Saturation of acyl chains converts cardiolipin from an antagonist to an activator of Toll-like receptor-4

Abstract: Cardiolipins (CLs) are tetra-acylated diphosphatidylglycerols found in bacteria, yeast, plants, and animals. In healthy mammals, CLs are unsaturated, whereas saturated CLs are found in blood cells from Barth syndrome patients and in some Gram-positive bacteria. Here, we show that unsaturated but not saturated CLs block LPS-induced NF-κB activation, TNF-α and IP-10 secretion in human and murine macrophages, as well as LPS-induced TNF-α and IL-1β release in human blood mononuclear cells. Using HEK293 cells transfected with Toll-like receptor 4 (TLR4) and its co-receptor Myeloid Differentiation 2 (MD2), we demonstrate that unsaturated CLs compete with LPS for binding TLR4/MD2 preventing its activation, whereas saturated CLs are TLR4/MD2 agonists. As a consequence, saturated CLs induce a pro-inflammatory response in macrophages characterized by TNF-α and IP-10 secretion, and activate the alternative NLRP3 inflammasome pathway in human blood-derived monocytes. Thus, we identify that double bonds discriminate between anti- and pro-inflammatory properties of tetra-acylated molecules, providing a rationale for the development of TLR4 activators and inhibitors for use as vaccine adjuvants or in the treatment of TLR4-related diseases. Graphical abstract

P2X7 receptor induces mitochondrial failure in monocytes and compromises NLRP3 inflammasome activation during sepsis

International audienceSepsis is characterized by a systemic inflammatory response followed by immunosuppres-sion of the host. Metabolic defects and mitochondrial failure are common in immunocom-promised patients with sepsis. The NLRP3 inflammasome is important for establishing an inflammatory response after activation by the purinergic P2X7 receptor. Here, we study a cohort of individuals with intra-abdominal origin sepsis and show that patient monocytes have impaired NLRP3 activation by the P2X7 receptor. Furthermore, most sepsis-related deaths are among patients whose NLRP3 activation is profoundly altered. In monocytes from sepsis patients, the P2X7 receptor is associated with mitochondrial dysfunction. Furthermore, activation of the P2X7 receptor results in mitochondrial damage, which in turn inhibits NLRP3 activation by HIF-1α. We show that mortality increases in a mouse model of sepsis when the P2X7 receptor is activated in vivo. These data reveal a molecular mechanism initiated by the P2X7 receptor that contributes to NLRP3 impairment during infection

A high concentration of TGF-β correlates with opportunistic infection in liver and kidney transplantation

Transforming growth factor-β (TGF-β) has been associated with numerous human infections, but its role in the occurrence of opportunistic infection (OI) after solid organ transplantation remains unexplored. This study aimed to assess the utility of the TGF-β following in vitro stimulation of whole peripheral blood (WPB) as a surrogate biomarker of post-transplant OI in a cohort of liver and kidney recipients. Thirty liver and thirty-one kidney transplant recipients were recruited to be prospectively monitored for one-year post-transplantation. Enzyme-linked immunosorbent assay (ELISA) was performed to calculate IFN-γ, IL-17, IL-10 and TGF-β concentration in the supernatant from the activated WPB. Recipients showed higher TGF-β concentrations compared to IFN-γ, IL-17, IL-10 at baseline, although these differences were not significant between INF and NoINF. However, recipients who developed an OI within the first sixth months had a higher concentration of TGF-β than those without OI. A concentration of TGF-β > 363.25 pg/ml in liver and TGF-β > 808.51 pg/ml in kidney recipients were able to stratify patients at high risk of OI with a sensitivity and specificity above 70% in both types of solid organ transplantations. TGF-β could provide valuable information for the management of liver and kidney recipients at risk of post-transplant infection.Our work was possible thanks to the support and funding obtained from the “Instituto de Salud Carlos III (ISCIII)”, Spanish Ministry of Health (Grant Number PI15/01370 and P19/01194); and co-funding by the European Union from the European Regional Development Fund (ERDF) with the principle of “A manner to build Europe”

Purinergic P2X7 receptor expression increases in leukocytes from intra-abdominal septic patients

