Klemen Strle

IL-1β impairs insulin-like growth factor I-induced differentiation and downstream activation signals of the insulin-like growth factor I receptor in myoblasts

Proinflammatory cytokines are elevated in disorders characterized by muscle wasting and weakness, such as inflammatory myopathies and AIDS wasting. We recently demonstrated that TNF-α impairs the ability of insulin-like growth factor (IGF)-I to promote protein synthesis in muscle precursor cells. In this study we extend these findings by showing that low concentrations of IL-1β impair IGF-I-dependent differentiation of myoblasts, as assessed by expression of the muscle specific protein, myosin heavy chain. In the absence of exogenous IGF-I, IL-1β (1 ng/ml) did not impair muscle cell development. However, in the presence of IGF-I, 100-fold lower concentrations of IL-1β (0.01 ng/ml) significantly suppressed myoblast differentiation, protein synthesis, and myogenin expression. Increasing IL-1β to 1 ng/ml completely blocked the anabolic actions of IGF-I in murine C2C12 myoblasts. Similarly, IL-1β inhibited IGF-I-stimulated protein synthesis in primary porcine myoblasts. IL-1β impaired the actions of IGF-I at a point distal to the IGF receptor, and this was not due to IL-1β-induced cell death. Instead, IL-1β inhibited the ability of IGF-I to phosphorylate tyrosine residues on both of its downstream docking proteins, insulin receptor substrate 1 and insulin receptor substrate 2. These data establish that physiological concentrations of IL-1β block the ability of IGF-I to promote protein synthesis, leading to reduced expression of the myogenic transcription factor, myogenin, and the subsequent development of more mature differentiated cells that express myosin heavy chain. Collectively, the results are consistent with the notion that very low concentrations of IL-1β significantly impair myogenesis, but they are unable to do so in the absence of the growth factor IGF-I.

Association of a Toll-like receptor 1 polymorphism with heightened Th1 inflammatory responses and antibiotic-refractory Lyme arthritis

OBJECTIVE Single-nucleotide polymorphisms (SNPs) that alter immune function, inflammatory responses, and disease susceptibility have been identified in several genes encoding Toll-like receptors (TLRs). The TLR SNPs with the best evidence of an effect on immune function are those in TLR1 (1805GG), TLR2 (2258GA), and TLR5 (1174CT). This study was undertaken to assess the frequency and functional outcomes of these polymorphisms in patients with Lyme disease. METHODS SNP frequencies and functional outcomes were assessed in 248 patients with Lyme disease. Cytokine and chemokine levels were determined using multiplex assays in the serum of patients with erythema migrans (EM), joint fluid of patients with Lyme arthritis, and supernatants of Borrelia burgdorferi-stimulated peripheral blood mononuclear cells (PBMCs) from patients with Lyme arthritis. RESULTS The frequency of the TLR1-1805GG polymorphism was greater in patients with antibiotic-refractory arthritis compared with patients with EM or those with antibiotic-responsive arthritis. Early in the illness, patients with EM carrying 1805GG, primarily those infected with B burgdorferi 16S-23S ribosomal spacer RNA intergenic type 1 (RST1) strains, had higher serum levels of interferon-γ (IFNγ), CXCL9, and CXCL10 and had more severe infection than EM patients carrying the 1805TG/TT polymorphism. These inflammatory responses were amplified in patients with Lyme arthritis, and the highest responses were observed in patients with 1805GG in the antibiotic-refractory group who had been infected with RST1 strains. When PBMCs from patients with Lyme arthritis were stimulated with a B burgdorferi RST1 strain, the 1805GG group had a significantly larger fold increase in the levels of IFNγ, CCL2, CXCL9, and CXCL10 compared to the 1805TG/TT group. In contrast, the TLR2 and TLR5 polymorphisms did not vary in frequency or function among the groups. CONCLUSION The TLR1-1805GG polymorphism in B burgdorferi RST1-infected patients was associated with stronger Th1-like inflammatory responses, an environment that may set the stage for antibiotic-refractory arthritis.

