技术资料

Mast cells (MCs) are tissue-resident immune cells known to degranulate in response to FcεRI crosslinking or MRGPRX2 engagement. MCs are found close to nerves,but the mechanisms that regulate this privileged localization remain unclear. Here,we investigated MRGPRX2 expression patterns and specific activities in MCs. We show that MRGPRX2 expression is heterogeneous in human MC (hMC) progenitors and mature MCs. Substance P (SP) is a rapid and specific activator of MRGPRX2,and long-term supplementation of MCs with SP expands MRGPRX2-expressing cells. While high concentrations of SP induce rapid MC degranulation,low concentrations prompt immature MC chemotaxis. Lastly,we demonstrate that in inflammatory skin conditions like psoriasis,the number of MRGPRX2 + MCs is increased,and during in vitro skin reinnervation,MRGPRX2 + MCs preferentially reside in proximity to and migrate toward SP + nerve fibers (NFs). This indicates that SP-MRGPRX2 signaling defines MC positioning and relocation within tissues and promotes immune cell-NF communication. Subject areas: Immunology,Molecular biology,Cell biology View Publication

Long-term culture of primary lymphocytes and hematopoietic stem and progenitor cells (HSPCs) is pivotal to their expansion and study. Furthermore,genetic engineering of the above-mentioned primary human cells has several safety needs,including the requirement of efficient in vitro assays for unwanted tumorigenic events. In this work,we tested and optimized the Miniaturized Optically Accessible Bioreactor (MOAB) platform. The MOAB consists of a millifluidic cell culture device with three optically-accessible culture chambers. Inside the MOAB,we inserted a silk-based framework that resembles some properties of the bone marrow environment and cultivated in this device either CD4 + T lymphocytes isolated from healthy donor buffy coat or cord blood-derived hematopoietic CD34 + cells. A fraction of these cells is viable for up to 3 months. Next,we tested the capability of the MOAB to detect tumorigenic events. Serial dilutions of engineered fluorescent tumor cells were mixed with either CD4 + or CD34 + primary cells,and their growth was followed. By this approach,we successfully detected as little as 100 tumorigenic cells mixed with 100,000 primary cells. We found that non-tumorigenic primary cells colonized the silk environment,whereas tumor cells,after an adaptation phase,expanded and entered the circulation. We conclude that the millifluidic platform allows the detection of rare tumorigenic events in the long-term culture of human cells. View Publication

Macrolides are widely used antibiotics for the treatment of bacterial airway infections. Due to its elevated minimum inhibitory concentration in standardized culture media,Pseudomonas aeruginosa is considered intrinsically resistant and,therefore,antibiotic susceptibility testing against macrolides is not performed. Nevertheless,due to macrolides’ immunomodulatory effect and suppression of virulence factors,they are used for the treatment of persistent P. aeruginosa infections. Here,we demonstrate that macrolides are,instead,effective antibiotics against P. aeruginosa airway infections in an Air-Liquid Interface (ALI) infection model system resembling the human airways. Importantly,macrolide treatment in both people with cystic fibrosis and primary ciliary dyskinesia patients leads to the accumulation of uL4 and uL22 ribosomal protein mutations in P. aeruginosa which causes antibiotic resistance. Consequently,higher concentrations of antibiotics are needed to modulate the macrolide-dependent suppression of virulence. Surprisingly,even in the absence of antibiotics,these mutations also lead to a collateral reduction in growth rate,virulence and pathogenicity in airway ALI infections which are pivotal for the establishment of a persistent infection. Altogether,these results lend further support to the consideration of macrolides as de facto antibiotics against P. aeruginosa and the need for resistance monitoring upon prolonged macrolide treatment. Subject terms: Antimicrobials,Clinical microbiology,Pathogens,Antimicrobial resistance View Publication