Inflammation is a tightly coordinated response of the host immune system to bacterial and viral infections, triggered by the production of inflammatory cytokines. Sepsis is defined as a systemic inflammatory response followed by immunosuppression of the host and organ dysfunction. This imbalance of the immune response increases the risk of mortality of patients with sepsis, making it a major problem for critical care units worldwide. The P2X7 receptor plays a crucial role in activating the immune system by inducing the activation of peripheral blood mononuclear cells. In this study, we analyzed a cohort of abdominal origin septic patients and found that the expression of the P2X7 receptor in the plasma membrane is elevated in the different subsets of lymphocytes. We observed a direct relationship between the percentage of P2X7-expressing lymphocytes and the early inflammatory response in sepsis. Additionally, in patients whose lymphocytes presented a higher percentage of P2X7 surface expression, the total lymphocytes populations proportionally decreased. Furthermore, we found a correlation between elevated soluble P2X7 receptors in plasma and inflammasome-dependent cytokine IL-18. In summary, our work demonstrates that P2X7 expression is highly induced in lymphocytes during sepsis, and this correlates with IL-18, along with other inflammatory mediators such as IL-6, IL-8, and procalcitonin

NLRP3 inflammasome activation and symptom burden in KRAS-mutated CMML patients is reverted by IL-1 blocking therapy

Chronic myelomonocytic leukemia (CMML) is frequently associated with mutations in the rat sarcoma gene (RAS), leading to worse prognosis. RAS mutations result in active RAS-GTP proteins, favoring myeloid cell proliferation and survival and inducing the NLRP3 inflammasome together with the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), which promote caspase-1 activation and interleukin (IL)-1(3 release. Here, we report, in a cohort of CMML patients with mutations in KRAS, a constitutive activation of the NLRP3 inflammasome in monocytes, evidenced by ASC oligomerization and IL-1(3 release, as well as a specific inflammatory cytokine signature. Treatment of a CMML patient with a KRASG12D mutation using the IL-1 receptor blocker anakinra inhibits NLRP3 inflammasome activation, reduces monocyte count, and improves the patient's clinical status, enabling a stem cell transplant. This reveals a basal inflammasome activation in RAS-mutated CMML patients and suggests potential therapeutic applications of NLRP3 and IL-1 blockers

Estudio del polimorfismo de los genes KIR y NKG2D y de sus ligandos HLA clase I y MICA en pacientes con melanoma

