The recruitment of CD4+CD25+Foxp3+T (Treg) cells is one of the most important mechanisms by which parasites down-regulate the immune system.
We compared the effects of Treg cells from Trichinella spiralis-infected mice and uninfected mice on experimental allergic airway inflammation in order to understand the functions of parasite-induced Treg cells. After four weeks of T. spiralis infection, we isolated Foxp3-GFP-expressing cells from transgenic mice using a cell sorter. We injected CD4+Foxp3+ cells from T. spiralis-infected [Inf(+)Foxp3+] or uninfected [Inf(-)Foxp3+] mice into the tail veins of C57BL/6 mice before the induction of inflammation or during inflammation. Inflammation was induced by ovalbumin (OVA)-alum sensitization and OVA challenge. The concentrations of the Th2-related cytokines IL-4, IL-5, and IL-13 in the bronchial alveolar lavage fluid and the levels of OVA-specific IgE and IgG1 in the serum were lower in mice that received intravenous application of Inf(+)Foxp3+ cells [IV(inf):+(+) group] than in control mice. Some features of allergic airway inflammation were ameliorated by the intravenous application of Inf(-)Foxp3+ cells [IV(inf):+(-) group], but the effects were less distinct than those observed in the IV(inf):+(+) group. We found that Inf(+)Foxp3+ cells migrated to inflammation sites in the lung and expressed higher levels of Treg-cell homing receptors (CCR5 and CCR9) and activation markers (Klrg1, Capg, GARP, Gzmb, OX40) than did Inf(-)Foxp3+ cells.
T. spiralis infection promotes the proliferation and functional activation of Treg cells. Parasite-induced Treg cells migrate to the inflammation site and suppress immune responses more effectively than non-parasite-induced Treg cells. The adoptive transfer of Inf(+)Foxp3+ cells is an effective method for the treatment and prevention of allergic airway diseases in mice and is a promising therapeutic approach for the treatment of allergic airway diseases.
Many studies have investigated the down-regulation of the immune system by parasite infection. CD4+CD25+Foxp3+T (Treg) cells are key players in parasite-mediated immune downregulation. Our previous study suggested that Treg cells recruited by Trichinella spiralis infection were the key cells mediating the amelioration of allergic airway inflammation in mice. In the present study, we investigated the functions of parasite-induced Treg cells using mice expressing GFP-tagged Foxp3. T. spiralis infection increased the number of Treg cells. Adoptive transfer of the parasite-induced Treg cells to mice with allergic airway inflammation ameliorated allergic airway inflammation. The transferred cells were recruited to inflammation sites in the lung. Cells from parasite-infected mice expressed higher levels of Treg-cell homing receptors and activation markers than did cells from uninfected mice. This study might help explain why immune disorders (often of unknown cause) are more prevalent among people in developed countries (areas with low parasite infection) than among those in developing countries (areas with parasite epidemics). Our finding might improve current cell therapy techniques and facilitate the development of new techniques that use parasites or parasite-borne materials to treat diverse immune disorders.
Recent evidence suggests that IL-17 contributes to airway hyperresponsiveness (AHR); however, the mechanisms that suppress the production of this cytokine remain poorly defined.
We sought to understand the cellular and molecular basis for suppression of established, IL-17-dependent allergic airways disease.
Mice were sensitized by airway instillations of ovalbumin (OVA) together with low levels of lipopolysaccharide. Leukocyte recruitment to the lung and AHR were assessed following daily challenges with aerosolized OVA. Flow cytometry and gene targeted mice were used to identify naturally-arising subsets of regulatory T cells (Tregs) and their cytokines required for the suppression of established allergic airway disease.
Allergic sensitization through the airway primed both effector and regulatory responses. Effector responses were initially dominant and led to airway inflammation and IL-17-dependent AHR. However, after multiple daily allergen challenges, IL-17 production and AHR declined, even though pulmonary levels of Th17 cells remained high. This loss of AHR was reversible and required the expansion of a Treg subset expressing both Foxp3 and inducible co-stimulator (ICOS). These Tregs also expressed the regulatory cytokines, IL-10, TGF-beta and IL-35. Whereas IL-10 and TGF-beta were dispensable for suppression of airway hyperresponsiveness, IL-35 was required. Analysis of human ICOS+ Tregs revealed that they also selectively expressed IL-35.
IL-35 production by ICOS+ Tregs can suppress IL-17 production and thereby reverse established, IL-17-dependent AHR in mice. The production of IL-35 by human ICOS+ Tregs suggests that targeting this pathway might be of therapeutic value for treating allergic asthma in humans.
Asthma; airway hyperresponsiveness; AHR; IL-17; Th17; Th2; IL-35; ICOS; ovalbumin
It is well established that female sex hormones have a pivotal role in inflammation. For instance, our group has previously reported that estradiol has proinflammatory actions during allergic lung response in animal models. Based on these findings, we have decided to further investigate whether T regulatory cells are affected by female sex hormones absence after ovariectomy. We evaluated by flow cytometry the frequencies of CD4+Foxp3+ T regulatory cells (Tregs) in central and peripheral lymphoid organs, such as the thymus, spleen and lymph nodes. Moreover, we have also used the murine model of allergic lung inflammation a to evaluate how female sex hormones would affect the immune response in vivo. To address that, ovariectomized or sham operated female Balb/c mice were sensitized or not with ovalbumin 7 and 14 days later and subsequently challenged twice by aerosolized ovalbumin on day 21. Besides the frequency of CD4+Foxp3+ T regulatory cells, we also measured the cytokines IL-4, IL-5, IL-10, IL-13 and IL-17 in the bronchoalveolar lavage from lungs of ovalbumine challenged groups. Our results demonstrate that the absence of female sex hormones after ovariectomy is able to increase the frequency of Tregs in the periphery. As we did not observe differences in the thymus-derived natural occurring Tregs, our data may indicate expansion or conversion of peripheral adaptive Tregs. In accordance with Treg suppressive activity, ovariectomized and ovalbumine-sensitized and challenged animals had significantly reduced lung inflammation. This was observed after cytokine analysis of lung explants showing significant reduction of pro-inflammatory cytokines, such as IL-4, IL-5, IL-13 and IL-17, associated to increased amount of IL-10. In summary, our data clearly demonstrates that OVA sensitization 7 days after ovariectomy culminates in reduced lung inflammation, which may be directly correlated with the expansion of Tregs in the periphery and further higher IL-10 secretion in the lungs.
