whether neutrophils exert an important role in the pathogenesis of organ tissue damage in SLE. Methods We used lupus-prone mouse model and model of lupus serum-induced tissue inflammation in mouse to investigate the role of neutrophils in the organ damage of SLE. Results We found that there was a little neutrophil infiltration in the inflammatory sites of skin, liver, brain and joint in lupus-prone mice. We also found that there was also little neutrophil infiltration in the site of skin inflammation induced by lupus serum in normal mouse. The severity of skin inflammation induced by lupus serum was not significantly decreased in mice with neutrophil depletion compared to ones without neutrophil depletion. But we found that neutrophils were actually involved in tissue injury induced by lupus IgG. Further studies showed that lupus IgG stimulated and activated neutrophils, and cause the death of neutrophils. Studies also confirmed that Fas plays an important role in neutrophils apoptosis. Conclusions Our study indicates that neutrophils participate in the early stage of lupus organ damage, and then they died through activation-mediated apoptosis. These findings promote the understanding of the role of neutrophils in the tissue injury with SLE.

found and develop a protocol to ensure that source documents are filed as patients enroll in the clinic.

Background: Cystic fibrosis (CF) transmembrane conductance regulator protein (CFTR) modulators are effective in reducing the disease burden of CF, and a little more than 90% of patients are eligible for CFTR modulators with the expansion of eligibility to those with a single copy of a F508del mutation.This broad success creates logistical challenges for clinical trials of new CF therapeutics for the small number of patients that remain ineligible for modulators.New logistically feasible trial designs must be developed to assess efficacy of CF therapeutics and CFTR restorative therapies.Novel study designs that use readily available external data sources, such as previous trial controls, must be rigorously developed and understood to meet these modern challenges.Methods: In the context of a pulmonary exacerbation simulation study, we will examine an adaptive trial design and three analytic approaches that use historical data.The adaptive trial methodology contains a planned interim analysis at which congruency between the historical and active controls is assessed.If congruent, historical controls will be used in the analysis to reduce the need for active controls and reduce the active trial sample size.If incongruent, the historical controls will be discarded and more active controls recruited.The three analytic methods include inverse probability weighting, propensity score-based power priors, and commensurate priors.We will assess a simulated time-to-event outcome with Poisson regression and compare analytic approaches regarding their ability to estimate treatment effect accurately and reduce required active trial sample size.Results: Early, limited simulation shows that the adaptive trial design and all three analytic approaches can be an improvement over naïve approaches under certain conditions.Inverse probability weighting can achieve unbiased results when most covariates measuring active trial and historical population differences were available.A hazard ratio of 0.63 (95% CI, 0.50-0.80 was obtained when historical participants were used exclusively as the control arm.The expected hazard ratio was 0.55 (95% CI, 0.34-0.86).Additionally, failure to adjust for differences between current and historical study populations results in biased estimates of efficacy and poor coverage.All methods will be compared in terms of coverage, power, and bias.Conclusions: Evaluation of trial designs and analytic approaches that leverage historical data is critical because these tools will be necessary to meet the challenges of modern CF clinical trials.As traditional placebocontrolled trials become increasingly less feasible, it will be critical for researchers to make use of efficient and accurate alternative designs.

antagonists show auspicious activity in mouse models of autoimmunity and both anti-MIF (Imalumab) and anti-MIF receptor antibodies (Milatuzamab) have advanced into phase II human clinical testing. The MIF promoter polymorphism comprises a unique fournucleotide microsatellite repeat (CATT5–8), with higher repeat number producing increased MIF expression. Because there is no information about the transcriptional regulation of these common alleles, we sought to identify the nuclear protein(s) regulating expression at this functional promoter polymorphism. Materials and methods We utilised DNA affinity chromatography and liquid chromatography-mass spectrometry analysis to identify unique nuclear proteins that interact with the 794 CATT5–8 MIF promoter polymorphism. Functional knockout, ectopic expression, and 794 CATT-length dependent transcriptional assays and tissue microarray studies confirmed findings. Results Proteomic analysis identified the transcription factor ICBP90, previously implicated in oncogenesis, as a unique 794 CATT5–8 microsatellite interacting protein. Phosphorylated ICBP90 bound to the MIF promoter in a CATT-length dependent manner and upregulated MIF expression in monocytes, and B and T lymphocytes. Strong correlation was observed between ICBP90 and MIF expression in human inflammatory tissue, with a noteworthy overlap between downstream transcripts regulated by ICBP or MIF. Conclusions ICBP90 regulates MIF transcription at the 794 MIF CATT5–8 susceptibility locus. Pharmacologic targeting of the ICBP90:CATTx interaction is underway to inhibit MIF promoter overactivity and provide for a structurally-defined, pharmacogenomic approach to treatment.

travel and time, making care more convenient and possibly increasing the number of encounters.There are confounding factors such as social distancing, masking, and the approval of elexacaftor/tezacaftor/ivacaftor in late 2019 for 37% of our patients that may have affected results.However, we have shown that video visits during a pandemic are a viable, costeffective, safe method of delivering CF care.Further analysis in years to come will help determine if video visits are effective outside of pandemic conditions.

