Actinic cheilitis (AC) is an oral potentially malignant lesion which is the counterpart of actinic keratosis of the skin and has potential to develop into squamous cell carcinoma. Regulatory T cells (Tregs) have a critical role in modulating the antitumor immune responses. The presence of regulatory T cells in potentially malignant lesions has not been described. We chose investigate the involvement of regulatory T cells in potentially malignant lesions.
Chronic inflammation affects most stages of tumorigenesis, including initiation, promotion, malignant differentiation, invasion and metastasis. Inflammasomes have been described as involved with persistent inflammation and are known to exert both pro and antitumour effects. We evaluated the influence of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase (CASP)-1 in the antitumor immune response using a multistage model of squamous cell carcinoma (SCC) development. Absence of ASC and CASP-1 resulted in an earlier incidence and increased number of papilloma. Loss of inflammassome function in mice resulted in decreased presence of natural killer (NK), dendritic (DC), CD4(+), CD8(+) and CD45RB(+) T cells in the tumor lesions as well as in lymph nodes (LN) compared with WT mice. Increased percentage of CD4(+)CD25(+)Foxp3(+) T cells was associated with association with inflammasome loss of function. Moreover, significant differences were also found with neutrophils and macrophage infiltrating the lesions. Myeloperoxidase (MPO), but not elastase (ELA), activity oscillated among the groups during the SCC development. Levels of proinflammatory cytokines IL-1?, IL-18, Tumor Necrosis Factor (TNF)-? and Interferon (IFN)-? were decreased in the tumor microenvironment in the absence of inflammasome proteins. These observations suggest a link between inflammasome function and SCC tumorigenesis, indicating an important role for inflammasome activation in the control of SCC development.
Engagement of programmed death-1 (PD-1) with its two ligands [programmed death ligand-1 (PD-L1) and PD-L2] has been associated with the suppression of tumor-reactive T cells; however, the underlying mechanism for this T-cell dysfunction is not clear. We hypothesized that PD-1 and PD-L1 signals are, in part, responsible for squamous cell carcinoma (SCC) escape from immune antitumor regulation by modulation of the tumor environment. In the present study, we used a multistage model of SCC to examine the role of PD-1/PD-L1 activation during tumor development. Tumor sites presented an increased percentage of CD4(+) and CD8(+) T cells expressing PD-1 when compared with non-tumorigenic control mice, whereas the expression of PD-L1 was particularly increased in F4/80(+) macrophages in tumor sites. Further, the systemic immune neutralization of PD-1 resulted in a decreased number and delayed incidence rate of papillomas followed by a differential expression of cytokeratins, suggesting that the PD-1-PD-L1 interaction contributes to the progression of SCC by downregulation of antitumor responses. In fact, blocking PD-1 increased the percentage of CD8(+) and CD4(+) T cells, and the levels of interferon-? in the tumor sites. Our results indicated involvement of PD-1(+) T cells in SCC development and in the modulation of the inflammatory immune response.Summary: Our results showed that the immune neutralization of PD-1(+) cells resulted in decreased papilloma incidence associated with CD4(+) and CD8(+) T cells infiltrate, higher levels of interferon-? and decreased levels of transforming growth factor-? in the tumor lesions. These findings support the role of PD-1 blockade as a promising component of immunotherapy against SCC.