Airway hyperresponsiveness (AHR) is one of the most prominent features of asthma, however, precise mechanisms for its induction have not been fully elucidated. We previously reported that systemic antigen sensitization alone directly induces AHR before development of eosinophilic airway inflammation in a mouse model of allergic airway inflammation, which suggests a critical role of antigen-specific systemic immune response itself in the induction of AHR. In the present study, we examined this possibility by cell transfer experiment, and then analyzed which cell source was essential for this process.
BALB/c mice were immunized with ovalbumin (OVA) twice. Spleen cells were obtained from the mice and were transferred in naive mice. Four days later, AHR was assessed. We carried out bronchoalveolar lavage (BAL) to analyze inflammation and cytokine production in the lung. Fluorescence and immunohistochemical studies were performed to identify T cells recruiting and proliferating in the lung or in the gut of the recipient. To determine the essential phenotype, spleen cells were column purified by antibody-coated microbeads with negative or positive selection, and transferred. Then, AHR was assessed.
Transfer of spleen cells obtained from OVA-sensitized mice induced a moderate, but significant, AHR without airway antigen challenge in naive mice without airway eosinophilia. Immunization with T helper (Th) 1 elicited antigen (OVA with complete Freund's adjuvant) did not induce the AHR. Transferred cells distributed among organs, and the cells proliferated in an antigen free setting for at least three days in the lung. This transfer-induced AHR persisted for one week. Interleukin-4 and 5 in the BAL fluid increased in the transferred mice. Immunoglobulin E was not involved in this transfer-induced AHR. Transfer of in vitro polarized CD4+ Th2 cells, but not Th1 cells, induced AHR. We finally clarified that CD4+CD62Llow memory/effector T cells recruited in the lung and proliferated, thus induced AHR.
These results suggest that antigen-sensitized memory/effector Th2 cells themselves play an important role for induction of basal AHR in an antigen free, eosinophil-independent setting. Therefore, regulation of CD4+ T cell-mediated immune response itself could be a critical therapeutic target for allergic asthma.
Tuberculosis is one of the world’s leading killers, stealing 1.4 million lives and causing 8.7 million new and relapsed infections in 2011. The only vaccine against tuberculosis is BCG which demonstrates variable efficacy in adults worldwide. Human infection with Mycobacterium tuberculosis results in the influx of inflammatory cells to the lung in an attempt to wall off bacilli by forming a granuloma. Gr1intCD11b+ cells are called myeloid-derived suppressor cells (MDSC) and play a major role in regulation of inflammation in many pathological conditions. Although MDSC have been described primarily in cancer their function in tuberculosis remains unknown. During M. tuberculosis infection it is crucial to understand the function of cells involved in the regulation of inflammation during granuloma formation. Understanding their relative impact on the bacilli and other cellular phenotypes is necessary for future vaccine and drug design.
We compared the bacterial burden, lung pathology and Gr1intCD11b+ myeloid-derived suppressor cell immune responses in M. tuberculosis infected NOS2-/-, RAG-/-, C3HeB/FeJ and C57/BL6 mice. Gr-1+ cells could be found on the edges of necrotic lung lesions in NOS2-/-, RAG-/-, and C3HeB/FeJ, but were absent in wild-type mice. Both populations of Gr1+CD11b+ cells expressed high levels of arginase-1, and IL-17, additional markers of myeloid derived suppressor cells. We then sorted the Gr1hi and Gr1int populations from M. tuberculosis infected NOS-/- mice and placed the sorted both Gr1int populations at different ratios with naïve or M. tuberculosis infected splenocytes and evaluated their ability to induce activation and proliferation of CD4+T cells. Our results showed that both Gr1hi and Gr1int cells were able to induce activation and proliferation of CD4+ T cells. However this response was reduced as the ratio of CD4+ T to Gr1+ cells increased. Our results illustrate a yet unrecognized interplay between Gr1+ cells and CD4+ T cells in tuberculosis.
In humans, the bacterial product lipopolysaccharide (LPS) has been associated with protection from allergic diseases such as asthma. However, in mouse models of allergic asthma, differential effects of LPS have been noted based on the dose. A low dose of LPS promotes Th2 responses and allergic disease but a high dose has been associated with suppression of allergic airway inflammation. Our recent work has described the ability of LPS to increase the frequency of CD11b+Gr1intF4/80+ (abbreviated as Gr1int cells) cells in the lung tissue of mice in a dose-dependent fashion that is dependent on TLR4 and the TLR adaptor protein, MyD88. Both phenotypically and morphologically, the cells were found to have similarities with myeloid-derived suppressor cells. Adoptive transfer of LPS-induced Gr1int cells suppressed allergen-induced airway inflammation suggesting that these myeloid cells may have regulatory functions in allergic asthma. Although the Gr1int cells are readily detectable in the lung tissue of LPS-treated mice, they are barely detectable in the lung-draining lymph nodes (LNs) or in the airway lumen. This causes selective enrichment of these cells over dendritic cells (DCs) in the tissue since migratory DCs are induced by LPS to migrate to the draining LNs for presentation of antigen to LN T cells. The Gr1int cells were found to blunt the ability of lung DCs to upregulate GATA-3 or to promote STAT5 activation in primed Th2 cells, both transcription factors having critical roles in Th2 effector function. Thus, a complete understanding of the generation and regulation of the Gr1int cells would provide new avenues to either promote or delete these cells for disease-specific immunoregulation.
