Abstract Background Staphylococcus aureus ( S. aureus ) is an increasingly recognized intracellular pathogen, yet infection outcomes vary with bacterial isolate and host cell type. The mechanisms underlying these differences remain poorly understood. This study investigates how distinct intracellular S. aureus isolates influence host signaling programs and infection outcomes by modulating cell death pathways and TNF-R1 dependent regulation of host cell fates across different human cell lines. Methods Four S. aureus isolates were analyzed for intracellular localization using transmission electron microscopy (TEM), structured illumination microscopy (SIM), serial block-face scanning electron microscopy (SBF-SEM), and imaging flow cytometry. Transcriptional reprogramming of infected U937 monocytes was examined by mRNA sequencing. Infection outcomes were characterized and compared to A549 and SaOS-2 cell lines employing Luminex cytokine assays, flow cytometry and Western blot analysis to characterize host cell death mechanisms in both wild-type and TNF-R1 deficient backgrounds. Results All S. aureus isolates localized to endolysosomal and cytosolic compartments but also peri⍰ and putatively intranuclearly, revealing an unexpected intracellular niche. In U937 monocytes, infection induced a conserved stress signature alongside isolate⍰specific transcriptional programs divergently affecting inflammation, metabolism, and cell fate, which was markedly attenuated in response to the chronic⍰infection isolate EDCC 5464. Cell death outcomes were likewise isolate⍰dependent, involving intrinsic and extrinsic apoptosis, mitochondrial depolarization, and caspase-1 activation at distinct temporal dynamics. TNF⍰R1 loss initially delayed but exacerbated late, isolate-independent cytotoxicity, identifying TNF⍰R1 as a key regulator of U937 infection outcome. SaOS⍰2 and A549 cell death was far less affected by isolate or TNF-R1 deficiency. Conclusions These results highlight the multilayered determinants governing intracellular S. aureus survival, non-canonical intracellular localization, and host cell susceptibility. The TNF/TNF-R1 axis is identified to critically determine regulated host defense during early infection stages in a tissue-specific manner. Together with distinct isolate-driven gene expression profiles, infection risks under TNF-targeted therapies and the contribution of S. aureus heterogeneity should be considered in the design of future host-directed treatment strategies. Plain English summary The bacterium Staphylococcus aureus ( S. aureus ) often lives harmlessly in humans but can cause severe or recurrent infections when the skin barrier is broken or the immune system is weakened. A major reason for its persistence is its ability to hide inside human cells, where it is shielded from immune attacks and antibiotics. To effectively target such bacteria, it is crucial to understand that infections vary depending on both the bacterial strain and the infected cell type. Many reasons behind these differences are still puzzling. We explored how different types of S. aureus (collected from different disease types) change how human cells respond to infection. We focused on how the different strains influence the way immune cells adjust their gene activity during infection, and how a receptor called TNF-R1 is involved in managing cell death responses. Bacteria were found not only in compartments meant to destroy them but also near and even inside the cell nucleus, an unexpected location. All strains triggered a similar stress response but also distinct patterns influencing inflammation, metabolism, and cell survival. A strain linked to chronic infection caused weaker responses, suggesting greater stealth. Cells lacking TNF-R1 initially survived longer but later showed greater damage, indicating this receptor’s role in infection control. In lung and bone cells, these effects were less pronounced. Concludingly, S. aureus occupies unexpected niches inside human cells and uses varying survival strategies. TNF-R1 is a key regulator of host infection responses in the analyzed immune cells, highlighting that both bacterial diversity and host factors must be considered when developing targeted treatments. Graphical Abstract Peri- and intranuclear localization early after S. aureus uptake across host cell lines, with isolate-specific modulation of host fates and a critical role for TNF-R1 to mediate regulated death responses of U937 cells. At 2 hpi, intracellular S. aureus not only localizes in (LAMP-1 decorated) membrane-enclosed compartments or directly in the cytosol, but within invaginations of the nuclear surface and intranuclearly with or without being surrounded by a vesicular membrane in U937 wt , SaOS-2 wt , and A549 wt cells. At 4 hpi, S. aureus triggers differential gene expression in ( A ) U937 wt cells to an isolate-specific extent, with both unique and shared transcriptomic signatures across the four isolates, that is muted for the chronic infection isolate EDCC 5464. Apoptotic cell death is induced to an isolate-dependent extent involving extrinsic initiator caspase-8, intrinsic initiator caspase-9 (EDCC 5055 only), and variable effector caspase-3/-7 activity in the earlier stages of infection (6 hpi), which then barely increases (24 hpi) in U937 wt cells. S. aureus -induced cell death and caspase activation is abolished in ( B ) U937 ΔTNF-R1 at 6 hpi, but is significantly reinforced at 24 hpi with diminished isolate-specificity. Correspondingly, mitochondrial trans-membrane potential (ΔΨm) is disrupted for all isolates upon TNF-R1 knockout, as well as caspase-1 activity, suggesting pyroptotic pathway activation at later stages of infection. ( C ) SaOS-2 wt cells show moderate caspase-3/-7 and -1 activation, while infection induces detachment of ( D ) A549 wt cells with minimal caspase activation. Infection induces an isolate- and cell line-dependent cytokine release. Coloured arrows indicate the mean proportion of effector-positive cells (↑ ∼20-40%, ↑ ↑ 40-60%, ↑ ↑ ↑ >60%) representing each S. aureus isolate. Grayed signaling arrows indicate the hypothesis by which TNF-R1 activation and internalization is required to kill lysosomal S. aureus via activation of anti-microbial enzymes and downstream regulated death pathway activation. Created with BioRender.com .

