William F. Carson

Regulatory Role of B Cells in a Murine Model of Allergic Airway Disease

Mice sensitized to OVA and subjected to acute OVA aerosol exposures develop allergic airway disease (AAD). However, chronic continuous Ag exposure results in resolution of AAD and the development of local inhalational tolerance (LIT). Because we have previously observed the persistence of B cells in the bronchoalveolar lavage (BAL) and hilar lymph nodes (HLN) at the resolution stage of this model, we investigated the role of B cells in the modulation of AAD. Although B cell-deficient mice developed LIT, adoptive transfer of HLN B cells from LIT mice to OVA-sensitized recipients resulted in attenuated AAD following subsequent OVA aerosol exposure, as determined by reduced BAL leukocytosis and eosinophilia, decreased tissue inflammation, and absent methacholine hyper-responsiveness. In similar adoptive transfer studies, HLN B cells from AAD mice were without effect. The protection transferred by LIT HLN B cells was Ag specific and was associated with accumulation of Foxp3+ T regulatory cells regionally in BAL and HLN, but not systemically in the spleen. Fluorescent labeling of LIT HLN B cells before adoptive transfer demonstrated that these cells had the capacity to migrate to local inflammatory sites. In vitro assessment demonstrated that the LIT HLN B cells exerted this regulatory effect via TGF-β induced conversion of CD4+CD25− T effector cells into functionally suppressive CD4+CD25+Foxp3+ T regulatory cells. These findings illustrated a novel regulatory role for regional B cells in AAD and suggested a possible contributory role of B cells, along with other cell types, in the establishment of LIT.

Accumulation of regulatory T cells in local draining lymph nodes of the lung correlates with spontaneous resolution of chronic asthma in a murine model.

Background: Mice sensitized to ovalbumin develop allergic airway disease (AAD) with short-term aerosol challenge; however, airway inflammation resolves with long-term aerosol challenge, referred to as local inhalational tolerance (LIT). Methods: We sought to determine if resolution of airway inflammation correlated with increases in lymphocyte subsets in local lung compartments, including putative regulatory T cells. Results: At the AAD stage, total numbers of T and B lymphocytes in bronchoalveolar lavage (BAL) were significantly increased above controls; however, at LIT, T and B lymphocytes were significantly reduced compared to AAD. In the lung tissue, the only alteration was a significant increase in CD4+ CD25+ T cells at AAD. In the hilar lymph node (HLN), CD4+ and CD4+ CD25+ T cells were significantly increased at AAD and LIT. In addition, CD8+ T cells were significantly elevated in the HLN at LIT, and CD19+ B cells were significantly increased at AAD. Adoptive transfer of HLN lymphocytes to lymphopenic mice confirmed that AAD lymphocytes could induce airway inflammation in response to aerosol challenge, whereas LIT lymphocytes were unable to do so. Depletion of CD4+ CD25+ T cells in vivo resulted in exacerbation of inflammation at AAD and LIT. CD4+ CD25+ T cells in the HLN also displayed suppressive activity in vitro. Additionally, T cells expressing Foxp3 were increased in the BAL and HLN during LIT. Conclusions: These results indicate that lymphocytes with regulatory functions are increased and sustained in local lung compartments at LIT and that their appearance correlates with the resolution of lung inflammation.

Oral Bromelain Attenuates Inflammation in an Ovalbumin-induced Murine Model of Asthma.

Bromelain, a widely used pineapple extract with cysteine protease activity, has been shown to have immunomodulatory effects in a variety of immune system models. The purpose of the present study was to determine the effects of orally administered bromelain in an ovalbumin (OVA)-induced murine model of acute allergic airway disease (AAD). To establish AAD, female C57BL/6J mice were sensitized with intraperitoneal (i.p.) OVA/alum and then challenged with OVA aerosols for 3 days. Mice were gavaged with either (phosphate buffered saline)PBS or 200u2009mg/kg bromelain in PBS, twice daily for four consecutive days, beginning 1u2009day prior to OVA aerosol challenge. Airway reactivity and methacholine sensitivity, bronchoalveolar lavage (BAL) cellular differential, Th2 cytokines IL-5 and IL-13, and lung histology were compared between treatment groups. Oral bromelain-treatment of AAD mice demonstrated therapeutic efficacy as evidenced by decreased methacholine sensitivity (P ≤ 0.01), reduction in BAL eosinophils (P ≤ 0.02) and IL-13 concentrations (P ≤ 0.04) as compared with PBS controls. In addition, oral bromelain significantly reduced BAL CD19+ B cells (P ≤ 0.0001) and CD8+ T cells (P ≤ 0.0001) in AAD mice when compared with controls. These results suggest that oral treatment with bromelain had a beneficial therapeutic effect in this murine model of asthma and bromelain may also be effective in human conditions.

STAT3-mediated IL-17 production by postseptic T cells exacerbates viral immunopathology of the lung.

