Priscila Ribeiro Andrade

CD163 favors Mycobacterium leprae survival and persistence by promoting anti-inflammatory pathways in lepromatous macrophages

Lepromatous macrophages possess a regulatory phenotype that contributes to the immunosuppression observed in leprosy. CD163, a scavenger receptor that recognizes hemoglobin–haptoglobin complexes, is expressed at higher levels in lepromatous cells, although its functional role in leprosy is not yet established. We herein demonstrate that human lepromatous lesions are microenvironments rich in IDO+CD163+. Cells isolated from these lesions were CD68+IDO+CD163+ while higher levels of sCD163 in lepromatous sera positively correlated with IL‐10 levels and IDO activity. Different Myco‐bacterium leprae (ML) concentrations in healthy monocytes likewise revealed a positive correlation between increased concentrations of the mycobacteria and IDO, CD209, and CD163 expression. The regulatory phenotype in ML‐stimulated monocytes was accompanied by increased TNF, IL‐10, and TGF‐β levels whereas IL‐10 blockade reduced ML‐induced CD163 expression. The CD163 blockade reduced ML uptake in human monocytes. ML uptake was higher in HEK293 cells transfected with the cDNA for CD163 than in untransfected cells. Simultaneously, increased CD163 expression in lepromatous cells seemed to be dependent on ML uptake, and contributed to augmented iron storage in lepromatous macrophages. Altogether, these results suggest that ML‐induced CD163 expression modulates the host cell phenotype to create a favorable environment for myco‐bacterial entry and survival.

Effect of Apoptotic Cell Recognition on Macrophage Polarization and Mycobacterial Persistence

ABSTRACT Intracellular Mycobacterium leprae infection modifies host macrophage programming, creating a protective niche for bacterial survival. The milieu regulating cellular apoptosis in the tissue plays an important role in defining susceptible and/or resistant phenotypes. A higher density of apoptotic cells has been demonstrated in paucibacillary leprosy lesions than in multibacillary ones. However, the effect of apoptotic cell removal on M. leprae-stimulated cells has yet to be fully elucidated. In this study, we investigated whether apoptotic cell removal (efferocytosis) induces different phenotypes in proinflammatory (Mϕ1) and anti-inflammatory (Mϕ2) macrophages in the presence of M. leprae. We stimulated Mϕ1 and Mϕ2 cells with M. leprae in the presence or absence of apoptotic cells and subsequently evaluated the M. leprae uptake, cell phenotype, and cytokine pattern in the supernatants. In the presence of M. leprae and apoptotic cells, Mϕ1 macrophages changed their phenotype to resemble the Mϕ2 phenotype, displaying increased CD163 and SRA-I expression as well as higher phagocytic capacity. Efferocytosis increased M. leprae survival in Mϕ1 cells, accompanied by reduced interleukin-15 (IL-15) and IL-6 levels and increased transforming growth factor beta (TGF-β) and IL-10 secretion. Mϕ1 cells primed with M. leprae in the presence of apoptotic cells induced the secretion of Th2 cytokines IL-4 and IL-13 in autologous T cells compared with cultures stimulated with M. leprae or apoptotic cells alone. Efferocytosis did not alter the Mϕ2 cell phenotype or cytokine secretion profile, except for TGF-β. Based on these data, we suggest that, in paucibacillary leprosy patients, efferocytosis contributes to mycobacterial persistence by increasing the Mϕ2 population and sustaining the infection.

Type 1 reaction in leprosy: a model for a better understanding of tissue immunity under an immunopathological condition.

Type 1 reaction (T1R) or reversal reaction is the leading cause of physical disabilities and deformities in leprosy. Leprosy patients, even after being considered cured and released from treatment, may suffer from reactional episodes for long periods of time. Early diagnosis is a great challenge for effectively treating and managing T1R. There is an urgent need to identify the most significant biomarkers to prevent recurrent T1R and to differentiate late T1R from relapse. T1R continues to be treated with corticosteroids and complications due to iatrogenic treatment remain frequent. This review aims to provide a framework from which to approach the great challenges that still persist in T1R management and debate key issues in order to reduce the distance between basic research and the clinic.

