Xenophagy as a Strategy for Mycobacterium leprae Elimination during Type 1 or Type 2 Leprosy Reactions: A Systematic Review

Background: Mycobacterium leprae is an intracellular bacillus that causes leprosy, a neglected disease that affects macrophages and Schwann cells. Leprosy reactions are acute inflammatory responses to mycobacterial antigens, classified as type1 (T1R), a predominant cellular immune response, or type2 (T2R), a humoral phenomenon, leading to a high number of bacilli in infected cells and nerve structures. Xenophagy is a type of selective autophagy that targets intracellular bacteria for lysosomal degradation; however, its immune mechanisms during leprosy reactions are still unclear. This review summarizes the relationship between the autophagic process and M. leprae elimination during leprosy reactions. Methods: Three databases, PubMed/Medline (n = 91), Scopus (n = 73), and ScienceDirect (n = 124), were searched. After applying the eligibility criteria, articles were selected for independent peer reviewers in August 2023. Results: From a total of 288 studies retrieved, eight were included. In multibacillary (MB) patients who progressed to T1R, xenophagy blockade and increased inflammasome activation were observed, with IL-1β secretion before the reactional episode occurrence. On the other hand, recent data actually observed increased IL-15 levels before the reaction began, as well as IFN-γ production and xenophagy induction. Conclusion: Our search results showed a dichotomy in the T1R development and their relationship with xenophagy. No T2R studies were found.


Introduction
Mycobacterium leprae is an intracellular acid-fast bacillus that causes leprosy, a disease that affects the peripheral nerves, skin, eyes, and respiratory tract [1].Despite medical advancements, leprosy is still an important public health problem in Brazil and worldwide due to its severe consequences [2].Factors that contribute to this include the stigma related to the disease, a lack of understanding and knowledge, failure in early detection, a sub-notification of cases, and bacterial resistance to dapsone and rifampicin [3].
The Ridley-Jopling classification (1966) is one of the most widely used systems to classify leprosy, which divides patients into five groups, according to their clinical and immunological status [4].Tuberculoid leprosy (TT) is characterized by a robust cellular immune response against M. leprae antigenic determinants, the presence of a single lesion, well-developed granulomas, and negative or rare bacilli [5].In contrast, lepromatous leprosy (LL) is characterized by a strong humoral response that does not prevent bacterial proliferation and tends to clinically manifest with skin lesions and high bacterial load [6].
Most patients present borderline phenotypes, which are immunologically unstable: borderline tuberculoid (BT), borderline borderline (BB), and borderline lepromatous (BL).Indeterminate (II) cases are considered to represent the initial stage of the disease.These cases eventually move toward one of the poles but progression can be halted with treatment [7].However, in 1998, the World Health Organization's Leprosy Expert Committee established a practical and easy classification for treatment: paucibacillary (PB) cases are those in which the cutaneous lesions number does not exceed five, including TT forms, while multibacillary (MB) cases present six or more skin lesions, including LL forms [7].
During the course of the disease, patients may experience exacerbated inflammatory responses known as leprosy reactions, which can be classified into two distinct types [8].Type 1 reactions (also called reversal reaction/RR) are characterized by an exacerbation of the cellular immune response against M. leprae antigenic determinants, with CD4+ T lymphocytes and CD163+ macrophage infiltration, and tend to occur more frequently in patients presenting the PB clinical forms, but RR can also affect BB and BL patients.On the other hand, type 2 reactions (erythema nodosum leprosum/ENL) are an exacerbation of the humoral immune response with tissue deposition of immune complexes and neutrophil infiltration, which are more commonly observed in patients presenting the BL and LL forms [9,10].These reactions are the primary cause of irreversible nerve damage and anatomical deformities related to leprosy and may arise spontaneously in up to 50% of patients before, during, and after treatment [8].
Autophagy (Greek: autos = self + phaguein = eating) is the process through which cellular components are degraded or recycled within the lysosome [11].Xenophagy (Greek: xenos = strange + phaguein = eating) is a specific type of selective autophagy related to the identification and removal of intracellular bacteria [12], aiding in the activation of the host's innate and adaptive immune system as a way to limit exacerbated inflammation and control infection [13].
When bacteria infect host cells, they can be recognized among others by Pattern Recognition Receptors (PRRs) and subsequently labeled by ubiquitin in the cytosol [14].The ubiquitinated pathogen is then recognized by a group of adaptors featuring a ubiquitinbinding domain and a LC3-interacting region motif, such as p62/SQSTM1 (sequestosome 1), optineurin (OPTN), and NDP52 (nuclear domain 10 protein 52), which bind the ubiquitinated cargo to LC3 (microtubule-associated protein 1 light chain 3) on autophagosomes [14,15].Then, the maturation of the autophagosome occurs through the autophagyrelated protein complex (ATG): ATG12-ATG5-ATG16L1 and other components [16].LC3 is the main indicator of autophagic activity [17].During the autophagy process, the cytosolic form of LC3 (LC3-I) conjugates with phosphatidylethanolamine (PE) through the ATG3 to form the LC3-phosphatidylethanolamine conjugate (LC3-II), which is located in preautophagosomes and autophagosomes, making this protein an autophagosome marker [17].Furthermore, the adaptors can also target bacteria-residing vacuoles or damaged vacuolar membranes in a ubiquitin-independent manner.In this scenario, the adaptors can respond to a wide variety of protein-, lipid-or sugar-base signals, which encompass galectin, complementing protein C3 and nucleotide-binding oligomerization domain (NOD) proteins [14].
Xenophagy has been shown to play an immunological role in M. leprae control [18].Specifically, patients with the TT form of leprosy have been found to exhibit higher levels of xenophagy compared to LL-form patients [19].Inhibition of macrophage xenophagy and a strong anti-inflammatory immune response could contribute to the bacillus persistence in LL patients [5].On the other hand, MB patients who developed type 1 reactions showed a restoration of autophagic flux, leading to a limited form of this episode [19].However, studies on the role of xenophagy in M. leprae elimination remain limited.In this systematic review, we will explore the relationship between the autophagic process and M. leprae elimination during type 1 and type 2 leprosy reactions.

