Irma Joosten

Psoriasis (Pso) is a chronic inflammatory skin disease, and up to 30% of Pso patients develop psoriatic arthritis (PsA), which can lead to irreversible joint damage. Early detection of PsA in Pso patients is crucial for timely treatment but difficult for dermatologists to implement. We, therefore, aimed to find disease-specific immune profiles, discriminating Pso from PsA patients, possibly facilitating the correct identification of Pso patients in need of referral to a rheumatology clinic. The phenotypes of peripheral blood immune cells of consecutive Pso and PsA patients were analyzed, and disease-specific immune profiles were identified via a machine learning approach. This approach resulted in a random forest classification model capable of distinguishing PsA from Pso (mean AUC = 0.95). Key PsA-classifying cell subsets selected included increased proportions of differentiated CD4+CD196+CD183-CD194+ and CD4+CD196-CD183-CD194+ T-cells and reduced proportions of CD196+ and CD197+ monocytes, memory CD4+ and CD8+ T-cell subsets and CD4+ regulatory T-cells. Within PsA, joint scores showed an association with memory CD8+CD45RA-CD197- effector T-cells and CD197+ monocytes. To conclude, through the integration of in-depth flow cytometry and machine learning, we identified an immune cell profile discriminating PsA from Pso. This immune profile may aid in timely diagnosing PsA in Pso.

Background: Infection with SARS-CoV-2 manifests itself as a mild respiratory tract infection in the majority of individuals, which progresses to a severe pneumonia and acute respiratory distress syndrome (ARDS) in 10-15% of patients. Inflammation plays a crucial role in the pathogenesis of ARDS, with immune dysregulation in severe COVID-19 leading to a hyperinflammatory response. A comprehensive understanding of the inflammatory process in COVID-19 is lacking. Methods: In this prospective, multicenter observational study, patients with PCR-proven or clinically presumed COVID-19 admitted to the intensive care unit (ICU) or clinical wards were included. Demographic and clinical data were obtained and plasma was serially collected. Concentrations of IL-6, TNF-α, complement components C3a, C3c and the terminal complement complex (TCC) were determined in plasma by ELISA. Additionally, 269 circulating biomarkers were assessed using targeted proteomics. Results were compared between ICU and non ICU patients. Findings: A total of 119 (38 ICU and 91 non ICU) patients were included. IL-6 plasma concentrations were elevated in COVID-19 (ICU vs. non ICU, median 174.5 pg/ml [IQR 94.5-376.3] vs. 40.0 pg/ml [16.5-81.0]), whereas TNF-α concentrations were relatively low and not different between ICU and non ICU patients (median 24.0 pg/ml [IQR 16.5-33.5] and 21.5 pg/ml [IQR 16.0-33.5], respectively). C3a and terminal complement complex (TCC) concentrations were significantly higher in ICU vs. non ICU patients (median 556.0 ng/ml [IQR 333.3-712.5]) vs. 266.5 ng/ml [IQR 191.5-384.0] for C3a and 4506 mAU/ml [IQR 3661-6595] vs. 3582 mAU/ml [IQR 2947-4300] for TCC) on the first day of blood sampling. Targeted proteomics demonstrated that IL-6 (logFC 2.2), several chemokines and hepatocyte growth factor (logFC 1.4) were significantly upregulated in ICU vs. non ICU patients. In contrast, stem cell factor was significantly downregulated (logFC −1.3) in ICU vs. non ICU patients, as were DPP4 (logFC −0.4) and protein C inhibitor (log FC −1.0), the latter two factors also being involved in the regulation of the kinin-kallikrein pathway. Unsupervised clustering pointed towards a homogeneous pathogenetic mechanism in the majority of patients infected with SARS-CoV-2, with patient clustering mainly based on disease severity. Interpretation: We identified important pathways involved in dysregulation of inflammation in patients with severe COVID-19, including the IL-6, complement system and kinin-kallikrein pathways. Our findings may aid the development of new approaches to host-directed therapy. Funding: Vidi grant (F.L.v.d.V.) and Spinoza grant (M.G.N.) from the Netherlands Organization for Scientific Research, and ERC Advanced Grant (#833247 to M.G.N.).

