Bronchoconstriction damages airway epithelia by crowding-induced excess cell extrusion.

Asthma is deemed an inflammatory disease, yet the defining diagnostic feature is mechanical bronchoconstriction. We previously discovered a conserved process called cell extrusion that drives homeostatic epithelial cell death when cells become too crowded. In this work, we show that the pathological crowding of a bronchoconstrictive attack causes so much epithelial cell extrusion that it damages the airways, resulting in inflammation and mucus secretion in both mice and humans. Although relaxing the airways with the rescue treatment albuterol did not affect these responses, inhibiting live cell extrusion signaling during bronchoconstriction prevented all these features. Our findings show that bronchoconstriction causes epithelial damage and inflammation by excess crowding-induced cell extrusion and suggest that blocking epithelial extrusion, instead of the ensuing downstream inflammation, could prevent the feed-forward asthma inflammatory cycle.

Bronchoconstriction damages airway epithelia by excess crowding-induced extrusion.

Asthma is deemed an inflammatory disease, yet the defining diagnostic symptom is mechanical bronchoconstriction. We previously discovered a conserved process that drives homeostatic epithelial cell death in response to mechanical cell crowding called cell extrusion(1, 2). Here, we show that the pathological crowding of a bronchoconstrictive attack causes so much epithelial cell extrusion that it damages the airways, resulting in inflammation and mucus secretion. While relaxing airways with the rescue treatment albuterol did not impact these responses, inhibiting live cell extrusion signaling during bronchoconstriction prevented all these symptoms. Our findings propose a new etiology for asthma, dependent on the mechanical crowding of a bronchoconstrictive attack. Our studies suggest that blocking epithelial extrusion, instead of ensuing downstream inflammation, could prevent the feed-forward asthma inflammatory cycle.

SARS-CoV-2 Infection Induces Psoriatic Arthritis Flares and Enthesis Resident Plasmacytoid Dendritic Cell Type-1 Interferon Inhibition by JAK Antagonism Offer Novel Spondyloarthritis Pathogenesis Insights.

Objective: Bacterial and viral infectious triggers are linked to spondyloarthritis (SpA) including psoriatic arthritis (PsA) development, likely via dendritic cell activation. We investigated spinal entheseal plasmacytoid dendritic cells (pDCs) toll-like receptor (TLR)-7 and 9 activation and therapeutic modulation, including JAK inhibition. We also investigated if COVID-19 infection, a potent TLR-7 stimulator triggered PsA flares. Methods: Normal entheseal pDCs were characterized and stimulated with imiquimod and CpG oligodeoxynucleotides (ODN) to evaluate TNF and IFNα production. NanoString gene expression assay of total pDCs RNA was performed pre- and post- ODN stimulation. Pharmacological inhibition of induced IFNα protein was performed with Tofacitinib and PDE4 inhibition. The impact of SARS-CoV2 viral infection on PsA flares was evaluated. Results: CD45+HLA-DR+CD123+CD303+CD11c- entheseal pDCs were more numerous than blood pDCs (1.9 ± 0.8% vs 0.2 ± 0.07% of CD45+ cells, p=0.008) and showed inducible IFNα and TNF protein following ODN/imiquimod stimulation and were the sole entheseal IFNα producers. NanoString data identified 11 significantly upregulated differentially expressed genes (DEGs) including TNF in stimulated pDCs. Canonical pathway analysis revealed activation of dendritic cell maturation, NF-κB signaling, toll-like receptor signaling and JAK/STAT signaling pathways following ODN stimulation. Both tofacitinib and PDE4i strongly attenuated ODN induced IFNα. DAPSA scores elevations occurred in 18 PsA cases with SARS-CoV2 infection (9.7 ± 4 pre-infection and 35.3 ± 7.5 during infection). Conclusion: Entheseal pDCs link microbes to TNF/IFNα production. SARS-CoV-2 infection is associated with PsA Flares and JAK inhibition suppressed activated entheseal plasmacytoid dendritic Type-1 interferon responses as pointers towards a novel mechanism of PsA and SpA-related arthropathy.

Cytokine "fine tuning" of enthesis tissue homeostasis as a pointer to spondyloarthritis pathogenesis with a focus on relevant TNF and IL-17 targeted therapies.

