Evidence that autophagy, but not the unfolded protein response, regulates the expression of IL-23 in the gut of patients with ankylosing spondylitis and subclinical gut inflammation.

OBJECTIVES: Interleukin (IL)-23 has been implicated in the pathogenesis of ankylosing spondylitis (AS). The aim of the study was to clarify the mechanisms underlying the increased IL-23 expression in the gut of AS patients. METHODS: Consecutive gut biopsies from 30 HLA-B27(+) AS patients, 15 Crohn's disease (CD) patients and 10 normal subjects were obtained. Evidence for HLA-B27 misfolding was studied. Unfolded protein response (UPR) and autophagy were assessed by RT-PCR and immunohistochemistry. The contribution of UPR and autophagy in the regulation of IL-23 expression was evaluated in in vitro experiments on isolated lamina propria mononuclear cells (LPMCs). RESULTS: Intracellular colocalisation of SYVN1 and FHCs but not a significant overexpression of UPR genes was observed in the gut of AS patients. Conversely, upregulation of the genes involved in the autophagy pathway was observed in the gut of AS and CD patients. Immunohistochemistry showed an increased expression of LC3II, ATG5 and ATG12 but not of SQSTM1 in the ileum of AS and CD patients. LC3II was expressed among infiltrating mononuclear cells and epithelial cells resembling Paneth cells (PC) and colocalised with ATG5 in AS and CD. Autophagy but not UPR was required to modulate the expression of IL-23 in isolated LPMCs of AS patients with chronic gut inflammation, CD patients and controls. CONCLUSIONS: Our data suggest that HLA-B27 misfolding occurs in the gut of AS patients and is accompanied by activation of autophagy rather than a UPR. Autophagy appears to be associated with intestinal modulation of IL-23 in AS.

Macrophage phenotype in the subclinical gut inflammation of patients with ankylosing spondylitis.

OBJECTIVE: Long-term evolution of subclinical gut inflammation to overt Crohn's disease (CD) has been described in AS patients. The aim of this study was to evaluate macrophage polarization occurring in the inflamed gut of patients with AS. METHODS: Twenty-seven HLA-B27(+) AS patients, 20 CD patients and 17 normal controls were consecutively enrolled. Classic M1 (iNOS(+)IL-10(-)), resolution phase (iNOS(+)IL-10(+)), M2 and CD14(+) macrophages were characterized by immunohistochemistry and flow cytometry. Quantitative gene expression analysis of IFN-γ, IL-4, IL-5, IL-33 and STAT6 was performed by real time PCR. RESULTS: Classic M1 macrophages were expanded in CD and AS, where resolution phase macrophages predominate. A large increase in CD163(+) (M2) macrophages was observed in AS strictly correlated with the expression of IL-33, a Th2 cytokine involved in M2 polarization. Unlike in CD, CD14(+) macrophages were virtually absent in the gut of AS patients and controls. CONCLUSION: The absence of CD14(+) macrophages together with the expansion of resolution phase and M2 macrophages is the immunological signature of subclinical ileal inflammation in AS.

Increased expression of interleukin-32 in the inflamed ileum of ankylosing spondylitis patients.

OBJECTIVE: To study the mRNA expression and protein tissue distribution of IL-32 in ileal biopsy specimens from patients with AS. METHODS: Quantitative gene expression analysis, by real-time PCR, of IL-32, IL-1ß, IL-10, TNF-α and IFN-γ was performed on ileal biopsies of 15 AS and 15 Crohn's disease (CD) patients and 10 healthy subjects (HSs). IL-32 tissue distribution was evaluated by immunohistochemistry. The effect of IL-32 on the production of IL-10 by intestinal epithelial cell lines was also evaluated. RESULTS: In the ileal specimens of patients with AS and intestinal chronic inflammation, significant up-regulation of IL-32 at both the mRNA and protein levels was found as compared with non-inflamed AS patients and controls. IL-32 over-expression in AS was accompanied by a significant increase of IL-10 but not of cytokines involved in IL-32 induction. IL-32 stimulates intestinal epithelial cell lines in vitro to produce IL-10. CONCLUSION: Our findings suggest IL-32 as an important cytokine probably involved in the innate immune response occurring in early phases of intestinal inflammation, where it seems to play a prevalent protective role.

