Simultaneous Assessment of mTORC1, JAK/STAT, and NLRP3 Inflammasome Activation Pathways in Patients with Sarcoidosis.

The unknown etiology of sarcoidosis, along with the variability in organ involvement and disease course, complicates the effective treatment of this disease. Based on recent studies, the cellular inflammatory pathways involved in granuloma formation are of interest regarding possible new treatment options, such as the mechanistic (formerly mammalian) target of rapamycin complex 1 (mTORC1) pathway, the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, and the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome pathway. The aim of this study was to explore the potential coexpression of these three inflammatory pathways in patients with sarcoidosis and see whether possible differences were related to disease outcome. The tissue of 60 patients with sarcoidosis was used to determine the activity of these three signaling pathways using immunohistochemistry. The activation of NLRP3 was present in 85% of all patients, and the activation of mTORC1 and JAK/STAT was present in 49% and 50% of patients, respectively. Furthermore, the presence of NLRP3 activation at diagnosis was associated with a chronic disease course of sarcoidosis. Our finding of different new conceptual inflammatory tissue phenotypes in sarcoidosis could possibly guide future treatment studies using the available inhibitors of either NLRP3, JAK-STAT, and mTORC1 inhibitors in a more personalized medicine approach.

Vedolizumab Induces Endoscopic and Histologic Remission in Patients With Crohn's Disease.

BACKGROUND & AIMS: We evaluated the ability of vedolizumab to induce endoscopic and histologic remission in patients with Crohn's disease (CD). METHODS: We performed a prospective study of 110 patients with active CD, based on CD activity index (CDAI) scores >220 and mucosal ulcerations, who received open-label vedolizumab (300 mg) infusions at weeks 0, 2, and 6, and every 8 weeks thereafter through week 52 at tertiary centers in Europe. Patients received an additional infusion at week 10 if their CDAI score had not decreased by 70 points. Patients underwent ileocolonoscopy with collection of biopsies at baseline and weeks 26 and 52; a local and central reader determined simple endoscopic index for CD (SES-CD) scores. Histologic features were assessed by a blinded pathologist at week 26. Serum concentrations of vedolizumab were measured at serial time points. The primary outcome was endoscopic and histologic remission in patients with active CD treated with vedolizumab for 52 weeks. RESULTS: At weeks 26 and 52, 36 patients (29%) and 34 patients (31%), respectively, were in corticosteroid-free clinical remission (CDAI score <150), respectively. Based on intent-to-treat analysis, endoscopic remission (SES-CD score <4) was achieved by 36 patients (33%) and 40 patients (36%) at weeks 26 and 52. Endoscopic responses (decrease in SES-CD score ≥50%) occurred in 44 patients (40%) at week 26 and 5 patients (45%) at week 52. Serum concentrations of vedolizumab were higher at weeks 2, 10, and 22 in patients with lower SES-CD scores. Histologic remission at week 26 was observed in 43 (64%) of 67 patients based on Geboes Score and 37 (66%) of 56 patients based on Robarts Histopathology Index scores in analyses of paired biopsies with inflammation at baseline. Serum concentrations of vedolizumab above 10 mg/L at week 22 were associated with endoscopic remission at week 26. CONCLUSIONS: In a prospective trial, we found that approximately one-third of patients with CD achieve endoscopic remission after 52 weeks of treatment with vedolizumab and two-thirds achieve histologic remission at week 26. Higher serum concentrations of vedolizumab were associated with better outcomes. EUDRACT no: 2014-005376-29.

Cutaneous Toxicity After Chemoradiotherapy and PD-L1 Inhibition in Two Patients with Esophageal Adenocarcinoma: More than Meets the Eye.

Dermatological adverse events have frequently been reported after immune checkpoint inhibition. When an adverse event occurs during combination of immune checkpoint inhibition with chemotherapy, the question arises which agent is responsible. Unnecessary withdrawal of either chemotherapy or immunotherapy could lead to suboptimal treatment outcomes. Here we report on two patients who developed a cutaneous drug reaction with fever during treatment with paclitaxel, carboplatin, radiotherapy, and PD-L1 inhibition (atezolizumab) for resectable esophageal adenocarcinoma. In the first case atezolizumab was suspected, and in the second paclitaxel. We discuss the clinical manifestation, treatment, and pathophysiology underlying both cases.

