Biology subjects

Ludwig, R. J.

Publications and source records attributed to Ludwig, R. J..

9 recordsLinked to original sources

bioRxiv2026

γδ T cell-derived IL-17A aggravates experimental epidermolysis bullosa acquisita in response to IL-1β

Epidermolysis bullosa acquisita (EBA) is a mucocutaneous autoimmune blistering disorder mediated by autoantibodies against type VII collagen and subsequent splitting of the dermal-epidermal junction. Interleukin (IL)-17A promotes the production of chemokines and cytokines involved in neutrophil recruitment and activation, suggesting that IL-17A may contribute to EBA pathogenesis. We aimed to clarify the role of IL-17A in experimental EBA. Using an antibody transfer-induced EBA mouse model, we demonstrate that IL-17A promotes disease development, as pharmacological blockade of IL-17A with a neutralizing antibody ameliorated skin lesions. Gamma-delta ({gamma}{delta}) T cells were identified as the predominant cellular source of IL-17A, supported by the attenuated disease phenotype observed in {gamma}{delta}TCR-deficient mice. In addition, CD4+ T cells and neutrophils contributed to IL-17A production in experimental EBA. Mechanistically, IL-1-beta, rather than IL-23, was identified as a critical mediator of IL-17A production by {gamma}{delta} T cells in vitro, consistent with our in vivo findings. Whereas EBA severity was not reduced in IL-23p19-deficient mice, disease was markedly attenuated in IL-1R-deficient animals. Together, our study provides the first evidence that {gamma}{delta} T cell-derived IL-17A plays a pivotal role in experimental EBA and identifies a previously unrecognized IL-1{beta}/{gamma}{delta} T cell/IL-17A axis that expands current understanding of EBA immunopathogenesis.

immunology↗

bioRxiv2026

Targeting neutrophil signaling networks in immune complex-mediated autoimmune disease

Fragment crystallizable gamma receptor (Fc{gamma}R)-induced signaling is a crucial process that determines the cellular response to immune complexes (IC) in autoimmune diseases. In several diseases including pemphigoid diseases (PD), such as epidermolysis bullosa acquisita (EBA), or rheumatoid arthritis (RA), neutrophils are prominently involved as effector cells, while others such as immune thrombocytopenia (ITP) are independent of neutrophils. At the same time, most diseases are commonly treated by broad-range immunosuppression accompanied by severe risk for adverse effects. Signal transduction inhibitors (STIs) have been successfully applied in cancer therapy. However, their use in autoimmune diseases is an emerging, but so far understudied potential treatment avenue. Therefore, we screened a target-selective compound library consisting of 155 STIs in a neutrophil-based assay and conducted a multiplex kinase activity profiling with IC-stimulated neutrophils. Thus, we found novel potential therapeutic targets that were validated both in vitro in functional neutrophil assays and in vivo in murine models of EBA Here, we demonstrate that both systemic and topical treatment with several individual STIs is effective in a prophylactic approach in these models. Furthermore, therapeutic treatment with the BTK inhibitor ibrutinib in the immunization-induced EBA model reduced disease severity by approximately 85 % and showed efficacy in additional experimental models of EBA, arthritis, and ITP. Together, the present study contributes to the elucidation of Fc{gamma}R-dependent signaling in neutrophils and identifies multiple novel promising treatment options including inhibition of PLC, PDK-1, PKC, p38, DNA-PK, KSP, c-Met, TBK-1 and BTK for IC-mediated autoimmune diseases.

immunology↗

bioRxiv2026

Mechanical Skin Stress-Induced Lesion Development via ATP-Amplified Neutrophil Extracellular Trap Formation

