Innate lymphoid cells regulate CD4+ T-cell responses to intestinal commensal bacteria.

Innate lymphoid cells (ILCs) are a recently characterized family of immune cells that have critical roles in cytokine-mediated regulation of intestinal epithelial cell barrier integrity. Alterations in ILC responses are associated with multiple chronic human diseases, including inflammatory bowel disease, implicating a role for ILCs in disease pathogenesis. Owing to an inability to target ILCs selectively, experimental studies assessing ILC function have predominantly used mice lacking adaptive immune cells. However, in lymphocyte-sufficient hosts ILCs are vastly outnumbered by CD4(+) T cells, which express similar profiles of effector cytokines. Therefore, the function of ILCs in the presence of adaptive immunity and their potential to influence adaptive immune cell responses remain unknown. To test this, we used genetic or antibody-mediated depletion strategies to target murine ILCs in the presence of an adaptive immune system. We show that loss of retinoic-acid-receptor-related orphan receptor-γt-positive (RORγt(+)) ILCs was associated with dysregulated adaptive immune cell responses against commensal bacteria and low-grade systemic inflammation. Remarkably, ILC-mediated regulation of adaptive immune cells occurred independently of interleukin (IL)-17A, IL-22 or IL-23. Genome-wide transcriptional profiling and functional analyses revealed that RORγt(+) ILCs express major histocompatibility complex class II (MHCII) and can process and present antigen. However, rather than inducing T-cell proliferation, ILCs acted to limit commensal bacteria-specific CD4(+) T-cell responses. Consistent with this, selective deletion of MHCII in murine RORγt(+) ILCs resulted in dysregulated commensal bacteria-dependent CD4(+) T-cell responses that promoted spontaneous intestinal inflammation. These data identify that ILCs maintain intestinal homeostasis through MHCII-dependent interactions with CD4(+) T cells that limit pathological adaptive immune cell responses to commensal bacteria.

Cytokine pathways in allergic disease.

Cytokines are critical in allergic intercellular communication networks, and they contribute to disease pathology through the recruitment and activation of pro-inflammatory leukocytes and in chronic disease to pro-fibrotic/remodeling events. Th2 cytokines predominate primarily in mild to moderate allergic asthma, although clinical trials with inhibitors of IL-4 and IL-5 have not provided the robust efficacy observed in animal models of allergy. These results not only highlight the complexity of allergic disease, but they also point to the importance of other cytokine networks in driving pathology. The heterogeneous nature of the disease is emphasized by the fact that the Th2/Th1/Th17 cytokine balance can be influenced by the initiating allergic trigger. For example, the house dust mite allergen Der p 2 mimics the activity of MD-2 by presenting lipopolysaccharide to Toll-like receptor-4 for the activation of inflammatory genes including innate-type cytokines. Here we discuss the functions of the novel cytokine players, thymic stromal lymphopoetin (TSLP), IL-33, IL-25, and IL-9 and delineate nonredundant roles for IL-4 and IL-13 in allergic disease. Persistent efforts in the characterization of these and other cytokine networks will be essential for understanding the complex pathogenic mechanisms that underpin allergic disease and for guiding targeted therapeutic interventions.

IL-22 induces an acute-phase response.

IL-22 is made by a unique set of innate and adaptive immune cells, including the recently identified noncytolytic NK, lymphoid tissue-inducer, Th17, and Th22 cells. The direct effects of IL-22 are restricted to nonhematopoietic cells, its receptor expressed on the surface of only epithelial cells and some fibroblasts in various organs, including parenchymal tissue of the gut, lung, skin, and liver. Despite this cellular restriction on IL-22 activity, we demonstrate that IL-22 induces effects on systemic biochemical, cellular, and physiological parameters. By utilizing adenoviral-mediated delivery of IL-22 and systemic administration of IL-22 protein, we observed that IL-22 modulates factors involved in coagulation, including fibrinogen levels and platelet numbers, and cellular constituents of blood, such as neutrophil and RBC counts. Furthermore, we observed that IL-22 induces thymic atrophy, body weight loss, and renal proximal tubule metabolic activity. These cellular and physiological parameters are indicative of a systemic inflammatory state. We observed that IL-22 induces biochemical changes in the liver including induction of fibrinogen, CXCL1, and serum amyloid A that likely contribute to the reported cellular and physiological effects of IL-22. Based on these findings, we propose that downstream of its expression and impact in local tissue inflammation, circulating IL-22 can further induce changes in systemic physiology that is indicative of an acute-phase response.

