RESUMEN

When organisms are exposed to external stimuli, misfolded proteins accumulate continuously, resulting in endoplasmic reticulum (ER) stress. Autophagy is of great significance for eliminating aggregated proteins and maintaining cellular homeostasis. However, the molecular mechanism of activating autophagy in response to ER stress in sea cucumber is remain unclear. In the current study, we demonstrated that the pathogen Vibrio splendidus can cause ER stress in Apostichopus japonicus coelomocytes and identified a Ca2+ binding partner calreticulin (designated as AjCRT), which increased with the occurrence of ER stress. The nucleotide sequence analysis showed that the open reading frame of AjCRT was 1242 bp and encoded a 413-amino-acid residue polyprotein with calreticulin domains. The spatial expression analysis revealed that AjCRT was ubiquitously expressed in all examined tissues with large magnitude in the coelomocytes and was minimally expressed in muscle. Furthermore, silencing AjCRT in vivo could significantly exacerbate ER stress induced by V. splendidus and resulted in the significant reduction of coelomocyte autophagy. These findings indicate a calreticulin-based mechanism that positively regulates autophagy in response to ER stress induced by pathogen infection. The results will provide a basis for understanding the way of host alleviating ER stress through autophagy, and pharmacological approaches may have potential for managing ER stress induced by pathogen and related cellular disorders.

Asunto(s)

Autofagia , Calreticulina , Estrés del Retículo Endoplásmico , Stichopus , Vibrio , Animales , Calreticulina/genética , Calreticulina/inmunología , Vibrio/fisiología , Stichopus/inmunología , Stichopus/genética , Stichopus/microbiología , Regulación de la Expresión Génica/inmunología , Secuencia de Aminoácidos , Filogenia , Perfilación de la Expresión Génica/veterinaria , Alineación de Secuencia/veterinaria , Inmunidad Innata/genética

RESUMEN

Immunoblotting, also termed western blotting, is a powerful method for detection and characterization of proteins separated by various electrophoretic techniques. The combination of sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE), having high separating power, immunoblotting to synthetic membranes, and detection with highly specific peptide antibodies, is especially useful for studying individual proteins in relation to cellular processes, disease mechanisms, etc. Here, we describe a protocol for the sequential detection of various forms of an individual protein using peptide antibodies, exemplified by the characterization of antibody specificity for different forms of the protein calreticulin by double SDS-PAGE immunoblotting.

Asunto(s)

Anticuerpos , Electroforesis en Gel de Poliacrilamida , Péptidos , Electroforesis en Gel de Poliacrilamida/métodos , Péptidos/química , Péptidos/inmunología , Anticuerpos/química , Anticuerpos/inmunología , Western Blotting/métodos , Humanos , Calreticulina/química , Calreticulina/inmunología , Calreticulina/metabolismo , Immunoblotting/métodos , Especificidad de Anticuerpos , Animales

RESUMEN

BACKGROUND: Primary Sjögren syndrome (pSjS) is one of the most prevalent systemic autoimmune diseases and characterized with hyperactivation of B cell and the abundant presence of autoantibodies in sera. The salivary gland epithelial cells (SGECs) release autoantigens to evoke autoimmunity through releasing elevated apoptosis or secreting autoantigen-containing exosomes, thus identifying autoantibodies directly to SGECs might provide insights into disease related biomarkers as well as further elucidating pathogenesis mechanisms. The present study was undertaken to identify autoantibodies to SGECs and to evaluate its clinical values in Chinese pSjS. METHODS: Cell-based indirect immunofluorescence and immunostaining, two-dimensional electrophoresis and liquid chromatograph-tandem mass spectrometry were conducted to identify the autoantibodies to human salivary gland cell line A253 in pSjS sera. Enzyme-linked immunosorbent assay (ELISA) was applied to identify autoantibody titer in pSjS cohort and healthy controls. The prevalence and clinical significance of the identified autoantibodies was further assessed in pSjS population. RESULTS: Anti-calreticulin (CALR) antibody was identified as a new autoantibody directly to SGECs in sera from pSjS patients. Anti-CALR antibody were detected in 37 of 120 pSjS patients (30.83 %) and 1 of 54 healthy controls (1.85 %). It was found in 40.85 % pSjS with anti-SSA positive, 53.85 % with anti-SSB positive, and 14.7 % in sero-negative pSjS. Anti-CALR antibody was associated with clinical manifestations including weight loss(p = 0.045), vasculitis (p = 0.031), and laboratory parameters including erythrocyte sedimentation rate (ESR) (r = 0.056, p = 0.021), Krebs von den Lungen-6 (KL-6) (r = 0.121, p = 0.035), IgG (r = 0.097, p < 0.001), IgG2 (r = 0.142, p = 0.022), IgG3 (r = 0.287, p < 0.001), fibrinogen (r = 0.084, p = 0.016), D-Dimer (r = 0.086, p = 0.012) and fibrinogen degradation production (r = 0.150, p = 0.002). The expression of CALR in salivary glands was related to lymphocytes infiltration into salivary glands in pSjS patients (r = 0.7076, p = 0.0034). CONCLUSION: To our knowledge, this was the first study to investigate the prevalence and clinical significance of anti-CALR antibody in Chinses pSjS patients. The present study identified an autoimmune antibody, anti-CALR antibody, as a good autoimmune biomarker for sero-negative pSjS.

