New cytokine therapeutics for inflammatory bowel disease

Conventional therapy for inflammatory bowel diseases rely on corticosteroids and 5-aminosalicylates combined with immunosuppressive agents for maintenance. These drugs are not always effective and may inflict serious side effects. Other therapies are therefore awaited. Infliximab, a monoclonal antibody against the pro-inflammatory cytokine TNF-alpha has been successfully applied as a treatment for Crohn's disease. The mechanism of action of this drug extends beyond the level of TNF-alpha scavenging and includes induction of apoptosis of effector cells. Numerous anti-TNF antibodies have been developed and are currently evaluated in clinical trials. Other targets for monoclonal antibodies include integrins and cytokines involved in T-cell differentiation and activation. Likewise recombinant proteins that moderate TNF bioactivity and lymphocyte function have been developed. The therapeutic effect of recombinant interleukin-10 seems to be dependent on local delivery of the protein. Antisense therapy targeting lymphocyte migration has also been tested in IBD. Finally, the conventional drug thalidomide and possibly MAP-kinase inhibitors may become novel treatment entities for IBD.     

Combination of IL-21 and IL-15 enhances tumour-specific cytotoxicity and cytokine production of TCR-transduced primary T cells

IL-21, and to a lesser extent IL-15, inhibits differentiation of antigen-primed CD8 T cells and promotes their homeostasis and anti-tumour activity. Here, we investigated molecular mechanisms behind tumour-specific responses of primary murine T lymphocytes engineered to express a TCR directed against human gp100/HLA-A2 following short-term exposure to IL-15 and/or IL-21. We demonstrated that IL-15 + IL-21, and to a lesser extent IL-21, enhanced antigen-specific T-cell cytotoxicity, which was related to enhanced expression of granzymes A and B, and perforin 1. Furthermore, IL-15 + IL-21 synergistically enhanced release levels and kinetics of T-cell IFNγ and IL-2, but not IL-10. Enhanced secretion of IFNγ was accompanied by increased gene expression and cytosolic protein content, and was restricted to effector memory T cells. To summarize, we show that IL-15 + IL-21 improves antigen-specific responses of TCR-transduced effector T cells at multiple levels, which provides a rationale to treat T cells with a combination of these cytokines prior to their use in adoptive TCR gene therapy.     

Glycolysis-dependent histone deacetylase 4 degradation regulates inflammatory cytokine production

Activation of the inflammatory response is accompanied by a metabolic shift to aerobic glycolysis. Here we identify histone deacetylase 4 (HDAC4) as a new component of the immunometabolic program. We show that HDAC4 is required for efficient inflammatory cytokine production activated by lipopolysaccharide (LPS). Surprisingly, prolonged LPS treatment leads to HDAC4 degradation. LPS-induced HDAC4 degradation requires active glycolysis controlled by GSK3β and inducible nitric oxide synthase (iNOS). Inhibition of GSK3β or iNOS suppresses nitric oxide (NO) production, glycolysis, and HDAC4 degradation. We present evidence that sustained glycolysis induced by LPS treatment activates caspase-3, which cleaves HDAC4 and triggers its degradation. Of importance, a caspase-3–resistant mutant HDAC4 escapes LPS-induced degradation and prolongs inflammatory cytokine production. Our findings identify the GSK3β-iNOS-NO axis as a critical signaling cascade that couples inflammation to metabolic reprogramming and a glycolysis-driven negative feedback mechanism that limits inflammatory response by triggering HDAC4 degradation.     

A novel heterodimeric cytokine consisting of IL-17 and IL-17F regulates inflammatory responses

CD4＋ helper T （TH） cells play crucial roles in immune responses. Recently a novel subset of TH cells, termed THIL-17, TH 17 or inflammatory TH （THi）, has been identified as critical mediators of tissue inflammation. These cells produce IL-17 （also called IL-17A） and IL-17F, two most homologous cytokines sharing similar regulations. Here we report that when overexpressed in 293T cells, IL-17 and IL-17F form not only homodimers but also heterodimers, which we name as IL-17A/F. Fully differentiated mouse THi cells also naturally secrete IL-17A/F as well as IL-17 and IL-17F homodimeric cytokines. Recombinant IL-17A/F protein exhibits intermediate levels of potency in inducing IL-6 and KC （CXCL 1） as compared to homodimeric cytokines. IL-17A/F regulation of IL-6 and KC expression is dependent on IL-17RA and TRAF6. Thus, IL-17A/F cytokine represents another mechanism whereby T cells regulate inflammatory responses and may serve as a novel target for treating various immune-mediated diseases.     

