Innate immunity and its control in the pathogenesis of inflammatory rheumatic diseases

The pathogenesis of inflammatory rheumatic diseases, including rheumatoid arthritis (RA) and spondyloarthropathies (SpAs) such as reactive arthritis (ReA), is incompletely understood. ReA is a sterile joint inflammation, which may follow a distal infection caused by Gram-negative bacteria that have lipopolysaccharide (LPS) in their outer membrane. The functions of innate immunity that may affect the pathogenesis, prognosis and treatment of these diseases were studied in this thesis. When compared with healthy controls, whole blood monocytes of healthy subjects with previous ReA showed enhanced capacity to produce TNF, an essential proinflammatory cytokine, in response to adherent conditions (mimicking vascular endothelium made adherent by inflammatory signals) and non-specific protein kinase C stimulation. Also, blood neutrophils of these subjects showed high levels of CD11b, an important adhesion molecule, in response to adherence or LPS. Thus, high responsiveness of monocytes and neutrophils when encountering inflammatory stimuli may play a role in the pathogenesis of ReA. The results also suggested that the known risk allele for SpAs, HLA-B27, may be an additive contributor to the observed differences. The promoter polymorphisms TNF 308A and CD14 (gene for an LPS receptor component) 159T were found not to increase the risk of acute arthritis. However, all female patients who developed chronic SpA had 159T and none of them had 308A, possibly reflecting an interplay between hormonal and inflammatory signals in the development of chronic SpA. Among subjects with early RA, those having the polymorphic TLR4 +896G allele (causing the Asp299Gly change in TLR4, another component of LPS receptor) required a combination of disease-modifying antirheumatic drugs to achieve remission. It is known that rapid treatment response is essential in order to maintain the patients work ability. Hence, +896G might be a candidate marker for identifying the patients who need combination treatment. The production of vascular endothelial growth factor (VEGF), which strongly promotes vascular permeability and angiogenesis that takes place e.g. early in rheumatic joints, was induced by LPS and inhibited by interferon (IFN)-alpha in peripheral blood mononuclear cells. These long-living cells might provide a source of VEGF when stimulated by LPS and migrating to inflamed joints, and the effect of IFN-alpha may contribute to the clinical efficacy of this cytokine in inhibiting joint inflammation.Luonnollinen immuunijärjestelmä käsittää kehon epäspesifiset vieraan materiaalin torjuntakeinot, jotka käynnistyvät nopeasti ennen spesifisen opitun immuniteetin aktivaatiota. Yksi tärkeimmistä luonnollisen immuniteetin toiminnoista on veren syöjäsolujen tarttuminen (adheesio) tulehduksen esiintuomiin molekyyleihin verisuonten seinämissä ja siirtyminen kudokseen hävittämään tulehduksen aiheuttajaa. Luonnollisen immuniteetin solujen keskeisiä reseptoreja ovat adheesiomolekyyli CD11b/CD18 sekä CD14/TLR4/MD-2, joka sitoo Gram-negatiivisten bakteerien LPS-rakennetta käynnistäen solussa mm. tulehdusvälittäjäaine TNF:n tuotannon. Tulehduksellisten reumasairauksien kuten reaktiivisen artriitin (ReA:n) ja nivelreuman (RA:n) patogeneesi tunnetaan puutteellisesti. ReA on spondyloartropatia (SpA) -tautiryhmään kuuluva niveltulehdus, jota edeltää nivelen ulkopuolinen, tyypillisesti Gram-negatiivisen bakteerin aiheuttama infektio. Tutkimuksessa havaittiin, että ReA:n sairastaneiden koehenkilöiden veren syöjäsolujen reaktio adheesioon (TNF-tuotannon käynnistyminen ja CD11b-tason nousu) tai LPS:ään (CD11b-tason nousu) oli voimakkaampi kuin ReA:a sairastamattomien. Täten syöjäsolujen reaktiivisuus voi liittyä ReA:n patogeneesiin. Luonnollisen immuniteetin geenien säätelyalueiden polymorfismit kuten CD14 -159C/T ja TNF -308G/A voivat vaikuttaa geenien luennan aktiivisuuteen ja tulehdusvasteeseen. Tutkimuksessa kaikilla naispotilailla, joilla SpA kroonistui, oli -159T eikä heistä kellään ollut -308A:ta, mikä voi kertoa hormoni- ja tulehdussignaalien yhteisvaikutuksesta SpA:ssa. Keskeistä RA-potilaan työkyvyn säilyttämiseksi on saada nopeasti hyvä hoitovaste. Tulosten perusteella TLR4 +896A/G -polymorfismi voi olla hyödyllinen hoitostrategian suunnittelussa: potilaat, joilla oli G-alleeli, eivät saavuttaneet remissiota yhdellä antireumaattisella lääkkeellä vaan tarvitsivat lääkeyhdistelmää. VEGF on kasvutekijä, joka lisää sekä verisuonten läpäisevyyttä että niiden kasvua, jota tapahtuu esim. reumaattisessa nivelessä. LPS:n havaittiin edistävän veren valkosolujen VEGF-tuottoa, mitä taas alfa-interferoni esti. Täten LPS:n stimuloima VEGF-tuotanto kudokseen siirtyvissä valkosoluissa voi olla turvotusta ja verisuonten kasvua edistävä mekanismi, ja tämän esto voi osin selittää alfa-interferonin tehoa esim. syövän kasvun ja nivelten tulehtuneisuuden vähentämisessä. Tutkimus edustaa lääketieteellistä perustutkimusta, jonka merkittäviä sovelluksia ovat tulehduksellisten reumasairauksien diagnostiikan, ennusteen ja hoidon kehittäminen

