The cellular architecture of the antimicrobial response network in human leprosy granulomas.

Granulomas are complex cellular structures composed predominantly of macrophages and lymphocytes that function to contain and kill invading pathogens. Here, we investigated the single-cell phenotypes associated with antimicrobial responses in human leprosy granulomas by applying single-cell and spatial sequencing to leprosy biopsy specimens. We focused on reversal reactions (RRs), a dynamic process whereby some patients with disseminated lepromatous leprosy (L-lep) transition toward self-limiting tuberculoid leprosy (T-lep), mounting effective antimicrobial responses. We identified a set of genes encoding proteins involved in antimicrobial responses that are differentially expressed in RR versus L-lep lesions and regulated by interferon-γ and interleukin-1ß. By integrating the spatial coordinates of the key cell types and antimicrobial gene expression in RR and T-lep lesions, we constructed a map revealing the organized architecture of granulomas depicting compositional and functional layers by which macrophages, T cells, keratinocytes and fibroblasts can each contribute to the antimicrobial response.

Immune Complex-Driven Generation of Human Macrophages with Anti-Inflammatory and Growth-Promoting Activity.

To maintain homeostasis, macrophages must be capable of assuming either an inflammatory or an anti-inflammatory phenotype. To better understand the latter, we stimulated human macrophages in vitro with TLR ligands in the presence of high-density immune complexes (IC). This combination of stimuli resulted in a broad suppression of inflammatory mediators and an upregulation of molecules involved in tissue remodeling and angiogenesis. Transcriptomic analysis of TLR stimulation in the presence of IC predicted the downstream activation of AKT and the inhibition of GSK3. Consequently, we pretreated LPS-stimulated human macrophages with small molecule inhibitors of GSK3 to partially phenocopy the regulatory effects of stimulation in the presence of IC. The upregulation of DC-STAMP and matrix metalloproteases was observed on these cells and may represent potential biomarkers for this regulatory activation state. To demonstrate the presence of these anti-inflammatory, growth-promoting macrophages in a human infectious disease, biopsies from patients with leprosy (Hanseniasis) were analyzed. The lepromatous form of this disease is characterized by hypergammaglobulinemia and defective cell-mediated immunity. Lesions in lepromatous leprosy contained macrophages with a regulatory phenotype expressing higher levels of DC-STAMP and lower levels of IL-12, relative to macrophages in tuberculoid leprosy lesions. Therefore, we propose that increased signaling by FcγR cross-linking on TLR-stimulated macrophages can paradoxically promote the resolution of inflammation and initiate processes critical to tissue growth and repair. It can also contribute to infectious disease progression.

Immunohistochemical characterization of the M4 macrophage population in leprosy skin lesions.

BACKGROUND: Since macrophages are one of the major cell types involved in the Mycobacterium leprae immune response, roles of the M1 and M2 macrophage subpopulations have been well defined. However, the role of M4 macrophages in leprosy or other infectious diseases caused by mycobacteria has not yet been clearly characterized. This study aimed to investigate the presence and potential role of M4 macrophages in the immunopathology of leprosy. METHODS: We analyzed the presence of M4 macrophage markers (CD68, MRP8, MMP7, IL-6, and TNF-α) in 33 leprosy skin lesion samples from 18 patients with tuberculoid leprosy and 15 with lepromatous leprosy by immunohistochemistry. RESULTS: The M4 phenotype was more strongly expressed in patients with the lepromatous form of the disease, indicating that this subpopulation is less effective in the elimination of the bacillus and consequently is associated with the evolution to one of the multibacillary clinical forms of infection. CONCLUSION: M4 macrophages are one of the cell types involved in the microbial response to M. leprae and probably are less effective in controlling bacillus replication, contributing to the evolution to the lepromatous form of the disease.

Leprosy - an overview of clinical features, diagnosis, and treatment.

