Ronald L. Hornung

Both IL-12p70 and IL-23 Are Synthesized During Active Crohn's Disease and Are Down-regulated by Treatment with Anti-IL-12 p40 Monoclonal Antibody

Background: Interleukin (IL)‐12p70 and IL‐23 are key T helper‐1 (TH1) cytokines that drive the inflammation seen in numerous models of intestinal inflammation. These molecules contain an identical p40 chain that is bound to a p35 chain in IL‐12 and a p19 chain in IL‐23, making both potentially susceptible to modulation by an anti‐IL‐12p40 monoclonal antibody (mAb). Methods: In the present study, we sought to determine whether active inflammation in Crohns disease (CD) is associated with the increased synthesis of both of these cytokines and whether patients treated with an anti‐IL‐12p40 mAb down‐regulate IL‐23 as well as IL‐12p70 as previous reported. Results: To this end we initially determined that IL‐12p70 secretion by control and CD antigen‐presenting cells (macrophages) in lamina propria mononuclear populations is optimized by stimulation with CD40L and interferon‐&ggr;. In subsequent studies using these stimulation conditions we found that patients with CD manifested both increased IL‐12p70 and IL‐23 secretion before anti‐IL‐12p40 mAb treatment and normal levels of secretion of these cytokines following cessation of treatment. Antigen‐presenting cells in lamina propria mononuclear cells from ulcerative colitis patients, in contrast, produced only baseline levels of IL‐23. Finally, we found that IL‐23‐induced T cell production of IL‐17 and IL‐6 are also greatly reduced after antibody treatment. The latter data are parallel to those from previous studies showing that anti‐IL‐12p40 down‐regulates IFN‐&ggr; and tumor necrosis factor‐&agr; secretion. Conclusions: We conclude that CD but not ulcerative colitis is associated with high levels of both IL‐12p70 and IL‐23 secretion as well as the secretion of downstream effector cytokines, and that this cytokine production is down‐regulated following administration of IL‐12p40 mAb.

Suppression of graft-versus-host disease and amplification of graft-versus-tumor effects by activated natural killer cells after allogeneic bone marrow transplantation.

Bone marrow transplantation (BMT) is currently used for the treatment of a variety of neoplastic diseases. However, significant obstacles limiting the efficacy of allogeneic BMT are the occurrence of graft-versus-host disease (GvHD) and tumor relapse. Natural killer (NK) cells exert a variety of immunologic and homoeostatic functions. We examined whether adoptive transfer of activated NK cells of donor type would prevent GvHD after allogeneic BMT in mice. Lethally irradiated C57BL/6 (H-2(b)) mice, were transplanted with MHC incompatible BALB/c (H-2(d)) bone marrow cells and spleen cells and rapidly succumbed to acute GvHD. In contrast, mice that also received activated NK cells of donor type exhibited significant increases in survival. In determining the mechanism by which the NK cells prevented GvHD, mice were concurrently treated with a neutralizing antibodies to the immunosuppressive cytokine TGFbeta. Anti-TGFbeta completely abrogated the protective effects of the activated donor NK cells indicating that TGFbeta plays an important role in the prevention of GvHD by NK cells. We then examined whether activated NK cells of donor type after allogeneic BMT would induce graft-versus-tumor (GvT) effects without GvHD in mice bearing a murine colon adenocarcinoma (MCA-38). 10 d after receiving the tumor, in which the mice had demonstrable lung metastases, recipients received an allogeneic BMT with or without activated NK cells. Administration of activated NK cells resulted in significant GvT effects after allogeneic BMT as evidenced by increases in median survival and fewer lung metastasis. No evidence of GVHD was detected compared with recipients receiving spleen cells alone which also developed fewer lung metastases but in which all had succumbed to GVHD. Thus, our findings suggest that adoptive immunotherapy using activated donor NK cells combined with allogeneic BMT inhibits GvHD and promotes GvT in advanced tumor-bearing mice. These results also suggest that GvT and GvHD can be dissociable phenomena.