STAT6 and STAT1 Pathway Activation in Circulating Lymphocytes and Monocytes as Predictor of Treatment Response in Rheumatoid Arthritis

Objective To find novel predictors of treatment response to disease-modifying antirheumatic drugs (DMARDs), we studied activation of STAT (signal transducers and activators of transcription) 6 and 1 in circulating leukocytes of patients with rheumatoid arthritis (RA). Methods 19 patients with untreated recent-onset RA, 16 patients with chronic RA irresponsive to synthetic DMARDs and 37 healthy volunteers provided blood samples for whole blood flow cytometric determination of intracellular STAT6 and STAT1 phosphorylation, expressed as relative fluorescence units, in response to IL-4 and IFN-gamma, respectively. Phosphorylation was restudied and treatment response (according to European League Against Rheumatism) determined after 1-year treatment with synthetic DMARDs in recent-onset RA and with biological DMARD in synthetic DMARD-irresponsive RA. Estimation-based exact logistic regression was used to investigate relation of baseline variables to treatment response. 95% confidence intervals of means were estimated by bias-corrected bootstrapping and the significance between baseline and follow-up values was calculated by permutation test. Results At baseline, levels of phosphorylated STAT6 (pSTAT6) induced by IL-4 in monocytes were higher in those who achieved good treatment response to synthetic DMARDs than in those who did not among patients with untreated RA (OR 2.74, 95% CI 1.05 to 9.47), and IFN-gamma-stimulated lymphocyte pSTAT1 levels were higher in those who achieved good treatment response to a biological drug than in those who did not among patients with chronic RA (OR 3.91, 95% CI 1.12 to 20.68). During follow-up, in recent-onset RA patients with good treatment response to synthetic DMARDS, the lymphocyte pSTAT6 levels decreased (p = 0.011), and, consequently, the ratio of pSTAT1/pSTAT6 in lymphocytes increased (p = 0.042). Conclusion Cytokine-stimulated STAT6 and STAT1 phosphorylation in circulating leukocytes was associated with treatment response to DMARDs in this pilot study. The result, if confirmed in larger studies, may aid in developing personalized medicine in RA.Peer reviewe

