Anàlisi de l’expressió del receptor scavenger cd36 en els leucòcits: conseqüències funcionals en la inflamació

La tesi que es presenta per optar al grau de doctor en biologia titulada: “Anàlisi de l’expressió del receptor scavenger CD36 en els leucòcits: conseqüències funcionals en la inflamació” ha estat acceptada per ser presentada per compendi de publicacions. Aquesta consta de dos articles: • Functional consequences of CD36 downregulation by TLR signals. Carlos Zamora et al. Cytokine 2012 Oct; 60(1): 257-65. • Functional consequences of platelet binding to T lymphocytes in inflammation. Carlos Zamora et al. Journal of Leukocyte Biology 2013 Sep; 94(3): 521-9. La inflamació és un conjunt de processos biològics que tenen com a finalitat comú protegir a l’organisme dels patògens i dany. Durant el procés inflamatori, intervenen diferents components cel·lulars a través de diferents receptors de membrana i citoquines proinflamatòries i antiinflamatòries. Un dels receptors involucrats en aquest procés inflamatori és el receptor scavenger CD36. En els monòcits, el CD36 és una molècula capaç de reconèixer i captar lipoproteïnes modificades, patògens, neutròfils apoptòtics i actuar com a coreceptor dels Toll-like receptors (TLRs) perquè es doni una eficient resposta inflamatòria. Fins al moment es desconeixia si els TLRs modulaven el CD36 en els monòcits i les conseqüències funcionals comportarà aquesta modulació. Per altra banda, es desconeixia si l’expressió de CD36 en limfòcits era extrínseca a ells i quines funcions podria tenir la subpoblació de limfòcits T que l’expressen. En el primer article, vam observar que els lligands de TLR2 i TLR4 disminuïen l’expressió de CD36 en la superfície de les diferents subpoblacions de monòcits, internalitzant-se cap al interior d’aquests. Aquesta, era parcialment deguda al TNFα produït en els cultius de TLRs. La reducció de l’expressió de CD36 en els monòcits va donar lloc a una disminució de la fagocitosis de neutròfils apoptòtics, un procés clau en el manteniment de l’homeòstasi en els teixits. En el segon article, vam observar que l’expressió de CD36 en una petita subpoblació de limfòcits T CD4+ era extrínseca a ells, provenint de les plaquetes unides a ells que presentaven. Aquesta subpoblació de limfòcits T CD4+ amb plaquetes unides presentava una disminució de la capacitat proliferativa i de producció de citoquines inflamatòries (IL-17 i IFNγ). En els pacients d’artritis reumatoide, s’observa un grup de pacients amb un augment de limfòcits T CD4+ amb plaquetes unides. Aquet grup de pacients van ser els que van presentar un fenotip menys sever de la malaltia, suggerint així que la unió de plaquetes als limfòcits era un mecanisme immunoregulador.Thesis presented to obtain the degree of Biology PhD entitled: “Analysis of scavenger receptor CD36 in leukocytes: functional consequences in inflammation” has been accepted to be presented to collection of published articles. This thesis contain two articles: • Functional consequences of CD36 downregulation by TLR signals. Carlos Zamora et al. Cytokine 2012 Oct; 60(1): 257-65. • Functional consequences of platelet binding to T lymphocytes in inflammation. Carlos Zamora et al. Journal of Leukocyte Biology 2013 Sep; 94(3): 521-9. Inflammation is a set of biological processes that aim to protect from the harmful effects of pathogens and cellular damage. During inflammatory process, different cells are involved, such as monocytes and lymphocytes, through different membrane receptors and pro- and antiinflammatory cytokines. One of the receptors involved in this inflammatory process is scavenger receptor CD36. In monocytes, CD36 molecule is able to recognize and capture modified lipoproteins, pathogens, apoptotic neutrophils, and act as a coreceptor of Toll-like receptors (TLRs) to give an efficient inflammatory response. Until now, it was not known whether TLRs modulate CD36 on monocytes and functional consequences that involve modulation in these cells. Moreover, it was not known whether the expression of CD36 on lymphocytes was extrinsic to them and how this T lymphocyte CD36+ subpopulation functions. In the first article, we observed that TLR2 and TLR4 ligands decreased the expression of CD36 on the surface of different subpopulations of monocytes, internalizing towards the inside of them. This was partially due to the TNFα production after TLRs cultures. Reduced expression of CD36 on monocytes resulted in a decrease of phagocytosis of apoptotic neutrophils, a key process in the maintenance of tissue homeostasis. In the second article, we observed that CD36 expression in a small subpopulation of CD4+ T lymphocytes was extrinsic to them, deriving from platelet bind to them. This subpopulation of CD4+ T cells with bound platelets showed a decreased proliferate capacity and inflammatory cytokine production (IL-17 and IFNγ). In rheumatoid arthritis patients, we observed a group with increased CD4+ T cells with bound platelets. This group of patients was those who had a less severe phenotype of the disease, suggesting that the binding of platelets to lymphocytes was an immunoregulador mechanism