Natural history of autoimmune lymphoproliferative syndrome associated with FAS gene mutations

Autoimmune lymphoproliferative syndrome (ALPS) presents in childhood with nonmalignant lymphadenopathy and splenomegaly associated with a characteristic expansion of mature CD4 and CD8 negative or double negative T-cell receptor αβ(+) T lymphocytes. Patients often present with chronic multilineage cytopenias due to autoimmune peripheral destruction and/or splenic sequestration of blood cells and have an increased risk of B-cell lymphoma. Deleterious heterozygous mutations in the FAS gene are the most common cause of this condition, which is termed ALPS-FAS. We report the natural history and pathophysiology of 150 ALPS-FAS patients and 63 healthy mutation-positive relatives evaluated in our institution over the last 2 decades. Our principal findings are that FAS mutations have a clinical penetrance of <60%, elevated serum vitamin B12 is a reliable and accurate biomarker of ALPS-FAS, and the major causes of morbidity and mortality in these patients are the overwhelming postsplenectomy sepsis and development of lymphoma. With longer follow-up, we observed a significantly greater relative risk of lymphoma than previously reported. Avoiding splenectomy while controlling hypersplenism by using corticosteroid-sparing treatments improves the outcome in ALPS-FAS patients. This trial was registered at www.clinicaltrials.gov as #NCT00001350.

Suppression of inflammation in ulcerative colitis by interferon-β-1a is accompanied by inhibition of IL-13 production

Objective Ulcerative colitis is associated with increased interleukin 13 (IL-13) production by natural killer T cells. Taking advantage of the inhibitory actions of interferon β on IL-13 expression, this proof-of-concept study aimed to show that decreasing IL-13 production is associated with clinical improvement of ulcerative colitis symptoms. Design Open-label interventional drug trial. Setting Outpatient clinical research hospital. Patients Adult patients with active ulcerative colitis (Short Clinical Colitis Activity Index (SCCAI)≥5). Interventions Treatment with 30 μg IM interferon-β-1a (Avonex) weekly for 12 weeks with 6 month follow-up. Main outcome measures Clinical response was defined as ≥3 point drop in the SCCAI for at least two consecutive monitoring visits, and cytokine production was measured in cultured peripheral blood and lamina propria mononuclear cells (LPMC) before and after treatment. Results 11 of 16 patients were clinical responders, and 4 were in remission (SCCAI ≤ 2) at the end of treatment. Rectal bleeding subscores improved dramatically by week 4 (38% with frank bleeding vs 87% pretreatment). Increased IL-13 production by LPMC T cells fell significantly in clinical responders (690±99 vs 297±58 pg/ml p=0.015) but was unchanged in non-responders (542±83 vs 510±39 pg/ml). In addition, non-responders had significantly higher production of IL-17 and IL-6 pre-treatment compared to responders. Conclusions Interferon-β-1a induces clinical response and remission in a large subset of patients with ulcerative colitis that is associated with significant inhibition of IL-13 production. In addition, increased IL-17 and IL-6 production is associated with no response to interferon-β. These data provide a proof-of-concept that IL-13 is an effector cytokine in ulcerative colitis and should be a target for novel therapies.

Somatic FAS mutations are common in patients with genetically undefined autoimmune lymphoproliferative syndrome

Autoimmune lymphoproliferative syndrome (ALPS) is characterized by childhood onset of lymphadenopathy, hepatosplenomegaly, autoimmune cytopenias, elevated numbers of double-negative T (DNT) cells, and increased risk of lymphoma. Most cases of ALPS are associated with germline mutations of the FAS gene (type Ia), whereas some cases have been noted to have a somatic mutation of FAS primarily in their DNT cells. We sought to determine the proportion of patients with somatic FAS mutations among a group of our ALPS patients with no detectable germline mutation and to further characterize them. We found more than one-third (12 of 31) of the patients tested had somatic FAS mutations, primarily involving the intracellular domain of FAS resulting in loss of normal FAS signaling. Similar to ALPS type Ia patients, the somatic ALPS patients had increased DNT cell numbers and elevated levels of serum vitamin B(12), interleukin-10, and sFAS-L. These data support testing for somatic FAS mutations in DNT cells from ALPS patients with no detectable germline mutation and a similar clinical and laboratory phenotype to that of ALPS type Ia. These findings also highlight the potential role for somatic mutations in the pathogenesis of nonmalignant and/or autoimmune hematologic conditions in adults and children.

