Mechanism and clinical evidence of immunotherapy in allergic rhinitis.

Allergic rhinitis is a common upper airway disease caused by hypersensitivity to various aeroallergens. It causes increased inflammation throughout the body and may be complicated by other otolaryngological pathologies such as chronic hyperplastic eosinophilic sinusitis, nasal polyposis, and serous otitis media. Allergic rhinitis is an IgE-mediated disease and immunotherapy can be a possible approach for patients to limit the use of antihistamines and corticosteroids. There is evidence that allergen immunotherapy can prevent the development of new sensitizations and reduce the risk of later development of asthma in patients with allergic rhinitis. However, some patients do not benefit from this approach and the efficacy of immunotherapy in reducing the severity and relapse of symptoms is still a matter of debate. This review highlights new aspects of allergic rhinitis with a particular focus on the impact of sexual dimorphism on the disease manifestation and efficacy to the allergen specific immunotherapy.

Cancer Vaccines: From the State of the Art to the Most Promising Frontiers in the Treatment of Colorectal Cancer.

Colorectal cancer represents 10% of all new cancer cases each year and accounts for almost 10% of all cancer deaths. According to the WHO, by 2040 there will be a 60% increase in colorectal cancer cases. These data highlight the need to explore new therapeutic strategies. Classical interventions include surgical resection, chemotherapy and radiotherapy, which are invasive strategies that have many side effects on the patients and greatly affect their quality of life. A great advance in the treatment of this cancer type, as well as of all the others, could be the development of a vaccination strategy preventing the onset, the progression or the relapse of the pathology. In this review, we summarize the main vaccination strategies that are being studied for the treatment of colorectal cancer (CRC) and finally explore the possibility of using B-cells for the development of a new type of vaccine.

LPS Guides Distinct Patterns of Training and Tolerance in Mast Cells.

Mast cells (MCs) are tissue-resident, long lived innate immune cells with important effector and immunomodulatory functions. They are equipped with an eclectic variety of receptors that enable them to sense multiple stimuli and to generate specific responses according on the type, strength and duration of the stimulation. Several studies demonstrated that myeloid cells can retain immunological memory of their encounters - a process termed 'trained immunity' or 'innate immune memory'. As MCs are among the one of first cells to come into contact with the external environment, it is possible that such mechanisms of innate immune memory might help shaping their phenotype and effector functions; however, studies on this aspect of MC biology are still scarce. In this manuscript, we investigated the ability of MCs primed with different stimuli to respond to a second stimulation with the same or different ligands, and determined the molecular and epigenetic drivers of these responses. Our results showed that, while the stimulation with IgE and ß-glucan failed to induce either tolerant or trained phenotypes, LPS conditioning was able to induce a profound and long-lasting remodeling of the signaling pathways involved in the response against LPS or fungal pathogens. On one side, LPS induced a strong state of unresponsiveness to secondary LPS stimulation due to the impairment of the PI3K-AKT signaling pathway, which resulted in the reduced activation of NF-κB and the decreased release of TNF-α and IL-6, compared to naïve MCs. On the other side, LPS primed MCs showed an increased release of TNF-α upon fungal infection with live Candida albicans, thus suggesting a dual role of LPS in inducing both tolerance and training phenotypes depending on the secondary challenge. Interestingly, the inhibition of HDAC during LPS stimulation partially restored the response of LPS-primed MCs to a secondary challenge with LPS, but failed to revert the increased cytokine production of these cells in response to C. albicans. These data indicate that MCs, as other innate immune cells, can develop innate immune memory, and that different stimulatory environments can shape and direct MC specific responses towards the dampening or the propagation of the local inflammatory response.

Purification of Murine and Human IL-10-Producing B Cells from Different Anatomical Compartments.

