Differences in IDO1+ dendritic cells and soluble CTLA-4 are associated with differential clinical responses to methotrexate treatment in rheumatoid arthritis.

Objective: Antigen-presenting dendritic cells (DCs) and monocytes play an essential role in rheumatoid arthritis (RA) pathogenesis, however, their tolerogenic potential remains unclear. Herein, the tolerogenic profiles of DCs are characterized in treatment-naïve RA patients to determine their role to inflammatory arthritis management. Methods: Thirty-six treatment-naïve RA patients were enrolled, of which 62% were non-responders to methotrexate (MTX) monotherapy based on disease activity score (DAS) after 6-months of therapy. DC and monocyte subset frequencies, activation (CD40, CD86, CD209 expression), and tolerogenic profile (intracellular indoleamine-2,3-dioxygenase [IDO1] and cytotoxic T lymphocyte antigen 4 [CTLA-4] expression) were examined in the baseline peripheral blood by multicolor flow-cytometry. Soluble CTLA-4 (sCTLA-4) levels in plasma were measured. Results: DC subsets were decreased in RA compared to healthy controls (HC), and the frequency of conventional DCs (cDC) inversely correlated with inflammatory markers and improvement in disease activity. CD141+ cDC1s were the major IDO1-expressing cells. IDO1+cDC1s were reduced in RA patients compared to HC. The baseline frequency of IDO1+cDC1s inversely correlated with improvement in disease activity. CTLA-4 expression in CD1c+ cDC2s and monocytes was lower in RA patients compared to HC. Moreover, MTX-responders had a significantly lower frequency of IDO1+cDC1 cells and higher level of sCTLA-4 in the plasma compared to MTX non-responders. There was a strong predictive association of low IDO1+cDC1 cells, low sCTLA-4 and non-response to MTX. Conclusions: Our findings reveal altered DC and monocytes immunophenotypes that are associated with RA pathology and treatment response. The frequencies of tolerogenic IDO1+cDC1s and the low level of sCTLA-4 are strongly associated with MTX non-responsiveness and therapeutic outcome. These results suggest that investigation of the association IDO1+cDC1 and sCTLA-4 with response to treatment may be more generalizable to other autoimmune diseases.

MicroRNA-27a-3p enhances the inflammatory phenotype of Juvenile Idiopathic Arthritis fibroblast-like synoviocytes.

BACKGROUND: Juvenile Idiopathic Arthritis (JIA) is the most prevalent chronic pediatric rheumatic disorder. In joints of JIA patients, aggressive phenotypic changes in fibroblast-like synoviocytes (FLS) of the synovial lining play a key role in inflammation. MicroRNAs are dysregulated in rheumatoid arthritis and JIA, including miR-27a-3p. However, it is not understood if miR-27a-3p, enriched in JIA synovial fluid (SF) and leukocytes, alters FLS function. METHODS: Primary JIA FLS cells were transfected with a miR-27a-3p mimic or a negative control microRNA (miR-NC) and stimulated with pooled JIA SF or inflammatory cytokines. Viability and apoptosis were analyzed by flow cytometry. Proliferation was evaluated using a 3H-thymidine incorporation assay. Cytokine production was assessed by qPCR and ELISA. Expression of TGF-ß pathway genes was determined using a qPCR array. RESULTS: MiR-27a-3p was constitutively expressed in FLS. Overexpression of miR-27a-3p caused increased interleukin-8 secretion in resting FLS, and interleukin-6 was elevated in SF-activated FLS compared to miR-NC. Furthermore, stimulation with pro-inflammatory cytokines augmented FLS proliferation in miR-27a-3p-transfected FLS relative to miR-NC. Expression of multiple TGF-ß pathway genes was modulated by overexpression of miR-27a-3p. CONCLUSIONS: MiR-27a-3p significantly contributes to FLS proliferation and cytokine production, making it a potential candidate for epigenetic therapy that targets FLS in arthritis.

Differentially Expressed Inflammation-Regulating MicroRNAs in Oligoarticular Juvenile Idiopathic Arthritis.

