Loss of 24-hydroxylated catabolism increases calcitriol and fibroblast growth factor 23 and alters calcium and phosphate metabolism in fetal mice.

Calcitriol circulates at low levels in normal human and rodent fetuses, in part due to increased 24-hydroxylation of calcitriol and 25-hydroxyvitamin D by 24-hydroxylase (CYP24A1). Inactivating mutations of CYP24A1 cause high postnatal levels of calcitriol and the human condition of infantile hypercalcemia type 1, but whether the fetus is disturbed by the loss of CYP24A1 is unknown. We hypothesized that loss of Cyp24a1 in fetal mice will cause high calcitriol, hypercalcemia, and increased placental calcium transport. The Cyp24a1+/- mice were mated to create pregnancies with wildtype, Cyp24a1+/-, and Cyp24a1 null fetuses. The null fetuses were hypercalcemic, modestly hypophosphatemic (compared to Cyp24a1+/- fetuses only), with 3.5-fold increased calcitriol, 4-fold increased fibroblast growth factor 23 (FGF23), and unchanged parathyroid hormone. The quantitative RT-PCR confirmed the absence of Cyp24a1 and 2-fold increases in S100g, sodium-calcium exchanger type 1, and calcium-sensing receptor in null placentas but not in fetal kidneys; these changes predicted an increase in placental calcium transport. However, placental 45Ca and 32P transport were unchanged in null fetuses. Fetal ash weight and mineral content, placental weight, crown-rump length, and skeletal morphology did not differ among the genotypes. Serum procollagen 1 intact N-terminal propeptide and bone expression of sclerostin and Blgap were reduced while calcitonin receptor was increased in nulls. In conclusion, loss of Cyp24a1 in fetal mice causes hypercalcemia, modest hypophosphatemia, and increased FGF23, but no alteration in skeletal development. Reduced incorporation of calcium into bone may contribute to the hypercalcemia without causing a detectable decrease in the skeletal mineral content. The results predict that human fetuses bearing homozygous or compound heterozygous inactivating mutations of CYP24A1 will also be hypercalcemic in utero but with normal skeletal development.

PTP1B phosphatase dampens iPSC-derived neutrophil motility and antimicrobial function.

Neutrophils are rapidly recruited to sites of infection and are critical for pathogen clearance. Therapeutic use of primary neutrophils has been limited, as they have a short lifespan and are not amenable to genetic manipulation. Human induced pluripotent stem cells (iPSCs) can provide a robust source of neutrophils for infusion and are genetically tractable. However, current work has indicated that dampened intracellular signaling limits iPSC-derived neutrophil (iNeutrophil) cellular activation and antimicrobial response. Here, we show that protein tyrosine phosphatase 1B (PTP1B) inhibits intracellular signaling and dampens iNeutrophil effector function. Deletion of the PTP1B phosphatase increased PI3K and ERK signaling and was associated with increased F-actin polymerization, cell migration, and phagocytosis. In contrast, other effector functions like NETosis and reactive oxygen species production were reduced. PTP1B-deficient neutrophils were more responsive to Aspergillus fumigatus and displayed rapid recruitment and control of hyphal growth. Accordingly, depletion of PTP1B increased production of inflammatory factors including the neutrophil chemokine interleukin-8. Taken together, these findings suggest that PTP1B limits iNeutrophil motility and antimicrobial function.

Neutrophil motility is regulated by both cell intrinsic and endothelial cell ARPC1B.