Asunto(s)

Autoanticuerpos , Calreticulina , Glándulas Salivales , Síndrome de Sjögren , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Biomarcadores/sangre , Calreticulina/inmunología , China , Pueblos del Este de Asia , Ensayo de Inmunoadsorción Enzimática , Glándulas Salivales/inmunología , Síndrome de Sjögren/inmunología , Síndrome de Sjögren/sangre

RESUMEN

Helminths produce calreticulin (CRT) to immunomodulate the host immune system as a survival strategy. However, the structure of helminth-derived CRT and the structural basis of the immune evasion process remains unclarified. Previous study found that the tissue-dwelling helminth Trichinella spiralis produces calreticulin (TsCRT), which binds C1q to inhibit activation of the complement classical pathway. Here, we used x-ray crystallography to resolve the structure of truncated TsCRT (TsCRTΔ), the first structure of helminth-derived CRT. TsCRTΔ was observed to share the same binding region on C1q with IgG based on the structure and molecular docking, which explains the inhibitory effect of TsCRT on C1q-IgG-initiated classical complement activation. Based on the key residues in TsCRTΔ involved in the binding activity to C1q, a 24 amino acid peptide called PTsCRT was constructed that displayed strong C1q-binding activity and inhibited C1q-IgG-initiated classical complement activation. This study is the first to elucidate the structural basis of the role of TsCRT in immune evasion, providing an approach to develop helminth-derived bifunctional peptides as vaccine target to prevent parasite infections or as a therapeutic agent to treat complement-related autoimmune diseases.

Asunto(s)

Calreticulina , Complemento C1q , Evasión Inmune , Trichinella spiralis , Trichinella spiralis/inmunología , Complemento C1q/inmunología , Complemento C1q/metabolismo , Complemento C1q/química , Animales , Calreticulina/inmunología , Calreticulina/química , Calreticulina/metabolismo , Cristalografía por Rayos X , Unión Proteica , Simulación del Acoplamiento Molecular , Proteínas del Helminto/inmunología , Proteínas del Helminto/química , Activación de Complemento/inmunología , Inmunoglobulina G/inmunología , Humanos , Antígenos Helmínticos/inmunología , Antígenos Helmínticos/química , Triquinelosis/inmunología , Triquinelosis/parasitología , Vía Clásica del Complemento/inmunología , Conformación Proteica

RESUMEN

Natural killer (NK) cell kill infected, transformed and stressed cells when an activating NK cell receptor is triggered1. Most NK cells and some innate lymphoid cells express the activating receptor NKp46, encoded by NCR1, the most evolutionarily ancient NK cell receptor2,3. Blockage of NKp46 inhibits NK killing of many cancer targets4. Although a few infectious NKp46 ligands have been identified, the endogenous NKp46 cell surface ligand is unknown. Here we show that NKp46 recognizes externalized calreticulin (ecto-CRT), which translocates from the endoplasmic reticulum (ER) to the cell membrane during ER stress. ER stress and ecto-CRT are hallmarks of chemotherapy-induced immunogenic cell death5,6, flavivirus infection and senescence. NKp46 recognition of the P domain of ecto-CRT triggers NK cell signalling and NKp46 caps with ecto-CRT in NK immune synapses. NKp46-mediated killing is inhibited by knockout or knockdown of CALR, the gene encoding CRT, or CRT antibodies, and is enhanced by ectopic expression of glycosylphosphatidylinositol-anchored CRT. NCR1)-deficient human (and Nrc1-deficient mouse) NK cells are impaired in the killing of ZIKV-infected, ER-stressed and senescent cells and ecto-CRT-expressing cancer cells. Importantly, NKp46 recognition of ecto-CRT controls mouse B16 melanoma and RAS-driven lung cancers and enhances tumour-infiltrating NK cell degranulation and cytokine secretion. Thus, NKp46 recognition of ecto-CRT as a danger-associated molecular pattern eliminates ER-stressed cells.