Venetoclax plus azacitidine treatment is clinically beneficial for elderly and unfit acute myeloid leukemia (AML) patients. However,the treatment is rarely curative,and relapse due to resistant disease eventually emerges. Since no current clinically feasible treatments are known to be effective at the state of acquired venetoclax resistance,this is becoming a major challenge in AML treatment. Studying venetoclax-resistant AML cell lines,we observed that venetoclax induced sublethal apoptotic signaling and DNA damage even though cell survival and growth were unaffected. This effect could be due to venetoclax inducing a sublethal degree of mitochondrial outer membrane permeabilization. Based on these results,we hypothesized that the sublethal apoptotic signaling induced by venetoclax could constitute a vulnerability in venetoclax-resistant AML cells. This was supported by screens with a broad collection of drugs,where we observed a synergistic effect between venetoclax and PARP inhibition in venetoclax-resistant cells. Additionally,the venetoclax-PARP inhibitor combination prevented the acquisition of venetoclax resistance in treatment naïve AML cell lines. Furthermore,the addition of azacitidine to the venetoclax-PARP inhibitor combination enhanced venetoclax induced DNA damage and exhibited exceptional sensitivity and long-term responses in the venetoclax-resistant AML cell lines and samples from AML patients that had clinically relapsed under venetoclax-azacitidine therapy. In conclusion,we mechanistically identify a new vulnerability in acquired venetoclax-resistant AML cells and identify PARP inhibition as a potential therapeutic approach to overcome acquired venetoclax resistance in AML. Subject terms: Acute myeloid leukaemia,Acute myeloid leukaemia View Publication

Despite a record number of clinical studies investigating various anti-myeloma treatments,the 5-year survival rate for multiple myeloma (MM) patients in the US is only 55%,and almost all patients relapse. Poor patient outcomes demonstrate that myeloma cells are “born to survive” which means they can adapt and evolve following treatment. Thus,new therapeutic approaches to combat survival mechanisms and target treatment resistance are required. Importantly,Mcl-1,anti-apoptotic protein,is required for the development of MM and treatment resistance. This study looks at the possibility of KS18,a selective Mcl-1 inhibitor,to treat MM and overcome resistance. Our investigation demonstrates that KS18 effectively induces cell death in MM by dual regulatory mechanisms targeting the Mcl-1 protein at both transcriptional and post-translational levels. Specifically,KS18 suppresses Mcl-1 activation via STAT-3 pathway and promotes Mcl-1 phosphorylation/ubiquitination/proteasome-dependent protein degradation (UPS). Significantly,KS18 triggered caspase-dependent apoptosis in MM patient samples and bortezomib-resistant cells,synergizing with venetoclax to boost apoptosis. KS18 promises to overcome bortezomib and venetoclax resistance and re-sensitize myeloma cells to chemotherapy. Furthermore,the study shows the tremendous impact of KS18 in inhibiting colony formation in bortezomib-resistant cells and demonstrates significant tumor shrinkage in KS18-treated NSG mice without notable toxicity signs after 4 weeks of therapy with a single acceptable dose each week,indicating its powerful anti-neoplastic and anti-resistance characteristics. This study strongly implies that KS18 may treat MM and provide new hope to patients who are experiencing recurrence or resistance. View Publication

Mesenchymal stromal cells (MSCs) are promising candidates for cartilage repair therapy due to their self-renewal,chondrogenic,and immunomodulatory capacities. It is widely recognized that a shift from fetal bovine serum (FBS)-containing medium toward a fully chemically defined serum-free (SF) medium would be necessary for clinical applications of MSCs to eliminate issues such as xeno-contamination and batch-to-batch variation. However,there is a notable gap in the literature regarding the evaluation of the chondrogenic ability of SF-expanded MSCs (SF-MSCs). In this study,we compared the in vivo regeneration effect of FBS-MSCs and SF-MSCs in a rat osteochondral defect model and found poor cartilage repair outcomes for SF-MSCs. Consequently,a comparative analysis of FBS-MSCs and SF-MSCs expanded using two SF media,MesenCult™-ACF (ACF),and Custom StemPro™ MSC SFM XenoFree (XF) was conducted in vitro. Our results show that SF-expanded MSCs constitute variations in morphology,surface markers,senescence status,differentiation capacity,and senescence/apoptosis status. Highly proliferative MSCs supported by SF medium do not always correlate to their chondrogenic and cartilage repair ability. Prior determination of the SF medium’s ability to support the chondrogenic ability of expanded MSCs is therefore crucial when choosing an SF medium to manufacture MSCs for clinical application in cartilage repair. View Publication