Macrophages (M?) play an essential role in innate immune responses and can either display a pro-inflammatory, classically activated phenotype (M1) or undergo an alternative activation program (M2) promoting immune regulation. M-CSF is used to differentiate monocytes into M? and IFN-? or IL-4+IL-13 to further polarize these cells towards M1 or M2, respectively. Recently, differentiation using only GM-CSF or M-CSF has been described to induce a M1- or M2-like phenotype, respectively. In this study, we combined both approaches by differentiating human M? in GM-CSF or M-CSF followed by polarization with either IFN-? or IL-4+IL-13. We describe the phenotypic differences between CD14(hi) CD163(hi) CD206(int) FOLR2-expressing M-CSF M? and CD14(lo) CD163(lo) CD206(hi) GM-CSF M? but show that both macrophage populations reacted similarly to further polarization with IFN-? or IL-4+IL-13 with up- and down-regulation of common M1 and M2 marker genes. We also show that high expression of the mannose receptor (CD206), a marker of alternative activation, is a distinct feature of GM-CSF M?. Changes of the chromatin structure carried out by chromatin modification enzymes (CME) have been shown to regulate myeloid differentiation. We analyzed the expression patterns of CME during M? polarization and show that M1 up-regulate the histone methyltransferase MLL and demethylase KDM6B, while resting and M2 M? were characterized by DNA methyltransferases and histone deacetylases. We demonstrate that MLL regulates CXCL10 expression and that this effect could be abrogated using a MLL-Menin inhibitor. Taken together we describe the distinct phenotypic differences of GM-CSF or M-CSF M? and demonstrate that M? polarization is regulated by specific epigenetic mechanisms. In addition, we describe a novel role for MLL as marker for classical activation. Our findings provide new insights into M? polarization that could be helpful to distinguish M? activation states.
Toll-like receptor (TLR) activation has been implicated in acetaminophen (APAP)-induced hepatotoxicity. Herein, we hypothesize that TLR3 activation significantly contributed to APAP-induced liver injury. In fasted wildtype (WT) mice, APAP caused significant cellular necrosis, edema, and inflammation in the liver, and the de novo expression and activation of TLR3 was found to be necessary for APAP-induced liver failure. Specifically, liver tissues from similarly fasted TLR3-deficient (tlr3(-/-) ) mice exhibited significantly less histological and biochemical evidence of injury after APAP challenge. Similar protective effects were observed in WT mice in which TLR3 was targeted through immunoneutralization at 3 h post-APAP challenge. Among three important death ligands (i.e. TNF?, TRAIL, and FASL) known to promote hepatocyte death after APAP challenge, TNF? was the only ligand that was significantly reduced in APAP-challenged tlr3(-/-) mice compared with APAP-challenged WT controls. In vivo studies demonstrated that TLR3 activation contributed to TNF? production in the liver presumably via F4/80(+) and CD11c(+) immune cells. In vitro studies indicated that there was cooperation between TNF? and TLR3 in the activation of JNK signaling in isolated and cultured liver epithelial cells (i.e. nMuLi). Moreover, TLR3 activation enhanced the expression of phosphorylated JNK in APAP injured livers. Thus, the current study demonstrates that TLR3 activation contributes to APAP-induced hepatotoxicity.
TLRs are a family of receptors that mediate immune system pathogen recognition. In the respiratory system, TLR activation has both beneficial and deleterious effects in asthma. For example, clinical data indicate that TLR6 activation exerts protective effects in asthma. Here, we explored the mechanism or mechanisms through which TLR6 mediates this effect using mouse models of Aspergillus fumigatus-induced and house dust mite antigen-induced (HDM antigen-induced) chronic asthma. Tlr6-/- mice with fungal- or HDM antigen-induced asthma exhibited substantially increased airway hyperresponsiveness, inflammation, and remodeling compared with WT asthmatic groups. Surprisingly, whole-lung levels of IL-23 and IL-17 were markedly lower in Tlr6-/- versus WT asthmatic mice. Tlr6-/- DCs generated less IL-23 upon activation with lipopolysaccharide, zymosan, or curdlan. Impaired IL-23 generation in Tlr6-/- mice also corresponded with lower levels of expression of the pathogen-recognition receptor dectin-1 and expansion of Th17 cells both in vivo and in vitro. Exogenous IL-23 treatment of asthmatic Tlr6-/- mice restored IL-17A production and substantially reduced airway hyperresponsiveness, inflammation, and lung fungal burden compared with that in untreated asthmatic Tlr6-/- mice. Together, our data demonstrate that TLR6 activation is critical for IL-23 production and Th17 responses, which both regulate the allergic inflammatory response in chronic fungal-induced asthma. Thus, therapeutics targeting TLR6 activity might prove efficacious in the treatment of clinical asthma.