Asunto(s)

Calreticulina , Estrés del Retículo Endoplásmico , Células Asesinas Naturales , Receptor 1 Gatillante de la Citotoxidad Natural , Animales , Humanos , Ratones , Alarminas/metabolismo , Calreticulina/inmunología , Calreticulina/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Inmunidad Innata , Sinapsis Inmunológicas , Células Asesinas Naturales/metabolismo , Neoplasias Pulmonares/metabolismo , Melanoma Experimental/metabolismo , Receptor 1 Gatillante de la Citotoxidad Natural/metabolismo , Virus Zika/fisiología

RESUMEN

OBJECTIVE: Pancreatic cancer stem-like cells (P-CSLCs) are thought to be associated with poor prognosis. Previously, we used proteomic analysis to identify a chaperone pro-phagocytic protein calreticulin (CALR) as a P-CSLC-specific protein. This study aimed to investigate the association between CALR and P-CSLC. METHODS: PANC-1-Lm cells were obtained as P-CSLCs from a human pancreatic cancer cell line, PANC-1, using a sphere induction medium followed by long-term cultivation on laminin. To examine the cancer stem cell properties, subcutaneous injection of the cells into immune-deficient mice and sphere formation assay were performed. Cell surface expression analysis was performed using flow cytometry. RESULTS: PANC-1-Lm showed an increased proportion of cell surface CALR-positive and side-population fractions compared with parental cells. PANC-1-Lm cells also had higher frequency of xenograft tumor growth and sphere formation than PANC-1 cells. Moreover, sorted CALRhigh cells from PANC-1-Lm had the highest sphere formation frequency among tested cells. Interestingly, the number of programmed death-ligand 1-positive cells among CALRhigh cells was increased as well, whereas that of human leukocyte antigen class I-positive cells decreased. CONCLUSION: In addition to the cancer stem cell properties, the P-CSLC, which showed elevated CALR expression on the cell surface, might be associated with evasion of immune surveillance.

Asunto(s)

Calreticulina/inmunología , Vigilancia Inmunológica/inmunología , Células Madre Neoplásicas/inmunología , Neoplasias Pancreáticas/inmunología , Animales , Calreticulina/metabolismo , Línea Celular Tumoral , Humanos , Masculino , Ratones Endogámicos NOD , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/inmunología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/inmunología , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Células Madre Neoplásicas/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Fosforilación , Trasplante Heterólogo

RESUMEN

Myeloproliferative neoplasms (MPNs) are characterized by a pathologic expansion of myeloid lineages. Mutations in JAK2, CALR and MPL genes are known to be three prominent MPN disease drivers. Mutant CALR (mutCALR) is an oncoprotein that interacts with and activates the thrombopoietin receptor (MPL) and represents an attractive target for targeted therapy of CALR mutated MPN. We generated a transgenic murine model with conditional expression of the human mutant exon 9 (del52) from the murine endogenous Calr locus. These mice develop essential thrombocythemia like phenotype with marked thrombocytosis and megakaryocytosis. The disease exacerbates with age showing prominent signs of splenomegaly and anemia. The disease is transplantable and mutCALR stem cells show proliferative advantage when compared to wild type stem cells. Transcriptome profiling of hematopoietic stem cells revealed oncogenic and inflammatory gene expression signatures. To demonstrate the applicability of the transgenic animals for immunotherapy, we treated mice with monoclonal antibody raised against the human mutCALR. The antibody treatment lowered platelet and stem cell counts in mutant mice. Secretion of mutCALR did not constitute a significant antibody sink. This animal model not only recapitulates human MPN but also serves as a relevant model for testing immunotherapeutic strategies targeting epitopes of the human mutCALR.

Asunto(s)

Anticuerpos Monoclonales/uso terapéutico , Calreticulina/antagonistas & inhibidores , Modelos Animales de Enfermedad , Células Madre Hematopoyéticas/metabolismo , Terapia Molecular Dirigida , Trombocitemia Esencial/terapia , Animales , Anticuerpos Monoclonales/inmunología , Plaquetas/inmunología , Plaquetas/metabolismo , Calreticulina/genética , Calreticulina/inmunología , Calreticulina/fisiología , Exones/genética , Mutación del Sistema de Lectura , Técnicas de Sustitución del Gen , Inmunoterapia , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Quimera por Radiación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Esplenomegalia/etiología , Trombocitemia Esencial/sangre , Trombocitemia Esencial/complicaciones , Trombocitemia Esencial/genética , Transcriptoma

RESUMEN

The Philadelphia-negative myeloproliferative neoplasms (MPNs) are malignancies of the hematopoietic stem cell (HSC) arising as a consequence of clonal proliferation driven by somatically acquired driver mutations in discrete genes (JAK2, CALR, MPL). In recent years, along with the advances in molecular characterization, the role of immune dysregulation has been achieving increasing relevance in the pathogenesis and evolution of MPNs. In particular, a growing number of studies have shown that MPNs are often associated with detrimental cytokine milieu, expansion of the monocyte/macrophage compartment and myeloid-derived suppressor cells, as well as altered functions of T cells, dendritic cells and NK cells. Moreover, akin to solid tumors and other hematological malignancies, MPNs are able to evade T cell immune surveillance by engaging the PD-1/PD-L1 axis, whose pharmacological blockade with checkpoint inhibitors can successfully restore effective antitumor responses. A further interesting cue is provided by the recent discovery of the high immunogenic potential of JAK2V617F and CALR exon 9 mutations, that could be harnessed as intriguing targets for innovative adoptive immunotherapies. This review focuses on the recent insights in the immunological dysfunctions contributing to the pathogenesis of MPNs and outlines the potential impact of related immunotherapeutic approaches.

