Dysregulated TRAF3 and BCL2 Expression Promotes Multiple Classes of Mature Non-hodgkin B Cell Lymphoma in Mice

TNF-Receptor Associated Factor (TRAF)-3 is a master regulator of B cell homeostasis and function. TRAF3 has been shown to bind and regulate various proteins involved in the control of innate and adaptive immune responses. Previous studies showed that TRAF3 overexpression renders B cells hyper-reactive to antigens and Toll-like receptor (TLR) agonists, while TRAF3 deficiency has been implicated in the development of a variety of B cell neoplasms. In this report, we show that transgenic mice overexpressing TRAF3 and BCL2 in B cells develop with high incidence severe lymphadenopathy, splenomegaly and lymphoid infiltrations into tissues and organs, which is the result of the growth of monoclonal and oligoclonal B cell neoplasms, as demonstrated by analysis of VHDJH gene rearrangement. FACS and immunohistochemical analyses show that different types of mature B cell neoplasms arise in TRAF3/BCL2 double-transgenic (tg) mice, all of which are characterized by the loss of surface IgM and IgD expression. However, two types of lymphomas are predominant: (1) mature B cell neoplasms consistent with diffuse large B cell lymphoma and (2) plasma cell neoplasms. The Ig isotypes expressed by the expanded B-cell clones included IgA, IgG, and IgM, with most having undergone somatic hypermutation. In contrast, mouse littermates representing all the other genotypes (TRAF3-/BCL2-; TRAF3+/BCL2-, and TRAF3-/BCL2+) did not develop significant lymphadenopathy or clonal B cell expansions within the observation period of 20 months. Interestingly, a large representation of the HCDR3 sequences expressed in the TRAF3-tg and TRAF3/BCL2-double-tg B cells are highly similar to those recognizing pathogen-associated molecular patterns and damage-associated molecular patterns, strongly suggesting a role for TRAF3 in promoting B cell differentiation in response to these antigens. Finally, allotransplantation of either splenocytes or cell-containing ascites from lymphoma-bearing TRAF3/BCL2 mice into SCID/NOD immunodeficient mice showed efficient transfer of the parental expanded B-cell clones. Altogether, these results indicate that TRAF3, perhaps by promoting exacerbated B cell responses to certain antigens, and BCL2, presumably by supporting survival of these clones, cooperate to induce mature B cell neoplasms in transgenic mice

Specialized proresolving mediators protect against experimental autoimmune myocarditis by modulating Ca2+ handling and NRF2 activation

Specialized proresolving mediators and, in particular, 5(S), (6)R, 7-trihydroxyheptanoic acid methyl ester (BML-111) emerge as new therapeutic tools to prevent cardiac dysfunction and deleterious cardiac damage associated with myocarditis progression. The cardioprotective role of BML-111 is mainly caused by the prevention of increased oxidative stress and nuclear factor erythroid-derived 2-like 2 (NRF2) down-regulation induced by myocarditis. At the molecular level, BML-111 activates NRF2 signaling, which prevents sarcoplasmic reticulum–adenosine triphosphatase 2A down-regulation and Ca2+ mishandling, and attenuates the cardiac dysfunction and tissue damage induced by myocarditis.This work was supported by the Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund (SAF-2017-84777R), Instituto de Salud Carlos III (ISCIII) (PI17/01093, PI17/01344, and PI20/01482), Sociedad Española de Cardiología, Proyecto Traslacional 2019 and Asociación del Ritmo Cardiaco (SEC, España), Proyecto Asociación Insuficiencia Cardiaca (Trasplante Cardiaco) 2020, Fondo Europeo de Desarrollo Regional, Fondo Social Europeo, and CIBERCV, a network funded by ISCIII, Spanish Ministry of Science, Innovation and Universities (PGC2018-097019-B-I00), Ministerio de Economía, Industria y Competitividad/Agencia Estatal de Investigación 10.13039/501100011033 PID2020-113238RB-I00, PID2019-105600RB-I00, the Instituto de Salud Carlos III (Fondo de Investigación Sanitaria grant PRB3 [PT17/0019/0003-ISCIII-SGEFI/ERDF, ProteoRed]), and “la Caixa” Foundation (project code HR17-00247). The Centro Nacional de Investigaciones Cardiovasculares is supported by the ISCIII, the Ministerio de Ciencia, Innovación y Universidades. Dr Ruiz-Hurtado is Miguel Servet I researcher of ISCIII (CP15/00129 Carlos III Health Institute). Dr Tamayo and R.I. Jaén, and M. Gil-Fernández were or currently are PhD students funded by the Formación de Profesorado Universitario program of the Spanish Ministry of Science, Innovation and Universities (FPU17/06135; FPU16/00827, FPU1901973)