Interleukin-22 and interleukin-22-producing NKp44+ natural killer cells in subclinical gut inflammation in ankylosing spondylitis.

OBJECTIVE: The intestinal inflammation observed in patients with ankylosing spondylitis (AS) is characterized by an overexpression of interleukin-23 (IL-23). IL-23 is known to regulate IL-22 production through lamina propria NKp44+ natural killer (NK) cells, which are thought to be involved in protective mucosal mechanisms. This study was undertaken to evaluate the frequency of NKp44+ NK cells and the expression of IL-22 in the ileum of AS patients. METHODS: Tissue NKp44+ NK cells, NKp46+ NK cells, and IL-22-producing cells were analyzed by flow cytometry. Quantitative gene expression analysis of IL-22, IL-23, IL-17, STAT-3, and mucin 1 (MUC-1) was performed by reverse transcriptase-polymerase chain reaction on ileal samples from 15 patients with AS, 15 patients with Crohn's disease (CD), and 15 healthy controls. NKp44, pSTAT-3, and IL-22 expression was analyzed by immunohistochemistry. RESULTS: The frequency of NKp44+ but not NKp46+ NK cells was increased in the inflamed ileum of AS patients compared to CD patients and controls. The frequency of NKp46+ NK cells was significantly increased only in CD patients. Among CD4+ lymphocytes and NKp44+ NK cell subsets, the latter were the major source of IL-22 on lamina propria mononuclear cells from AS patients. Significant up-regulation of IL-22, IL-23p19, MUC-1, and STAT-3 transcripts in the terminal ileum of patients with AS was observed. Immunohistochemical analysis confirmed the increased IL-22 and pSTAT-3 expression in inflamed mucosa from AS and CD patients. CONCLUSION: Our findings indicate that overexpression of IL-22, together with an increased number of IL-22-producing NKp44+ NK cells, occurs in the gut of AS patients, where it appears to play a tissue-protective role.

Expression of interleukin-32 in the inflamed arteries of patients with giant cell arteritis.

OBJECTIVE: Giant cell (temporal) arteritis (GCA) is a vasculitis that mainly affects the large and medium arteries, especially the branches of the proximal aorta. Interleukin-32 (IL-32) is a recently described Th1 proinflammatory cytokine, and is mainly induced by interferon-γ (IFNγ), IL-1ß, and tumor necrosis factor α (TNFα). This study was undertaken to investigate the expression and tissue distribution of IL-32 in artery biopsy specimens from patients with GCA. METHODS: Quantitative gene expression analysis of IL-32, IL-1ß, TNFα, IFNγ, IL-6, and IL-27 was performed in artery biopsy specimens obtained from 18 patients with GCA and 15 controls. Immunohistochemistry analysis was performed to evaluate IL-32 tissue distribution and identify IL-32-producing cells. Circulating Th1 lymphocytes were evaluated by flow cytometry. RESULTS: We demonstrated a strong and significant up-regulation of IL-32 at both the messenger RNA and protein levels in the artery biopsy samples from patients with GCA. IL-32 was abundantly expressed by vascular smooth muscle cells of inflamed arteries and neovessels within inflammatory infiltrates. IL-32 expression strongly correlated with the intensity of the systemic inflammatory response. IL-32 overexpression was accompanied by strong overexpression of Th1 cytokines, such as IFNγ and IL-27p28, in inflamed arteries from GCA patients. The Th1 lymphocyte population was also expanded among peripheral blood mononuclear cells from GCA patients and produced higher amounts of IL-32 compared to controls. CONCLUSION: Our findings indicate that overexpression of IL-32 together with a clear Th1 response immunologically characterizes the inflammatory response in GCA. In particular, IL-32 seems to be an important mediator of artery inflammation in GCA.

Expansion of intestinal CD4+CD25(high) Treg cells in patients with ankylosing spondylitis: a putative role for interleukin-10 in preventing intestinal Th17 response.