Neutrophil-directed motility is necessary for host defense, but its dysregulation can also cause collateral tissue damage. Actinopathies are monogenic disorders that affect the actin cytoskeleton and lead to immune dysregulation. Deficiency in ARPC1B, a component of the Arp2/3 complex, results in vascular neutrophilic inflammation; however, the mechanism remains unclear. Here, we generated human induced pluripotent stem cell (iPSC)-derived neutrophils (denoted iNeutrophils) that are deficient in ARPC1B and show impaired migration and a switch from forming pseudopodia to forming elongated filopodia. We show, using a blood vessel on a chip model, that primary human neutrophils have impaired movement across an endothelium deficient in APRC1B. We also show that the combined deficiency of ARPC1B in iNeutrophils and endothelium results in further reduction in neutrophil migration. Taken together, these results suggest that ARPC1B in endothelium is sufficient to drive neutrophil behavior. Furthermore, the findings provide support for using the iPSC system to understand human neutrophil biology and model disease in a genetically tractable system.

Liquid-liquid interfaces enable tunable cell confinement to recapitulate surrounding tissue deformations during neutrophil interstitial migration in vivo.

Cell migration is regulated by an interplay between both chemical and mechanical cues. Immune cells navigate through interstitial spaces and generate forces to deform surrounding cells, which in turn exert opposing pressures that regulate cell morphology and motility mechanisms. Current in vitro systems to study confined cell migration largely utilize rigid materials orders of magnitude stiffer than surrounding cells, limiting insights into how these local physical interactions regulate interstitial cell motility. Here, we first characterize mechanical interactions between neutrophils and surrounding cells in larval zebrafish and subsequently engineer in vitro migration channels bound by a deformable liquid-liquid interface that responds to cell generated pressures yielding a gradient of confinement across the length of a single cell. Tuning confining pressure gradients replicates mechanical interactions with surrounding cells during interstitial migration in vivo . We find that neutrophils favor a bleb-based mechanism of force generation to deform a barrier applying cell-scale confining forces. This work introduces a biomimetic material interface that enables new avenues of exploring the influence of mechanical forces on cell migration.

Constitutively active Lyn kinase causes a cutaneous small vessel vasculitis and liver fibrosis syndrome.

Neutrophilic inflammation is a hallmark of many monogenic autoinflammatory diseases; pathomechanisms that regulate extravasation of damaging immune cells into surrounding tissues are poorly understood. Here we identified three unrelated boys with perinatal-onset of neutrophilic cutaneous small vessel vasculitis and systemic inflammation. Two patients developed liver fibrosis in their first year of life. Next-generation sequencing identified two de novo truncating variants in the Src-family tyrosine kinase, LYN, p.Y508*, p.Q507* and a de novo missense variant, p.Y508F, that result in constitutive activation of Lyn kinase. Functional studies revealed increased expression of ICAM-1 on induced patient-derived endothelial cells (iECs) and of ß2-integrins on patient neutrophils that increase neutrophil adhesion and vascular transendothelial migration (TEM). Treatment with TNF inhibition improved systemic inflammation; and liver fibrosis resolved on treatment with the Src kinase inhibitor dasatinib. Our findings reveal a critical role for Lyn kinase in modulating inflammatory signals, regulating microvascular permeability and neutrophil recruitment, and in promoting hepatic fibrosis.

Cell Type-Specific Transcriptome Profiling Reveals a Role for Thioredoxin During Tumor Initiation.

Neutrophils in the tumor microenvironment exhibit altered functions. However, the changes in neutrophil behavior during tumor initiation remain unclear. Here we used Translating Ribosomal Affinity Purification (TRAP) and RNA sequencing to identify neutrophil, macrophage and transformed epithelial cell transcriptional changes induced by oncogenic RasG12V in larval zebrafish. We found that transformed epithelial cells and neutrophils, but not macrophages, had significant changes in gene expression in larval zebrafish. Interestingly, neutrophils had more significantly down-regulated genes, whereas gene expression was primarily upregulated in transformed epithelial cells. The antioxidant, thioredoxin (txn), a small thiol that regulates reduction-oxidation (redox) balance, was upregulated in transformed keratinocytes and neutrophils in response to oncogenic Ras. To determine the role of thioredoxin during tumor initiation, we generated a zebrafish thioredoxin mutant. We observed an increase in wound-induced reactive oxygen species signaling and neutrophil recruitment in thioredoxin-deficient zebrafish. Transformed keratinocytes also showed increased proliferation and reduced apoptosis in thioredoxin-deficient larvae. Using live imaging, we visualized neutrophil behavior near transformed cells and found increased neutrophil recruitment and altered motility dynamics. Finally, in the absence of neutrophils, transformed keratinocytes no longer exhibited increased proliferation in thioredoxin mutants. Taken together, our findings demonstrate that tumor initiation induces changes in neutrophil gene expression and behavior that can impact proliferation of transformed cells in the early tumor microenvironment.

