Expression and variation of lymphocyte scavenger receptors in inflammatory diseases and cancer

[eng] The immune system comprises cellular and humoral components that protect the organism against external and internal aggressions, such as pathogens and cancer. Therefore, it can distinguish healthy, self structures from exogenous or damaged structures. This is achieved with the so-called pattern recognition receptors (PRR) belonging to several protein superfamilies. These are specialized in the recognition of microbial-associated molecular patterns (MAMP) and damage-associated molecular patterns (DAMP). The present thesis focuses on a superfamily of family PRRs known as scavenger receptors, in particular, the CD5 and CD6 lymphocyte receptors. CD5 and CD6 have extracellular domains with scavenger receptor cysteine-rich (SRCR) structures. They are expressed on all T lymphocytes and a subset of B lymphocytes (B1a) involved in the production of polyreactive natural antibodies. Both receptors bind to the antigen-specific receptor complex of T (TCR) and B (BCR) cells. They transduce signals and modulate intracellular activation and differentiation signals mediated by the TCR and BCR. While the nature of CD5’s endogenous ligand is controversial, it is well known that CD6 binds to the adhesion molecule ALCAM/CD166, as well as to other proteins. CD5 and CD6 also act as PRRs by recognizing MAMPs from bacterial, fungal, viral, and parasitic origin. CD5 and CD6 play relevant roles in inflammatory diseases and cancer, as shown by association of single nucleotide polymorphisms (SNP) from at the CD5 and CD6 loci with the clinical course of lupus, rheumatoid arthritis, multiple sclerosis, psoriasis, melanoma and chronic lymphocytic leukemia. On this basis, it was hypothesized that changes in the expression level or the amino acid sequence of CD5 and CD6 would impact the susceptibility to or the phenotypical characteristics of immune mediated disorders, such as inflammatory diseases and cancer. Therefore, the objective of the present thesis is to assess the immunomodulatory effect of CD5 and CD6 gene expression and variation in experimental models of immune-mediated and in cohorts of immune-mediated disorders patients. To this end, CD5 and CD6 deficient mice were subjected to dextran sulphate sodium (DSS)-induced colitis, a mouse model of inflammatory bowel disease (IBD). Additionally, the effect of CD5, CD6 and CD166/ALCAM SNPs was analyzed on cohorts of IBD (n=1352 in Crohn’s disease and n=1013 in ulcerative colitis), primary Sjögren’s syndrome (n=212) and prostate cancer (n=376) patients by means of generalized linear models. CD5 and CD6 deficiency resulted in attenuated and exacerbated forms, respectively, of DSS-induced colitis, supporting their involvement in this experimental IBD model. Analyses in IBD patient cohorts showed association of CD5 SNPs with Crohn’s disease location (rs2241002) and requirement of biological therapies (rs2241002-rs2229177 haplotypes), and with poor ulcerative colitis prognosis (rs2241002-rs2229177 haplotypes), and association of CD6 SNPs with Crohn’s disease location (rs17824933) and prognosis (rs12360861), ulcerative colitis extent, and IBD extraintestinal manifestations (rs17824933). In primary Sjögren’s syndrome patients, analyses of individual SNPs showed association of CD5 rs2241002 with anti-Ro/La positivity, CD6 rs17824933 with neutropenia, and CD6 rs11230563 with leukopenia, neutropenia and peripheral nervous system involvement. Further analyses showed association of haplotypes from CD5 (rs2241002-rs2229177) with anemia and thrombocytopenia, CD6 (rs17824933-rs11230563-rs12360861) with cutaneous involvement, and CD166/ALCAM (rs6437585-rs579565-rs1044243 and rs6437585-rs579565-rs1044243) with disease susceptibility and several analytical parameters. In prostate cancer patients, the CD6 rs12360861 and CD166/ALCAM rs579565 SNPs were associated with biochemical recurrence, and CD5 rs2241002-rs2229177 haplotypes were associated with International Society of Urological Pathology grade ≥2. In summary, the results obtained in this thesis support a role for the CD5 and CD6 lymphocyte receptors, as well as the CD6 ligand CD166/ALCAM, in the pathogenesis of IBD, primary Sjögren’s syndrome and prostate cancer. These is in line with previous reports and position these receptors as relevant regulators of the immune system.[cat] CD5 i CD6 són receptors transmembrana de tipus scavenger expressats en tots els limfòcits T i un subgrup de limfòcits B (B1a). Transdueixen senyals i modulen les senyals generades pels receptors clonotípics de cèl·lules T (TCR) i cèl·lules B (BCR). Si bé hi ha controvèrsia respecte la natura del lligand endogen de CD5, se sap que CD6 s’uneix a la molècula d’adhesió ALCAM/CD166, així com a altres proteïnes. A més, CD5 i CD6 reconeixen estructures conservades presents en bacteris, fongs, virus i paràsits. CD5 i CD6 juguen papers rellevants en algunes malalties inflamatòries i càncers, com es dedueix de l’associació de polimorfismes dels loci CD5 i CD6 amb el curs clínic del lupus, artritis reumatoide, esclerosi múltiple, psoriasi, melanoma i leucèmia limfàtica crònica. L’objectiu de la tesi és determinar l’efecte immunomodulador de l’expressió i variació de CD5 i CD6 en desordres immunomitjançats. Amb aquesta finalitat, es van sotmetre ratolins deficients en CD5 o CD6 a colitis induïda per dextransulfat de sodi (DSS), un model de malaltia inflamatòria intestinal (MII). A més, es va analitzar l’efecte de polimorfismes de CD5, CD6 i ALCAM/CD166 en cohorts de pacients de MII, síndrome de Sjögren primària i càncer de pròstata. La deficiència de CD5 i CD6 va resultar en formes atenuades i exacerbades, respectivament, de la colitis induïda per DSS, donant suport al seu paper en aquest model experimental de MII. A més, es va observar associació de polimorfismes de CD5 i CD6 amb la localització de la malaltia de Crohn, requeriment de teràpies biològiques, pronòstic de la colitis ulcerosa i presència de manifestacions extraintestinals. En pacients de síndrome de Sjögren primària, polimorfismes de CD5, CD6 i ALCAM/CD166 es van associar amb la susceptibilitat a la malaltia, presència de citopènies hematològiques, afectació nerviosa i cutània, i diversos paràmetres analítics. En càncer de pròstata polimorfismes de CD5, CD6 i ALCAM/CD166 es van associar amb la recidiva bioquímica i el grau ISUP. En resum, aquests resultats donen suport a un paper de CD5, CD6 i el lligand de CD6 CD166/ALCAM en la patogènesi de desordres immunomitjançats i els posicionen com a reguladors rellevants del sistema immunitari