Influenza A viral infections have been identified as the etiologic agents for historic pandemics, and contribute to the annual mortality associated with acute viral pneumonia. While both innate and acquired immunity are important in combating influenza virus infection, the mechanism connecting these arms of the immune system remains unknown. Recent data have indicated that the Notch system is an important bridge between antigen-presenting cells (APCs) and T cell communication circuits and plays a central role in driving the immune system to overcome disease. In the present study, we examine the role of Notch signaling during influenza H1N1 virus infection, focusing on APCs. We demonstrate here that macrophages, but not dendritic cells (DCs), increased Notch ligand Delta-like 1 (Dll1) expression following influenza virus challenge. Dll1 expression on macrophages was dependent on retinoic acid-inducible gene-I (RIG-I) induced type-I IFN pathway, and not on the TLR3-TRIF pathway. We also found that IFN?-Receptor knockout mice failed to induce Dll1 expression on lung macrophages and had enhanced mortality during influenza virus infection. Our results further showed that specific neutralization of Dll1 during influenza virus challenge induced higher mortality, impaired viral clearance, and decreased levels of IFN-?. In addition, we blocked Notch signaling by using ?-secretase inhibitor (GSI), a Notch signaling inhibitor. Intranasal administration of GSI during influenza infection also led to higher mortality, and higher virus load with excessive inflammation and an impaired production of IFN-? in lungs. Moreover, Dll1 expression on macrophages specifically regulates IFN-? levels from CD4(+)and CD8(+)T cells, which are important for anti-viral immunity. Together, the results of this study show that Dll1 positively influences the development of anti-viral immunity, and may provide mechanistic approaches for modifying and controlling the immune response against influenza H1N1 virus infection.
PD-1 and PD-L1 can be involved in tumor escape, and little is known about the role of these molecules in oral tumors or pre-malignant lesions. In the present study, we investigated the expression of PD-1 and PD-L1 in the blood and lesion samples of patients with actinic cheilitis (AC) and oral squamous cell carcinoma (OSCC). Our results showed that lymphocytes from peripheral blood and tissue samples exhibited high expression of PD-1 in both groups analyzed. Patients with AC presented higher percentage as well as the absolute numbers of CD4+PD-1+ and CD8+PD-1+ lymphocytes in peripheral blood mononuclear cells (PBMC) than healthy individuals, while patients with OSCC presented an increased frequency of CD8+PD1+ in PBMC when compared with controls. On the other hand, increased frequency of CD4+ and CD8+ T cells expressing PD-1(+) accumulate in samples from OSCC, and the expression of PD-L1 was intense in OSCC and moderate in AC lesion sites. Lower levels of IFN-? and higher levels of TGF-? were detected in OSCC samples. Our data demonstrate that PD-1 and PD-L1 molecules are present in blood and samples of AC and OSCC patients. Further studies are required to understand the significance of PD-1 and PD-L1 in oral tumors microenvironment.
Chemokines are key mediators of leukocyte recruitment during pathogenic insult and also play a prominent role in homeostasis. While most chemokine receptors bind to multiple chemokines, CCR6 is unique in that this receptor is one of only a few that can bind only a single chemokine ligand, CCL20. CCR6 is an important receptor that is involved in regulating several aspects of mucosal immunity, including the ability to mediate the recruitment of immature dendritic cells (DCs) and mature DCs, and professional antigen presenting cells (APCs) to the sites of epithelial inflammation. Further, CCR6 mediates the homing of both CD4(+) T (T-helper; Th) cells and DCs to the gut mucosal lymphoid tissue. DCs, which are known to be essential immune cells in innate immunity and in the initiation of adaptive immunity, play a central role in initiating a primary immune response. Herein, we summarize the role of CCR6 in immune responses at epithelial and mucosal sites in both the lung and gut based on a review of the current literature.
