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.

Mycobacterium lepraemurium uses TLR-6 and MR, but not lipid rafts or DC-sign, to gain access into mouse macrophages.

OBJECTIVE/BACKGROUND: Mycobacterium lepraemurium (MLM), the etiologic agent of murine leprosy, is an intracellular parasite of macrophages; the mechanism used by this bacterium to enter macrophages is not known. The fate of the MLM phagosome inside macrophages is also unknown. This study was conducted to investigate how MLM enters macrophages and to define the maturation process of MLM phagosome inside macrophages. MATERIALS AND METHODS: Peritoneal macrophages were incubated in the presence of mannan-bovine serum albumin (BSA), and antibodies to known macrophage receptors, including, anti-FcγRIII/RII (anti-CD16/32), anti-CD35 (anti-CR1), anti-TLR2, anti-TLR4, anti-TLR6, anti-CD14, and anti-dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN). Then, macrophages were challenged with Iris Fuchsia-stained MLM, at a multiplicity of infection of 50:1. The blocking effect of the antibodies (and mannan-BSA) used was analyzed using direct microscopy and flow cytometry. The maturation process of MLM phagosomes was visualized by their interaction with antibodies to Rab5, Rab7, proton ATPase, and cathepsin D, by confocal microscopy. RESULTS: Only mannan-BSA and anti-TLR6 antibody significantly blocked the entry of MLM into macrophages. None of the other antibodies, including that for DC-SIGN, meaningfully inhibited the endocytic process. We also found that MLM is a fusiogenic mycobacterium. This was deduced from the orderly association of MLM phagosomes with Rab5, Rab7, Proton ATPase, and lysosomes (cathepsin D). CONCLUSION: Fusion of MLM phagosomes with lysosomes seems to be a necessary event for the intracellular multiplication of MLM; similar to Mycobacterium leprae, this microorganism hardly grows on artificial, synthetic, bacteriologic media.

Expression of CD64 on Circulating Neutrophils Favoring Systemic Inflammatory Status in Erythema Nodosum Leprosum.

Erythema Nodosum Leprosum (ENL) is an immune reaction in leprosy that aggravates the patient´s clinical condition. ENL presents systemic symptoms of an acute infectious syndrome with high leukocytosis and intense malaise clinically similar to sepsis. The treatment of ENL patients requires immunosuppression and thus needs to be early and efficient to prevent both disabilities and permanent nerve damage. Some patients experience multiple episodes of ENL and prolonged use of immunosuppressive drugs may lead to serious adverse effects. Thalidomide treatment is extremely effective at ameliorating ENL symptoms. Several mechanisms have been proposed to explain the efficacy of thalidomide in ENL, including the inhibition of TNF production. Given its teratogenicity, thalidomide is prohibitive for women of childbearing age. A rational search for molecular targets during ENL episodes is essential to better understand the disease mechanisms involved, which may also lead to the discovery of new drugs and diagnostic tests. Previous studies have demonstrated that IFN-γ and GM-CSF, involved in the induction of CD64 expression, increase during ENL. The aim of the present study was to investigate CD64 expression during ENL and whether thalidomide treatment modulated its expression. Leprosy patients were allocated to one of five groups: (1) Lepromatous leprosy, (2) Borderline leprosy, (3) Reversal reaction, (4) ENL, and (5) ENL 7 days after thalidomide treatment. The present study demonstrated that CD64 mRNA and protein were expressed in ENL lesions and that thalidomide treatment reduced CD64 expression and neutrophil infiltrates-a hallmark of ENL. We also showed that ENL blood neutrophils exclusively expressed CD64 on the cell surface and that thalidomide diminished overall expression. Patient classification based on clinical symptoms found that severe ENL presented high levels of neutrophil CD64. Collectively, these data revealed that ENL neutrophils express CD64, presumably contributing to the immunopathogenesis of the disease.

Differential trafficking of TLR1 I602S underlies host protection against pathogenic mycobacteria.

We recently identified I602S as a frequent single-nucleotide polymorphism of human TLR1 that greatly inhibits cell surface trafficking, confers hyporesponsiveness to TLR1 agonists, and protects against the mycobacterial diseases leprosy and tuberculosis. Because mycobacteria are known to manipulate the TLR system to their advantage, we hypothesize that the hyporesponsive 602S variant may confer protection by enabling the host to overcome this immune subversion. We report that primary human monocytes and macrophages from homozygous TLR1 602S individuals are resistant to mycobacterial-induced downregulation of macrophage MHC class II, CD64, and IFN-γ responses compared with individuals who harbor the TLR1 602I variant. Additionally, when challenged with mycobacterial agonists, macrophages from TLR1 602S/S individuals resist induction of host arginase-1, an enzyme that depletes cellular arginine stores required for the production of antimicrobial reactive nitrogen intermediates. The differences in cell activation mediated by TLR1 602S and TLR1 602I are observed upon stimulation with soluble mycobacterial-derived agonists but not with whole mycobacterial cells. Taken together, these results suggest that the TLR1 602S variant protects against mycobacterial disease by preventing soluble mycobacterial products, perhaps released from granulomas, from disarming myeloid cells prior to their encounter with whole mycobacteria.

