Biology subjects

Loehning, M.

Publications and source records attributed to Loehning, M..

9 recordsLinked to original sources

bioRxiv2026

TLR-mediated activation of synovial fibroblasts from osteoarthritis patients promotes chondrocyte dysfunction

Objective: Toll like receptor (TLR) activation by cartilage derived damage-associated molecular patterns contributes to osteoarthritis (OA) pathogenesis, but the role of synovial fibroblasts in this process remains incompletely understood. We investigated the TLR responsiveness of human OA synovial fibroblasts and determined how TLR activation influences fibroblast-mediated regulation of chondrocyte function. Design: Primary synovial fibroblasts isolated from OA patients were characterized for TLR expression and stimulated with agonists targeting TLR1/2 to TLR9. Inflammatory mediator production, matrix metalloproteinase expression, mitochondrial respiration, and transcriptomic responses were assessed. Functional effects on cartilage homeostasis were evaluated using autologous chondrocyte spheroid co-cultures. Results: Approximately 50% of cells within OA synovial membranes were fibroblasts (CD45-CD31-PDPN+). Synovial fibroblasts expressed multiple TLRs at both the transcript and protein levels. Activation of TLR1/2, TLR4, TLR5, and TLR2/6 induced robust expression of IL-6, IL-8, G-CSF, MMP3, and MMP10, with minimal effects on mitochondrial respiratory function. Transcriptomic analysis revealed activation of inflammatory pathways together with enrichment of antigen presentation and protein translation programs. In autologous co-culture, TLR1/2-activated synovial fibroblasts promoted inflammatory and catabolic gene expression, suppressed anabolic gene expression, and impaired chondrocyte spheroid growth. Conclusions: Human OA synovial fibroblasts are highly responsive to TLR activation and acquire a pro-inflammatory, cartilage-degrading phenotype that directly impairs chondrocyte homeostasis. These findings identify synovial fibroblasts as key effectors of innate immune signalling within the OA joint and support targeting shared TLR-mediated pathways in joint-resident cells as a potential disease-modifying therapeutic strategy.

immunology↗

bioRxiv2025

Stable characteristics of intrapopulation heterogeneity in virus-specific Th1 cells during chronic viral challenge infection

Virus-specific CD4+ T cells typically undergo T helper (Th) 1 differentiation and contribute to a type 1 immune response in infection with lymphocytic choriomeningitis virus (LCMV). Using this model pathogen, we performed an in-depth analysis of the quantitative expression stability of the Th1 key transcription factor T-bet. Previously, it was shown that virus-specific Th1 cells arising in acute infections expressed T-bet at distinct intensities and maintained their T-bet expression differences after viral clearance as memory cells for weeks in the steady state. However, it was unclear whether differential T-bet expression was associated with heterogeneity inside the Th1 population and if the quantitative T-bet memory, particularly of those cells expressing T-bet at low levels, could withhold the strong and continuous stimulation present during chronic infection. Using T-bet-ZsGreen reporter mice, virus-specific Th1 cells were characterized phenotypically at protein, RNA, and DNA/chromatin accessibility levels. The Th1 cells arising during acute LCMV Armstrong infection showed a continuous spectrum of T-bet expression, ranging from cells with very high T-bet to cells with low T-bet. Even though the cells with low T-bet expression clearly possessed Th1 characteristics, they additionally showed certain Tfh-like features at protein and RNA level. When virus-specific Th1 cells were sorted according to T-bet-ZsGreen reporter expression intensity, adoptively transferred, and rechallenged by infecting the host animals with the chronic Clone 13 strain of LCMV, they maintained quantitative differences in T-bet reporter and IFN-{gamma} expression levels. A subpopulation of the progeny of the former T-betlow cells still showed a mild Tfh-associated phenotype. Independent of their past and present T-bet expression level, all virus-reactive CD4+ T cells acquired phenotypic signs of exhaustion as characterized by upregulation of PD-1, LAG3, and TOX and vast absence of effector cytokine co-expression in the chronic infection environment. Collectively, our findings highlight the heterogeneity of T-bet+ antiviral CD4+ T cells and the stability of quantitative differences in individual virus-specific CD4+ T cells during chronic viral challenge infection.

immunology↗

bioRxiv2025

D-Lactate drives cytotoxicity in Th1 cells by limiting mitochondrial ROS production through enhanced glyoxalase system activity