Anàlisi de l'expressió del receptor scavenger cd36 en els leucòcits: conseqüències funcionals en la inflamació

La tesi que es presenta per optar al grau de doctor en biologia titulada: "Anàlisi de l'expressió del receptor scavenger CD36 en els leucòcits: conseqüències funcionals en la inflamació" ha estat acceptada per ser presentada per compendi de publicacions. Aquesta consta de dos articles: • Functional consequences of CD36 downregulation by TLR signals. Carlos Zamora et al. Cytokine 2012 Oct; 60(1): 257-65. • Functional consequences of platelet binding to T lymphocytes in inflammation. Carlos Zamora et al. Journal of Leukocyte Biology 2013 Sep; 94(3): 521-9. La inflamació és un conjunt de processos biològics que tenen com a finalitat comú protegir a l'organisme dels patògens i dany. Durant el procés inflamatori, intervenen diferents components cel·lulars a través de diferents receptors de membrana i citoquines proinflamatòries i antiinflamatòries. Un dels receptors involucrats en aquest procés inflamatori és el receptor scavenger CD36. En els monòcits, el CD36 és una molècula capaç de reconèixer i captar lipoproteïnes modificades, patògens, neutròfils apoptòtics i actuar com a coreceptor dels Toll-like receptors (TLRs) perquè es doni una eficient resposta inflamatòria. Fins al moment es desconeixia si els TLRs modulaven el CD36 en els monòcits i les conseqüències funcionals comportarà aquesta modulació. Per altra banda, es desconeixia si l'expressió de CD36 en limfòcits era extrínseca a ells i quines funcions podria tenir la subpoblació de limfòcits T que l'expressen. En el primer article, vam observar que els lligands de TLR2 i TLR4 disminuïen l'expressió de CD36 en la superfície de les diferents subpoblacions de monòcits, internalitzant-se cap al interior d'aquests. Aquesta, era parcialment deguda al TNFα produït en els cultius de TLRs. La reducció de l'expressió de CD36 en els monòcits va donar lloc a una disminució de la fagocitosis de neutròfils apoptòtics, un procés clau en el manteniment de l'homeòstasi en els teixits. En el segon article, vam observar que l'expressió de CD36 en una petita subpoblació de limfòcits T CD4+ era extrínseca a ells, provenint de les plaquetes unides a ells que presentaven. Aquesta subpoblació de limfòcits T CD4+ amb plaquetes unides presentava una disminució de la capacitat proliferativa i de producció de citoquines inflamatòries (IL-17 i IFNγ). En els pacients d'artritis reumatoide, s'observa un grup de pacients amb un augment de limfòcits T CD4+ amb plaquetes unides. Aquet grup de pacients van ser els que van presentar un fenotip menys sever de la malaltia, suggerint així que la unió de plaquetes als limfòcits era un mecanisme immunoregulador.Thesis presented to obtain the degree of Biology PhD entitled: "Analysis of scavenger receptor CD36 in leukocytes: functional consequences in inflammation" has been accepted to be presented to collection of published articles. This thesis contain two articles: • Functional consequences of CD36 downregulation by TLR signals. Carlos Zamora et al. Cytokine 2012 Oct; 60(1): 257-65. • Functional consequences of platelet binding to T lymphocytes in inflammation. Carlos Zamora et al. Journal of Leukocyte Biology 2013 Sep; 94(3): 521-9. Inflammation is a set of biological processes that aim to protect from the harmful effects of pathogens and cellular damage. During inflammatory process, different cells are involved, such as monocytes and lymphocytes, through different membrane receptors and pro- and antiinflammatory cytokines. One of the receptors involved in this inflammatory process is scavenger receptor CD36. In monocytes, CD36 molecule is able to recognize and capture modified lipoproteins, pathogens, apoptotic neutrophils, and act as a coreceptor of Toll-like receptors (TLRs) to give an efficient inflammatory response. Until now, it was not known whether TLRs modulate CD36 on monocytes and functional consequences that involve modulation in these cells. Moreover, it was not known whether the expression of CD36 on lymphocytes was extrinsic to them and how this T lymphocyte CD36+ subpopulation functions. In the first article, we observed that TLR2 and TLR4 ligands decreased the expression of CD36 on the surface of different subpopulations of monocytes, internalizing towards the inside of them. This was partially due to the TNFα production after TLRs cultures. Reduced expression of CD36 on monocytes resulted in a decrease of phagocytosis of apoptotic neutrophils, a key process in the maintenance of tissue homeostasis. In the second article, we observed that CD36 expression in a small subpopulation of CD4+ T lymphocytes was extrinsic to them, deriving from platelet bind to them. This subpopulation of CD4+ T cells with bound platelets showed a decreased proliferate capacity and inflammatory cytokine production (IL-17 and IFNγ). In rheumatoid arthritis patients, we observed a group with increased CD4+ T cells with bound platelets. This group of patients was those who had a less severe phenotype of the disease, suggesting that the binding of platelets to lymphocytes was an immunoregulador mechanism