CD123, the plasmacytoid dendritic cell phenotypic marker, is abundant in leprosy type 1 reaction.

DEAR EDITOR, The chronic course of leprosy is aggravated by the emergence of reactional states such as type 1 reaction (T1R). T1R is deemed to occur as a sudden and heightened inflammatory response that eventually compromises the skin and peripheral nerves. T1R occurs more frequently among the unstable borderline forms of leprosy [borderline tuberculoid (BT), borderline borderline (BB) and borderline lepromatous (BL)], but has also been known to occur in patients with lepromatous leprosy (LL). Even though the mechanisms involved in the development of T1R remain unknown, it is believed that, during the episode, the BL and LL clinical forms undergo an immunological shift towards the tuberculoid pole of leprosy, favouring the emergence of a T helper 1 response profile. A prominent T1R feature is an abrupt infiltration of the skin lesions by polymorphic monocytic cell populations, including epithelioid cells, which constitute loose and immature granulomas. Epithelioid cells are considered nonphagocytes of unknown ontogeny with a high secretory capacity. It has been suggested that plasmacytoid monocytes (PMs), whose phenotypic profile includes dendritic cell (DC) and macrophagic markers, are in fact epithelioid-cell precursors. As PMs produce high levels of interferon a/b and express CD123, the a subunit of the interleukin (IL)-3 receptor, they are also thought to represent a prior immature state of plasmacytoid DCs (pDCs). Although many studies have shed light on the pathogenesis of T1R, they have had little impact on treatment policies, which still advocate corticoid therapy alone. Therefore, the aim of our study was to explore new possible intervention pathways by focusing on the investigation of CD123 cell populations and the epithelioid-cell phenotype in T1R skin lesions of patients with BL and LL. These patients were chosen in order to exclude the possibility of any prior immune responsiveness. Clinical data of groups are provided in Table S1 (see Supporting Information). Full details of the methods used in this study are provided in Data S1 (see Supporting Information). The L-lep (BL and LL combined, before T1R) skin lesion undergoes drastic morphological changes at the outset of T1R, displaying different cell populations and a new organizational pattern (Fig. 1a). Evaluation of cell phenotypes through immunohistochemistry analysis showed the presence of a diverse set of DC markers (data not shown). The pDC marker CD123 displayed intense staining in the central area of the granuloma while labelling epithelioid cells (Fig. 1b), and was more strongly expressed in the reactional skin lesion cells (> 50% of positive cells) compared with the L-lep group. The predominance of CD123 cells in T1R was confirmed by the increase of CD123 mRNA in this group in relation to the patients with L-lep (Fig. 1c). The longitudinal analysis of the CD123 gene expression of each patient before and at the outset of the T1R episode revealed this phenomenon more clearly (Fig. 1c). Moreover, flow cytometry analysis of isolated lesional cells also confirmed the higher percentage of CD123 cells in the T1R lesions than in the L-lep group (Fig. 1d). As CD123 is the low-affinity receptor for IL-3, the gene expression of this cytokine was evaluated in skin lesions of both groups. Significantly, IL-3 mRNA was observed in five of the eight T1R samples (Fig. 1e), but was not detected in any of the eight L-lep specimens analysed. To clarify the CD123 population phenotypes, doubleimmunofluorescence assays were performed. Although CD123 colocalized with some CD68 cells in both groups, the T1R lesions exhibited a higher percentage of double-positive cells than the L-lep group (Fig. 2a–e). Blood dendritic cell antigen (BDCA)4 and BDCA2, pDC markers in blood, were expressed by all CD123 cells in both groups (Fig. 2b–e). Regarding the epithelioid-cell populations, double-immunofluorescence assays showed that CD123BDCA4 and CD123BDCA2 cells were found in T1R granuloma (Fig. 2b–e). In addition, double-positive cells of both populations showed a higher density in T1R than in L-lep lesions (Fig. 2e). Regarding DC populations, CD1a was expressed in the CD123 cells located in the dermis in both groups, whereas a higher frequency was noted in the T1R lesions (Fig. 2c–e). CD123CD83 and CD123CD86 were identified in the skin lesions of both groups, with higher expression among patients with T1R (Fig. 2e). In the reactional skin lesions, the percentage of CD123/CD11c cells was higher than in the L-lep group (Fig. 2e). Moreover, CD209 colocalized more often with CD123 in both lesions in the T1R group (Fig. 2e). Meanwhile, CD123Toll-like receptor 2 cells were more numerous in the T1R skin lesions than in the L-lep group (Fig. 2e).