Using biomarkers to predict the presence of FAS mutations in patients with features of the autoimmune lymphoproliferative syndrome

To the Editor: The autoimmune lymphoproliferative syndrome (ALPS) is characterized by chronic lymphadenopathy, splenomegaly, autoimmune cytopenias, and expansion of T cell receptor (TCR) αβ+ CD3+CD4−CD8− (αβ-double-negative [DNT]) cells (see this article’s Table E1 in the Online Repository at www.jacionline.org). Approximately two thirds of the patients with ALPS symptoms are genetically characterized, and most have germline (ALPS Ia) or somatic (ALPS Ia-s) TNFRSF6 (FAS) mutations. A small number of patients have defects in genes encoding Fas ligand (ALPS Ib), caspase-10 (ALPS II), or neuroblatoma-RAS (NRAS) viral oncogene homolog (ALPS IV). In addition, a large group of patients with ALPS findings remain genetically uncharacterized (ALPS III), and yet another has an undefined ALPS-like syndrome (ALPS-phenotype; Table I).1,2 Given the clinical similarities among all these groups, we sought to develop a biomarkers-based algorithm to predict the presence or absence of FAS mutations in this setting. TABLE I Description of patients with ALPS and control groups included in the study To this end, we investigated 26 parameters including immunophenotyping, eosinophil and monocyte counts, serum or plasma vitamin B12 (B12), soluble FAS ligand (sFASL), immunoglobulins, and levels of 14 cytokines in 562 subjects classified into 6 categories (Tables I and E1). The number of measurements, medians, and first and third quartiles are presented in this article’s Tables E2 and E3 in the Online Repository at www.jacionline.org. A full description of the Methods can be found in the Online Repository at www.jacionline.org. Elevated αβ-DNT cells are a hallmark of ALPS, but their utility for predicting FAS mutations had not been previously evaluated. 3 Patients with ALPS Ia and Ia-s had a high percentage of αβ-DNT cells, with median values 5.1% and 7.7%, respectively, compared with 0.5% for control mutation-negative relatives (MNRs; P 4% found in 60% (90/152) of patients with type Ia and in the majority of patients with type Ia-s (7/9), but in only 13% (11/85) of patients with ALPS type III and ALPS-phenotype (Fig 1, A). This value was associated with a positive likelihood ratio (LR) of 5.0 and a posttest probability of 89.3% for harboring FAS mutations. Conversely, the presence of αβ-DNT cells in the 1% to 2% range decreased the posttest probability to 25%, with a LR of 0.19 (Fig 2, B and C; see this article’s Table E4 in the Online Repository at www.jacionline.org). FIG 1 Biomarkers in patients with ALPS and control groups. Dashed lines represent cut-off values used to calculate likelihood ratios. Bars denote median values. P values for the differences between groups were obtained by Mann-Whitney test and are shown above ... FIG 2 sFASL levels and combinations of biomarkers accurately predict FAS mutations. A, Scatter plot showing sFASL levels. Increasing (B) and decreasing (C) probabilities for having a FAS mutation according to the percentage of αβ-DNT cells, ... In line with previous reports, patients with ALPS, regardless of mutation status, had 16% made the diagnosis of ALPS very unlikely (LR = 0.17). Other described abnormalities including increased CD3+HLA-DR+ to CD3+CD25+ ratio and high number of B cells had no additional diagnostic utility.4 We also evaluated serum B12 levels in patients with ALPS and found very elevated median levels in ALPS Ia and Ia-s (2259 ng/L; 1653 ng/L) compared with control MNRs (474 ng/L; P 1500 ng/L was 4.0, with a posttest probability of 87%. In contrast, having B12 levels <1000 ng/L diminished the posttest probability to 35% (Fig 2, B and C; Table E4). Analysis of