Pancreatic cancer is associated with aberrant monocyte function and successive differentiation into macrophages with inferior anti-tumour characteristics

Background/objectives: Inflammation is related to the development and progression of pancreatic cancer (PC). Locally, anti-inflammatory macrophages (M2), and systemically, high levels of certain inflammation-modulating cytokines associate with poor prognosis in PC. The detailed effects of systemic inflammation on circulating monocytes and macrophage polarisation remain unknown. We aimed to find out how intracellular signalling of peripheral blood monocytes is affected by the systemic inflammatory state in PC patients and how it affects their differentiation into macrophages. Methods: Monocytes were isolated from 50 consenting PC patients and 20 healthy controls (HC). The phosphorylation status of the signalling molecules was assessed by flow cytometry both from unstimulated and appropriately stimulated monocytes. Monocytes derived from HC and PC patients were cocultured with cancer cells (MIA PaCa-2 and HPAF-II) in media supplemented with autologous serum, and the CD marker expression of the obtained macrophages was assessed by flow cytometry. Results: Phosphorylation levels of unstimulated STAT2, STAT3 and STAT6 were higher (p <0.05) and those of stimulated NF-kB (p = 0.004) and STAT5 (p = 0.006) were lower in patients than in controls. The expression of CD86, a proinflammatory (Ml) marker, was higher in control- than patient-derived cocultured macrophages (p = 0.029). Conclusions: Circulating monocytes from PC patients showed constitutive phosphorylation and weaker response to stimuli, indicating aberrant activation and immune suppression. When co-culturing the patient-derived monocytes with cancer cells, they differentiated into macrophages with reduced levels of M1 macrophage marker CD86, suggesting compromised anti-tumour features. The results highlight the need for global management of tumour-associated immune aberrations in PC treatment. (C) 2021 Published by Elsevier B.V. on behalf of IAP and EPC.Peer reviewe

Tofacitinib treatment modulates the levels of several inflammation-related plasma proteins in rheumatoid arthritis and baseline levels of soluble biomarkers associate with the treatment response

Sixteen patients with active rheumatoid arthritis, despite conventional synthetic disease-modifying antirheumatic drugs, started additional tofacitinib treatment. Blood samples were drawn at baseline and at 3-month follow-up visits, and plasma levels of 92 inflammation-related proteins were measured with proximity extension assay. Levels of 21 proteins changed substantially (>20%) and statistically significantly upon tofacitinib treatment, and the baseline levels of interleukin-6, C-C motif chemokine 11 and Delta and Notch-like epidermal growth factor-related receptor associated with the treatment response. The data on the effects of tofacitinib on soluble proteins in patients with rheumatoid arthritis (RA) is currently very limited. We analyzed how tofacitinib treatment and thus inhibition of the Janus kinase-signal transducer and activation of transcription pathway affects the in vivo levels of inflammation-related plasma proteins in RA patients. In this study, 16 patients with active RA [28-joint disease activity score (DAS28) >3.2] despite treatment with conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) started tofacitinib treatment 5 mg twice daily. Levels of 92 inflammation-related plasma proteins were determined by proximity extension assay at baseline and at 3 months. Tofacitinib treatment for 3 months, in csDMARD background, decreased the mean DAS28 from 4.4 to 2.6 (P 20%) and statistically significant (P 50%) decrease was observed for interleukin-6 (IL-6), C-X-C motif chemokine ligand 1, matrix metalloproteinase-1, and AXIN1. Higher baseline levels of IL-6 and lower levels of C-C motif chemokine 11 and Delta and Notch-like epidermal growth factor-related receptors were associated with DAS28 improvement. Our results indicate that tofacitinib downregulates several proinflammatory plasma proteins that may contribute to the clinical efficacy of tofacitinib. In addition, soluble biomarkers may predict the treatment response to tofacitinib.Peer reviewe