Leprosy is a chronic infectious disease caused by Mycobacterium (M.) leprae. Worldwide, 210,758 new cases were diagnosed in 2015. The highest incidence is found in India, Brazil, and Indonesia. While the exact route of transmission remains unknown, nasal droplet infection is thought to be most likely. The pathogen primarily affects the skin and peripheral nervous system. The disease course is determined by individual host immunity. Clinically, multibacillary lepromatous variants are distinguished from paucibacillary tuberculoid forms. Apart from the various characteristic skin lesions, the condition is marked by damage to the peripheral nervous system. Advanced disease is characterized by disfiguring mutilations. Current treatment options are based on WHO recommendations. Early treatment frequently results in complete remission without sequelae. While paucibacillary forms are treated with rifampicin and dapsone for at least six months, multibacillary leprosy is treated for at least twelve months, additionally requiring clofazimine. Leprosy reactions during therapy may considerably aggravate the disease course. Besides individual treatment, WHO-supported preventive measures and strategies play a key role in endemic areas.

Induction and treatment of anergy in murine leprosy.

Leprosy is a disease consisting of a spectrum of clinical, bacteriological, histopathological and immunological manifestations. Tuberculoid leprosy is frequently recognized as the benign polar form of the disease, while lepromatous leprosy is regarded as the malignant form. The different forms of leprosy depend on the genetic and immunological characteristics of the patient and on the characteristics of the leprosy bacillus. The malignant manifestations of lepromatous leprosy result from the mycobacterial-specific anergy that develops in this form of the disease. Using murine leprosy as a model of anergy in this study, we first induced the development of anergy to Mycobacterium lepraemurium (MLM) in mice and then attempted to reverse it by the administration of dialysable leucocyte extracts (DLE) prepared from healthy (HLT), BCG-inoculated and MLM-inoculated mice. Mice inoculated with either MLM or BCG developed a robust cell-mediated immune response (CMI) that was temporary in the MLM-inoculated group and long-lasting in the BCG-inoculated group. DLE were prepared from the spleens of MLM- and BCG-inoculated mice at the peak of CMI. Independent MLM intradermally-inoculated groups were treated every other day with HLT-DLE, BCG-DLE or MLM-DLE, and the effect was documented for 98 days. DLE administered at a dose of 1.0 U (1 × 10(6) splenocytes) did not affect the evolution of leprosy, while DLE given at a dose of 0.1 U showed beneficial effects regardless of the DLE source. The dose but not the specificity of DLE was the determining factor for reversing anergy.

Galectin-3 regulates the innate immune response of human monocytes.

Galectin-3 is a ß-galactoside-binding lectin widely expressed on epithelial and hematopoietic cells, and its expression is frequently associated with a poor prognosis in cancer. Because it has not been well-studied in human infectious disease, we examined galectin-3 expression in mycobacterial infection by studying leprosy, an intracellular infection caused by Mycobacterium leprae. Galectin-3 was highly expressed on macrophages in lesions of patients with the clinically progressive lepromatous form of leprosy; in contrast, galectin-3 was almost undetectable in self-limited tuberculoid lesions. We investigated the potential function of galectin-3 in cell-mediated immunity using peripheral blood monocytes. Galectin-3 enhanced monocyte interleukin 10 production to a TLR2/1 ligand, whereas interleukin 12p40 secretion was unaffected. Furthermore, galectin-3 diminished monocyte to dendritic cell differentiation and T-cell antigen presentation. These data demonstrate an association of galectin-3 with unfavorable host response in leprosy and a potential mechanism for impaired host defense in humans.

The role of indoleamine 2, 3-dioxygenase in lepromatous leprosy immunosuppression.