Myeloid-derived growth factor regulates neutrophil motility in interstitial tissue damage.

Neutrophil recruitment to tissue damage is essential for host defense but can also impede tissue repair. The cues that differentially regulate neutrophil responses to tissue damage and infection remain unclear. Here, we report that the paracrine factor myeloid-derived growth factor (MYDGF) is induced by tissue damage and regulates neutrophil motility to damaged, but not infected, tissues in zebrafish larvae. Depletion of MYDGF impairs wound healing, and this phenotype is rescued by depleting neutrophils. Live imaging and photoconversion reveal impaired neutrophil reverse migration and inflammation resolution in mydgf mutants. We found that persistent neutrophil inflammation in tissues of mydgf mutants was dependent on the HIF-1α pathway. Taken together, our data suggest that MYDGF is a damage signal that regulates neutrophil interstitial motility and inflammation through a HIF-1α pathway in response to tissue damage.

Generation of Human Neutrophils from Induced Pluripotent Stem Cells in Chemically Defined Conditions Using ETV2 Modified mRNA.

This protocol describes a rapid and efficient feeder-, serum-, and xeno-free method for neutrophil generation from hiPSCs using ETV2 modified mRNA (mmRNA), which directs hematoendothelial programming of hiPSCs. Hematoendothelial progenitors were cultured with GM-CSF, FGF-2, and UM171 to expand myelomonocytic progenitors, followed by treatment with G-CSF and retinoic acid agonist Am580 to induce neutrophil maturation. This protocol is suitable for generating functional neutrophils from iPSCs to interrogate the role of genes in a neutrophil development and function. For complete details on the use and execution of this protocol, please refer to Brok-Volchanskaya et al. (2019).

Citrullination regulates wound responses and tissue regeneration in zebrafish.

Calcium is an important early signal in wound healing, yet how these early signals promote regeneration remains unclear. Peptidylarginine deiminases (PADs), a family of calcium-dependent enzymes, catalyze citrullination, a post-translational modification that alters protein function and has been implicated in autoimmune diseases. We generated a mutation in the single zebrafish ancestral pad gene, padi2, that results in a loss of detectable calcium-dependent citrullination. The mutants exhibit impaired resolution of inflammation and regeneration after caudal fin transection. We identified a new subpopulation of cells displaying citrullinated histones within the notochord bead following tissue injury. Citrullination of histones in this region was absent, and wound-induced proliferation was perturbed in Padi2-deficient larvae. Taken together, our results show that Padi2 is required for the citrullination of histones within a group of cells in the notochord bead and for promoting wound-induced proliferation required for efficient regeneration. These findings identify Padi2 as a potential intermediary between early calcium signaling and subsequent tissue regeneration.

Depleted iron stores in voluntary blood donors: A three-center cross-sectional study in Ghana.