CD4+ T lymphocytes with cytotoxic activity are an understudied T helper (Th) cell population with the capacity to orchestrate immune responses and kill target cells in a TCR-restricted manner. Particularly, the mechanisms that promote the differentiation of cytotoxic Th cells remain elusive. Here, by supplementing Th1 cells with different lactate stereoisomers, we identify a previously unrecognized stereospecific adaptation that reinforces cytotoxic and proinflammatory programs. Although both D- and L-lactate impacted histone acetylation and metabolic activity, only D-lactate increased IFN-{gamma} and TNF- production and most potently promoted cytotoxic function. D-lactate also selectively enhanced mTORC1 and ERK1/ERK2 signaling, although these changes were not sufficient to fully explain the distinctive phenotype. Instead, the decisive feature of D-lactate supplementation was a reduction in mitochondrial reactive oxygen species (ROS), associated with electron transport chain (ETC) remodeling and enhanced glyoxalase-dependent buffering. Together, these findings identify a stereospecific lactate redox axis promoting cytotoxic Th1 cell differentiation.

immunology↗

bioRxiv2023

IL-33 controls IL-22-dependent antibacterial defense by modulating the microbiota

IL-22 plays a critical role in defending against mucosal infections, but how IL-22 production is regulated is incompletely understood. Here, we show that mice lacking IL-33 or its receptor ST2 (IL-1RL1) were more resistant to Streptococcus pneumoniae lung infection than wild-type animals, and that single nucleotide polymorphisms in IL33 and IL1RL1 were associated with pneumococcal pneumonia in humans. The effect of IL-33 on S. pneumoniae infection was mediated by negative regulation of IL-22 production in innate lymphoid cells (ILCs), but independent of ILC2s as well as IL-4 and IL-13 signaling. Moreover, IL-33s influence on antibacterial defense was dependent on housing conditions of the mice, and mediated by the modulatory effect of IL-33 on the microbiota. Collectively, we provide insight into the bidirectional crosstalk between the innate immune system and the microbiota. We identify a mechanism, dependent on both genetic and environmental factors, that impacts the efficacy of antibacterial immune defense and thus susceptibility to pneumonia. SIGNIFICANCE STATEMENTLower respiratory tract infections are the fifth leading cause of death. Here, we describe a mechanism influenced by genetic and environmental factors that affects the efficacy of pulmonary antibacterial immune responses. We show that IL-33 controls antibacterial defense by regulating the production of IL-22, a cytokine with known functions in antimicrobial immunity in lungs. The effect of IL-33 on IL-22-dependent defense was influenced by the hygienic status of the mice and mediated by IL-33s modulatory effect on the animal microbiota. In addition, genetic variation in genes involved in IL-33 signaling was associated with bacterial pneumonia in humans. Our findings may be important for our understanding of the factors influencing predisposition to lower respiratory tract infections.

immunology↗

bioRxiv2022

Statistical Inference of Enhancer-Gene Networks Reveals Pivotal Role of T-bet Expression Intensity for T Helper Cell Fate

Mammalian genomes harbor many more enhancers than genes, which greatly complicates the elucidation of cell-state-specific regulatory networks. Here, we developed a computational framework for learning enhancer-based gene networks from joint data on enhancer activity and transcript abundance. Dissecting the developmental plasticity of T helper (Th) cells with this approach, we uncovered a highly connected enhancer-gene network that supports graded Th-cell differentiation states, rather than mutual exclusivity of type-1 and type-2 immunity. Machine learning identifies a small number of regulatory enhancer types as network hubs. Hub enhancers in Th1 cells integrate as inputs the expression level of the master-regulator transcription factor, T-bet, and STAT signals governed by the cytokine environment. The quantitative balance between cell-intrinsic T-bet, driving phenotypic stability, and environmental cues enabling plasticity explains the heterogeneous reprogramming capacities of individual Th1 cells differentiating during natural infections in vivo. Moreover, we provide a framework for elucidating genome-scale regulatory networks based on enhancer activity.

systems biology↗

bioRxiv2022

Plasticity and lineage commitment of individual Th1 cells are determined by stable T-bet expression quantities

T helper 1 (Th1) cell identity is defined by the expression of the lineage-defining transcription factor T-bet. Here, we examine the influence of T-bet expression heterogeneity on subset plasticity by leveraging cell sorting of distinct in vivo-differentiated Th1 cells based on their quantitative expression of T-bet and interferon-{gamma}. Heterogeneous T-bet expression states were regulated by virus-induced type-I interferons and were stably maintained even after secondary viral infection. Exposed to Th2-polarizing conditions, the sorted subpopulations exhibited graded levels of plasticity: T-bet quantities were inversely correlated with the ability to express the Th2 lineage-specifying transcription factor GATA-3 and Th2 cytokines. Reprogramed Th1 cells acquired graded, but stable mixed Th1+2 phenotypes with a hybrid epigenetic landscape. Continuous presence of T-bet in differentiated Th1 cells was essential to ensure Th1 cell stability. Thus, innate cytokine signals regulate Th1 cell plasticity via an individual cell-intrinsic rheostat to enable T cell subset adaptation to subsequent challenges. HIGHLIGHTSO_LIType-I interferons triggered by infection determine T-bet expression states in Th1 cells C_LIO_LIT-bet and IFN-{gamma} expression states indicate the plasticity of individual Th1 cells C_LIO_LIIndividual T-bet expression states and plasticity persist after secondary infection C_LIO_LIReprogramming yields stable Th1+2 phenotypes and a mixed epigenetic landscape C_LI

immunology↗

bioRxiv2022

A Buprenorphine depot formulation provides effective sustained post-surgical analgesia for 72h in mouse femoral fracture models