ABSTRACT Survivors of severe sepsis exhibit increased morbidity and mortality in response to secondary infections. Although bacterial secondary infections have been widely studied, there remains a paucity of data concerning viral infections after sepsis. In an experimental mouse model of severe sepsis (cecal ligation and puncture [CLP]) followed by respiratory syncytial virus (RSV) infection, exacerbated immunopathology was observed in the lungs of CLP mice compared with RSV-infected sham surgery mice. This virus-associated immunopathology was evidenced by increased mucus production in the lungs of RSV-infected CLP mice and correlated with increased IL-17 production in the lungs. Respiratory syncytial virus–infected CLP mice exhibited increased levels of TH2 cytokines and reduced interferon &ggr; in the lungs and lymph nodes compared with RSV-infected sham mice. In addition, CD4 T cells from CLP mice produced increased IL-17 in vitro irrespective of the presence of exogenous cytokines or blocking antibodies. This increased IL-17 production correlated with increased STAT3 transcription factor binding to the IL-17 promoter in CD4 T cells from CLP mice. Furthermore, in vivo neutralization of IL-17 before RSV infection led to a significant reduction in virus-induced mucus production and TH2 cytokines. Taken together, these data provide evidence that postseptic CD4+ T cells are primed toward IL-17 production via increased STAT3-mediated gene transcription, which may contribute to the immunopathology of a secondary viral infection.

Subcutaneous late phase responses are augmented during local inhalational tolerance in a murine asthma model

Acute exposure of sensitized mice to antigen elicits allergic airway disease (AAD) characterized by Th2 cytokine‐dependent pulmonary eosinophilia, methacholine hyperresponsiveness and antigen‐specific IgE elevation. However, chronic exposure induces a local inhalational tolerance (LIT), with resolution of the airway responses but persistent systemic IgE production. To further determine if systemic immunologic responses were maintained during LIT, we assessed subcutaneous late phase responses to ovalbumin in this model. Sensitized and AAD mice developed small subcutaneous responses to ovalbumin, with footpad thickness increasing to 113.7 and 113.6% of baseline, respectively. In comparison, LIT mice developed marked foot swelling (141.6%). Histologic examination confirmed increased inflammation in the chronic animals, with a significant contribution by eosinophils. Thus, the resolution of airway inflammatory responses with chronic antigen inhalation is a localized response, not associated with loss of systemic responses to antigen.

Monocytes to functional dendritic cells is often a bridge too far for cancer therapy