Autophagy Is an Innate Mechanism Associated with Leprosy Polarization

Leprosy is a chronic infectious disease that may present different clinical forms according to the immune response of the host. Levels of IFN-γ are significantly raised in paucibacillary tuberculoid (T-lep) when compared with multibacillary lepromatous (L-lep) patients. IFN-γ primes macrophages for inflammatory activation and induces the autophagy antimicrobial mechanism. The involvement of autophagy in the immune response against Mycobacterium leprae remains unexplored. Here, we demonstrated by different autophagic assays that LC3-positive autophagosomes were predominantly observed in T-lep when compared with L-lep lesions and skin-derived macrophages. Accumulation of the autophagic receptors SQSTM1/p62 and NBR1, expression of lysosomal antimicrobial peptides and colocalization analysis of autolysosomes revealed an impairment of the autophagic flux in L-lep cells, which was restored by IFN-γ or rapamycin treatment. Autophagy PCR array gene-expression analysis revealed a significantly upregulation of autophagy genes (BECN1, GPSM3, ATG14, APOL1, and TPR) in T-lep cells. Furthermore, an upregulation of autophagy genes (TPR, GFI1B and GNAI3) as well as LC3 levels was observed in cells of L-lep patients that developed type 1 reaction (T1R) episodes, an acute inflammatory condition associated with increased IFN-γ levels. Finally, we observed increased BCL2 expression in L-lep cells that could be responsible for the blockage of BECN1-mediated autophagy. In addition, in vitro studies demonstrated that dead, but not live M. leprae can induce autophagy in primary and lineage human monocytes, and that live mycobacteria can reduce the autophagy activation triggered by dead mycobacteria, suggesting that M. leprae may hamper the autophagic machinery as an immune escape mechanism. Together, these results indicate that autophagy is an important innate mechanism associated with the M. leprae control in skin macrophages.

Cell-type deconvolution with immune pathways identifies gene networks of host defense and immunopathology in leprosy

Transcriptome profiles derived from the site of human disease have led to the identification of genes that contribute to pathogenesis, yet the complex mixture of cell types in these lesions has been an obstacle for defining specific mechanisms. Leprosy provides an outstanding model to study host defense and pathogenesis in a human infectious disease, given its clinical spectrum, which interrelates with the host immunologic and pathologic responses. Here, we investigated gene expression profiles derived from skin lesions for each clinical subtype of leprosy, analyzing gene coexpression modules by cell-type deconvolution. In lesions from tuberculoid leprosy patients, those with the self-limited form of the disease, dendritic cells were linked with MMP12 as part of a tissue remodeling network that contributes to granuloma formation. In lesions from lepromatous leprosy patients, those with disseminated disease, macrophages were linked with a gene network that programs phagocytosis. In erythema nodosum leprosum, neutrophil and endothelial cell gene networks were identified as part of the vasculitis that results in tissue injury. The present integrated computational approach provides a systems approach toward identifying cell-defined functional networks that contribute to host defense and immunopathology at the site of human infectious disease.

