Role of Citrullinated Collagen in Autoimmune Arthritis.

Citrullination of proteins plays an important role in protein function and it has recently become clear that citrullinated proteins play a role in immune responses. In this study we examined how citrullinated collagen, an extracellular matrix protein, affects T-cell function during the development of autoimmune arthritis. Using an HLA-DR1 transgenic mouse model of rheumatoid arthritis, mice were treated intraperitoneally with either native type I collagen (CI), citrullinated CI (cit-CI), or phosphate buffered saline (PBS) prior to induction of autoimmune arthritis. While the mice given native CI had significantly less severe arthritis than controls administered PBS, mice receiving cit-CI had no decrease in the severity of autoimmune arthritis. Using Jurkat cells expressing the inhibitory receptor leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1), Western blot analysis indicated that while CI and cit-CI bound to LAIR-1 with similar affinity, only CI induced phosphorylation of the LAIR ITIM tyrosines; cit-CI was ineffective. These data suggest that cit-CI acts as an antagonist of LAIR-1 signaling, and that the severity of autoimmune arthritis can effectively be altered by targeting T cells with citrullinated collagen.

Leukocyte-associated immunoglobulin-like receptor 1 inhibits T-cell signaling by decreasing protein phosphorylation in the T-cell signaling pathway.

Multiple observations implicate T-cell dysregulation as a central event in the pathogenesis of rheumatoid arthritis. Here, we investigated mechanisms for suppressing T-cell activation via the inhibitory receptor leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1). To determine how LAIR-1 affects T-cell receptor (TCR) signaling, we compared 1) T cells from LAIR-1-sufficient and -deficient mice, 2) Jurkat cells expressing either LAIR-1 mutants or C-terminal Src kinase (CSK) mutants, and 3) T cells from mice that contain a CSK transgene susceptible to chemical inhibition. Our results indicated that LAIR-1 engagement by collagen or by complement C1q (C1Q, which contains a collagen-like domain) inhibits TCR signaling by decreasing the phosphorylation of key components in the canonical T-cell signaling pathway, including LCK proto-oncogene SRC family tyrosine kinase (LCK), LYN proto-oncogene SRC family tyrosine kinase (LYN), ζ chain of T-cell receptor-associated protein kinase 70 (ZAP-70), and three mitogen-activated protein kinases (extracellular signal-regulated kinase, c-Jun N-terminal kinase 1/2, and p38). The intracellular region of LAIR-1 contains two immunoreceptor tyrosine-based inhibition motifs that are both phosphorylated by LAIR-1 activation, and immunoprecipitation experiments revealed that Tyr-251 in LAIR-1 binds CSK. Using CRISPR/Cas9-mediated genome editing, we demonstrate that CSK is essential for the LAIR-1-induced inhibition of the human TCR signal transduction. T cells from mice that expressed a PP1 analog-sensitive form of CSK (CskAS) corroborated these findings, and we also found that Tyr-251 is critical for LAIR-1's inhibitory function. We propose that LAIR-1 activation may be a strategy for controlling inflammation and may offer a potential therapeutic approach for managing autoimmune diseases.

1,25-Dihydroxyvitamin D3 and 20-Hydroxyvitamin D3 Upregulate LAIR-1 and Attenuate Collagen Induced Arthritis.

Vitamin D plays a crucial role in regulation of the immune response. However, treatment of autoimmune diseases with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] doses sufficient to be effective is prohibitive due to its calcemic and toxic effects. We use the collagen-induced arthritis (CIA) model to analyze the efficacy of the noncalcemic analog of vitamin D, 20S-hydroxyvitamin D3 [20S(OH)D3], as well as 1,25(OH)2D3, to attenuate arthritis and explore a potential mechanism of action. Mice fed a diet deficient in vitamin D developed a more severe arthritis characterized by enhanced secretion of T cell inflammatory cytokines, compared to mice fed a normal diet. The T cell inflammatory cytokines were effectively suppressed, however, by culture of the cells with 20S(OH)D3. Interestingly, one of the consequences of culture with 1,25(OH)2D3 or 20S(OH)D3, was upregulation of the natural inhibitory receptor leukocyte associated immunoglobulin-like receptor-1 (LAIR-1 or CD305). Polyclonal antibodies which activate LAIR-1 were also capable of attenuating arthritis. Moreover, oral therapy with active forms of vitamin D suppressed arthritis in LAIR-1 sufficient DR1 mice, but were ineffective in LAIR-1-/- deficient mice. Taken together, these data show that the effect of vitamin D on inflammation is at least, in part, mediated by LAIR-1 and that non-calcemic 20S(OH)D3 may be a promising therapeutic agent for the treatment of autoimmune diseases such as Rheumatoid Arthritis.