OBJECTIVE: To evaluate microRNA expression in synovial fluid (SF), plasma, and leukocytes from patients with juvenile idiopathic arthritis (JIA). METHODS: MicroRNA expression in pooled JIA plasma and SF was assessed by absolute quantitative droplet digital PCR array. The results were validated in individual patient samples. MicroRNA content in leukocytes and extracellular vesicles was evaluated by real-time PCR in JIA blood and SF. Blood microRNA expression was compared with healthy controls (HCs). Principal component analysis was used to profile JIA plasma and SF microRNAs, and the potential biological consequences of microRNA dysregulation were investigated by pathway analysis. RESULTS: MiR-15a-5p and miR-409-3p levels were higher in JIA plasma than in HC plasma. JIA SF contained elevated levels of miR-21-5p, miR-27a-3p, miR-146b-5p, miR-155-5p, and miR-423-5p, and decreased miR-192-5p and miR-451a, compared to JIA plasma. Extracellular vesicle analysis demonstrated variable encapsulation among selected microRNAs, with only miR-155-5p being represented substantially in extracellular vesicles. SF leukocytes also had higher expression of miR-21-5p, miR-27a-3p, miR-146b-5p, and miR-155-5p, and lower expression of miR-409-3p and miR-451a, relative to blood. No differences were observed between JIA and HC blood leukocytes. Clusters of microRNAs were commonly altered in JIA joint fluid and leukocytes compared to JIA blood samples. In silico analysis predicted that differentially expressed microRNAs in JIA target the transforming growth factor (TGF)-ß pathway. CONCLUSION: The expression of multiple microRNAs is dysregulated in JIA both locally and systemically, which may inhibit the TGF-ß pathway. These findings advance our knowledge of JIA immunopathogenesis and may lead to the development of targeted therapies.

Prolidase deficiency, a rare inborn error of immunity, clinical phenotypes, immunological features, and proposed treatments in twins.

BACKGROUND: Prolidase deficiency (PD) is an autosomal recessive inborn multisystemic disease caused by mutations in the PEPD gene encoding the enzyme prolidase D, leading to defects in turnover of proline-containing proteins, such as collagen. PD is categorized as a metabolic disease, but also as an inborn error of immunity. PD presents with a range of findings including dysmorphic features, intellectual disabilities, recurrent infections, intractable skin ulceration, autoimmunity, and splenomegaly. Despite symptoms of immune dysregulation, only very limited immunologic assessments have been reported and standard therapies for PD have not been described. We report twin females with PD, including comprehensive immunologic profiles and treatment modalities used. CASE PRESENTATION: Patient 1 had recurrent infections in childhood. At age 13, she presented with telangiectasia, followed by painful, refractory skin ulcerations on her lower limbs, where skin biopsy excluded vasculitis. She had typical dysmorphic features of PD. Next-generation sequencing revealed pathogenic compound heterozygous mutations (premature stop codons) in the PEPD gene. Patient 2 had the same mutations, typical PD facial features, atopy, and telangiectasias, but no skin ulceration. Both patients had imidodipeptiduria. Lymphocyte subset analysis revealed low-normal frequency of Treg cells and decreased frequency of expression of the checkpoint molecule CTLA-4 in CD4+ TEM cells. Analysis of Th1, Th2, and Th17 profiles revealed increased inflammatory IL-17+ CD8+ TEM cells in both patients and overexpression of the activation marker HLA-DR on CD4+ TEM cells, reflecting a highly activated proinflammatory state. Neither PD patient had specific antibody deficiencies despite low CD4+CXCR5+ Tfh cells and low class-switched memory B cells. Plasma IL-18 levels were exceptionally high. CONCLUSIONS: Immunologic abnormalities including skewed frequencies of activated inflammatory CD4+ and CD8+ TEM cells, decreased CTLA-4 expression, and defects in memory B cells may be a feature of immune dysregulation associated with PD; however, a larger sample size is required to validate these findings. The high IL-18 plasma levels suggest underlying autoinflammatory processes.

High Dose Intravenous IgG Therapy Modulates Multiple NK Cell and T Cell Functions in Patients With Immune Dysregulation.

Intravenous immunoglobulin (IVIG) is an effective immunomodulatory treatment for immune dysregulation diseases. However, the mechanisms by which it reduces systemic inflammation are not well understood. NK cell cytotoxicity is decreased by IVIG in women with reduced fertility, but IVIG effects on NK cells in immune dysregulation are less clear. We hypothesized that IVIG modulation of lymphocyte function, especially in NK cells, is important for resolution of inflammation. Our aim was to identify IVIG-induced changes in a cohort of patients with Kawasaki disease (KD) and those that occur broadly in pediatric patients with various immune dysregulatory diseases. Peripheral blood mononuclear cells (PBMCs) of patients with KD or autoimmune/inflammatory diseases were phenotyped pre and post high dose IVIG treatment by flow cytometry. In KD patients, after IVIG infusion Treg cell frequency and the proportion of activated CD25+ immunoregulatory CD56bright NK cells was increased, and multiple lymphocyte subsets showed increased expression of the lymphoid tissue homing receptor CD62L. Importantly, IVIG treatment decreased the frequency of cells expressing the degranulation marker CD107a among cytotoxic CD56dim NK cells, which was reflected in a significant reduction in target cell killing and in decreased production of multiple pro-inflammatory mediators. Interestingly, the activating receptor CD336 was expressed on a higher proportion of CD56bright NK cells after IVIG in both KD and autoimmune/inflammatory patients while other NK receptors were increased differentially in each cohort. In autoimmune/inflammatory patients IVIG induced the proliferation marker CD71 on a higher percentage of CD56dim NK cells, and in contrast to KD patients, CD107a+ cells were increased in this subset. Furthermore, when PBMCs were stimulated ex vivo with IL-2 or Candida antigen in autologous plasma, more of the CD4+ T cells of KD patients expressed CD25 after IVIG therapy but fewer cytotoxic T cells were degranulated based on CD107a expression. In summary, IVIG treatment in patients with immune dysregulation has multiple effects, especially on NK cell subsets and CD4+ T cells, which are compatible with promoting resolution of inflammation. These novel findings provide insight into the immunomodulatory actions of IVIG in autoimmune and inflammatory conditions.

