Genetic variations in A20 DUB domain provide a genetic link to citrullination and neutrophil extracellular traps in systemic lupus erythematosus

Objectives: Genetic variations in TNFAIP3 (A20) de-ubiquitinase (DUB) domain increase the risk of systemic lupus erythematosus (SLE) and rheumatoid arthritis. A20 is a negative regulator of NF-κB but the role of its DUB domain and related genetic variants remain unclear. We aimed to study the functional effects of A20 DUB-domain alterations in immune cells and understand its link to SLE pathogenesis. Methods: CRISPR/Cas9 was used to generate human U937 monocytes with A20 DUB-inactivating C103A knock-in (KI) mutation. Whole genome RNA-sequencing was used to identify differentially expressed genes between WT and C103A KI cells. Functional studies were performed in A20 C103A U937 cells and in immune cells from A20 C103A mice and genotyped healthy individuals with A20 DUB polymorphism rs2230926. Neutrophil extracellular trap (NET) formation was addressed ex vivo in neutrophils from A20 C103A mice and SLE-patients with rs2230926. Results: Genetic disruption of A20 DUB domain in human and murine myeloid cells did not give rise to enhanced NF-κB signalling. Instead, cells with C103A mutation or rs2230926 polymorphism presented an upregulated expression of PADI4, an enzyme regulating protein citrullination and NET formation, two key mechanisms in autoimmune pathology. A20 C103A cells exhibited enhanced protein citrullination and extracellular trap formation, which could be suppressed by selective PAD4 inhibition. Moreover, SLE-patients with rs2230926 showed increased NETs and increased frequency of autoantibodies to citrullinated epitopes. Conclusions: We propose that genetic alterations disrupting the A20 DUB domain mediate increased susceptibility to SLE through the upregulation of PADI4 with resultant protein citrullination and extracellular trap formation

Long-Term Follow-Up of Newborns with 22q11 Deletion Syndrome and Low TRECs.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked DownloadBackground: Population-based neonatal screening using T-cell receptor excision circles (TRECs) identifies infants with profound T lymphopenia, as seen in cases of severe combined immunodeficiency, and in a subgroup of infants with 22q11 deletion syndrome (22q11DS). Purpose: To investigate the long-term prognostic value of low levels of TRECs in newborns with 22q11DS. Methods: Subjects with 22q11DS and low TRECs at birth (22q11Low, N=10), matched subjects with 22q11DS and normal TRECs (22q11Normal, N=10), and matched healthy controls (HC, N=10) were identified. At follow-up (median age 16 years), clinical and immunological characterizations, covering lymphocyte subsets, immunoglobulins, TRECs, T-cell receptor repertoires, and relative telomere length (RTL) measurements were performed. Results: At follow-up, the 22q11Low group had lower numbers of naïve T-helper cells, naïve T-regulatory cells, naïve cytotoxic T cells, and persistently lower TRECs compared to healthy controls. Receptor repertoires showed skewed V-gene usage for naïve T-helper cells, whereas for naïve cytotoxic T cells, shorter RTL and a trend towards higher clonality were found. Multivariate discriminant analysis revealed a clear distinction between the three groups and a skewing towards Th17 differentiation of T-helper cells, particularly in the 22q11Low individuals. Perturbations of B-cell subsets were found in both the 22q11Low and 22q11Normal group compared to the HC group, with larger proportions of naïve B cells and lower levels of memory B cells, including switched memory B cells. Conclusions: This long-term follow-up study shows that 22q11Low individuals have persistent immunologic aberrations and increased risk for immune dysregulation, indicating the necessity of lifelong monitoring. Clinical implications: This study elucidates the natural history of childhood immune function in newborns with 22q11DS and low TRECs, which may facilitate the development of programs for long-term monitoring and therapeutic choices. Keywords: 22q11.2 deletion syndrome; DiGeorge syndrome; T lymphopenia; TREC; long-term outcome; newborn screening; severe combined immunodeficiency.University of Gothenburg Regional research grant Region Halland Swedish Research Council European Commission Queen Silvia Jubilee Foundation Swedish Primary Immunodeficiency Organization Sparbanken Foundation Varberg Frimurare Barnhusdirektionen Foundation Gothenburg Medical Society Medical Faculty at Umea University Cancer Research Foundation in Northern Sweden Swedish government county councils, the ALF-agreement Umea University Vasterbottens County Counci