Asunto(s)

Células Madre Hematopoyéticas/inmunología , Inmunoterapia/métodos , Inflamación/inmunología , Trastornos Mieloproliferativos/terapia , Linfocitos T/inmunología , Microambiente Tumoral/inmunología , Calreticulina/genética , Calreticulina/inmunología , Calreticulina/metabolismo , Células Madre Hematopoyéticas/metabolismo , Humanos , Inflamación/genética , Janus Quinasa 2/genética , Janus Quinasa 2/inmunología , Janus Quinasa 2/metabolismo , Mutación/inmunología , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/inmunología , Cromosoma Filadelfia , Linfocitos T/metabolismo , Microambiente Tumoral/genética

RESUMEN

Because of its capacity to increase a physiologic inflammatory response, to stimulate phagocytosis, to promote cell lysis and to enhance pathogen immunogenicity, the complement system is a crucial component of both the innate and adaptive immune responses. However, many infectious agents resist the activation of this system by expressing or secreting proteins with a role as complement regulatory, mainly inhibitory, proteins. Trypanosoma cruzi, the causal agent of Chagas disease, a reemerging microbial ailment, possesses several virulence factors with capacity to inhibit complement at different stages of activation. T. cruzi calreticulin (TcCalr) is a highly-conserved, endoplasmic reticulum-resident chaperone that the parasite translocates to the extracellular environment, where it exerts a variety of functions. Among these functions, TcCalr binds C1, MBL and ficolins, thus inhibiting the classical and lectin pathways of complement at their earliest stages of activation. Moreover, the TcCalr/C1 interaction also mediates infectivity by mimicking a strategy used by apoptotic cells for their removal. More recently, it has been determined that these Calr strategies are also used by a variety of other parasites. In addition, as reviewed elsewhere, TcCalr inhibits angiogenesis, promotes wound healing and reduces tumor growth. Complement C1 is also involved in some of these properties. Knowledge on the role of virulence factors, such as TcCalr, and their interactions with complement components in host-parasite interactions, may lead toward the description of new anti-parasite therapies and prophylaxis.

Asunto(s)

Calreticulina/inmunología , Complemento C1/inmunología , Interacciones Huésped-Parásitos/inmunología , Parásitos/patogenicidad , Animales , Activación de Complemento , Humanos , Evasión Inmune , Parásitos/inmunología , Trypanosoma cruzi/inmunología , Trypanosoma cruzi/patogenicidad , Factores de Virulencia/inmunología

RESUMEN

Calreticulin (CRT) is a multifunctional ubiquitous protein that is widely presented in all cells in eukaryotes except erythrocytes. CRT is well known for diverse cellular functions such as endoplasmic reticulum (ER)-specialized protein quality control during protein synthesis and folding, in-vivo Ca2+ homeostasis, antigen presentation, phagocytosis, wound-healing, proliferation, adhesion, and migration of cells. In the current study, we identified CRT from Hippocampus abdominalis (HaCRT) and analyzed expression profiles and functional properties. The cDNA sequence of HaCRT was identified with an open reading frame of 1226 bp. The molecular weight of HaCRT was estimated as 49 kDa. The in-silico study revealed conserved sequence arrangements such as two CRT signature motifs (5'-KHEQSIDCGGGYVKVF-3' and 5'-LMFGPDICG-3'), triplicate repeats (5'-IKDPEAKKPEDWD-3', 5'-IPDPDDTKPEDWD-3', 5'-IPDPDAKKPDDWD-3'), signal peptide and an ER-targeting 5'-KDEL-3' sequence of HaCRT. Close sequence similarity of HaCRT was observed with Hippocampus comes from phylogenetic analysis and pairwise sequence comparison. From quantitative polymerase chain reaction (qPCR) results, HaCRT was ubiquitously distributed in all tested tissues and expression levels of HaCRT were significantly modulated in blood, liver and gill tissues after stimulation with Streptococcus iniae, Edwardsiella tarda, polyinosinic:polycytidylic acid, and lipopolysaccharides. Bacterial- and pathogen-associated molecular patterns-binding activities were observed with recombinant HaCRT (rHaCRT). The treatment of murine macrophages with rHaCRT induced the expression of immune genes, such as tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), inducible nitric oxide synthase (iNOS), and interleukin-1ß (IL-1ß). Furthermore, rHaCRT exhibited wound-healing ability. Based on the results from the above study, we suggest that HaCRT play an indispensable role in the immunity of big-belly seahorses by recognition and elimination of pathogens as well as the tissue repairing process.