mPGES1-Dependent Prostaglandin E2 (PGE2) Controls Antigen-Specific Th17 and Th1 Responses by Regulating T Autocrine and Paracrine PGE2 Production.

The integration of inflammatory signals is paramount in controlling the intensity and duration of immune responses. Eicosanoids, particularly PGE2, are critical molecules in the initiation and resolution of inflammation and in the transition from innate to acquired immune responses. Microsomal PGE synthase 1 (mPGES1) is an integral membrane enzyme whose regulated expression controls PGE2 levels and is highly expressed at sites of inflammation. PGE2 is also associated with modulation of autoimmunity through altering the IL-23/IL-17 axis and regulatory T cell (Treg) development. During a type II collagen-CFA immunization response, lack of mPGES1 impaired the numbers of CD4+ regulatory (Treg) and Th17 cells in the draining lymph nodes. Ag-experienced mPGES1-/- CD4+ cells showed impaired IL-17A, IFN-γ, and IL-6 production when rechallenged ex vivo with their cognate Ag compared with their wild-type counterparts. Additionally, production of PGE2 by cocultured APCs synergized with that of Ag-experienced CD4+ T cells, with mPGES1 competence in the APC compartment enhancing CD4+ IL-17A and IFN-γ responses. However, in contrast with CD4+ cells that were Ag primed in vivo, exogenous PGE2 inhibited proliferation and skewed IL-17A to IFN-γ production under Th17 polarization of naive T cells in vitro. We conclude that mPGES1 is necessary in vivo to mount optimal Treg and Th17 responses during an Ag-driven primary immune response. Furthermore, we uncover a coordination of autocrine and paracrine mPGES1-driven PGE2 production that impacts effector T cell IL-17A and IFN-γ responses.

N-acetylcysteine targets 5 lipoxygenase-derived, toxic lipids and can synergize with prostaglandin E2 to inhibit ferroptosis and improve outcomes following hemorrhagic stroke in mice.

OBJECTIVES: N-acetylcysteine (NAC) is a clinically approved thiol-containing redox modulatory compound currently in trials for many neurological and psychiatric disorders. Although generically labeled as an "antioxidant," poor understanding of its site(s) of action is a barrier to its use in neurological practice. Here, we examined the efficacy and mechanism of action of NAC in rodent models of hemorrhagic stroke. METHODS: Hemin was used to model ferroptosis and hemorrhagic stroke in cultured neurons. Striatal infusion of collagenase was used to model intracerebral hemorrhage (ICH) in mice and rats. Chemical biology, targeted lipidomics, arachidonate 5-lipoxygenase (ALOX5) knockout mice, and viral-gene transfer were used to gain insight into the pharmacological targets and mechanism of action of NAC. RESULTS: NAC prevented hemin-induced ferroptosis by neutralizing toxic lipids generated by arachidonate-dependent ALOX5 activity. NAC efficacy required increases in glutathione and is correlated with suppression of reactive lipids by glutathione-dependent enzymes such as glutathione S-transferase. Accordingly, its protective effects were mimicked by chemical or molecular lipid peroxidation inhibitors. NAC delivered postinjury reduced neuronal death and improved functional recovery at least 7 days following ICH in mice and can synergize with clinically approved prostaglandin E2 (PGE2 ). INTERPRETATION: NAC is a promising, protective therapy for ICH, which acted to inhibit toxic arachidonic acid products of nuclear ALOX5 that synergized with exogenously delivered protective PGE2 in vitro and in vivo. The findings provide novel insight into a target for NAC, beyond the generic characterization as an antioxidant, resulting in neuroprotection and offer a feasible combinatorial strategy to optimize efficacy and safety in dosing of NAC for treatment of neurological disorders involving ferroptosis such as ICH. Ann Neurol 2018;84:854-872.