To elucidate further the possible role of the tryptophan, rate-limiting enzyme indoleamine 2, 3-dioxygenase (IDO) in leprosy, the distribution of IDO-positive cells and IDO activity in the skin biopsies and sera of these patients representing the entire spectrum of the disease were studied. An increased number of macrophages/dendritic cells (DC-lineage IDO(+) cells were found in lepromatous (LL) compared to tuberculoid (BT) and reversal reaction (RR) patients. IDO-positive cells showing CD68 and CD86 surface markers predominated in LL lesions, while higher levels of IDO activity were observed in the sera of LL versus BT patients. Tests revealed an increased IDO message in Mycobacterium leprae-stimulated peripheral blood mononuclear cells (PBMC) by real-time polymerase chain reaction (PCR) and increased IDO expression in M. leprae-stimulated CD14(+) cells of both healthy controls (HC) and LL patients, as evaluated via flow cytometry. Increased M. leprae-induced IDO-protein synthesis was also confirmed by Western blot. Based on our in vitro studies, it was confirmed that M. leprae up-regulated IDO expression and activity in HC and LL monocytes. Interferon (IFN)-γ synergized with M. leprae in promoting IDO expression and activity in monocytes. IDO expression induced by both IFN-γ and M. leprae was abrogated by 1-methyltryptophan (1-MT). Our data suggest that M. leprae chronic infection activates the suppressive molecule IDO which, in turn, contributes to the specific immunosuppression observed in LL leprosy.

T-cell release of granulysin contributes to host defense in leprosy.

A novel mechanism by which T cells contribute to host defense against microbial pathogens is release of the antimicrobial protein granulysin. We investigated the role of granulysin in human infectious disease using leprosy as a model. Granulysin-expressing T cells were detected in cutaneous leprosy lesions at a six-fold greater frequency in patients with the localized tuberculoid as compared with the disseminated lepromatous form of the disease. In contrast, perforin, a cytolytic molecule that colocalizes with granulysin in cytotoxic granules, was expressed at similar levels across the spectrum of disease. Within leprosy lesions, granulysin colocalized in CD4+ T cells and was expressed in CD4+ T-cell lines derived from skin lesions. These CD4+ T-cell lines lysed targets by the granule exocytosis pathway and reduced the viability of mycobacteria in infected targets. Given the broad antimicrobial spectrum of granulysin, these data provide evidence that T-cell release of granulysin contributes to host defense in human infectious disease.

Interleukin-4 regulates the expression of CD209 and subsequent uptake of Mycobacterium leprae by Schwann cells in human leprosy.

The ability of microbial pathogens to target specific cell types is a key aspect of the pathogenesis of infectious disease. Mycobacterium leprae, by infecting Schwann cells, contributes to nerve injury in patients with leprosy. Here, we investigated mechanisms of host-pathogen interaction in the peripheral nerve lesions of leprosy. We found that the expression of the C-type lectin, CD209, known to be expressed on tissue macrophages and to mediate the uptake of M. leprae, was present on Schwann cells, colocalizing with the Schwann cell marker, CNPase (2',3'-cyclic nucleotide 3'-phosphodiesterase), along with the M. leprae antigen PGL-1 in the peripheral nerve biopsy specimens. In vitro, human CD209-positive Schwann cells, both from primary cultures and a long-term line, have a higher binding of M. leprae compared to CD209-negative Schwann cells. Interleukin-4, known to be expressed in skin lesions from multibacillary patients, increased CD209 expression on human Schwann cells and subsequent Schwann cell binding to M. leprae, whereas Th1 cytokines did not induce CD209 expression on these cells. Therefore, the regulated expression of CD209 represents a common mechanism by which Schwann cells and macrophages bind and take up M. leprae, contributing to the pathogenesis of leprosy.

Selection of a human T helper type 1-like T cell subset by mycobacteria.