CD11b+Gr1intF4/80+ regulatory cells; Dendritic cells; Allergic airway inflammation; LPS
Induction of endogenous regulatory T cells (Tregs) represents an exciting new potential modality for treating allergic diseases such as asthma. Tregs have been implicated in the regulation of asthma but the anatomic location where they exert their regulatory function, and the mechanisms controlling their migration necessary for their suppressive function in asthma are not known. Understanding these aspects of Treg biology will be important for harnessing their power in the clinic.
To determine the anatomic location where Tregs exert their regulatory function in the sensitization and effector phases of allergic asthma, and to determine the chemokine receptors that control the migration of Tregs to these sites in vivo in mice and in humans.
The clinical efficacy and the anatomic location of adoptively transferred chemokine receptor-deficient CD4+CD25+ Foxp3+ Tregs was determined in the sensitization and effector phases of allergic airway inflammation in mice. The chemokine receptor expression profile was determined on Tregs recruited into the human airway following bronchoscopic segmental allergen challenge of subjects with asthma.
We show that CCR7, but not CCR4, is required on Tregs to suppress allergic airway inflammation during the sensitization phase. In contrast, CCR4, but not CCR7, is required on Tregs to suppress allergic airway inflammation during the effector phase. Consistent with our murine studies, humans with allergic asthma had an increase in CCR4 expressing functional Tregs in the lung following segmental allergen challenge.
The location of Treg function differs during allergic sensitization and during allergen-induced recall responses in the lung, and that this differential localization is critically dependent on differential chemokine function.
Asthma; regulatory T cells; chemokines; CCR4; CCR7; segmental allergen challenge
We previously reported in an ovalbumin-induced model of allergic asthma that Fms-like tyrosine kinase 3 ligand (Flt3-L) reversed airway hyperresponsiveness (AHR) and airway inflammation, and increased the number of regulatory CD11chighCD8αhighCD11blow dendritic cells in the lung. In this study, we investigated the effect of Flt3-L in a clinically relevant aeroallergen-induced asthma on the phenotypic expression of lung T cells. Balb/c mice were sensitized and challenged with cockroach antigen (CRA), and AHR to methacholine was established. These mice received three intraperitoneal injections of anti-CD25 antibody (PC61; 250 μg) and Flt3-L (3 μg) daily for 10 days. Cytokines and Ig levels in the serum were measured and differential bronchoalveolar lavage fluid (BALF) cell counts were examined. Flt3-L reversed AHR to methacholine to the control level. Flt3-L significantly decreased levels of BALF IL-5, IFN-γ, eosinophilia and substantially increased IL-10 and the number of CD4+CD25+ Forkhead winged helix transcription factor box P3 (Foxp3+) IL-10+ T cells in the lung. Administration of PC61 antibody blocked the effect of Flt3-L and substantially increased AHR, eosinophilia, and BALF IL-5 and IFN-γ levels, and decreased BALF IL-10 levels and the number of CD4+CD25+Foxp3+IL-10+ T cells. Flt3-L significantly decreased CD62-L, but increased inducible costimulatory molecule and Foxp3 mRNA expression in the CD4+CD25+ T cells isolated from lungs of Flt3-L–treated, CRA-sensitized mice compared to CRA-sensitized mice without Flt3-L treatment and PBS control group. Flt3-L significantly inhibited the effect of CRA sensitization and challenge to increase GATA3 expression in lung CD4+CD25+ T cells. Collectively, these data suggest that the therapeutic effect of Flt3-L is mediated by increased density of naturally occurring CD4+CD25+Foxp3+IL-10+ICOS+ T-regulatory cells in the lung. Flt3-L could be a therapeutic strategy for the management and prevention of allergic asthma.
airway hyperresponsiveness; anti-CD25 antibody; Fms-like tyrosine kinase 3 ligand; Forkhead winged helix transcription factor box P3; naturally occurring CD4+CD25+ T-regulatory cells
We have previously shown that non-myeloablative total lymphoid irradiation/rabbit anti-thymocyte serum (TLI/ATS) conditioning facilitates potent donor-recipient immune tolerance following bone marrow transplantation (BMT) across major histocompatibility complex (MHC) barriers via recipient invariant natural killer T cell (iNKT cell)-derived IL-4-dependent expansion of donor Foxp3+ naturally occurring Treg (nTreg). Here we report a more specific mechanism. Wild-type (WT) BALB/c (H-2d) hosts were administered TLI/ATS and BMT from WT or STAT6−/− C57BL/6 (H-2b) donors. Donor nTreg following STAT6−/− BMT demonstrated no loss of proliferation in vivo, indicating that an IL-4 responsive population in the recipient rather than the donor drives donor nTreg proliferation. In GVHD target organs, three recipient CD11b+ cell subsets (Gr-1highCD11cneg; Gr-1intCD11cneg; and Gr-1lowCD11c+) were enriched early after TLI/ATS + BMT versus TBI/ATS + BMT. Gr-1lowCD11c+ cells induced potent H-2Kb+CD4+Foxp3+ nTreg proliferation in vitro in 72-hr MLR. Gr-1lowCD11c+ cells were significantly reduced in STAT6−/− and iNKT cell-deficient Jα18−/− BALB/c recipients after TLI/ATS + BMT. Depletion of CD11b+ cells resulted in severe acute GVHD, and adoptive transfer of WT Gr-1lowCD11c+ cells to Jα18−/− BALB/c recipients of TLI/ATS + BMT restored day 6 donor Foxp3+ nTreg proliferation and protection from CD8 effector T cell-mediated GVHD. Blockade of PD-L1 or PD-L2, but not CD40, TGF-β, Arginase 1, or iNOS inhibited nTreg proliferation in co-cultures of recipient-derived Gr-1lowCD11c+ cells with donor nTreg. Through iNKT-dependent Th2 polarization, myeloid-derived immunomodulatory DCs are expanded after non-myeloablative TLI/ATS conditioning and allogeneic BMT, induce PD-1 ligand dependent donor nTreg proliferation, and maintain potent graft-versus-host immune tolerance.
