Leprosy neuropathy is a chronic neurological condition, caused by the infection of the nerve by its etiologic agents, Mycobacterium leprae and Mycobacterium lepromatosis (1–5). M. leprae infection provokes early pathological changes in the host cell that are, to some extent, associated with the late degenerative appearance of the infected nerves (6, 7). Schwann cells, the glial cells of the peripheral nervous system, are the preferable host for M. leprae entry, persistence, and replication within the nerve (8–11). Therefore, much attention has been given to the molecular and cellular alterations driven by leprosy bacilli once inside Schwann cells in order to identify the underlying reasons that culminate in the severe neuropathy seen in patients.

The immune response elicited in the nerve microenvironment against the bacilli is a key component that may lead to the distinct clinical manifestations (4). Infected Schwann cells produce a broad panel of inflammatory chemokines and cytokines, that accompanied with cell-mediated immune response, have been linked to the manifestation of neural pain and leprosy neuritis in patients (12–15). Of importance, this immunomodulation profile seen in infected Schwann cells was reported to occur before the reprogramming of the Schwann cells to the immature phenotype, highlighting the crucial role of the immune signaling network in the context of the early stages of M. leprae infection (16, 17). Additionally, M. leprae infection of Schwann cells has been associated with alterations in the glucose/lactate metabolic pathway (18, 19), lipid/cholesterol accumulation (20, 21), mitochondrial dysfunction (11), and myelin dismantling (22–24). Some of these changes were also confirmed in leprosy patients, and are suggested to cause the ongoing neuropathy and the observed tissue fibrosis and loss of nerve function experienced by leprosy individuals (14, 25). However, while these events may arise from modifications of the host Schwann cell's supportive function of the nerve, the accompanying changes in gene expression profile are largely unknown.

Studies have shown that the phenolic glycolipid 1 (PGL-1), a major M. leprae cell wall pathogenic component, is essential for M. leprae internalization into Schwann cells and has also been extensively attributed to induce pathology in vitro and in experimental infectious models (10, 23, 26). The understanding of the immunopathogenic mechanisms related to nerve damage in leprosy patients is pivotal for the development of new therapeutic strategies to control leprosy neuropathy. The treatment of nerve damage with steroids is effective but at least 40% of patients relapse and require a further course of steroids (27, 28).

Because leprosy neuropathy is an intricate complex disease, in which both viable and dead M. leprae may have a detrimental role for disease progression, it is necessary to fully understand and decipher the contribution of viable and dead bacteria in altering Schwann cell biology. For example, it was suggested that dead bacilli, unlike viable M. leprae, make Schwann cells susceptible to attack by killer cells (29). Moreover, dead bacteria and its components, such as lipoarabinomannan (LAM), were also reported to cause neural damage via modulation of the autophagic flux (30) and the complement attack of the nerve (31).

Despite these observations, the early effects of viable and dead M. leprae on the global Schwann cell gene expression profile that may be linked to primary neural leprosy are still largely unknown. Therefore, in the present study, we analyzed the expression profile of transcripts involved in neuropathy, glial cell plasticity, nerve repair, and the inflammatory network in leprosy and non-leprosy nerve biopsies and after challenging Schwann cells with viable and dead M. leprae independently. Our utmost goal was to provide novel evidence of how viable and dead bacteria modulate Schwann cell gene expression responses along with a detailed statistical correspondence to several histopathological findings commonly observed in nerve biopsies from leprosy and non-leprosy patients.

The comprehension of Schwann cell-M. leprae pathological interactions is vital to fully understand disease progression in experimental models as well as in leprosy patients. Moreover, how viable and dead bacteria affect Schwann cell biology is largely unknown and poorly explored in the field. Therefore, in the present work, we used the in vitro host-pathogen interaction model to determine how viable and dead bacteria modify the global gene expression profile as soon as the bacteria comes in contact with the Schwann cells. Furthermore, we extended these early changes in the Schwann cell gene profile in response to infection to the analysis of patient nerve biopsies, establishing a potential link to the early response pattern during the disease progression in leprosy individuals.

We initially performed an exploratory, broad gene expression analysis of Schwann cells challenged with viable or dead bacteria, along with data obtained from nerve biopsies. The following families of genes involved in the nerve response to injury were investigated: (i) peripheral neuropathy, (ii) inflammatory network, (iii) Schwann cell plasticity/dedifferentiation, and (iv) nerve regeneration support.