Hypothesis: an alternative pathway for the regulation of inflammation.

Regulation of inflammation is a crucial event since its alteration, such as in sepsis and chronic autoimmune (i.e. rheumatoid arthritis, lupus erythematosus) or infectious diseases (i.e. tuberculosis, leprosy), determines severe tissue damage. Although there is a general consensus that regulation of inflammation results from a balance between proinflammatory and antiinflammatory pathways, we arrived at the conclusion that well known chemoattractants/proinflammatory molecules such as bacterial formyl peptides or immune complexes (IC), could induce, paradoxically, strong antiinflammatory effects. Thus, we demonstrated that N-formyl-methionyl-leucyl-phenylalanine (FMLP) exerted a drastic antiinflammatory effect, inhibiting the secretion of tumor necrosis alpha (TNF-alpha) induced by lipopolysaccharides, a potent TNF-alpha inducer. We also determined that in human neutrophils FMLP and IC induced the downregulation of receptors for the Fc portion of IgG (FcgammaRII and FcgammaRIIIB). Moreover, FMLP inhibited interferon gamma (IFN-gamma)-induced FcgammaRI expression and IC downregulate class II molecules of the major histocompatibility complex on monocytes. Part of these effects were mediated by the release of aspartic-, serin-, or metalloproteases. All these results favor the postulation of a new concept on the regulation of inflammation carried out through an alternative and non conventional pathway, in which a chemoattractant/proinflammatory agent could, under certain circumstances, act as an antiinflammatory molecule.

Hypotesis: an alternative pathway for the regulation of inflammation

Hipótesis: una vía alternativa de regulación de procesos inflamatorios. La regulación de mecanismos inflamatorios es un evento crucial debido a que una alteración de los mismos, como sucede por ejemplo, en la sepsis, en enfermedades autoinmunes crónicas (artritis reumatoidea, lupus eritematoso) o en enfermedades infecciosas (tuberculosis, lepra), genera daños tisulares severos. Aunque hay un consenso general de que la regulación de procesos inflamatorios resulta de un balance entre vías proinflamatorias y antiinflamatorias, nosotros arribamos a la conclusión de que moléculas quimioatractantes / proinflamatorias como, por ejemplo, péptidos formilados bacterianos o complejos inmunes (CI), pueden también inducir, paradójicamente, potentes efectos ntiinflamatorios. Así, demostramos que el péptido formilado prototipo N-formilmetionil- leucil-fenilalanina (FMLP), ejerce un drástico efecto antiinflamatorio, inhibiendo la secreción de factor de necrosis tumoral alfa (TNF-α) inducido por lipopolisacáridos, un potente inductor de la secreción de TNF-α. También determinamos que el FMLP y los CI inducen la disminución de la expresión de receptores para el fragmento Fc de IgG (FcγRII and FcγRIIIB) en neutrófilos humanos. Más aún, el FMLP inhibe la inducción de la expresión de los FcγRI por interferón gamma (IFN-γ) y los CI disminuyen la expresión de moléculas de clase II del complejo mayor de histocompatibilidad en monocitos humanos. Parte de esos efectos fueron mediados por la liberación de aspártico-, serino-, o metaloproteasas. Todos estos resultados nos permiten especular sobre un nuevo concepto en el cual la regulación de los procesos inflamatorios también puede llevarse a cabo por una vía alternativa, no convencional, en la cual un agente quimioatractante / proinflamatorio, bajo determinadas circunstancias, puede actuar como una molécula antiinflamatoria.

Hypotesis: an alternative pathway for the regulation of inflammation

Hipótesis: una vía alternativa de regulación de procesos inflamatorios. La regulación de mecanismos inflamatorios es un evento crucial debido a que una alteración de los mismos, como sucede por ejemplo, en la sepsis, en enfermedades autoinmunes crónicas (artritis reumatoidea, lupus eritematoso) o en enfermedades infecciosas (tuberculosis, lepra), genera daños tisulares severos. Aunque hay un consenso general de que la regulación de procesos inflamatorios resulta de un balance entre vías proinflamatorias y antiinflamatorias, nosotros arribamos a la conclusión de que moléculas quimioatractantes / proinflamatorias como, por ejemplo, péptidos formilados bacterianos o complejos inmunes (CI), pueden también inducir, paradójicamente, potentes efectos ntiinflamatorios. Así, demostramos que el péptido formilado prototipo N-formilmetionil- leucil-fenilalanina (FMLP), ejerce un drástico efecto antiinflamatorio, inhibiendo la secreción de factor de necrosis tumoral alfa (TNF-α) inducido por lipopolisacáridos, un potente inductor de la secreción de TNF-α. También determinamos que el FMLP y los CI inducen la disminución de la expresión de receptores para el fragmento Fc de IgG (FcγRII and FcγRIIIB) en neutrófilos humanos. Más aún, el FMLP inhibe la inducción de la expresión de los FcγRI por interferón gamma (IFN-γ) y los CI disminuyen la expresión de moléculas de clase II del complejo mayor de histocompatibilidad en monocitos humanos. Parte de esos efectos fueron mediados por la liberación de aspártico-, serino-, o metaloproteasas. Todos estos resultados nos permiten especular sobre un nuevo concepto en el cual la regulación de los procesos inflamatorios también puede llevarse a cabo por una vía alternativa, no convencional, en la cual un agente quimioatractante / proinflamatorio, bajo determinadas circunstancias, puede actuar como una molécula antiinflamatoria.(AU)

Lysis of autologous macrophages pulsed with hsp10 from Mycobacterium leprae is associated to the absence of bacilli in leprosy.