The Role of Leukocyte-Associated Ig-like Receptor-1 in Suppressing Collagen-Induced Arthritis.

Several observations implicate a critical role for T cell dysregulation as a central problem in rheumatoid arthritis. We investigated a mechanism for suppressing T cell activation by stimulating a natural inhibitory receptor called leukocyte-associated Ig-like receptor-1 (LAIR-1). The collagen-induced arthritis (CIA) model and DR-1 transgenic mice were used to study the importance of LAIR-1 in autoimmune arthritis. Splenocytes from wild-type or LAIR-1-/- mice were stimulated with soluble anti-CD3 Ab in the presence or absence of α1(II) and supernatants were collected for cytokine analysis. B6.DR1 mice were immunized with type II collagen/CFA to induce arthritis and were treated with either the stimulatory mAb to LAIR-1 or a hamster IgG control. Finally, B6.DR1/LAIR-1-/- and B6.DR1/LAIR-1+/+ mice were challenged for CIA and mean severity scores were recorded thrice weekly. Using splenocytes or purified CD4+ cells that were sufficient in LAIR-1, CD3-induced cytokine secretion was significantly suppressed in the presence of collagen, whereas LAIR-1-deficient splenocytes had no attenuation. Treatment with a stimulatory mAb to LAIR-1 also significantly attenuated CIA in the LAIR+/+ mice. When B6.DR1/LAIR-1-/- mice were immunized with type II collagen they developed more severe arthritis and had a greater percentage of affected limbs than the wild-type mice. These data demonstrate that collagen can suppress the T cell cytokine response through the action of LAIR-1. Treatment with stimulating LAIR-1 Abs suppresses CIA whereas B6.DR1/LAIR-1-/- mice develop more severe arthritis than wild-type controls. These data suggest that LAIR-1 may be a potential therapeutic target for suppressing rheumatoid arthritis.

The role of Syk in peripheral T cells.

The aim of this study was to understand how Syk affects peripheral T cell function. T cells from Syk-/- chimeric mice and DR1 Sykfl/fl CD4cre conditional mice gave strong CD3-induced Th1, Th2, and Th17 cytokine responses. However, an altered peptide ligand (APL) of human CII (256-276) with two substitutions (F263N, E266D), also called A12, elicited only Th2 cytokine responses from Sykfl/fl T cells but not Sykfl/fl-CD4cre T cells. Western blots revealed a marked increase in the phosphorylation of Syk, JNK and p38 upon A12/DR1 activation in WT or Sykfl/fl T cells but not in Sykfl/flCD4-cre cells. We demonstrate that Syk is required for the APL- induction of suppressive cytokines. Chemical Syk inhibitors blocked activation of GATA-3 by peptide A12/DR1. In conclusion, this study provides novel insights into the role that Syk plays in directing T cell activity, and may shape therapeutic approaches for autoimmune diseases.

Characterization of the Syk-Dependent T Cell Signaling Response to an Altered Peptide.

Rheumatoid arthritis is an autoimmune disorder characterized by T cell dysregulation. We have shown that an altered peptide ligand (A9) activates T cells to use an alternate signaling pathway that is dependent on FcRγ and spleen tyrosine kinase, resulting in downregulation of inflammation. In the experiments described in this study, we have attempted to determine the molecular basis of this paradox. Three major Src family kinases found in T cells (Lck, Fyn, and Lyn) were tested for activation following stimulation by A9/I-Aq Unexpectedly we found they are not required for T cell functions induced by A9/I-Aq, nor are they required for APL stimulation of cytokines. On the other hand, the induction of the second messenger inositol trisphosphate and the mobilization of calcium are clearly triggered by the APL A9/I-Aq stimulation and are required for cytokine production, albeit the cytokines induced are different from those produced after activation of the canonical pathway. DBA/1 mice doubly deficient in IL-4 and IL-10 were used to confirm that these two cytokines are important for the APL-induced attenuation of arthritis. These studies provide a basis for exploring the effectiveness of analog peptides and the inhibitory T cells they induce as therapeutic tools for autoimmune arthritis.

