Osteopontin protects from autoimmunity-driven diffuse large B cell lymphoma development

Several studies have highlighted the association between certain autoimmune diseases and increased occurrence of hematological malignancies, such as Systemic lupus erythematosus (SLE). Patients affected by SLE have indeed a high risk of developing non-Hodgkin lymphoma (NHL), mostly diffuse large B cell lymphoma (DLBCL). Osteopontin (OPN) has been associated with SLE pathogenesis, as SLE patients have increased serum levels of OPN and often polymorphisms in SPP1, the gene encoding for OPN. To study the role of this molecule in autoimmunity-associated lymphomagenesis, the Faslpr/lpr mutation of the autoimmunity-prone mouse strain has been transferred onto a OPN-deficient background. Interestingly, OPN-/-Faslpr/lpr mice showed a significantly higher incidence of splenic B cell lymphomas in comparison to Faslpr/lpr mice. Indeed, in OPN-deficient autoimmune mice we observed an expansion of mature CD19+IgMCD23-CD21/35- cells, which showed the typical signature of the activated type of DLBCL (ABC-DLBCL), BCL6-, BCL2+, c-MYC+, IRF4+, Ki67hi, and a striking activation of the STAT3 pathway. Additionally, OPN-deficient autoimmune CD19+ B cells showed a baseline hyper-activation of Toll-like receptor 9 (TLR9)-dependent MYD88 signaling pathway, and were more activated when stimulated with the TLR9 agonist CpG 1826, which mimics double strand-DNA circulating in autoimmune conditions. Immunohistochemistry on splenic tissues and immunofluorescence staining on CD19+ cells showed that CD19+ B cells from Faslpr/lpr mice express the intracellular isoform of OPN at the endosomal surface, where TLR9 is also located. To gain mechanistic insights, I genetically modified ABC-DLBCL cell lines, derived from lymphomatous spleens of OPN-/- mice, to over-express either the soluble (sOPN) or the intracellular isoform of OPN (iOPN). Remarkably, in vitro experiments showed that iOPN was able to prevent the CpG-mediated activation of STAT3 and, partially, of NFkB pathway, suggesting a negative regulatory role for iOPN in this setting. Additionally, when injected in vivo, while the over-expression of sOPN conferred a growth advantage over control cells, iOPN-expressing cells were less tumorigenic. Interestingly, other than B cell-intrinsic, the molecular mechanisms at the basis of the aggressive lymphomagenesis observed in OPN-/-Faslpr/lpr mice were also likely stroma related. Indeed, mainly myeloid cell subsets of the splenic microenvironment of OPNdeficient autoimmune mice expressed more Tnfsf13B (encoding for BAFF) and Il6, both key factors for B cell survival and proliferation, in comparison with OPN-competent counterparts. Interestingly, in lymphomatous areas of the spleen from OPN-deficient mice, a high number of infiltrating CD8+ T cells with an activated morphology was notable. However, their analysis by flow cytometry showed markers of exhaustion (TIM3 and PD1), suggesting that, despite being recruited at the tumour site, they were likely dysfunctional and unable to contrast tumour expansion. The translational relevance of these findings comes from the analysis of ABC-DLBCL biopsies that expressed a lower SPP1/OPN level than GC-DLBCL counterparts, suggesting how a diminished expression of B-cell intrinsic OPN may be associated with the development of the most aggressive DLBCL. However, the existence of a specific intracellular form and/or localization of OPN in humans remains to be investigated. Altogether, my data suggest that B cell-intrinsic iOPN is contrasting autoimmunity driven ABC-DLBCL development, also in concert with microenvironment-related dynamics, which in an OPN-competent host seem to contribute in preventing lymphomagenesis

Concomitant reduction of c-Myc expression and PI3K/AKT/mTOR signaling by quercetin induces a strong cytotoxic effect against Burkitt's lymphoma

Burkitt’s lymphoma is an aggressive B cell lymphoma whose pathogenesis involves mainly c-Myc translocation and hyperexpression, in addition to antigen-independent BCR signaling and, in some cases, EBV infection. As result of BCR signaling activation, the PI3K/AKT/mTOR pathway results constitutively activated also in the absence of EBV, promoting cell survival and counterbalancing the pro-apoptotic function that c-Myc may also exert. In this study we found that quercetin, a bioflavonoid widely distributed in plant kingdom, reduced c-Myc expression and inhibited the PI3K/AKT/mTOR activity in BL, leading to an apoptotic cell death. We observed a higher cytotoxic effect against the EBV-negative BL cells in comparison with the positive ones, suggesting that this oncogenic gammaherpesvirus confers an additional resistance to the quercetin treatment. Besides cell survival, PI3K/AKT/mTOR pathway also regulates autophagy: we found that quercetin induced a complete autophagic flux in BL cells, that contributes to c-Myc reduction in some of these cells. Indeed, autophagy inhibition by chloroquine partially restored c-Myc expression in EBV-positive (Akata) and EBV-negative (2A8) cells that harbor c-Myc mutation. Interestingly, chloroquine did not affect the quercetin-mediated reduction of c-Myc expression in Ramos cells, that have no c-Myc mutation in the coding region, although autophagy was induced. These results suggest that mutant c-Myc could be partially degraded through autophagy in BL cells, as previously reported for other mutant oncogenic proteins