Histologic evidence that mast cells contribute to local tissue inflammation in peripheral spondyloarthritis by regulating interleukin-17A content.

OBJECTIVES: Synovial mast cells contain IL-17A, a key driver of tissue inflammation in SpA. A recent in vitro study showed that tissue-derived mast cells can capture and release exogenous IL-17A. The present study aimed to investigate if this mechanism could contribute to tissue inflammation in SpA. METHODS: Potential activation of mast cells by IL-17A was assessed by gene expression analysis of the Laboratory of Allergic Diseases 2 (LAD2) mast cell line. The presence of IL-17A-positive mast cells was assessed by immunohistochemistry in synovial tissue obtained before and after secukinumab treatment, as well as in skin and gut tissues from SpA-related conditions. RESULTS: IL-17A did not induce a pro-inflammatory response in human LAD2 mast cells according to the canonical IL-17A signalling pathway. In SpA synovial tissue, the percentage of IL-17A-positive mast cells increased upon treatment with secukinumab. IL-17A-positive mast cells were also readily detectable in non-inflamed barrier tissues such as skin and gut. In non-inflamed dermis and gut submucosa, IL-17A-positive mast cells are the most prevalent IL-17A-positive cells in situ. Compared with non-inflamed tissues, both total mast cells and IL-17A-positive mast cells were increased in psoriatic skin dermis and in submucosa from inflammatory bowel disease gut. In contrast, the proportion of IL-17A-positive mast cells was strikingly lower in the inflamed compared with non-inflamed gut lamina propria. CONCLUSION: IL-17A-positive mast cells are present across SpA target tissues and correlate inversely with inflammation, indicating that their IL-17A content can be regulated. Tissue-resident mast cells may act as IL-17A-loaded sentinel cells, which release IL-17A to amplify tissue inflammation.

ER stress abrogates the immunosuppressive effect of IL-10 on human macrophages through inhibition of STAT3 activation.

OBJECTIVE AND DESIGN: To determine whether ER stress affects the inhibitory pathways of the human immune system, particularly the immunosuppressive effect of IL-10 on macrophages. MATERIAL OR SUBJECTS: In vitro stimulation of human monocyte-derived macrophages. TREATMENT: Cells were stimulated with TLR ligands and IL-10, while ER stress was induced using thapsigargin or tunicamycin. METHODS: mRNA expression was determined using qPCR, while cytokine protein production was measured using ELISA. Protein expression of receptors and transcription factors was determined using flow cytometry. Student's t test was used for statistics. RESULTS: While under normal conditions IL-10 potently suppresses pro-inflammatory cytokine production by LPS-stimulated macrophages, we demonstrate that ER stress counteracts the immunosuppressive effects of IL-10, leading to increased pro-inflammatory cytokine production. We identified that ER stress directly interferes with IL-10R signaling by reducing STAT3 phosphorylation on Tyr705, which thereby inhibits the expression of SOCS3. Moreover, we show that ER stress also inhibits STAT3 activation induced by other receptors such as IL-6R. CONCLUSIONS: Combined, these data uncover a new general mechanism by which ER stress promotes inflammation. Considering its potential involvement in the pathogenesis of diseases such as Crohn's disease and spondyloarthritis, targeting of this mechanism may provide new opportunities to counteract inflammation.

New phenotyping questionnaire for diagnosing sarcoidosis-associated small fiber neuropathy.