Implemented twice-monthly meetings with combined pediatric and adult teams; 6) Adult team agreed to meet patients 6 months before date of transition, and patients were offered a tour of the adult center prior to transition; 70 Surveyed recently transitioned adult patients about their experience.Major issues reported included the need for a designated inpatient unit for CF patients and for inpatient staff to be educated on CF and disease process.Results: By October 2020, all 10 patients in our 18-21-year-old target group were transitioned successfully.Post-transition survey scores improved to 3.3 (n = 3; Figure 1).By managing up and meeting with hospital administration and the Office of Patient Experience, we were able to create an adult CF inpatient unit in 2019.Additionally, adult staff on that unit were then educated by our pediatric CF nurse educator.Conclusion: By formalizing our transition process, we were able to increase patient and families' knowledge and readiness for transition, increase patients' comfort in transition to the adult center, and work with hospital administration to designate a much needed adult inpatient unit.This had led to a 100% patient retention rate in our center during this time period.

5,811,097 A 9, 1998 Allison et al. ... ... 424,144.1 5,855,887 A 1/1999 Allison et al. ... ... 424,144.1 6,051,227 A 4/2000 Allison et al. ... ... 424,144.1 6,120,765 A * 9/2000 Hibino et al. ............ 424/94.63 6,150,584 A 11/2000 Kucherlapati et al. ......... 800/18 6,613,327 B1 * 9/2003 Ling et al. ......... ... 424,134.1 6,682,736 B1 1/2004 Hanson et al. .. ... 424,144.1 7.229,628 B1* 6/2007 Allison et al. ............ 424,278.1

Background and aims Neutrophils play a crucial role in host defense through mechanisms including phagocytosis and formation of neutrophil extracellular traps (NETs), a recently identified neutrophil cell death process in which DNA is extruded together with cytoplasmic and granular content to trap and eliminate extracellular pathogens. Although beneficial from a host-pathogen perspective, exaggerated neutrophil activation has been linked to autoimmunity, in particular the rheumatic disease systemic lupus erythematosus (SLE) where nucleic acid-containing immune complexes (IC) drive inflammation. Toll-like receptor (TLR) agonists, such as nucleic acids, are important components of pathogens, enabling enhanced phagocytosis by macrophages and dendritic cells, but the role of TLR signalling in processing of SLE ICs and downstream inflammatory neutrophil effector functions is not known. Methods Standard Methods. Results We observed that both FcgR- and TLR8-engagement were required for induction of NETosis, whereas TLR8 activation, through the RNA component of the ICs, suppressed further IC-mediated phagocytosis. Mechanistically, TLR8 ligation induced PI3K-dependent ROS generation through NADPH oxidase, and subsequent furin-dependent proteolytic cleavage of the N-terminal part of FcgRIIA shifting neutrophils away from phagocytosis of ICs toward NETosis. TLR8 activated neutrophils promoted cleavage of FcgRIIA also on plasmacytoid dendritic cells and monocytes resulting in impaired overall clearance of ICs and increased complement C5a generation. Importantly, ex vivo derived activated neutrophils from SLE patients demonstrated a similar cleavage of FcgRIIA that was correlated with markers of disease activity as well as complement activation. Conclusions Therapeutic approaches aimed at blocking TLR8 activation would be predicted to increase phagocytosis of circulating ICs while disarming their inflammatory potential.

Neutrophils play a crucial role in host defense. However, neutrophil activation is also linked to autoimmune diseases such as systemic lupus erythematosus (SLE), where nucleic acid–containing immune complexes (IC) drive inflammation. The role of Toll-like receptor (TLR) signaling in processing of SLE ICs and downstream inflammatory neutrophil effector functions is not known. We observed that TLR7/8 activation leads to a furin-dependent proteolytic cleavage of the N-terminal part of FcgRIIA, shifting neutrophils away from phagocytosis of ICs toward the programmed form of necrosis, NETosis. TLR7/8-activated neutrophils promoted cleavage of FcgRIIA on plasmacytoid dendritic cells and monocytes, resulting in impaired overall clearance of ICs and increased complement C5a generation. Importantly, ex vivo derived activated neutrophils from SLE patients demonstrated a similar cleavage of FcgRIIA that was correlated with markers of disease activity, as well as complement activation. Therapeutic approaches aimed at blocking TLR7/8 activation would be predicted to increase phagocytosis of circulating ICs, while disarming their inflammatory potential.