Cytochrome P450 1B1 Contributes to the Development of Angiotensin II-Induced Aortic Aneurysm in Male Apoe(-/-) Mice.

Cytochrome P450 (CYP) 1B1 is implicated in vascular smooth muscle cell migration, proliferation, and hypertension. We assessed the contribution of CYP1B1 to angiotensin (Ang) II-induced abdominal aortic aneurysm (AAA). Male Apoe(-/-)/Cyp1b1(+/+) and Apoe(-/-)/Cyp1b1(-/-) mice were infused with Ang II or its vehicle for 4 weeks; another group of Apoe(-/-)/Cyp1b1(+/+) mice was coadministered the CYP1B1 inhibitor 2,3',4,5'-tetramethoxystilbene (TMS) every third day for 4 weeks. On day 28 of Ang II infusion, AAAs were analyzed by ultrasound and ex vivo by Vernier calipers, mice were euthanized, and tissues were harvested. Ang II produced AAAs in Apoe(-/-)/Cyp1b1(+/+) mice; mice treated with TMS or Apoe(-/-)/Cyp1b1(-/-) mice had reduced AAAs. Ang II enhanced infiltration of macrophages, T cells, and platelets and increased platelet-derived growth factor D, Pdgfrb, Itga2, and matrix metalloproteinases 2 and 9 expression in aortic lesions; these changes were inhibited in mice treated with TMS and in Apoe(-/-)/Cyp1b1(-/-) mice. Oxidative stress resulted in cyclooxygenase-2 expression in aortic lesions. These effects were minimized in Apoe(-/-)/Cyp1b1(+/+) mice treated with TMS and in Apoe(-/-)/Cyp1b1(-/-) mice and by concurrent treatment with the superoxide scavenger 4-hydroxyl-2,2,6,6-tetramethylpiperidine-1-oxyl. CYP1B1 contributed to the development of Ang II-induced AAA and associated pathogenic events in mice, likely by enhancing oxidative stress and associated signaling events. Thus, CYP1B1 may serve as a target for therapeutic agents for AAA in males.

Critical role of all-trans retinoic acid in stabilizing human natural regulatory T cells under inflammatory conditions.

Recent studies have demonstrated that thymus-derived naturally occurring CD4(+)Foxp3(+) regulatory T cells (Tregs) in human and mouse may be unstable and dysfunctional in the presence of proinflammatory cytokines. All-trans RA (atRA), the active derivative of vitamin A, has been shown to regulate Treg and T effector cell differentiation. We hypothesize atRA stabilizes human natural Tregs (nTregs) under inflammatory conditions. atRA prevents human nTregs from converting to Th1 and/or Th17 cells and sustains their Foxp3 expression and suppressive function in vitro or in vivo following encounters with IL-1 and IL-6. Interestingly, adoptive transfer of human nTregs pretreated with atRA significantly enhanced their suppressive effects on xenograft-vs.-host diseases (xGVHDs), and atRA- but not rapamycin-pretreated nTregs sustained the functional activity against xGVHD after stimulation with IL-1/IL-6. atRA suppresses IL-1 receptor (IL-1R) up-regulation, accelerates IL-6R down-regulation, and diminishes their signaling events as well as prevents the up-regulation of STIP1 homology and U-Box containing protein 1 on Foxp3(+) cells following IL-1/IL-6 stimulation. atRA also increases histone acetylation on Foxp3 gene promoter and CpG demethylation in the region of Foxp3 locus (i.e., Treg-specific demethylated region). These results strongly implicate that nTregs primed with atRA may represent a novel treatment strategy to control established chronic immune-mediated autoimmune and inflammatory diseases.

TGF-ß-induced CD4+Foxp3+ T cells attenuate acute graft-versus-host disease by suppressing expansion and killing of effector CD8+ cells.