Small fiber neuropathy is a common complication in patients with sarcoidosis and its prevalence is estimated at 40-86%. The underlying mechanism influences the presentation of small fiber neuropathy. For example, patients with metabolic diseases are often associated with a classic length-dependent small fiber neuropathy pattern, while patients with inflammatory diseases are more often present with a non-length-dependent small fiber neuropathy. Detailed phenotyping may be useful to improve diagnostic efficiency, as a clue to underlying mechanisms and as a precondition for personalized medicine. This study examined four phenotypes distinguishing between length-dependent and non-length-dependent presentation with a new subdivision for continuous and intermittent presentation. Forty-eight sarcoid patients with symptoms and at least two clinical signs of small fiber neuropathy and normal nerve conduction studies were classified as having probable small fiber neuropathy. A new small fiber neuropathy phenotyping questionnaire has been developed that allows patients to mark the anatomical locations of pain at three different levels: the skin, muscles, and joints. The location of symptoms was used to define length dependence, and two colors were used to distinguish continuous (red) from intermittent (blue) symptoms. In addition, skin biopsy, corneal confocal microscopy, Sudoscan and water immersion skin wrinkling were used to investigate a correlation between the four phenotypes, sensory function, nerve fiber density, and autonomic nerve function. Overall, 35% of patients with probable small fiber neuropathy showed length-dependent symptoms and 44% showed non-length-dependent symptoms while 21% suffered from non-neuropathic musculoskeletal pain. The distinction between intermittent and continuous symptoms showed significantly less continuous than intermittent non-length-dependent symptoms (odds ratio = 0.3, P = 0.01). Moreover, continuous length-dependent symptoms were the only phenotype that correlated with thermal threshold testing (R = 0.3; P = 0.02) and the small fiber neuropathy screening list (R = 0.3; P = 0.03). In addition, thermal threshold testing (TTT) also correlated with the small fiber neuropathy (SFN) screening list (R = 0.3; P = 0.03). Other diagnostic methods showed no correlation with any of the four defined phenotypes. A novel finding is that TTT is only associated with continuous length-dependent pain, suggesting that TTT could result in more false negatives in patients with other pain phenotypes. Determining the pathophysiologic mechanisms could help develop new diagnostic methods. If patients suspected of SFN show symptoms without a length-dependent continuous presentation, the diagnosis should focus less on the diagnostic methods used.

Efficacy and safety of adalimumab for the treatment of peripheral arthritis in spondyloarthritis patients without ankylosing spondylitis or psoriatic arthritis.

OBJECTIVES: To evaluate the efficacy and safety of adalimumab in patients with peripheral spondyloarthritis (SpA) not fulfilling the criteria for ankylosing spondylitis (AS) or psoriatic arthritis (PsA). METHODS: 40 patients with active peripheral SpA fulfilling the European Spondyloarthropathy Study Group or Amor criteria but not the criteria for AS or PsA were included in a randomised, double-blind, placebo-controlled clinical trial. Patients were treated 1 : 1 with adalimumab or placebo for 12 weeks, followed by an open label extension up to week 24. Safety and efficacy measurements were performed every 6 weeks, with the patient's global assessment of disease activity at week 12 as the primary endpoint. RESULTS: At week 12, the patient's and physician's global assessment of disease activity, swollen joint count, Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Ankylosing Spondylitis Disease Activity Score (ASDAS) and erythrocyte sedimentation rate improved significantly in the adalimumab group compared with the baseline values and compared with placebo. A similar improvement was seen upon adalimumab treatment from weeks 12 to 24 in the patients originally randomised to placebo, whereas the clinical response was maintained or even augmented at week 24 in the patients who received adalimumab from the start. ASDAS inactive disease and BASDAI50 responses were met in 42% of the adalimumab group versus 0%-5% in the placebo group at week 12 (p=0.001 and p=0.008, respectively), and were further increased at week 24. The number of adverse events was not different between the adalimumab and placebo groups. CONCLUSIONS: Adalimumab appears to be effective and well tolerated in SpA patients with peripheral arthritis, also in those patients not fulfilling the AS or PsA criteria.

Undifferentiated spondyloarthritis vs ankylosing spondylitis and psoriatic arthritis: a real-life prospective cohort study of clinical presentation and response to treatment.