A curious feature of axial disease in ankylosing spondylitis (AS) and related non-radiographic axial spondyloarthropathy (nrAxSpA) is that spinal inflammation may ultimately be associated with excessive entheseal tissue repair with new bone formation. Other SpA associated target tissues including the gut and the skin have well established paradigms on how local tissue immune responses and proven disease relevant cytokines including TNF and the IL-23/17 axis contribute to tissue repair. Normal skeletal homeostasis including the highly mechanically stressed entheseal sites is subject to tissue microdamage, micro-inflammation and ultimately repair. Like the skin and gut, healthy enthesis has resident immune cells including ILCs, γδ T cells, conventional CD4+ and CD8+ T cells and myeloid lineage cells capable of cytokine induction involving prostaglandins, growth factors and cytokines including TNF and IL-17 that regulate these responses. We discuss how human genetic studies, animal models and translational human immunology around TNF and IL-17 suggest a largely redundant role for these pathways in physiological tissue repair and homeostasis. However, disease associated immune system overactivity of these cytokines with loss of tissue repair "fine tuning" is eventually associated with exuberant tissue repair responses in AS. Conversely, excessive biomechanical stress at spinal enthesis or peripheral enthesis with mechanically related or degenerative conditions is associated with a normal immune system attempts at cytokine fine tuning, but in this setting, it is commensurate to sustained abnormal biomechanical stressing. Unlike SpA, where restoration of aberrant and excessive cytokine "fine tuning" is efficacious, antagonism of these pathways in biomechanically related disease may be of limited or even no value.

IL-17A and TNF Modulate Normal Human Spinal Entheseal Bone and Soft Tissue Mesenchymal Stem Cell Osteogenesis, Adipogenesis, and Stromal Function.

OBJECTIVE: The spondylarthritides (SpA) are intimately linked to new bone formation and IL-17A and TNF pathways. We investigated spinal soft tissue and bone mesenchymal stem cell (MSC) responses to IL-17A and TNF, including their osteogenesis, adipogenesis, and stromal supportive function and ability to support lymphocyte recruitment. METHODS: Normal spinal peri-entheseal bone (PEB) and entheseal soft tissue (EST) were characterized for MSCs by immunophenotypic, osteogenic, chondrogenic, and adipogenic differentiation criteria. Functional and gene transcriptomic analysis was carried out on undifferentiated, adipo- differentiated, and osteo-differentiated MSCs. The enthesis C-C Motif Chemokine Ligand 20-C-C Motif Chemokine Receptor 6 (CCL20-CCR6) axis was investigated at transcript and protein levels to ascertain whether entheseal MSCs influence local immune cell populations. RESULTS: Cultured MSCs from both PEB and EST displayed a tri-lineage differentiation ability. EST MSCs exhibited 4.9-fold greater adipogenesis (p < 0.001) and a 3-fold lower osteogenic capacity (p < 0.05). IL-17A induced greater osteogenesis in PEB MSCs compared to EST MSCs. IL-17A suppressed adipogenic differentiation, with a significant decrease in fatty acid-binding protein 4 (FABP4), peroxisome proliferator-activated receptor gamma (PPARγ), Cell Death Inducing DFFA Like Effector C (CIDEC), and Perilipin-1 (PLIN1). IL-17A significantly increased the CCL20 transcript (p < 0.01) and protein expression (p < 0.001) in MSCs supporting a role in type 17 lymphocyte recruitment. CONCLUSIONS: Normal spinal enthesis harbors resident MSCs with different in vitro functionalities in bone and soft tissue, especially in response to IL-17A, which enhanced osteogenesis and CCL20 production and reduced adipogenesis compared to unstimulated MSCs. This MSC-stromal-enthesis immune system may be a hitherto unappreciated mechanism of "fine tuning" tissue repair responses at the enthesis in health and could be relevant for SpA understanding.

Multi-pronged approach to human mesenchymal stromal cells senescence quantification with a focus on label-free methods.

Human mesenchymal stromal cells (hMSCs) have demonstrated, in various preclinical settings, consistent ability in promoting tissue healing and improving outcomes in animal disease models. However, translation from the preclinical model into clinical practice has proven to be considerably more difficult. One key challenge being the inability to perform in situ assessment of the hMSCs in continuous culture, where the accumulation of the senescent cells impairs the culture's viability, differentiation potential and ultimately leads to reduced therapeutic efficacies. Histochemical [Formula: see text]-galactosidase staining is the current standard for measuring hMSC senescence, but this method is destructive and not label-free. In this study, we have investigated alternatives in quantification of hMSCs senescence, which included flow cytometry methods that are based on a combination of cell size measurements and fluorescence detection of SA-[Formula: see text]-galactosidase activity using the fluorogenic substrate, C[Formula: see text]FDG; and autofluorescence methods that measure fluorescence output from endogenous fluorophores including lipopigments. For identification of senescent cells in the hMSC batches produced, the non-destructive and label-free methods could be a better way forward as they involve minimum manipulations of the cells of interest, increasing the final output of the therapeutic-grade hMSC cultures. In this work, we have grown hMSC cultures over a period of 7 months and compared early and senescent hMSC passages using the advanced flow cytometry and autofluorescence methods, which were benchmarked with the current standard in [Formula: see text]-galactosidase staining. Both the advanced methods demonstrated statistically significant values, (r = 0.76, p [Formula: see text] 0.001 for the fluorogenic C[Formula: see text]FDG method, and r = 0.72, p [Formula: see text] 0.05 for the forward scatter method), and good fold difference ranges (1.120-4.436 for total autofluorescence mean and 1.082-6.362 for lipopigment autofluorescence mean) between early and senescent passage hMSCs. Our autofluroescence imaging and spectra decomposition platform offers additional benefit in label-free characterisation of senescent hMSC cells and could be further developed for adoption for future in situ cellular senescence evaluation by the cell manufacturers.