Hereditary spastic paraplegias are rare genetic disorders characterized by corticospinal tract impairment. Spastic paraplegia 83 (SPG83) is associated with biallelic mutations in the HPDL gene,leading to varied severities from neonatal to juvenile onset. The function of HPDL is unclear,though it is speculated to play a role in alternative coenzyme Q10 biosynthesis. Here,we report the generation of hiPS lines from primary skin fibroblasts derived from three SPG83 patients with different HPDL mutations,using episomal reprogramming. The patients’ clinical characteristics are carefully listed. The hiPS lines were meticulously characterized,demonstrating typical pluripotent characteristics through immunofluorescence assays for stemness markers (OCT4,TRA1-60,NANOG,and SSEA4) and RT-PCR for endogenous gene expression. Genetic integrity and identity were confirmed via Sanger sequencing and short tandem repeat analysis. These hiPS cells displayed typical pluripotent characteristics and were able to differentiate into neocortical neurons via a dual SMAD inhibition protocol. In addition,HPDL mutant neurons assessed via long-term culturing were able to achieve effective maturation,similarly to their wild-type counterparts. The HPDL hiPS lines we generated will provide a valuable model for studying SPG83,offering insights into its molecular mechanisms and potential for developing targeted therapies. View Publication

Induced pluripotent stem cell (iPSC)-derived mesenchymal stromal cells (iMSCs) offer a promising alternative to primary mesenchymal stromal cells (MSCs) and their derivatives,particularly extracellular vesicles (EVs),for use in advanced therapy medicinal products. In this study we evaluated the immunomodulatory and regenerative potential of iMSCs as well as iMSC-EVs,alongside primary human umbilical cord-derived mesenchymal stromal cells (hUCMSCs). Our findings demonstrate that iMSCs exhibit comparable abilities to hUCMSCs in regulating lymphocyte proliferation and inducing an anti-inflammatory phenotype in monocytes. We also observed decreased TNFα levels and increased IL-10 induction,indicating a potential mechanism for their immunomodulatory effects. Furthermore,iMSC-EVs also showed effective immunomodulation by inhibiting T cell proliferation and inducing macrophage polarization similar to their parental cells. Additionally,iMSC-EVs exhibited pro-regenerative potential akin to hUCMSC-EVs in in vitro scratch assays. Notably,priming iMSCs with pro-inflammatory cytokines significantly enhanced the immunomodulatory potential of iMSC-EVs. These results underscore the considerable promise of iMSCs and iMSCs-EVs as an alternate source for MSC-derived therapeutics,given their potent immunomodulatory and regenerative properties. The online version contains supplementary material available at 10.1038/s41598-024-75956-3. View Publication

Translational medicine provides insight into novel drugs and predicts unwanted effects. In well-characterized pathways (e.g.,cytokine-Janus kinase [JAK]-signal transducers and activators of transcription [STAT]),a variety of in vitro assessments were used to estimate selectivity of effects on different potential targets (i.e.,JAK1,JAK2,JAK3,and tyrosine kinase 2 [TYK2]). Several approved drugs were characterized as selective for the JAK family. These assessments are challenged by a lack of compounds that only inhibit one JAK family member. Deucravacitinib is a first-in-class,oral,selective,allosteric inhibitor of TYK2,a kinase required for IL-12,IL-23,and Type I interferon signaling. Unlike deucravacitinib,which selectively binds to the TYK2 regulatory domain,JAK1,2,3 inhibitors target the catalytic domain,contributing to nonselective targeting of JAK1,2,3. Cytokines associated with JAK1,2,3 signaling are required for both immune and nonimmune functions. A similar laboratory abnormality profile was observed in clinical trials using JAK1,2,3 inhibitors that has not been observed with deucravacitinib. In vitro testing of JAK1,2,3 inhibitors has relied upon assays of signal transduction,such as those measuring STAT phosphorylation,for estimates of potency and selectivity. These assay systems can be effective in estimating in vivo efficacy; however,they may not provide insight into downstream outcomes of receptor signaling,which may be more relevant for evaluating safety aspects. Assay systems assessing functional outcomes from cells may yield a more useful translational evaluation. Here,deucravacitinib was assessed for potency and selectivity versus three representatives of the JAK inhibitor class (tofacitinib,baricitinib,and upadacitinib) based on functional assays. JAK inhibitors had suppressive activity against JAK2-dependent hematopoietic colony-forming assays modeling thrombopoiesis,erythropoiesis,and myelopoiesis; however,deucravacitinib did not. Deucravacitinib had limited potency against NK cells,cytotoxic T cells,T-helper cells,and regulatory T cells activated by JAK1/JAK3-dependent common gamma chain cytokines. These data are consistent with the biologic role of JAK1,2,3 and pharmacodynamic changes in clinical laboratory abnormalities. Against TYK2-dependent cytokines,deucravacitinib selectively inhibited Type I interferon stimulation of monocytes and dendritic cells and was a more potent inhibitor than JAK inhibitors. IL-12 and IL-23 functional outputs were similarly potently inhibited by deucravacitinib. Results are consistent with deucravacitinib selectively inhibiting TYK2. View Publication