The use of TGF-ß-induced CD4(+)Foxp3(+) T cells (induced regulatory T cells [iTregs]) is an important prevention and treatment strategy in autoimmune diseases and other disorders. However, the potential use of iTregs as a treatment modality for acute graft-versus-host disease (aGVHD) has not been realized because they may be unstable and less suppressive in this disease. We restudied the ability of iTregs to prevent and treat aGVHD in two mouse models. Our results showed that, as long as an appropriate iTreg-generation protocol is used, these iTregs consistently displayed a potent ability to control aGVHD development and reduce mortality in the aGVHD animal models. iTreg infusion markedly suppressed the engraftment of donor CD8(+) cells and CD4(+) cells, the expression of granzyme A and B, the cytotoxic effect of donor CD8(+) cells, and the production of T cell cytokines in aGVHD. Therefore, we conclude that as long as the correct methods for generating iTregs are used, they can prevent and even treat aGVHD.

In Vivo Dual Fluorescence Imaging to Detect Joint Destruction.

Diagnosis of cartilage damage in early stages of arthritis is vital to impede the progression of disease. In this regard, considerable progress has been made in near-infrared fluorescence (NIRF) optical imaging technique. Arthritis can develop due to various mechanisms but one of the main contributors is the production of matrix metalloproteinases (MMPs), enzymes that can degrade components of the extracellular matrix. Especially, MMP-1 and MMP-13 have main roles in rheumatoid arthritis and osteoarthritis because they enhance collagen degradation in the process of arthritis. We present here a novel NIRF imaging strategy that can be used to determine the activity of MMPs and cartilage damage simultaneously by detection of exposed type II collagen in cartilage tissue. In this study, retro-orbital injection of mixed fluorescent dyes, MMPSense 750 FAST (MMP750) dye and Alexa Fluor 680 conjugated monoclonal mouse antibody immune-reactive to type II collagen, was administered in the arthritic mice. Both dyes were detected with different intensity according to degree of joint destruction in the animal. Thus, our dual fluorescence imaging method can be used to detect cartilage damage as well as MMP activity simultaneously in early stage arthritis.

Peptide ligand structure and I-Aq binding avidity influence T cell signaling pathway utilization.

Factors that drive T cells to signal through differing pathways remain unclear. We have shown that an altered peptide ligand (A9) activates T cells to utilize an alternate signaling pathway which is dependent upon FcRγ and Syk. However, it remains unknown whether the affinity of peptide binding to MHC drives this selection. To answer this question we developed a panel of peptides designed so that amino acids interacting with the p6 and p9 predicted MHC binding pockets were altered. Analogs were tested for binding to I-A(q) using a competitive binding assay and selected analogs were administered to arthritic mice. Using the collagen-induced arthritis (CIA) model, arthritis severity was correlated with T cell cytokine production and molecular T cell signaling responses. We establish that reduced affinity of interaction with the MHC correlates with T cell signaling through the alternative pathway, leading ultimately to secretion of suppressive cytokines and attenuation of arthritis.

Engineered regulatory T cells coexpressing MHC class II:peptide complexes are efficient inhibitors of autoimmune T cell function and prevent the development of autoimmune arthritis.

Regulatory T cells (Tregs) are critical homeostatic components in preventing the development of autoimmunity, and are a major focus for their therapeutic potential for autoimmune diseases. To enhance the efficacy of Tregs in adoptive therapy, we developed a strategy for generating engineered Tregs that have the capacity to target autoimmune T cells in an Ag-specific manner. Using a retroviral expression system encoding Foxp3 and HLA-DR1 covalently linked to the immunodominant peptide of the autoantigen type II collagen (DR1-CII), naive T cells were engineered to become Tregs that express DR1-CII complexes on their surface. When these cells were tested for their ability to prevent the development of collagen induced arthritis, both the engineered DR1-CII-Foxp3 and Foxp3 only Tregs significantly reduced the severity and incidence of disease. However, the mechanism by which these two populations of Tregs inhibited disease differed significantly. Disease inhibition by the DR1-CII-Foxp3 Tregs was accompanied by significantly lower numbers of autoimmune CII-specific T cells in vivo and lower levels of autoantibodies in comparison with engineered Tregs expressing Foxp3 alone. In addition, the numbers of IFN-γ- and IL-17-expressing T cells in mice treated with DR1-CII-Foxp3 Tregs were also significantly reduced in comparison with mice treated with Foxp3 engineered Tregs or vector control cells. These data indicate that the coexpression of class II autoantigen-peptide complexes on Tregs provides these cells with a distinct capacity to regulate autoimmune T cell responses that differs from that used by conventional Tregs.