Among the global gene expression, HLA-DRB1, MAPK14, GAP43, FABP7, NTN1, and LRRTM4 were upregulated in nerve biopsies from PNL patients and in viable and dead M. leprae-infected Schwann cells, in comparison with the respective control groups (Figure 2). It has been described that human leukocyte antigen (HLA) alleles affect the host immune response against M. leprae (36, 37). HLA-DRB1 is one of the most upregulated genes in multiple sclerosis, having a special role in inducing demyelination (38). Moreover, this transcript is consistently related to the tuberculoid leprosy (T-Lep) clinical form, while HLA-DQB1 has a strong relation with the L-Lep clinical form (39). In the present study, HLA-DQB1 was downregulated in PNL patients, in comparison with the uninfected nerve specimens (Figure 1A). Additionally, receptors for the axon guidance molecule, netrin-1 (NTN1), are expressed by Schwann cells and play a role in peripheral regeneration and induce the regeneration phenotype (40).

When evaluating the set of genes related to Schwann cell plasticity and dedifferentiation, some serine/threonine kinases, such as mitogen-activated protein kinase 14 (MAPK14), widely known for its role at the inflammasome pathways in the neuroglia (41), were found to be upregulated in PNL patients. However, there is a lack of information regarding MAPK14 in leprosy. GAP43, the major protein of the axon growth-cone that plays a role in axon growth (42) and regeneration was also upregulated, which suggests a tentative role for axonal regrowth in the injured infected nerve. SHH, WNK, and KCNJ10 were upregulated in leprosy nerve biopsies and in dead M. leprae-infected Schwann cells (Figure 2). The sonic hedgehog gene (SHH), which has been previously reported in nerve damage (43, 44), may also trigger regeneration and induce Schwann cell proliferation, as an attempt to counteract the harm caused by the bacilli (44). KCNJ10 encodes the inward-rectifying potassium channel (Kir4.1). Alteration of KCNJ10 gene expression is related to neuropathies, such as Huntington's disease East/SeSAME syndrome, by elevating the extracellular K⁽⁺⁾, which consequently leads to abnormal neuron excitability (45, 46). It was demonstrated in mice that inflammation can silence (Kir4.1) channels, leading to hyperalgesia and trigeminal pain (47). Despite the absence of studies relating the KCNJ10 gene and M. leprae infection, it seems this may be a potential pathway to be explored for leprosy neuritis in the future. cJUN was the only gene statistically upregulated in PNL biopsies and viable M. leprae-infected Schwann cells in comparison to the respective controls (Figure 2). cJUN is a master Schwann cell regulator involved in the transition of a differentiated phenotype toward a progenitor/stem-cell like stage (48) and has also previously been reported to be upregulated, among other developmental and neural crest genes, during M. leprae infection in mouse Schwann cells (35).

PLP1, SOSTDC1, RAF1, ARTN, OLIG1, MAPK1, IFNB, and TYRP1 were upregulated in viable and dead M. leprae-infected Schwann cells, but not in the PNL biopsies. The tyrosinase-related protein 1 (TYRP1) plays a role in the melanin biosynthetic pathway, being mostly expressed by melanocytes (49). It has been documented that leprosy patients may present hyper or de-pigmented lesions due to a defective transfer of melanin (50). The melanocytes have been recently associated with the innate immune response, by producing inflammatory cytokines, such as IL-1β (50). But the role of the TYRP1 pathway in the peripheral nerve requires further investigation. It was documented that interferon beta (IFNB) is increased during cell stress conditions, but the lack of this protein leads to neuroinflammation (51, 52). IFNB is essential to start the cell fate pathway driven by NUPR1, a gene signature that contributes to a progressive infection in human cells (53). Oligodendrocyte transcription factor 1 (OLIG1) is an important protein of the remyelination phenotype, usually upregulated after a disturbance in the cell microenvironment (54). It is well described that during diabetic neuropathy, the lack of insulin leads to the demyelination phenotype due to a downregulation of OLIG1 (54). On the other hand, in this study, some genes like MAPK3 (ERK1), an important extracellular signal-regulated kinase and Schwann cell migration and proliferation molecule (55) was downregulated in PNL patients. Likewise, ErbB2, a well-known demyelination inducer and Connexin43—GJA1—were also significantly reduced in PNL patients. Despite the lack of information about GJA1 in leprosy, this has been described in neural impairment, like in the pathogenesis of Alzheimer's disease, where GJA1 downregulation leads to reduced levels of ApoE (56, 57).

While we know that the polarity of leprosy can result from the host's inflammatory response profile, multiple features of neurological involvement may also occur. For example, polymorphisms in certain genes such as IL10, ninjurin 1 and TNF have been associated with leprosy susceptibility (58–60).