Helminths are known to elicit a wide range of immunomodulation characterized by dominant Th2-type immune responses. Our group previously showed that a DNA vaccine encoding the mycobacterial 65-kDa heat shock protein (DNA-hsp65) showed immunomodulatory properties. We also showed, using a helminth-tuberculosis (TB) co-infection model, that the DNA-hsp65 vaccine protected mice against TB. We next investigated the mechanistic role of the vaccine during helminth-TB co-infection. Clinically, helminth infection causes type 2 granulomas in the lung. Mice were immunized with DNA-hsp65 while they were submitted to the type 2 granuloma induction protocol by Schistosoma mansoni eggs infusion. In this work we investigated the effects of DNA-hsp65 on the pathology and immune response during the development of type 2 granuloma induced by S. mansoni eggs. Histologic analyses of lung parenchyma showed that the DNA-hsp65 vaccine protected mice against exacerbated fibrosis induced by Schistosoma eggs, and decreased the size of the granulomas. These changes were correlated with a reduction in the number of T cells specific for the egg antigens in the lung and also with modulation of Th2 cytokine expression. Taken together, our results showed that the adjuvant properties of the DNA-hsp65 vaccine regulated the immune response in this Th2 model, and resulted in a preserved lung parenchyma.
Studies in humans and animal models indicate that profound immunosuppression is one of the chronic consequences of severe sepsis. This immune dysfunction encompasses deficiencies in activation of cells in both the myeloid and lymphoid cell lineages. As a result, survivors of severe sepsis are at risk of succumbing to infections perpetrated by opportunistic pathogens that are normally controlled by a fully functioning immune system. Recent studies have indicated that epigenetic mechanisms may be one driving force behind this immunosuppression, through suppression of proinflammatory gene production and subsequent immune cell activation, proliferation and effector function. A better understanding of epigenetics and post-septic immunosuppression can improve our diagnostic tools and may be an important potential source of novel molecular targets for new therapies. This review will discuss important pathways of immune cell activation affected by severe sepsis, and highlight pathways of epigenetic regulation that may be involved in post-septic immunosuppression.
Previous epidemiological studies in humans and experimental studies in animals indicate that survivors of severe sepsis exhibit deficiencies in the activation and effector function of immune cells. In particular, CD4+ T lymphocytes can exhibit reduced proliferative capacity and improper cytokine responses following sepsis. To further investigate the cell-intrinsic defects of CD4+ T cells following sepsis, splenic CD4+ T cells from sham surgery and post-septic mice were transferred into lymphopenic mice. These recipient mice were then subjected to both TH1-(purified protein derivative) and TH2-(Schistosoma mansoni egg antigen) driven models of granulomatous lung inflammation. Post-septic CD4+ T cells mediated smaller TH1 and larger TH2 lung granulomas as compared to mice receiving CD4+ T cells from sham surgery donors. However, cytokine production by lymph node cells in antigen restimulation assays indicated increased pan-specific cytokine expression by post-septic CD4+ T cell recipient mice in both TH1 and TH2 granuloma models. These include increased production of T(H)2 cytokines in TH1 inflammation, and increased production of T(H)1 cytokines in TH2 inflammation. These results suggest that cell-intrinsic defects in CD4+ T cell effector function can have deleterious effects on inflammatory processes post-sepsis, due to a defect in the proper regulation of TH-specific cytokine expression.
The thermally dimorphic fungus Paracoccidioides brasiliensis (Pb) is the causative agent of paracoccidioidomycosis (PCM), one of the most frequent systemic mycosis that affects the rural population in Latin America. PCM is characterized by a chronic inflammatory granulomatous reaction, which is consequence of a Th1-mediated adaptive immune response. In the present study we investigated the mechanisms involved in the immunoregulation triggered after a prior contact with cell-free antigens (CFA) during a murine model of PCM. The results showed that the inoculation of CFA prior to the infection resulted in disorganized granulomatous lesions and increased fungal replication in the lungs, liver and spleen, that paralleled with the higher levels of IL-4 when compared with the control group. The role of IL-4 in facilitating the fungal growth was demonstrated in IL-4-deficient- and neutralizing anti-IL-4 mAb-treated mice. The injection of CFA did not affect the fungal growth in these mice, which, in fact, exhibited a significant diminished amount of fungus in the tissues and smaller granulomas. Considering that in vivo anti-IL-4-application started one week after the CFA-inoculum, it implicates that IL-4-CFA-induced is responsible by the mediation of the observed unresponsiveness. Further, the characterization of CFA indicated that a proteic fraction is required for triggering the immunosuppressive mechanisms, while glycosylation or glycosphingolipids moieties are not. Taken together, our data suggest that the prior contact with soluble Pb antigens leads to severe PCM in an IL-4 dependent manner.