plasma cytokines revealed 2 additional biomarkers for ALPS: IL-18 and TNF-α. Median plasma IL-18 levels were elevated in patients with ALPS Ia and Ia-s compared with control MNRs (1041 pg/mL, 1526 pg/mL, and 208 pg/mL, respectively; P < .0001). Patients with ALPS III and ALPS-phenotype had median values of 521 pg/mL and 702 pg/mL, respectively (P < .001 compared with MNRs; Fig 1, D). Furthermore, IL-18 <500 pg/mL was rarely seen in patients with ALPS and FAS mutations (7/56), with an associated negative LR of 0.19. TNF-α levels were higher in all ALPS groups with median values of 5 pg/mL for ALPS Ia (P < .0001), 9 pg/mL for ALPS Ia-s (P < .05), 8 pg/mL for ALPS III (P < .0001), and 7 pg/mL for ALPS-phenotype (P < .0001) compared with 1.3 pg/mL for MNRs (Fig 1, E). As previously reported, IL-10 was markedly elevated in ALPS Ia and Ia-s compared with MNRs (P 40 pg/mL, contrasting with 26% (10/38) of patients with ALPS III and ALPS-phenotype. For levels of IL-10 >40 ng/mL, the positive LR was 3.8, with a posttest probability of 85% for having a FAS mutation. Notably, only 20% (29/141) of patients with ALPS Ia and no patients with ALPS Ia-s had IL-10 values <20 pg/mL, giving a negative LR of 0.31 and a posttest probability of 33% for FAS mutations (Fig 2, B and C; Table E4). A recent report documented high levels of sFASL in patients with ALPS.7We expanded these findings analyzing more than 200 patients and controls. Ninety-seven percent of patients with ALPS Ia (136/140) and all patients with ALPS Ia-s had plasma sFASL >200 pg/mL, with median values of 1114 pg/mL and 1329 pg/mL, respectively, compared with control MNR levels of 104 pg/mL (P <.0001 for both groups). Only modest elevations of sFASL were seen in patients with ALPS III and ALPS-phenotype, as well as healthy mutation-positive relatives, with median values of 208 pg/mL, 174 pg/mL, and 207 pg/mL, respectively (Fig 2, A). These findings make sFASL the most sensitive biomarker to rule out a FAS mutation, with values <200 pg/mL associated with a negative LR of 0.05 and a posttest probability of 7.7% (Fig 2, C; Table E4). Soluble FASL also showed a strong positive correlation with IL-10 (r = 0.8;P < .0001) and a moderate correlation with αβ-DNTcells (r = 0.6; P < .0001) and B12 levels (r = 0.69; P < .0001; see this article’s Fig E1, A, in the Online Repository at www.jacionline.org). The area under the ROC curve for sFASL, αβ-DNT cells, B12, and IL-10 levels were calculated to evaluate how well they discriminate patients with a FAS mutation from those without (Fig E1, B). The area under the curve for sFASL was 0.9 (defines an excellent test) and for αβ-DNT cells was 0.81. B12 and IL-10 exhibited areas significantly less than sFASL (P < .05), with values of 0.76 and 0.77. We next evaluated whether combinations of αβ-DNT cells, B12, IL-10, IL-18, and sFASL would have increased power to predict or exclude FAS mutations in patients suspected of ALPS (Fig 2, B; Table E4). The combination of αβ-DNT cells >4% with B12 >1500 ng/L or IL-10 >40 pg/mL or IL-18 >500 ng/mL or sFASL >300 pg/mL was associated with >95% probability of having a FAS mutation. Conversely, having αβ-DNT cells <2% in combination with IL-10 <20 pg/mL or B12 <1000 ng/L or IL-18 <500 ng/mL decreased the probability of a FAS mutation to less than 10% (Fig 2, C; Table E4). Finally, finding αβ-DNT cells <2% and sFASL <200 pg/mL resulted in <2% probability for a FAS mutation. In conclusion, the biomarkers described should aid in the selection of patients with findings of ALPS for further diagnostic workup. In addition, the presence of a combination of markers strongly suggestive of a FAS mutation in the setting of a negative genetic test should prompt a search for somatic mutations in sorted αβ-DNT cells.