Association between Changes in the Patterns of Antinuclear Autoantibodies during Immune Checkpoint Inhibition Therapy and the Development of Severe Immune Related Adverse Events

Immune-related adverse events (irAEs) are unpredictable autoimmune-like toxicities induced by immune checkpoint inhibitors (ICI). irAEs are a consequence of a breakdown in self-tolerance. ICIs can induce autoantibody formation, and the presence of antinuclear autoantibodies (ANAs) has been reported in patients who developed irAEs. Our goal was to compare ANA patterns by indirect immunofluorescence at different timepoints before (baseline) and after the initiation of ICI treatment and to analyze the role of ANA pattern changes as predictors of irAEs. This is a 2-year-follow-up prospective study of 152 consecutive patients with solid tumors treated with anti-PD-(L)1 blockade agents. They were included from September 2018 until March 2020 in the Hospital de la Santa Creu I Sant Pau (Barcelona, Spain). We grouped patients into three groups: ANA de novo (patients who showed new ANA patterns at any time after ICI initiation), ANA (ANA positive at baseline without changes in the ANA patterns after initiation of treatment) and non-ANA (ANA negative at baseline and after ICI initiation). We did not find any association between the appearance of ANAs and irAE rates or the number and types of irAEs. However, patients in the ANA de novo group showed higher severe irAE rates (grade ≥ 3) than the other groups. Additionally, in most of the patients with severe irAEs (83.3%), changes in ANA patterns preceded irAE onset. In conclusion, we found ANA induction during ICI therapies in 22 patients and our results suggest that the appearance of ANAs may predict the severity of the irAE