Suppression of inflammation is critical for effective therapy of many infectious diseases. However, the high rates of mortality caused by sepsis attest to the need to better understand the basis of the inflammatory sequelae of sepsis and to develop new options for its treatment. In mice, inflammatory responses to host danger-associated molecular patterns (DAMPs), but not to microbial pathogen-associated molecular patterns (PAMPs), are repressed by the interaction [corrected] of CD24 and SiglecG (SIGLEC10 in human). Here we use an intestinal perforation model of sepsis to show that microbial sialidases target the sialic acid-based recognition of CD24 by SiglecG/10 to exacerbate inflammation. Sialidase inhibitors protect mice against sepsis by a mechanism involving both CD24 and Siglecg, whereas mutation of either gene exacerbates sepsis. Analysis of sialidase-deficient bacterial mutants confirms the key contribution of disrupting sialic acid-based pattern recognition to microbial virulence and supports the clinical potential of sialidase inhibition for dampening inflammation caused by infection.
Studies have shown that Notch is essential for the maintenance of a T cell Th2 phenotype in vivo. It has also been shown that Notch ligands have diverse functions during T cell activation. We chose to investigate the role of Notch ligands during the Th2 response.
Aspergillus fumigatus conidia aggravate asthmatic responses. Lung macrophages normally kill fungal conidia, but the presence of type 2 cytokines during asthma contributes to the alternative (or M2) activation of these cells, which secrete proallergic factors and exhibit impaired innate immunity.
Immunosuppression following severe sepsis remains a significant human health concern, as long-term morbidity and mortality rates of patients who have recovered from life-threatening septic shock remain poor. Mouse models of severe sepsis indicate this immunosuppression may be partly due to alterations in myeloid cell function; however, the effect of severe sepsis on subsequent CD4(+) T-cell responses remains unclear. In the present study, CD4(+) T cells from mice subjected to an experimental model of severe sepsis (cecal ligation and puncture (CLP)) were analyzed in vitro. CD4(+)CD62L(+) T cells from CLP mice exhibited reduced proliferative capacity and altered gene expression. Additionally, CD4(+)CD62L(+) T cells from CLP mice exhibit dysregulated cytokine production after in vitro skewing with exogenous cytokines, indicating a decreased capability of these cells to commit to either the T(H)1 or T(H)2 lineage. Repressive histone methylation marks were also evident at promoter regions for the T(H)1 cytokine IFN-gamma and the T(H)2 transcription factor GATA-3 in naïve CD4(+) T cells from CLP mice. These results provide evidence that CD4(+) T-cell subsets from post-septic mice exhibit defects in activation and effector function, possibly due to chromatin remodeling proximal to genes involved in cytokine production or gene transcription.
One of the more insidious outcomes of patients who survive severe sepsis is profound immunosuppression. In this study, we addressed the hypothesis that post septic immune defects were due, in part, to the presence and/or expansion of regulatory T cells (Tregs). After recovery from severe sepsis, mice exhibited significantly higher numbers of Tregs, which exerted greater in vitro suppressive activity compared with controls. The expansion of Tregs was not limited to CD25(+) cells, because Foxp3 expression was also detected in CD25(-) cells from post septic mice. This latter group exhibited a significant increase of chromatin remodeling at the Foxp3 promoter, because a marked increase in acetylation at H3K9 was associated with an increase in Foxp3 transcription. Post septic splenic dendritic cells promoted Treg conversion in vitro. Using a solid tumor model to explore the function of Tregs in an in vivo setting, we found post septic mice showed an increase in tumor growth compared with sham-treated mice with a syngeneic tumor model. This observation could mechanistically be related to the ability of post septic Tregs to impair the antitumor response mediated by CD8(+) T cells. Together, these data show that the post septic immune system obstructs tumor immunosurveillance, in part, by augmented Treg expansion and function.