Methods
The present systematic review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines.

Study Selection and Eligibility Criteria
Eligibility criteria were previously used to minimize the risk of bias.The inclusion and exclusion criteria followed the PICOS (Population/Intervention/Control/ Outcomes/Study) (Table 1).There were no restrictions on language or publication date.Articles that did not meet the following eligibility criteria were excluded: (a) studies that use only mice and rats from different species; (b) studies that do not have a control group or comparator; (c) studies in animal models and/or other organisms; as well as reviews, letters for editors, duplicates, and the presence of data used in different studies.

Selection and Data Collection
The screening of studies was performed through reading the titles, abstracts, and full texts.The selection of studies was performed by two independent researchers (D.D.N.C. and M.S.d.S.F.).Discrepancies were resolved by a third rater (P.d.A.S.) (Figure 1).

Data Items
Within the included articles, information related to authors, year of publication, study design, group, number of participants (n), sex, average age, average bacillary index (BI), and logarithmic bacillary index of skin lesion (LBI) was extracted.Furthermore, information about patients with leprosy and their treatment status was also extracted.Finally, information was obtained about the cell types used and the outcomes linked to autophagy/xenophagy in in vitro studies and in humans with leprosy.

Risk of Bias Assessment
The recommendations of the Cochrane risk of bias assessment tool were used [20].Each study was categorized according to the percentage of positive responses to the questions corresponding to the assessment instrument (Figures 2 and 3).Risk of bias was analyzed using RevMan 5.3.0 software developed for Systematic Reviews, available for free download (https://training.cochrane.org/online-learning/core-softwareCochrane%20reviews/revman/revman-5-to%20go%20down, accessed on 24 July 2023).This program was used to detect intervening factors based on the 7 judgment criteria provided by the program, which are: (1) random sequence generation, (2) allocation concealment,

Data Items
Within the included articles, information related to authors, year of public study design, group, number of participants (n), sex, average age, average bacillary (BI), and logarithmic bacillary index of skin lesion (LBI) was extracted.Furthermo formation about patients with leprosy and their treatment status was also extracte 20reviews/revman/revman-5-to%20go%20down, accessed on 24 July 2023).This program was used to detect intervening factors based on the 7 judgment criteria provided by the program, which are: (1) random sequence generation, (2) allocation concealment, (3) subject and staff blinding, (4) blinding evaluation procedures, (5) incomplete results data, (6) selective reporting, and (7) other biases.

Search Results
A total of 288 studies were identified between searches in the databases Pub-Med/Medline (n = 91), Scopus (n = 73), and ScienceDirect (n = 124).After the removal of

Search Results
A total of 288 studies were identified between searches in the databases Pub-Med/Medline (n = 91), Scopus (n = 73), and ScienceDirect (n = 124).After the removal of

Search Results
A total of 288 studies were identified between searches in the databases PubMed/ Medline (n = 91), Scopus (n = 73), and ScienceDirect (n = 124).After the removal of duplicates (n = 61), 227 articles were screened for the inclusion process.Then, 217 publications were excluded after observing the title/abstract, and the remaining 10 studies were selected for reading the full text.Finally, eight studies were included in the present systematic review.The process of search, selection, and inclusion of studies was summarized in the flow diagram of the PRISMA statement (Figure 1).

Yang et al., 2014b [25]
Lepromatous lepromatous (LL), borderline lepromatous (BL), borderline tuberculoid (BT), and tuberculoid (TT) CD4+ T cells, monocytes, and monocyte-derived macrophages from PBMCs ↑ IRGM protein and mRNA levels in monocytes and macrophages upon stimulation with M. leprae.↑ IRGM in monocytes from TT type, then BT type, BL type, and followed by LL type, showing inverse correlation with the severity of the disease.