Interleukin-19 impairment in active Crohn's disease patients

The exact function of interleukin-19 (IL-19) on immune response is poorly understood. In mice, IL-19 up-regulates TNFalpha and IL-6 expression and its deficiency increases susceptibility to DSS-induced colitis. In humans, IL-19 favors a Th2 response and is elevated in several diseases. We here investigate the expression and effects of IL-19 on cells from active Crohn"s disease (CD) patient. Twenty-three active CD patients and 20 healthy controls (HC) were included. mRNA and protein IL-19 levels were analyzed in monocytes. IL-19 effects were determined in vitro on the T cell phenotype and in the production of cytokines by immune cells. We observed that unstimulated and TLR-activated monocytes expressed significantly lower IL-19 mRNA in active CD patients than in HC (logFC =21.97 unstimulated; 21.88 with Pam3CSK4; and 21.91 with FSL-1; p<0.001). These results were confirmed at protein level. Exogenous IL-19 had an anti-inflammatory effect on HC but not on CD patients. IL-19 decreased TNFalpha production in PBMC (850.7675.29 pg/ml vs 2626.06350 pg/ml; p<0.01) and increased CTLA4 expression (22.0461.55% vs 13.9862.05%; p<0.05) and IL-4 production (32.568.9 pg/ml vs 13.562.9 pg/ml; p<0.05) in T cells from HC. IL-10 regulated IL-19 production in both active CD patients and HC. We observed that three of the miRNAs that can modulate IL-19 mRNA expression, were up-regulated in monocytes from active CD patients. These results suggested that IL-19 had an anti-inflammatory role in this study. Defects in IL-19 expression and the lack of response to this cytokine could contribute to inflammatory mechanisms in active CD patients

IL-19 production after TLR activation in PBMC cultures from active CD patients (n = 3) and HC (n = 6).

<p>PBMC were cultured 24(A) and 48 h (B) with different TLR ligands (LPS, Pam3CSK4 or FSL-1) and IL-19 production was determined by ELISA as described in Materials and Methods. The viability of PBMC was determined by Trypan Blue (>99%). *p<0.05; **p<0.01; ***p<0.001.</p

rhIL-19 up-regulates IL-4 production in activated T cells.

<p>PBMC from HC (n = 6) and active CD (n = 6) were activated and expanded with anti-CD3+anti-CD2+anti-CD28 as described in Materials and Methods. Supernatants were removed and IL-4 and IFNγ content were analyzed by ELISA. A) A) Up-regulation of IL-4 by rhIL-19 in HC cultures. B) IFNγ content in HC and CD patients in the presence or absence of rhIL-19.*p<0.05.</p

rhIL-19 up-regulates CTLA4 expression on CD4+CD25+CD127

<p>− <b>cells.</b> PBMC from HC (n = 4) and active CD (n = 3) were activated with anti-CD3+anti-CD2+anti-CD28 as described in Materials and Methods. The expression of CTLA4 (A) and GITR (B) was determined on CD4+CD25+CD127− cells by flow cytometry. *p<0.05.</p

Heat map image of IL-10 family cytokines.

<p>Monocytes mRNA was extracted from HC (n = 3) and active CD patients (n = 3) after 6 h of culture with medium in the absence or presence of Pam3CSK4 and FSL-1. A) Green denoted down-regulated and red up-regulated genes. B) logFC of IL-19 mRNA expression between CD and HC are shown. **p<0.01; ***p<0.001.</p

IL-10 regulates IL-19 expression.

<p>PBMC from A) HC (n = 3) and B) active CD patients (n = 3) were activated 24 h with TLR ligands (LPS, Pam3CSK4 and FSL-1) in the presence of rhIL-10 (200 ng/ml) and blocking anti-IL10 (500 ng/ml). Results are expressed as: (IL19 production with TLR+rhIL-10 or anti-IL10-IL-19 production with TLR)/IL-19 production with TLR.</p

rhIL-19 down-regulates TNFα production in cultures from HC after LPS activation.

<p>PBMC from HC and active CD patients were cultured with LPS (HC n = 10 and CD n = 5), Pam3CSK4 (HC n = 10 and CD n = 5) and FSL-1 (HC n = 5 and CD n = 3) for 24 h with or without rhIL-19 (400 ng/ml). A and B) TNFα and IL-10 production in cultures from HC after TLR activation with or without rhIL-19. D and E) TNFα and IL-10 production in cultures from active CD patients after TLR activation with or without rhIL-19. C and F) TNFα production from HC and active CD patients cultured with rhIL-10 (200 ng/ml). *p<0.05.</p