IL-10 is the best known and most studied anti-inflammatory cytokine and, in the last 20 years, it has acquired even greater fame as it has been associated with the regulatory phenotype of B cells. Indeed, although great efforts have been made to find a unique marker, to date IL-10 remains the main way to follow both murine and human regulatory B cells, hence the need of precise and reproducible methods to identify and purify IL-10-producing B cells for both functional and molecular downstream assays. In this chapter, we present our protocols to isolate these cells from the murine spleen and peritoneum and from human peripheral blood. Since the production of IL-10 by B cells is not only a weapon to counteract the adverse effect of pro-inflammatory cytokines but also a response to cellular activation, we focused on those B cells that are prone to IL-10 production and detectable following a short-term stimulation with phorbol-12-myristate-13-acetate, ionomycin, and lipopolysaccharide (murine system) or CpG (human system).

il-10 Gene Locus DNA Methylation in Regulatory B Cells.

Epigenetic studies are becoming increasingly common in the immunology field thanks to the support of cutting edge technology and to their potential of providing a large amount of data at the single cell level. Moreover, epigenetic modifications were shown to play a role in autoimmune/inflammatory disorders, paving the way for the possibility of using the results of epigenetic studies for therapeutic purposes. In recent years, epigenetic marks such as DNA methylation, histone modifications and nucleosome positioning were shown to regulate B cell fate and function during an immune response, but very little has been done in the context of one of the most recently discovered B cell subsets, that is regulatory B cells. Although no consensus has yet been found on the identity of these immunosuppressive B cells, the role of the IL-10 cytokine is consolidated, both in the murine and human setting. In this chapter we will focus on the analysis of the methylation profile of a gene of interest and we will specifically describe cloning and pyrosequencing bisulphite sequencing PCR (BSP). Given the specific context, we will provide tips and tricks for the analysis of the il-10 gene locus. Nonetheless, the methods presented are valid for the study of any gene of interest.

Mast cells crosstalk with B cells in the gut and sustain IgA response in the inflamed intestine.

B lymphocytes are among the cell types whose effector functions are modulated by mast cells (MCs). The B/MC crosstalk emerged in several pathological settings, notably the colon of inflammatory bowel disease (IBD) patients is a privileged site in which MCs and IgA+ cells physically interact. Herein, by inducing conditional depletion of MCs in red MC and basophil (RMB) mice, we show that MCs control B cell distribution in the gut and IgA serum levels. Moreover, in dextran sulfate sodium (DSS)-treated RMB mice, the presence of MCs is fundamental for the enlargement of the IgA+ population in the bowel and the increase of systemic IgA production. Since both conventional B-2 and peritoneal-derived B cells populate the intestine and communicate with MCs in physiological conditions and during inflammation, we further explored this interplay through the use of co-cultures. We show that MCs finely regulate different aspects of splenic B cell biology while peritoneal B cells are unresponsive to the supporting effects provided by MCs. Interestingly, peritoneal B cells induce a pro-inflammatory skewing in MCs, characterized by increased ST2 and TNF-α expression. Altogether, this study uncovers the versatility of the B/MC liaison and highlights key aspects for the resolution of intestinal inflammation.

IL-10-producing B cells are characterized by a specific methylation signature.

Among the family of regulatory B cells, the subset able to produce interleukin-10 (IL-10) is the most studied, yet its biology is still a matter of investigation. The DNA methylation profiling of the il-10 gene locus revealed a novel epigenetic signature characterizing murine B cells ready to respond through IL-10 synthesis: a demethylated region located 4.5 kb from the transcription starting site (TSS), that we named early IL10 regulatory region (eIL10rr). This feature allows to distinguish B cells that are immediately prone and developmentally committed to IL-10 production from those that require a persistent stimulation to exert an IL-10-mediated regulatory function. These late IL-10 producers are instead characterized by a delayed IL10 regulatory region (dIL10rr), a partially demethylated DNA portion located 9 kb upstream from the TSS. A demethylated region was also found in human IL-10-producing B cells and, very interestingly, in some B-cell malignancies, such as chronic lymphocytic leukemia and mantle cell lymphoma, characterized by an immunosuppressive microenvironment. Our findings define murine and human regulatory B cells as an epigenetically controlled functional state of mature B cell subsets and open a new perspective on IL-10 regulation in B cells in homeostasis and disease.

