Activation of RIPK1 controls TNF-mediated apoptosis, necroptosis and inflammatory pathways1. Cleavage of human and mouse RIPK1 after residues D324 and D325, respectively, by caspase-8 separates the RIPK1 kinase domain from the intermediate and death domains. The D325A mutation in mouse RIPK1 leads to embryonic lethality during mouse development2,3. However, the functional importance of blocking caspase-8-mediated cleavage of RIPK1 on RIPK1 activation in humans is unknown. Here we identify two families with variants in RIPK1 (D324V and D324H) that lead to distinct symptoms of recurrent fevers and lymphadenopathy in an autosomal-dominant manner. Impaired cleavage of RIPK1 D324 variants by caspase-8 sensitized patients' peripheral blood mononuclear cells to RIPK1 activation, apoptosis and necroptosis induced by TNF. The patients showed strong RIPK1-dependent activation of inflammatory signalling pathways and overproduction of inflammatory cytokines and chemokines compared with unaffected controls. Furthermore, we show that expression of the RIPK1 mutants D325V or D325H in mouse embryonic fibroblasts confers not only increased sensitivity to RIPK1 activation-mediated apoptosis and necroptosis, but also induction of pro-inflammatory cytokines such as IL-6 and TNF. By contrast, patient-derived fibroblasts showed reduced expression of RIPK1 and downregulated production of reactive oxygen species, resulting in resistance to necroptosis and ferroptosis. Together, these data suggest that human non-cleavable RIPK1 variants promote activation of RIPK1, and lead to an autoinflammatory disease characterized by hypersensitivity to apoptosis and necroptosis and increased inflammatory response in peripheral blood mononuclear cells, as well as a compensatory mechanism to protect against several pro-death stimuli in fibroblasts. View Publication

Hepatocyte death during acetaminophen (APAP) intoxication elicits a reactive inflammatory response, with hepatic recruitment of neutrophils and monocytes, which further aggravates liver injury. Neutrophil elastase (NE), secreted by activated neutrophils, carries degradative and cytotoxic functions and maintains a proinflammatory state. We investigated NE as a therapeutic target in acetaminophen-induced liver injury (AILI). C57BL/6 mice were administered a toxic dose of APAP, 2 h prior to receiving the NE inhibitor sivelestat, N-acetylcysteine (NAC), or a combination therapy, and were euthanized after 24 and 48 h. Upon APAP overdose, neutrophils and monocytes infiltrate the injured liver, accompanied by increased levels of NE. Combination therapy of NAC and sivelestat significantly limits liver damage, as evidenced by lower serum transaminase levels and less hepatic necrosis compared to mice that received APAP only, and this to a greater extent than NAC monotherapy. Lower hepatic expression of proinflammatory markers was observed in the combination treatment group, and flow cytometry revealed significantly less monocyte influx in livers from mice treated with the combination therapy, compared to untreated mice and mice treated with NAC only. The potential of NE to induce leukocyte migration was confirmed in vitro. Importantly, sivelestat did not impair hepatic repair. In conclusion, combination of NE inhibition with sivelestat and NAC dampens the inflammatory response and reduces liver damage following APAP overdose. This strategy exceeds the standard of care and might represent a novel therapeutic option for AILI. View Publication

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex disease with no known cause or mechanism. There is an increasing appreciation for the role of immune and metabolic dysfunction in the disease. ME/CFS has historically presented in outbreaks, often has a flu-like onset, and results in inflammatory symptoms. Patients suffer from severe fatigue and post-exertional malaise. There is little known about the metabolism of specific immune cells in ME/CFS patients. To investigate immune metabolism in ME/CFS, we isolated CD4+ and CD8+ T cells from 53 ME/CFS patients and 45 healthy controls. We analyzed glycolysis and mitochondrial respiration in resting and activated T cells, along with markers related to cellular metabolism, and plasma cytokines. We found that ME/CFS CD8+ T cells have reduced mitochondrial membrane potential compared to healthy controls. Both CD4+ and CD8+ T cells from ME/CFS patients had reduced glycolysis at rest, while CD8+ T cells also had reduced glycolysis following activation. ME/CFS patients had significant correlations between measures of T cell metabolism and plasma cytokine abundance that differed from healthy control subjects. Our data indicate that patients have impaired T cell metabolism consistent with ongoing immune alterations in ME/CFS that may illuminate the mechanism behind this disease. View Publication