Discussion
Leprosy reactions are the most common source of persistent neuropathy, deformity and disability induced by M. leprae infection.They are characterized by an inflammatory immune response to degraded components of the bacillus that mostly appear with treatment [27].Therefore, the identification of biomarkers that can predict the T1R or T2R development in individuals with leprosy is extremely important, contributing to better decisions on treatment strategies and control of irreversible complications [28].As xenophagy is widely related to the immune response and intracellular pathogen elimination, understanding its relationship with type 1 and type 2 leprosy reactions presents advances in the search for markers for a better prognosis and sequelae prevention.
When multibacillary (MB) patients were followed up for 24 months and classified according to the occurrence or not of reversal reaction (T1R), it was observed that MB patients who developed reversal reactional episodes in the future presented xenophagy blockade and increased inflammasome activation.The xenophagy impairment in the T1R group was associated with an increased expression of the NLR family pyrin domain containing 3 (NLRP3), caspase-1 (p10), and IL-1β production, with IL-1β secretion already being observed at a diagnosis time point, 2-20 months before the reactional episode occurrence [20].The same results previously observed with another mycobacteria, Mycobacterium tuberculosis (MTB), showed that blocking xenophagy inhibited tumor necrosis factor-α (TNF-α) while enhancing IL-1β production in peripheral blood mononuclear cells stimulated with MTB [29].
The investigation into the processes involved in autophagy/xenophagy and cell death mechanisms, including apoptosis, necroptosis, and pyroptosis, in the cutaneous lesions of patients with leprosy, and the possible relationship of these mechanisms with leprosy and its clinical progression showed that M. leprae can adapt and modulate immune evasion strategies, facilitating its proliferation and reducing immunological surveillance [22].The results indicated that apoptotic and necrotic marker (FasL, Casp8, RIP1, RIP3, MLKL, BAX, and Casp3) expressions were higher in the lepromatous (LL) than in the tuberculoid (TT) and indeterminate (II) forms.On the other hand, when the xenophagy marker Beclin-1 was analyzed, protein expression was found to be increased in the TT but decreased in the LL form [30]. Suggesting that in severe forms of the disease, the action of cytokines that strongly inhibit macrophage activity, such as IL-10, inhibit the formation of autophagolysosomes, corroborating the results obtained in T1R patients in which impaired xenophagy is directly related to inflammasome activation.
When analyzing the treatment of macrophages with live or killed M. leprae stimuli, a difference in xenophagy induction was observed.While killed M. leprae preferentially induced proinflammatory cytokines, live M. leprae resulted in anti-inflammatory T cell responses, characterized by high IL-10 production, which suppressed xenophagy in a negative feedback loop and allowed the persistence of M. leprae [19].In a second study using live or killed M. leprae strains isolated from tuberculoid leprosy (T-lep) and lepromatous leprosy (L-lep) patients, the authors found that live M. leprae (regardless of the patient's clinical form) promotes M2 macrophage differentiation.This skewing was associated with a downregulated IRGM expression, an organizer of the core xenophagy machinery, and reduced autophagosome formation, with lower caspase 3 and caspase 9 activity [26].Studies showed that IRGM polymorphism was associated with the increased susceptibility to leprosy by affecting inflammatory cytokines, with T-lep patients showing the highest expression, whereas L-lep had the lowest expression of IRGM [24,25].Moreover, live M. leprae-infected macrophages prevented efficient phagocytosis, suppressed inflammation, and inhibited xenophagy and apoptosis [26].