Association of a Type 2-Polarized T Cell Phenotype With Methotrexate Nonresponse in Patients With Rheumatoid Arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is a chronic inflammatory disease mediated through complex immunologic pathways. Among RA patients receiving low-dose methotrexate (MTX) monotherapy, approximately one-half exhibit a meaningful clinical response within the first 6 months of starting treatment. Whether baseline immune phenotypes differ between subsequent MTX responders and nonresponders is unknown. This study utilized comprehensive T cell immunophenotyping to identify specific immunologic pathways associated with MTX-nonresponsive joint inflammation in patients with RA. METHODS: In total, 32 patients with recent-onset RA were treated with MTX therapy. After 6 months, 15 patients were categorized as responders and 17 as nonresponders. Comprehensive blood T cell immunophenotyping, using multiparameter immunofluorescence flow cytometry analyses, was performed at baseline and following 6 months of treatment. RESULTS: Baseline measures of disease activity (Disease Activity Score in 28 joints [DAS28], C-reactive protein level, and erythrocyte sedimentation rate) did not differ between MTX responders and nonresponders following MTX treatment. Frequencies of CD4+ and CD8+ T cells were skewed to favor higher CD4:CD8 T cell ratios in MTX responders compared to nonresponders (P < 0.05). The proportion of inducible costimulator-expressing Treg cells was significantly greater among MTX nonresponders. Interleukin-13 (IL-13)-producing, but not interferon-γ- or IL-17-producing, CD4+ effector memory T (Tem) cells were significantly more frequent in MTX nonresponders (P < 0.05). The ratio of IL-13+:IL-17+ Tem cells among CD4+ Tem cells was 1.9-fold higher in MTX nonresponders compared to responders (P < 0.05). Both the CD4:CD8 T cell ratio and the frequency of IL-13+CD4+ Tem cells correlated with changes in the DAS28 score following MTX treatment, whereas T cell expression of immune checkpoint inhibitor markers (CTLA-4, programmed death 1, and T cell immunoglobulin and mucin domain-containing protein 3) did not differ between MTX responders and nonresponders. CONCLUSION: We observed a bias toward type 2-polarized T cell inflammatory responses in the peripheral blood of MTX-nonresponsive RA patients. Targeting the IL-13+CD4+ T cell pathway could be a new therapeutic strategy in RA patients whose disease remains resistant to MTX.

RNA sensors enable human mast cell anti-viral chemokine production and IFN-mediated protection in response to antibody-enhanced dengue virus infection.

Dengue hemorrhagic fever and/or dengue shock syndrome represent the most serious pathophysiological manifestations of human dengue virus infection. Despite intensive research, the mechanisms and important cellular players that contribute to dengue disease are unclear. Mast cells are tissue-resident innate immune cells that play a sentinel cell role in host protection against infectious agents via pathogen-recognition receptors by producing potent mediators that modulate inflammation, cell recruitment and normal vascular homeostasis. Most importantly, mast cells are susceptible to antibody-enhanced dengue virus infection and respond with selective cytokine and chemokine responses. In order to obtain a global view of dengue virus-induced gene regulation in mast cells, primary human cord blood-derived mast cells (CBMCs) and the KU812 and HMC-1 mast cell lines were infected with dengue virus in the presence of dengue-immune sera and their responses were evaluated at the mRNA and protein levels. Mast cells responded to antibody-enhanced dengue virus infection or polyinosinicpolycytidylic acid treatment with the production of type I interferons and the rapid and potent production of chemokines including CCL4, CCL5 and CXCL10. Multiple interferon-stimulated genes were also upregulated as well as mRNA and protein for the RNA sensors PKR, RIG-I and MDA5. Dengue virus-induced chemokine production by KU812 cells was significantly modulated by siRNA knockdown of RIG-I and PKR, in a negative and positive manner, respectively. Pretreatment of fresh KU812 cells with supernatants from dengue virus-infected mast cells provided protection from subsequent infection with dengue virus in a type I interferon-dependent manner. These findings support a role for tissue-resident mast cells in the early detection of antibody-enhanced dengue virus infection via RNA sensors, the protection of neighbouring cells through interferon production and the potential recruitment of leukocytes via chemokine production.