Lupus-like disease is driven by NETs enriched in mitochondrial DNA. Neutrophil extracellular traps (NETs) are implicated in autoimmunity, but how they are generated and their roles in sterile inflammation remain unclear. Ribonucleoprotein immune complexes (RNP ICs), inducers of NETosis, require mitochondrial reactive oxygen species (ROS) for maximal NET stimulation. After RNP IC stimulation of neutrophils, mitochondria become hypopolarized and translocate to the cell surface. Extracellular release of oxidized mitochondrial DNA is proinflammatory in vitro, and when this DNA is injected into mice, it stimulates type I interferon (IFN) signaling through a pathway dependent on the DNA sensor STING. Mitochondrial ROS are also necessary for spontaneous NETosis of low-density granulocytes from individuals with systemic lupus erythematosus. This was also observed in individuals with chronic granulomatous disease, who lack NADPH oxidase activity but still develop autoimmunity and type I IFN signatures. Mitochondrial ROS inhibition in vivo reduces disease severity and type I IFN responses in a mouse model of lupus. Together, these findings highlight a role for mitochondria in the generation not only of NETs but also of pro-inflammatory oxidized mitochondrial DNA in autoimmune diseases.

Background Protein citrullination, the post-translational conversion of arginine to citrulline, is thought to participate in the development of anti-citrullinated protein antibodies (ACPA) in patients with rheumatoid arthritis (RA). Recently, synovial fluid cells from RA patients were found to have citrullination of several intracellular proteins, a process named hypercitrullination. This unique form of citrullination did not occur in other types of cell death such as apoptosis or NETosis, but was proposed to be induced only through pore-forming membranolytic pathways, including complement-mediated cell lysis through the membrane attack complex. Objectives To explore the naturally occurring humoral mechanisms required for induction of complement-mediated neutrophil lysis in the RA joint. Methods Neutrophils (PMNs) isolated from synovial fluid (SF) (n=4) or peripheral blood (PB) (n=25) of ACPA-positive RA patients as well as PB of healthy controls (HC) (n=12) were analyzed for anti-PMN binding IgG, C3bi and C5b-9 complement deposition, as well as PMN viability using Annexin V and propidium iodide staining by flow cytometry analysis. In addition, PMNs isolated from HC were incubated with 4 matched RA serum and SF samples as well as 7 additional unmatched RA SF samples, or normal human serum (NHS); complement deposition, complement regulatory protein CD46 expression, and PMN viability were then measured. Finally, the kinetics by which HC PMNs underwent cell death upon stimulation with complement-fixing antibodies (Abs) and NHS were analyzed using the IncuCyte ZOOM® live cell imaging system. Results In the periphery, RA PMNs had highly increased mean fluorescence intensity (MFI) ratio for C3bi cell-surface deposition as compared to HC (p=0.0183), but no differences were seen for C5b-9 deposition. However, in SF, RA PMNs had elevated MFI ratio for both C5b-9 deposition (p=0.0121) as well as anti-PMN binding IgG deposition (p=0.0011) as compared to PB PMNs from HC, or paired PB PMNs from RA patients (p=0.0177, p=0.0113, respectively). Consistent with the selective increase in complement C5b-9 deposition in SF, we observed a decrease in the expression of CD46 in SF, but not PB PMNs of RA patients (p=0.0270), suggesting a potential mechanism through which SF PMNs are sensitized to complement-mediated lysis. Further, PMNs isolated from SF were, to a higher extent than PB PMNs, undergoing secondary necrosis, supporting the notion of complement-mediated lysis of SF PMNs in vivo. Finally, adding RA SF, but not RA serum, to HC PMNs induced markedly elevated C3bi and C5b-9 deposition, reduction in CD46 levels and PMN cell death, clearly demonstrating the PMN cytotoxic properties of RA SF in 10/11 samples, as well as suggesting an important role of complement activation in this process. IgG- and complement-mediated lysis of PMNs was also verified in vitro by live cell imaging. Conclusions RA SF PMNs demonstrate increased complement deposition associated with PMN cell death both in vivo and in vitro. Elucidating the cause of complement deposition and the role of CD46 in promoting PMN lysis may help to uncover a mechanism for hypercitrullination, ACPA formation and RA disease propagation. References Holers, V.M. Annu. Rev. Immunol (2014) 32:433–59. Romero, V. et al. Sci Transl Med (2013) 5 (209):1–10. Disclosure of Interest None declared