Background: Antibody-mediated rejection in solid organ transplantation is an important immunological barrier to successful long-term graft survival. Next to complement activation, natural killer (NK) cells have been implicated in the process. Human cytomegalovirus (CMV), independently associated with decreased graft survival, has a strong imprint on the immune response. Here, we assessed the effect of CMV status on alloreactive NK cell reactivity. Methods: We compared antibody-mediated NK cell cytolytic activity (CD107a expression) and IFNγ production between healthy CMV-seropositive (n = 8) and CMV-seronegative (n = 11) individuals, in cocultures of NK cells with anti-HLA class I or rituximab (control) antibody-coated Raji cells. Results: First, we showed that within the NKG2C+ NK cells, it is specifically the NKG2C+/A− subset that is enriched in CMV+ individuals. We then observed that in particular the NK cell antibody-dependent cell mediated cytotoxicity (ADCC), but also non-ADCC alloreactivity toward HLA-positive target cells was increased in CMV+ individuals as compared to CMV− ones. This enhanced ADCC as well as non-ADCC NK cell reactivity in CMV+ individuals was particularly characterized by a significantly higher number of ILT2+ and NKG2C+ NK cells that possessed cytolytic activity and/or produced IFNγ in response to HLA-positive target cells. Conclusions: With regard to organ transplantation, these data suggest that CMV infection enhances NK cell alloreactivity, which may pose an additional adverse effect on graft survival, especially in the presence of donor specific antibodies.

Age-related changes of the immune system, termed immunosenescence, may underlie the increased risk of infections and morbidity in the elderly. Little is known about the effects of acute exercise on peripheral immune parameters in octogenarians. Therefore, we investigated acute exercise-induced changes in phenotype and function of the immune system in octogenarians participating in the 2013 edition of the Nijmegen Four Days Marches. Blood sampling was performed at baseline and immediately after 4 days of the walking exercise (30 km/day). A comprehensive set of adaptive and innate immune traits were enumerated and analyzed by flow-cytometry. Peripheral blood mononuclear cells, isolated before and after walking were stimulated with LPS and supernatants were analysed for IL-1β, IL-6, IL-8 and TNF-α concentrations by ELISA. CMV serostatus was determined by ELISA. The walking exercise induced a clear leucocytosis with numerical increases of granulocytes, monocytes and lymphocytes. These exercise-induced changes were most profound in CMV seropositive subjects. Within lymphocytes, numerical increases of particularly CD4+ T cells were noted. Further T cell differentiation analysis revealed profound increases of naïve CD4+ T cells, including naïve Treg. Significant increases were also noted for CD4+ memory T cell subsets. In contrast, only slight increases in naïve and memory CD8+ T cell subsets were detected. Exercise did not affect markers of immune exhaustion in memory T cell subsets. NK cells demonstrated a numerical decline and a change in cellular composition with a selective decrease of the mature CD56dim NK cells. The latter was seen in CMV seronegative subjects only. Also, a higher IL-6 and IL-8 production capacity of LPS-stimulated PBMC was seen after walking. In this exceptional cohort of octogenarian walkers, acute exercise induced changes in immune cell numbers and functions. A clear response of CD4+ T cells, rather than CD8+ T cells or NK cells was noted. Remarkably, the response to exercise within the CD4+ T cell compartment was dominated by naïve CD4+ subsets.

Introduction: Schnitzler’s syndrome (SchS) is a disabling autoinflammatory disorder, characterized by a chronic urticarial rash, an M-protein, arthralgia, and other signs of systemic inflammation. Anti-interleukin-1 (IL-1) beta antibodies are highly effective, but the pathophysiology is still largely unknown. Here we studied the effect of in-vivo IL-1 inhibition on serum markers of inflammation and cellular immune responses. Methods: Eight patients with SchS received monthly subcutaneous (s.c.) injections with 150 mg canakinumab for six months. Blood was drawn for measurement of serum markers of inflammation (12 times per patient) and for functional and phenotypic analysis of both freshly isolated and toll-like receptor (TLR)-ligand-stimulated peripheral blood mononuclear cells (PBMCs) (five times per patient). All data were compared to results of healthy controls. Results: IL-6 levels in serum and in lysates of freshly isolated PBMCs and serum myeloid-related protein (MRP8)/14 and S100A12 levels correlated with disease activity. In vitro, LPS stimulation resulted in higher IL-6 and IL-1 beta production in PBMCs from symptomatic SchS patients compared to healthy controls, whereas patient cells were relatively hyporesponsive to poly:IC and Pam3Cys. The mRNA microarray of PBMCs showed distinct transcriptomes for controls, symptomatic patients and anti-IL-1-treated patients. Numbers of T- and B-cell subsets as well as M-protein concentrations were not affected by IL-1 inhibition. Free light chain levels were elevated in 4 out of 8 patients. Conclusions: In conclusion, patient PBMCs are hyperresponsive to LPS, and clinical efficacy of IL-1 beta inhibition in patients with SchS is associated with in-vivo and ex-vivo suppression of inflammation. Interestingly, patient PBMCs showed divergent responses to TLR2/6, TLR3 and TLR4 ligands. Our data underscore that IL-1 beta plays a pivotal role in SchS.