Asunto(s)

Calreticulina/genética , Calreticulina/inmunología , Proteínas de Peces/genética , Smegmamorpha/genética , Smegmamorpha/inmunología , Secuencia de Aminoácidos , Animales , Calreticulina/química , Proteínas de Peces/química , Proteínas de Peces/inmunología , Perfilación de la Expresión Génica/veterinaria , Filogenia

RESUMEN

CD47 acts as a "don't eat me" signal that protects cells from phagocytosis by binding and activating its receptor SIPRA on macrophages. CD47 suppresses multiple different pro-engulfment "eat me" signals, including immunoglobulin G (IgG), complement, and calreticulin, on distinct target cells. This complexity has limited understanding of how the "don't eat me" signal is transduced biochemically. Here, we utilized a reconstituted system with a defined set of signals to interrogate the mechanism of SIRPA activation and its downstream targets. CD47 ligation altered SIRPA localization, positioning SIRPA for activation at the phagocytic synapse. At the phagocytic synapse, SIRPA inhibited integrin activation to limit macrophage spreading across the surface of the engulfment target. Chemical reactivation of integrin bypassed CD47-mediated inhibition and rescued engulfment, similar to the effect of a CD47 function-blocking antibody. Thus, the CD47-SIRPA axis suppresses phagocytosis by inhibiting inside-out activation of integrin signaling in the macrophage, with implications to cancer immunotherapy applications.

Asunto(s)

Antígeno CD47/metabolismo , Integrinas/metabolismo , Macrófagos/inmunología , Fagocitosis/inmunología , Receptores Inmunológicos/metabolismo , Animales , Calreticulina/inmunología , Línea Celular , Proteínas del Sistema Complemento/inmunología , Células HEK293 , Humanos , Inmunoglobulina G/inmunología , Activación de Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosfatidilserinas/inmunología , Células RAW 264.7 , Transducción de Señal/inmunología

RESUMEN

American Trypanosomiasis, a parasitic disease produced by Trypanosoma cruzi (T. cruzi), endemic in Latin America, infects about 6 million people. During the chronic stage of the infection, approximately 30% of infected people will develop Chagas Disease, the clinical manifestation. Few decades ago it was reported that, during the chronic stage, the parasite interferes with the development of solid tumors. However, the identification of parasite molecules responsible for such effects remained elusive. Years later, we described T.cruzi Calreticulin (TcCalr), an endoplasmic reticulum resident chaperone that infective trypomastigotes translocate to the parasite exterior, where it displays anticomplement activities. Most likely, at least some of these activities are related with the antitumor properties of TcCalr, as shown in in vitro, ex vivo, in ovum, and in vivo models. In this context we, we have seen that in vivo subcutaneous peritumoral inoculation of rTcCalr enhances local infiltration of T cells and slows tumor development. Based on these precedents, we propose that in vitro treatment of a mammary adenocarcinoma (TA3 cell line) with rTcCalr, will enhance tumor immunogenicity. In agreement with this proposal, we have shown that: i). rTcCalr binds to TA3 cells in a concentration-dependent fashion, ii). C1q binds to TA3 cells in an rTcCalr-dependent fashion, confirmed by the reversion attained using anti-TcS (a central TcCalr domain that binds C1) F(ab')2 antibody fragments, iii). incubation of TA3 cells with rTcCalr, promotes cell phagocytosis by murine macrophages and, iv). rTcCalr decreases the membrane expression of MHC class II, m-Dectin-1, Galectin-9 and PD-L1, while increasing the expression of Rae-1γ. In synthesis, herein we show that in vitro treatment of a murine mammary adenocarcinoma with rTcCalr enhances phagocytosis and modulates the expression of a variety of membrane molecules that correlates with increased tumor immunogenicity.

Asunto(s)

Adenocarcinoma/inmunología , Antígenos de Protozoos/inmunología , Calreticulina/inmunología , Neoplasias Mamarias Experimentales/inmunología , Animales , Línea Celular Tumoral , Ratones , Fagocitosis/inmunología , Trypanosoma cruzi