OBJECTIVE: SpA is a phenotypically heterogeneous disease, with AS and PsA as its best studied subtypes. This study aimed to investigate whether, despite a different phenotypic presentation, patients with undifferentiated SpA (uSpA) have similar disease activity and response to treatment to those with AS and PsA. METHODS: 175 patients presenting at a dedicated SpA outpatient clinic were recruited in a real-life prospective cohort with follow-up every 3 months. Clinical characteristics, disease activity at presentation and response to treatment of uSpA were compared with AS and PsA. RESULTS: Twenty-three per cent (n = 40) of the patients were classified as uSpA. These patients were younger and tended to have a shorter disease duration than AS and PsA patients. uSpA patients exhibited a mixed axial (inflammatory back pain in 87.5%) and peripheral (peripheral arthritis in 62.5%) phenotype, with almost half of the patients having low-grade sacroiliitis on conventional X-ray. The overall disease activity in uSpA was similar to AS and higher than in PsA, also when analysing only anti-TNF naive patients. Initiation of TNF blockade significantly decreased disease activity in uSpA, with a similar amplitude to that in AS and PsA. CONCLUSION: uSpA is a frequent, severe and anti-TNF-responsive phenotypic subtype of SpA. In agreement with the new ASAS classification criteria for axial and peripheral SpA and emerging data on TNF blockade in non-radiographic axial SpA and peripheral uSpA, these data emphasize the need for early diagnosis and optimal treatment of not only AS and PsA but also other SpA subforms.

Angiopoietin-2 promotes inflammatory activation of human macrophages and is essential for murine experimental arthritis.

BACKGROUND: Angiopoietin (Ang)-1 and Ang-2, and their shared receptor Tie2, are expressed in rheumatoid arthritis (RA) synovial tissue, but the cellular targets of Ang signalling and the relative contributions of Ang-1 and Ang-2 to arthritis are poorly understood. OBJECTIVES: To determine the cellular targets of Ang signalling in RA synovial tissue, and the effects of Ang-2 neutralisation in murine collagen-induced arthritis (CIA). METHODS: RA and psoriatic arthritis (PsA) synovial biopsies were examined for expression of Tie2 and activated phospho (p)-Tie2 by quantitative immunohistochemistry and immunofluorescent double staining. Human monocyte and macrophage Tie2 expression was determined by flow cytometry and quantitative PCR. Regulation of macrophage intracellular signalling pathways and gene expression were examined by immunoblotting and ELISA. CIA was assessed in mice treated with saline, control antibody, prednisolone or neutralising anti-Ang-2 antibody. RESULTS: Expression of synovial Tie2 and p-Tie2 was similar in RA and PsA. Tie2 activation in RA patient synovial tissue was predominantly localised in synovial macrophages and was expressed by human macrophage. Ang-1 and Ang-2 stimulated activation of multiple intracellular signalling pathways, and cooperated with tumour necrosis factor to induce macrophage interleukin 6 and macrophage inflammatory protein 1α production. Ang-2 selectively suppressed macrophage thrombospondin-2 production. Ang-2 neutralisation significantly decreased disease severity, synovial inflammation, neo-vascularisation and joint destruction in established CIA. CONCLUSIONS: The authors identify synovial macrophages as primary targets of Ang signalling in RA, and demonstrate that Ang-2 promotes the pro-inflammatory activation of human macrophages. Ang-2 makes requisite contributions to pathology in CIA, indicating that targeting Ang-2 may be of therapeutic benefit in the treatment of RA.

Transmembrane TNF drives osteoproliferative joint inflammation reminiscent of human spondyloarthritis.

TNF plays a key role in immune-mediated inflammatory diseases including rheumatoid arthritis (RA) and spondyloarthritis (SpA). It remains incompletely understood how TNF can lead to different disease phenotypes such as destructive peripheral polysynovitis in RA versus axial and peripheral osteoproliferative inflammation in SpA. We observed a marked increase of transmembrane (tm) versus soluble (s) TNF in SpA versus RA together with a decrease in the enzymatic activity of ADAM17. In contrast with the destructive polysynovitis observed in classical TNF overexpression models, mice overexpressing tmTNF developed axial and peripheral joint disease with synovitis, enthesitis, and osteitis. Histological and radiological assessment evidenced marked endochondral new bone formation leading to joint ankylosis over time. SpA-like inflammation, but not osteoproliferation, was dependent on TNF-receptor I and mediated by stromal tmTNF overexpression. Collectively, these data indicate that TNF can drive distinct inflammatory pathologies. We propose that tmTNF is responsible for the key pathological features of SpA.