IntroductionOrthobiologics, such as autologous nanofat, are emerging as a potential treatment option for osteoarthritis (OA), a common degenerative joint causing pain and disability in the elderly. Nanofat, a minimally processed human fat graft rich in stromal vascular fraction (SVF) secretory factors, has shown promise in relieving pain. This study aimed to elucidate the molecular mechanisms underlying nanofat treatment of OA-affected cells and compare two filtration systems used for nanofat preparation.MethodsChondrocytes and synoviocytes were isolated from articular cartilage and synovium of 22 OA-patients. Lipoaspirates from 13 OA-patients were emulsified using the Adinizer® or Lipocube™ Nano filter systems to generate nanofat. The fluid phase of SVF from both filtered and unfiltered lipoaspirates was applied to OA-affected cells. Luminex multiplex ELISA were performed with lipoaspirates and cell supernatants alongside functional assays evaluating cell migration, proliferation, metabolic activity, and senescence.ResultsA total of 62 cytokines, chemokines, growth factors, neuropeptides, matrix-degrading enzymes, and complement components were identified in lipoaspirates. Among these, significant concentration differences were observed for TIMP-2, TGF-ß3, and complement component C3 between the filtered and unfiltered samples. Nanofat enhanced chondrocyte proliferation and migration, as well as synoviocyte migration and metabolic activity, while reducing chondrocyte metabolic activity. Pain-related factors like β-NGF, MCP-1, Substance P, VEGF, and αCGRP were reduced, while anti-inflammatory TGF-β1+3 increased and pro-inflammatory cytokines (IL-5, IL-7, IL-15, and IFN-γ) decreased. Nanofat also elevated secretion of complement components and TIMPs in both cell types. Notably, our results revealed no significant differences in cellular effects between sSVF filtered using the Adinizer® and Lipocube™ Nano systems, as well as compared to unfiltered sSVF.DiscussionHere, we provide first insights into how autologous nanofat therapy may ameliorate OA by enhancing chondrocyte proliferation and synoviocyte migration while modulating inflammatory and pain-related factors. However, further research is needed to determine its effects on cartilage regeneration.