RESUMEN

The scavenger receptor SR-F1 binds to and mediates the internalization of a wide range of ligands, and is involved in several immunological processes. We produced recombinant SR-F1 ectodomain and fragments deleted from the last 2 or 5 C-terminal epidermal growth factor-like modules and investigated their role in the binding of acetylated low density lipoprotein (AcLDL), complement C1q, and calreticulin (CRT). C1q measured affinity was in the 100 nM range and C1q interaction occurs via its collagen-like region. We identified two different binding regions on SR-F1: the N-terminal moiety interacts with C1q and CRT whereas the C-terminal moiety binds AcLDL. The role of SR-F1 N-linked glycans was also tested by mutating each of the three glycosylated asparagines. The three mutants retained binding activities for both AcLDL and C1q. A stable THP-1 cell line overexpressing SR-F1 was generated and C1q was shown to bind more strongly to the surface of SR-F1 overexpressing macrophages, with C1q/SR-F1 colocalization observed in some membrane areas. We also observed a higher level of CRT internalization for THP-1 SR-F1 cells. Increasing SR-F1 negatively modulated the uptake of apoptotic cells. Indeed, THP-1 cells overexpressing SR-F1 displayed a lower phagocytic capacity as compared with mock-transfected cells, which could be partially restored by addition of C1q in the extracellular milieu. Our data shed some light on the role of SR-F1 in efferocytosis, through its capacity to bind C1q and CRT, two proteins involved in this process.

Asunto(s)

Apoptosis/inmunología , Complemento C1q/inmunología , Macrófagos/inmunología , Fagocitosis/inmunología , Receptores Depuradores de Clase F/inmunología , Calreticulina/inmunología , Comunicación Celular/inmunología , Complemento C1q/metabolismo , Humanos , Receptores Depuradores de Clase F/metabolismo , Células THP-1

RESUMEN

To successfully infect, Trypanosoma cruzi evades and modulates the host immune response. T. cruzi calreticulin (TcCalr) is a multifunctional, endoplasmic reticulum (ER)-resident chaperone that, translocated to the external microenvironment, mediates crucial host-parasite interactions. TcCalr binds and inactivates C1 and mannose-binding lectin (MBL)/ficolins, important pattern- recognition receptors (PRRs) of the complement system. Using an apoptotic mimicry strategy, the C1-TcCalr association facilitates the infection of target cells. T. cruzi infection also seems to confer protection against tumorigenesis. Thus, recombinant TcCalr has important antiangiogenic properties, detected in vitro, ex vivo, and in ovum, most likely contributing at least in part, to its antitumor properties. Consequently, TcCalr is useful for investigating key issues of host-parasite interactions and possible new immunological/pharmacological interventions in the areas of Chagas' disease and experimental cancer.

Asunto(s)

Calreticulina/inmunología , Carcinogénesis/inmunología , Enfermedad de Chagas/complicaciones , Enfermedad de Chagas/inmunología , Interacciones Huésped-Parásitos/inmunología , Neoplasias/etiología , Trypanosoma cruzi/patogenicidad , Animales , Enfermedad de Chagas/parasitología , Enfermedad de Chagas/patología , Humanos , Evasión Inmune/inmunología , Neoplasias/inmunología , Trypanosoma cruzi/fisiología , Factores de Virulencia/inmunología

RESUMEN

Tumor cells treated by immunogenic cell death (ICD) inducers emit danger associated molecular patterns (DAMP), including but not limited to calreticulin (CALR), which translocates from the ER lumen to the surface of the cellular membrane where it serves as de novo uptake signal for antigen presenting cells of the immune system. CALR is exposed at an early stage of ICD and dictates tumor antigen transfer and therefore the immunogenicity of cancer cell death. Here, we provide a bi-color flow cytometry protocol for the quantification of ICD-associated CALR cell surface exposure in fixed samples. As compared to the detection of surface exposed CALR by confocal microscopy, the present flow cytometry-based analysis is cost-efficient and does not require sophisticated equipment. Moreover, the staining panel can be extended to a multicolor analysis for the parallel assessment of additional parameters.

Asunto(s)

Antineoplásicos Inmunológicos/farmacología , Calreticulina/análisis , Citometría de Flujo/métodos , Muerte Celular Inmunogénica/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Animales , Calreticulina/inmunología , Línea Celular Tumoral , Humanos , Ratones , Microscopía Confocal/métodos , Neoplasias/inmunología , Neoplasias/patología , Coloración y Etiquetado/métodos

RESUMEN

Immunogenic cell death (ICD), a functionally peculiar type of apoptosis, represents a unique way to deliver danger-associated molecular patterns (DAMPs) to the tumor microenvironment. Once emitted by dying cancer cells, DAMPs orchestrate antigen-specific immune responses by acting on both innate and adaptive components of the immune system. Accumulating preclinical and clinical evidence indicates that one of these DAMPs, calreticulin (CALR) represents a novel powerful prognostic biomarker, reflecting the activation of a clinically relevant anticancer immune response in different cancer malignancies. Therefore, the assessment of CALR emission can provide a therapeutic tool for the stratification of cancer patients and the identification of individuals that are intrinsically capable to respond to a particular treatment. Here we describe methods for the quantification of CALR exposure in the tumor microenvironment of cancer patients by flow cytometry and immunohistochemistry.