Neutrophilic skin diseases, including Behcet disease, Sweet syndrome, pyoderma gangrenosum (PG), and epidermolysis bullosa acquisita (EBA), are characterized by an exaggerated inflammatory response following mechanical skin stimulation, yet the underlying mechanisms remain unclear. We identify adenosine triphosphate (ATP) released from keratinocytes as a key mediator of this phenomenon, promoting neutrophil extracellular trap (NET) formation. Using an EBA murine model as a model of neutrophilic skin disease, where scratching (a prototypic mechanical stimulation) exacerbates lesional severity, we observed abundant NET deposition in lesional skin. Degradation of these NETs with DNase1 reduced clinical and histopathological severities. In vitro, purified NET components increased IL-8 secretion from keratinocytes and fibroblasts, suggesting that NETs amplify inflammation via a self-amplifying loop of neutrophil recruitment. In the EBA mouse, scratch restriction with neck collars not only attenuated clinical and histological disease severities but also decreased lesional NETosis and neutrophils. Mechanistically, keratinocytes released ATP in response to mechanical stress in vitro, and pharmacologic purinergic blockade in the EBA mice with suramin phenocopied the protective effects of scratch restriction. While ATP alone did not induce NETosis, ATP enhanced complement component 5a (C5a)-induced NET formation in vitro. These findings indicate that keratinocyte-derived ATP, released in response to mechanical stress, contributes to NETosis in a C5a-dependent manner, thereby exaggerating neutrophilic inflammation, leading to blistering and further NETosis. Histopathological analyses of EBA and PG cases also demonstrated NETs accumulation localized to the upper dermis, suggesting a conserved ATP-NET axis. Targeting this pathway may represent a promising therapeutic strategy for neutrophilic skin diseases.

immunology↗

bioRxiv2025

Murine CMV Infection Unmasks Macrophage-Driven Inflammatory Cardiomyopathy in Pkp2, but not in Ttn Mutant Mice

2.BackgroundGenetic cardiomyopathies display variable penetrance and phenotypic expression, highlighting the influence of environmental modulators. Myocarditis, commonly triggered by cardiotropic viruses, overlaps clinically with genetic cardiomyopathies. Consequently, these infections are implicated as secondary factors that accelerate disease onset and progression, yet their precise impact in specific genetic settings remains unexplored. MethodsTo interrogate this, genetic mouse models heterozygous for a mutant allele of desmosomal plakophilin-2 (Pkp2+/-) or sarcomeric titin (Ttn+/-), genes frequently linked to acute myocarditis, were challenged with murine cytomegalovirus (MCMV) to determine how latent infection influences myocardial inflammation, tissue remodeling, and cardiac performance. Integrated experimental approaches, including echocardiography, histology, flow cytometry, single-cell RNA sequencing, as well as cytokine and kinome analyses, defined immune and signaling responses in infected versus non-infected hearts. ResultsAcute, MCMV-induced viral myocarditis and subsequent latent MCMV infection unmasked early disease onset in Pkp2+/- animals, leading to progressive systolic impairment, whereas in Ttn+/- mice cardiac structure and function remained preserved throughout infection. Cardiac immune profiling uncovered infection- and genotype-specific divergence: both genetic models showed a stable myocardial effector-memory CD8+ T-cell response to MCMV, but only Pkp2+/- hearts recruited additional Ly6C+ CCR2+ monocytes and macrophages with distinct inflammatory signatures. In the absence of infection, Pkp2 insufficiency initiated subclinical CCL2 secretion and subsequent recruitment of CCR2+ cells, reflecting early immune activation preceding age-associated functional and structural decline. At this stage, cytokine and kinase evaluations indicated a balance between proinflammatory and compensatory signals. However, with aging or following MCMV challenge, this balance shifted towards persistent inflammation, evidenced by chronic upregulation of cytokines and activation of signaling pathways, which ultimately led to adverse effects and myocardial dysfunction. ConclusionsManifestation of genetic cardiomyopathies depends on interactions between inherited susceptibility and environmental stressors. Here, we show that cytomegalovirus infection intensifies inflammation in PKP2-related cardiomyopathy. In contrast, TTN-linked cardiomyopathy does not exhibit increased inflammation under the same conditions. For individuals carrying desmosomal variants, infection control and tailored anti-inflammatory strategies may attenuate or delay disease manifestation and progression.

immunology↗

bioRxiv2025

CDK7, CDK9, or CDK11 Inhibition Reduces Neutrophil-Driven Inflammation and Tissue Damage in Experimental Autoimmune Models