Background: Factor H (FH) and FH-Related (FHR) proteins are crucial complement regulators. Their roles may extend beyond extracellular functions, potentially influencing cellular processes in inflammatory diseases like Osteoarthritis (OA) and malignancies such as Head and Neck Cancer (HNC). However, comparative expression profiles across different disease contexts and their intracellular presence require detailed investigation. Methods: We quantified FH and FHRs (−2, −3, −4, −5) levels via ELISA in synovial fluid (SF) from OA patients (n = 175), non-OA controls (n = 15), and in serum from HNC patients (n = 20) pre- and post-tumor removal versus controls. RNA sequencing and mendelian randomization (MR) analyses compared gene expression of CFH and CFHRs in OA vs non-OA tissues. To investigate subcellular protein distribution in HNC tumors, FH, FHR-1, FHR-2, FHR-3, and FHR-5 was assessed in cellular fractions (total lysate, membrane, cytoplasm, cytoskeleton, nucleus, chromatin), followed by protein identification and abundance measurement using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results: Distinct protein profiles emerged significant elevated FHR-4 levels in OA-SF in male and female patients, while FHR-5 (males) and FH (females) also showed significant increased levels. Notably, FHR-2 and FHR-3 levels were unchanged in OA-SF compared to non-OA-SF. Conversely, sera from HNC patients exhibited significantly elevated FHR-2, FHR-3, FHR-4, and FHR-5 pre-operatively compared to healthy controls, which remained elevated post-surgery. Mass spectrometry analysis of subcellular fractions revealed widespread distribution. Remarkably, FH, FHR-1, FHR-2 and FHR-5 were found in the nucleus, indicating possible nuclear functions. MR analysis indicated that genetically predicted higher expression of CFH was associated with an increased risk of knee-OA. Conclusion: FH-family proteins display context-dependent dysregulation: specific elevations (notably FHR-4) typify the local OA joint environment, contrasting with broad systemic FHR increases in HNC. The confirmed intracellular, particularly nuclear, localization points towards non-canonical functions of FHRs. Together, these findings reveal disease-specific molecular signatures and potential genetic contributions involving FH/FHR pathways in OA and HNC pathogenesis, highlighting avenues for biomarker development and therapeutic targeting.

Liposomes are a well-established carrier and controlled release system in medicine and bioanalysis. Their biomimetic capabilities are harnessed for the development of a reliable homogeneous assay platform technology that lends itself to high-throughput screening and point-of-care applications since no wash or separation steps are needed. It was developed for fluorescent, chemiluminescent, and electrochemical detection strategies and applied to antibodies directed against small or polymeric molecules and peptides as model analytes. The simplicity of the approach is achieved as mere binding of analytes or analyte-associated entities to the liposome surface leads to the activation of the complement system, which in turn lyses the liposomes. Released encapsulated marker molecules are quantified and directly correlated to the analytes. Control over the liposome chemistry, including cholesterol content, surface chemistry, and encapsulants, was identified to be key to ensure their general serum and storage stability (more than 40 months at 4 °C and up to 4 weeks at 37 °C) and their efficient and specific response to complement activity. Additional assay conditions of relevance included the concentration of liposomes and their ratio to serum proteins, the amount of complement trigger per liposome, and the activity of complement proteins. Understanding and being able to control the liposomes enable various analysis strategies including the quantification of analytes, determination of complement activity, and evaluation of the therapeutic application potential of antibodies. A time-resolved version of the assay even allows the study of the complex actions of the complement system. Graphical Abstract