Asunto(s)

Calreticulina/inmunología , Citometría de Flujo/métodos , Muerte Celular Inmunogénica , Inmunohistoquímica/métodos , Neoplasias/inmunología , Animales , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/inmunología , Calreticulina/análisis , Humanos , Neoplasias/patología , Microambiente Tumoral

RESUMEN

Calreticulin (CALR) has anti-tumor effects by increasing dendritic cell maturation and tumor antigen presentation. However, whether CALR affects macrophages and modulates progression of acute respiratory distress syndrome/acute lung injury (ARDS/ALI) remains unknown. In this study, we discovered that CALR protein was highly expressed in the mice with LPS-induced ALI and CALR expression level was positively correlated to the severity of ALI. Commercial anti-CALR antibody (aCALR) can neutralize recombinant CALR (rCALR) and suppress the expression of TNF-alpha and IL-6 in the rCALR-treated macrophages. Blocking CALR activity by intraperitoneal (i.p.) administration of aCALR significantly suppressed ALI, accompanied with lower total cell counts, neutrophil and T cell infiltration in bronchoalveolar lavage (BAL) and lung tissues. The expression of CXCL15, IL-6, IL-1beta, TNF-alpha, and CALR were significantly reduced, in association with more polarization of Siglec F+CD206+M2 subtype macrophages in the aCALR-treated mice. Pre-depletion of circulating monocytes did not abolish the aCALR-mediated suppression of ALI. Further analysis in bone marrow-derived macrophages (BMDMs) showed that aCALR suppressed the expression of CD80, IL-6, IL-1beta, IL-18, NLRP3, and p-p38 MAPK; but enhanced the expression of CD206 and IL-10. In addition, we observed more expression and phosphorylation of STAT6 in the aCALR-treated BMDM. Lack of STAT6 resulted in comparable and slightly higher expression of CALR, TNF-alpha and IL-6 in the aCALR-treated STAT6-/- BMDMs than the untreated cells. Therefore, we conclude that CALR is a novel biomarker in the evaluation of ALI. Blocking CALR activity by aCALR effectively suppressed ALI independent of circulating monocytes. Siglec F+CD206+M2 subtype macrophages and p38 MAPK/STAT6 signaling pathway played important role in the immune regulation of aCALR. Blocking CALR activity is a promising therapeutic approach in the treatment of ARDS/ALI.

Asunto(s)

Lesión Pulmonar Aguda/tratamiento farmacológico , Anticuerpos/administración & dosificación , Anticuerpos/inmunología , Calreticulina/antagonistas & inhibidores , Calreticulina/inmunología , Polaridad Celular/efectos de los fármacos , Macrófagos/inmunología , Lesión Pulmonar Aguda/sangre , Lesión Pulmonar Aguda/inducido químicamente , Animales , Calreticulina/sangre , Polaridad Celular/inmunología , Citocinas/sangre , Modelos Animales de Enfermedad , Inyecciones Intraperitoneales , Lipopolisacáridos/efectos adversos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Síndrome de Dificultad Respiratoria/tratamiento farmacológico , Factor de Transcripción STAT6/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo

RESUMEN

Tumor-associated macrophages (TAM) promote triple-negative breast cancer (TNBC) progression. Here, we report BRCA1-IRIS-overexpressing (IRISOE) TNBC cells secrete high levels of GM-CSF in a hypoxia-inducible factor-1α (HIF1α)- and a NF-κB-dependent manner to recruit macrophages to IRISOE cells and polarize them to protumor M2 TAMs. GM-CSF triggered TGFß1 expression by M2 TAMs by activating STAT5, NF-κB, and/or ERK signaling. Despite expressing high levels of TGFß1 receptors on their surface, IRISOE TNBC cells channeled TGFß1/TßRI/II signaling toward AKT, not SMAD, which activated stemness/EMT phenotypes. In orthotopic and syngeneic mouse models, silencing or inactivating IRIS in TNBC cells lowered the levels of circulating GM-CSF, suppressed TAM recruitment, and decreased the levels of circulating TGFß1. Coinjecting macrophages with IRISOE TNBC cells induced earlier metastasis in athymic mice accompanied by high levels of circulating GM-CSF and TGFß1. IRISOE TNBC cells expressed low levels of calreticulin (the "eat me" signal for macrophages) and high levels of CD47 (the "do not eat me" signal for macrophages) and PD-L1 (a T-cell inactivator) on their surface. Accordingly, IRISOE TNBC tumors had significantly few CD8+/PD-1+ cytotoxic T cells and more CD25+/FOXP3+ regulatory T cells. These data show that the bidirectional interaction between IRISOE cells and macrophages triggers an immunosuppressive microenvironment within TNBC tumors that is favorable for the generation of immune-evading/stem-like/IRISOE TNBC metastatic precursors. Inhibiting this interaction may inhibit disease progression and enhance patients' overall survival. SIGNIFICANCE: The BRCA1-IRIS oncogene promotes breast cancer aggressiveness by recruiting macrophages and promoting their M2 polarization.