Gene variation at immunomodulatory and cell adhesion molecules Loci impacts primary Sjögren's Syndrome

Primary Sjögren's syndrome (pSS) is an autoimmune disease triggered by a combination of environmental and host genetic factors, which results in the focal lymphocytic infiltration of exocrine glands causing eye and mouth dryness. Glandular infiltrates include T and B cell subsets positive for CD5 and/or CD6, two surface scavenger receptors involved in the fine-tuning of intracellular signals mediated by the antigen-specific receptor complex of T (TCR) and B (BCR) cells. Moreover, the epithelial cells of inflamed glands overexpress CD166/ALCAM, a CD6 ligand involved in homo and heterotypic cell adhesion interactions. All this, together with the reported association of functionally relevant single nucleotide polymorphisms (SNPs) of CD5, CD6, and CD166/ALCAM with the risk or prognosis of some immune-mediated inflammatory disorders, led us to investigate similar associations in a local cohort of patients with pSS. The logistic regression analyses of individual SNPs showed the association of CD5 rs2241002T with anti-Ro/La positivity, CD6 rs17824933C with neutropenia, and CD6 rs11230563T with increased leukopenia and neutropenia but decreased peripheral nervous system EULAR Sjögren's syndrome disease activity index (ESSDAI). Further analyses showed the association of haplotypes from CD5 (rs2241002T-rs2229177C) with anemia and thrombocytopenia, CD6 (rs17824933G-rs11230563C-rs12360861G) with cutaneous ESSDAI, and CD166/ALCAM (rs6437585C-rs579565A-rs1044243C and rs6437585C-rs579565G-rs1044243T) with disease susceptibility and several analytical parameters (anti-nuclear antibodies, neurological ESSDAI, and hematologic cytopenias). These results support the relevance of gene variation at loci coding for cell surface receptors involved in the modulation of T and B lymphocyte activation (CD5, CD6) and epithelial-immune cell adhesion (CD166/ALCAM) in modulating the clinical and analytical outcomes in patients with pSS.Peer ReviewedPostprint (published version

Gene variation at immunomodulatory and cell adhesion molecules loci impacts primary Sjögren's syndrome