Unraveling Molecular Signatures of Immunostimulatory Adjuvants in the Female Genital Tract through Systems Biology

Sexually transmitted infections (STIs) unequivocally represent a major public health concern in both industrialized and developing countries. Previous efforts to develop vaccines for systemic immunization against a large number of STIs in humans have been unsuccessful. There is currently a drive to develop mucosal vaccines and adjuvants for delivery through the genital tract to confer protective immunity against STIs. Identification of molecular signatures that can be used as biomarkers for adjuvant potency can inform rational development of potent mucosal adjuvants. Here, we used systems biology to study global gene expression and signature molecules and pathways in the mouse vagina after treatment with two classes of experimental adjuvants. The Toll-like receptor 9 agonist CpG ODN and the invariant natural killer T cell agonist alpha-galactosylceramide, which we previously identified as equally potent vaginal adjuvants, were selected for this study. Our integrated analysis of genome-wide transcriptome data determined which signature pathways, processes and networks are shared by or otherwise exclusive to these 2 classes of experimental vaginal adjuvants in the mouse vagina. To our knowledge, this is the first integrated genome-wide transcriptome analysis of the effects of immunomodulatory adjuvants on the female genital tract of a mammal. These results could inform rational development of effective mucosal adjuvants for vaccination against STIs

La gD2 coadministrada con el AFCo1 por vía intranasal induce inmunidad protectora contra virus de herpes simple tipo 2 en ratones

La infección por virus herpes simple tipo 2 (VHS-2) continúa siendo un problema de salud mundial. Esta infección es transmitida sexualmente y es la principal causa de úlceras genitales. La prevención de esta enfermedad requiere de la utilización de vacunas mucosales, pues las vacunas parenterales no han sido exitosas. Por otra parte, no existen adyuvantes mucosales, por lo que el desarrollo de estos es esencial para la estrategia de estas vacunas. La administración intranasal (IN) de la glicoproteína D del VHS-2 (gD2), coadministrada con el cocleato (AFCo1+gD2) sería igualmente efectiva con la gD2 incluida (AFCo1-gD2). Se inocularon ratones hembras C57BL/6 por la vía IN con gD2, contenida dentro del cocleato, coadministrada con el cocleato o gD2 sola. Se determinaron los niveles de IgG anti gD2 en suero y lavado vaginal, así como las subclases de IgG anti gD2 por ELISA. Se determinó la respuesta linfoproliferativa en células de bazo, el perfil de citoquinas Th1/Th2, los signos de la enfermedad y la protección frente al reto viral. Se observaron altos títulos de IgG e IgG2c anti gD2 en el suero de los animales inoculados con la gD2 y el AFCo1 como adyuvante. No se observaron diferencias significativas (p>0,05) entre los grupos que recibieron AFCo1+gD2 y los que recibieron AFCo1-gD2. Se observó un perfil de citoquinas tipo Th1 y un 100% de sobrevida en los grupos que recibieron el AFCo1 como adyuvante de la gD2, mientras que en el grupo que recibió la gD2 sola no se observó protección. Estos resultados indican que la gD2 puede ser utilizada coadministrada con AFCo1 por vía IN como un potencial candidato vacunal contra VHS-2

Nasal immunization with AFCo1 induces immune response to N. gonorrhoea in mice

Neisseria gonorrhoeae infections are common sexually transmitted diseases. Increased antibiotic-resistant of N. gonorrhoeae strains were reported. N. meningitidis is another human restricted bacterium transmitted through mucosa. However, the induction of systemic specific IgG antibody against some proteins between the two species is known, but the mucosal immune response to these pathogens is not clear. We hypothesized that N. meningitidis could induce immune response against N. gonorrhoeae. Therefore, serogroup B Proteoliposome (PL) was transformed into AFCo1 (Adjuvant Finlay Cochleate 1) and used for nasal immunization of C57Bl/6 mice. The specific IgG and IgG subclasses against both antigens in sera and vaginal extraction were measured by ELISA. Specific proliferation (3H incorporation) of spleen cells and lymph node recall in vitro with PL or N. gonorrhoeae total antigens was measured. Serum and vaginal extraction anti N. meningitidis and N. gonorrhoeae IgG as well as the induction of specific IgG subclasses were detected. N. gonorrhoeae induces specific proliferation of spleen, cervical lymph node (cLN), and mediestinal (meLN) cells from immunized mice. In conclusion, AFCo1 induce anti N. meningitidis immune responses that recognized N. gonorrhoeae antigens in mice