Background Cytomegalovirus(CMV) infections have a significant effect on morbidity and mortality in kidney transplants. We conducted a study to ascertain the association of natural killer cell killer immunoglobulin-like receptors and human leukocyte antigen (HLA) genotype with risk of CMV disease. Methods The 90 CMV-negative patients receiving a first renal transplantation from a CMV-positive donor in this study received triple immunosuppressive therapy and prophylactic CMV treatment for up to 3 months after transplantation. Results We observed a 43.3% incidence rate of CMV disease within the first year after transplantation. Twenty-seven recipients experienced a rejection episode, 14 of which had CMV disease, mostly after rejection, suggesting that in this group, CMV disease is not a risk factor for rejection. KIR gene or genotype distribution were similar between the CMV diseased and CMV disease-free group. Twenty-seven recipients (30%) carried KIR-AA genotype, of which nine (33%) had CMV disease. Of the remaining 63 (70%) recipients with KIR-BX genotype, 30 (48%) had CMV disease. There was no significant difference between the two genotype groups with regard to occurrence of CMV disease, although there was a trend toward a lower incidence of CMV disease in recipients carrying the KIR-AA genotype. For CMV disease, we found no significant risk associated with the number of activating or inhibitory KIRs. Neither was missing KIR ligands for the inhibitory KIRs (HLA-C1/C2/Bw4) in recipients associated with lower rates of CMV disease. Conclusion In CMV-negative recipients, genotypic analysis of KIR repertoire and HLA ligands does not provide risk factors for primary CMV disease after renal transplantation.

The HIV co-receptors CXCR4 and CCR5 play an important role in HIV infection and replication. Therefore we hypothesize that long-term non-progressors (LTNP) with viral control have lower expression of CCR5 and CXCR4 on CD4+ cells, specifically on memory T-lymphocytes since they are the primary target cells of HIV. In this cross-sectional study, we included five HIV-infected LTNP with viral control (CD4 > 750 cell/μl & HIV < 50 copies for ≥2 years), thirteen HIV-infected and seven HIV-uninfected individuals at Radboud UMC Nijmegen, the Netherlands. We determined the CCR5 and CXCR4 expression among CD4+ and CD8+ lymphocyte subsets; memory (CD45RO+), naïve (CD45RA+) cells and regulatory T-cells (CD4+CD25highFoxP3+). In addition, CCR5∆32 polymorphism is related with disease progression and was therefore determined using polymerase chain reaction. The percentage of CCR5-expressing CD4+ cells of LTNP was comparable with healthy controls; whereas HIV-infected individuals showed more CCR5-expressing cells. This was observed in memory and naïve CD4+ cells, but not in regulatory T-cells. The mean fluorescence intensity of CCR5-expressing CD4+ cells was similar in all groups. All groups had comparable percentages of CXCR4-expressing cells. The mean fluorescence intensity of CXCR4-expressing cells was significantly higher in HIV-infected normally progressors in both memory and naïve CD4+ cells, but not in CD8+ cells. The CCR5∆32 polymorphism was not related to group. We show that HIV affects -directly or indirectly- the expression of CCR5 in CD4+ T-lymphocytes; yet this effect is not seen in LTNP with viral control. Avoiding upregulation of CCR5 could be an important method via which LTNP counteracts the effects of HIV and suppresses viral replication. Exploring how LTNP suppress the upregulation of CCR5 could be an important step for discovering new therapeutics.