Genomic locus on chromosome 1 regulates susceptibility to spontaneous arthritis in mice deficiency of IL-1RA.

BACKGROUND: To understand the role of genetic factors on chromosome 1 in the regulation of spontaneous arthritis in mice deficient in IL-1 receptor antagonist protein (IL_1RA), we previously used speed congenic breeding to transfer the QTL region from DBA/1(-/-) mice that are resistant to spontaneous arthritis into BALB/c(-/-) mice which are susceptible. We were able to establish two congenic strains which exhibited a delayed onset and reduced severity of disease. In this study, we asked a different set of questions. How will the QTL region from BALB/c(-/-) interact with the rest of the genome in the DBA/1(-/-) background? Will the DBA/1(-/-) mice become susceptible to spontaneous arthritis if the QTL genomic region on chromosome 1 was replaced with the genomic fragment of the same region from BALB/c(-/-)? We conducted the congenic breeding with the similar procedure as that of congenic strains with BALB/c(-/-) background. RESULT: Instead of BALB/c(-/-), DBA/1(-/-) was used as the recurrent parent while BALB/c(-/-) was used as the donor parent. By the 6(th) generation we determined that all of the chromosomes in the progeny were of DBA/1(-/-) origin with the exception of the QTL portion of chromosome 1 which is heterozygous of BALB/c(-/-) and DBA/1(-/-) origin. We then intercrossed selected mice to produce homozygous strains containing the homozygous genomic region of BALB/c(-/-) on chromosome 1, while the rest of genome are homozygous DBA/1(-/-). This strain was observed for the development of spontaneous arthritis. Up to 9 weeks of age, both congenic strain and DBA/1(-/-) did not develop arthritis. However, after 9 weeks, the congenic strain started to exhibit signs of arthritis, while the DBA/1(-/-) remained free from disease. CONCLUSION: The result indicates a strong influence of genetic factor(s) on the QTL of chromosome 1 on the susceptibility to spontaneous arthritis. Identification of genetic factors within this QTL region in the future will significantly enhance our understanding of molecular mechanism of spontaneous arthritis.

Adoptive transfer of human gingiva-derived mesenchymal stem cells ameliorates collagen-induced arthritis via suppression of Th1 and Th17 cells and enhancement of regulatory T cell differentiation.

OBJECTIVE: Current approaches offer no cures for rheumatoid arthritis (RA). Accumulating evidence has revealed that manipulation of bone marrow-derived mesenchymal stem cells (BM-MSCs) may have the potential to control or even prevent RA, but BM-MSC-based therapy faces many challenges, such as limited cell availability and reduced clinical feasibility. This study in mice with established collagen-induced arthritis (CIA) was undertaken to determine whether substitution of human gingiva-derived mesenchymal stem cells (G-MSCs) would significantly improve the therapeutic effects. METHODS: CIA was induced in DBA/1J mice by immunization with type II collagen and Freund's complete adjuvant. G-MSCs were injected intravenously into the mice on day 14 after immunization. In some experiments, intraperitoneal injection of PC61 (anti-CD25 antibody) was used to deplete Treg cells in arthritic mice. RESULTS: Infusion of G-MSCs in DBA/1J mice with CIA significantly reduced the severity of arthritis, decreased the histopathology scores, and down-regulated the production of inflammatory cytokines (interferon-γ and interleukin-17A). Infusion of G-MSCs also resulted in increased levels of CD4+CD39+FoxP3+ cells in arthritic mice. These increases were noted early after infusion in the spleens and lymph nodes, and later after infusion in the synovial fluid. The FoxP3+ Treg cells that were increased in frequency mainly consisted of Helios-negative cells. When Treg cells were depleted, infusion of G-MSCs partially interfered with the progression of CIA. Pretreatment of G-MSCs with a CD39 or CD73 inhibitor significantly reversed the protective effect of G-MSCs on CIA. CONCLUSION: The role of G-MSCs in controlling the development and severity of CIA mostly depends on CD39/CD73 signals and partially depends on the induction of CD4+CD39+FoxP3+ Treg cells. G-MSCs provide a promising approach for the treatment of autoimmune diseases.

