state of skin macrophage populations (65, 66). Facchetti et al. (67) described a cell type they called plasmocytoid monocytes (PM) and suggested, based on ultrastructure and immunohistochemical data, that they are the precursors of the epithelioid cells (68). These cells' phenotypic profile includes DCs and macrophage markers, being identified as a CD3−, CD11c−, CD14−, CD20−, CD36+, CD56−, CD68+, CD123+, BDCA2+ population (69). Since PMs produce high levels of type I IFN and express CD123, they are also thought to be a previous immature state of the plasmocytoid DC (pDC) (67).

Although efforts to identify cell markers and inflammatory mediators *in situ* the immunopathogenesis of leprosy is not fully understand. The high heterogeneity and the existence of mixed cell phenotypes in different timepoints of infection that are influenced by the mediators produced in tissue microenvironment together with the inexistence of antibodies highly specific to clearly differentiate human cells contribute to the difficulty of establish a precise role of each cell type in leprosy immunopathogenesis.

#### Macrophages

Macrophages have been identified as key players in the pathogenesis of leprosy. It has been demonstrated that during an inflammatory response, bone marrow derived monocytes enter the tissue in large numbers and take part in the defense against the pathogens. In a very elegant study, Kibbie et al. (70) demonstrated that unstimulated endothelial cells trigger monocytes to become M2 macrophages and that IFN-γ activates endothelial cells to induce monocyte to differentiate into M1 macrophages by a mechanism regulated by Jagged 1 (JAG1), a protein localized in the vascular endothelium. It is known that tissue macrophage populations have a mixed embryonic and postnatal bone marrow origin, but the exact mechanisms of differentiation and activation is not understood. There are a lot of evidences that a significant percentage of tissue macrophages are independent from blood monocytes and different phenotypes or functions are the result of different macrophages origin (71). Therefore, it is not possible to differentiate resident-tissue macrophages and recruited monocyte-macrophages once they coexist in a common environment (63).

The heterogeneity of tissue resident macrophages during homeostasis and inflammation shows that macrophages cannot be correctly classified as M1 or M2 when in a specific tissue. Although too simplistic, this nomenclature has been used in order to establish the pivotal role of macrophages in the establishment of the different forms of the disease. Each clinical presentation in leprosy is associated with a different macrophagic population in host tissue. Macrophages can present a proinflammatory M1 phenotype in which vitamin D-dependent antimicrobial pathway predominates, as observed in the paucibacillary lesions and in the onset of reversal reaction (72, 73); through to anti-inflammatory M2 phenotype in which there is an upregulation of phagocytic pathways as found in lepromatous skin tissues (72–75).

Immunohistochemistry analysis demonstrated a high expression of Galectin-3 on macrophages found in skin lesions of lepromatous patients; in contrast, it is almost undetectable in tuberculoid lesions. The increase of Galectin-3 in lepromatous cells contributes for reduced T cell activation in these patients (76).

de Sousa et al. (74) have demonstrated that the understanding of the role of cytokines, arginase 1, and costimulatory molecules in macrophages may contribute for the comprehension of innate immunity function in the establishment of the polar forms of leprosy. In addition, Teles et al. demonstrated that in macrophages present in lepromatous skin cells there is an upregulation of IL-27 (77), a paradoxal cytokine that may activate IFN-β and IL-10 that contribute for the blockade of antimicrobial pathways (78).

Although the predominance of specific cell markers of M1 or M2 in the different clinical forms of leprosy, there is a continuum of phenotypes between these ranges with some cells sharing phenotypes of both M1 and M2 macrophages. Lepromatous macrophages, while predominantly expressing M2 markers like CD163, indoleamine 2,3 dioxygenase (IDO), arginase, and SRA-I (16, 61, 79–81), have some M1 characteristics like increased iron storage and a diminished expression of the iron exporter Ferroportin (Fpn-1) as well, which indicates that augmented iron deposits may favor *M. leprae* survival inside the foamy macrophages (79) (**Figure 1**).

Besides iron, *M. leprae* incorporates cholesterol and converts it to cholestenone; however, it does not use cholesterol as a nutritional source (82), although cholesterol colocalizes to *M. leprae*containing phagosomes, and the blockade of cholesterol decreases the bacterial survival (83). Previous studies have demonstrated that *M. leprae* induces lipid body biogenesis and cholesterol accumulation in host cells (84). In lepromatous lesions, hostderived oxidized phospholipids were detected in macrophages, and one specific oxidized phospholipid, 1-palmitoyl-2-(5, 6 epoxyisoprostane E2)-sn-glycero-3-phosphorylcholine accumulate in macrophages infected with live mycobacteria (85). Normal HDL, a scavenger of oxidized phospholipids, may revert the inhibition of innate immune responses caused by mycobacterial infection. However, this effect was not observed when they have used HDL from lepromatous patients (85).

#### Dendritic Cells

In skin, DCs are located in the epidermis, as Langerhans cells and in the dermis, as dermal DCs (59). Langerhans cells in leprosy skin lesions express CD1a and langerin. These cells efficiently present antigens to T cells as part of the host response to *M. leprae* (86).

Previous reports have demonstrated that Langerhans cells are dendritic cells; however, recent findings that evaluate the