Los resultados y conclusiones del presente trabajo son los siguientes: 1. El genotipo KIR2DL3+/C1+ se asocia a protección frente al desarrollo de melanoma y de metástasis en ganglio centinela en pacientes diagnosticados de MES y MN. 2. La ausencia de KIR2DL3 (KIR2DL2 en homocigosis) en pacientes portadores de ligandos C1 podría representar un factor de riesgo para el desarrollo de melanoma nodular y para la progresión a la ulceración de la lesión tumoral. 3. El genotipo KIR2DL1+2DS1-C2C2 podría ser considerado como un factor de riesgo para el desarrollo de melanoma y de metástasis en ganglio centinela en individuos diagnosticados de MES. 4. El aumento de clones de células NK KIR2DS1+ observado en pacientes de melanoma sugiere un papel importante de dicha población celular en la respuesta inmunitaria frente al melanoma cutáneo. 5. Los SNPs de la región NKC estudiados individualmente no parece presentar asociación con el desarrollo y/o pronóstico del melanoma cutáneo. 6. El haplotipo NK-3 del bloque hb-2 de la región génica NKC parece estar asociado con un mayor riesgo de desarrollo de melanoma cutáneo. 7. La expresión aumentada del receptor NKG2D en células NK y linfocitos T CD8+ observada en los pacientes con melanoma cutáneo parece indicar un estado de activación de las mismas. 8. El alelo MICA*009 parece estar asociado con un mayor riesgo de desarrollo de melanoma cutáneo. No obstante, se requieren estudios con series más amplias para confirmar esta asociación. 9. El dimorfismo en posición 80 del gen MICA no parece estar asociado con el desarrollo del melanoma cutáneo. 10. La variante MICA-129Met está asociada con un mayor nivel de MICA soluble en plasma y una peor supervivencia de los pacientes de melanoma cutáneo. OBJECTIVES 1. To study the gene polymorphism of KIR receptors and their HLA class I ligands in patients with melanoma and in a control population. 2. To analyze the subtypes of NK cells and CD8 + T lymphocytes that express KIR receptors in patients with melanoma and in a control population. 3. To study the gene polymorphism of the NKG2D receptor and its MICA ligands in patients with melanoma and in a control population. 4. To analyze the expression of the NKG2D receptor in NK cells and CD8 + T lymphocytes of patients with melanoma and a control population. 5. To study the level of soluble MICA in plasma samples from melanoma patients at diagnosis and from a control population. METHODOLOGY The KIR, HLA-A, B, C, and MICA genes typing was performed by SSO-PCR, reverse-SSO-PCR and SSP-PCR techniques, and the NKC gene region typing by an allelic discrimination assay with Taqman probes and by Sanger sequencing. These trials were carried out in 233 patients diagnosed with cutaneous melanoma and 200 healthy controls. The flow cytometry study to evaluate the expression of KIR receptors in NK cells and peripheral blood CD8 + T lymphocytes was performed in 35 patients diagnosed with melanoma and 24 healthy individuals, while the expression of NKG2D was evaluated in 48 patients with melanoma. and 37 healthy individuals. The determination of soluble MICA protein was carried out using a "sandwich" ELISA technique in the plasma of 30 patients diagnosed with cutaneous melanoma and 26 healthy individuals. RESULTS AND CONCLUSIONS The results and conclusions of the present work were the following: 1. The KIR2DL3+/C1+ genotype is associated with protection against the development of melanoma and sentinel lymph node metastasis in patients diagnosed with SSM and NM. 2. The absence of KIR2DL3 (KIR2DL2 in homozygosis) in patients carrying C1 ligands could represent a risk factor for the development of nodular melanoma and for the progression to the ulceration of the tumor lesion. 3. The KIR2DL1+2DS1-C2C2 genotype could be considered as a risk factor for the development of melanoma and sentinel lymph node metastasis in individuals diagnosed with SSM. 4. The increase of KIR2DS1+ NK cell clones observed in melanoma patients suggests an important role of this cell population in the immune response against the cutaneous melanoma. 5. The NKC region SNPs studied individually does not seem to be associated with the development and/or prognosis of cutaneous melanoma. 6. The NK-3 haplotype of the hb-2 block of the NKC gene region seems to be associated with an increased risk of cutaneous melanoma development. 7. The increased expression of NKG2D receptor observed in NK cells and CD8+ T lymphocytes from patients with cutaneous melanoma seems to indicate an activation state of them. 8. The MICA*009 allele seems to be associated with an increased risk of cutaneous melanoma development. However, studies with larger series are needed to confirm this association. 9. The position 80 MICA gene dimorphism does not seem to be associated with the development of cutaneous melanoma. 10. The MICA-129Met variant is associated with a higher level of soluble MICA in plasma and a worse survival of cutaneous melanoma patients

Saturation of acyl chains converts cardiolipin from an antagonist to an activator of Toll-like receptor-4

Abstract: Cardiolipins (CLs) are tetra-acylated diphosphatidylglycerols found in bacteria, yeast, plants, and animals. In healthy mammals, CLs are unsaturated, whereas saturated CLs are found in blood cells from Barth syndrome patients and in some Gram-positive bacteria. Here, we show that unsaturated but not saturated CLs block LPS-induced NF-κB activation, TNF-α and IP-10 secretion in human and murine macrophages, as well as LPS-induced TNF-α and IL-1β release in human blood mononuclear cells. Using HEK293 cells transfected with Toll-like receptor 4 (TLR4) and its co-receptor Myeloid Differentiation 2 (MD2), we demonstrate that unsaturated CLs compete with LPS for binding TLR4/MD2 preventing its activation, whereas saturated CLs are TLR4/MD2 agonists. As a consequence, saturated CLs induce a pro-inflammatory response in macrophages characterized by TNF-α and IP-10 secretion, and activate the alternative NLRP3 inflammasome pathway in human blood-derived monocytes. Thus, we identify that double bonds discriminate between anti- and pro-inflammatory properties of tetra-acylated molecules, providing a rationale for the development of TLR4 activators and inhibitors for use as vaccine adjuvants or in the treatment of TLR4-related diseases. Graphical abstract