Oral squamous cell carcinoma (OSCC) is a cancerous lesion with high incidence worldwide. The immunoregulatory events leading to OSCC persistence remain to be elucidated. Our hypothesis is that regulatory T cells (Tregs) are important to obstruct antitumor immune responses in patients with OSCC. In the present study, we investigated the frequency, phenotype, and activity of Tregs from blood and lesions of patients with OSCC. Our data showed that >80% of CD4(+)CD25(+) T cells isolated from PBMC and tumor sites express FoxP3. Also, these cells express surface Treg markers, such as GITR, CD45RO, CD69, LAP, CTLA-4, CCR4, and IL-10. Purified CD4(+)CD25(+) T cells exhibited stronger suppressive activity inhibiting allogeneic T-cell proliferation and IFN-gamma production when compared with CD4(+)CD25(+) T cells isolated from healthy individuals. Interestingly, approximately 25% of CD4(+)CD25(-) T cells of PBMC from patients also expressed FoxP3 and, although these cells weakly suppress allogeneic T cells proliferative response, they inhibited IFN-gamma and induced IL-10 and TGF-beta secretion in these co-cultures. Thus, our data show that Treg cells are present in OSCC lesions and PBMC, and these cells appear to suppress immune responses both systemically and in the tumor microenvironment.
Alternatively activated (M2) macrophages play critical roles in diverse chronic diseases, including parasite infections, cancer, and allergic responses. However, little is known about the acquisition and maintenance of their phenotype. We report that M2-macrophage marker genes are epigenetically regulated by reciprocal changes in histone H3 lysine-4 (H3K4) and histone H3 lysine-27 (H3K27) methylation; and the latter methylation marks are removed by the H3K27 demethylase Jumonji domain containing 3 (Jmjd3). We found that continuous interleukin-4 (IL-4) treatment leads to decreased H3K27 methylation, at the promoter of M2 marker genes, and a concomitant increase in Jmjd3 expression. Furthermore, we demonstrate that IL-4-dependent Jmjd3 expression is mediated by STAT6, a major transcription factor of IL-4-mediated signaling. After IL-4 stimulation, activated STAT6 is increased and binds to consensus sites at the Jmjd3 promoter. Increased Jmjd3 contributes to the decrease of H3K27 dimethylation and trimethylation (H3K27me2/3) marks as well as the transcriptional activation of specific M2 marker genes. The decrease in H3K27me2/3 and increase in Jmjd3 recruitment were confirmed by in vivo studies using a Schistosoma mansoni egg-challenged mouse model, a well-studied system known to support an M2 phenotype. Collectively, these data indicate that chromatin remodeling is mechanistically important in the acquisition of the M2-macrophage phenotype.
Squamous cell carcinoma (SCC) constitutes a microenvironment that could modulate the antitumor immune response. Also, tumor-infiltrating lymphocytes are believed to play complex regulatory roles in antitumor immunity against SCC. The presence of regulatory T cells (Tregs) has been associated with the suppression of tumor-reactive T cells. However, the underlying mechanism for this T cell dysfunction is not clear. We used a multistage model of SCC to examine the role of Treg cells during tumor development. 7,12-dimethylbenz[a]-anthracene/phorbol 12-myristate 13-acetate treatment and systemic depletion of Treg cells using an anti-CD25 monoclonal antibody (PC61) resulted in a decrease in the number and incidence of papilloma. Furthermore, CD25 depletion increased the proportion of CD8(+) and CD4(+) T cells that were isolated from tumor lesions. The levels of interleukin (IL)-1?, IL-10, IL-12, IL-13, interferon-?, transforming growth factor-? and tumor necrosis factor-?, but not IL-17, were increased in the tumor microenvironment after Treg depletion. Therefore, our results indicated involvement of CD25(+) T cells in SCC development and in the suppression of the inflammatory immune response.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.