BACKGROUND: Blood donation is frequently associated with iron deficiency. Although iron deficiency is endemic in Ghana, there is a scarcity of data on iron stores in blood donors to inform donor recruitment policy. This study determined the prevalence and factors predictive of depleted iron stores in blood donors. MATERIALS AND METHODS: This cross-sectional study recruited 287 blood donors from three regions in Ghana. Venous blood samples were collected for estimation of C-reactive protein, full blood count, and serum ferritin. Questionnaires were used to capture sociodemographic data. Data were analyzed using SPSS or GraphPad Prism. Multivariate logistic regression and receiver operator characteristics (ROC) analyses were, respectively, used to determine the factors associated with depleted iron stores or sensitivities of calculated red cell indices in predicting depleted iron stores in the participants. RESULTS: Whereas 27.4% of the blood donors had depleted iron stores (ferritin <15 ng/dL), only 11% took iron supplementation. While ferritin levels significantly increased with age, 49.5% of the blood donors were aged 20-29 years. Whereas 39.5% of participants had never donated blood, 24.9% had donated ≥3 units of whole blood in the past 2 years. Female (adjusted odds ratio [aOR]: 7.407, P = 0.005), multiple previous donations (1-2 [aOR: 1.846, P = 0.431]; ≥3 [aOR: 6.297, P = 0.016]), no iron supplementation (aOR: 17.553, P = 0.078), or platelet count ≥150 × 109/L (aOR: 2.689, P = 0.354) significantly associated with iron depletion. ROC analyses showed that whereas mean cell hemoglobin (MCH) density (area under the curve [AUC]: 0.735, P < 0.01), MCH (AUC: 0.772, P < 0.01) or Shine and Lal (AUC: 0.736, P < 0.01) fairly predicted iron depletion, combined cell index (AUC: 0.660, P < 0.01) or Green and King (AUC: 0.603, P < 0.01) indices poorly predicted iron depletion. CONCLUSIONS: More than quarter of voluntary blood donors suffers postdonation sideropenia. Calculated red cell indices should be investigated in different settings to validate usefulness in detecting iron depletion.

Neutrophil phagocyte oxidase activity controls invasive fungal growth and inflammation in zebrafish.

Neutrophils are primary phagocytes of the innate immune system that generate reactive oxygen species (ROS) and mediate host defense. Deficient phagocyte NADPH oxidase (PHOX) function leads to chronic granulomatous disease (CGD) that is characterized by invasive infections, including those by the generally non-pathogenic fungus Aspergillus nidulans The role of neutrophil ROS in this specific host-pathogen interaction remains unclear. Here, we exploit the optical transparency of zebrafish to image the effects of neutrophil ROS on invasive fungal growth and neutrophil behavior in response to Aspergillus nidulans In a wild-type host, A. nidulans germinates rapidly and elicits a robust inflammatory response with efficient fungal clearance. PHOX-deficient larvae have increased susceptibility to invasive A. nidulans infection despite robust neutrophil infiltration. Expression of subunit p22phox (officially known as CYBA), specifically in neutrophils, does not affect fungal germination but instead limits the area of fungal growth and excessive neutrophil inflammation and is sufficient to restore host survival in p22phox-deficient larvae. These findings suggest that neutrophil ROS limits invasive fungal growth and has immunomodulatory activities that contribute to the specific susceptibility of PHOX-deficient hosts to invasive A. nidulans infection.

Effective and Rapid Generation of Functional Neutrophils from Induced Pluripotent Stem Cells Using ETV2-Modified mRNA.

Human induced pluripotent stem cells (hiPSCs) can serve as a versatile and scalable source of neutrophils for biomedical research and transfusion therapies. Here we describe a rapid efficient serum- and xenogen-free protocol for neutrophil generation, which is based on direct hematoendothelial programming of hiPSCs using ETV2-modified mRNA. Culture of ETV2-induced hematoendothelial progenitors in the presence of GM-CSF, FGF2, and UM171 led to continuous production of generous amounts of CD34+CD33+ myeloid progenitors which could be harvested every 8-10 days for up to 30 days of culture. Subsequently, myeloid progenitors were differentiated into neutrophils in the presence of G-CSF and the retinoic acid agonist Am580. Neutrophils obtained in these conditions displayed a typical somatic neutrophil morphology, produced reactive oxygen species, formed neutrophil extracellular traps and possessed phagocytic and chemotactic activities. Overall, this technology offers an opportunity to generate a significant number of neutrophils as soon as 14 days after initiation of differentiation.