Adequate pain management is essential for ethical and scientific reasons in animal experiments and should completely cover the period of expected pain without the need for frequent re-application. However, current depot formulations of Buprenorphine are only available in the USA and have limited duration of action. Recently, a new microparticulate Buprenorphine formulation (BUP-Depot) for sustained release has been developed as an alternative product within Europe. Pharmacokinetics indicate a potential effectiveness for about 72h. Here, we investigated whether the administration of the BUP-Depot ensures continuous and sufficient analgesia in two mouse femoral fracture models and could therefore serve as a potent alternative to the application of Tramadol via drinking water. Both protocols were examined for analgesic effectiveness, side effects on experimental readout, and effects on fracture healing outcomes in male and female C57BL/6N mice. The BUP-Depot provided effective analgesia for 72h, comparable to the effectiveness of Tramadol in drinking water. Fracture healing outcome was not different between analgesic regimes. The availability of a Buprenorphine depot formulation for laboratory animals in Europe would be a beneficial addition for extended pain relief in mice, thereby increasing animal welfare.

animal behavior and cognition↗

bioRxiv2022

High-resolution kinetic gene expression analysis of T helper cell differentiation reveals a STAT-dependent, unique transcriptional program in Th1/2 hybrid cells

Selective differentiation of CD4+ T helper (Th) cells into specialized subsets such as Th1 and Th2 cells is a key element of the adaptive immune system driving appropriate immune responses. Besides those canonical Th cell lineages, hybrid phenotypes such as Th1/2 cells arise in vivo, and their generation could be reproduced in vitro. While master-regulator transcription factors like T-bet for Th1 and GATA-3 for Th2 cells drive and maintain differentiation into the canonical lineages, the transcriptional architecture of hybrid phenotypes is less well understood. In particular, it has remained unclear whether a hybrid phenotype implies a mixture of the effects of several canonical lineages for each gene, or rather a bimodal behavior across genes. Th cell differentiation is a dynamic process in which the regulatory factors are modulated over time, but longitudinal studies of Th cell differentiation are sparse. Here, we present a dynamic transcriptome analysis following Th cell differentiation into Th1, Th2 and Th1/2 hybrid cells. We identified an early bifurcation point in gene expression programs, and we found that only a minority of [~]20% of Th cell-specific genes showed mixed effects from both Th1 and Th2 cells on Th1/2 hybrid cells. While most genes followed either Th1 or Th2 cell gene expression, another fraction of [~]20% of genes followed a Th1 and Th2 cell-independent transcriptional program under control of the transcription factors STAT1 and STAT4. Overall, our results emphasize the key role of high-resolution longitudinal data for the characterization of cellular phenotypes.

immunology↗

bioRxiv2020

HIF-stabilization prevents delayed fracture healing

The initial phase of fracture healing decides on success of bone regeneration and is characterized by an inflammatory milieu and low oxygen tension (hypoxia). Negative interference with or prolongation of this fine-tuned initiation phase will ultimately lead to a delayed or incomplete healing such as non-unions which then requires an effective and gentle therapeutic intervention. Common reasons include a dysregulated immune response, immunosuppression or a failure in cellular adaptation to the inflammatory hypoxic milieu of the fracture gap and a reduction in vascularizing capacity by environmental noxious agents (e.g. rheumatoid arthritis, smoking). The hypoxia-inducible factor (HIF)-1α is responsible for the cellular adaptation to hypoxia, activating angiogenesis and supporting cell attraction and migration to the fracture gap. Here, we hypothesized that stabilizing HIF-1α could be a cost-effective and low-risk prevention strategy of fracture healing disorders. Therefore, we combined a well-known HIF-stabilizer – deferoxamine (DFO) – and a less known HIF-enhancer – macrophage migration inhibitory factor (MIF) – to synergistically induce improved fracture healing. Stabilization of HIF-1α enhanced calcification and osteogenic differentiation of MSCs in vitro. In vivo, the application of DFO with or without MIF during the initial healing phase accelerated callus mineralization and vessel formation in a clinically relevant mouse-osteotomy-model in a compromised healing setting. Our findings provide support for a promising preventive strategy towards bone healing disorders in patients with a higher risk due to e.g. delayed neovascularization by accelerating fracture healing using DFO and MIF to stabilize HIF-1α.Competing Interest StatementThe authors have declared no competing interest.View Full Text

bioengineering↗