Graft-versus-host disease; transplantation; tolerance; T cells; monocytes
► A worm-derived product, ES-62, protects against allergic airway inflammation induced by ovalbumin in mice. ► Protection is associated with resetting of the Th1/Th2 balance and correlates with suppression of Th17 responses. ► The study provides important information on the mechanism of action of a parasitic helminth-derived immunomodulator. ► The immunomodulator offers novel and safe therapeutic potential in the treatment of allergic diseases.
We previously demonstrated inhibition of ovalbumin-induced allergic airway hyper-responsiveness in the mouse using ES-62, a phosphorylcholine-containing glycoprotein secreted by the filarial nematode, Acanthocheilonema viteae. This inhibition correlated with ES-62-induced mast cell desensitisation, although the degree to which this reflected direct targeting of mast cells remained unclear as suppression of the Th2 phenotype of the inflammatory response, as measured by eosinophilia and IL-4 levels in the lungs, was also observed. We now show that inhibition of the lung Th2 phenotype is reflected in ex vivo analyses of draining lymph node recall cultures and accompanied by a decrease in the serum levels of total and ovalbumin-specific IgE. Moreover, ES-62 also suppresses the lung infiltration by neutrophils that is associated with severe asthma and is generally refractory to conventional anti-inflammatory therapies, including steroids. Protection against Th2-associated airway inflammation does not reflect induction of regulatory T cell responses (there is no increased IL-10 or Foxp3 expression) but rather a switch in polarisation towards increased Tbet expression and IFNγ production. This ES-62-driven switch in the Th1/Th2 balance is accompanied by decreased IL-17 responses, a finding in line with reports that IFNγ and IL-17 are counter-regulatory. Consistent with ES-62 mediating its effects via IFNγ-mediated suppression of pathogenic Th2/Th17 responses, we found that neutralising anti-IFNγ antibodies blocked protection against airway inflammation in terms of pro-inflammatory cell infiltration, particularly by neutrophils, and lung pathology. Collectively, these studies indicate that ES-62, or more likely small molecule analogues, could have therapeutic potential in asthma, in particular for those subtypes of patients (e.g. smokers, steroid-resistant) who are refractory to current treatments.
Asthma; Airway inflammation; Parasitic helminth; ES-62; IFNγ; IL-4; IL-17; Neutrophil
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).
We sought to determine if resolution of airway inflammation correlated with increases in lymphocyte subsets in local lung compartments, including putative regulatory T cells.
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.
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.
Adoptive transfer; Allergic airway inflammation; Foxp3; Lymphocytes; Mouse; Ovalbumin; Regulatory T cells
NF-kB is a critical transcription factor for the production of many inflammatory cytokines. It is activated in the airway epithelium of human asthmatics and in mice after allergic stimulation. To examine the role of NF-kB activation in allergic inflammation we generated transgenic mouse lines that allowed for the inducible stimulation of NF-kB in airway epithelial cells. After allergic sensitization with ovalbumin and alum, mice were challenged daily with ovalbumin aerosols and NF-kB was activated in airway epithelium by administration of doxycycline. Enhancement of airway epithelial NF-kB expression alone did not lead to increased airway responsiveness to methacholine. However induction of epithelial NF-kB during allergic inflammation caused airway hyperresponsiveness, increased airway neutrophilic and lymphocytic inflammation and goblet cell hyperplasia. Accompanying the exaggerated inflammation was an increase in the cytokines, G-CSF, IL-15, and KC. Interestingly, the counter regulatory interleukin, IL-10, was suppressed by NF-kB activation. The epithelial NF-kB dependent modulation of these cytokines provides a plausible explanation for the increased inflammation seen with overexpression of NF-kB. Modulation of airway epithelial NF-kB activation enhances the airway hyperresponsiveness and mucus secretion found in the mouse lung during allergic inflammation. NF-kB represents a potential target for pharmacologic intervention in human asthma.
We have analyzed the human B-cell tumors that arise spontaneously in SCID mice who have been given transplants of peripheral blood lymphocytes from Epstein-Barr virus (EBV)-seropositive donors to determine if patterns of EBV gene expression are correlated with phenotypic changes in the tumor B cells. Tumor cells were separated into two B-cell subsets by cell sorting on the basis of differential coexpression of membrane CD23 and CD38. One subset showed intermediate levels of CD23 and CD38 expression (CD23intCD38int), while a second subset had low-level CD23 but high-level CD38 expression (CD23loCD38hi). The CD23intCD38int cells had a high proliferative index and secreted little immunoglobulin in vitro; the CD23loCD38hi cells had a low proliferative index and high-level immunoglobulin secretion. We next analyzed the sorted cells for viral transcripts associated with latency (EBNA-1, EBNA-2, and LMP-1) or lytic cycle replication (ZEBRA and gp350 envelope protein). Only latent cycle transcripts were found in CD23intCD38int cells, whereas lytic cycle transcripts and transforming virus were present in the CD23loCD38hi cells. Finally, we generated short-term cell lines from the sorted CD23intCD38int cells and transferred these cells to SCID recipients. The resulting secondary tumors were predominantly CD23loCD38hi, suggesting that the CD23intCD38int lymphoblastoid cells are precursors to the well-differentiated, plasmacytoid CD23loCD38hi cells. These observations are discussed in the context of a three-step model for EBV-associated lymphomagenesis in humans.