Isobaric labeling-based mass spectrometry (ILMS) has been widely used to quantify,on a proteome-wide scale,the relative protein abundance in different biological conditions. However,large-scale ILMS data sets typically involve multiple runs of mass spectrometry,bringing great computational difficulty to the integration of ILMS samples. We present zMAP,a toolset that makes ILMS intensities comparable across mass spectrometry runs by modeling the associated mean-variance dependence and accordingly applying a variance stabilizing z-transformation. The practical utility of zMAP is demonstrated in several case studies involving the dynamics of cell differentiation and the heterogeneity across cancer patients. The online version contains supplementary material available at 10.1186/s13059-024-03382-9. View Publication

Dysregulated innate immune responses underlie multiple inflammatory diseases,but clinical translation of preclinical innate immunity research in mice is hampered by the difficulty of studying human inflammatory reactions in an in vivo context. We therefore sought to establish in vivo human inflammatory responses in NSG-QUAD mice that express four human myelopoiesis transgenes to improve engraftment of a human innate immune system. We reconstituted NSG-QUAD mice with human hematopoietic stem and progenitor cells (HSPCs),after which we evaluated human myeloid cell development and subsequent human responses to systemic and local lipopolysaccharide (LPS) challenges. NSG-QUAD mice already displayed engraftment of human monocytes,dendritic cells and granulocytes in peripheral blood,spleen and liver at 6 weeks after HSPC reconstitution,in which both classical,intermediate and non-classical monocytes were present. These huNSG-QUAD mice responded to intraperitoneal and intranasal LPS challenges with production of NF-κB-dependent human cytokines,a human type I interferon response,as well as inflammasome-mediated production of human IL-1β and IL-18. The latter were specifically abrogated by the NLRP3 inhibitor MCC950,while LPS-induced human monocyte death was not altered. Besides providing proof-of-principle for small molecule testing of human inflammatory reactions in huNSG-QUAD mice,this observation suggests that LPS-induced in vivo release of human NLRP3 inflammasome-generated cytokines occurs in a cell death-independent manner. HuNSG-QUAD mice are competent for the NF-κB,interferon and inflammasome effectors of human innate immunity,and can thus be utilized to investigate signaling mechanisms and pharmacological targeting of human inflammatory responses in an in vivo setting. View Publication

The increasing scarcity of organs and the significant morbidity linked to dialysis require the development of engineered kidney tissues from human-induced pluripotent stem cells. Integrative approaches that synergize scalable kidney organoid differentiation,tissue biomanufacturing,and comprehensive assessment of their immune response and host integration are essential to accomplish this. Here,we create engineered human kidney tissues composed of organoid building blocks (OBBs) and transplant them into mice reconstituted with allogeneic human immune cells. Tissue-infiltrating human immune cells are composed of effector T cells and innate cells. This immune infiltration leads to kidney tissue injury characterized by reduced microvasculature,enhanced kidney cell apoptosis,and an inflammatory gene signature comparable to kidney organ transplant rejection in humans. Upon treatment with the immunosuppressive agent rapamycin,the induced immune response is greatly suppressed. Our model is a translational platform to study engineered kidney tissue immunogenicity and develop therapeutic targets for kidney rejection. Subject areas: Health sciences,Immunology,Bioengineering,Tissue engineering View Publication