IL-22 is required for Th17 cell-mediated pathology in a mouse model of psoriasis-like skin inflammation.

Psoriasis is a chronic skin disease resulting from the dysregulated interplay between keratinocytes and infiltrating immune cells. We report on a psoriasis-like disease model, which is induced by the transfer of CD4(+)CD45RB(hi)CD25(-) cells to pathogen-free scid/scid mice. Psoriasis-like lesions had elevated levels of antimicrobial peptide and proinflammatory cytokine mRNA. Also, similar to psoriasis, disease progression in this model was dependent on the p40 common to IL-12 and IL-23. To investigate the role of IL-22, a Th17 cytokine, in disease progression, mice were treated with IL-22-neutralizing antibodies. Neutralization of IL-22 prevented the development of disease, reducing acanthosis (thickening of the skin), inflammatory infiltrates, and expression of Th17 cytokines. Direct administration of IL-22 into the skin of normal mice induced both antimicrobial peptide and proinflammatory cytokine gene expression. Our data suggest that IL-22, which acts on keratinocytes and other nonhematopoietic cells, is required for development of the autoreactive Th17 cell-dependent disease in this model of skin inflammation. We propose that IL-22 antagonism might be a promising therapy for the treatment of human psoriasis.

Deletion of active ADAMTS5 prevents cartilage degradation in a murine model of osteoarthritis.

Human osteoarthritis is a progressive disease of the joints characterized by degradation of articular cartilage. Although disease initiation may be multifactorial, the cartilage destruction appears to be a result of uncontrolled proteolytic extracellular matrix destruction. A major component of the cartilage extracellular matrix is aggrecan, a proteoglycan that imparts compressive resistance to the tissue. Aggrecan is cleaved at a specific 'aggrecanase' site in human osteoarthritic cartilage; this cleavage can be performed by several members of ADAMTS family of metalloproteases. The relative contribution of individual ADAMTS proteases to cartilage destruction during osteoarthritis has not been resolved. Here we describe experiments with a genetically modified mouse in which the catalytic domain of ADAMTS5 (aggrecanase-2) was deleted. After surgically induced joint instability, there was significant reduction in the severity of cartilage destruction in the ADAMTS5 knockout mice compared with wild-type mice. This is the first report of a single gene deletion capable of abrogating the course of cartilage destruction in an animal model of osteoarthritis. These results demonstrate that ADAMTS5 is the primary 'aggrecanase' responsible for aggrecan degradation in a murine model of osteoarthritis, and suggest rational strategies for therapeutic intervention in osteoarthritis.

Epithelial-intrinsic IKKα expression regulates group 3 innate lymphoid cell responses and antibacterial immunity.

Innate lymphoid cells (ILCs) are critical for maintaining epithelial barrier integrity at mucosal surfaces; however, the tissue-specific factors that regulate ILC responses remain poorly characterized. Using mice with intestinal epithelial cell (IEC)-specific deletions in either inhibitor of κB kinase (IKK)α or IKKß, two critical regulators of NFκB activation, we demonstrate that IEC-intrinsic IKKα expression selectively regulates group 3 ILC (ILC3)-dependent antibacterial immunity in the intestine. Although IKKß(ΔIEC) mice efficiently controlled Citrobacter rodentium infection, IKKα(ΔIEC) mice exhibited severe intestinal inflammation, increased bacterial dissemination to peripheral organs, and increased host mortality. Consistent with weakened innate immunity to C. rodentium, IKKα(ΔIEC) mice displayed impaired IL-22 production by RORγt(+) ILC3s, and therapeutic delivery of rIL-22 or transfer of sort-purified IL-22-competent ILCs from control mice could protect IKKα(ΔIEC) mice from C. rodentium-induced morbidity. Defective ILC3 responses in IKKα(ΔIEC) mice were associated with overproduction of thymic stromal lymphopoietin (TSLP) by IECs, which negatively regulated IL-22 production by ILC3s and impaired innate immunity to C. rodentium. IEC-intrinsic IKKα expression was similarly critical for regulation of intestinal inflammation after chemically induced intestinal damage and colitis. Collectively, these data identify a previously unrecognized role for epithelial cell-intrinsic IKKα expression and TSLP in regulating ILC3 responses required to maintain intestinal barrier immunity.