Primary Sjögren's syndrome (pSS) is an autoimmune disease triggered by a combination of environmental and host genetic factors, which results in the focal lymphocytic infiltration of exocrine glands causing eye and mouth dryness. Glandular infiltrates include T and B cell subsets positive for CD5 and/or CD6, two surface scavenger receptors involved in the fine-tuning of intracellular signals mediated by the antigen-specific receptor complex of T (TCR) and B (BCR) cells. Moreover, the epithelial cells of inflamed glands overexpress CD166/ALCAM, a CD6 ligand involved in homo and heterotypic cell adhesion interactions. All this, together with the reported association of functionally relevant single nucleotide polymorphisms (SNPs) of CD5, CD6, and CD166/ALCAM with the risk or prognosis of some immune-mediated inflammatory disorders, led us to investigate similar associations in a local cohort of patients with pSS. The logistic regression analyses of individual SNPs showed the association of CD5 rs2241002T with anti-Ro/La positivity, CD6 rs17824933C with neutropenia, and CD6 rs11230563T with increased leukopenia and neutropenia but decreased peripheral nervous system EULAR Sjögren's syndrome disease activity index (ESSDAI). Further analyses showed the association of haplotypes from CD5 (rs2241002T-rs2229177C) with anemia and thrombocytopenia, CD6 (rs17824933G-rs11230563C-rs12360861G) with cutaneous ESSDAI, and CD166/ALCAM (rs6437585C-rs579565A-rs1044243C and rs6437585C-rs579565G-rs1044243T) with disease susceptibility and several analytical parameters (anti-nuclear antibodies, neurological ESSDAI, and hematologic cytopenias). These results support the relevance of gene variation at loci coding for cell surface receptors involved in the modulation of T and B lymphocyte activation (CD5, CD6) and epithelial-immune cell adhesion (CD166/ALCAM) in modulating the clinical and analytical outcomes in patients with pS

Novel tumor-targeted self-nanostructured and compartmentalized water-in-oil-in-water polyurethane-polyurea nanocapsules for cancer theragnosis

Encapsulation of water-soluble bioactive compounds for enabling specific accumulation in tumor locations, while avoiding premature clearance and/or degradation in the bloodstream, is one of the main hallmarks in nanomedicine, especially that of NIR fluorescent probes for cancer theragnosis. The herein reported technology furnishes water-dispersible double-walled polyurethane-polyurea hybrid nanocapsules (NCs) loaded with indocyanine green (ICG-NCs), using a versatile and highly efficient one-pot and industrially scalable synthetic process based on the use of two different prepolymers to set up the NCs walls. Flow cytometry and confocal microscopy confirmed that both ICG-loaded NCs internalized in monocyte-derived dendritic cells (moDCs). The in vivo analysis of xenograft A375 mouse melanoma model revealed that amphoteric functionalization of NCs' surface promotes the selective accumulation of ICG-NCs in tumor tissues, making them promising agents for a less-invasive theragnosis of cancer

Conserved bacterial-binding peptides of the scavenger-like lymphocyte receptor CD6 protect from mouse experimental sepsis

Sepsis is an unmet clinical need constituting one of the most important causes of death worldwide, a fact aggravated by the appearance of multidrug resistant strains due to indiscriminate use of antibiotics. Host innate immune receptors involved in pathogen-associated molecular patterns (PAMPs) recognition represent a source of broad-spectrum therapies alternative or adjunctive to antibiotics. Among the few members of the ancient and highly conserved scavenger receptor cysteine-rich superfamily (SRCR-SF) sharing bacterial-binding properties there is CD6, a lymphocyte-specific surface receptor. Here, we analyze the bacterial-binding properties of three conserved short peptides (11-mer) mapping at extracellular SRCR domains of human CD6 (CD6.PD1, GTVEVRLEASW; CD6.PD2 GRVEMLEHGEW; and CD6.PD3, GQVEVHFRGVW). All peptides show high binding affinity for PAMPs from Gram-negative (lipopolysaccharide; Kd from 3.5 to 3,000 nM) and Gram-positive (lipoteichoic acid; Kd from 36 to 680 nM) bacteria. The CD6.PD3 peptide possesses broad bacterial-agglutination properties and improved survival of mice undergoing polymicrobial sepsis in a dose- and time-dependent manner. Accordingly, CD6.PD3 triggers a decrease in serum levels of both pro-inflammatory cytokines and bacterial load. Interestingly, CD6.PD3 shows additive survival effects on septic mice when combined with Imipenem/Cilastatin. These results illustrate the therapeutic potential of peptides retaining the bacterial-binding properties of native CD6

Polygenic Innate Immunity Score to Predict the Risk of Cytomegalovirus Infection in CMV D+/R- Transplant Recipients. A Prospective Multicenter Cohort Study