Autologous haematopoietic stem cell transplantation has been tried as one experimental strategy for the treatment of patients with aggressive multiple sclerosis refractory to other immunotherapies. The procedure is aimed at ablating and repopulating the immune repertoire by sequentially mobilizing and harvesting haematopoietic stem cells, administering an immunosuppressive conditioning regimen, and re-infusing the autologous haematopoietic cell product. ‘Non-myeloablative’ conditioning regimens to achieve lymphocytic ablation without marrow suppression have been proposed to improve safety and tolerability. One trial with non-myeloablative autologous haematopoietic stem cell transplantation reported clinical improvement and inflammatory stabilization in treated patients with highly active multiple sclerosis. The aim of the present study was to understand the changes in the reconstituted immune repertoire bearing potential relevance to its mode of action. Peripheral blood was obtained from 12 patients with multiple sclerosis participating in the aforementioned trial and longitudinally followed for 2 years. We examined the phenotype and function of peripheral blood lymphocytes by cell surface or intracellular staining and multi-colour fluorescence activated cell sorting alone or in combination with proliferation assays. During immune reconstitution post-transplantation we observed significant though transient increases in the proportion of CD4+FoxP3+ T cells and CD56high natural killer cell subsets, which are cell subsets associated with immunoregulatory function. CD8+CD57+ cytotoxic T cells were persistently increased after therapy and were able to suppress CD4+ T cell proliferation with variable potency. In contrast, a CD161high proinflammatory CD8+ T cell subset was depleted at all time-points post-transplantation. Phenotypic characterization revealed that the CD161highCD8+ T cells were mucosal-associated invariant T cells, a novel cell population originating in the gut mucosa but expressing the central nervous system-homing receptor CCR6. Detection of mucosal-associated invariant T cells in post-mortem multiple sclerosis brain white matter active lesions confirmed their involvement in the disease pathology. Intracellular cytokine staining demonstrated interferon γ and interleukin 17 production and lack of interleukin 10 production, a pro-inflammatory profile. Mucosal-associated invariant T cell frequency did not change in patients treated with interferon β; and was more depleted after autologous haematopoietic stem cell transplantation than in patients who had received high-dose cyclophosphamide (n = 7) or alemtuzumab (n = 21) treatment alone, suggesting an additive or synergistic effect of the conditioning regime components. We propose that a favourably modified balance of regulatory and pro-inflammatory lymphocytes underlies the suppression of central nervous system inflammation in patients with multiple sclerosis following non-myeloablative autologous haematopoietic stem cell transplantation with a conditioning regimen consisting of cyclophosphamide and alemtuzumab.
stem cells; multiple sclerosis; T cells; proinflammatory cytokines; immune regulation
The continual rise of asthma in industrialised countries stands in strong contrast to the situation in developing lands. According to the modified Hygiene Hypothesis, helminths play a major role in suppressing bystander immune responses to allergens, and both epidemiological and experimental studies suggest that the tropical parasitic trematode Schistosoma mansoni elicits such effects. The focus of this study was to investigate which developmental stages of schistosome infection confer suppression of allergic airway inflammation (AAI) using ovalbumin (OVA) as a model allergen. Moreover, we assessed the functional role and localization of infection-induced CD4+Foxp3+ regulatory T cells (Treg) in mediating such suppressive effects. Therefore, AAI was elicited using OVA/adjuvant sensitizations with subsequent OVA aerosolic challenge and was induced during various stages of infection, as well as after successful anti-helminthic treatment with praziquantel. The role of Treg was determined by specifically depleting Treg in a genetically modified mouse model (DEREG) during schistosome infection. Alterations in AAI were determined by cell infiltration levels into the bronchial system, OVA-specific IgE and Th2 type responses, airway hyper-sensitivity and lung pathology. Our results demonstrate that schistosome infection leads to a suppression of OVA-induced AAI when mice are challenged during the patent phase of infection: production of eggs by fecund female worms. Moreover, this ameliorating effect does not persist after anti-helminthic treatment, and depletion of Treg reverts suppression, resulting in aggravated AAI responses. This is most likely due to a delayed reconstitution of Treg in infected-depleted animals which have strong ongoing immune responses. In summary, we conclude that schistosome-mediated suppression of AAI requires the presence of viable eggs and infection-driven Treg cells. These data provide evidence that helminth derived products could be incorporated into treatment strategies that specifically target suppression of immune responses in AAI by inducing Treg cells.
Infections with schistosomes, such as S. mansoni, S. japonicum and S. haematobium, are considered a major public health concern. Morbidity arises through granulomatous responses to eggs that become trapped in infected tissues. Interestingly, schistosomes belong to the group of helminths that have been shown to reduce allergy or autoimmunity. Indeed, the evidence provided by epidemiological surveys and experimental animal models has been so overwhelming that such helminths are now included in the Hygiene Hypothesis. However, since helminths provoke immunological responses that are similar to those seen in allergy (increased eosinophilia and IgE) it is suggested that additional mechanisms dampen such allergic responses. Helminth-induced regulatory T cells (Treg) are considered a component of these modulatory networks. Using an allergic airway inflammation model, we have elucidated that schistosome-mediated protection requires patency, that is, active egg production from fecund female worms. In addition, protection was shown to be mediated by infection-induced Treg. Interestingly, in endemic countries it is usually individuals with strong patent infections that show reduced allergic prevalence. Thus, further research into the immunomodulatory capacity of schistosome-egg derived factors may elucidate novel drug candidates or enhance treatment strategies to reduce allergic responses on the cellular level.