Gp120-alum boosting of a Gag-Pol-Env DNA/MVA AIDS vaccine: poorer control of a pathogenic viral challenge.

Envelope protein immunogens may improve DNA or live-vectored HIV vaccines by complementing antiviral cellular responses with Env antibodies. We tested this concept by administering two immunizations of alum-adjuvanted HIV-1 89.6 gp120 to macaques being primed at weeks 0 and 8 with SHIV 89.6 Gag-Pol-Env DNA and boosted at week 24 with SHIV-89.6 Gag-Pol-Env recombinant modified vaccinia Ankara (MVA). Three hundred micrograms of gp120 was delivered with the second DNA prime and the MVA booster. Eight months after vaccination, all animals were challenged intrarectally with the related, yet serologically distinct, SHIV-89.6P. The gp120 immunizations raised binding, but not neutralizing antibody for the challenge virus, and allowed testing of whether gp120 vaccines that fail to raise neutralizing antibody can improve protection. Following the second gp120 immunization, the plus-gp120 group showed >10 times higher levels of binding antibody than the minus-gp120 group. These levels fell and were overall similar in both groups at the time of challenge. Following the second challenge, both groups had similar temporal patterns and heights of binding and neutralizing antibodies. However, the plus-gp120 group had less consistent control of viremia and higher levels of plasma viral RNA for the first year postchallenge. Assays for complement-dependent enhancing antibody revealed a trend toward higher levels of activity in the plus-gp120 group. This trend did not reach significance in our animal groups of 8. We conclude that gp120 inoculations that fail to raise neutralizing antibody do not improve the efficacy of Gag-Pol-Env DNA/MVA vaccines.

The pathology and immunology of atopic dermatitis.

Atopic dermatitis (AD) is a pruritic chronic inflammatory disease of the skin that is triggered by an underlying complicated interplay between the genetics of the individual and stimulation by allergens. Patients with AD demonstrate compromised barrier function that leads to activation of keratinocytes and immune cells which favor a strong Th2 bias. As a result of this immunological bias such patients also suffer from secondary pathogenic infections. A wide array of cytokines and chemokines interact to yield symptoms characteristic of AD. In addition, the involvement of different immunological cell types compounds our difficulty in understanding its immunopathogenesis. The use of various mouse models and transgenics has allowed us to intricately examine the functioning of the various molecules identified to play a role in AD. Such mouse models have also aided in the testing and development of various therapeutics for AD. This review is focused on examining the various factors contributing to the pathogenesis and exacerbation of AD as well as current treatments for AD. There is scope for improving the therapy of AD patients and thereby allowing them a better quality of life.

Inter-regulation of Th17 cytokines and the IL-36 cytokines in vitro and in vivo: implications in psoriasis pathogenesis.

Accumulating evidence indicates that IL-1 family members and Th17 cytokines have a pathogenic role in psoriasis. We investigated the regulatory interactions of the IL-1-like IL-36 cytokine family and the Th17 cytokines in the context of skin inflammation. We observed increased gene expression of all three IL-36 cytokines in a Th17-dominant psoriasis-like animal model. The induction was downregulated by neutralizing IL-22. Expression of the IL-36s was also induced in cultured primary human keratinocytes (KC) by IL-17A and tumor necrosis factor (TNF)-α, and IL-22 synergized with IL-17A and TNF-α. Furthermore, the IL-36s directly induced their own expression and the production of proinflammatory mediators (TNF-α, IL-6, IL-8) in KC. These functions were markedly enhanced with the addition of IL-17A or TNF-α to the cultures. Similarly, IL-36α and IL-36ß augmented IL-17A-mediated induction of antibacterial peptides. Finally, we show that the increased gene expression of IL-36 correlated with Th17 cytokines in the lesions of psoriatic patients. Our results indicate that the IL-36 cytokines are not only regulated by Th17 cytokines, but that they themselves can regulate the expression and enhance the function of Th17 cytokines. We propose that a feedback loop between the IL-36 and Th17 cytokines is involved in driving cytokine expression in psoriatic tissues.