IL-10 Inhibits Apoptosis of Promyeloid Cells by Activating Insulin Receptor Substrate-2 and Phosphatidylinositol 3′-Kinase

IL-10 is well known to be a potent inhibitor of the synthesis of proinflammatory cytokines, but noninflammatory hemopoietic cells also express IL-10Rs. Here we show that IL-10 directly affects progenitor myeloid cells by protecting them from death following the removal of growth factors. Murine factor-dependent cell progenitors cultured in the absence of growth factors were 43 ± 1% apoptotic after 12 h. Addition of IL-10 at a concentration as low as 100 pg/ml significantly reduced the apoptotic population to 32 ± 3%. At 10 ng/ml, IL-10 caused a 4-fold reduction in the apoptotic population (11 ± 1%). The anti-apoptotic activity of IL-10 was significantly inhibited with a neutralizing IL-10R Ab. Factor-dependent cell progenitor promyeloid cells expressed functional IL-10Rs, as assessed by precipitation of a 110-kDa protein with an Ab to the IL-10R and by the ability of IL-10 to activate Jak1 and Tyk2 and to phosphorylate tyrosine 705 on Stat-3. IL-10 increased tyrosyl phosphorylation of insulin receptor substrate-2 and stimulated the enzymatic activity of both phosphatidylinositol 3′-kinase and Akt. The anti-apoptotic activity of IL-10 was blocked by inhibition of phosphatidylinositol 3′-kinase. Wortmannin and LY294002 also totally inhibited activation of extracellular signal-related kinase (ERK)1/2 by IL-10. Direct inhibition of ERK1/2 with the mitogen-activated protein kinase/ERK kinase inhibitor PD98059 partially, but significantly, impaired the anti-apoptotic activity of IL-10. These data establish that activation of the IL-10R promotes survival of progenitor myeloid cells. This survival-promoting activity is totally due to IL-10 stimulating the insulin receptor substrate-2/PI 3-kinase/Akt pathway, which increases the anti-apoptotic activity of ERK1/2.

Borrelia burgdorferi RST1 (OspC Type A) Genotype Is Associated with Greater Inflammation and More Severe Lyme Disease

Evidence is emerging for differential pathogenicity among Borrelia burgdorferi genotypes in the United States. By using two linked genotyping systems, ribosomal RNA intergenic spacer type (RST) and outer surface protein C (OspC), we studied the inflammatory potential of B. burgdorferi genotypes in cells and patients with erythema migrans or Lyme arthritis. When macrophages were stimulated with 10 isolates of each RST1, RST2, or RST3 strain, RST1 (OspC type A)-stimulated cells expressed significantly higher levels of IL-6, IL-8, chemokine ligand (CCL) 3, CCL4, tumor necrosis factor, and IL-1β, factors associated with innate immune responses. In peripheral blood mononuclear cells, RST1 strains again stimulated significantly higher levels of these mediators. Moreover, compared with RST2, RST1 isolates induced significantly more interferon (IFN)-α, IFN-γ, and CXCL10, which are needed for adaptive immune responses; however, OspC type I (RST3) approached RST1 (OspC type A) in stimulating these adaptive immune mediators. Similarly, serum samples from patients with erythema migrans who were infected with the RST1 genotype had significantly higher levels of almost all of these mediators, including exceptionally high levels of IFN-γ-inducible chemokines, CCL2, CXCL9, and CXCL10; and this pronounced inflammatory response was associated with more symptomatic infection. Differences among genotypes were not as great in patients with Lyme arthritis, but those infected with RST1 strains more often had antibiotic-refractory arthritis. Thus, the B. burgdorferi RST1 (OspC type A) genotype, followed by the RST3 (OspC type I) genotype, causes greater inflammation and more severe disease, establishing a link between spirochetal virulence and host inflammation.

A novel human autoantigen, endothelial cell growth factor, is a target of T and B cell responses in patients with Lyme disease.