Background: Differences in DNA methylation have been reported in B and T lymphocyte populations, including CD4+ T cells, isolated from rheumatoid arthritis (RA) patients when compared to healthy controls. CD4+ T cells are a heterogeneous cell type with subpopulations displaying distinct DNA methylation patterns. In this study, we investigated DNA methylation using reduced representation bisulfite sequencing in two CD4+ T cell populations (CD4+ memory and na{\{i}}ve cells) in three groups: newly diagnosed disease modifying antirheumatic drugs (DMARD) na{\"{i}}ve RA patients (N = 11) methotrexate (MTX) treated RA patients (N = 18) and healthy controls (N = 9) matched for age gender and smoking status. Results: Analyses of these data revealed significantly more differentially methylated positions (DMPs) in CD4+ memory than in CD4+ na{\""{i}}ve T cells (904 vs. 19 DMPs) in RA patients compared to controls. The majority of DMPs (72{\%}) identified in newly diagnosed and DMARD na{\""{i}}ve RA patients with active disease showed increased DNA methylation (39 DMPs) whereas most DMPs (80{\%}) identified in the MTX treated RA patients in remission displayed decreased DNA methylation (694 DMPs). Interestingly we also found that about one third of the 101 known RA risk loci overlapped (±500 kb) with the DMPs. Notably introns of the UBASH3A gene harbor both the lead RA risk SNP and two DMPs in CD4+ memory T cells. Conclusion: Our results suggest that RA associated DNA methylation differences vary between the two T cell subsets but are also influenced by RA characteristics such as disease activity disease duration and/or MTX treatment.""" View Publication

Cyclosporine A (CsA) is a powerful immunosuppressant, but it is an ineffective stand-alone treatment for systemic lupus erythematosus (SLE) due to poor target tissue distribution and renal toxicity. We hypothesized that CD71 (transferrin receptor 1)-directed delivery of CsA to the lymphatic system would improve SLE outcomes in a murine model. We synthesized biodegradable, ligand-conjugated nanoparticles [P2Ns-gambogic acid (GA)] targeting CD71. GA conjugation substantially increased nanoparticle association with CD3+ or CD20+ lymphocytes and with intestinal lymphoid tissues. In orally dosed MRL-lpr mice, P2Ns-GA-encapsulated CsA increased lymphatic drug delivery 4- to 18-fold over the ligand-free formulation and a commercial CsA capsule, respectively. Improved lymphatic bioavailability of CsA was paralleled by normalization of anti-double-stranded DNA immunoglobulin G titer, plasma cytokines, and glomerulonephritis. Thus, this study demonstrates the translational potential of nanoparticles that enhance the targeting of lymphatic tissues, transforming CsA into a potent single therapeutic for SLE. View Publication