Osteoarthritis (OA) is a degenerative joint disease causing loss of articular cartilage and structural damage in all joint tissues. Given the limited regenerative capacity of articular cartilage, methods to support the native structural properties of articular cartilage are highly anticipated. The aim of this study was to infiltrate zwitterionic monomer solutions into human OA-cartilage explants to replace lost proteoglycans. The study included polymerization and deposition of methacryloyloxyethyl-phosphorylcholine- and a novel sulfobetaine-methacrylate-based monomer solution within ex vivo human OA-cartilage explants and the encapsulation of isolated chondrocytes within hydrogels and the corresponding effects on chondrocyte viability. The results demonstrated that zwitterionic cartilage–hydrogel networks are formed by infiltration. In general, cytotoxic effects of the monomer solutions were observed, as was a time-dependent infiltration behavior into the tissue accompanied by increasing cell death and penetration depth. The successful deposition of zwitterionic hydrogels within OA cartilage identifies the infiltration method as a potential future therapeutic option for the repair/replacement of OA-cartilage extracellular suprastructure. Due to the toxic effects of the monomer solutions, the focus should be on sealing the OA-cartilage surface, instead of complete infiltration. An alternative treatment option for focal cartilage defects could be the usage of monomer solutions, especially the novel generated sulfobetaine-methacrylate-based monomer solution, as bionic for cell-based 3D bioprintable hydrogels.

The programmed cell death 1 ligand 1 (PD‐L1)/programmed cell death protein 1 (PD‐1) axis is primarily associated with immunosuppression in cytotoxic T lymphocytes (CTLs). However, mounting evidence is supporting the thesis that PD‐L1 not only functions as a ligand but mediates additional cellular functions in tumor cells. Moreover, it has been demonstrated that PD‐L1 is not exclusively localized at the cellular membrane. Subcellular fractionation revealed the presence of PD‐L1 in various cellular compartments of six well‐characterized head and neck cancer (HNC) cell lines, including the nucleus. Via Western blotting, we detected PD‐L1 in its well‐known glycosylated/deglycosylated state at 40–55 kDa. In addition, we detected previously unknown PD‐L1 variants with a molecular weight at approximately 70 and > 150 kDa exclusively in nuclear protein fractions. These in vitro findings were confirmed with primary tumor samples from head and neck squamous cell carcinoma (HNSCC) patients. Furthermore, we demonstrated that nuclear PD‐L1 variant expression is cell‐cycle‐dependent. Immunofluorescence staining of PD‐L1 in different cell cycle phases of synchronized HNC cells supported these observations. Mechanisms of nuclear PD‐L1 trafficking remain less understood; however, proximity ligation assays showed a cell‐cycle‐dependent interaction of the cytoskeletal protein vimentin with PD‐L1, whereas vimentin could serve as a potential shuttle for nuclear PD‐L1 transportation. Mass spectrometry after PD‐L1 co‐immunoprecipitation, followed by gene ontology analysis, indicated interaction of nuclear PD‐L1 with proteins involved in DNA remodeling and messenger RNA (mRNA) splicing. Our results in HNC cells suggest a highly complex regulation of PD‐L1 and multiple tumor cell‐intrinsic functions, independent of immune regulation. These observations bear significant implications for the therapeutic efficacy of immune checkpoint inhibition.

Bone marrow-derived mesenchymal stromal cells (BMSCs) respond to a variety of tumor cell-derived signals, such as inflammatory cytokines and growth factors. As a result, the inflammatory tumor microenvironment may lead to the recruitment of BMSCs. Whether BMSCs in the tumor environment are more likely to promote tumor growth or tumor suppression is still controversial. In our experiments, direct 3D co-culture of BMSCs with tumor cells from the head and neck region (HNSCC) results in strong expression and secretion of MMP-9. The observed MMP-9 secretion mainly originates from BMSCs, leading to increased invasiveness. In addition to our in vitro data, we show in vivo data based on the chorioallantoic membrane (CAM) model. Our results demonstrate that MMP-9 induces hemorrhage and increased perfusion in BMSC/HNSCC co-culture. While we had previously outlined that MMP-9 expression and secretion originate from BMSCs, our data showed a strong downregulation of MMP-9 promoter activity in HNSCC cells upon direct contact with BMSCs using the luciferase activity assay. Interestingly, the 2D and 3D models of direct co-culture suggest different drivers for the downregulation of MMP-9 promoter activity. Whereas the 3D model depicts a BMSC-dependent downregulation, the 2D model shows cell density-dependent downregulation. In summary, our data suggest that the direct interaction of HNSCC cells and BMSCs promotes tumor progression by significantly facilitating angiogenesis via MMP-9 expression. On the other hand, data from 3D and 2D co-culture models indicate opposing regulation of the MMP-9 promoter in tumor cells once stromal cells are involved.