OBJECTIVE Autoantigen presentation by HLA-DR molecules is thought to be a central component of many autoimmune diseases, but identifying disease-relevant autoantigens has been a difficult challenge. In this study we aimed to identify autoantigens in patients with antibiotic-refractory Lyme arthritis, in which infection-induced autoimmunity is thought to play an important role. METHODS Using tandem mass spectrometry, naturally presented HLA-DR self peptides from a patients synovium were identified, synthesized, and reacted with his peripheral blood mononuclear cells (PBMCs). Immunoreactive peptides and their source proteins were then tested for T and B cell responses using large numbers of patient cells or sera. RESULTS Of 120 HLA-DR-presented self peptides identified from one patient, one peptide derived from endothelial cell growth factor (ECGF) caused his PBMCs to proliferate. T and B cell responses to ECGF occurred systemically in ∼10-30% of patients with early or late manifestations of Lyme disease, primarily in those with refractory arthritis-associated HLA-DR alleles, such as DRB1*0101 and 0401. Compared with patients with antibiotic-responsive arthritis, those with antibiotic-refractory arthritis had significantly higher concentrations of ECGF in synovial fluid (P<0.0001) and more often had ECGF antibody reactivity. Among non-antibiotic-treated historical patients who developed arthritis, 26% had ECGF reactivity, which often developed before the onset of arthritis and was associated with significantly longer courses of arthritis. CONCLUSION T and B cell responses to ECGF occur in a subset of patients with Lyme disease, particularly in those with antibiotic-refractory arthritis, providing the first direct evidence of autoimmune T and B cell responses in this illness.

Borrelia burgdorferi Stimulates Macrophages to Secrete Higher Levels of Cytokines and Chemokines than Borrelia afzelii or Borrelia garinii

To delineate the inflammatory potential of the 3 pathogenic species of Borrelia burgdorferi sensu lato, we stimulated monocyte-derived macrophages from healthy human donors with 10 isolates each of B. burgdorferi, Borrelia afzelii, or Borrelia garinii recovered from erythema migrans skin lesions of patients with Lyme borreliosis from the United States or Slovenia. B. burgdorferi isolates from the United States induced macrophages to secrete significantly higher levels of interleukin (IL)-8, CCL3, CCL4, IL-6, IL-10, and tumor necrosis factor than B. garinii or B. afzelii isolates. Consistent with this response in cultured macrophages, chemokine and cytokine levels in serum samples of patients from whom the isolates were obtained were significantly greater in B. burgdorferi-infected patients than in B. afzelii- or B. garinii-infected patients. These results demonstrate in vitro and in vivo that B. burgdorferi has greater inflammatory potential than B. afzelii and B. garinii, which may account in part for variations in the clinical manifestations of Lyme borreliosis.

IL-10 promotes survival of microglia without activating Akt

IL-10 is an anti-inflammatory cytokine that has recently been shown to promote survival of neurons and glia. Here we establish that IL-10 induces phosphorylation of Stat3 on Tyr(705) and serves as a survival factor for N13 microglial cells. Recombinant IL-10 (10 ng/ml) decreases growth factor withdrawal-induced apoptosis by 50%, as assessed by TUNEL. In contrast to IL-10, IGF-I increases enzymatic activity of PI 3-kinase and causes phosphorylation on serine(473) of Akt but does not prevent microglial apoptosis. These data establish that IL-10 activates Stat3 and inhibits the mitochondrial pathway of cell death without activating the Akt cell survival pathway.

Performance of United States Serologic Assays in the Diagnosis of Lyme Borreliosis Acquired in Europe

BACKGROUND Physicians in the United States sometimes need to evaluate a patient for suspected Lyme borreliosis (LB) who may have acquired the infection in Europe. Using serum samples from European LB patients, we compared the performance of European and US serodiagnostic tests, including newer-generation assays containing Vmp-like sequence, expressed or its C6 peptide. METHODS The sensitivity of each assay was determined using 64 serum samples from LB patients with early or late disease manifestations who acquired the infection in Europe. Specificity was measured using 100 sera from healthy subjects from a nonendemic area. RESULTS For the detection of European-acquired infection, conventional 2-tiered testing (enzyme-linked immunosorbent assay [ELISA] followed by immunoblotting) using US assays had an overall sensitivity and specificity of 52% and 100%, compared with 81% (P = .0007) and 99% (P = 1.00) using analogous European tests. The sensitivity of a US C6 ELISA used as a stand-alone test (88% overall) was statistically comparable to that of conventional 2-tiered testing using European tests (P = .47) and was 100% specific. Similarly, an alternative 2-tiered algorithm using a standard US ELISA followed by a C6 ELISA was comparably sensitive (84% overall) compared with conventional 2-tiered testing using European assays (P = .82), and specificity remained 100%. CONCLUSIONS European assays outperformed analogous US assays in a conventional 2-tiered testing algorithm. However, a C6 ELISA used as a stand-alone test or in the second tier of a 2-tiered algorithm performed comparably to conventional 2-tiered testing using European assays, and can be used for evaluation of any patient, regardless of travel history.