Roles of swallowing and belching in different phenotypes of gastroesophageal reflux disease.

BACKGROUND: The contributions of swallowing and belching to specific gastroesophageal reflux disease (GERD) phenotypes are unclear. METHODS: This study retrospectively analyzed esophageal pH/impedance studies, comparing reflux events preceded by gastric belching (GB), supragastric belching (SGB), air swallowing, and liquid/solid swallowing based on reflux position, lower esophageal sphincter (LES) pressure, and acid exposure time (AET). KEY RESULTS: 20 GERD patients and 10 controls were studied. Upright GERD patients and controls had a higher proportion of reflux events with a preceding swallow or belch (0.64, 0.64) than the supine group (0.38, p = 0.043). The upright group and controls trended toward a higher proportion of reflux events preceded by overall swallowing (0.61, 0.50) and air swallowing (0.55, 0.48) than the supine group (0.32, 0.31 p = 0.064, p = 0.11), but the three groups had similar rates of liquid/solid swallowing (0.032, 0.024, 0.017, p = 0.69). LES pressure did not correlate with reflux events preceded by swallowing (R2 = 0.021, p = 0.44). There was a higher rate of events preceded by gastric belching in the control group (0.14) than in the upright (0.032) and supine groups (0.066, p = 0.049). LES pressure did not correlate with the rate of events preceded by belching (R2 = 0.000093, p = 0.96). Normal AET patients had a higher rate of events preceded by GB (0.12) than those with increased acid exposure (0.030, p = 0.0083), but the two groups had similar rates of preceding air (0.43, 0.47, p = 0.68), liquid/solid (0.018, 0.032, p = 0.30), and overall swallowing (0.44, 0.53, p = 0.38). CONCLUSIONS AND INFERENCES: Swallowing more than belching is a dominant mechanism for reflux irrespective of GERD position, LES pressure, and AET.

C-reactive protein lowers the serum level of IL-17, but not TNF-α, and decreases the incidence of collagen-induced arthritis in mice.

The biosynthesis of C-reactive protein (CRP) in the liver is increased in inflammatory diseases including rheumatoid arthritis. Previously published data suggest a protective function of CRP in arthritis; however, the mechanism of action of CRP remains undefined. The aim of this study was to evaluate the effects of human CRP on the development of collagen-induced arthritis (CIA) in mice which is an animal model of autoimmune inflammatory arthritis. Two CRP species were employed: wild-type CRP which binds to aggregated IgG at acidic pH and a CRP mutant which binds to aggregated IgG at physiological pH. Ten CRP injections were given on alternate days during the development of CIA. Both wild-type and mutant CRP reduced the incidence of CIA, that is, reduced the number of mice developing CIA; however, CRP did not affect the severity of the disease in arthritic mice. The serum levels of IL-17, IL-6, TNF-α, IL-10, IL-2 and IL-1ß were measured: both wild-type and mutant CRP decreased the level of IL-17 and IL-6 but not of TNF-α, IL-10, IL-2 and IL-1ß. These data suggest that CRP recognizes and binds to immune complexes, although it was not clear whether CRP functioned in its native pentameric or in its structurally altered pentameric form in the CIA model. Consequently, ligand-complexed CRP, through an as-yet undefined mechanism, directly or indirectly, inhibits the production of IL-17 and eventually protects against the initiation of the development of arthritis. The data also suggest that IL-17, not TNF-α, is critical for the development of autoimmune inflammatory arthritis.

A programmable arthritis-specific receptor for guided articular cartilage regenerative medicine.