Virus stimulation of human mast cells results in the recruitment of CD56⁺ T cells by a mechanism dependent on CCR5 ligands.

The trafficking of effector cells to sites of infection is crucial for antiviral responses. However, the mechanisms of recruitment of the interferon-γ-producing and cytotoxic CD56(+) T cells are poorly understood. Human mast cells are sentinel cells found in the skin and airway and produce selected proinflammatory mediators in response to multiple pathogen-associated signals. The role of human mast cell-derived chemokines in T-cell recruitment to virus infection was examined. Supernatants from primary human cord blood-derived mast cells (CBMCs) infected with mammalian reovirus were examined for chemokine production and utilized in chemotaxis assays. Virus-infected CBMCs produced several chemokines, including CCL3, CCL4, and CCL5. Supernatants from reovirus-infected CBMCs selectively induced the chemotaxis of CD8(+) T cells (10±1%) and CD3(+)CD56(+) T cells (19±5%). CD56(+) T-cell migration was inhibited by pertussis toxin (65±9%) and met-RANTES (56±7%), a CCR1/CCR5 antagonist. CD56(+) T cells expressed CCR5, but little CCR1. The depletion of CCL3, CCL4, and CCL5 from reovirus-infected CBMC supernatants significantly (41±10%) inhibited CD56(+) T-cell chemotaxis. This study demonstrates a novel role for mast cells and CCR5 in CD56(+) T-cell trafficking and suggests that human mast cells enhance immunity to viruses through the selective recruitment of cytotoxic effector cells to virus infection sites. These findings could be exploited to enhance local T-cell responses in chronic viral infection and malignancies at mast cell-rich sites.

The effect of bacterial, viral and fungal infection on mast cell reactivity in the allergic setting.

Mast cells are well known for their role in allergic inflammation where, upon aggregation of the high-affinity immunoglobulin E receptor, they release mediators such as histamine that cause classical allergic symptoms. Mast cells are located in almost all tissues and are especially numerous in organs that interface with the environment. Given this strategic location and the more recent notion that they are endowed with receptors that recognize endogenous and exogenous danger signals such as pathogens, it is not surprising that they function as important cells in immune surveillance. When mast cells are activated by pathogens they modulate innate and adaptive immune responses. In allergy, infections might cause exacerbation of the allergic reaction by affecting the reactivity of mast cells. With new developments within the field of mast cell biology, we will better understand how mast cells execute their effector functions. This knowledge will also help to improve the management of allergic diseases.

Enhancement of mast cell IL-6 production by combined toll-like and nucleotide-binding oligomerization domain-like receptor activation.

BACKGROUND: Mast cells respond to bacterial infection by producing mediators that recruit and activate leukocytes, mediate vasodilation and induce bronchoconstriction. These mast cell-driven responses play a crucial role in protective immunity against bacterial infection, but may contribute to bacterial exacerbation of allergic diseases. Bacterial components including peptidoglycan (PGN) and lipopeptides are known to activate receptors such as Toll-like receptors (TLR) and nucleotide-binding oligomerization domain-like receptors (NLR). Since the consequences of mast cell activation by individual or combinations of bacterial components have not been fully characterized, we determined the effects of TLR2 and NLR activation, alone or in combination, on human mast cell mediator production. METHODS: Cord blood-derived human mast cells were activated by bacterial PGN, the lipopeptide Pam(3)CSK(4) and NLR agonists alone or in combination. Mast cell degranulation, LTC(4) production and the production of cytokines were assessed. RESULTS: PGN and the lipopeptide Pam(3)CSK(4) induced human mast cells to produce the pro-inflammatory mediators IL-1ß, IL-6, CXCL8 and LTC(4) in addition to anti-inflammatory IL-10. NLR agonists alone did not induce these responses, but significantly and selectively increased Pam(3)CSK(4)-mediated mast cell IL-6 production. PGN- and Pam(3)CSK(4)-induced mast cell IL-6, but not IL-1ß, production was dependent on adenylyl cyclase activity and could be partially inhibited by the cyclooxygenase inhibitor naproxen. CONCLUSIONS: Increased mast cell IL-6 production in response to combined TLR2 and NLR activation could play a role in the protection against bacterial infection, but potentially exacerbate inflammation-dependent conditions. In addition, mast cell IL-6 production is dependent on adenylyl cyclase activity.