Mycobacteria elicit a cellular immune response in their hosts. This response usually leads to protective immunity, but may sometimes be accompanied by immunopathology due to delayed type hypersensitivity (DTH). A striking example in man is tuberculoid leprosy, which is characterized by high cellular immunity to Mycobacterium leprae and immunopathology due to DTH. Skin lesions of patients suffering from this disease have the characteristics of DTH reactions in which macrophages and CD4+ T lymphocytes predominate. In animal models, it has been shown that DTH responses are associated with the presence of a particular subset of CD4+ T cells (T helper type 1 [Th1]) that secrete only certain cytokines, such as interleukin 2 (IL-2), interferon gamma (IFN-gamma), and lymphotoxin, but no IL-4 or IL-5. We studied the cytokine release of activated M. leprae-reactive CD4+ T cell clones derived from tuberculoid leprosy patients. These T cell clones, which were reactive with mycobacterial heat shock proteins, exhibited a Th1-like cytokine secretion pattern with very high levels of IFN-gamma. Half of these clones secreted low levels of IL-4 and IL-5, but the ratio of IFN-gamma to IL-4 and IL-5 was much higher than that of T cell clones reactive with nonmycobacterial antigens. A Th1-like cytokine secretion pattern was also observed for T cell clones and polyclonal T cell lines from control individuals that recognized both heat shock and other mycobacterial antigens. The levels of IFN-gamma secreted by these clones were, however, significantly less than those of patient-derived T cell clones. This Th1-like pattern was not found with T cell clones from the same patients and healthy individuals generated in the same manner, but reactive with nonmycobacterial antigens. Our data thus indicate that mycobacteria selectively induce human T cells with a Th1-like cytokine secretion profile.

Arginine at positions 13 or 70-71 in pocket 4 of HLA-DRB1 alleles is associated with susceptibility to tuberculoid leprosy.

Evaluation of human histocompatibility leukocyte antigen (HLA) class II genes in 54 cases of tuberculoid leprosy (TL) and 44 controls has shown a positive association with HLA-DRB1 alleles that contain Arg13 or Arg70-Arg71. Among TL patients, 87% carry specific alleles of DRB1 Arg13 or Arg70-Arg71 as compared to 43% among controls (p = 5 x 10(-6)) conferring a relative risk of 8.8. Thus, susceptibility to TL involves three critical amino acid positions of the beta chain, the side chains of which, when modeled on the DR1 crystal structure, line a pocket (pocket 4) accommodating the side chain of a bound peptide. This study suggests that disease susceptibility may be determined by the independent contribution of polymorphic residues participating in the formation of a functional arrangement (i.e., pocket) within the binding cleft of an HLA molecule.

Presence of intestinal helminths decreases T helper type 1 responses in tuberculoid leprosy patients and may increase the risk for multi-bacillary leprosy.

Resistance to intracellular pathogens such as Mycobacterium leprae is dependent upon an effective T helper type 1 (Th1)-type immune response. On the other hand, intestinal helminths are known to subvert the host's immune response towards to either a Th2-type immune response or a regulatory T cell up-regulation, which may affect the host's ability to mount an effective response to mycobacteria. Here, we report a significant association between intestinal helminth infections and lepromatous leprosy [odds ratio (OR), 10.88; confidence interval (CI) 95%: 4.02-29.4; P<0.001]. We also observed that the frequency of intestinal helminths correlated strongly with the mycobacterial index (r=0.982, P<0.01). Corroborating with our hypothesis, intracellular levels of interferon-gamma were decreased significantly in leprosy patients co-infected with intestinal helminths when compared to leprosy patients without worms. Conversely, lepromatous leprosy patients with intestinal worms produced higher levels of both interleukin (IL)-4 and IL-10. Our results suggest that a pre-existing infection by intestinal helminths may facilitate the establishment of M. leprae infection or its progression to more severe forms of leprosy.

Colonization by Helicobacter pylori of leprosy patients in Spain: immunomodulation to low molecular weight antigens of H. pylori.

We studied the prevalence of Helicobacter pylori in patients with leprosy and the effects of co-infection on the immune response to Helicobacter antigens in the polar groups of leprosy (lepromatous and tuberculoid). We showed that there is no difference in the prevalence of H. pylori in patients with leprosy as compared to a non-leprosy population. We also demonstrated that the immune response to low molecular weight H. pylori antigens (35, 26 and 19 kDa) differs in patients with lepromatous as compared to those with tuberculoid leprosy. In lepromatous leprosy, we show that there is a higher prevalence of the 35 and 26 kDa antigens, but a lower prevalence of the 19 kDa antigen. These immunological results are consistent with previous histopathological studies illustrating a more severe gastrointestinal inflammation in lepromatous patients; importantly, a response to the 35 kDa antigen is recognized as a marker for the development of ulcerative disease.