Suppressor of cytokine signaling 1 stringently regulates distinct functions of IL-7 and IL-15 in vivo during T lymphocyte development and homeostasis

SOCS1(-/-) mice accumulate within the thymus and periphery CD8(+) lymphocytes that express memory cell markers and display heightened in vitro responses to common gamma-chain cytokines. To investigate whether dysregulated homeostasis of T lymphocytes and acquisition of memory phenotype by CD8(+) cells in SOCS1(-/-) mice were mediated by IL-7 and/or IL-15 in vivo, we have generated SOCS1(-/-)IL-7(-/-), SOCS1(-/-)IL-15(-/-) and SOCS1(-/-)IL-7(-/-)IL-15(-/-) mice. We observed that in mice lacking SOCS1, either IL-7 or IL-15 skewed thymocyte development toward CD8 lineage, whereas IL-15 is the principal mediator of dysregulated homeostasis in the periphery. Homeostatic proliferation of SOCS1(-/-) CD8(+) lymphocytes in Rag1(-/-), Rag1(-/-)IL-7(-/-), Rag1(-/-)IL-15(-/-), and Rag1(-/-)IL-7(-/-)IL-15(-/-) mice showed that SOCS1 deficiency did not overcome the requirement for IL-7 and IL-15 to sustain homeostatic expansion. Differential expression of memory phenotype markers CD44, CD122, and Ly6C by SOCS1(-/-)IL-15(-/-) CD8(+) lymphocytes suggest that multiple signals contributed to the memory cell differentiation program. To address whether increased IL-15 responsiveness of SOCS1(-/-) CD8(+) lymphocytes required prior TCR sensitization, we generated SOCS1(-/-) H-Y TCR transgenic (Tg) mice. Using female SOCS1(-/-) H-Y TCR(tg) mice in Rag1(+/+) and Rag1(-/-) backgrounds, we show that acquisition of the memory phenotype by SOCS1-deficient CD8(+) lymphocytes did not require prior antigenic stimulation, but required the presence of activated T cells. SOCS1 deficiency accelerated the maturation of CD8 single-positive thymocytes expressing Tg TCR, but did not compromise negative selection in HY-TCR(tg) males. Our findings illustrate distinct functions for IL-7 and IL-15 in T lymphocyte development and homeostasis, and stringent regulation of these processes by SOCS1.     

STAT4 isoforms differentially regulate Th1 cytokine production and the severity of inflammatory bowel disease

STAT4, a critical regulator of inflammation in vivo, can be expressed as two alternative splice forms, a full-length STAT4alpha, and a STAT4beta isoform lacking a C-terminal transactivation domain. Each isoform is sufficient to program Th1 development through both common and distinct subsets of target genes. However, the ability of these isoforms to mediate inflammation in vivo has not been examined. Using a model of colitis that develops following transfer of CD4(+) CD45RB(high) T cells expressing either the STAT4alpha or STAT4beta isoform into SCID mice, we determined that although both isoforms mediate inflammation and weight loss, STAT4beta promotes greater colonic inflammation and tissue destruction. This correlates with STAT4 isoform-dependent expression of TNF-alpha and GM-CSF in vitro and in vivo, but not Th1 expression of IFN-gamma or Th17 expression of IL-17, which were similar in STAT4alpha- and STAT4beta-expressing T cells. Thus, higher expression of a subset of inflammatory cytokines from STAT4beta-expressing T cells correlates with the ability of STAT4beta-expressing T cells to mediate more severe inflammatory disease.     