Peripheral blood mononuclear cells from leprosy patients and normal individuals were analysed for their ability to lyse autologous macrophages pulsed with the Mycobacterium leprae 10 kDa heat shock protein (hsp10), an antigen considered to have an important role in the protective responses in leprosy. Strong cytotoxic responses, with an involvement of gammadelta T and class-I and class-II restricted alphabeta T cells and/or CD16+56+ cells, were observed in normal individuals, paucibacillary (PB) and those multibacillary (MB) patients with undetectable bacillary load. On the contrary, only a weak class-II restricted cytotoxic response was observed in those MB patients with positive bacillary load (MB(+)). Simultaneous addition of IFNgamma plus TNFalpha and IL-12 during hsp10 stimulation could partially upregulate the low cytotoxic response observed in MB(+) by enhancing class-II restricted T cell activity and by development of gammadelta T and/or CD16+56+ cell activity. Our results suggest that the ability to mount an effective cytotoxic response against hsp10-pulsed macrophages in leprosy patients is closely related to the patient's bacterial load and not to the clinical form of the disease.

T lymphocyte subpopulations in leprosy patients and their relation with circulating immune complexes.

The possible relationship between circulating immune complexes (CIC) and peripheral T lymphocyte populations was studied in thirteen active multibacillary leprosy (10 lepromatous--LL--and 3 borderline lepromatous--BL--) and 19 matched controls. Theophylline-resistant T cells (The-R, a lymphocyte subpopulation displaying helper activity on B cells) and total T cells were assessed by means of the E rosette technique, with and without previous theophylline incubation, 1h 37 degrees C, respectively. CIC were quantified by 125I-C1q binding test. Although leprosy patients showed a statistical non significant light depression in total T cells the remarkable variability in circulating levels of The-R T cells enabled us to separate them into two well delineated groups (in relation to this variable p less than 0.001) with no difference in age, sex and bacteriologic state: a) leprosy patients with The-R T cells proportionally conserved (6LL and 2BL); b) leprosy patients with The-R T cells proportionally depressed (4LL and 1BL). Patients belonging to the latter group showed the highest statistically significant levels of CIC. Even though we do not discard an unknown factor being responsible for our findings, we believe that this inverse relationship between elevated CIC and depressed The-R circulating T cells might be representing a lower helper activity on antibody synthesis intending to reduce its excessive production.

Lepromin-induced lymphoproliferative response of experimental leprosy monkeys: regulatory role of monocyte and lymphocyte subsets.

We investigated the immunological status of seven normal, control Mangabey monkeys and 23 Mangabey monkeys experimentally inoculated with Mangabey-origin Mycobacterium leprae. Clinically, these monkeys were divided into three broad groups: a recently inoculated group, a resistant group, and a susceptible group. The resistant group included 11 monkeys, seven of which showed no clinical sign of disease to date and four of which had shown local disease that partially regressed spontaneously. The susceptible group included eight monkeys, five of which have disseminated disease and three with local but stable disease. When peripheral blood mononuclear cells of these monkeys were cultured with Dharmendra-type human lepromin, one of seven normal monkeys, four of four of the recently inoculated group, seven of 10 resistant monkeys, and three of eight susceptible monkeys showed significant responses. In this experimental monkey model, we studied possible regulatory mechanisms by using OKT4- and OKT8-enriched lymphocytes, and Fc receptor-positive (FcR+) and FcR- monocyte (M phi) subsets. The OKT4+ subset was the main lepromin-responsive cell type. High percentages of OKT8+ cells showed a good negative correlation with the lymphoproliferative responses of T-enriched cells supplemented with unfractionated M phi. But the depletion of OKT8+ cells could not increase the response of nonresponding monkeys' lymphocytes. The resistant group and susceptible group did not differ in their percentages of OKT8+ cells. Because OKT8+ cells negatively regulate the response of lymphocytes and OKT4+ cells are the main responding cells, OKT8+ cells are phenotypically and functionally suppressor cells and OKT4+ cells are the helper/inducer cell population in this system. The FcR- M phi population mainly includes antigen-presenting activity, but high percentages of FcR- M phi showed a significant negative correlation with lymphoproliferative responses in the resistant group. A weak but significant lymphocyte response to Dharmendra lepromin was obtained by depleting FcR+ M phi from cultures of some susceptible monkeys, whereas lymphocytes of other susceptible monkeys remained unresponsive to lepromin. By these criteria, we could find an array of immunological defects in monkeys with experimental leprosy. The data suggest that some immunological defects may exist in the OKT4+ lymphocytes or FcR- M phi of leprosy monkeys.