Abstract Background Immune complex (IC)-mediated formation of neutrophil extracellular traps (NETs) has emerged as a mechanism to increase the autoantigenic burden as well as promote the prominent type I interferon (IFN) signature seen in patients with the autoimmune disease systemic lupus erythematosus (SLE). The aim of the current study was to explore properties of ICs regulating their ability to induce phagocytosis and NETosis. Results Whereas immobilized IgG, mimicking tissue-deposited ICs, induced NETs in a TLR-independent manner, both FcgR- and TLR-engagement were required for induction of NETosis by soluble RNA-ICs, as demonstrated by FcgR blocking antibodies as well as RNase-treatment. Despite that degradation of RNA inhibited NETosis, removal of the TLR ligand by RNase markedly increased the phagocytosis of RNA-ICs by neutrophils suggesting that TLR activation suppressed phagocytosis. Consistent with this hypothesis, addition of a TLR8 agonist (R848) inhibited phagocytosis of ICs, but not beads, in neutrophils. Mechanistically, we observed that TLR8 induced ROS-dependent activation of the serine protease furin, participating in shedding of cell surface FcgRIIA reducing the phagocytic capacity but promoting progression into NETosis. Conclusions i) In the fluid phase, both FcgRIIA and TLR8 ligation are necessary for IC-mediated NETosis; ii) TLR8 activation induces furin-mediated shedding of FcgRIIA, thereby inhibiting further phagocytosis of ICs but enabling NETosis. Therapeutic approaches aimed at degrading the TLR8 ligand, such as RNases, would be predicted to increase uptake of circulating ICs, while disarming their inflammatory potential and ability to induce NETs.

Background Neutrophils participate in host defence through mechanisms including phagocytosis and formation of neutrophil extracellular traps (NETs), a neutrophil cell death process in which DNA is extruded together with cytoplasmic and granular content to trap and eliminate pathogens. Immune complex (IC)-mediated NET formation has emerged as a mechanism that may increase the autoantigenic burden as well as promote type I interferon production in patients with the autoimmune disease systemic lupus erythematosus (SLE). Although TLR agonists, such as nucleic acids, have been shown to enhance phagocytosis by macrophages and dendritic cells, the role of TLR signalling in neutrophil phagocytosis of RNA-containing SLE ICs has not been extensively studied. The aim of the current study was to explore the cross-talk between TLRs and FcgRs in the regulation of IC-mediated phagocytosis and NETosis. Materials and methods Neutrophils, isolated from healthy individuals were incubated with RNA-containing ICs and analysed for phagocytosis and NETosis by flow cytometry and fluorimetry, respectively, in the presence of blocking antibodies or TLR8 inhibitors (oligodinucleotides, RNase). Neutrophils from healthy controls (n = 7) and SLE patients (n = 19) were analysed for FcgRIIA expression by flow cytometry, using two antibody clones, recognising full-length or shed FcgRIIA, and the results related to clinical data. Results Both FcgRIIA- and TLR8-engagement were required for induction of NETosis by RNA-ICs, as demonstrated by FcgR blocking antibodies as well as RNase treatment. Although degradation of RNA inhibited NETosis, removal of the TLR ligand by RNase markedly increased the phagocytosis of RNA-ICs by neutrophils (p < 0.0001), suggesting that TLR activation suppressed phagocytosis. Consistent with this hypothesis, addition of TLR8 agonist (R848) inhibited phagocytosis of ICs (p < 0.0001), but not beads, in neutrophils. Mechanistically, TLR8 activation mediated furin-dependent proteolytic cleavage of the most N-terminal part of FcgRIIA reducing the phagocytic capacity while promoting progression into NETosis. Importantly, ex vivo isolated neutrophils from SLE patients demonstrated increased shedding of neutrophil FcgRIIA (p < 0.0001), which was correlated with neutrophil activation (r = −0.73, p = 0.003) and the presence of anti-Sm/RNP antibodies (p < 0.001). Conclusions Neutrophils are not terminally differentiated cells but could shift into phagocytic or NETosing cells, partly regulated by a cross-talk between TLR8 and FcgRIIA. SLE patients have ongoing shedding of neutrophil FcgRIIA related to neutrophil activation and anti-RNA antibodies, demonstrating the in vivo relevance of our observation. Therapeutic approaches aimed at degrading the TLR8 ligand would be predicted to increase uptake of circulating ICs, while disarming their inflammatory potential and ability to induce NETs.