OBJECTIVE: Subclinical gut inflammation has been demonstrated in patients with ankylosing spondylitis (AS). This study was undertaken to determine the frequency of regulatory CD4+CD25(high) T cells (Treg cells) and to evaluate Treg cell-related cytokines (interleukin-2 [IL-2], transforming growth factor ß [TGFß], and IL-10) and transcription factors (FoxP3 and STAT-5) in the ileum of patients with AS. METHODS: Quantitative gene expression analysis, by reverse transcriptase-polymerase chain reaction, of Treg-related cytokines (IL-2, TGFß, and IL-10) and transcription factors (STAT-5 and FoxP3) was performed on ileal biopsy specimens from 18 patients with AS, 15 patients with active Crohn's disease (CD), and 15 healthy subjects. Tissue and circulating Treg cells were also analyzed by flow cytometry. RESULTS: A significant up-regulation of IL-2, TGFß, FoxP3, STAT-5, and IL-10 transcripts in the terminal ileum of AS patients displaying chronic ileal inflammation was observed. Flow cytometric analysis of Treg cells showed significant peripheral expansion in both patients with AS and chronic inflammation and patients with CD (mean ± SD 1.08 ± 0.4% and 1.05 ± 0.3%, respectively) as compared with healthy subjects (0.25 ± 0.12%) (P < 0.05). Interestingly, a 5-fold increase in the proportion of Treg cells was observed in the gut of patients with AS (5 ± 3%) as compared with healthy subjects (1.2 ± 0.4%) (P < 0.001), with 70-80% of these cells also producing IL-10. In vitro studies showed that blocking IL-10 was sufficient to induce Th17 polarization on lamina propria mononuclear cells isolated from AS patients. CONCLUSION: Our findings provide the first evidence that an active Treg cell response, mainly dominated by IL-10 production, occurs in the gut of AS patients and is probably responsible for the absence of a clear Th17 polarization in the ileum of AS patients.

Phenotype and functional changes of Vgamma9/Vdelta2 T lymphocytes in Behçet's disease and the effect of infliximab on Vgamma9/Vdelta2 T cell expansion, activation and cytotoxicity.

INTRODUCTION: Infliximab is a chimeric monoclonal antibody against tumor necrosis factor alpha (TNF-alpha) that has been introduced recently for Behçet's disease (BD) patients who were resistant to standard treatment. The aim of this study was to analyse the functional changes of Vgamma9/Vdelta2 T lymphocytes in both active and inactive disease and the effect of infliximab on Vgamma9/Vdelta2 T cell expansion, activation and cytotoxicity. METHODS: We investigated 1) cell expansion, 2) expression of TNFRII receptor, 3) perforin and gamma interferon (IFN) content, 4) release of granzyme A (GrA) and 5) phenotype changes, in vitro and in vivo, in Vgamma9/Vdelta2 T lymphocytes by means of fluorescence-activated cell sorter analysis of lymphocyte cultures from patients with active and inactive BD and healthy subjects. RESULTS: Cell expansion, expression of TNFRII, perforin and gamma IFN content and release of granzyme A were significantly higher in active patients. In vitro and ex vivo treatment with infliximab resulted in a significant reduction of all parameters together with changes in the phenotype of Vgamma9/Vdelta2 T cells. CONCLUSIONS: All together these data indicate that infliximab is capable of interfering with Vgamma9/Vdelta2 T cell function in BD and although cell culture models cannot reliably predict all potential effects of the drug in vivo, our results present the possibility that this drug may find use in a range of immunological disorders, characterized by dysregulated cell-mediated immunity.

NF-kappaB protects Behçet's disease T cells against CD95-induced apoptosis up-regulating antiapoptotic proteins.