Quercetin induces apoptosis and autophagy in primary effusion lymphoma cells by inhibiting PI3K/AKT/mTOR and STAT3 signaling pathways

Quercetin, a bioflavonoid contained in several vegetables daily consumed, has been studied for long time for its antiinflammatory and anticancer properties. Quercetin interacts with multiple cancer-related pathways such as PI3K/AKT, Wnt/β-catenin and STAT3. These pathways are hyperactivated in primary effusion lymphoma (PEL), an aggressive B cell lymphoma whose pathogenesis is strictly linked to the oncogenic virus Kaposis' Sarcoma-associated Herpesvirus (KSHV). In this study, we found that quercetin inhibited PI3K/AKT/mTOR and STAT3 pathways in PEL cells, and as a consequence, it down-regulated the expression of the prosurvival cellular proteins such as c-FLIP, cyclin D1 and cMyc. It also reduced the release of IL-6 and IL-10 cytokines, leading to PEL cell death. Moreover, quercetin induced a prosurvival autophagy in these cells and increased the cytotoxic effect of bortezomib, a proteasomal inhibitor, against them. Interestingly, quercetin decreased also the expression of latent and lytic KSHV proteins involved in PEL tumorigenesis and up-regulated the surface expression of HLA-DR and calreticulin, rendering the dying cells more likely detectable by the immune system. The results obtained in this study indicate that quercetin, which does not exert any cytotoxicity against normal B cells, may represent a good candidate for the treatment of this aggressive B cell lymphoma, especially in combination with autophagy inhibitors or with bortezomib

Intracellular osteopontin protects from autoimmunity-driven lymphoma development inhibiting TLR9-MYD88-STAT3 signaling

Background: Autoimmune disorders, including Systemic Lupus Erythematosus (SLE), are associated with increased incidence of hematological malignancies. The matricellular protein osteopontin (OPN) has been linked to SLE pathogenesis, as SLE patients show increased serum levels of OPN and often polymorphisms in its gene. Although widely studied for its pro-tumorigenic role in different solid tumours, the role of OPN in autoimmunity-driven lymphomagenesis has not been investigated yet. Methods: To test the role of OPN in the SLE-associated lymphomagenesis, the SLE-like prone Fas(lpr/lpr) mutation was transferred onto an OPN-deficient background. Spleen from Fas(lpr/lpr) and OPN-/-Fas(lpr/lpr) mice, as well as purified B cells, were analysed by histopathology, flow cytometry, Western Blot, immunohistochemistry, immunofluorescence and gene expression profile to define lymphoma characteristics and investigate the molecular mechanisms behind the observed phenotype. OPN cellular localization in primary splenic B cells and mouse and human DLBCL cell lines was assessed by confocal microscopy. Finally, gain of function experiments, by stable over-expression of the secreted (sOPN) and intracellular OPN (iOPN) in OPN-/-Fas(lpr/lpr) -derived DLBCL cell lines, were performed for further validation experiments. Results: Despite reduced autoimmunity signs, OPN-/-Fas(lpr/lpr) mice developed splenic lymphomas with higher incidence than Fas(lpr/lpr) counterparts. In situ and ex vivo analysis featured such tumours as activated type of diffuse large B cell lymphoma (ABC-DLBCL), expressing BCL2 and c-MYC, but not BCL6, with activated STAT3 signaling. OPN-/-Fas(lpr/lpr) B lymphocytes showed an enhanced TLR9-MYD88 signaling pathway, either at baseline or after stimulation with CpG oligonucleotides, which mimic dsDNA circulating in autoimmune conditions. B cells from Fas(lpr/lpr) mice were found to express the intracellular form of OPN. Accordingly, gene transfer-mediated re-expression of iOPN, but not of its secreted isoform, into ABC-DLBCL cell lines established from OPN-/-Fas(lpr/lpr) mice, prevented CpG-mediated activation of STAT3, suggesting that the intracellular form of OPN may represent a brake to TLR9 signaling pathway activation. Conclusion: These data indicate that, in the setting of SLE-like syndrome in which double strand-DNA chronically circulates and activates TLRs, B cell intracellular OPN exerts a protective role in autoimmunity-driven DLBCL development, mainly acting as a brake in the TLR9-MYD88-STAT3 signaling pathway

Intracellular osteopontin protects from autoimmunity-driven lymphoma development inhibiting TLR9-MYD88-STAT3 signaling