Regulation of entheseal IL-23 expression by IL-4 and IL-13 as an explanation for arthropathy development under dupilumab therapy.

OBJECTIVES: Dupilumab blocks the IL-4 receptor (IL-4R) and thus signalling of the 'Th2' cytokines IL-4 and IL-13. It has a license to treat atopic eczema and was recently linked to emergent enthesitis and psoriasis. We investigated the cellular and functional basis for how IL-4/IL-13 regulates the IL-23-IL-17 axis in entheseal stromal, myeloid and lymphocyte cells. METHODS: Immunohistochemistry was performed on healthy enthesis samples from patients undergoing elective spinal surgery to investigate entheseal tissue IL-4R expression and cytokine expression by intracellular flow cytometry for IL-4 and IL-13. Digested human enthesis samples were stimulated with lipopolysaccharide (LPS) for IL-23 induction, either alone or with IL-4 or IL-13. Enthesis fibroblasts were stimulated with TNF and IL-17 with and without IL-4 or IL-13 to assess the effect on CCL20 secretion. Synovial fluid samples from PsA patients were also analysed by ELISA for levels of IL-4 and IL-13. RESULTS: The IL-4/IL-13 receptor was present in both the peri-entheseal bone and enthesis soft tissue, and entheseal-derived T cells produced basal levels of IL-4, but not IL-13. Both IL-4 and IL-13 attenuated LPS-induced entheseal IL-23 production. IL-4 also downregulated secretion of TNF/IL-17A-induced CCL20 from entheseal fibroblasts. Both IL-13 and IL-4 were also detectable in the synovial fluid of PsA patients. We also noted a seronegative inflammatory oligoarthritis whilst under dupilumab therapy. CONCLUSION: Our findings suggest a previously unknown protective role for IL-4/IL-13 in entheseal induction of the IL-23-IL-17 axis. These findings point towards a novel explanation for IL-13 pathway single nucleotide polymorphisms in PsA and also a molecular explanation for why anti-IL-4/IL-13 therapy may induce musculoskeletal entheseal pathology as recently reported.

Normal human enthesis harbours conventional CD4+ and CD8+ T cells with regulatory features and inducible IL-17A and TNF expression.

BACKGROUND: The human enthesis conventional T cells are poorly characterised. OBJECTIVES: To study the biology of the conventional T cells in human enthesis. METHODS: CD4+ and CD8+ T cells were investigated in 25 enthesis samples using immunofluorescence, cytometrically, bulk RNAseq and quantitative real-time PCR following anti-CD3/CD28 bead stimulation to determine interleukin (IL)-17A and tumour necrosis factor (TNF) levels. T-cell receptor (TCR) repertoires were characterised and a search for putative T-cell reactivity was carried out using TCR3 database. The impact of pharmacological antagonism with retinoic acid receptor-related orphan nuclear receptor gamma t inhibitor (RORγti), methotrexate and phosphodiesterase type 4 inhibitor (PDE4i) was investigated. RESULTS: Immunofluorescence and cytometry suggested entheseal resident CD4+ and CD8+ T cells with a resident memory phenotype (CD69+/CD45RA-) and tissue residency gene transcripts (higher NR4A1/AhR and lower KLF2/T-bet transcripts). Both CD4+ and CD8+ T cells showed increased expression of immunomodulatory genes including IL-10 and TGF-ß compared with peripheral blood T cells with entheseal CD8+ T cells having higher CD103, CD49a and lower SIPR1 transcript that matched CD4+ T cells. Following stimulation, CD4+ T cells produced more TNF than CD8+ T cells and IL-17A was produced exclusively by CD4+ T cells. RNAseq suggested both Cytomegalovirus and influenza A virus entheseal resident T-cell clonotype reactivity. TNF and IL-17A production from CD4+ T cells was effectively inhibited by PDE4i, while RORγti only reduced IL-17A secretion. CONCLUSIONS: Healthy human entheseal CD4+ and CD8+ T cells exhibit regulatory characteristics and are predicted to exhibit antiviral reactivity with CD8+ T cells expressing higher levels of transcripts suggestive of tissue residency. Inducible IL-17A and TNF production can be robustly inhibited in vitro.