Altered cytokine expression by macrophages from HLA-B27-positive spondyloarthritis patients without evidence of endoplasmic reticulum stress.

OBJECTIVES: We investigated endoplasmic reticulum (ER) stress and cytokine expression in peripheral blood-derived macrophages and synovial tissue from HLA-B27+ SpA patients. METHODS: Macrophages from healthy donors, SpA and RA patients were polarized with IFN-γ or IL-10 and activated with lipopolysaccharide. Expression of ER stress markers (BiP, CHOP, ERdj4) and cytokines (IL-23, IL-12, TNF, IL-10) was measured by qRT-PCR. Expression of ER stress markers and cytokines in synovial tissue from SpA patients was evaluated by microarray analysis. RESULTS: Macrophages from HLA-B27+ SpA patients did not show elevated ER stress markers. However, the expression of IL-23 and IL-12 by peripheral blood-derived macrophages was higher in HLA-B27+ SpA in comparison with healthy donors. Synovial tissue from HLA-B27+ SpA patients showed higher expression of TNF compared with HLA-B27- SpA patients. CONCLUSION: HLA-B27+ SpA patients showed increased expression of IL-23, IL-12 and TNF without evidence of ER stress.

Coronary Artery Vessel Healing Pattern, Short and Long Term, After Implantation of the Everolimus-Eluting Bioresorbable Vascular Scaffold.

BACKGROUND: Although the Absorb bioresorbable vascular scaffold is increasingly used in daily clinical practice for the treatment of coronary artery disease, the exact vascular healing pattern and the resorption process in humans is unknown because histological data are derived only from animal studies. METHODS AND RESULTS: We have obtained 4 autopsies (5 scaffolds) since August 2013. Duration of bioresorbable vascular scaffold implantation ranged from 3 to 501 days. All autopsies and histological assessments were performed by dedicated cardiovascular pathologists. At 1 week after bioresorbable vascular scaffold implantation, struts were covered with a fine layer of fibrin and platelets. At 113 days, the scaffold struts were fully covered with smooth muscle cells. Hyaline eosinophilic and proteoglycan material infiltrating the scaffold struts was observed at 501 days after implantation. At all time points, we observed the presence of multinuclear foreign body giant cells adjacent to the scaffold struts. CONCLUSIONS: Resorption and healing processes after bioresorbable vascular scaffold implantation in human patients mirror those observed in porcine models. The presence of multinucleated foreign body giant cells at both short- and long-term follow-up needs further investigation and may be related to a low-grade absorptive inflammatory response to the polymer.

Tie2 signaling cooperates with TNF to promote the pro-inflammatory activation of human macrophages independently of macrophage functional phenotype.

Angiopoietin (Ang) -1 and -2 and their receptor Tie2 play critical roles in regulating angiogenic processes during development, homeostasis, tumorigenesis, inflammation and tissue repair. Tie2 signaling is best characterized in endothelial cells, but a subset of human and murine circulating monocytes/macrophages essential to solid tumor formation express Tie2 and display immunosuppressive properties consistent with M2 macrophage polarization. However, we have recently shown that Tie2 is strongly activated in pro-inflammatory macrophages present in rheumatoid arthritis patient synovial tissue. Here we examined the relationship between Tie2 expression and function during human macrophage polarization. Tie2 expression was observed under all polarization conditions, but was highest in IFN-γ and IL-10 -differentiated macrophages. While TNF enhanced expression of a common restricted set of genes involved in angiogenesis and inflammation in GM-CSF, IFN-γ and IL-10 -differentiated macrophages, expression of multiple chemokines and cytokines, including CXCL3, CXCL5, CXCL8, IL6, and IL12B was further augmented in the presence of Ang-1 and Ang-2, via Tie2 activation of JAK/STAT signaling. Conditioned medium from macrophages stimulated with Ang-1 or Ang-2 in combination with TNF, sustained monocyte recruitment. Our findings suggest a general role for Tie2 in cooperatively promoting the inflammatory activation of macrophages, independently of polarization conditions.