Tumor necrosis factor alpha blockade exacerbates murine psoriasis-like disease by enhancing Th17 function and decreasing expansion of Treg cells.

OBJECTIVE: Patients with psoriasis and psoriatic arthritis respond well to tumor necrosis factor alpha (TNFalpha) blockers in general; however, there is now mounting evidence that a small cohort of patients with rheumatoid arthritis who receive TNFalpha blockers develop psoriasis. This study was undertaken to explore the mechanisms underlying TNFalpha blockade-induced exacerbation of skin inflammation in murine psoriasis-like skin disease. METHODS: Skin inflammation was induced in BALB/c scid/scid mice after they received CD4+CD45RB(high)CD25- (naive CD4) T cells from donor mice. These mice were treated with either anti-interleukin-12 (anti-IL-12)/23p40 antibody or murine TNFRII-Fc fusion protein and were examined for signs of disease, including histologic features, various cytokine levels in the serum, and cytokine or FoxP3 transcripts in the affected skin and draining lymph node (LN) cells. In a separate study, naive CD4+ T cells were differentiated into Th1 or Th17 lineages with anti-CD3/28 magnetic beads and appropriate cytokines in the presence or absence of TNFalpha. Cytokine gene expression from these differentiated cells was also determined. RESULTS: Neutralization of TNFalpha exacerbated skin inflammation and markedly enhanced the expression of the proinflammatory cytokines IL-1beta, IL-6, IL-17, IL-21, and IL-22 but suppressed FoxP3 expression in the skin and reduced the number of FoxP3-positive Treg cells in the draining LNs. TNFalpha also demonstrated a divergent role during priming and reactivation of naive T cells. CONCLUSION: These results reveal a novel immunoregulatory role of TNFalpha on Th17 and Treg cells in some individuals, which may account for the exacerbation of skin inflammation in some patients who receive anti-TNF treatments.

Blockade of the interleukin-21/interleukin-21 receptor pathway ameliorates disease in animal models of rheumatoid arthritis.

OBJECTIVE: Interleukin-21 (IL-21) is a T cell-derived cytokine that modulates T cell, B cell, and natural killer cell responses. In this study, the effects of blocking IL-21 were examined in 2 rodent models of rheumatoid arthritis (RA) to determine whether IL-21 contributes to their pathologic processes. METHODS: DBA/1 mice were immunized with bovine type II collagen and then treated with murine IL-21 receptor Fc fusion protein (IL-21R.Fc), which was initiated after the onset of arthritis symptoms in 10% of the cohort. The mice were assessed 3 times per week for signs of disease, including histologic features as well as serum cytokine, Ig, and cytokine messenger RNA (mRNA) levels in the paws. In a separate experiment, Lewis rats were immunized with Freund's complete adjuvant followed by administration of IL-21R.Fc at the peak of inflammation in the joints. Rats were assessed daily for histologic features and for scoring of arthritis severity. In addition, the effects of IL-21R.Fc on the production of interferon-gamma (IFNgamma) by T cells were examined. RESULTS: Treatment of DBA/1 mice with IL-21R.Fc reduced the clinical and histologic signs of collagen-induced arthritis. Nonspecific IgG1 levels were decreased in response to treatment. The levels of IL-6 mRNA in the paws and the serum IL-6 levels were decreased after treatment with IL-21R.Fc. IFNgamma mRNA levels were increased in the paws, and the addition of IL-21R.Fc to collagen-activated lymph node cultures enhanced the levels of IFNgamma. Collagen-specific spleen cell responses in IL-21R.Fc-treated mice were observed as reduced levels of IFNgamma and increased levels of IL-6. Treatment of Lewis rats with IL-21R.Fc after induction of adjuvant-induced arthritis resulted in reversal of disease signs and improvements in histologic parameters. CONCLUSION: These findings demonstrate a pathogenic role for IL-21 in animal models of RA, and support consideration of IL-21 as a therapeutic target in human RA.