The Glioblastoma (GBM) immune microenvironment plays a critical role in tumor development, progression, and prognosis. A comprehensive understanding of the intricate milieu and its interactions remains unclear, and single-cell analysis is crucially needed. Leveraging mass cytometry (CyTOF), we analyzed immunocytes from 13 initial and three recurrent GBM samples and their matched peripheral blood mononuclear cells (pPBMCs). Using a panel of 30 markers, we provide a high-dimensional view of the complex GBM immune microenvironment. Hematoxylin and eosin staining and polychromatic immunofluorescence were used for verification of the key findings. In the initial and recurrent GBMs, glioma-associated microglia/macrophages (GAMs) constituted 59.05 and 27.87{\%} of the immunocytes, respectively; programmed cell death-ligand 1 (PD-L1), T cell immunoglobulin domain and mucin domain-3 (TIM-3), lymphocyte activation gene-3 (LAG-3), interleukin-10 (IL-10) and transforming growth factor-$\beta$ (TGF$\beta$) demonstrated different expression levels in the GAMs among the patients. GAMs could be subdivided into different subgroups with different phenotypes. Both the exhausted T cell and regulatory T (Treg) cell percentages were significantly higher in tumors than in pPBMCs. The natural killer (NK) cells that infiltrated into the tumor lesions expressed higher levels of CXC chemokine receptor 3 (CXCR3), as these cells expressed lower levels of interferon-$\gamma$ (IFN$\gamma$). The immune microenvironment in the initial and recurrent GBMs displayed similar suppressive changes. Our study confirmed that GAMs, as the dominant infiltrating immunocytes, present great inter- and intra-tumoral heterogeneity and that GAMs, increased exhausted T cells, infiltrating Tregs, and nonfunctional NK cells contribute to local immune suppressive characteristics. Recurrent GBMs share similar immune signatures with the initial GBMs except the proportion of GAMs decreases. View Publication

SCOPE Necrotizing enterocolitis (NEC) is a devastating disease that is highly lethal in premature infants. Human milk oligosaccharides (HMOs) efficiently reduce the incidence of NEC. However, the protective mechanism of HMO treatment is unknown. It is hypothesized that HMOs protect against NEC by inhibiting the damage to intestinal epithelial cells. METHODS AND RESULTS C57BL/6 pups are challenged with hypoxia and cold stress to induce NEC. All pups are sacrificed after 72 h. It is found that HMO administration reduces the concentrations of IL-8 in the serum and ileum of all NEC mice. Ileum toll-like receptor 4 (TLR4) protein expression and nuclear factor kappa-B (NF$\kappa$B) pathway activation are inhibited. The proliferative ability of enterocytes in the ileum is restored as determined by labeling with proliferation markers (Ki67, SOX9). In a 3D culture intestinal crypt organoids study, HMO treatment improves the maturation of organoid cells and increases the ratio of proliferative cells under lipopolysaccharides (LPS) treatment. HMO treatment downregulates TLR4 expression in the organoid cells, thus reducing the effect of LPS. CONCLUSION HMOs protect intestinal epithelial cells from injury by accelerating the turnover of crypt cells by reducing the expression of TLR4 on intestinal epithelial cells. View Publication

Rationale: Mesenchymal stem cells (MSC) hold great promise in the treatment of various diseases including autoimmune diseases, inflammatory diseases, etc., due to their pleiotropic properties. However, largely incongruent data were obtained from different MSC-based clinical trials, which may be partially due to functional heterogeneity among MSC. Here, we attempt to derive homogeneous mesenchymal stem cells with neuromesodermal origin from human pluripotent stem cells (hPSC) and evaluate their functional properties. Methods: Growth factors and/or small molecules were used for the differentiation of human pluripotent stem cells (hPSC) into neuromesodermal progenitors (NMP), which were then cultured in animal component-free and serum-free induction medium for the derivation and long-term expansion of MSC. The resulted NMP-MSC were detailed characterized by analyzing their surface marker expression, proliferation, migration, multipotency, immunomodulatory activity and global gene expression profile. Moreover, the in vivo therapeutic potential of NMP-MSC was detected in a mouse model of contact hypersensitivity (CHS). Results: We demonstrate that NMP-MSC express posterior HOX genes and exhibit characteristics similar to those of bone marrow MSC (BMSC), and NMP-MSC derived from different hPSC lines show high level of similarity in global gene expression profiles. More importantly, NMP-MSC display much stronger immunomodulatory activity than BMSC in vitro and in vivo, as revealed by decreased inflammatory cell infiltration and diminished production of pro-inflammatory cytokines in inflamed tissue of CHS models. Conclusion: Our results identify NMP as a new source of MSC and suggest that functional and homogeneous NMP-MSC could serve as a candidate for MSC-based therapies. View Publication