Indoleamine 2,3-dioxygenase and iron are required for Mycobacterium leprae survival

Our previous study has demonstrated that IL-10 may modulate both indoleamine 2,3-dioxygenase (IDO) and CD163 expression in lepromatous leprosy (LL) cells, favoring Mycobacterium leprae persistence through induction of regulatory pathways and iron storage. Here, we observed that in LL lesion cells there is an increase in the expression of proteins involved in iron metabolism such as hemoglobin (Hb), haptoglobin, heme oxygenase 1 and transferrin receptor 1 (TfR1) when compared to tuberculoid leprosy (BT) cells. We also found increased iron deposits and diminished expression of the iron exporter ferroportin 1 in LL lesion cells. Hemin, but not FeSO4 stimulation, was able to enhance M. leprae viability by a mechanism that involves IDO. Analysis of cell phenotype in lesions demonstrated a predominance of M2 markers in LL when compared with BT lesion cells. A positive correlation between CD163 and PPARG with the bacillary index (BI) was observed. In contrast, TNF, STAT1 and CSF2 presented a negative correlation with the BI. In summary, this study demonstrates that iron may regulate IDO expression by a mechanism that involves IL-10, which may contribute for the predominance of M2-like phenotype in LL lesions that favors the phagocytosis and maintenance of M. leprae in host cells.

Inflammatory Cytokines Are Involved in Focal Demyelination in Leprosy Neuritis

Mycobacterium leprae (ML) infection causes nerve damage that often leads to permanent loss of cutaneous sensitivity and limb deformities, but understanding of the pathogenesis of leprous neuropathy that would lead to more effective treatments is incomplete. We studied reactional leprosy patients with (n = 9) and without (n = 8) acute neuritis. Nerve conduction studies over the course of the reactional episode showed the findings of demyelination in all patients with neuritis. Evaluation of patient sera revealed no correlation of the presence of antibodies against gangliosides and the clinical demyelination. In nerve biopsies of 3 patients with neuritis, we identified tumor necrosis factor (TNF), TNF receptors, and TNF-converting enzyme in Schwann cells (SCs) using immunofluorescence. To elucidate immunopathogenetic mechanisms, we performed experiments using a human SC line. ML induced transmembrane TNF and TNF receptor 1 expression in the SCs; TNF also induced interleukin (IL)-6 and IL-8 production by the SCs; and ML induced IL-23 secretion, indicating involvement of this previously unrecognized factor in leprosy nerve damage. These data suggest that ML may contribute to TNF-mediated inflammation and focal demyelination by rendering SCs more sensitive to TNF within the nerves of patients with leprous neuropathy.

Autophagy Impairment Is Associated With Increased Inflammasome Activation and Reversal Reaction Development in Multibacillary Leprosy

Leprosy reactions are responsible for incapacities in leprosy and represent the major cause of permanent neuropathy. The identification of biomarkers able to identify patients more prone to develop reaction could contribute to adequate clinical management and the prevention of disability. Reversal reaction may occur in unstable borderline patients and also in lepromatous patients. To identify biomarker signature profiles related with the reversal reaction onset, multibacillary patients were recruited and classified accordingly the occurrence or not of reversal reaction during or after multidrugtherapy. Analysis of skin lesion cells at diagnosis of multibacillary leprosy demonstrated that in the group that developed reaction (T1R) in the future there was a downregulation of autophagy associated with the overexpression of TLR2 and MLST8. The autophagy impairment in T1R group was associated with increased expression of NLRP3, caspase-1 (p10) and IL-1β production. In addition, analysis of IL-1β production in serum from multibacillary patients demonstrated that patients who developed reversal reaction have significantly increased concentrations of IL-1β at diagnosis, suggesting that the pattern of innate immune responses could predict the reactional episode outcome. In vitro analysis demonstrated that the blockade of autophagy with 3-methyladenine (3-MA) in Mycobacterium leprae-stimulated human primary monocytes increased the assembly of NLRP3 specks assembly, and it was associated with an increase of IL-1β and IL-6 production. Together, our data suggest an important role for autophagy in multibacillary leprosy patients to avoid exacerbated inflammasome activation and the onset of reversal reaction.