B-cell depletion inhibits arthritis in a collagen-induced arthritis (CIA) model, but does not adversely affect humoral responses in a respiratory syncytial virus (RSV) vaccination model.

We report the development of a mouse B cell-depleting immunoconjugate (anti-CD22 monoclonal antibody [mAb] conjugated to calicheamicin) and its in vivo use to characterize the kinetics of CD22+ B-cell depletion and reconstitution in murine primary and secondary lymphoid tissues. The effect of B-cell depletion was further studied in a murine collagen-induced arthritis (CIA) model and a respiratory syncytial virus (RSV) vaccination model. Our results show that (1) the immunoconjugate has B-cell-specific in vitro and in vivo cytotoxicity; (2) B-cell reconstitution starts in the bone marrow and spleen around day 30 after depletion and is completed in all tissues tested by day 50; (3) B-cell depletion inhibits the development of clinical and histologic arthritis in the CIA model; (4) depletion of type II collagen antibody levels is not necessary for clinical and histologic prevention of CIA; and (5) B-cell depletion does not adversely affect memory antibody responses after challenge nor clearance of infectious virus from lungs in the RSV vaccination model. These results demonstrate for the first time that only B-cell reduction but not type II collagen antibody levels correlate with the prevention of arthritis and represent key insights into the role of CD22-targeted B-cell depletion in mouse autoimmunity and vaccination models.

Comparable in vivo efficacy of CD28/B7, ICOS/GL50, and ICOS/GL50B costimulatory pathways in murine tumor models: IFNgamma-dependent enhancement of CTL priming, effector functions, and tumor specific memory CTL.

Increasing evidence suggests that B7/CD28 interactions are important in clonal expansion and effector function of nai;ve CD4(+) T cells, whereas ICOS/GL50 interactions may optimize the responses of recently activated T(H) cells. In tumor models, it has been shown that engagement of ICOS, like CD28, by its ligands can be effective in enhancing tumor immunity. In this report, we have directly compared the in vivo efficacy of CD28 vs ICOS activation in the MethA fibrosarcoma and B16F1 melanoma tumor models. We studied the efficacy of systemic treatment of tumors with murine B7.2-IgG or GL50-IgG fusion proteins, and the therapeutic potential of B7.1 or GL50 vaccines given during various phases of the antitumor responses. In addition, we compare the efficacy of ICOS-ligand splice variants GL50 and GL50B in promoting tumor immunity. We find that each of these pathways is equally effective in promoting tumor immunity and that the efficacy of both GL50 and B7.1 vaccines is IFN-gamma but not IL-10 dependent. Our results suggest that CD28 or ICOS costimulation-based strategies may be equally efficacious as adjuvants to conventional cancer treatment.

Tumor cells secreting IL-13 but not IL-13Ralpha2 fusion protein have reduced tumorigenicity in vivo.

IL-13 is a Th2 cytokine that plays crucial roles in the pathophysiology of allergy, asthma and helminth infection. The high affinity receptor for IL-13, IL-13Ralpha2, may act as a decoy receptor for IL-13. The anti-tumor effect of IL-13 and its soluble receptor IL-13Ralpha2 have been examined in different tumor systems. Previous studies have shown that IL-13 enhances anti-tumor responses in some model systems, whereas IL-13Ralpha2Fc prevents IL-13 mediated suppression of tumor immuno-surveillance in a different model system. In this study, we have used a cytokine (receptor) gene therapy approach and studied the immune responses mediated by IL-13 and IL-13Ralpha2Fc in poorly immunogenic B16F1 melanoma and immunogenic MethA fibrosarcoma tumor models. We find that IL-13 reduces the tumorigenicity of B16F1 melanoma and MethA fibrosarcoma cells in vivo, most likely through the recruitment of neutrophils and macrophages. IL-13 mediated anti-tumor responses do not lead to the generation of tumor-specific T cells. Neither IL-13Ralpha2Fc gene transduction nor in vivo treatment with soluble IL-13Ralpha2Fc has a statistically significant effect of tumor growth. IL-13Ralpha2 deficient host background does not alter tumor growth, suggesting that endogenous levels of IL-13 do not contribute to an anti-tumor response in these models. We conclude that IL-13, but not soluble IL-13Ralpha2, has anti-tumor activity in the models described here, possibly by enhancing innate anti-tumor immunity.