Successful, long-term treatment of articular cartilage injuries is important for the prevention of osteoarthritis but remains a major challenge. Three studies in 2022 highlight new approaches to improving articular cartilage regeneration.

Introduction Factor H-related proteins (FHRs) have emerged as novel players in complement-mediated diseases, as they exhibit structural resemblances to factor H but lack the regulatory domains, enabling them to antagonize factor H and increase complement activation through several activities. Despite the widely importance of the complement system in kidney transplantation, FHRs have not been studied in this context. Utilizing a novel monoclonal antibody, we investigated the presence of FHR-3 in kidney allografts. Methods The RTEC-2 monoclonal antibody was validated using immunohistochemistry, Western Blot analysis, and immunoprecipitation combined with mass spectrometry. FHR-3 deposition, localization, and the relationships to complement activation were analyzed in human kidney biopsies obtained pre-transplantation from living and deceased donors, and post-transplantation in cases with acute tubular necrosis, acute cellular and vascular rejection, or chronic rejection. Results Glomerular FHR-3 deposition was detected in kidneys from deceased, but not living, donors before transplantation. Additionally, we observed FHR-3 deposition in post-transplant settings, both in cases of rejection and non-rejection. While tubular and vascular deposition of FHR-3 was observed in some cases, FHR-3 was predominantly seen in the glomeruli, where it was primarily localized to podocytes. Moreover, co-localization of FHR-3 and C3d was rarely detected, with most cases exhibiting separate and non-overlapping staining patterns for both antigens, However, there was a moderate correlation between the staining intensity of the FHR-3 and C3d in the kidney biopsies ( r =0.38, P=0.01). Conclusion We detected FHR-3 deposition in kidney allografts under inflammatory conditions, primarily colocalizing with podocytes in both the presence and absence of complement activation.

Osteoarthritis is highly heterogeneous, so effective therapies will need to target clearly defined molecular endotypes, restore mechanical joint function and reduce pain; thus, a 'one-size-fits-all' approach is unlikely to succeed.

Oxytocin (OXT) is a neuropeptide that has been associated with neurological diseases like autism, a strong regulating activity on anxiety and stress-related behavior, physiological effects during pregnancy and parenting, and various cellular effects in neoplastic tissue. In this study, we aimed to unravel the underlying mechanism that OXT employs to regulate cell-cell contacts, spheroid formation, and cellular migration in a 3D culture model of human MLS-402 cells. We have generated a labeled OXT receptor (OXTR) overexpressing cell line cultivated in spheroids that were treated with the OXTR agonists OXT, Atosiban, and Thr4-Gly7-oxytocin (TGOT); with or without a pre-treatment of antisense oligos (Gapmers) that induce exon skipping in the human OXTR gene. This exon skipping leads to the exclusion of exon 4 and therefore a receptor that lost its intracellular G-protein-binding domain. Sensitive digital PCR (dPCR) provided us with the means to differentiate between wild type and truncated OXTR in our cellular model. OXTR truncation differentially activated intracellular signaling cascades related to cell-cell attachment and proliferation like Akt, ERK1/2-RSK1/2, HSP27, STAT1/5, and CREB, as assessed by a Kinase Profiler Assay. Digital and transmission electron microscopy revealed increased tight junction formation and well-organized cellular protrusions into an enlarged extracellular space after OXT treatment, resulting in increased cellular survival. In summary, OXT decreases cellular migration but increases cell-cell contacts and therefore improves nutrient supply. These data reveal a novel cellular effect of OXT that might have implications for degenerating CNS diseases and tumor formation in various tissues.