Chemokine receptor (CCR) 5 is expressed on dendritic cells, macrophages, CD8 cells, memory CD4 T cells, and stromal cells, and is frequently used as a marker of T helper type 1 cells. Interventions that abrogate CCR5 or interfere with its ligand binding have been shown to alter T helper type 2–induced inflammatory responses. The role of CCR5 on allergic airway responses is not defined. CCR5-deficient (CCR5−/−) and wild-type (CCR5+/+) mice were sensitized and challenged with ovalbumin (OVA) and allergic airway responses were monitored 48 hours after the last OVA challenge. Cytokine levels in lung cell culture supernatants were also assessed. CCR5−/− mice showed significantly lower airway hyperresponsiveness (AHR) and lower numbers of total cells, eosinophils, and lymphocytes in bronchoalveolar lavage (BAL) fluid compared with CCR5+/+ mice after sensitization and challenge. The levels of IL-4 and IL-13 in BAL fluid of CCR5−/− mice were lower than in CCR5+/+ mice. Decreased numbers of lung T cells were also detected in CCR5−/− mice after sensitization and challenge. Transfer of OVA-sensitized T cells from CCR5+/+, but not transfer of CCR5−/− cells, into CCR5−/− mice restored AHR and numbers of eosinophils in BAL fluid after OVA challenge. Accordingly, the numbers of airway-infiltrating donor T cells were significantly higher in the recipients of CCR5+/+ T cells. Taken together, these data suggest that CCR5 plays a pivotal role in allergen-induced AHR and airway inflammation, and that CCR5 expression on T cells is essential to the accumulation of these cells in the airways.
rodent; T cells; cytokines; chemokines; lung
Viral and bacterial respiratory tract infections in early-life are linked to the development of allergic airway inflammation and asthma. However, the mechanisms involved are not well understood. We have previously shown that neonatal and infant, but not adult, chlamydial lung infections in mice permanently alter inflammatory phenotype and physiology to increase the severity of allergic airway disease by increasing lung interleukin (IL)-13 expression, mucus hyper-secretion and airway hyper-responsiveness. This occurred through different mechanisms with infection at different ages. Neonatal infection suppressed inflammatory responses but enhanced systemic dendritic cell:T-cell IL-13 release and induced permanent alterations in lung structure (i.e., increased the size of alveoli). Infant infection enhanced inflammatory responses but had no effect on lung structure. Here we investigated the role of hematopoietic cells in these processes using bone marrow chimera studies.
Neonatal (<24-hours-old), infant (3-weeks-old) and adult (6-weeks-old) mice were infected with C. muridarum. Nine weeks after infection bone marrow was collected and transferred into recipient age-matched irradiated naïve mice. Allergic airway disease was induced (8 weeks after adoptive transfer) by sensitization and challenge with ovalbumin. Reconstitution of irradiated naïve mice with bone marrow from mice infected as neonates resulted in the suppression of the hallmark features of allergic airway disease including mucus hyper-secretion and airway hyper-responsiveness, which was associated with decreased IL-13 levels in the lung. In stark contrast, reconstitution with bone marrow from mice infected as infants increased the severity of allergic airway disease by increasing T helper type-2 cell cytokine release (IL-5 and IL-13), mucus hyper-secretion, airway hyper-responsiveness and IL-13 levels in the lung. Reconstitution with bone marrow from infected adult mice had no effects.
These results suggest that an infant chlamydial lung infection results in long lasting alterations in hematopoietic cells that increases the severity of allergic airway disease in later-life.
CD4+CD25+Foxp3+ regulatory T cells (Tregs) regulate disease-associated immunity and excessive inflammatory responses, and numbers of CD4+CD25+Foxp3+ Tregs are increased during malaria infection. The mechanisms governing their generation, however, remain to be elucidated. In this study we investigated the role of commonly accepted factors for Foxp3 induction, TCR stimulation and cytokines such as IL-2, TGFβ and IL-10, in the generation of human CD4+CD25+Foxp3+ T cells by the malaria parasite Plasmodium falciparum. Using a co-culture system of malaria-infected red blood cells (iRBCs) and peripheral blood mononuclear cells from healthy individuals, we found that two populations of Foxp3hi and Foxp3int CD4+CD25hi T cells with a typical Treg phenotype (CTLA-4+, CD127low, CD39+, ICOS+, TNFRII+) were induced. Pro-inflammatory cytokine production was confined to the Foxp3int subset (IFNγ, IL-4 and IL-17) and inversely correlated with high relative levels of Foxp3hi cells, consistent with Foxp3hi CD4 T cell–mediated inhibition of parasite-induced effector cytokine T cell responses. Both Foxp3hi and Foxp3int cells were derived primarily from proliferating CD4+CD25− T cells with a further significant contribution from CD25+Foxp3+ natural Treg cells to the generation of the Foxp3hi subset. Generation of Foxp3hi, but not Foxp3int, cells specifically required TGFβ1 and IL-10. Add-back experiments showed that monocytes expressing increased levels of co-stimulatory molecules were sufficient for iRBC-mediated induction of Foxp3 in CD4 T cells. Foxp3 induction was driven by IL-2 from CD4 T cells stimulated in an MHC class II–dependent manner. However, transwell separation experiments showed that direct contact of monocytes with the cells that acquire Foxp3 expression was not required. This novel TCR-independent and therefore antigen-non specific mechanism for by-stander CD4+CD25hiFoxp3+ cell induction is likely to reflect a process also occurring in vivo as a consequence of immune activation during malaria infection, and potentially a range of other infectious diseases.
Infection with the malaria parasite Plasmodium falciparum affects 300–600 million people each year. Regulatory T cells (Tregs) expressing the transcription factor Foxp3, which drives genes involved in immunosuppression, are specialized immune cells that can inhibit both protective and harmful inflammatory responses during malaria. While Treg numbers are increased during malaria infection, little is known about how they are induced by the parasite. We addressed this question using an in vitro culture system to model the interaction of the malaria parasite with human immune cells. We found that the parasite induced soluble immune mediators, including the T cell growth-factor IL-2 and the regulatory proteins IL-10 and TGFβ, which drive the induction and expansion of Tregs. These Tregs expressed high levels of Foxp3 and suppressed the production of inflammation and protective immunity-driving mediators by concurrently induced effector T cells. Importantly, we demonstrate that induction of Tregs by the malaria parasite did not necessarily require direct contact with antigen-presenting cells. Our findings suggest that the parasite induces Tregs in an antigen non-specific manner, which may explain why malarial immunosuppression is not confined to malaria-specific immune responses, and provide new insights into the mechanisms governing Treg induction during malaria infection, and potentially other infectious diseases.