Saturation of acyl chains converts cardiolipin from an antagonist to an activator of Toll-like receptor-4

Abstract: Cardiolipins (CLs) are tetra-acylated diphosphatidylglycerols found in bacteria, yeast, plants, and animals. In healthy mammals, CLs are unsaturated, whereas saturated CLs are found in blood cells from Barth syndrome patients and in some Gram-positive bacteria. Here, we show that unsaturated but not saturated CLs block LPS-induced NF-κB activation, TNF-α and IP-10 secretion in human and murine macrophages, as well as LPS-induced TNF-α and IL-1β release in human blood mononuclear cells. Using HEK293 cells transfected with Toll-like receptor 4 (TLR4) and its co-receptor Myeloid Differentiation 2 (MD2), we demonstrate that unsaturated CLs compete with LPS for binding TLR4/MD2 preventing its activation, whereas saturated CLs are TLR4/MD2 agonists. As a consequence, saturated CLs induce a pro-inflammatory response in macrophages characterized by TNF-α and IP-10 secretion, and activate the alternative NLRP3 inflammasome pathway in human blood-derived monocytes. Thus, we identify that double bonds discriminate between anti- and pro-inflammatory properties of tetra-acylated molecules, providing a rationale for the development of TLR4 activators and inhibitors for use as vaccine adjuvants or in the treatment of TLR4-related diseases.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Evaluating the Link between BAFF System Gene Expression and Acute Rejection Development in Kidney Transplantation

B-cell activating factor (BAFF) system signaling is critical for B-cell homeostasis, effector functions, and tolerance maintenance in transplants, but it has not been studied in kidney transplant recipients (KTRs). The aim was to analyze the changes in BAFF system expression in KTRs with/without acute rejection (AR/NAR). The BAFF system expression was analyzed by qPCR in 40 KTRs. A meta-analysis of BAFF system expression and histological renal damage was identified by the Chronic Allograft Damage Index (CADI) and performed from the GEO database. Proliferation-inducing ligand (APRIL) expression increased at three- and six-months post-KT (p = 0.014 and p p = 0.038). BAFF expression remained stable in NAR-KTRs, but was increased in CADI concerning the No-CADI group at one year (p = 0.008). BCMA expression increased in the CADI group at one- (p = 0.001) and six-years post-KT (p = 0.024). At three months, the transmembrane activator and calcium modulator interactor (TACI) gene significantly elevated KTRs with DSAs (donor-specific antibody; p = 0.034). KTRs with DSAs significantly increase the B-cell activating factor receptor (R-BAFF; p = 0.021) and TACI (p = 0.018) between pre- and three-month post-KT. Changes in the expression of the BAFF system increase during post-KTR in the development of AR and chronic allograft damage, and could be an important pathological tool to detect and prevent kidney graft outcomes

All That Glitters in cfDNA Analysis Is Not Gold or Its Utility Is Completely Established Due to Graft Damage: A Critical Review in the Field of Transplantation

In kidney transplantation, a biopsy is currently the gold standard for monitoring the transplanted organ. However, this is far from an ideal screening method given its invasive nature and the discomfort it can cause the patient. Large-scale studies in renal transplantation show that approximately 1% of biopsies generate major complications, with a risk of macroscopic hematuria greater than 3.5%. It would not be until 2011 that a method to detect donor-derived cell-free DNA (dd-cfDNA) employing digital PCR was devised based on analyzing the differences in SNPs between the donor and recipient. In addition, since the initial validation studies were carried out at the specific moments in which rejection was suspected, there is still not a good understanding of how dd-cfDNA levels naturally evolve post-transplant. In addition, various factors, both in the recipient and the donor, can influence dd-cfDNA levels and cause increases in the levels of dd-cfDNA themselves without suspicion of rejection. All that glitters in this technology is not gold; therefore, in this article, we discuss the current state of clinical studies, the benefits, and disadvantages