Asunto(s)

Proteína BRCA1/metabolismo , Macrófagos/inmunología , Neoplasias de la Mama Triple Negativas/inmunología , Escape del Tumor/inmunología , Microambiente Tumoral/inmunología , Animales , Proteína BRCA1/genética , Calreticulina/inmunología , Calreticulina/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Macrófagos/metabolismo , Ratones , Células Madre Neoplásicas/inmunología , Células Madre Neoplásicas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transducción de Señal/inmunología , Análisis de Supervivencia , Linfocitos T Citotóxicos/inmunología , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta1/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/mortalidad , Neoplasias de la Mama Triple Negativas/patología , Microambiente Tumoral/genética , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Recombinant calreticulin from Trypanosoma cruzi (rTcCalr), the parasite responsible for Chagas' disease, binds to Canine Transmissible Venereal Tumor (CTVT) cells from primary cultures and to a canine mammary carcinoma cell line. A Complement-binding assay indicated that interaction of the first component C1q with these tumor cells operated independently of the rTcCalr-presence. This apparent independence could be explained by the important structural similarities that exist among rTcCarl, endogenous normal canine and/or mutated calreticulins present in several types of cancer. In phagocytosis assays, tumor cells treated with rTcCalr were readily engulfed by macrophages and, co-cultured with DCs, accelerated their maturation. In addition, DCs maturation, induced by tumor cells co-cultured with rTcCalr, activated T cells more efficiently than DCs, treated or not with LPS. In an apparent paradox, a decrease in MHC Class I expression was observed when these tumor cells were co-cultivated with rTcCalr. This decrease may be related to a down regulation signaling promoting the rescue of MHC I. Possibly, these in vitro assays may be valid correlates of in vivo sceneries. Based on these results, we propose that rTcCalr improves in vitro the immunogenicity of two widely different tumor cell lines, thus suggesting that the interesting properties of rTcCalr to boost immune responses warrant future studies.

Asunto(s)

Formación de Anticuerpos/inmunología , Biomarcadores de Tumor/inmunología , Calreticulina/inmunología , Competencia Celular/inmunología , Enfermedad de Chagas/inmunología , Proteínas Protozoarias/inmunología , Trypanosoma cruzi/inmunología , Animales , Línea Celular Tumoral , Enfermedad de Chagas/parasitología , Proteínas del Sistema Complemento/inmunología , Perros , Regulación hacia Abajo/inmunología , Femenino , Humanos , Activación de Linfocitos/inmunología , Macrófagos/inmunología , Macrófagos/parasitología , Masculino , Ratones

RESUMEN

Before 2013, the diagnosis of about 30% to 45% cases of primary myelofibrosis (PMF) and essential thrombocythemia (ET) posed a diagnostic difficulty because of the missing reliable clonal marker. Calreticulin (CALR) mutation was identified as a recurrent mutation in about 60% to 88% of JAK2/MPL-negative PMF and ET. Molecular methods like Sanger sequencing and polymerase chain reaction (PCR) are considered gold standard, but they have limited availability, complex techniques, and labor intensive. In contrast to molecular methods, immunohistochemistry (IHC) is a widely available, rapid, simple, and cost-effective option. There are only few studies evaluating the utility of IHC for CALR mutation detection. Hence, we studied the role of IHC in CALR mutation detection and compared it with PCR. Thirty-one JAK2V617F-negative PMF and ET were evaluated for CALR mutation status. PCR was done and interpreted by comparing bands with the expected product size. The bone marrow biopsy was simultaneously put up for IHC using antimutated CALR monoclonal antibody (CAL2). CALR mutation was detected in 64.5% (20/31) cases. Prevalence of CALR mutation in JAK2-negative PMF and ET was 60.9% (14/23) and 75% (6/8), respectively. Sensitivity, specificity, positive predictive value, and negative predictive value of IHC analyzed were 89.4%, 100%, 100%, and 84.6%, respectively. A very good level of agreement (κ=0.86) was observed between PCR and IHC. We suggest that IHC is the best screening test to detect CALR mutation in resource limited countries with limited availability and affordability of molecular methods.

Asunto(s)

Calreticulina/genética , Calreticulina/inmunología , Inmunohistoquímica/métodos , Mielofibrosis Primaria/genética , Trombocitemia Esencial/genética , Adolescente , Adulto , Anciano , Anticuerpos Monoclonales , Biopsia , Médula Ósea/metabolismo , Humanos , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa/métodos , Mielofibrosis Primaria/metabolismo , Trombocitemia Esencial/metabolismo