Reciprocal influence of B cells and tumor macro and microenvironments in the ApcMin/+ model of colorectal cancer.

One of the most fascinating aspects of the immune system is its dynamism, meant as the ability to change and readapt according to the organism needs. Following an insult, we assist to the spontaneous organization of different immune cells which cooperate, locally and at distance, to build up an appropriate response. Throughout tumor progression, adaptations within the systemic tumor environment, or macroenvironment, result in the promotion of tumor growth, tumor invasion and metastasis to distal organs, but also to dramatic changes in the activity and composition of the immune system. In this work, we show the changes of the B-cell arm of the immune system following tumor progression in the ApcMin/+ model of colorectal cancer. Tumor macroenvironment leads to an increased proportion of total and IL-10-competent B cells in draining LNs while activates a differentiation route that leads to the expansion of IgA+ lymphocytes in the spleen and peritoneum. Importantly, serum IgA levels were significantly higher in ApcMin/+ than Wt mice. The peculiar involvement of IgA response in the adenomatous transformation had correlates in the gut-mucosal compartment where IgA-positive elements increased from normal mucosa to areas of low grade dysplasia while decreasing upon overt carcinomatous transformation. Altogether, our findings provide a snapshot of the tumor education of B lymphocytes in the ApcMin/+ model of colorectal cancer. Understanding how tumor macroenvironment affects the differentiation, function and distribution of B lymphocytes is pivotal to the generation of specific therapies, targeted to switching B cells to an anti-, rather than pro-, tumoral phenotype.

Regulatory B cells are induced by gut microbiota-driven interleukin-1ß and interleukin-6 production.

Regulatory B cells (Breg cells) differentiate in response to inflammation and subsequently restrain excessive immune responses via the release of interleukin-10 (IL-10). However, the precise inflammatory signals governing their differentiation remain to be elucidated. Here we show that the gut microbiota promotes the differentiation of Breg cells in the spleen as well as in the mesenteric lymph nodes. Perturbation of the gut microbiome imposed either by antibiotic treatment or by changes in the sterility of housing conditions reduces the number and function of Breg cells. Following the induction of arthritis, IL-1ß and IL-6 are produced only in conventionally housed mice and both cytokines directly promote Breg cell differentiation and IL-10 production. Mice lacking IL-6 receptor (IL-6R) or IL-1 receptor 1 (IL-1R1) specifically on B cells have a reduced number of IL-10-producing B cells and develop exacerbated arthritis compared to control animals. Thus, in response to inflammatory signals induced by both the gut flora and arthritis, Breg cells increase in number and restrain excessive inflammation.

IL-10 production by B cells is differentially regulated by immune-mediated and infectious stimuli and requires p38 activation.

IL-10 is an immune suppressive cytokine with pleiotropic effects on B cell biology. IL-10 production has a pivotal role for the regulatory suppressive functions that B cells exert in many physiological and pathological settings. Several exogenous stimuli and endogenous immune mediators can trigger IL-10-producing B cell maturation. To clarify and gain a better insight into the mechanisms of IL-10 production by B cells, we first compared the effects of LPS, CpG, agonistic CD40 mAb and BAFF on IL-10 production, and then we investigated the signal transduction mechanisms responsible for these responses. While infectious/danger stimuli determine the rapid production and release of IL-10 by B cells, a limited subset of CD40-poised, IL-10-competent B cells produce IL-10 in response to a later antigenic or infectious signal. Although BAFF is able to induce a similar subset of IL-10-competent B cells, these cells do not similarly respond to the same antigenic or infectious signals. Importantly, by using specific inhibitors of the MAP kinase pathways, we found that while il-10 gene expression triggered by the TLR agonists LPS and CpG is strongly dependent on p38 activity, the induction of IL-10 competence in CD40-activated B cells does not depend on ERK1/2, p38 or JNK pathways.