BackgroundCyclin-dependent kinases (CDKs) are involved in basic cellular processes like regulation of cell-cycle progression and transcription. However, recent data also indicate a specific role in terminally differentiated neutrophils by promoting reactive oxygen species (ROS) release, degranulation, neutrophil extracellular trap formation, or apoptosis. Since these mechanisms are implicated in multiple autoimmune diseases, we aimed to delineate the role of CDKs in IC-mediated autoimmune diseases both in vitro and in vivo. MethodsWe analyzed CDK gene expression in unstimulated and immune complex (IC)-stimulated neutrophils. Subsequently, we investigated the effect of pharmacological CDK inhibition on IC-activated neutrophil functions. To analyze the inhibitors in a more translational approach, we proceeded with the systemic and topical application of the effective inhibitors in a murine antibody transfer-induced local epidermolysis bullosa acquisita (EBA) model. The most efficient inhibitor, MC180295, was validated in two other IC-mediated models of autoimmune disease: Serum-transfer arthritis (STA), which also, but not exclusively, depends on neutrophils and immune thrombocytopenia (ITP), which is considered less neutrophil-dependent. ResultsWe found 14 CDKs expressed in unstimulated cells, while the IC-stimulation showed an upregulation of CDK2 and CDK4 expression. Inhibitors selectively targeting CDK1, CDK2, CDK4/6, CDK7, CDK9, CDK11, and CDK12 showed effects on different neutrophil functions (surface activation marker expression, ROS release, adhesion, apoptosis) in vitro. In the predominantly neutrophil-driven EBA model, we observed a reduction of disease severity upon treatment with CDK7, CDK9, or CDK11 inhibitors. Inhibiting these CDKs with topical THZ2, MC180295, or OTS964, respectively, also improved the clinical phenotype. In line with our hypothesis, MC180295 impaired the development of STA, but not ITP. ConclusionsCDK7, CDK11, and especially CDK9 inhibition show therapeutic potential in IC-driven neutrophil-mediated diseases such as rheumatoid arthritis and EBA.

immunology↗

bioRxiv2025

CD19xCD3 bispecific T cell engager treatment induces remission in experimental pemphigoid disease

Pemphigoid diseases (PDs) are autoimmune disorders marked by autoantibodies (Aab) against skin and mucous membrane proteins, causing muco-cutaneous blistering in predominantly elderly patients. Since current therapeutics are often insufficient, PDs impose a significant morbidity and mortality burden. CD19xCD3 bispecific T cell engagers (TCEs) - originally developed for B cell malignancies - have shown promise in treatment-refractory autoimmune diseases like systemic sclerosis and rheumatoid arthritis. To explore their potential in PDs, we tested a murine CD19xCD3 TCE in a mouse model of epidermolysis bullosa acquisita (EBA), a prototypical PD. The TCE selectively depleted B cells in blood and bone marrow, but not spleen, of healthy mice. Mice with clinically manifest immunization-induced EBA were randomized to receive either the CD19xCD3 TCE or control treatment upon reaching a predefined disease severity. After 13 weeks, 45% of TCE-treated mice achieved remission, versus 23% of controls. This improvement correlated with reduced antigen-specific B cells, though total and antigen-specific IgG levels were unchanged. These findings suggest that CD19xCD3 TCE preferentially target autoreactive B cells and may be effective at lower doses than those used in oncology. Our data support the potential of CD19xCD3 bispecific T cell engagers as a therapeutic strategy for PDs. eTOC SynopsisGross, Jochimsen, Drager and colleagues demonstrate that CD19xCD3 bispecific T cell engagers, can selectively target autoreactive B cells and induce remission in a mouse model of pemphigoid disease, highlighting their potential as a novel therapeutic strategy for autoimmune blistering disorders.

immunology↗

bioRxiv2025

In pemphigus, cell detachment, but not autoantibody binding, induces cell-wide, long-lasting transcriptomic and proteomic changes

Desmoglein 1 (DSG1) and desmoglein 3 (DSG3) are adhesion molecules that maintain intercellular connections between epidermal, hair follicle, and mucosal keratinocytes. Autoantibodies (AAbs) targeting these molecules ultimately lead to the blister formation characteristic of pemphigus vulgaris (PV) or pemphigus foliaceus (PF). To investigate the molecular events following autoantibody binding up to 48 hours, we quantified transcriptome and proteome dynamics during split formation in a human skin organ culture (HSOC) model for PV and in a 2D cell-culture model for PV and endemic PF. Treatment of the cells in 2D culture with PX43, a single-chain variable fragment targeting DSG1/3, or with endemic PF anti-DSG1 IgG yielded neither a significant transcriptome nor a proteome response over time relative to the respective IgG controls. When treating the HSOC model with mouse antibody AK23 (targeting DSG3) or with PX43, only the latter induced split formation. In the absence of split formation, no differentially regulated pathways were detected at the transcriptomic level. Split formation, observed as early as 5 hours post-injection, was associated with significant and sustained upregulation of IFN{gamma} and TNF-related genes, mediated by upstream NF{kappa}B, MAPK, and JAK-STAT pathways. The gene expression changes, corroborated by proteomics data, were strongly correlated with early wounding and keratinocyte detachment, as well as the transcriptome profile in the skin from PV patients, while inversely associated with keratinocyte differentiation and cell stretching. The co-occurrence of well-wide and long-lasting transcriptome and proteome responses with split formation suggests that PF-IgG and PV-related AAbs neither induce downstream transcriptome nor proteome changes directly. Rather, these changes appear to be secondary effects resulting from reduced adhesion and mechanical-stress-induced split formation of keratinocytes in vitro and most likely in patient skin in vivo.