The novel cytokine Metrnl/IL-41 is elevated in Psoriatic Arthritis synovium and inducible from both entheseal and synovial fibroblasts.

Meteorin-like(IL-41), is a novel cytokine that is thought to be immunoregulatory and is highly expressed in psoriatic skin. We investigated IL-41 protein expression in synovial tissue in RA(Rheumatoid Arthritis), PsA(Psoriatic Arthritis) and OA(Osteoarthritis) patients and evaluated IL-41 production from healthy enthesis samples, as the enthesis represent the primary inflammatory site in PsA. IL-41 was measured in synovial fluid from PsA, RA and OA patients. Synovial biopsies were stained for IL-41 by immunohistochemistry. IL-41 was highly expressed in the synovial fluid and synovial tissue of PsA patients (median = 7722 pg/ml) when compared to OA patients (median = 5044 pg/ml). We found that entheseal stromal cells were the dominant producer of IL-41 from the enthesis. Moreover, stromal derived IL-41, could be further induced by IL-17A/F and TNF. In conclusion, IL-41 is expressed in PsA synovium and is present and inducible at the enthesis. Its functional effect in psoriatic inflammation remains to be fully elucidated.

Identification of myeloid cells in the human enthesis as the main source of local IL-23 production.

OBJECTIVE: We investigated whether the normal human spinal enthesis contained resident myeloid cell populations, capable of producing pivotal proinflammatory cytokines including tumour necrosis factor (TNF) and interleukin (IL)-23 and determined whether these could be modified by PDE4 inhibition. METHODS: Normal human enthesis soft tissue (ST) and adjacent perientheseal bone (PEB) (n=15) were evaluated using immunohistochemistry (IHC), digested for myeloid cell phenotyping, sorted and stimulated with different adjuvants (lipopolysaccharide and mannan). Stimulated enthesis fractions were analysed for inducible production of spondyloarthropathy disease-relevant mediators (IL-23 full protein, TNF, IL-1ß and CCL20). Myeloid populations were also compared with matched blood populations for further mRNA analysis and the effect of PDE4 inhibition was assessed. RESULTS: A myeloid cell population (CD45+ HLADR+ CD14+ CD11c+) phenotype was isolated from both the ST and adjacent PEB and termed 'CD14+ myeloid cells' with tissue localisation confirmed by CD14+ IHC. The CD14- fraction contained a CD123+ HLADR+ CD11c- cell population (plasmacytoid dendritic cells). The CD14+ population was the dominant entheseal producer of IL-23, IL-1ß, TNF and CCL20. IL-23 and TNF from the CD14+ population could be downregulated by a PDE4I and other agents (histamine and 8-Bromo-cAMP) which elevate cAMP. Entheseal CD14+ cells had a broadly similar gene expression profile to the corresponding CD14+ population from matched blood but showed significantly lower CCR2 gene expression. CONCLUSIONS: The human enthesis contains a CD14+ myeloid population that produces most of the inducible IL-23, IL-1ß, TNF and CCL20. This population has similar gene expression profile to the matched blood CD14+ population.

The Early Phases of Ankylosing Spondylitis: Emerging Insights From Clinical and Basic Science.

In our paper, we discuss how the early phases of ankylosing spondylitis (AS) are linked to peri-firbocartilagenous osteitis in the sacroiliac joint and entheseal bone related anchoring sites. This skeletal proclivity is linked to an abnormal immunological response to skeletal biomechanical stress and associated microdamage. A key event in the early stages of AS appears to be the association with subclinical Crohn's-like colitis with this gut inflammation being pivotal to the osteitis reaction. Whether this osteitis is consequent to non-specific intestinal innate immune activation or adaptive immune responses against specific microbiotal or self-antigens is unknown. Recurrent iritis is an HLA-B27 associated feature that may predate AS and pursues a course independent of joint involvement, and points toward the pivotal role of organ specific immunology over generalized systemic immune responses in disease expression. Human genetics and animal model studies strongly incriminate the IL23/17 axis and TNF-α in disease pathogenesis. Preliminary work shows a strong convergence of innate immune cells including type 3 innate lymphoid-cells (ILC3) and γδ T-cells in skin, gut, entheseal, and eye inflammation. Despite the HLA-B27 association, the role of adaptive immunity, especially CD8+ T-cells mediated responses remains unproven and alternative theories have been proposed. The emerging non-dependence of axial inflammation on IL-23 but dependence on IL-17A is an unexpected new twist that awaits full explanation. In this mini-review, we discuss the key events in the early stages of human AS from clinical and basic science aspects, which could be crucial for attempted disease prevention studies in at risk subjects.