Spontaneous in vitro IL-6 production in various intestinal segments in patients with inflammatory bowel disease

Interleukin-6 (IL-6) plays an important role in regulation of intestinal inflammatory processes in inflammatory bowel disease (IBD). The levels of IL-6 in media from cultured biopsy samples were determined by ELISA in 14 Crohn’s disease (CD) patients, 17 patients with ulcerative colitis (UC), and 24 healthy controls in terminal ileum, cecum, and rectum. Results were confirmed by measuring mRNA expression in selected patients. In CD patients, there were increased levels of IL-6 (expressed in picograms per milligram of biopsy tissue mass) in terminal ileum compared with controls (median, 617 vs. 90.4; p < 0.001). High IL-6 levels were found in the rectum of CD patients with active disease but normal endoscopic findings (791 vs. 131; p < 0.05). This result was confirmed by mRNA expression. There was a substantial increase of IL-6 levels in cultured cecal (median, 327 vs. 94.0; p < 0.001) and rectal mucosa (median, 282 vs.131; p < 0.05) but not in ileal mucosa of UC patients. In conclusion, IL-6 production was higher in IBD patients than in controls; it correlated with disease activity and varied among different intestinal segments. In clinically active CD patients without rectal involvement, high IL-6 levels in cultured rectal mucosa suggest immune stimulation even in the absence of macroscopic inflammation.     

Distinct roles of IL-22 in human psoriasis and inflammatory bowel disease

IL-22, an IL-10 family cytokine, is produced by different leukocyte subsets, including T cells, NK cells and lymphoid tissue inducer (LTi) cells. IL-22 mediates the crosstalk between leukocytes and tissue epithelia because its receptor is preferentially expressed on various tissue epithelial cells. IL-22 is essential for host defense against infections of extracellular pathogens, such as bacteria and yeasts, by eliciting various innate defensive mechanisms from tissue epithelial cells and promoting wound-healing responses. In autoimmune diseases, however, diverse tissue microenvironments and underlying pathogenic mechanisms may result in opposing contributions of IL-22 in disease progression. For example, in psoriasis, IL-22 can synergize with other proinflammatory cytokines to induce many of the pathogenic phenotypes from keratinocytes and exacerbate disease progression. In contrast, IL-22 plays a beneficial role in IBD by enhancing barrier integrity and epithelial innate immunity of intestinal tract.     

Suppressor of cytokine signaling 1 regulates IL-15 receptor signaling in CD8+CD44high memory T lymphocytes

T lymphocyte survival, proliferation, and death in the periphery are dependent on several cytokines. Many of these cytokines induce the expression of suppressor of cytokine signaling-1 (SOCS1), a feedback inhibitor of JAK kinases. However, it is unclear whether the cytokines that regulate T lymphocyte homeostasis are critically regulated by SOCS1 in vivo. Using SOCS1(-/-)IFN-gamma(-/-) mice we show that SOCS1 deficiency causes a lymphoproliferative disorder characterized by decreased CD4/CD8 ratio due to chronic accumulation of CD8+CD44(high) memory phenotype T cells. SOCS1-deficient CD8+ T cells express elevated levels of IL-2Rbeta, show increased proliferative response to IL-15 and IL-2 in vitro, and undergo increased bystander proliferation and vigorous homeostatic expansion in vivo. Sorted CD8+CD44(high) T cells from SOCS1(-/-)IFN-gamma(-/-) mice respond 5 times more strongly than control cells, indicating that SOCS1 is a critical regulator of IL-15R signaling. Consistent with this idea, IL-15 stimulates sustained STAT5 phosphorylation in SOCS1-deficient CD8+ T cells. IL-15 strongly induces TNF-alpha production in SOCS1-deficient CD8+ T cells, indicating that SOCS1 is also a critical regulator of CD8+ T cell activation by IL-15. However, IL-15 and IL-2 induce comparable levels of Bcl-2 and Bcl-x(L) in SOCS1-deficient and SOCS1-sufficient CD8+ T cells, suggesting that cytokine receptor signals required for inducing proliferation and cell survival signals are not identical. These results show that SOCS1 differentially regulates common gamma-chain cytokine signaling in CD8+ T cells and suggest that CD8+ T cell homeostasis is maintained by distinct mechanisms that control cytokine-mediated survival and proliferation signals.     