Phenotypical microRNA screen reveals a noncanonical role of CDK2 in regulating neutrophil migration.

Neutrophil migration is essential for inflammatory responses to kill pathogens; however, excessive neutrophilic inflammation also leads to tissue injury and adverse effects. To discover novel therapeutic targets that modulate neutrophil migration, we performed a neutrophil-specific microRNA (miRNA) overexpression screen in zebrafish and identified 8 miRNAs as potent suppressors of neutrophil migration. Among those, miR-199 decreases neutrophil chemotaxis in zebrafish and human neutrophil-like cells. Intriguingly, in terminally differentiated neutrophils, miR-199 alters the cell cycle-related pathways and directly suppresses cyclin-dependent kinase 2 (Cdk2), whose known activity is restricted to cell cycle progression and cell differentiation. Inhibiting Cdk2, but not DNA replication, disrupts cell polarity and chemotaxis of zebrafish neutrophils without inducing cell death. Human neutrophil-like cells deficient in CDK2 fail to polarize and display altered signaling downstream of the formyl peptide receptor. Chemotaxis of primary human neutrophils is also reduced upon CDK2 inhibition. Furthermore, miR-199 overexpression or CDK2 inhibition significantly improves the outcome of lethal systemic inflammation challenges in zebrafish. Our results therefore reveal previously unknown functions of miR-199 and CDK2 in regulating neutrophil migration and provide directions in alleviating systemic inflammation.

Mammalian Actin-binding Protein-1/Hip-55 Interacts with FHL2 and Negatively Regulates Cell Invasion.

Mammalian actin-binding protein-1 (mAbp1) is an adaptor protein that binds actin and modulates scission during endocytosis. Recent studies suggest that mAbp1 impairs cell invasion; however, the mechanism for the inhibitory effects of mAbp1 remain unclear. We performed a yeast two-hybrid screen and identified the adaptor protein, FHL2, as a novel binding partner that interacts with the N-terminal actin depolymerizing factor homology domain (ADFH) domain of mAbp1. Here we report that depletion of mAbp1 or ectopic expression of the ADFH domain of mAbp1 increased Rho GTPase signaling and breast cancer cell invasion. Moreover, cell invasion induced by the ADFH domain of mAbp1 required the expression of FHL2. Taken together, our findings show that mAbp1 and FHL2 are novel binding partners that differentially regulate Rho GTPase signaling and MTLn3 breast cancer cell invasion.

Integrin associated proteins differentially regulate neutrophil polarity and directed migration in 2D and 3D.

Directed neutrophil migration in blood vessels and tissues is critical for proper immune function; however, the mechanisms that regulate three-dimensional neutrophil chemotaxis remain unclear. It has been shown that integrins are dispensable for interstitial three-dimensional (3D) leukocyte migration; however, the role of integrin regulatory proteins during directed neutrophil migration is not known. Using a novel microfluidic gradient generator amenable to 2D and 3D analysis, we found that the integrin regulatory proteins Kindlin-3, RIAM, and talin-1 differentially regulate neutrophil polarization and directed migration to gradients of chemoattractant in 2D versus 3D. Both talin-1-deficient and RIAM-deficient neutrophil-like cells had impaired adhesion, polarization, and migration on 2D surfaces whereas in 3D the cells polarized but had impaired 3D chemotactic velocity. Kindlin-3 deficient cells were able to polarize and migrate on 2D surfaces but had impaired directionality. In a 3D environment, Kindlin-3 deficient cells displayed efficient chemotaxis. These findings demonstrate that the role of integrin regulatory proteins in cell polarity and directed migration can be different in 2D and 3D.

Focal adhesion kinase is required for synovial fibroblast invasion, but not murine inflammatory arthritis.