Amino acid sequence of B-cell epitope of N-terminal region of ESAT-6 Mycobacterium leprae role as specific antigen for diagnosis of leprosy.

The objective of this study was to find a specific B-cell epitope of N-terminal region of antigen L-ESAT-6 from leprosy patients, healthy individuals and healthy nurses working for more than 10 years in the leprosy ward of Dr. A. Rivai Abdullah Leprosy Hospital, Palembang, Indonesia. Fifty subjects were enrolled in this study, comprising 10 subjects with LL type leprosy, 10 subjects with BB type leprosy, 10 subjects with TT type leprosy, 10 healthy nurses from leprosy ward and 10 healthy individuals as control group. The amino acid sequence of residues 11-36 of the N-terminal region of L-ESAT-6 were divided into a series of 18 peptides each consisting of 9-mer peptides with an overlap of 8-mers and an offset of one amino acid. The series of 18 peptides were synthesized in the form of biotinylated peptides and used to screen sera of 50 subjects using an indirect ELISA method. Our study identified at the N-terminal of L-ESAT-6, LEQCQES, VNELQG and IDALLE as epitope marker for LL and BB type of leprosy, epitope marker for TT type of leprosy and for (protective epitope marker) healthy nurses working for more than 10 years in the leprosy ward, respectively. These antigens can be used in immunochromatographic test for the early diagnosis of leprosy.

HLA-DR and HLA-DQ alleles in patients from the south of Brazil: markers for leprosy susceptibility and resistance.

BACKGROUND: Many epidemiological studies have shown that the genetic factors of the host play a role in the variability of clinical response to infection caused by M. leprae. With the purpose of identifying genes of susceptibility, the present study investigated the possible role of HLA-DRB1 and DQA1/DQB1 alleles in susceptibility to leprosy, and whether they account for the heterogeneity in immune responses observed following infection in a Southern Brazilian population. METHODS: One hundred and sixty-nine leprosy patients and 217 healthy controls were analyzed by polymerase chain reaction amplification and reverse hybridization with sequence-specific oligonucleotide probes and sequence-specific primers (One Lambda, CA, USA). RESULTS: There was a positive association of HLA-DRB1*16 (*1601 and *1602) with leprosy per se (7.3% vs. 3.2%, P = 0.01, OR = 2.52, CI = 1.26-5.01), in accord with previous serological studies, which showed DR2 as a marker of leprosy. Although, HLA-DQA1*05 frequency (29.8% vs. 20.9%, P = 0.0424, OR = 1.61, CI = 1.09-2.39) was higher in patients, and HLA-DQA1*02 (3.0% vs. 7.5%, P = 0.0392, OR = 0.39, CI = 0.16 - 0.95) and HLA-DQA1*04 (4.0% vs. 9.1%, P = 0.0314, OR = 0.42, CI = 0.19 - 0.93) frequencies lower, P-values were not significant after the Bonferroni's correction. Furthermore, HLA-DRB1*1601 (9.0% vs. 1.8%; P = 0.0016; OR = 5.81; CI = 2.05-16.46) was associated with susceptibility to borderline leprosy compared to control group, and while HLA-DRB1*08 (11.2% vs. 1.2%; P = 0.0037; OR = 12.00; CI = 1.51 - 95.12) was associated with susceptibility to lepromatous leprosy, when compared to tuberculoid leprosy, DRB1*04 was associated to protection. CONCLUSION: These data confirm the positive association of HLA-DR2 (DRB1*16) with leprosy per se, and the protector effect of DRB1*04 against lepromatous leprosy in Brazilian patients.

A quantitative and morphometric study of tryptase-positive mast cells in cutaneous leprosy lesions.