The emergence of regulatory T cells (Tregs) as central mediators of peripheral tolerance in the immune system has led to an important area of clinical investigation to target these cells for the treatment of autoimmune diseases such as type 1 diabetes. We have demonstrated earlier that in vitro treatment of T cells from healthy individuals with TX527, a low-calcemic analog of bioactive vitamin D, can promote a CD4+CD25highCD127low regulatory profile and imprint a migratory signature specific for homing to sites of inflammation. Towards clinical application of vitamin D-induced Tregs in autologous adoptive immunotherapy for type 1 diabetes, we show here that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and TX527 similarly imprint T cells from type 1 diabetes patients with a CD4+CD25highCD127low regulatory profile, modulate surface expression of skin- and inflammation-homing receptors, and increase expression of CTLA-4 and OX-40. Also, 1,25(OH)2D3 and TX527 treatment inhibit the production of effector cytokines IFN-γ, IL-9, and IL-17. Importantly, 1,25(OH)2D3 and TX527 promote the induction of IL-10-producing CD4+CD25highCD127low T cells with a stable phenotype and the functional capacity to suppress proliferation of autologous responder T cells in vitro. These findings warrant additional validation of vitamin D-induced Tregs in view of future autologous adoptive immunotherapy in type 1 diabetes.
Airway eosinophilia and Th2 lymphocytes-recruitment to the lung are one of the main pathological features of asthma. It is clear now that the axis chemokine/chemokine receptors have a role in controlling leukocyte recruitment and development of the inflammatory process observed in asthma. Although it has been reported that CCR9 receptor is expressed in asthmatic patients, it is not known whether CCR9 may have a regulatory role of the development of this disease. Our aim was to analyze the expression of CCR9 in a murine model of allergic airway inflammation (WT) and compared to CCR9 deficient (KO) mice.
Four groups of 6 to 8 weeks female CCR9-deficient mice were sensitized by intraperitoneal injections of 10 micrograms of ovalbumin (OVA) in alum (ALOH3) diluted in PBS, on days 1 and 8 of the established sensitization protocol. Aerosolised OVA was administered (1% in PBS) on days 15, 20 and 34. 24 hours after last OVA exposure, mice were sacrificed and bronchoalveolar lavage (BAL) fluid and cells were obtained. Total and differential cell numbers were obtained and characterized cell subpopulations by FACS analysis. Cytokine/chemokine levels were quantified by ELISA and qRT-PCR respectively.
Total cell numbers in BAL were no significantly different between WT and KO mice. Interestingly, reduction in the numbers of eosinophils was observed in CCR9 KO mice compared to WT mice. Histological analysis of lung tissue demonstrated a reduction in the granulocytic population (eosinophils) in CCR9 KO mice. Analysis of cell subpopulations by FACS demonstrated that CD4+ lymphocytes were significantly reduced but CD8+ and CD19+ lymphocytes numbers were not different between WT and CCR9-deficient mice. A population of CCR9+ Gr1+ was altered in KO mice and it correlated with cytological analysis. Furthermore, histological analysis demonstrated alteration in mucus production in allergic airway in CCR9 deficient mice, accompanied with a no-significant reduction of OVA-specific anti-IgE antibodies in serum at the time of analysis.
Altogether, these results suggest that CCR9 may be involved in recruitment of granulocytic cell subpopulation into the allergic airways and have an impact in the regulation of the chronic inflammatory process.
CD4+ T follicular helper (TFH) cells guide development and maturation of B cells and are crucial for effective antibody responses. Here we found rhesus macaque TFH cells, defined as CXCR5+CD4 T cells, contain two major populations: PD-1INT and PD-1HIGH cells. Of these, PD-1HIGHCD4+ T cells highly co-express ICOS but little CCR7, and reside in lymph node germinal centers (GCs), but not in blood. These cells secrete IL-21 and express transcriptional factor Bcl-6 at higher levels than CXCR5+PD-1INTCD4+ T cells. In addition, the frequency of PD-1HIGHCD4+ T cells is low in lymph nodes of newborns, but increases with age. Levels of PD-1HIGHCD4+ T cells correlate with mature B cells in lymph nodes, and PD-1 blockade in PD-1HIGHCD4+ T and B cell co-cultures significantly inhibits IgG production. In summary, PD-1HIGHCD4+ T cells residing in GC represent a specific TFH subset that contributes to maturation of B cells and IgG production.
follicular CD4 T helper cells; PD-1; B cell; IgG; lymph nodes
Antigen-specific immunotherapy (SIT) has been widely practiced in treating allergic diseases such as asthma. However, this therapy may induce a series of allergic adverse events during treatment. Peptide immunotherapy (PIT) was explored to overcome these disadvantages. We confirmed that multiple antigen peptides (MAPs) do not cause autoimmune responses, which led to the presumption that MAPs intervention could alleviate allergic airway inflammation without inducing adverse effects.
In this study, synthesized OVA323-339MAP octamers were subcutaneously injected into ovalbumin (OVA)-sensitized and -challenged Balb/c mice to observe its effect on allergic airway inflammation, Th2 immune response, and immune regulating function. It was confirmed that OVA sensitization and challenge led to significant peritracheal inflammatory, cell infiltration, and intensive Th2 response. Treatment of OVA323-339MAP octomers in the airway inflammation mice model increased CD4+CD25+Foxp3+ T regulatory (Treg) cells and their regulatory function in peripheral blood, mediastinal draining lymph nodes, and the spleen. Furthermore, OVA323-339MAP increased IL-10 levels in bronchial alveolar lavage fluid (BALF); up-regulated the expression of IL-10, membrane-bound TGF-β1, as well as Foxp3 in lung tissues; and up-regulated programmed death-1 (PD-1) and cytotoxic T lymphocyte associated antigen 4 (CTLA-4) on the surface of Treg cells. These results were further correlated with the decreased OVA specific immunoglobulin E (sIgE) level and the infiltration of inflammatory cells such as eosinophils and lymphocytes in BALF. However, OVA323-339 peptide monomers did not show any of the mentioned effects in the same animal model.