INTRODUCTION: Synovial fibroblasts invade cartilage and bone, leading to joint destruction in rheumatoid arthritis. However, the mechanisms that regulate synovial fibroblast invasion are not well understood. Focal adhesion kinase (FAK) has been implicated in cellular invasion in several cell types, and FAK inhibitors are in clinical trials for cancer treatment. Little is known about the role of FAK in inflammatory arthritis, but, given its expression in synovial tissue, its known role in invasion in other cells and the potential clinical availability of FAK inhibitors, it is important to determine if FAK contributes to synovial fibroblast invasion and inflammatory arthritis. METHODS: After treatment with FAK inhibitors, invasiveness of human rheumatoid synovial fibroblasts was determined with Matrigel invasion chambers. Migration and focal matrix degradation, two components of cellular invasion, were assessed in FAK-inhibited rheumatoid synovial fibroblasts by transwell assay and microscopic examination of fluorescent gelatin degradation, respectively. Using mice with tumor necrosis factor α (TNFα)-induced arthritis in which fak could be inducibly deleted, invasion and migration by FAK-deficient murine arthritic synovial fibroblasts were determined as described above and arthritis was clinically and pathologically scored in FAK-deficient mice. RESULTS: Inhibition of FAK in human rheumatoid synovial fibroblasts impaired cellular invasion and migration. Focal matrix degradation occurred both centrally and at focal adhesions, the latter being a novel site for matrix degradation in synovial fibroblasts, but degradation was unaltered with FAK inhibitors. Loss of FAK reduced invasion in murine arthritic synovial fibroblasts, but not migration or TNFα-induced arthritis severity and joint erosions. CONCLUSIONS: FAK inhibitors reduce synovial fibroblast invasion and migration, but synovial fibroblast migration and TNFα-induced arthritis do not rely on FAK itself. Thus, inhibition of FAK alone is unlikely to be sufficient to treat inflammatory arthritis, but current drugs that inhibit FAK may inhibit multiple factors, which could increase their efficacy in rheumatoid arthritis.

The F-BAR protein PSTPIP1 controls extracellular matrix degradation and filopodia formation in macrophages.

PSTPIP1 is a cytoskeletal adaptor and F-BAR protein that has been implicated in autoinflammatory disease, most notably in the PAPA syndrome: pyogenic sterile arthritis, pyoderma gangrenosum, and acne. However, the mechanism by which PSTPIP1 regulates the actin cytoskeleton and contributes to disease pathogenesis remains elusive. Here, we show that endogenous PSTPIP1 negatively regulates macrophage podosome organization and matrix degradation. We identify a novel PSTPIP1-R405C mutation in a patient presenting with aggressive pyoderma gangrenosum. Identification of this mutation reveals that PSTPIP1 regulates the balance of podosomes and filopodia in macrophages. The PSTPIP1-R405C mutation is in the SRC homology 3 (SH3) domain and impairs Wiskott-Aldrich syndrome protein (WASP) binding, but it does not affect interaction with protein-tyrosine phosphatase (PTP)-PEST. Accordingly, WASP inhibition reverses the elevated F-actin content, filopodia formation, and matrix degradation induced by PSTPIP1-R405C. Our results uncover a novel role for PSTPIP1 and WASP in orchestrating different types of actin-based protrusions. Our findings implicate the cytoskeletal regulatory functions of PSTPIP1 in the pathogenesis of pyoderma gangrenosum and suggest that the cytoskeleton is a rational target for therapeutic intervention in autoinflammatory disease.

The focal adhesion kinase inhibitor PF-562,271 impairs primary CD4+ T cell activation.