Study protocol : establishment of a multicentre pre-eclampsia database and biobank in Sweden: GO PROVE and UP MOST, a prospective cohort study

Introduction Pre-eclampsia, a multisystem disorder in pregnancy, is one of the most common causes of maternal morbidity and mortality worldwide. However, we lack methods for objective assessment of organ function in pre-eclampsia and predictors of organ impairment during and after pre-eclampsia. The women’s and their partners’ experiences of pre-eclampsia have not been studied in detail. To phenotype different subtypes of the disorder is of importance for prediction, prevention, surveillance, treatment and follow-up of pre-eclampsia. The aim of this study is to set up a multicentre database and biobank for pre-eclampsia in order to contribute to a safer and more individualised treatment and care. Methods and analysis This is a multicentre cohort study. Prospectively recruited pregnant women ≥18 years, diagnosed with pre-eclampsia presenting at Sahlgrenska University Hospital, Uppsala University Hospital and at Södra Älvsborgs Hospital, Sweden, as well as normotensive controls are eligible for participation. At inclusion and at 1-year follow-up, the participants donate biosamples that are stored in a biobank and they are also asked to participate in various organ-specific evaluations. In addition, questionnaires and interviews regarding the women’s and partner’s experiences are distributed at follow-up. Ethics and dissemination By creating a database and biobank, we will provide the means to explore the disorder in a broader sense and allow clinical and laboratory discoveries that can be translated to clinical trials aiming at improved care of women with pre-eclampsia. Further, to evaluate experiences and the psychological impact of being affected by pre-eclampsia can improve the care of pregnant women and their partners. In case of incidental pathological findings during examinations performed, they will be handled in accordance with clinical routine. Data are stored in a secure online database. Biobank samples are identified through the women’s personal identification number and pseudonymised after identification in the biobank before analysis. This study was approved by the regional ethical review board in Gothenburg on 28 December 2018 (approval number 955-18) and by the Swedish Ethical Review Authority on 27 February 2019 (approval number 2019-00309). Results from the study will be published in international peer-reviewed journals. Trial registration number ISRCTN1306076

Long-Term Follow-Up of Newborns with 22q11 Deletion Syndrome and Low TRECs

Background: Population-based neonatal screening using T-cell receptor excision circles (TRECs) identifies infants with profound T lymphopenia, as seen in cases of severe combined immunodeficiency, and in a subgroup of infants with 22q11 deletion syndrome (22q11DS). Purpose: To investigate the long-term prognostic value of low levels of TRECs in newborns with 22q11DS. Methods: Subjects with 22q11DS and low TRECs at birth (22q11Low, N=10), matched subjects with 22q11DS and normal TRECs (22q11Normal, N=10), and matched healthy controls (HC, N=10) were identified. At follow-up (median age 16 years), clinical and immunological characterizations, covering lymphocyte subsets, immunoglobulins, TRECs, T-cell receptor repertoires, and relative telomere length (RTL) measurements were performed. Results: At follow-up, the 22q11Low group had lower numbers of naïve T-helper cells, naïve T-regulatory cells, naïve cytotoxic T cells, and persistently lower TRECs compared to healthy controls. Receptor repertoires showed skewed V-gene usage for naïve T-helper cells, whereas for naïve cytotoxic T cells, shorter RTL and a trend towards higher clonality were found. Multivariate discriminant analysis revealed a clear distinction between the three groups and a skewing towards Th17 differentiation of T-helper cells, particularly in the 22q11Low individuals. Perturbations of B-cell subsets were found in both the 22q11Low and 22q11Normal group compared to the HC group, with larger proportions of naïve B cells and lower levels of memory B cells, including switched memory B cells. Conclusions: This long-term follow-up study shows that 22q11Low individuals have persistent immunologic aberrations and increased risk for immune dysregulation, indicating the necessity of lifelong monitoring. Clinical Implications: This study elucidates the natural history of childhood immune function in newborns with 22q11DS and low TRECs, which may facilitate the development of programs for long-term monitoring and therapeutic choices