Inflammatory bowel disease: the role of inflammatory cytokine gene polymorphisms

The mechanisms responsible for development of inflammatory bowel disease (IBD) have not been fully elucidated, although the main cause of disease pathology is attributed to up-regulated inflammatory processes. The aim of this study was to investigate frequencies of polymorphisms in genes encoding pro-inflammatory and anti-inflammatory markers in IBD patients and controls. We determined genotypes of patients with IBD (n= 172) and healthy controls (n= 389) for polymorphisms in genes encoding various cytokines (interleukin (IL)-1beta, IL-6, tumour necrosis factor (TNF), IL-10, IL-1 receptor antagonist). Association of these genotypes to disease incidence and pathophysiology was investigated. No strong association was found with occurrence of IBD. Variation was observed between the ulcerative colitis study group and the control population for the TNF-alpha-308 polymorphism (p= 0.0135). There was also variation in the frequency of IL-6-174 and TNF-alpha-308 genotypes in the ulcerative colitis group compared with the Crohn's disease group (p= 0.01). We concluded that polymorphisms in inflammatory genes are associated with variations in IBD phenotype and disease susceptibility. Whether the polymorphisms are directly involved in regulating cytokine production, and consequently pathophysiology of IBD, or serve merely as markers in linkage disequilibrium with susceptibility genes remains unclear.     

Immunization of mice with T cell-dependent antigens promotes IL-6 and TNF-alpha production in muscle cells

IL-6 and TNF-alpha are proinflammatory cytokines involved in various inflammatory or non-inflammatory disorders characterized by muscle wasting. While infiltrating leukocytes are known to be the major source of these cytokines, it is unclear whether muscle cells also contribute to local inflammation. In this study, we first showed that cultured muscle cells and naive mouse muscle tissue were capable of producing IL-6 and TNF-alpha. We demonstrated an increased expression of IL-6 and TNF-alpha on muscle sections of C57BL/6J mice immunized with acetylcholine receptor (AChR) in the complete Freund's adjuvant (CFA) or with CFA only. In the presence of IL-6 or TNF-alpha, cultured AChR-expressing mouse (G-8) and human (TE671) skeletal muscle cells showed significantly decreased alpha-bungarotoxin-binding sites as measured by cellular ELISA. Moreover, IL-6- or TNF-alpha-treated muscle cells displayed a considerable increase in apoptotic cell ratios. Collectively, this study suggests a direct role for these two cytokines in muscle cell destruction and a possibility of muscle cell damage via an autocrine fashion.     

Allium sativum (garlic) suppresses leukocyte inflammatory cytokine production in vitro: potential therapeutic use in the treatment of inflammatory bowel disease

BACKGROUND: Cytokines involved in inflammatory bowel disease (IBD) direct a predominantly cell-mediated T- helper-1 (Th1) immune response. The nonspecific anti-inflammatory treatment being used in the management of patients with IBD has not changed much since the 1970s and new therapeutic agents are keenly sought. Several compounds isolated from Allium sativum (garlic) modulate leukocyte cell proliferation and cytokine production. METHODS: To investigate the possible therapeutic effects of garlic in the treatment of patients with IBD, whole blood and peripheral blood mononuclear cells (PBMCs) were stimulated in the presence of various concentrations of garlic extract and the effect on leukocyte cytokine production was determined in vitro using multiparameter flow cytometry. RESULTS: Monocyte interleukin (IL)-12 production was inhibited significantly in the presence of low concentrations of garlic extract (>or=0.1 microg/ml total protein). Monocyte IL-10 production increased significantly and monocyte tumor necrosis factor-alpha (TNF-alpha), IL-1alpha, IL-6, IL-8, T-cell interferon-gamma (IFN-gamma), IL-2, and TNF-alpha decreased significantly in the presence of >or=10 microg/ml garlic extract. Twenty to fifty percent of the immunomodulatory activity of garlic extract on cytokine production was acid labile. The inhibitory activity of methylprednisolone, a commonly used anti-inflammatory in IBD, with garlic on leukocyte cytokine production was additive. CONCLUSIONS: By inhibiting Th1 and inflammatory cytokines while upregulating IL-10 production, treatment with garlic extract may help to resolve inflammation associated with IBD. An in vivo animal model study needs to be undertaken to determine the significance of these in vitro findings.     