The focal adhesion kinase inhibitor, PF-562,271, is currently in clinical development for cancer, however it is not known how PF-562,271 affects T cell function. Here, we demonstrate inhibitory effects of PF-562,271 on the activation of primary human and mouse T cells. PF-562,271 inhibits T cell receptor signaling-induced T cell adhesion to intercellular adhesion molecule-1 and T cell interactions with antigen-presenting cells. An additional focal adhesion kinase inhibitor, PF-573,228, and genetic depletion of focal adhesion kinase also impair T cell conjugation with antigen-presenting cells. PF-562,271 blocks phosphorylation of the signaling molecules zeta chain associate protein of 70 kDa, linker of activated T cells, and extracellular signal-regulated kinase, and impairs T cell proliferation. The effects observed on T cell proliferation cannot solely be attributed to focal adhesion kinase inhibition, as genetic depletion did not alter proliferation. The effect of PF-562,271 on T cell proliferation is not rescued when proximal T cell receptor signaling is bypassed by stimulation with phorbol-12-myristate-13-acetate and ionomycin. Taken together, our findings demonstrate that focal adhesion kinase regulates integrin-mediated T cell adhesion following T cell receptor activation. Moreover, our findings suggest that PF-562,271 may have immunomodulatory effects that could impact its therapeutic applications.

Localized bacterial infection induces systemic activation of neutrophils through Cxcr2 signaling in zebrafish.

Neutrophils are the first line of defense against tissue damage and are rapidly mobilized to sites of bacterial infection. However, the signals that regulate neutrophil recruitment are not well defined. Here, using photolabel-enabled fate mapping in zebrafish larvae, we show that localized otic infection with Pseudomonas aeruginosa induces systemic activation and mobilization of neutrophils from the CHT through Cxcr2 signaling. We have cloned the zebrafish Cxcr1 and Cxcr2 receptors and show that Cxcr2 functions as a Cxcl8 receptor in live zebrafish. With the use of morpholino-mediated depletion, we show that infection-induced neutrophil mobilization from the CHT is mediated by Cxcr2 but not Cxcr1. By contrast, Cxcr2 depletion does not affect neutrophil recruitment to the chemoattractant LTB4. Taken together, our findings identify Cxcl8-Cxcr2 signaling as an infection-induced long-range cue that mediates neutrophil motility and mobilization from hematopoietic tissues, positioning Cxcr2 as a critical pathway that mediates infection-induced systemic activation of neutrophils.

Citrullination of fibronectin modulates synovial fibroblast behavior.

INTRODUCTION: Rheumatoid arthritis is an autoimmune arthritis characterized by joint destruction. Anti-citrullinated protein antibodies are pathologic in rheumatoid arthritis, but the role of the citrullinated proteins themselves is much less clear. Citrullination is the conversion of the arginine residues of a protein to citrulline. In the inflamed rheumatoid joint there is increased protein citrullination. Several proteins are citrullinated in rheumatoid arthritis, including collagen type II, fibrinogen, and fibronectin. Fibronectin is thought to mediate the adhesion of joint-invading synovial fibroblasts to the rheumatoid cartilage in addition to regulating other synovial fibroblast functions. However, the effect of citrullinated fibronectin on synovial fibroblasts is unknown. METHODS: To investigate the effect of citrullinated fibronectin on synovial fibroblast behavior, we cultured normal murine, arthritic murine, and human rheumatoid synovial fibroblasts. We then compared several synovial fibroblast functions in the presence of fibronectin versus citrullinated fibronectin. We assessed adhesion with time-lapse microscopy, migration with transwell assays, focal adhesion kinase and paxillin phosphorylation by western blot, and focal matrix degradation by fluorescent gelatin degradation. RESULTS: Normal synovial fibroblasts have impaired adhesion, spreading, migration, and integrin-mediated phosphorylation of focal adhesion kinase and paxillin on citrullinated fibronectin. Murine arthritic and human rheumatoid synovial fibroblasts also have impaired adhesion and spreading on citrullinated fibronectin, but focal matrix degradation is unaffected by citrullinated fibronectin. CONCLUSION: Citrullination of fibronectin alters synovial fibroblast behavior and may affect how these cells adhere to and invade the joint and travel through the bloodstream. This work suggests an important role for the interaction of synovial fibroblasts with citrullinated matrix in the pathophysiology of rheumatoid arthritis.