Elevated Levels of IL-23 in a Subset of Patients With Post–Lyme Disease Symptoms Following Erythema Migrans

BACKGROUND The causes of post-Lyme disease symptoms are unclear. Herein, we investigated whether specific immune responses were correlated with such symptoms. METHODS The levels of 23 cytokines and chemokines, representative of innate and adaptive immune responses, were assessed in sera from 86 antibiotic-treated European patients with erythema migrans, 45 with post-Lyme symptoms and 41 without symptoms, who were evaluated prior to treatment and 2, 6, and 12 months thereafter. RESULTS At study entry, significant differences between groups were observed for the type 1 helper T cell (TH1)-associated chemokines CXCL9 and CXCL10, which were associated with negative Borrelia cultures, and the type 17 helper T cell (TH17)-associated cytokine interleukin 23 (IL-23), which was associated with positive cultures and the development of post-Lyme symptoms (P ≤ .02). Moreover, of the 41 patients with detectable IL-23 levels, 25 (61%) developed post-Lyme symptoms, and all 7 with IL-23 levels ≥ 230 ng/mL had such symptoms. Furthermore, antibody responses to the ECGF autoantigen were more common in patients with post-Lyme symptoms (P = .07) and were correlated directly with IL-23 levels (P = .02). Despite the presence of post-Lyme symptoms, all posttreatment culture results were negative, antiborrelial antibody responses declined, and there were no objective signs of disseminated disease, suggesting that spirochetal eradication had occurred with treatment in all patients. CONCLUSIONS High TH1-associated responses correlated with more effective immune-mediated spirochetal killing, whereas high TH17-associated immune responses, often accompanied by autoantibodies, correlated with post-Lyme symptoms, providing a new paradigm for the study of postinfectious symptoms in a subset of patients with Lyme disease.

Dysregulation of CD4+CD25(high) T cells in the synovial fluid of patients with antibiotic-refractory Lyme arthritis.

OBJECTIVE To examine the role of immune dysregulation in antibiotic-refractory Lyme arthritis by comparing the phenotype, frequency, and function of CD4+ Teff cells and Treg cells in patients with antibiotic- responsive arthritis and patients with antibiotic-refractory arthritis. METHODS Matched peripheral blood and synovial fluid samples from 15 patients with antibiotic-responsive arthritis were compared with those from 16 patients with antibiotic-refractory arthritis, using flow cytometry, suppression assays, and cytokine assays. RESULTS Critical differences between the 2 patient groups were observed in the synovial fluid CD4+CD25(high) population, a cell subset usually composed of FoxP3-positive Treg cells. In patients with antibiotic-refractory arthritis, this cell population often had fewer FoxP3-positive cells and a greater frequency of FoxP3-negative (Teff) cells compared with patients with antibiotic-responsive arthritis. Moreover, the expression of glucocorticoid-induced tumor necrosis factor receptor and OX40 on CD4+CD25(high) cells was significantly higher in the antibody-refractory group. Suppression assays showed that CD4+CD25(high) cells in patients with antibiotic-refractory arthritis did not effectively suppress proliferation of CD4+CD25- cells or secretion of interferon-γ and tumor necrosis factor α, whereas those cells in patients with antibiotic-responsive arthritis did suppress proliferation of CD4+CD25- cells and secretion of interferon-γ and tumor necrosis factor α. Finally, in the antibiotic-refractory group, higher ratios of CD25(high) FoxP3-negative cells to CD25(high) FoxP3-positive cells correlated directly with a longer duration of arthritis after antibiotic treatment. CONCLUSION Patients with antibiotic-refractory Lyme arthritis often have lower frequencies of Treg cells, higher expression of activation coreceptors, and less effective inhibition of proinflammatory cytokines. This suggests that immune responses in these patients are excessively amplified, leading to immune dysregulation and antibiotic-refractory arthritis.