Efficient induction of primary and secondary T cell-dependent immune responses in vivo in the absence of functional IL-2 and IL-15 receptors

IL-2 and IL-15 are thought to be important cytokines for T cell-dependent immune responses. Mice deficient in IL-2, IL-2Ralpha, and IL-2Rbeta are each characterized by a rapid lethal autoimmune lymphoproliferative disorder that complicates their use in studies aimed at investigating the role of these cytokines and receptors for immune responses in vivo. We have previously characterized a novel transgenic (Tg) mouse on the IL-2Rbeta-/- genetic background (Tg-/- mice) that lacks autoimmune disease but still contains peripheral T cells that are nonresponsive to IL-2 and IL-15. In the present study, these mice were used to investigate the extent by which IL-2 and IL-15 are essential for T cell immunity in vivo. Tg-/- mice generated near normal primary and secondary Ab responses to OVA, readily mounted first and second set allogeneic skin graft rejection responses, and developed primary and recall CD8 T cell responses to vaccinia virus. However, Tg-/- mice generated a slightly lower level of IgG2a Abs to OVA, exhibited a somewhat delayed first set skin graft rejection response with lower allo-specific CTL, and developed a significantly lower number of IFN-gamma-producing vaccinia-specific CD8+ T cells. Thus, although T effector function is somewhat impaired, T cell immunity is largely functional in the absence of IL-2- and IL-15-induced signaling through IL-2Rbeta.     

IL-15 regulates CD8+ T cell contraction during primary infection

During the course of acute infection with an intracellular pathogen, Ag-specific T cells proliferate in the expansion phase, and then most of the T cells die by apoptosis in the following contraction phase, but the few that survive become memory cells and persist for a long period of time. Although IL-15 is known to play an important role in long-term maintenance of memory CD8+ T cells, the potential roles of IL-15 in CD8+ T cell contraction are not known. Using an adoptive transfer system of OT-I cells expressing OVA257-264/Kb-specific TCR into control, IL-15 knockout (KO) and IL-15 transgenic (Tg) mice followed by challenge with recombinant Listeria monocytogenes expressing OVA, we found that the survival of CD44+CD62L-CD127- effector OT-I cells during the contraction phase is critically dependent on IL-15. In correlation with the expression level of Bcl-2 in OT-I cells, the number of OT-I cells was markedly reduced in IL-15 KO mice but remained at a high level in IL-15 Tg mice during the contraction phase, compared with control mice. In vivo administration of rIL-15 during the contraction phase in IL-15 KO mice inhibited the contraction of effector OT-I cells accompanied by up-regulation of Bcl-2 expression. Furthermore, enforced expression of Bcl-2 protected the majority of effector OT-I cells from death in IL-15 KO mice after infection. These results suggest that IL-15 plays a critical role in protecting effector CD8+ T cells from apoptosis during the contraction phase following a microbial infection via inducing antiapoptotic molecules.     

The role of activated cytotoxic T cells in inflammatory bowel disease

The role of cell-mediated cytotoxicity in the pathogenesis of ulcerative colitis and Crohn's disease has been controversial since reports indicating either a decreased, or an increased, activity of cytotoxic T cells in active stages of inflammatory bowel disease exist. Some of these discrepancies may be attributed to the fact that so far mostly peripheral blood lymphocytes rather than intestinal T cells have been examined. To overcome some of these limitations we performed in situ hybridizations for the detection of perforin and granzyme A mRNA expressing cells, i.e. of cytotoxic cells activated in situ, in the affected intestinal mucosa. These studies revealed increased frequencies of activated, cytotoxic T cells in active stages of ulcerative colitis and Crohn's disease. Interestingly, activated perforin mRNA expressing T cells are present both in the CD4 and in the CD8 T cell subsets. In the latter T cell subset up to 60% of the mucosal T cells isolated from the affected sites express perforin mRNA at detectable levels. The elevated frequency of activated cytotoxic cells and their histological distribution also in close proximity to the epithelial cells may thus indicate an important role for cytotoxic cells in the pathogenesis of inflammatory bowel disease since activated cytotoxic T cells may further exacerbate the inflammatory process through the production of pro-inflammatory cytokines such as interferon-gamma or tumor necrosis factor-alpha, but also through the release of pro-inflammatory cytokines and chemokines upon lysis of epithelial cells and the increased influx of luminal antigens at the site of epithelial erosions.     