RNA polymerase III (Pol III) is an essential 17-subunit complex responsible for the transcription of small housekeeping RNAs such as transfer RNAs and 5S ribosomal RNA. Biallelic variants in four genes (POLR3A, POLR3B, and POLR1C and POLR3K) encoding Pol III subunits have previously been found in individuals with (neuro-) developmental disorders. In this report, we describe three individuals with biallelic variants in POLR3GL, a gene encoding a Pol III subunit that has not been associated with disease before. Using whole exome sequencing in a monozygotic twin and an unrelated individual, we detected homozygous and compound heterozygous POLR3GL splice acceptor site variants. RNA sequencing confirmed the loss of full-length POLR3GL RNA transcripts in blood samples of the individuals. The phenotypes of the described individuals are mainly characterized by axial endosteal hyperostosis, oligodontia, short stature, and mild facial dysmorphisms. These features largely fit within the spectrum of phenotypes caused by previously described biallelic variants in POLR3A, POLR3B, POLR1C, and POLR3K. These findings further expand the spectrum of POLR3-related disorders and implicate that POLR3GL should be included in genetic testing if such disorders are suspected. View Publication

Lupus nephritis is a potentially fatal autoimmune disease for which the current treatment is ineffective and often toxic. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with lupus nephritis and from healthy control subjects using single-cell RNA sequencing. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid cells, T cells, natural killer cells and B cells that demonstrated both pro-inflammatory responses and inflammation-resolving responses. We found evidence of local activation of B cells correlated with an age-associated B-cell signature and evidence of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1, were broadly expressed, implying a potentially central role in cell trafficking. Gene expression of immune cells in urine and kidney was highly correlated, which would suggest that urine might serve as a surrogate for kidney biopsies. View Publication

Preventing the immune escape of tumor cells by blocking inhibitory checkpoints, such as the interaction between programmed death ligand-1 (PD-L1) and programmed death-1 (PD-1) receptor, is a powerful anticancer approach. However, many patients do not respond to checkpoint blockade. Tumor PD-L1 expression is a potential efficacy biomarker, but the complex mechanisms underlying its regulation are not completely understood. Here, we show that the eukaryotic translation initiation complex, eIF4F, which binds the 5' cap of mRNAs, regulates the surface expression of interferon-$\gamma$-induced PD-L1 on cancer cells by regulating translation of the mRNA encoding the signal transducer and activator of transcription 1 (STAT1) transcription factor. eIF4F complex formation correlates with response to immunotherapy in human melanoma. Pharmacological inhibition of eIF4A, the RNA helicase component of eIF4F, elicits powerful antitumor immune-mediated effects via PD-L1 downregulation. Thus, eIF4A inhibitors, in development as anticancer drugs, may also act as cancer immunotherapies. View Publication

Memory T-cells are essential to maintain long-term immunological memory. It is widely thought that the bone marrow (BM) plays an important role in the long-term maintenance of memory T-cells. There is controversy however on the longevity and recirculating kinetics of BM memory T-cells. While some have proposed that the BM is a reservoir for long-lived, non-circulating memory T-cells, it has also been suggested to be the preferential site for memory T-cell self-renewal. In this study, we used in vivo deuterium labeling in goats to simultaneously quantify the average turnover rates-and thereby expected lifespans-of memory T-cells from BM, blood and lymph nodes (LN). While the fraction of Ki-67 positive cells, a snapshot marker for recent cell division, was higher in memory T-cells from blood compared to BM and LN, in vivo deuterium labeling revealed no substantial differences in the expected lifespans of memory T-cells between these compartments. Our results support the view that the majority of memory T-cells in the BM are self-renewing as fast as those in the periphery, and are continuously recirculating between the blood, BM, and LN. View Publication