Our study indicates that OVA323-339MAP had significant therapeutic effects on mice allergic airway inflammation by regulating the balance of Th1/Th2 response through Treg cells in vivo.
Allergic airway inflammation; Specific immunotherapy; Multiple antigen peptide
Asthma is estimated to affect as many as 300 million people worldwide and its incidence and prevalence are rapidly increasing throughout the world, especially in children and within developing countries. Recently, there has been a growing interest in the use of potentially beneficial bacteria for allergic diseases. This study is aimed at exploring the therapeutic effects of long-term treatment with two different beneficial bacterial strains (Bifidobacterium breve M-16 V and Lactobacillus rhamnosus NutRes1) and a glucocorticoid (budesonide), as a reference treatment, on inflammatory response in a murine model for chronic allergic asthma.
To mimic the chronic disease in asthmatic patients, we used the murine ovalbumin-induced asthma model combined with prolonged allergen exposure. Airway function; pulmonary airway inflammation; airway remodelling, mRNA expression of pattern recognition receptors, Th-specific cytokines and transcription factors in lung tissue; mast cell degranulation; in vitro T cell activation; and expression of Foxp3 in blood Th cells were examined.
Lactobacillus rhamnosus reduced lung resistance to a similar extent as budesonide treatment in chronically asthmatic mice. Pulmonary airway inflammation, mast cell degranulation, T cell activation and airway remodelling were suppressed by all treatments. Beneficial bacteria and budesonide differentially modulated the expression of toll-like receptors (TLRs), nod-like receptors (NLRs), cytokines and T cell transcription factors. Bifidobacterium breve induced regulatory T cell responses in the airways by increasing Il10 and Foxp3 transcription in lung tissue as well as systemic by augmenting the mean fluorescence intensity of Foxp3 in blood CD4+ T cells.
These findings show that Bifidobacterium breve M-16 V and Lactobacillus rhamnosus NutRes1 have strong anti-inflammatory properties that are comparable to budesonide and therefore may be beneficial in the treatment of chronic asthma.
Allergic asthma; Beneficial bacteria; Glucocorticoids; Regulatory T cell
Allergic asthma is a dysregulation of the immune system which leads to the development of Th2 responses to innocuous antigens (allergens). Some infections and microbial components can re-direct the immune response toward the Th1 response, or induce regulatory T cells to suppress the Th2 response, thereby inhibiting the development of allergic asthma. Since Acinetobacter baumannii infection can modulate lung cellular and cytokine responses, we studied the effect of A. baumannii in modulating airway eosinophilia in a mouse model of allergic asthma. Ovalbumin (OVA)-sensitized mice were treated with live A. baumannii or phosphate buffered saline (PBS), then intranasally challenged with OVA. Compared to PBS, A. baumannii treatment significantly reduced pulmonary Th2 cytokine and chemokine responses to OVA challenge. More importantly, the airway inflammation in A. baumannii-treated mice was strongly suppressed, as seen by the significant reduction of the proportion and the total number of eosinophils in the bronchoalveolar lavage fluid. In addition, A. baumannii-treated mice diminished lung mucus overproduction and pathology. However, A. baumannii treatment did not significantly alter systemic immune responses to OVA. Serum OVA-specific IgE, IgG1 and IgG2a levels were comparable between A. baumannii- and PBS-treated mice, and tracheobronchial lymph node cells from both treatment groups produced similar levels of Th1 and Th2 cytokines in response to in vitro OVA stimulation. Moreover, it appears that TLR-4 and IFN-γ were not directly involved in the A. baumannii-induced suppression of airway eosinophilia. Our results suggest that A. baumannii inhibits allergic airway inflammation by direct suppression of local pulmonary Th2 cytokine responses to the allergen.
Asthma is a chronic disease associated with airway hyperresponsiveness (AHR), airway obstruction, and airway remodeling. NF-κB is a transcriptional factor that regulates and co-ordinates the expression of various inflammatory genes. The NF-κB subunits, p50 and Rel-A, are translocated to the nucleus by importin α3 and importin α4. There is growing evidence that vitamin D is a potent immunomodulator. However, the evidence for beneficial or adverse effects of vitamin D in asthma is still unclear.
In this study, we examined the effect of vitamin D status on AHR, airway inflammation, and cytokines in the bronchoalveolar lavage fluid (BALF) in a murine model of allergic asthma.
Female BALB/c mice were fed with special vitamin D-deficient or vitamin D-sufficient (2,000 IU/kg) or vitamin D-supplemented (10,000 IU/kg) diet for 13 weeks. Mice were sensitized and challenged with ovalbumin. The effect of vitamin D on lung histology, AHR, T-regulatory cells and BALF cytokines was examined. The expression of importin-α3 and Rel-A in the lung of OVA-sensitized mice was analyzed using immunofluorescence.
Vitamin D deficiency was associated with higher AHR in OVA-sensitized and challenged mice than those in vitamin D-sufficient mice. This was accompanied with marked signs of airway remodeling, high BALF eosinophilia, increased BALF pro-inflammatory cytokines, reduced BALF IL-10 levels, reduced blood T-regulatory cells, increased expression of importin-α3 and Rel-A in the lung tissue. Vitamin D supplementation attenuated the pro-inflammatory effects, but did not completely reverse the features of allergic airway inflammation.
Conclusion and Clinical Relevance
Vitamin D could be beneficial as an adjunct therapy in the treatment of allergic asthma.