OBJECTIVE: To determine whether prolongation of the inflammatory reaction in patients with Behçet's disease (BD) is related to apoptosis resistance and is associated with the up-regulation of antiapoptotic factors. METHODS: The percentage of cell death was evaluated by flow cytometry in peripheral blood mononuclear cells from 35 patients with BD and 30 healthy volunteers. The expression levels of antiapoptotic factors and NF-kappaB regulatory proteins were measured using Western blotting and immunohistochemical analyses. To down-regulate NF-kappaB nuclear translocation, BD T lymphocytes were exposed in vitro to thalidomide and subjected to transfection with NF-kappaB small interfering RNA. RESULTS: Although CD95 is highly expressed in BD T cells, the absence of sensitivity to CD95-induced apoptosis observed may be attributable to the inhibitory action of antiapoptotic genes. Immunoblot analysis for major antiapoptotic proteins showed considerable up-regulation of the short form of cellular FLIP (cFLIP) and Bcl-x(L) in BD activated T cells, while levels of Bcl-2, caspase 3, and caspase 8 in activated T cells from patients with BD were comparable with those in activated T cells from normal donors. Moreover, expression of IKK and IkappaB was up-regulated, whereas NF-kappaB translocated to the nucleus in BD T cells, suggesting that NF-kappaB activation may modulate the expression of antiapoptotic genes. Interestingly, thalidomide and NF-kappaB small interfering RNA down-regulated cFLIP and Bcl-x(L) expression levels and sensitized BD activated T cells to CD95-induced apoptosis. CONCLUSION: Taken together, these results indicate that NF-kappaB contributes to the regulation of the apoptosis-related factors and death receptors leading to apoptosis resistance in BD T cell subsets. Our results suggest that NF-kappaB plays a crucial role in the pathogenesis of BD, and that its pharmacologic control could represent a key strategy in modulating specific immune-mediated disease.

Vgamma9/Vdelta2 T lymphocytes in Italian patients with Behçet's disease: evidence for expansion, and tumour necrosis factor receptor II and interleukin-12 receptor beta1 expression in active disease.

Behçet's disease is a multisystem disease in which there is evidence of immunological dysregulation. It has been proposed that gamma/delta T cells are involved in its pathogenesis. The aim of the present study was to assess the capacity of gamma/delta T cells with phenotype Vgamma9/Vdelta2, from a group of Italian patients with Behçet's disease, to proliferate in the presence of various phosphoantigens and to express tumour necrosis factor (TNF) and IL-12 receptors. Twenty-five patients and 45 healthy individuals were studied. Vgamma9/Vdelta2 T cells were analyzed by fluorescence activated cell sorting, utilizing specific monoclonal antibodies. For the expansion of Vgamma9/Vdelta2 T cells, lymphocytes were cultured in the presence of various phosphoantigens. The expression of TNF receptor II and IL-12 receptor beta1 was evaluated with the simultaneous use of anti-TNF receptor II phycoerythrin-labelled (PE) or anti-IL-12 receptor beta1 PE and anti-Vdelta2 T-cell receptor fluorescein isothiocyanate. There was a certain hierarchy in the response of Vgamma9/Vdelta2 T cells toward the different phosphoantigens, with the highest expansion factor obtained with dimethylallyl pyrophosphate and the lowest with xylose 1P. The expansion factor was fivefold greater in patients with active disease than in those with inactive disease or in control individuals. TNF receptor II and IL-12 receptor beta1 expressions were increased in both patients and control individuals. The proportion of Vgamma9/Vdelta2 T cells bearing these receptors was raised in active disease when Vgamma9/Vdelta2 T cells were cultured in the presence of dimethylallyl pyrophosphate. These results indicate that Vgamma9/Vdelta2 T cell activation is correlated with disease progression and probably involved in the pathogenesis.

Randomized study of subcutaneous low molecular weight heparin plus aspirin versus intravenous immunoglobulin in the treatment of recurrent fetal loss associated with antiphospholipid antibodies.

OBJECTIVE: To compare the 2 most efficacious therapeutic regimens, intravenous immunoglobulin (IVIG) and anticoagulation with low molecular weight (LMW) heparin plus low-dose aspirin, in women with recurrent pregnancy loss associated with antiphospholipid antibodies (aPL). METHODS: We examined 40 women with recurrent abortion (at least 3 occurrences) and repeatedly positive test results for anticardiolipin or lupus anticoagulant. The subjects were randomly assigned to treatment with IVIG or LMW heparin plus low-dose aspirin. Both therapies were started when the women were pregnant as documented by a positive urine test. IVIG was stopped at the thirty-first week of gestation, aspirin at the thirty-fourth week, and heparin at the thirty-seventh week. The primary outcome of interest was the rate of live births with the 2 treatments. RESULTS: The characteristics of the 2 groups were similar at the time of randomization. The women treated with LMW heparin plus low-dose aspirin had a higher rate of live births (84%) than those treated with IVIG (57%). CONCLUSION: Treatment with LMW heparin plus low-dose aspirin should be considered as the standard therapy for recurrent pregnancy loss due to aPL.