In the present work, we were able to correlate the pattern of histopathological impairments with alteration in the gene expression profile induced by M. leprae infection. The correlogram analysis demonstrated, for the first time, a statistically significant correlation between the gene signature from the early stages—before fibrosis—and the histopathological alterations of the nerve damage in leprosy neuropathy. Regarding the histopathological features, TYRP1, SHH, and MAPK14 expression were strongly correlated to onion bulb axons, axon regeneration, demyelination, and Schwann cell proliferation, while MCP1/CCL2 was strongly negatively correlated with nerve inflammatory infiltrates: epineurial, endoneurial, perineurial, and lymphocytic infiltrates. It is important to realize that the weak correlation with endoneurial, perineurial, and epineurial fibrosis was due to the inclusion criteria for this study, which selected patients with no signs of fibrosis.

The biserial correlation demonstrated that the monocyte chemoattractant protein 1 (MCP1/CCL2) was positively correlated with AFB and negatively correlated with PCR, which strongly indicates the correlation of increased expression of this gene by viable bacteria. This finding corroborates a previous study by our group, which showed activation of the ESX-1 mycobacterial system by the viable M. leprae, leading to the activation of the OASL gene and the induction of CCL2, impairing the host bactericidal response, which was not observed with dead M. leprae stimuli in THP-1 cells (61). More than that, independently, Schwann cells are responsible for the triggering response of nerve damage through initiating the clearance of the debris by myelinophagy, followed by macrophage recruitment, which is especially regulated by CCL2 (62). In this sense, we see the relevance of CCL2 gene activation not only as a protective mechanism in the maintenance of mycobacterial viability, but also as a biological marker indicative of positive AFB. CCL2 has been described as one of the innate immunity genes immediately activated in the context of infection in vitro (35). Thus, despite the relevant findings, there is still a way to go in terms of understanding the role of this chemokine in infection and nerve damage during leprosy.

To strengthen the data obtained from the gene signature of M. leprae infection, we evaluated the immunomodulatory profile secreted by Schwann cells without infection and challenged with viable and dead M. leprae. We observed a pro-inflammatory profile elicited by the dead bacilli, while an anti-inflammatory microenvironment appeared to be promoted by infection with the viable bacteria. Studies have shown that macrophage infection by viable M. leprae leads to a regulatory T cell response rather than a cytotoxic T cell response, which contributes to the persistence of the infection in the host (63, 64). This characteristic is already well demonstrated in patients with L-lep, which leads to a high bacillary load. In the present work, we demonstrated that Schwann cells also behave in a way to induce the anti- or pro-inflammatory phenotype according to the bacilli viability. Since in reactional episodes there is a mixture of viable and dead bacilli, it is interesting to point out the role of the Schwann cells, and not only the macrophages, as modulators of the reaction process in the nerve, which can even lead to leprosy neuritis (14).

In the present study, we identified the early impact of viable and dead bacteria, independently, on modifying the gene expression profile of human Schwann cells in vitro. We also described a molecular signature associated with neural damage in early stages of pure neural leprosy from patients (i.e., HLA-DRB1, MAPK14, GAP43, FABP7, NTN1, and LRRTM4). Leprosy is a complex intricate disease and the identification of this genetic profiling may contribute for the fully understanding of leprosy neuropathy pathogenesis with the long-term goal of identifying these pathways as targets for the development of effective therapeutic strategies. We acknowledge the relatively limited number of human nerve samples, as in our experimental design we narrowed our analysis to nerve biopsies of leprosy patients with no signs of neural fibrosis and at the initial stages of neural damage. Therefore, future work with additional nerve samples from leprosy individuals, at distinct clinical stages, are important to advance this host-pathogen interactions and associated genetic analysis in larger cohorts. In summary, these results open new perspectives for the understanding of the genetic signature of neural commitment in leprosy disease.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

The studies involving human participants were reviewed and approved by Oswaldo Cruz Foundation Ethics Committee (number of purports: 2.227.887). The Ethics Committee waived the requirement of written informed consent for participation.

BS, RP, and BM designed and carried out experiments, collected and interpreted data, and prepared illustrations. BS, GS, RP, and BM analyzed the data. BS, MM, GS, PS-R, MOM, MJ, ES, RP, and BM reviewed and edited the manuscript. ES, RP, and BM conceptualized and designed experiments, supervised the study, interpreted data, and obtained funding. BS, RP, and BM wrote the manuscript. PS-R provided viable M. leprae. All authors read and approved the final version of the manuscript.

This study was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Fundação Oswaldo Cruz (Fiocruz) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Dead M. leprae was obtained through BEI Resources, NIAID, NIH: Mycobacterium leprae, Strain NHDP, Gamma-Irradiated Whole Cells (lyophilized), NR-19326.

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.

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