Although live M. leprae isolated from T-lep or L-lep patients are able to downregulate the autophagic machinery, when analyzing the autophagic mechanisms in these two groups (T-lep and L-lep), large differences are observed that must be much more related to the cytokines produced and M1-M2 macrophage polarization.For example, levels of IFN-γ are significantly raised in paucibacillary T-lep when compared with multibacillary L-lep patients.IFN-γ primes macrophages for inflammatory activation and induces the xenophagy antimicrobial mechanism.LC3-positive autophagosomes and autophagic gene expression were predominantly observed in T-lep when compared with L-lep lesions and skin-derived macrophages.In L-lep skin lesion cells, high expression of BCL2 (a hindrance to autophagy) was observed together with an inhibition of the autophagic flux.Furthermore, an upregulation of autophagic genes (TPR, GFI1B and GNAI3) as well as LC3 levels was observed in cells of L-lep patients that developed RT1 episodes, an acute inflammatory condition associated with increased IFN-γ levels [5].This supports previous studies which found that the xenophagy inhibiting IL-10 cytokine is predominant in multibacillary leprosy (MB) compared to high levels of IL-26, IFN-γ, and TNF-α (xenophagy-inducing cytokines) found during paucibacillary tuberculoid leprosy (PB) [31,32].
The ability of activated human macrophages to eliminate intracellular mycobacteria involves the induction of both vitamin D-dependent and -independent antimicrobial responses [33].Activation of the vitamin D pathway leads to the induction of xenophagy and antimicrobial peptides such as cathelicidin and β-defensin 2, culminating in the bacteria elimination [34].T-lep lesions are characterized by the expression of antimicrobial genes and the presence of cells undergoing xenophagy [34].In contrast, in L-lep lesions M. leprae induces type I interferons and subsequently IL-10, which results in IFN-γ and vitamin D-dependent antimicrobial responses in macrophage inhibition, contributing to bacterial persistence [34].This corroborates the findings of previous studies that IFN-γ plays a central role in activating the autophagic pathway, especially in tuberculoid leprosy, leading to a more self-limited form.
Despite the fact that a previous study shows that multibacillary (MB) patients who develop T1R during treatment exhibit an xenophagy blockade in skin cells, which results in increased inflammasome activation [21], more recent results from the same research group show that MB patients who progress to T1R had increased levels of IL-15 even before the beginning of the reaction, leading them to hypothesize that IL-15 binds to the IL-15R on CD4+ T cells and contributes to IFN-γ production.Once established, IL-15 production is reduced and IFN-γ acts on host cells by inducing xenophagy [23].
Regarding the type 2 reaction (T2R), we were unable to find publications in our search relating to xenophagy and erythema nodosum leprosum (ENL).Study using qPCR to screen a panel of 90 genes related to the immune response in leprosy in RNA-derived peripheral leukocytes of patients with (n = 94) and without leprosy reactions (n = 57) observed that there is a marked signature for type 1 (reversal reactions) in the blood, comprising genes mostly related to the innate immune responses, including type I IFN components, autophagy/xenophagy, and Parkin and Toll like receptors.On the other hand, only Parkin was differentially expressed in the T2R (ENL) group [35].
In order to enhance its persistence within the host and evade the immune response, M. leprae promotes the polarization of macrophages toward an M2 phenotype, leading to the inhibition of xenophagy [8].This phenomenon is particularly characteristic of patients with multibacillary (MB) forms of leprosy, where there is a reduced control over bacillary replication.However, for patients who develop T1R, the data remain uncertain and appear to be more closely associated with the cytokine profile, macrophage polarization, and CD4+ T cell responses than with bacillary death.These findings suggest that xenophagy plays a role in the development of the less severe forms of leprosy and leprosy reactions.Nonetheless, further studies are required to establish a clearer understanding of the relationship between the autophagic process and type 1 leprosy reactions.As for the occurrence of T2R (ENL), our search did not yield any data regarding its association with the autophagic process.