IL-21 activates both innate and adaptive immunity to generate potent antitumor responses that require perforin but are independent of IFN-gamma.

IL-21 is a key factor in the transition between innate and adaptive immune responses. We have used the cytokine gene therapy approach to study the antitumor responses mediated by IL-21 in the B16F1 melanoma and MethA fibrosarcoma tumor models in mice. Retrovirally transduced tumor cells secreting biologically functional IL-21 have growth patterns in vitro similar to that of control green fluorescent protein-transduced cells, but are completely rejected in vivo. We show that IL-21 activates NK and CD8(+) T cells in vivo, thus mediating complete rejection of poorly immunogenic tumors. Rejection of IL-21-secreting tumors requires the presence of cognate IL-21R and does not depend on CD4(+) T cell help. Interestingly, perforin, but not IFN-gamma or other major Th1 and Th2 cytokines (IL-12, IL-4, or IL-10), is required for the IL-21-mediated antitumor response. Moreover, IL-21 results in 50% protection and 70% cure of nonimmunogenic tumors when given before and after tumor challenge, respectively, in C57BL/6 mice. We conclude that IL-21 immunotherapy warrants clinical evaluation as a potential treatment for cancer.

Characterization of and osteoarthritis susceptibility in ADAMTS-4-knockout mice.

OBJECTIVE: To determine the importance of the enzymatic activity of ADAMTS-4 in normal growth and development and to evaluate the role of ADAMTS-4 in the progression of osteoarthritis (OA). METHODS: We generated catalytic domain-deleted ADAMTS-4-transgenic mice and performed extensive gross and histologic analyses of various organs. The mice were challenged by surgical induction of joint instability leading to OA, to determine the importance of the enzymatic activity of ADAMTS-4 in the progression of the disease. The response of wild-type (WT) and ADAMTS-4-knockout (ADAMTS-4-KO) articular cartilage to interleukin-1 and retinoic acid challenge in vitro was also evaluated. RESULTS: ADAMTS-4-KO mice up to 1 year of age exhibited no gross or histologic abnormalities in 36 tissue sites examined. Despite evidence of ADAMTS-4 expression and activity in growth plates of WT mice, catalytic silencing of this proteinase caused no abnormalities in skeletal development, growth, or remodeling. There was no effect of ADAMTS-4 knockout on the progression or severity of OA 4 weeks or 8 weeks after surgical induction of joint instability. Enzymatic cleavage of aggrecan at the TEGE(373-374)ARGS site was clearly evident after exposure of articular cartilage from ADAMTS-4-KO mice to inflammatory cytokines. CONCLUSION: Although expression of the ADAMTS-4 gene has been found in many tissues throughout the body, deletion of enzymatic activity did not appear to have any effect on normal growth and physiology. Our study provides evidence that ADAMTS-4 is the primary aggrecanase in murine growth plates; however, deletion of its enzymatic activity did not affect normal long bone remodeling. Our results also lead to the hypothesis that, in the mouse, ADAMTS-4 is not the primary enzyme responsible for aggrecan degradation at the TEGE(373-374)ARGS site. The elucidation of the relative importance of ADAMTS-4 in the pathologic process of human OA will require examination of human OA tissues and evidence of disease modification in patients following therapeutic intervention.

Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine.

Heterologous prime/boost regimens have the potential for raising high levels of immune responses. Here, we report that DNA priming followed by a recombinant modified vaccinia Ankara (rMVA) booster has controlled a highly pathogenic immunodeficiency virus challenge in a Rhesus macaque model. Both the DNA and rMVA components of the vaccine expressed multiple immunodeficiency virus proteins. Two DNA inoculations at 0 and 8 weeks and a single rMVA booster at 24 weeks effectively controlled an intrarectal challenge administered 7 months after the booster. These highly promising findings provide hope that a relatively simple multiprotein DNA/MVA vaccine can help to control the AIDS epidemic.