Dysregulated TRAF3 and BCL2 expression promotes multiple classes of mature non-hodgkin B cell lymphoma in mice

TNF-Receptor Associated Factor (TRAF)-3 is a master regulator of B cell homeostasis and function. TRAF3 has been shown to bind and regulate various proteins involved in the control of innate and adaptive immune responses. Previous studies showed that TRAF3 overexpression renders B cells hyper-reactive to antigens and Toll-like receptor (TLR) agonists, while TRAF3 deficiency has been implicated in the development of a variety of B cell neoplasms. In this report, we show that transgenic mice overexpressing TRAF3 and BCL2 in B cells develop with high incidence severe lymphadenopathy, splenomegaly and lymphoid infiltrations into tissues and organs, which is the result of the growth of monoclonal and oligoclonal B cell neoplasms, as demonstrated by analysis of VHDJH gene rearrangement. FACS and immunohistochemical analyses show that different types of mature B cell neoplasms arise in TRAF3/BCL2 double-transgenic (tg) mice, all of which are characterized by the loss of surface IgM and IgD expression. However, two types of lymphomas are predominant: (1) mature B cell neoplasms consistent with diffuse large B cell lymphoma and (2) plasma cell neoplasms. The Ig isotypes expressed by the expanded B-cell clones included IgA, IgG, and IgM, with most having undergone somatic hypermutation. In contrast, mouse littermates representing all the other genotypes (TRAF3-/BCL2-; TRAF3+/BCL2-, and TRAF3-/BCL2+) did not develop significant lymphadenopathy or clonal B cell expansions within the observation period of 20 months. Interestingly, a large representation of the HCDR3 sequences expressed in the TRAF3-tg and TRAF3/BCL2-double-tg B cells are highly similar to those recognizing pathogen-associated molecular patterns and damage-associated molecular patterns, strongly suggesting a role for TRAF3 in promoting B cell differentiation in response to these antigens. Finally, allotransplantation of either splenocytes or cell-containing ascites from lymphoma-bearing TRAF3/BCL2 mice into SCID/NOD immunodeficient mice showed efficient transfer of the parental expanded B-cell clones. Altogether, these results indicate that TRAF3, perhaps by promoting exacerbated B cell responses to certain antigens, and BCL2, presumably by supporting survival of these clones, cooperate to induce mature B cell neoplasms in transgenic mice.Funding was from Instituto de Salud Carlos III (ISCIII) PI080170, PI12/01135, and PI16/00895 to JZ and from the National Institutes of Health (AI070859 to JZ and CA163743 to JR).The cost of this publication was paid in part by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI) and by funds from the European Fund for Economic and Regional Development (FEDER).Peer reviewe

Mitochondrial DAMPs induce endotoxin tolerance in human monocytes: an observation in patients with myocardial infarction.

Monocyte exposure to mitochondrial Danger Associated Molecular Patterns (DAMPs), including mitochondrial DNA (mtDNA), induces a transient state in which these cells are refractory to further endotoxin stimulation. In this context, IRAK-M up-regulation and impaired p65 activity were observed. This phenomenon, termed endotoxin tolerance (ET), is characterized by decreased production of cytokines in response to the pro-inflammatory stimulus. We also show that monocytes isolated from patients with myocardial infarction (MI) exhibited high levels of circulating mtDNA, which correlated with ET status. Moreover, a significant incidence of infection was observed in those patients with a strong tolerant phenotype. The present data extend our current understanding of the implications of endotoxin tolerance. Furthermore, our data suggest that the levels of mitochondrial antigens in plasma, such as plasma mtDNA, should be useful as a marker of increased risk of susceptibility to nosocomial infections in MI and in other pathologies involving tissue damage

M1/M2 score of ACS patients.

<p>(<b>A–B</b>) Percentage of patients classified according to the M1/M2 Score of UA (black bars), NSTEMI (dark gray bars) and STEMI (light grey bars) patients, calculated using mRNA expression levels of TNFα and IL10 in MΦs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095073#s2" target="_blank">Methods</a>). (<b>A</b>) At baseline: M1, M1/M0, M0, M0/M2, M2. (<b>B</b>) After LPS (3 h, 10 ng/ml): M1high, M1, M1/M2, M2. (<b>C–D</b>) Circulating mtDNA levels in the plasma of ACS patients compared with the M1/M2 score. Data are shown as Mean±SD. (<b>C</b>) Score at basal condition: M1 n = 2, M1/M0 n = 5, M0 n = 19, M0/M2 n = 28, M2 n = 21. (D) Score after LPS (3 h, 10 ng/ml): M1-high n = 5, M1 n = 27, M1/M2 n = 23, M2 n = 20.</p

Modulation of human monocytes by exposure to mitochondrial DAMPs.