Interferons are a class of pro-inflammatory cytokines with important immunological functions, especially in regards to host defense against viral infections. Interferons are also unique cytokines in their ability to both be produced by and activate numerous cell types, of both hematopoietic and stromal origin. For example, interferons stimulate the activation of immune cells through the upregulation of major histocompatibility complex (MHC) molecules, promoting antigen presentation and lymphocyte activation(1). In addition, interferon has potent anti-proliferative/pro-apoptotic effects, and can signal many cell types to degrade mRNA and limit protein synthesis, in an attempt to limit viral replication in infected (or potentially infected) cells(2). n nInterferon’s ability to both activate the immune system and limit protein synthesis in a wide range of target cells makes it an attractive immunotherapy option for cancer, where concurrent activation of the immune system and blockade of tumor proliferation would appear beneficial. Recombinant interferon treatment has been used for the past decade as an immunotherapy option, with efficacy against hematologic tumors (such as leukemia and lymphoma(3)) and malignancies of the skin (such as multiple myeloma and AIDS-related Kaposi’s sarcoma(4, 5)). Unfortunately, not all malignancies show responsiveness to interferon treatment; for example, treatment of cervical cancer patients with recombinant interferon α2β (IFNα2β) often lacks efficacy, especially when used in patients suffering from advanced malignancies(6). A better understanding of the mechanisms underlying the refractory nature of non-hematologic/non-cutaneous tumors to interferon treatment is essential for improving the therapeutic efficiency of interferon, as well as providing a better understanding of the mechanisms of interferon activity. n nIn this issue of Translational Research, Roy and colleagues look to study the effects of IFNα2β treatment on cervical cancer patients through studying immune responses in advanced cervical cancer patients (CaCx-IIIB), with an emphasis on the modulation of monocyte function as compared to healthy controls(7). The authors compare the phenotype and effector function of peripheral blood monocytes (PBMCs) from CaCx-IIIB patients with healthy controls, to determine if monocytes from CaCx-IIIB suffer from activation defects prior to activation with IFNα2β. Secondly, the authors used the PBMCs from both CaCx-IIIB patients and healthy controls to generate immature dendritic cells (DCs) in vitro, and then study their maturation using IFNα2β. The authors conclude that PBMCs from CaCx-IIIB patients exhibit decreased activation potential as compared to healthy control PBMCs, and that activation with IFNα2β fails to overcome this activation defect. In addition, DCs derived from CaCx-IIIB patients exhibit significantly decreased antigen-presenting function, as well as a deficiency in their ability to promote cytotoxic TH1-type responses in cognate T cells. The authors conclude that both the decreased activation potential of PBMCs and the refractory IFNα2β responsiveness of PBMC-derived DCs from CaCx-IIIB patients may in part explain IFNα2β’s ineffectiveness as a cervical cancer immunotherapy option. n nDendritic cells play a critical role in promoting antitumor immunity both through the activation of cytotoxic CD8+ T cells and cytokine-producing CD4+ T cells(8). To accomplish this, host dendritic cells must upregulate antigen presentation machinery (such as MHC class I and II) and costimulatory factors (such as CD80/86) to activate the anti-tumor effector functions of antigen-specific T cells(9). Type I interferons such as IFNα2β have the ability to mature human monocytes both in vitro and in vivo (studies summarized by Santini et al(10)), and as such they make a promising candidate for cancer immunotherapy. However, the results of the study by Roy et al suggest that monocytes from patients suffering from advanced malignancies may be unresponsive to immunomodulation with IFNα2β. This also results in a decreased ability of IFNα2β-stimulated DCs from CaCx-IIIB patients to promote cytotoxic T cell activation and proliferation in response to tumor-derived antigens (i.e. HeLa cell extract). This may explain the reduced efficacy of IFNa2β treatment in the treatment of cervical cancer patients, especially in those patients suffering from advanced stages of malignancy. In addition, the identification of an interferon-unresponsive phenotype in CaCx-IIIB patients may be used as a biomarker for the appropriate use of IFNα2β treatment; for example, simple assays of PBMCs from cervical cancer patients may be used to determine if a patient is a prime candidate for IFNα2β treatment. n nOf particular interest is the apparent refractory nature of PBMCs to IFNα2β stimulation, even in the context of in vitro maturation into DCs. These results suggest that there may be cell-intrinsic defects in the PBMCs of CaCx-IIIB patients that limit their ability to respond to exogenous IFNα2β. The authors present evidence for this possibility by analyzing the expression of IFNα receptors on ex vivo differentiated DCs from healthy and CaCx-IIIB patients, showing a decrease in receptor expression on PBMC-derived DCs from cancer patients. It is important to note that the observed differences in cell surface phenotype come after a lengthy in vitro cell culture protocol involving the addition of recombinant cytokines and optimized culture media; this suggests that the deficiencies observed are not reversed by removal of the PBMCs from the affected patient, or by conditioning of the PBMCs with optimized growth and differentiation stimuli. Previous studies in human patients and animal models indicate that malignancies have the ability to reprogram immune cells to promote tumor growth; one example of this phenomenon is the tumor-associated macrophage (TAM) which helps support the malignancy through the promotion of angiogenesis and the production of growth factors(11). However, this “reprogramming” effect is often observed in vivo in the context of the tumor; the present study indicates not only an effect on circulating immune cells far from the site of tumorigenesis, but also that this phenotype may be hard-wired in the progenitor cell and any daughter cells which may arise. n nRecent studies into the effect of inflammation on subsequent immune responses have identified epigenetic regulation of gene expression as a possible mechanism for the maintenance of immunosuppressive phenotypes in cells. For example, increases in repressive histone modifications (resulting in gene silencing) have been observed in immune cells following significant inflammatory events, such as severe sepsis. In particular, peripheral dendritic cells from post-septic mice exhibit decreased activation potential, evidenced by decreased IL-12 production, which then correlates with repressive histone modification events at the IL12 promoter in these cells(12). Additionally, CD4+ T cell subsets from post-septic animals exhibit increases in permissive histone modification events at the promoter regions of Foxp3, correlating in increased regulatory T cells in these animals, increased suppressive activity of post-septic regulatory T cells, and an increased susceptibility of post-septic animals to solid tumor challenge(13). Similar evidence of epigenetic regulation has been shown in TAMs as well, as decreases in permissive histone modifications correlates with decreased expression of antigen presentation machinery, specifically MHC II(14). It is intriguing to hypothesize that a similar phenomenon may be occurring in the PBMCs of CaCx-IIIB patients, whereby an as-yet unknown mechanism is driving the epigenetic repression of IFNα2β receptors, resulting in unresponsiveness to type 1 interferon treatment. In such a scenario, coupling IFNα2β treatment with other treatments aimed at modifying or reversing epigenetic marks may help rescue IFNα2β’s efficacy in unresponsive cancer patients. n nClearly, solid tumors and advanced malignancies have a powerful ability to modulate the immune system, resulting in promotion of tumor growth at the expense of protective anti-tumor immunity. It therefore becomes critical to properly identify which patients will respond positively to immunotherapy. Also, treatments aimed at promoting tumor immunity must be able to overcome the immunosuppression mediated by advanced malignancies. In the present study, Roy et al have identified an important phenotype of cervical cancer PBMCs, providing an explanation for the refractory nature of IFNα2β treatment in these patients, and providing a useful phenotype for identifying candidates for IFNα2β in a clinical setting.