Objective: Investigational cell therapies have been developed as disease-modifying agents for the treatment of osteoarthritis (OA), including those that inducibly respond to inflammatory factors driving OA progression. However, dysregulated inflammatory cascades do not specifically signify the presence of OA. Here, we deploy a synthetic receptor platform that regulates cell behaviors in an arthritis-specific fashion to confine transgene expression to sites characterized by cartilage degeneration. Methods: An scFv specific for type II collagen (CII) was used to produce a synthetic Notch (synNotch) receptor that enables "CII-synNotch" mesenchymal stromal cells (MSCs) to recognize CII fibers exposed in damaged cartilage. Engineered cell activation by both CII-treated culture surfaces and on primary tissue samples was measured via inducible reporter transgene expression. TGFß3-expressing cells were assessed for cartilage anabolic gene expression via qRT-PCR. In a co-culture with CII-synNotch MSCs engineered to express IL-1Ra, ATDC5 chondrocytes were stimulated with IL-1α, and inflammatory responses of ATDC5s were profiled via qRT-PCR and an NF-κB reporter assay. Results: CII-synNotch MSCs are highly responsive to CII, displaying activation ranges over 40-fold in response to physiologic CII inputs. CII-synNotch cells exhibit the capacity to distinguish between healthy and damaged cartilage tissue and constrain transgene expression to regions of exposed CII fibers. Receptor-regulated TGFß3 expression resulted in upregulation of Acan and Col2a1 in MSCs, and inducible IL-1Ra expression by engineered CII-synNotch MSCs reduced pro-inflammatory gene expression in chondrocytes. Conclusion: This work demonstrates proof-of-concept that the synNotch platform guides MSCs for spatially regulated, disease-dependent delivery of OA-relevant biologic drugs.

Infusion of GMSCs relieves autoimmune arthritis by suppressing the externalization of neutrophil extracellular traps via PGE2-PKA-ERK axis.

INTRODUCTION: Rheumatoid arthritis (RA) is a systemic autoimmune disease with limited treatment success, characterized by chronic inflammation and progressive cartilage and bone destruction. Accumulating evidence has shown that neutrophil extracellular traps (NETs) released by activated neutrophils are important for initiating and perpetuating synovial inflammation and thereby could be a promising therapeutic target for RA. K/B × N serum transfer-induced arthritis (STIA) is a rapidly developed joint inflammatory model that somehow mimics the inflammatory response in patients with RA. Human gingival-derived mesenchymal stem cells (GMSCs) have been previously shown to possess immunosuppressive effects in arthritis and humanized animal models. However, it is unknown whether GMSCs can manage neutrophils in autoimmune arthritis. OBJECTIVES: To evaluate whether infusion of GMSCs can alleviate RA by regulating neutrophils and NETs formation. If this is so, we will explore the underlying mechanism(s) in an animal model of inflammatory arthritis. METHODS: The effects of GMSCs on RA were assessed by comparing the symptoms of the K/B × N serum transfer-induced arthritis (STIA) model administered either with GMSCs or with control cells. Phenotypes examined included clinical scores, rear ankle thickness, paw swelling, inflammation, synovial cell proliferation, and immune cell frequency. The regulation of GMSCs on NETs was examined through immunofluorescence and immunoblotting in GMSCs-infused STIA mice and in an in vitro co-culture system of neutrophils with GMSCs. The molecular mechanism(s) by which GMSCs regulate NETs was explored both in vitro and in vivo by silencing experiments. RESULTS: We found in this study that adoptive transfer of GMSCs into STIA mice significantly ameliorated experimental arthritis and reduced neutrophil infiltration and NET formation. In vitro studies also showed that GMSCs inhibited the generation of NETs in neutrophils. Subsequent investigations revealed that GMSCs secreted prostaglandin E2 (PGE2) to activate protein kinase A (PKA), which ultimately inhibited the downstream extracellular signal-regulated kinase (ERK) pathway that is essential for NET formation. CONCLUSION: Our results demonstrate that infusion of GMSCs can ameliorate inflammatory arthritis mainly by suppressing NET formation via the PGE2-PKA-ERK signaling pathway. These findings further support the notion that the manipulation of GMSCs is a promising stem cell-based therapy for patients with RA and other autoimmune and inflammatory diseases.