Abstract Background Autoimmune disorders, including Systemic Lupus Erythematosus (SLE), are associated with increased incidence of hematological malignancies. The matricellular protein osteopontin (OPN) has been linked to SLE pathogenesis, as SLE patients show increased serum levels of OPN and often polymorphisms in its gene. Although widely studied for its pro-tumorigenic role in different solid tumours, the role of OPN in autoimmunity-driven lymphomagenesis has not been investigated yet. Methods To test the role of OPN in the SLE-associated lymphomagenesis, the SLE-like prone Faslpr/lpr mutation was transferred onto an OPN-deficient background. Spleen from Faslpr/lpr and OPN-/-Faslpr/lpr mice, as well as purified B cells, were analysed by histopathology, flow cytometry, Western Blot, immunohistochemistry, immunofluorescence and gene expression profile to define lymphoma characteristics and investigate the molecular mechanisms behind the observed phenotype. OPN cellular localization in primary splenic B cells and mouse and human DLBCL cell lines was assessed by confocal microscopy. Finally, gain of function experiments, by stable over-expression of the secreted (sOPN) and intracellular OPN (iOPN) in OPN-/-Faslpr/lpr -derived DLBCL cell lines, were performed for further validation experiments. Results Despite reduced autoimmunity signs, OPN-/-Faslpr/lpr mice developed splenic lymphomas with higher incidence than Faslpr/lpr counterparts. In situ and ex vivo analysis featured such tumours as activated type of diffuse large B cell lymphoma (ABC-DLBCL), expressing BCL2 and c-MYC, but not BCL6, with activated STAT3 signaling. OPN-/-Faslpr/lpr B lymphocytes showed an enhanced TLR9-MYD88 signaling pathway, either at baseline or after stimulation with CpG oligonucleotides, which mimic dsDNA circulating in autoimmune conditions. B cells from Faslpr/lpr mice were found to express the intracellular form of OPN. Accordingly, gene transfer-mediated re-expression of iOPN, but not of its secreted isoform, into ABC-DLBCL cell lines established from OPN-/-Faslpr/lpr mice, prevented CpG-mediated activation of STAT3, suggesting that the intracellular form of OPN may represent a brake to TLR9 signaling pathway activation. Conclusion These data indicate that, in the setting of SLE-like syndrome in which double strand-DNA chronically circulates and activates TLRs, B cell intracellular OPN exerts a protective role in autoimmunity-driven DLBCL development, mainly acting as a brake in the TLR9-MYD88-STAT3 signaling pathway

Inflammation as Prognostic Hallmark of Clinical Outcome in Patients with SARS-CoV-2 Infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is often characterized by a life-threatening interstitial pneumonia requiring hospitalization. The aim of this retrospective cohort study is to identify hallmarks of in-hospital mortality in patients affected by Coronavirus Disease 19 (COVID-19). A total of 150 patients admitted for COVID-19 from March to June 2021 to “F. Perinei” Murgia Hospital in Altamura, Italy, were divided into survivors (n = 100) and non-survivors groups (n = 50). Blood counts, inflammation-related biomarkers and lymphocyte subsets were analyzed into two groups in the first 24 h after admission and compared by Student’s t-test. A multivariable logistic analysis was performed to identify independent risk factors associated with in-hospital mortality. Total lymphocyte count and CD3+ and CD4+ CD8+ T lymphocyte subsets were significantly lower in non-survivors. Serum levels of interleukin-6 (IL-6), lactate dehydrogenase (LDH), C-reactive protein (CRP) and procalcitonin (PCT) were significantly higher in non-survivors. Age > 65 years and presence of comorbidities were identified as independent risk factors associated with in-hospital mortality, while IL-6 and LDH showed a borderline significance. According to our results, markers of inflammation and lymphocytopenia predict in-hospital mortality in COVID-19

Inflammation as Prognostic Hallmark of Clinical Outcome in Patients with SARS-CoV-2 Infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is often characterized by a life-threatening interstitial pneumonia requiring hospitalization. The aim of this retrospective cohort study is to identify hallmarks of in-hospital mortality in patients affected by Coronavirus Disease 19 (COVID-19). A total of 150 patients admitted for COVID-19 from March to June 2021 to “F. Perinei” Murgia Hospital in Altamura, Italy, were divided into survivors (n = 100) and non-survivors groups (n = 50). Blood counts, inflammation-related biomarkers and lymphocyte subsets were analyzed into two groups in the first 24 h after admission and compared by Student’s t-test. A multivariable logistic analysis was performed to identify independent risk factors associated with in-hospital mortality. Total lymphocyte count and CD3+ and CD4+ CD8+ T lymphocyte subsets were significantly lower in non-survivors. Serum levels of interleukin-6 (IL-6), lactate dehydrogenase (LDH), C-reactive protein (CRP) and procalcitonin (PCT) were significantly higher in non-survivors. Age > 65 years and presence of comorbidities were identified as independent risk factors associated with in-hospital mortality, while IL-6 and LDH showed a borderline significance. According to our results, markers of inflammation and lymphocytopenia predict in-hospital mortality in COVID-19