Intimal lining layer macrophages but not synovial sublining macrophages display an IL-10 polarized-like phenotype in chronic synovitis.

INTRODUCTION: Synovial tissue macrophages play a key role in chronic inflammatory arthritis, but the contribution of different macrophage subsets in this process remains largely unknown. The main in vitro polarized macrophage subsets are classically (M1) and alternatively (M2) activated macrophages, the latter comprising interleukin (IL)-4 and IL-10 polarized cells. Here, we aimed to evaluate the polarization status of synovial macrophages in spondyloarthritis (SpA) and rheumatoid arthritis (RA). METHODS: Expression of polarization markers on synovial macrophages, peripheral blood monocytes, and in vitro polarized monocyte-derived macrophages from SpA versus RA patients was assessed by immunohistochemistry and flow cytometry, respectively. The polarization status of the intimal lining layer and the synovial sublining macrophages was assessed by double immunofluorescence staining. RESULTS: The expression of the IL-10 polarization marker cluster of differentiation 163 (CD163) was increased in SpA compared with RA intimal lining layer, but no differences were found in other M1 and M2 markers between the diseases. Furthermore, no significant phenotypic differences in monocytes and in vitro polarized monocyte-derived macrophages were seen between SpA, RA, and healthy controls, indicating that the differential CD163 expression does not reflect a preferential M2 polarization in SpA. More detailed analysis of intimal lining layer macrophages revealed a strong co-expression of the IL-10 polarization markers CD163 and cluster of differentiation 32 (CD32) but not any of the other markers in both SpA and RA. In contrast, synovial sublining macrophages had a more heterogeneous phenotype, with a majority of cells co-expressing M1 and M2 markers. CONCLUSIONS: The intimal lining layer but not synovial sublining macrophages display an IL-10 polarized-like phenotype, with increased CD163 expression in SpA versus RA synovitis. These differences in the distribution of the polarized macrophage subset may contribute to the outcome of chronic synovitis.

Soluble immune complexes shift the TLR-induced cytokine production of distinct polarized human macrophage subsets towards IL-10.

BACKGROUND: Costimulation of murine macrophages with immune complexes (ICs) and TLR ligands leads to alternative activation. Studies on human myeloid cells, however, indicate that ICs induce an increased pro-inflammatory cytokine production. This study aimed to clarify the effect of ICs on the pro- versus anti-inflammatory profile of human polarized macrophages. MATERIALS AND METHODS: Monocytes isolated from peripheral blood of healthy donors were polarized for four days with IFN-γ, IL-4, IL-10, GM-CSF, M-CSF, or LPS, in the presence or absence of heat aggregated gamma-globulins (HAGGs). Phenotypic polarization markers were measured by flow cytometry. Polarized macrophages were stimulated with HAGGs or immobilized IgG alone or in combination with TLR ligands. TNF, IL-6, IL-10, IL-12, and IL-23 were measured by Luminex and/or RT-qPCR. RESULTS: HAGGs did not modulate the phenotypic polarization and the cytokine production of macrophages. However, HAGGs significantly altered the TLR-induced cytokine production of all polarized macrophage subsets, with the exception of MΦ(IL-4). In particular, HAGGs consistently enhanced the TLR-induced IL-10 production in both classically and alternatively polarized macrophages (M1 and M2). The effect of HAGGs on TNF and IL-6 production was less pronounced and depended on the polarization status, while IL-23p19 and IL-12p35 expression was not affected. In contrast with HAGGs, immobilized IgG induced a strong upregulation of not only IL-10, but also TNF and IL-6. CONCLUSION: HAGGs alone do not alter the phenotype and cytokine production of in vitro polarized human macrophages. In combination with TLR-ligands, however, HAGGs but not immobilized IgG shift the cytokine production of distinct macrophage subsets toward IL-10.