Borrelia burgdorferi Lipoprotein–Mediated TLR2 Stimulation Causes the Down-Regulation of TLR5 in Human Monocytes

Toll-like receptors (TLRs) trigger innate immune responses via the recognition of conserved pathogen-associated molecular patterns. Lipoproteins from Borrelia burgdorferi, the agent of Lyme disease, activate inflammatory cells through TLR2 and TLR1. We show that stimulation of human monocytes with B. burgdorferi lysate, lipidated outer surface protein A, and triacylated lipopeptide Pam3CysSerLys4 results in the up-regulation of both TLR2 and TLR1 but the down-regulation of TLR5, the receptor for bacterial flagellin, and that this effect is mediated via TLR2. TLR4 stimulation had no effect on TLR2, TLR1, and TLR5 expression. Human monocytes stimulated with TLR5 ligands (including p37 or flaA, the minor protein from B. burgdorferi flagella) up-regulated TLR5. In addition, TLR2 stimulation rendered cells hyporesponsive to a TLR5 agonist. These results indicate that diverse stimuli can cause differential TLR expression, and we hypothesize that these changes may be useful for either the pathogen and/or the host.

Serological Diagnosis of Human Herpes Simplex Virus Type 1 and 2 Infections by Luciferase Immunoprecipitation System Assay

ABSTRACT Highly quantitative and high-throughput serological tests for evaluation of humoral responses to herpes simplex virus 1 (HSV-1) and HSV-2 are not available. The efficacy of luciferase immunoprecipitation system (LIPS) assays for antibody profiling and serologic diagnosis of HSV-1 and HSV-2 infection was investigated using a panel of five recombinant HSV antigens. Plasma samples from subjects seropositive for HSV-1 and/or HSV-2 or seronegative for HSV-1 and HSV-2 that had previously been analyzed by Western blotting and the Focus Plexus immunoassay were evaluated. The LIPS test measuring anti-gG1 antibody titers was 96% sensitive and 96% specific for detecting HSV-1 infection, compared with the Focus immunoassay, and was 92% sensitive and 96% specific, compared with Western blotting. The results for the anti-gG2 LIPS test for HSV-2 precisely matched those for Western blotting, with 100% sensitivity and 100% specificity, and showed robust antibody titers in all the HSV-2-infected samples that were over 1,000 times higher than those in HSV-2-negative or HSV-1-positive samples. Antibodies to three additional HSV-2 proteins, gB, gD, and ICP8, were detected in many of the HSV-1- and/or HSV-2-infected plasma samples and showed preferentially higher immunoreactivity in HSV-2-infected plasma. The titers of antibodies to these three HSV-2 antigens also significantly correlated with each other (R = 0.75 to 0.81; P < 0.0001). These studies indicate that the robust anti-gG1 and anti-gG2 antibody responses detected by LIPS assays are useful for HSV-1 and HSV-2 detection and suggest that profiling of antibody responses to a panel of HSV proteins may be useful for characterizing individual humoral responses to infection and for monitoring responses to vaccines.

IL-27 inhibits HIV-1 infection in human macrophages by down-regulating host factor SPTBN1 during monocyte to macrophage differentiation

IL-27 promotes the differentiation of monocytes to HIV-resistant macrophages by down-regulating host factor SPTBN1.

Successful granulocyte-colony stimulating factor treatment of Crohn's disease is associated with the appearance of circulating interleukin-10-producing T cells and increased lamina propria plasmacytoid dendritic cells

Granulocyte‐colony stimulating factor (G‐CSF) has proved to be a successful therapy for some patients with Crohns disease. Given the known ability of G‐CSF to exert anti‐T helper 1 effects and to induce interleukin (IL)‐10‐secreting regulatory T cells, we studied whether clinical benefit from G‐CSF therapy in active Crohns disease was associated with decreased inflammatory cytokine production and/or increased regulatory responses. Crohns patients were treated with G‐CSF (5 µg/kg/day subcutaneously) for 4 weeks and changes in cell phenotype, cytokine production and dendritic cell subsets were measured in the peripheral blood and colonic mucosal biopsies using flow cytometry, enzyme‐linked immunosorbent assay and immunocytochemistry. Crohns patients who achieved a clinical response or remission based on the decrease in the Crohns disease activity index differed from non‐responding patients in several important ways: at the end of treatment, responding patients had significantly more CD4+ memory T cells producing IL‐10 in the peripheral blood; they also had a greatly enhanced CD123+ plasmacytoid dendritic cell infiltration of the lamina propria. Interferon‐γ production capacity was not changed significantly except in non‐responders, where it increased. These data show that clinical benefit from G‐CSF treatment in Crohns disease is accompanied by significant induction of IL‐10 secreting T cells as well as increases in plasmacytoid dendritic cells in the lamina propria of the inflamed gut mucosa.