Several genetic polymorphisms of the innate immune system have been described to increase the risk of cytomegalovirus (CMV) infection in transplant patients. The aim of this study was to assess the impact of a polygenic score to predict CMV infection and disease in high risk CMV transplant recipients (heart, liver, kidney or pancreas). On hundred and sixteen CMV-seronegative recipients of grafts from CMV-seropositive donors undergoing heart, liver, and kidney or pancreas transplantation from 7 centres were prospectively included for this purpose during a 2-year period. All recipients received 100-day prophylaxis with valganciclovir. CMV infection occurred in 61 patients (53%) at 163 median days from transplant, 33 asymptomatic replication (28%) and 28 CMV disease (24%). Eleven patients (9%) had recurrent CMV infection. Clinically and/or functionally relevant single nucleotide polymorphisms (SNPs) from TLR2, TLR3, TLR4, TLR7, TLR9, AIM2, MBL2, IL28, IFI16, MYD88, IRAK2 and IRAK4 were assessed by real time polymerase chain reaction (RT-PCR) or sequence-based typing (PCR-SBT). A polygenic score including the TLR4 (rs4986790/rs4986791), TLR9 (rs3775291), TLR3 (rs3775296), AIM2 (rs855873), TLR7 (rs179008), MBL (OO/OA/XAO), IFNL3/IL28B (rs12979860) and IFI16 (rs6940) SNPs was built based on the risk of CMV infection and disease. The CMV score predicted the risk of CMV disease with an AUC of the model of 0.68, with sensitivity and specificity of 64.3 and 71.6%, respectively. Even though further studies are needed to validate this score, its use would represent an effective model to develop more robust scores predicting the risk of CMV disease in donor/recipient mismatch (D+/R-) transplant recipients

CD6 deficiency impairs early immune response to bacterial sepsis

CD6 is a lymphocyte-specific scavenger receptor expressed on adaptive (T) and innate (B1a, NK) immune cells, which is involved in both fine-tuning of lymphocyte activation/differentiation and recognition of bacterial-associated molecular patterns (i.e., lipopolysaccharide). However, evidence on CD6's role in the physiological response to bacterial infection was missing. Our results show that induction of monobacterial and polymicrobial sepsis in Cd6 -/- mice results in lower survival rates and increased bacterial loads and pro-inflammatory cytokine levels. Steady state analyses of Cd6 -/- mice show decreased levels of natural polyreactive antibodies, concomitant with decreased cell counts of spleen B1a and marginal zone B cells. Adoptive transfer of wild-type B cells and mouse serum, as well as a polyreactive monoclonal antibody improve Cd6 -/- mouse survival rates post-sepsis. These findings support a nonredundant role for CD6 in the early response against bacterial infection, through homeostatic expansion and functionality of innate-related immune cells

Experimental and genetic evidence for the impact of CD5 and CD6 expression and variation in inflammatory bowel disease

Crohn's disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBD) resulting from the interaction of multiple environmental, genetic and immunological factors. CD5 and CD6 are paralogs encoding lymphocyte co-receptors involved in fine-tuning intracellular signals delivered upon antigen-specific recognition, microbial pattern recognition and cell adhesion. While CD5 and CD6 expression and variation is known to influence some immune-mediated inflammatory disorders, their role in IBD remains unclear. To this end, Cd5- and Cd6-deficient mice were subjected to dextran sulfate sodium (DSS)-induced colitis, the most widely used experimental animal model of IBD. The two mouse lines showed opposite results regarding body weight loss and disease activity index (DAI) changes following DSS-induced colitis, thus supporting Cd5 and Cd6 expression involvement in the pathophysiology of this experimental IBD model. Furthermore, DNA samples from IBD patients of the ENEIDA registry were used to test association of CD5 (rs2241002 and rs2229177) and CD6 (rs17824933, rs11230563, and rs12360861) single nucleotide polymorphisms with susceptibility and clinical parameters of CD (n=1352) and UC (n=1013). Generalized linear regression analyses showed association of CD5 variation with CD ileal location (rs2241002CC) and requirement of biological therapies (rs2241002C-rs2229177T haplotype), and with poor UC prognosis (rs2241002T-rs2229177T haplotype). Regarding CD6, association was observed with CD ileal location (rs17824933G) and poor prognosis (rs12360861G), and with left-sided or extensive UC, and absence of ankylosing spondylitis in IBD (rs17824933G). The present experimental and genetic evidence support a role for CD5 and CD6 expression and variation in IBD's clinical manifestations and therapeutic requirements, providing insight into its pathophysiology and broadening the relevance of both immunomodulatory receptors in immune-mediated disorders

Experimental and genetic evidence for the impact of CD5 and CD6 expression and variation in inflammatory bowel disease