CD4(+) T cell-dependent airway mucus production occurs in response to IL-5 expression in lung

The potential role of airway interleukin-5 (IL-5) expression in eliciting mucus production was demonstrated in a pulmonary IL-5 transgenic mouse model (NJ.1726) in which naive transgenic mice display comparable levels of airway mucus relative to allergen-sensitized and -challenged wild-type mice. The intrinsic mucus accumulation of NJ.1726 was abolished in compound transgenic-gene knockout mice deficient of either CD4(+) cells [NJ.1726/CD4(-/-)] or alphabeta T cell receptor-positive (TCR(+)) cells [NJ.1726/alphabeta TCR(-/-)]. In addition, mucus production in naive NJ.1726 was inhibited by >90% after administration of the soluble anti-IL-4 receptor alpha-subunit antagonist. The loss of mucus production in NJ.1726/CD4(-/-), NJ.1726/alphabeta TCR(-/-), and anti-IL-4 receptor alpha-subunit antagonist-treated mice occurred notwithstanding the significant pulmonary eosinophilia and expansion of airway B cells induced by ectopic IL-5 expression. Furthermore, the loss of mucus accumulation occurred in these mice despite elevated levels of airway and peripheral IL-5, indicating that IL-5 does not directly induce goblet cell metaplasia and mucus production. Thus pulmonary expression of IL-5 alone is capable of inducing CD4(+) T cell-dependent goblet cell metaplasia, apparently mediated by IL-4 receptor alpha-subunit-ligand interactions, and represents a previously unrecognized novel pathway for augmenting allergen-induced mucus production.     

Hyperexpression of CD40 ligand (CD154) in inflammatory bowel disease and its contribution to pathogenic cytokine production.

CD40 ligand (CD40L or CD154), a type II membrane protein with homology to TNF, is transiently expressed on activated T cells and known to be important for B cell Ig production and for activation and differentiation of monocytes and dendritic cells. Both Crohn's disease and ulcerative colitis are characterized by local production of cytokines such as TNF and by an influx of activated lymphocytes into inflamed mucosa. Herein, we investigated whether CD40L signaling participates in immune responses in these diseases. Our results demonstrated that CD40L was expressed on freshly isolated lamina propria T cells from these patients and was functional to induce IL-12 and TNF production by normal monocytes, especially after IFN-gamma priming. The inclusion of a blocking mAb to CD40L or CD40 in such cocultures significantly decreased monocyte IL-12 and TNF production. Moreover, lamina propria and peripheral blood T cells from these patients, after in vitro activation with anti-CD3, showed increased and prolonged expression of CD40L as compared with controls. Immunohistochemical analyses indicated that the number of CD40+ and CD40L+ cells was significantly increased in inflamed mucosa, being B cells/macrophages and CD4+ T cells, respectively. These findings suggest that CD40L up-regulation is involved in pathogenic cytokine production in inflammatory bowel disease and that blockade of CD40-CD40L interactions may have therapeutic effects for these patients.     

IL-12-independent IFN-gamma production by T cells in experimental Chagas' disease is mediated by IL-18.

IL-12p35-deficient (IL-12p35(-/-)) mice were highly susceptible to Trypanosoma cruzi infection and succumbed during acute infection, demonstrating the crucial importance of endogenous IL-12 in resistance to experimental Chagas' disease. Delayed immune responses were observed in mutant mice, although comparable IFN-gamma and TNF-alpha blood levels as in wild-type mice were detected 2 wk postinfection. In vivo and in vitro analysis demonstrated that T cells, but not NK cells, were recruited to infected organs. Analysis of mice double deficient in the recombinase-activating gene 2 (RAG2) and IL-12p35, as well as studies involving T cell depletion, identified CD4(+) T cells as the cellular source for IL-12-independent IFN-gamma production. IL-18 was induced in IL-12p35(-/-) mice and was responsible for IFN-gamma production, as demonstrated by in vivo IL-18 neutralization studies. In conclusion, evidence is presented for an IL-12-independent IFN-gamma production in experimental Chagas' disease that is T cell and IL-18 dependent.