Nekroptoosi infektioissa ja tulehdussairauksissa - solut viimeisellä portilla

Vertaisarvioitu. English summaryOhjelmoitunutta solukuolemaa tarvitaan elinten kehittymisessä, kudosten homeostaasin ja uusiutumiskyvyn ylläpidossa sekä puolustussolujen toiminnan säätelyssä. Sen ainoana muotona pidettiin pitkään kaspaasientsyymien säätelemää, tulehdusta lietsomatonta apoptoosia. Nykyään tiedetään, ettei tulehdus- ja soluvauriosignaalien laukaisema nekroosikaan ole vain kontrolloimaton solutuho, vaan siitä on aktivoitumiskykyisiä geneettisesti ohjelmoituneita muotoja, joista tunnetuin on nekroptoosi eli tulehduksellisesti ohjelmoitunut nekroottinen solukuolema. Keskeiset solunsisäisessä nekroptoosisignaloinnissa toimivat proteiinit ovat RIP (receptor-interacting protein kinase) 1, RIP3 ja MLKL (mixed lineage kinase domain-like protein). Niiden aktivoituessa, esimerkiksi virus- (muun muassa koronavirus) tai bakteeri-infektion yhteydessä, nekroptoottisesta solusta vapautuu vaarasignaalimolekyylejä. Nekroptoosi aiheuttaa tulehdusreaktion, jolla voi kudostuhon ohella olla regeneratiivisia vaikutuksia. Nekroptoositutkimus on avannut uusia näkymiä immuunipuolustuksen keinoihin ja tulehduksellisten sairauksien patogeneesiin sekä mahdollisuuksiin vaikuttaa näihin.Peer reviewe

Constitutive STAT3 Phosphorylation in Circulating CD4(+) T Lymphocytes Associates with Disease Activity and Treatment Response in Recent-Onset Rheumatoid Arthritis

The aim of the present study was to examine constitutive signal transducer and activator of transcription 3 (STAT3) phosphorylation in circulating leukocytes as a candidate biomarker in rheumatoid arthritis (RA). 25 patients with recent-onset, untreated RA provided samples for whole blood flow cytometric determination of intracellular STAT3 phosphorylation, expressed as relative fluorescence units. The occurrence of constitutive STAT3 phosphorylation was evaluated by determining proportion of STAT3-phosphorylated cells among different leukocyte subtypes. Plasma levels of interleukin (IL)-6, IL-17 and IL-21 were measured by immunoassay, radiographs of hands and feet were examined and disease activity score (DAS28) was determined. Biomarkers were restudied and treatment response (according to European League Against Rheumatism) was determined after 12 months of treatment with disease-modifying antirheumatic drugs. At baseline, constitutive phosphorylation of STAT3 occurred in CD4(+) T cells of 14 (56%) patients, CD8(+) T cells of 13 (52%) patients, in CD19+ B cells of 7 (28%) patients, and in CD14(+) monocytes of 12 (48%) patients. STAT3 phosphorylation levels of CD4(+) T cells associated with DAS28, and those of all leukocyte subtypes studied associated with erosive disease. The presence of constitutive STAT3 phosphorylation in CD4(+) T lymphocytes, pSTAT3 fluorescence intensity of CD4(+) and CD8(+) T cells and C-reactive protein (CRP) levels at baseline associated with good treatment response. In conclusion, constitutive STAT3 phosphorylation in circulating CD4(+) T cells is common in recent-onset untreated RA and associates with good treatment response in patients characterized by high disease activity and the presence of systemic inflammation.Peer reviewe

Association of Matrix Metalloproteinases-7,-8 and-9 and TIMP-1 with Disease Severity in Acute Pancreatitis. A Cohort Study