Conclusions and Future Perspectives
Our search results showed a dichotomy in the development of type 1 leprosy reactions and their relationship with xenophagy, with some data showing that MB patients who developed reversal reactional episodes in the future presented xenophagy blockade and increased inflammasome activation, with IL-1β secretion 2-20 months before the reactional episode occurrence.More recent data, noteworthy from the same research group, show that MB patients who progress to T1R had increased levels of IL-15 even before the reaction began, suggesting that IL-15 binds to the IL-15R on CD4+ T cells and contributes to IFN-γ production and induces xenophagy.As the other data presented suggest a better prognosis in patients where the xenophagy system is activated, would patients with stronger IL-15 production have a better outcome from T1R than those who previously produce IL-1β?
Our study demonstrated that although there are good publications on the type 1 reaction, they remain few and diverse.New studies on the relationship between xenophagy and leprosy reactions are necessary, especially when considering the type 2 reaction (erythema nodosum leprosum).

( 3 )Figure 1 .
Figure 1.PRISMA 2020 flow diagram for new systematic reviews, which included searches o bases and registers only.We considered, if feasible, reporting the number of records identifie each database or register searched (rather than the total number across all databases/register

Figure 1 .
Figure 1.PRISMA 2020 flow diagram for new systematic reviews, which included searches of databases and registers only.We considered, if feasible, reporting the number of records identified from each database or register searched (rather than the total number across all databases/registers).

Figure 2 .
Figure 2. Risk of bias graph: a review of authors' judgments about each risk of bias item presented as percentages across all included studies.

Figure 3 .
Figure 3. Risk of bias summary: a review of authors' judgments about each risk of bias item for each included study [5,19-25].

Figure 2 .
Figure 2. Risk of bias graph: a review of authors' judgments about each risk of bias item presented as percentages across all included studies.

Figure 2 .
Figure 2. Risk of bias graph: a review of authors' judgments about each risk of bias item presented as percentages across all included studies.

Figure 3 .
Figure 3. Risk of bias summary: a review of authors' judgments about each risk of bias item for each included study [5,19-25].

Figure 3 .
Figure 3. Risk of bias summary: a review of authors' judgments about each risk of bias item for each included study [5,19-25].

Table 1 .
Eligibility criteria based on the PICOS strategy.

Table 2 .
Basic characteristics of participants included in the human studies.

Table 3 .
Basic characteristics of samples included in in vitro studies.