While mast cells are known to induce differences in the matrix structures, microvascular patterns, and immune responses in a number of diseases, the possible role of mast cells in these same processes over the spectrum of leprosy has not yet been investigated. Thus, ascertaining the possible influence of mast cells in the outcome of the anti-leprosy response to Mycobacterium leprae is of major importance. In this study, 51 cutaneous biopsies of leprosy patients were stained with anti-tryptase antibody in order to quantify mast cells in leprosy lesions and compare the number and size of these cells in all the forms of leprosy. Biopsies were grouped according to an adapted Ridley-Jopling clinical-immunological classification (17 T, 17 B and 17 L). It was found that the L (lepromatous leprosy) group had the lowest dermal mast cell density values among the three groups studied. Furthermore, the average mast cell cross-sectional area was significantly higher in the L in comparison to the B (borderline-borderline) and T (tuberculoid) biopsies, suggesting mast cell functional differences within the groups. The higher mast cell density in the T and B groups was considered indirect evidence of the role of mast cells in the activated immune response to M. leprae infection.

Leukocyte antimicrobial function in patients with leprosy.

Patients with lepromatous leprosy are unresponsive to lepromin skin-test material and possess defective lymphocyte function in vitro, including impaired mitogenesis in response to antigens of Mycobacterium leprae. It has been claimed that their macrophages cannot digest M. leprae in vitro; such a defect could explain both lepromin nonreactivity and impaired lymphocyte function on the basis of failure of the afferent limb of the immune response (i.e., defective macrophage "processing" of M. leprae). The present studies indicate that macrophages from patients with lepromatous and tuberculoid leprosy and from normal donors do not differ in their ability to digest heat-killed M. leprae in vitro, or in their ability to sustain the viability of M. leprae in tissue culture; that monocytes, macrophages, and polymorphonuclear leukocytes of leprosy patients and controls possess equivalent microbicidal activity against Listeria monocytogenes, Escherichia coli, Proteus vulgaris, Staphylococcus aureus, and Candida albicans; and that polymorphonuclear leukocytes from patients with lepromatous leprosy iodinate ingested bacteria normally. Whether the basic immune defect leading to the development of lepromatous leprosy resides in the lymphocyte or in the macrophage remains to be determined. However, the present study shows that phagocytic cells from patients with either principal form of leprosy function normally in a variety of sophisticated tests of antimicrobial function.

Utility of measuring serum levels of anti-PGL-I antibody, neopterin and C-reactive protein in monitoring leprosy patients during multi-drug treatment and reactions.

OBJECTIVE: To verify the validity of measuring the levels of Mycobacterium leprae-specific anti-phenolic glycolipid (PGL)-I antibody, neopterin, a product of activated macrophages, and C-reactive protein (CRP), an acute phase protein, in serial serum samples from patients for monitoring the leprosy spectrum and reactions during the course of multi-drug treatment (MDT). METHODS: Twenty-five untreated leprosy patients, 15 multi-bacillary (MB) and 10 paucibacillary (PB), participated. Eight patients developed reversal reaction and five developed erythema nodosum leprosum (ENL) during follow-up. The bacterial index (BI) in slit-skin smears was determined at diagnosis and blood samples collected by venipuncture at diagnosis and after 2, 4, 6 and 12 months of MDT. PGL-I antibody and neopterin were measured by enzyme-linked immunosorbent assay, whereas the CRP levels were measured by the latex agglutination method. RESULTS: The levels of PGL-I antibodies and neopterin were higher in the sera of MB than PB patients, which correlated with the patients' BI. The serum levels of CRP did not differ significantly between the MB and PB patients. The serum levels of PGL-I and neopterin were no higher in reactional patients than non-reactional patients prone to such reactions. However, ENL patients had higher serum CRP levels than non-reactional MB patients. The serum PGL-I antibody levels declined significantly during MDT, in contrast to neopterin and CRP levels. CONCLUSION: Measuring the serum levels of PGL-I antibodies and neopterin appeared to be useful in distinguishing MB from PB patients, whereas monitoring the levels of PGL-I antibodies appeared to be useful in monitoring MB patients on MDT. Measuring serum CRP, although not useful in monitoring the patients, has limited significance in detecting ENL reactional patients.