immunology↗

bioRxiv2021

A severe form of maternal separation in rat consisting of nineteen-day 6-hour daily separation at unpredictable times minimally affects behavior across lifespan and possibly confers protection against some maladaptive outcomes

Maternal separation (MS), a type of early life stress, has been associated with adverse socioemotional and behavioral outcomes throughout the lifespan across multiple species. Comprehensive longitudinal biobehavioral characterization of MS in rats is sparse and conflicting, warranting more studies. We conducted an MS paradigm involving 6-hour daily separation at unpredictable start times from P2 to P21. We hypothesized this severe form of MS would lead to developmentally emerging maladaptive biobehavioral consequences from juvenile through adult periods compared to Controls (C), especially in social behaviors. We tested: (1) own dam odor preference shortly after weaning; (2) juvenile and adult anxiety-like, sociability, and play behaviors using the light-dark test, three-chambered social interaction test, and video-coded juvenile play behavior; and (3) adult coping behaviors and neuroendocrine response using the forced swim test and blood corticosterone responses. Our results show minimal effects on biobehavioral outcomes across the lifespan. Recently weaned MS male rats had a stronger preference for their dams odor. Juvenile MS females spent more time in rough-and-tumble play than C female rats. No differences in sociability were found in the juvenile or adult periods. MS promoted a decrease in anxiety-like behavior that persisted from juvenile to adult periods. Finally, MS led to deficits in coping behavior in male adults, but basal and reactive corticosterone levels were unaltered by MS. More studies are needed to validate our surprising findings and probe the neural mechanisms underlying some of the observed protective effects. Significance StatementMaternal separation (MS), a type of early life stress, has been shown to lead to short and long-term adverse socioemotional consequences in humans and biobehavioral outcomes in rodents. Available data on MS exposed rats are sparse, conflicting, and often lack longitudinal outcomes. We designed a 6-hour unpredictable daily MS paradigm from postnatal day (P2) to P21 to severely disrupt normal dam-pup interactions. Our results show MS leads to increased preference for own dam odor, persisting reductions in anxiety-like behaviors from juvenile to adulthood, and no effects on sociability. Adverse effects on coping behaviors were uniquely identified in males. The present study warrants reevaluation of the central dogma that MS leads to maladaptive biobehavioral outcomes.

neuroscience↗

bioRxiv2021

Maternal separation affects fronto-cortical activity in rat pups during dam-pup interactions and behavioral transitions

Early-life stress is known to impair neurodevelopment. Prior work from our group showed that prolonged physical and emotional separation necessitated by the medical needs of preterm infants (born <37 weeks) is associated with lower electroencephalogram (EEG) power in frontal areas, and that trend can be reversed by an intervention that enhances the physical and emotional contact between preterm infant and mother. Here we sought to model the changes in preterm infant frontal EEG power in a rodent model. We examined effects of daily maternal separation (MS) on frontal cortex electrophysiological (electrocorticography [EcoG]) activity in neonatal rats. We also explored the effects of dam-pup behavioral interactions on EcoG activity. From postnatal days (P) 2-10, rat pups were separated daily from their dam and isolated from their littermates for 3 hours. Control rats were normally reared. On P10, pups were implanted with telemetry devices and an electrode placed on the left frontal dura. EcoG activity was recorded during daily sessions over the next four days while pups remained in the home cage, as well as in response to a pup-dam isolation-reunion paradigm at P12. EcoG power was computed in 1 Hz frequency bins between 1-100 Hz. Dam and pup behavioral interactions during recording sessions were coded and synchronized to EcoG data. MS pups showed lower EcoG power during dam-pup interactions. These data parallel human and provide evidence of lower fronto-cortical activity as an early marker of early-life stress and possible mechanism for long-term effects of maternal separation on neurobehavioral development.

neuroscience↗