<p>(<b>A</b>) Workflow diagram: MΦs from HV were pre-exposed to mitochondrial DAMPs (mtLys, 20 µg/ml, white bars), LPS (10 ng/ml, gray bars) or left untreated (black bars) for 5 days, then stimulated for 24 h with LPS, as described in the scheme; <b>B–F</b>: (<b>B</b>) Protein levels of IL1β, (<b>C</b>) IL6, (<b>D</b>) IL12p70, (<b>E</b>) TNFα and (<b>F</b>) TGF-β in culture supernatants (measured by CBA). Data are shown as Mean±SD, n = 3; *p<0.01, ***p<0.001; ns: non-significant <i>vs.</i>corresponding control of untreated 5d +LPS stimulus (ANOVA/Dunn). (<b>G and H</b>): Histogram plots of surface HLA-DQ (up) and HLA-DR (down) expression on CD14+ MΦs evaluated by flow cytometry, after 24 h of LPS challenge in MΦs pre-exposed 5 days to mtLys (left panels) or LPS (right panels), compared to MΦs with no pre-treatment (gray filled, all panels). Mean Fold decrease of MFI compared to corresponding control with no pre-treatment is shown. (<b>I</b>) CD163+CD14+ frequencies in MΦs pre-exposed 5 days to mtLys (<b>central panel</b>), LPS (<b>right panel</b>) or medium (<b>left panel</b>). Representative dot plots of three independently performed experiments are shown. Values in the upper-left quadrants indicate the Mean frequencies of CD163+CD14+ subsets.</p

Modulation of human monocytes by exposure to mitochondrial DNA.

<p>(<b>A</b>) Workflow diagram: MΦs from HV were pre-exposed to mitochondrial DNA isolated from HeLa (mtDNA, 5 µg/ml, white bars), LPS (10 ng/ml, gray bars) or left untreated (black bars) for 5 days, then stimulated for 24 h with LPS, as described in the scheme. <b>B–F</b>: (<b>B</b>) Protein levels of IL1β, (<b>C</b>) IL6, (<b>D</b>) IL12p70, (<b>E</b>) TNFα and (<b>F</b>) TGF-β in culture supernatants (measured by CBA). Data are shown as Mean±SD, n = 3; *p<0.05,**p<0.01 ***p<0.001; ns: non-significant <i>vs.</i> corresponding control of untreated 5d +LPS stimulus (ANOVA/Dunn). (<b>G and H</b>): Histogram plots of surface HLA-DQ (up) and HLA-DR (down) expression on CD14+ MΦs evaluated by flow cytometry, after 24 h of LPS challenge in MΦs pre-exposed 5 days to mtDNA (left panels) or LPS (right panels), compared to MΦs with no pre-treatment (gray filled, all panels). Mean Fold decrease of MFI compared to corresponding control with no pre-treatment is shown. (<b>I</b>) CD163+CD14+ frequencies in MΦs pre-exposed 5 days to mtDNA (<b>central panel</b>), LPS (<b>right panel</b>) or medium (<b>left panel</b>). Representative dot plots of three independently performed experiments are shown. Values in the upper-left quadrants indicate the Mean frequencies of CD163+CD14+ subsets.</p

Levels of mtDNA in ACS patients' plasma and cytokine expression.

<p>Correlation between plasma mtDNA and TNFα after LPS challenge (3 h, 10 ng/ml, <b>A</b>) and IL10 mRNA basal levels (<b>B</b>) in MΦs from patients with ACS <i>r</i>: Spearman correlation coefficients are shown.</p

Modulation of IL10 production after exposure to mitochondrial DAMPs and mitochondrial DNA.

<p>MΦs from HV were pre-exposed to both mitochondrial DAMPs (mtLys, 20 µg/ml, white bars) and mitochondrial DNA isolated from HeLa (mtDNA, 5 µg/ml, white bars) or left untreated (black bars) for 5 days, then stimulated for 24 h with LPS, as described in the scheme of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095073#pone-0095073-g001" target="_blank">Figure 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095073#pone-0095073-g003" target="_blank">Figure 3</a>. Protein levels of IL10 in culture supernatants (measured by CBA) is given. Data are shown as Mean±SD, n = 3; *p<0.01 <i>vs.</i> corresponding control of untreated 5d+LPS stimulus (ANOVA/Dunn).</p

Mitochondrial DNA levels in plasma are increased in STEMI patients.

<p>Plasma mitochondrial DNA (mtDNA) levels of ACS patients (white bars) and HV (gray bar). Data are shown as box plots with Mean±SD. HV n = 20, patients with ACS: UA n = 30, NSTEMI n = 24, STEMI n = 21; **p<0.01, patients <i>vs.</i> HV (ANOVA/Dunn).</p