Crohn's disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBD) resulting from the interaction of multiple environmental, genetic and immunological factors. CD5 and CD6 are paralogs encoding lymphocyte co-receptors involved in fine-tuning intracellular signals delivered upon antigen-specific recognition, microbial pattern recognition and cell adhesion. While CD5 and CD6 expression and variation is known to influence some immune-mediated inflammatory disorders, their role in IBD remains unclear. To this end, Cd5- and Cd6-deficient mice were subjected to dextran sulfate sodium (DSS)-induced colitis, the most widely used experimental animal model of IBD. The two mouse lines showed opposite results regarding body weight loss and disease activity index (DAI) changes following DSS-induced colitis, thus supporting Cd5 and Cd6 expression involvement in the pathophysiology of this experimental IBD model. Furthermore, DNA samples from IBD patients of the ENEIDA registry were used to test association of CD5 (rs2241002 and rs2229177) and CD6 (rs17824933, rs11230563, and rs12360861) single nucleotide polymorphisms with susceptibility and clinical parameters of CD (n=1352) and UC (n=1013). Generalized linear regression analyses showed association of CD5 variation with CD ileal location (rs2241002CC) and requirement of biological therapies (rs2241002C-rs2229177T haplotype), and with poor UC prognosis (rs2241002T-rs2229177T haplotype). Regarding CD6, association was observed with CD ileal location (rs17824933G) and poor prognosis (rs12360861G), and with left-sided or extensive UC, and absence of ankylosing spondylitis in IBD (rs17824933G). The present experimental and genetic evidence support a role for CD5 and CD6 expression and variation in IBD's clinical manifestations and therapeutic requirements, providing insight into its pathophysiology and broadening the relevance of both immunomodulatory receptors in immune-mediated disorders.This work was supported by Spanish Ministerio de Economía y Competitividad (MINECO, SAF2016-80535-R) and Ministerio de Ciencia e Innovación (MCIN/AEI/10.13039/501100011033, PID2019-106658RB-I00), co-financed by European Development Regional Fund “A way to achieve Europe” ERDF, and Agència de Gestió d’Ajuts Universitaris i de Recerca from Generalitat de Catalunya (2017/SGR/1582). SC-L, MV-dA, CC, AL-P, and EC are recipients of fellowships from Spanish Ministerio de Educación, Cultura y Deporte (FPU15/02897), Spanish MINECO (BES-2014-069237 and BES-2017-082107), Chilean Agencia Nacional de Investigación y Desarrollo (2018-72190154), and European Community Seventh Framework Program (FP7/2007/2013; 229673), respectively. SC-L and JL are recipients of short-term fellowships from European Federation of Immunological Societies-Immunology Letters (EFIS-IL) and Erasmus+ from the European Union, respectively. The ENEIDA registry of GETECCU is supported by Biogen, Janssen, Takeda and Pfizer. The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication

Contribution of evolutionary selected immune gene polymorphism to immune-related disorders: the case of lymphocyte scavenger receptors CD5 and CD6

Pathogens are one of the main selective pressures that ancestral humans had to adapt to. Components of the immune response system have been preferential targets of natural selection in response to such pathogen-driven pressure. In turn, there is compelling evidence showing that positively selected immune gene variants conferring increased resistance to past or present infectious agents are today associated with increased risk for autoimmune or inflammatory disorders but decreased risk of cancer, the other side of the same coin. CD5 and CD6 are lymphocytic scavenger receptors at the interphase of the innate and adaptive immune responses since they are involved in both: (i) microbial-associated pattern recognition; and (ii) modulation of intracellular signals mediated by the clonotypic antigen-specific receptor present in T and B cells (TCR and BCR, respectively). Here, we review available information on CD5 and CD6 as targets of natural selection as well as on the role of CD5 and CD6 variation in autoimmunity and cancer.This research was funded by Spanish Ministerio de Economía y Competitividad (MINECO; SAF-2016-80535-R, PID2019-106658RB-I00 and PCIN-2015-070) co-financed by European Development Regional Fund (ERDF) “A way to achieve Europe”, Agencia Estatal de Investigación (AEI; PID2019-110933GB-I00/AEI/10.13039/501100011033 and CEX2018-000792-M) and Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR; 2017/SGR/1582 and 2017SGR00702) from Generalitat de Catalunya to F.L. and E.B. In addition, S.C.-L., M.V.-d.A., C.C. and A.L.-P. are recipients of fellowships from Ministerio de Educación, Cultura y Deporte (FPU15/02897) MINECO (BES-2014-069237; BES-2017-082107) and Chilean Agencia Nacional de Investigación y Desarrollo (ANID; 2018-72190154), respectively