Objectives Several biomarkers for early detection of severe acute pancreatitis (SAP) have been presented. Matrix metalloproteinases (MMP) and their tissue inhibitors (TIMP) are released early in inflammation. We aimed to assess levels of MMP-7, -8, -9 and TIMP-1 in acute pancreatitis (AP) and explore their ability to detect disease severity. Our second aim was to find an association between MMPs, TIMP and creatinine. Methods We collected plasma samples for MMP-7, -8, -9 and TIMP-1 analyses from 176 patients presenting within 96 h from onset of acute pancreatitis (AP) symptoms. We used samples from 32 control subjects as comparison. The revised Atlanta Classification was utilised to assess severity of disease. Receiver operating characteristic curve analysis and Spearman's Rho-test were utilised for statistical calculations. Results Compared with controls, patients showed higher levels of all studied markers. MMP-8 was higher in moderately severe AP than in mild AP (p = 0.005) and MMP-8, -9 and TIMP-1 were higher in severe than in mild AP (pPeer reviewe

Baseline JAK phosphorylation profile of peripheral blood leukocytes, studied by whole blood phosphospecific flow cytometry, is associated with 1-year treatment response in early rheumatoid arthritis

Background: We found recently that baseline signal transducer and activator of transcription 3 phosphorylation in peripheral blood CD4(+) T cells of patients with early rheumatoid arthritis (RA) is associated with treatment response to synthetic disease-modifying antirheumatic drugs (DMARDs). This prompted us to study the baseline phosphorylation profiles of Janus kinases (JAKs) in blood leukocytes with respect to treatment response in early RA. Methods: Thirty-five DMARD-naive patients with early RA provided blood samples for whole blood flow cytometric determination of phosphorylation of JAKs in CD4(+) and CD8(+) T cells, CD19(+) B cells, and CD14(+) monocytes. Treatment response was determined after 1 year of treatment with synthetic DMARDs, with remission defined as absence of tender and swollen joints and normal erythrocyte sedimentation rate. Exact logistic regression was used to investigate the association of baseline variables with treatment response. Ninety-five percent CIs of means were estimated by bias-corrected bootstrapping. Results: High JAK3 phosphorylation in CD4(+) and CD8(+) T cells, CD19(+) B cells, and CD14(+) monocytes and low JAK2 phosphorylation in CD14(+) monocytes were significantly associated with remission following treatment with synthetic DMARDs. Conclusions: Baseline JAK phosphorylation profile in peripheral blood leukocytes may provide a means to predict treatment response achieved by synthetic DMARDs among patients with early RA.Peer reviewe

Blood Leukocyte Signaling Pathways as Predictors of Severity of Acute Pancreatitis

Objectives Clinical practice lacks biomarkers to predict the severity of acute pancreatitis (AP). We studied if intracellular signaling of circulating leukocytes could predict persistent organ dysfunction (OD) and secondary infections in AP. Methods A venous blood sample was taken from 174 patients with AP 72 hours or less from onset of symptoms and 31 healthy controls. Phosphorylation levels (p) of appropriately stimulated signal transducer and activator of transcription 1 (STAT1), STAT6, nuclear factor-kappa B (NF-kappa B), Akt, and nonstimulated STAT3 in monocytes, neutrophils, and lymphocytes was measured using phosphospecific flow cytometry. Results The patients showed higher pSTAT3 and lower pSTAT1, pSTAT6, pNF-kappa B, and pAkt than healthy controls. pSTAT3 in all leukocyte subtypes studied increased, and pSTAT1 in monocytes and T cells decreased in an AP severity-wise manner. In patients without OD at sampling, high pSTAT3 in monocytes and T lymphocytes were associated with development of persistent OD. In patients with OD, low interleukin-4-stimulated pSTAT6 in monocytes and neutrophils and Escherichia coli-stimulated pNF-kappa B in neutrophils predicted OD persistence. High pSTAT3 in monocytes, CD8(+) T cells, and neutrophils; low pSTAT1 in monocytes and T cells; and low pNF-kappa B in lymphocytes predicted secondary infections. Conclusions Leukocyte STAT3, STAT1, STAT6, and NF-kappa Beta phosphorylations are potential predictors of AP severity.Peer reviewe