The Early-Career Complementologists (ECCO) is a task force that was established, in close collaboration with the European Complement Network (ECN) and the International Complement Society (ICS), with the specific mission to support and connect early-career researchers (ECRs) in the complement field. ECRs are junior scientists at the early stages of their training which include undergraduate as well as graduate students, Ph.D. graduates, and post-doctoral fellows. This unique population within the scientific community represents the next generation of scientific leaders. However, ECRs are faced with key challenges and the COVID-19 pandemic has disproportionately impacted them. In this paper, we provide further insight into specific needs and challenges of ECRs in the complement field. We surveyed 52 ECRs in the complement field and assessed their perceptions of 1) mentor and peer support, 2) working conditions as well as 3) career interests and prospects. Furthermore, we review the various activities carried out by ECCO over the past years such as our social media presence, social events, and newly-created awards. We also discuss the future activities and events to be carried out by ECCO. Through these initiatives and activities, ECCO strives to boost collaborations between ECRs, provide recognition, and improve the visibility of their work. In addition, continuous joint efforts must also be made by the scientific community, research institutes, and funding organizations to nurture and invest in ECRs.

Osteoarthritis (OA) affects more than 500 million people worldwide and is among the five diseases in Germany causing the highest suffering of the patients and cost for the society. The quality of life of OA patients is severely compromised, and adequate therapy is lacking owing to a knowledge gap that acts as a major barrier to finding safe and effective solutions. Chronic, low-grade inflammation plays a central role in OA pathogenesis and is associated with both OA pain and disease progression. Innate immune pathways, such as the complement- and pattern-recognition receptor pathways, are pivotal to the inflammation in OA and key components of the innate immune system implicated in OA include DAMP-TLR signaling, the complement system, carboxypeptidase B (CPB), and mononuclear cells. Anaphylatoxins C3a and C5a are small polypeptides (77 and 74 amino acids, respectively) which are released by proteolytic cleavage of the complement components C3 and C5. The alternative complement pathway seems to play a crucial role in OA pathogenesis as these complement components, mostly C3 and its activation peptide C3a, were detected at high levels in osteoarthritic cartilage, synovial membrane, and cultured chondrocytes. Targeting the complement system by using anti-complement drugs as a therapeutic option bears the risk of major side effects such as increasing the risk of infection, interfering with cell regeneration and metabolism, and suppressing the clearance of immune complexes. Despite those adverse effects, several synthetic complement peptide antagonists show promising effects in ameliorating inflammatory cell responses also in joint tissues.

Current animal-free methods to assess teratogenicity of drugs under development still deliver high numbers of false negatives. To improve the sensitivity of human teratogenicity prediction, we characterized the TeraTox test, a newly developed multilineage differentiation assay using 3D human-induced pluripotent stem cells. TeraTox produces primary output concentration-dependent cytotoxicity and altered gene expression induced by each test compound. These data are fed into an interpretable machine-learning model to perform prediction, which relates to the concentration-dependent human teratogenicity potential of drug candidates. We applied TeraTox to profile 33 approved pharmaceuticals and 12 proprietary drug candidates with known in vivo data. Comparing TeraTox predictions with known human or animal toxicity, we report an accuracy of 69% (specificity: 53%, sensitivity: 79%). TeraTox performed better than 2 quantitative structure-activity relationship models and had a higher sensitivity than the murine embryonic stem cell test (accuracy: 58%, specificity: 76%, and sensitivity: 46%) run in the same laboratory. The overall prediction accuracy could be further improved by combining TeraTox and mouse embryonic stem cell test results. Furthermore, patterns of altered gene expression revealed by TeraTox may help grouping toxicologically similar compounds and possibly deducing common modes of action. The TeraTox assay and the dataset described here therefore represent a new tool and a valuable resource for drug teratogenicity assessment.